METHOD FOR DETERMINING WHETHER AN INFECTION IS BACTERIAL AND/OR VIRAL, AND SIDE FLOW DEVICE FOR DETE

专利摘要:
method for determining whether an infection is bacterial and/or viral, side flow device for detecting an analyte in a sample, and, device for detecting a bacterial and/or viral marker in a sample. a lateral flow assay is capable of detecting and differentiating viral and bacterial infections. a combined point of test markers in viral infection treatment diagnostic device and markers for bacterial infection, to effectively help in the rapid differentiation of viral and bacterial infections. in some embodiments, bimodal methods and devices determine whether an infection is bacterial and/or viral. a dual use two strip sample analysis device includes a first two strip lateral flow chromatographic test strip to detect mxa and a low level of c-reactive protein and a second lateral flow chromatographic test strip to detect high levels of protein c reactive. in a preferred embodiment, the sample is a fingerstick blood sample.
公开号:BR112015021199B1
申请号:R112015021199-2
申请日:2014-03-03
公开日:2022-01-11
发明作者:Robert P. Sambursky；Robert W. Vandine；Uma Manesh Babu；Peter Condon
申请人:Rapid Pathogen Screening, Inc；
IPC主号:

专利说明:

REFERENCE TO RELATED ORDERS
[001] This order claims priority from the following pending orders:
[002] US Application Serial Number 13/788,616, filed March 7, 2013, entitled “MULTIPLANAR SIDE FLOW ASSAY WITH DIVERTING ZONE”;
[003] US Application Serial Number 13/790,125, filed March 8, 2013, entitled “METHOD AND DEVICE FOR COMBINED DETECTION OF VIRAL AND BACTERIAL INFECTIONS”.
[004] US Application Serial Number 13/790,160, filed March 8, 2013, entitled “METHOD AND DEVICE FOR COMBINED DETECTION OF VIRAL AND BACTERIAL INFECTIONS”. BACKGROUND OF THE INVENTION FIELD OF INVENTION
[005] The invention relates to the field of lateral flow immunoassays. More specifically, the invention relates to a lateral flow immunoassay that rapidly detects viral and bacterial infection. DESCRIPTION AND RELATED ART
[006] Fever is a common cause of children visiting urgent care centers in both family medicine and pediatric services. Most commonly, this relates to a respiratory infection or gastroenteritis. The high incidence of fever in children and the administration of unnecessary antibiotics as a precaution is the reason for the development of a rapid screening test for biomarkers that indicate viral and/or bacterial infection.
[007] It is often challenging to differentiate between viral and bacterial infections. This is especially true in young children who cannot verbalize their symptoms and in an outpatient setting, where access to laboratory diagnosis is expensive, time-consuming, and requires several days to produce a result. More recently, several new diagnostic markers have been identified. Several of these markers show great promise for differentiating bacterial viral infections. Two of these proteins include MxA and C-reactive protein (PCR). Most respiratory infections are related to pharyngitis of which 40% are caused by viruses and 25-50% by the beta hemolytic streptococcal group A. Minor causes are acute bronchiolitis and pneumonia.
[008] Severe community-acquired pneumonia is caused by bacterial infections in about 60% of cases, requiring admission to an intensive care unit (ICU) for about 10% of patients. The remaining 30% are related to respiratory viruses.
[009] About 80% of all antimicrobial agents are prescribed in primary care, and up to 80% of these are for respiratory tract indications. Respiratory infections are by far the most common cause of cough in primary care. Broad-spectrum antibiotics are often prescribed for coughs, including acute bronchitis, and many of these prescriptions will, at best, benefit patients only marginally and may cause side effects and promote antibiotic resistance. Factors that drive clinicians to give antibiotics include the lack of an adequate diagnostic marker for bacterial infections, concern over lack of patient follow-up, and time pressure.
[010] Mx proteins are members of the superfamily of high molecular weight GTPases. Suitably, these GTPases are upregulated by type I alpha/beta or type II interferons (IFN). GTPases Mx are expressed exclusively in cells treated with IFN alpha/beta but not IFN gamma. Type I interferons play an important role in innate immune responses and have immunomodulatory, antiproliferative, and antiviral functions. Human MxA, a 78 kDa protein, accumulates in the cytoplasm of IFN-treated cells and inhibits replication of a wide variety of viruses. The MxA protein may offer certain advantages as markers for viral infections over other induced proteins such as 2', 5'-oligoadenylate synthetase, because of its lower basal concentration, longer half-life (2.3 days) and induction. fast. MxA mRNA is detectable in isolated peripheral white blood cells stimulated with IFN within 1 to 2 h of IFN induction, and MxA protein begins to accumulate shortly thereafter.
[011] Studies have shown that the expression of MxA protein in peripheral blood is a sensitive and specific marker for viral infection. The higher levels of MxA in the viral infection group compared to the bacterial infection group can be explained by the fact that the MxA protein is induced exclusively by type I IFN and not by IFN-gamma, IL-1, TNF-alpha, or any of the other cytokines by bacterial infection. Serotype I IFN levels remain within normal limits, even in patients with severe bacterial infections.
[012] Likewise, most viral infections have been reported to cause little acute phase response, and low concentrations of C-Reactive Protein (CRP) have been used to distinguish diseases of viral origin from those of bacterial etiology. Since the plasma concentration of CRP increases rapidly after stimulation and decreases rapidly with a short half-life, CRP can be a very useful tool in the diagnosis and monitoring of infections and inflammatory diseases. In Scandinavia, point-of-care PCR testing is part of the routine evaluation of patients with respiratory infections in general practice, and its use has proven cost-effective. In general practice, PCR has been found to be valuable in diagnosing bacterial diseases and in differentiating bacterial and viral infections. The diagnostic value of CRP has often been found to be greater than that of the erythrocyte sedimentation rate (TSE) and greater than or equal to that of the white blood cell count (CGB).
[013] Clinically, this can be challenging to differentiate between certain systemic viral and bacterial infections. Bacterial cultures are usually performed in cases of serious infections, such as pneumonia, or when the consequence of missing a diagnosis can lead to serious complications, such as Strep throat. Crops are often difficult to obtain. Unfortunately, viral cultures are not routinely performed given the significant delay in receiving results. New PCR viral screening panels are useful but very expensive and do not provide point-of-care information. Thus, there continues to be a need for a simple, easy-to-use diagnostic test that is able to differentiate between viral and bacterial infections. SUMMARY OF THE INVENTION
[014] The present invention provides a lateral flow assay, which is capable of detecting and differentiating viral and bacterial infections. A combined point of test markers in viral infection treatment diagnostic device and markers for bacterial infection, to effectively help in the rapid differentiation of viral and bacterial infections. In a preferred embodiment, the bacterial marker is PCR. In another preferred embodiment, the viral marker is MxA. In some embodiments of the invention, it is not necessary to lyse cells in the sample prior to application to the device.
[015] In a preferred embodiment, a method determines whether an infection is bacterial and/or viral by first taking a sample. The sample is then transferred to a dual-use two-strip sample analyzer. The sample analysis device includes a first side flow chromatographic test strip with a first reagent zone and a second reagent zone. The first reagent zone includes at least one first reagent specific to a low level of C-reactive protein such that when the sample contacts the first reagent, a labeled first complex is formed if the low level of C-reactive protein is present. in the sample. The second reagent zone includes at least one second reagent specific for MxA such that when the sample contacts the second reagent, a second labeled complex is formed if MxA is present in the sample. The first lateral flow chromatographic test strip also includes a first detection zone comprising a first binding partner which binds the first labeled complex; and a second binding partner which binds the second labeled complex. The two-strip lateral flow tester also includes a second lateral flow test strip parallel in a lateral flow direction to a first lateral flow chromatographic test strip. The second lateral flow chromatographic test strip includes at least a third reagent zone including at least a third reagent specific to a high level of C-reactive protein such that when the sample contacts the third reagent, a third complex labeled if high level of C-reactive protein is present in the sample. The third reagent on the second lateral flow chromatographic test strip only detects a level of C-reactive protein that is higher than the level of C-reactive protein detected by the second reagent on the first lateral flow chromatographic test strip. The second lateral flow chromatographic test strip also includes a second detection zone with a third binding partner which binds the third labeled complex. The sample is also analyzed for the presence of the low level of C-reactive protein, MxA, and the high level of C-reactive protein.
[016] In another preferred embodiment, a dual-use, two-stranded lateral flow assay device detects a bacterial and/or viral marker in a sample. The device includes a first side flow chromatographic test strip with a first reagent zone and a second reagent zone. The first reagent zone includes at least one first reagent specific to a low level of C-reactive protein such that when the sample contacts the first reagent, a labeled first complex is formed if the low level of C-reactive protein is present. in the sample. The second reagent zone includes at least one second reagent specific for MxA such that when the sample contacts the second reagent, a second labeled complex is formed if MxA is present in the sample. The first lateral flow chromatographic test strip also includes a first detection zone comprising a first binding partner which binds the first labeled complex; and a second binding partner which binds the second labeled complex. The two-strip lateral flow tester also includes a second two-strip lateral flow test strip parallel to the lateral flow direction of the first lateral flow chromatographic test strip. The second lateral flow chromatographic test strip includes at least a third reagent zone comprising at least one third reagent specific for a high level of C-reactive protein such that when the sample contacts the third reagent, a third complex labeled if high level of C-reactive protein is present in the sample. The third reagent on the second lateral flow chromatographic test strip only detects a level of C-reactive protein that is higher than the level of C-reactive protein detected by the second reagent on the first lateral flow chromatographic test strip. The second lateral flow chromatographic test strip also includes a second detection zone with a third binding partner which binds the third labeled complex.
[017] Another preferred embodiment is a method of determining whether an infection is bacterial and/or viral, and includes the step of collecting a sample. The sample is then transferred to a sample analyzing device. The sample analysis device includes a sample compressor with a first reagent zone including at least one first reagent specific for a low level of C-reactive protein such that when the sample contacts the first reagent, a first reagent is formed. labeled complex if low level of C-reactive protein is present in the sample, and at least one second reagent specific for MxA so that when the sample contacts the second reagent, a second labeled complex is formed if MxA is present in the sample , and a second reagent zone including at least a third reagent specific for a high level of C-reactive protein, where the third reagent detects only a level of C-reactive protein that is higher than the level of C-reactive protein detected by the second reagent , so that when the sample contacts the third reagent, a labeled third complex is formed if the high level of C-reactive protein is present in the sample. The device also includes a first lateral flow chromatographic test strip which includes a first detection zone including a first binding partner which binds to the first labeled complex, a second binding partner which binds to the second labeled complex, and a first bypass zone located upstream of the first detection zone on the lateral flow chromatographic test strip. The first bypass zone stops the lateral flow on the first lateral flow chromatographic test strip. The device also includes a second lateral flow test strip parallel in a lateral flow direction to the first lateral flow chromatographic test strip. The second lateral flow chromatographic test strip includes a second detection zone including a third binding partner which binds to the third labeled complex and a second bypass zone located upstream of the first detection zone on the flow chromatographic test strip. side. The second bypass zone stops the lateral flow on the second lateral flow chromatographic test strip. The device also includes a first sample application zone where the sample is placed on the sample analyzing device. The first sample application zone is placed in a position selected from the group consisting of: i) the first side flow chromatographic test strip upstream of the detection zone and ii) the first reagent zone of the sample compressor. The device also includes a second sample application zone where the sample is placed over the sample analyzing device. The second sample application zone is placed in a position selected from the group consisting of: i) the second lateral flow chromatographic test strip upstream of the detection zone and ii) the second reagent zone of the sample compressor. The sample compressor is in a different plane than the side flow chromatographic test strip and a second side flow chromatographic test strip. The sample compressor's first reagent zone bridges the first bypass zone, and the sample compressor's second reagent zone bridges the second bypass zone, bypassing the flow to the sample compressor and returning flow to the sample compressor. the first chromatographic test strip and the second chromatographic test strips at the end of the first bypass zone and the second bypass zone. The sample is analyzed for the presence of the low level of C-reactive protein, MxA, and the high level of C-reactive protein.
[018] Another preferred embodiment is a lateral flow device for detecting an analyte in a sample. The device includes a sample compressor with a first reagent zone including at least a first reagent specific for a low level of C-reactive protein such that, when the sample contacts the first reagent, a labeled first complex is formed if the low level of C-reactive protein is present in the sample, and at least one second reagent specific for MxA so that when the sample contacts the second reagent, a second labeled complex is formed if MxA is present in the sample, and a second reagent zone including at least a third reagent specific for a high level of C-reactive protein, where the third reagent detects only a level of C-reactive protein that is higher than the level of C-reactive protein detected by the second reagent, so as to that when the sample contacts the third reagent, a third labeled complex is formed if the high level of C-reactive protein is present in the sample. The device also includes a first lateral flow chromatographic test strip which includes a first detection zone including a first binding partner which binds to the first labeled complex, a second binding partner which binds to the second labeled complex, and a first bypass zone located upstream of the first detection zone on the lateral flow chromatographic test strip. The first bypass zone stops the lateral flow on the first lateral flow chromatographic test strip. The device also includes a second lateral flow chromatographic test strip parallel in a lateral flow direction to a first lateral flow chromatographic test strip. The second lateral flow chromatographic test strip includes a second detection zone comprising a third binding partner which binds to the third labeled complex and a second bypass zone located upstream of the first detection zone on the flow chromatographic test strip. side. The second bypass zone stops the lateral flow on the second lateral flow chromatographic test strip. The device also includes a first sample application zone where the sample is placed on the sample analyzing device. The first sample application zone is placed in a position selected from the group consisting of: i) the first side flow chromatographic test strip upstream of the detection zone and ii) the first reagent zone of the sample compressor. The device also includes a second sample application zone where the sample is placed over the sample analyzing device. The second sample application zone is placed in a position selected from the group consisting of: i) the second lateral flow chromatographic test strip upstream of the detection zone and ii) the second reagent zone of the sample compressor. The sample compressor is in a different plane than the side flow chromatographic test strip and a second side flow chromatographic test strip. The sample compressor's first reagent zone bridges the first bypass zone, and the sample compressor's second reagent zone bridges the second bypass zone, bypassing the flow to the sample compressor and returning the flow. for the first chromatographic test strip and the second chromatographic test strips at the end of the first bypass zone and the second bypass zone.
[019] In another preferred embodiment, a method by simultaneously detecting at least one extracellular analyte and at least one intracellular analyte, collecting a sample and transferring the sample to a sample analysis device. The sample is also lysed and the extracellular analyte and intracellular analyte are simultaneously detected on the same sample analyzing device. In a preferred embodiment, the extracellular analyte is C-reactive protein and the intracellular analyte is MxA protein.
[020] In another preferred embodiment, a method for detecting MxA protein and C-reactive protein in a sample includes the steps of adding the sample to a mixture of an antibody against the MxA protein conjugated to a first label and an antibody to the protein C-reactive protein conjugated to a second marker different from the first marker, detect a presence of MxA protein by determining whether the antibody to MxA protein has agglutinated, and detect a presence of C-reactive protein conjugated by detecting whether the antibody against C-reactive protein has agglutinated .
[021] In another preferred embodiment, a method for detecting the presence of an unknown viral infection in a sample is to first collect the sample. The sample is then transferred to a sample application zone of a sample analyzing device. The sample analyzing device includes a conjugated zone including a sialic acid nanomycelle with a marker within the nanomycelle and a detection zone laterally downstream of the sample application zone which includes a sialic acid nanoparticle homologue. The sample is analyzed for a positive result in the detection zone.
[022] In another preferred embodiment, a method for detecting the presence of an unknown viral infection in a sample is to first collect the sample. The sample is then transferred to a sample application zone of a sample analyzing device. The sample analysis device includes a zone conjugated to a molecule selected from the group consisting of: a nanomicel including a binding partner for a specific virus causing viral infection and a marker and a homologous sialic acid micelle including a marker within the nanomicel. The sample analysis device also includes a detection zone laterally downstream of the sample application zone, with a nanoparticle specific to the virus causing the viral infection. The sample is analyzed for a positive result in the detection zone. BRIEF DESCRIPTION OF THE DRAWINGS
[023] Fig. 1 shows visual test results from the rapid screening test window to distinguish viral and bacterial infections and an interpretation of these results.
[024] Fig. 2 shows three test cassettes with different colored lines.
[025] Fig. 3 shows a comparison of a two-line detector, in which both lines are the same color, and an extra-sensitive two-line detector, where the two lines are of different colors.
[026] Fig. 4A shows a device with a corresponding test line for the presence of a viral marker and a second, separate test line that detects the presence of a bacterial marker in an embodiment of the present invention.
[027] Fig. 4B shows a device with a corresponding test line for the presence of a viral marker and a second, separate test line that detects the presence of a bacterial marker in another embodiment of the present invention.
[028] Fig. 5A shows a sample analysis device including a lysis zone located between an application zone and a reagent zone in an embodiment of the present invention.
[029] Fig. 5B shows a sample analysis device including a lysis zone overlapping a sample application zone in an embodiment of the present invention.
[030] Fig. 5C shows a sample analysis device including a lysis zone overlapping a reagent zone in an embodiment of the present invention.
[031] Fig. 5D shows a sample analysis device including a lysis zone located between an application zone and a reagent zone in an embodiment of the present invention.
[032] Fig. 6A shows a device with a corresponding test line for the presence of a bacterial marker such as CRP levels in an embodiment of the present invention.
[033] Fig. 6B shows a device with a corresponding test line for the presence of a bacterial marker such as high levels of PCR in another embodiment of the present invention.
[034] Fig. 7A shows a sample analysis device including a lysis zone located between an application zone and a reagent zone in an embodiment of the present invention.
[035] Fig. 7B shows a sample analysis device including a lysis zone overlapping a sample application zone in an embodiment of the present invention.
[036] Fig. 7C shows a sample analysis device including a lysis zone overlapping a reagent zone in an embodiment of the present invention.
[037] Fig. 7D shows a sample analysis device including a lysis zone overlapping between an application zone and a reagent zone in an embodiment of the present invention.
[038] Fig. 8A shows a fully open sample analyzing device with the dual test strips, as well as a conjugated zone and a sample application zone over a sample compressor in a separate plane from the test strips in an embodiment of the present invention.
[039] Fig. 8B shows the sample analyzing device of Fig. 8A with part of the housing closed, but the conjugate zone still visible on the left side of the device.
[040] Fig. 8C shows the sample analyzing device of Fig. 8A after the test has started.
[041] Fig. 9A shows a negative test result for both MxA and PCR in an embodiment of the present invention.
[042] Fig. 9B shows a positive test result for MxA and an embodiment of the present invention.
[043] Fig. 9C shows a positive test result for MxA in an embodiment of the present invention.
[044] Fig. 9D shows a positive test result for PCR and an embodiment of the present invention.
[045] Fig. 9E shows a positive test result for PCR and an embodiment of the present invention.
[046] Fig. 9F shows a negative test result for both PCR and MxA, indicating co-infection in an embodiment of the present invention.
[047] Fig. 10A shows a fully open sample analyzing device with dual test strips and a conjugated zone on a sample compressor in a separate plane from the test strips in an embodiment of the present invention.
[048] Fig. 10B shows the sample analyzing device of Fig. 10A with part of the housing closed, but the conjugate zone still visible on the left side of the device.
[049] Fig. 10C shows the sample analyzing device of Fig. 10A after the test has started.
[050] Fig. 11 shows a kit for analyzing samples using a sample analyzing device in an embodiment of the present invention.
[051] Fig. 12 shows a sample analysis device with dual test strips another embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION
[052] The present invention provides a lateral flow assay that is capable of differentiating between viral and bacterial infections. Rather than testing specific analytes for a specific bacterial or viral infection, the lateral flow assays described here test for diagnostic markers that are specifically produced in a host in response to a general, nonspecific bacterial infection and a nonspecific, viral infection. general. Diagnostic markers are preferably markers of an unspecified and/or unknown disease of bacterial or viral origin. In preferred embodiments, diagnostic markers are specific markers for an immune response to an unspecified and/or unknown disease of bacterial or viral origin.
[053] A combined point of treatment diagnostic device tests markers for both viral and bacterial infections and can effectively aid in the rapid differentiation of viral and bacterial infections, for example in the outpatient office or during an urgent care visit. This ability can dramatically reduce healthcare costs by limiting misdiagnosis and subsequent antibiotic overuse. Such a practice can limit antibiotic allergies, adverse events, and antibiotic resistance. The quick result obtained from the test also allows for a diagnosis while the patient is still being examined by the doctor. In a preferred embodiment, the test result is obtained in less than 10 minutes after applying the sample to the device, and is preferably read in approximately 10 minutes. On samples that are highly positive, the test strip is visible in approximately 1-5 minutes.
[054] In a preferred embodiment of the present invention, the lateral flow immunoassay device of the present invention includes a sample transport liquid, which may be a buffer, and a chromatographic test strip containing one or more materials from the present invention. fleece or membranes with capillary properties through which the sample flows. Some preferred materials and membranes for the test strip include, but are not limited to, polyethylene terephthalate (PET) fibers, such as Dacron® fibers, nitrocellulose, polyester, nylon, cellulose acetate, polypropylene, glass fibers, and combinations. of these materials and their supports. In some embodiments of the invention, it is not necessary to lyse the cells in the sample or treat the sample in any way before applying them to the test strip.
[055] A preferred method of the present invention uses a sample analysis device, for example a chromatographic test strip, to determine whether an infection is bacterial or viral. In this method, the sample is collected and transferred to the chromatographic test strip. In a preferred embodiment, the sample is a sample including leukocytes. Test strips include the reagent zone. The reagent zone preferably includes at least a first reagent specific for a bacterial label so that when the bacterial label present in the sample contacts the first reagent, a labeled first complex is formed. The reagent zone preferably also includes at least one second reagent specific for a viral marker so that when the viral marker present in the sample contacts the second reagent, a second labeled complex is formed. The detection zone includes both a bacterial marker binding partner which binds the first labeled complex and a viral marker binding partner which binds the second labeled complex. The sample is then analyzed for the presence of the viral marker and/or the bacterial marker.
[056] A preferred embodiment of a device of the present invention includes a sample application zone. The device also includes a reagent zone which includes at least one first reagent specific for a bacterial label so that when a bacterial label present in the sample contacts the first reagent, a labeled first complex and at least one second reagent specific for a viral marker so that when the viral marker present in the sample contacts the second reagent, a second labeled complex is formed. The detection zone in the device includes a bacterial marker binding partner that binds the first labeled complex and a viral marker binding partner that binds a second labeled complex. An example of a device that can be used is a chromatographic test strip. In other preferred embodiments, some of the device zones are on one or more chromatographic test strips, while other zones (e.g., the reagent zone, the sample application zone, and/or the control binding partner ) are on a sample compressor, separate from and in a different plane of the chromatographic test strip.
[057] In a preferred embodiment, the presence of the viral marker or bacterial marker is indicated by a test line visible to the naked eye. The presence of the viral marker can be indicated by a first test line while the presence of the bacterial marker is indicated by a second test line. In some embodiments, the first test line displays a first color when positive and the second test line displays a second color different from the first color when positive. In embodiments where both the first test line and the second test line are placed in the same space on the sample analyzing device, a third color is preferably formed when both the first test line and the second test line are positive. In other embodiments, the two test lines are spatially separated from each other on the device.
[058] Viral and bacterial infections are highly contagious and difficult to differentiate clinically due to a significant overlap in signs and symptoms, which often leads to overprescribing of systemic antibiotics and promotes antibiotic resistance. In developed countries, acute respiratory infections are the main cause of morbidity, accounting for: 20% of medical consultations, 30% of absences from work, and 75% of all antibiotic prescriptions. In the US, there are approximately 76 million visits to physicians' offices annually due to acute respiratory infection. The ability to detect an immune response to an infection aids in the clinical diagnostic ability to differentiate infections resulting from a viral and/or bacterial etiology.
[059] In a preferred embodiment, the bacterial marker is PCR. In another preferred embodiment, the viral marker is MxA. In some embodiments, the detection zone also includes a control line that is visible to the naked eye when the device is operating.
[060] In a preferred embodiment, the marker for viral infection is MxA and the marker for bacterial infection is C-reactive protein (CRP). High levels of MxA protein are strongly correlated with systemic viral infection and increased CRP is more associated with bacterial infections. The present invention includes a rapid infectious screening test to identify MxA and PCR in samples. MxA is present in leukocytes (white blood cells). Therefore, the sample can be taken anywhere leukocytes are available, for example in a sample of peripheral blood, nasopharyngeal aspirates, tears, spinal fluid, and middle ear aspirates.
[061] In some embodiments with a single test strip containing both PCR and MxA, the threshold concentration of PCR in a sample required to elicit a positive result is approximately 6-15 mg/L. In other preferred embodiments, the threshold concentration of MxA in a sample to elicit a positive result can be as low as approximately 15 ng/ml; however, the threshold concentration may be higher, ranging from approximately 20 ng/ml to approximately 250 ng/ml. The threshold concentration may depend on the size of the sample to be applied to the test strips, as well as their dilution, if applicable.
[062] In some embodiments, the device and methods described herein allow rapid, visual, qualitative in vitro detection of both MxA and PCR directly from peripheral whole blood. In a preferred embodiment, the test measures an immune response to a suspected viral and/or bacterial infection in patients over one year of age who experience an outbreak of fever within seven days, with respiratory symptoms consistent with respiratory disease. , and with suspected diagnosis of acute pharyngitis or community-acquired pneumonia. Negative results do not necessarily exclude respiratory infection and should not be used as the sole basis for diagnosis, treatment, or other management decisions. In some embodiments, the use of additional laboratory tests (e.g. bacterial and viral culture, immunofluorescence, viral polymerase chain reaction, and radiography) and clinical presentation is preferably additionally used to confirm whether or not there is a specific lower respiratory or pharyngeal pathogen.
[063] In addition, there are some conditions that lead to false positives or erroneous negatives. These include, but are not limited to, current use of immunosuppressive drugs by the patient providing the sample, current use of oral anti-infective drugs by the patient providing the sample, current use of interferon therapy (e.g. for multiple sclerosis, HIV, HBV, HCV) by the patient providing the sample and live viral immunization within the last 30 days by the patient providing the sample. Both false negatives and false positives are possible as levels may vary due to therapy.
[064] In preferred embodiments, the device and methods are intended for professional use in an outpatient clinic or urgent care clinic and should be used in conjunction with other clinical (laboratory or radiographic) and epidemiological information.
[065] In preferred embodiments, the dual use of dual chromatographic test strip assays detects the body's immune response and viral and/or bacterial infections in patients using a multiplexed pattern of results. In a specific preferred embodiment, the assay tests for resistance to Myxovirus A (MxA), low levels of C-reactive protein ("low" CRP), and high levels of C-reactive protein ("high" CRP). Two test strips are preferably used. In some embodiments, a sample compressor in a different plane than the chromatographic test strip is also used. The first test strip assays for MxA and low levels of C-reactive Protein, and the second test strip is an assay for high levels of C-reactive protein. The first test strip and/or sample compressor include reagents to detect MxA protein and a low level of C-reactive protein. The second test strip and/or sample compressor include reagents to detect a high level of C-reactive protein. The two test strips are preferably run side by side, and each strip preferably also includes a control line. Control reagents are preferably either on the test strips or in the sample compressor. These tests detect and classify biological infections as viral, bacterial, or a co-infection of viruses and bacteria. In some preferred embodiments, dual use of the dual chromatographic test strip assay is used to detect samples from patients with a febrile respiratory illness.
[066] In some embodiments with two test strips, over the first test strip, a threshold concentration of CRP ("low" level CRP) of approximately 6-15 mg/l (serum cut-off value) in the sample is required to elicit a positive result, and a concentration threshold of at least 15 ng/ml of MxA in a sample is required to elicit a positive result. In other preferred embodiments, the threshold concentration of MxA can be in a range from approximately 15 ng/ml to approximately 250 ng/ml to elicit a positive result. The threshold concentration may depend on the size of the sample to be applied to the test strips, as well as their dilution, if applicable. In a preferred embodiment, the low CRP threshold concentration, for example in the extracellular serum of a blood sample, is 7 mg/L for a fingerstick cut-off value, which is equivalent to 10 mg/L for a fingerstick cut-off value. of serum cut. In a preferred embodiment, the concentration of MxA, for example in the peripheral blood mononuclear cells of a blood sample, is 40 ng/ml for a fingerstick cut-off value, which is equivalent to 40 ng/ml for a cut-off value of venous blood. In the second test strip, a threshold concentration of PCR ("high" level of PCR) of approximately 60-100 mg/L in the sample is required to elicit a positive result in some embodiments. In a particularly preferred embodiment, a high CRP threshold concentration in the second test strip is approximately 80 mg/L at a fingerstick cut-off value.
[067] In other embodiments, other markers for viral infection and/or bacterial infection may be used. For example, approximately 12% of host genes change their expression after Lymphocyte Choriomeningitis Virus (LCMV) infection, and a subset of these genes can discriminate between virulent and non-virulent LCMV infection. Large transcriptional changes have given preliminary confirmation by quantitative PCR and protein studies and are potentially valuable candidates as biomarkers for arenavirus disease. Other markers for bacterial infection include, but are not limited to, procalcitonin, urinary trypsin inhibitor (uTi), lipopolysaccharide, IL-1, IL-6, IL-8, IL-10, ESR, and an elevated CGB count ( increased), lactate, troponin, vascular endothelial growth factor, platelet-derived growth factor, cortisol, proadrenomedullin, macrophage migratory inhibitory marker, activated protein C, CD 4,8,13,14, or 64, caspase, placenta-derived growth, calcitonin gene-related peptide, high mobility group 1, copeptin, naturietic peptides, lipopolysaccharide binding protein, tumor necrosis factor alpha, circulating endothelial progenitor cells, complement 3a, and trigger receptor expressed on myeloid cells (train-1).
[068] In one embodiment, the infections to be distinguished are respiratory infections. In other embodiments, other types of infections, which may be bacterial or viral in origin, are differentiated using the system of the present invention. Some examples include, but are not limited to, encephalitis, meningitis, gastroenteritis, febrile respiratory illness (including bronchitis, pharyngitis, pneumonia), sinusitis, otitis media, urinary tract infections, and conjunctivitis.
[069] Lateral flow devices are known, and are described in, for example, US Patent Application Publication Nos. 2005/0175992 and 2007/0059682. The contents of these two patents are incorporated herein by reference. Other lateral flow devices known in the art may alternatively be used with the systems and methods of the present invention.
[070] Published US Patent Application No. 2007/0059682 discloses the detection of an analyte and a sample that may also contain one or more interfering substances. This publication teaches the separation of analyte from interfering substances by capturing the interfering substances on the chromatographic support, and detecting the analyte with the carrier separated from the interfering substances.
[071] US Patent Application Publication No. 2005/0175992 discloses a method for detecting targets, such as pathogens and/or allergy-associated components, in a human body fluid when the body fluid sample is collected by a device collection, such as a swab member. Samples are transferred from the swab limb to a sample analysis device, where analysis of targets can take place by enzymatic or immunochemical means. The test result can be displayed in a very short period of time and can be read directly by the user. This allows for point-of-care testing with results available during a patient visit. The inventions described in this copending patent application are particularly advantageous for the diagnosis of conjunctivitis.
[072] In a method of the invention, the sample to be analyzed is applied to a chromatographic carrier. The carrier may be made from a single chromatographic material, or preferably, several capillary active materials made from the same or different materials and fixed to a carrier support. These materials are in close contact with each other to form a transport path along which a liquid driven by capillary forces flows from an application zone, past a reactant zone, towards one or more zones. detection and optionally a waste zone at the other end of the conveyor. In other embodiments, the liquid passes the reagent zone before flowing into a sample application zone. In an especially preferred embodiment, the carrier is a chromatographic test strip. In other preferred embodiments, the sample may be applied to a sample compressor in a plane other than the chromatographic test strip, and then transferred to the chromatographic test strip by the sample compressor.
[073] In some embodiments, the sample is applied directly to the conveyor by immersing the conveyor application zone in the sample. Alternatively, application of the sample to the carrier may be carried out by collecting the sample with a wet or dry cleaning element from which the sample may be transferred, optionally after wetting, to the application zone of the carrier. Usually, the cleaning element is sterile and may be dried or pretreated with a fluid prior to the collection step. Materials suitable for cleaning the elements according to the invention may comprise synthetic materials, fabrics or fibrous webs. Some examples of such wiper elements are described in German Patents DE 44 39 429 and DE 196 22 503, which are incorporated herein by reference. In other embodiments, the sample may be collected by a collection receptacle, such as a pipette, and transferred directly to the conveyor.
[074] Depending on the type of detection method, different reagents are present in the reagent zone of the carrier, which, in some embodiments, is preferably located between the application zone and the detection zone or, in other embodiments embodiment, is preferably located before the application zone. In still other embodiments, the reagents may be on the sample compressor separate from and in a different plane than the conveyor including the detection zone.
[075] In a sandwich immunoassay, it is preferable to have a non-immobilized, labeled reagent in the reagent zone that is specific for each bacterial and viral marker being detected. Thus, when a viral or bacterial marker present in the sample contacts the corresponding labeled viral or bacterial reagent present in the reagent zone, a labeled complex is formed between the label and the corresponding labeled reagent. The labeled complex in turn is capable of forming an additional complex with a viral or bacterial marker binding partner immobilized on the test line in the detection zone. In a competitive immunoassay, the reagent zone preferably contains a labeled, non-immobilized label analog that competes with the label for the label-binding partner immobilized in the detection zone. The tag-binding partners in the reagent zone and detection zone are preferably monoclonal, polyclonal or recombinant antibodies or antibody fragments capable of specific binding to the corresponding tag.
[076] In a preferred embodiment, the present invention provides for the reduction of interfering substances that may be present in the test sample. Since an interfering substance, for example a human anti-mouse antibody (HAMA), may also be able to form a complex with the labeled, non-immobilized reagent from the reagent zone and the immobilized binding partner from the detection zone, thereby indicating a positive result in the immunoassay, the carrier may additionally include at least one capture zone. Each capture zone containing an immobilized capture reagent specifically binds to a particular interfering substance, thereby immobilizing the interfering substance in the capture zone. As the capture zone is separated from the detection zone by space, and the sample begins to migrate over the reagent zone and capture zone before reaching the carrier detection zone, the method allows for the separation of the substance or substances interference from the analyte or analytes of interest. Preferably, the capture zone is located between the reagent zone and the detection zone. However, the capture zone can also be located between the application zone and the reagent zone.
[077] Marker detection can be achieved in a detection zone. The binding molecule immobilizes the labeled complex or labeled label analogue by immune reaction or other reaction in the detection zone, thus building a visible test line in the detection zone during the process. Preferably, the label is an optically detectable label. The formation of a complex on the test line concentrates and immobilizes the marker and the test line becomes visible to the naked eye, indicating a positive test result. Direct markers are particularly preferred, and more particularly gold markers which can be better recognized with the naked eye. Additionally, an electronic reading device (eg based on a photometric, acoustic, impedimetric, potentiometric and/or amperometric transducer) can be used to obtain more accurate results and a semi-quantitation of the analyte. Other labels can be latex, fluorophores or phosphophores.
[078] In one embodiment, the sensitivity of the visual readout side flow immunoassays is increased by adding a small amount of fluorescent dye or conjugated fluorescent latex beads to the conjugated starting material. When the visible spectrum test line is visibly present, the test result is observed and recorded. However, in the case of weak positives that do not give rise to a distinct visual test line, light of a suitable spectrum, such as a UV spectrum, is thrown onto the test line to excite and fluoresce the fluorescent latex granules that are connected on the test line to increase the visible color on the test line.
[079] In a preferred embodiment, the reagents are configured such that the visible test line corresponding to the presence of the viral marker will be separated from the test line corresponding to the presence of the bacterial marker. However, one can easily determine whether the sample contained bacterial or viral markers (or both) simply by locating the development of test lines in the detection zone. In another preferred embodiment, reagents can be chosen as well as test lines developed in different colors. That is, the presence of a viral marker will cause the development of a different colored line than that developed by the presence of a bacterial marker. For example, the label corresponding to the reagent that recognizes the viral marker may be red, while the label corresponding to the reagent that recognizes the bacterial marker may be green. Differently, colored labels are known that can be associated with the non-immobilized reagent. Some examples include, but are not limited to, colloidal gold, colloidal selenium, colloidal carbon, latex granules, paramagnetic granules, fluorescent and chemiluminescent labels, and mixtures thereof.
[080] Figures 4A and 4B show a chromatographic test strip (400) with a test line (402) corresponding to the presence of a viral marker and a second, separate test line (403) that detects the presence of a marker bacterian. The sample is applied to the application zone (401) of the chromatographic test strip (400). As shown in Figure 4A, the sample then passes a reagent zone (460) containing at least one labeled viral binding partner and at least one labeled bacterial binding partner which is eluted and then able to migrate with a carrier liquid (eg a buffer solution). Alternatively, as shown in Figure 4B, the reagent zone (460) is located upstream of a sample application zone (401) such that the labeled binding partners in the reagent zone are eluted by the carrier liquid. samples and travel to the sample. The labeled viral binding partner is capable of specifically binding a viral marker of interest to form a complex which in turn is capable of specifically binding another specific reagent or binding partner in the detection zone. The labeled viral binding partner is capable of specifically binding a bacterial marker of interest to form a complex which in turn is capable of specifically binding another specific reagent or binding partner in the detection zone. Although not shown in these Figures, an absorbent pad, as well as other known lateral flow immunoassay components including, but not limited to, a waste zone, a carrier support, a housing, and an opening in the housing for reading the result, may optionally also be a component of the test strips (400) in these embodiments.
[081] The test strips (400) also include a detection zone (405) containing at least a first section for detecting a viral marker, for example a test line (402), including an immobilized specific binding partner, complementary to the viral reagent complex formed by the viral tag and its labeled binding partner. Thus, at the test line (402), the binding partner detection zone traps the tagged viral binding partners from the reagent zone (460) along with their bound viral markers. This location of the viral marker with its labeled binding partners gives rise to an indication on the test line (402). In the test line (402), the presence of the viral marker is determined by qualitatively and/or quantitatively reading the test line (402) indication resulting from the accumulation of the labeled binding partners.
[082] The detection zone (405) also includes at least a second detection section of a bacterial marker, for example a test line (403), including an immobilized specific binding partner, complementary to the bacterial reagent complex formed by the bacterial marker and its labeled binding partner. Thus, at the test line (403), the binding partner detection zone traps the labeled bacterial binding partners of the reagent zone (460) along with their bound bacterial labels. This location of the bacterial marker with its labeled binding partners gives rise to an indication on the test line (403). In the test line (403), the presence of the bacterial marker is determined by qualitatively and/or quantitatively reading the test line (403) indication resulting from the accumulation of the labeled binding partners. While the test line (402) is upstream of the test line (403) with respect to the flow direction (408) in the figures, in alternative embodiments, the test line (403) is upstream of the test line (402). In yet another embodiment, test lines (402) and (403) are located at the same location on the test strips.
[083] Optionally, the detection zone (405) can contain other test lines to detect other viral and/or bacterial markers, as well as a control line (404). The control line (404) indicates that the labeled specific binding partner has traveled through the length of the assay, even though it may not have any viral or bacterial markers attached, thus confirming the proper operation of the assay. As shown in Figures 4A through 4B, the control zone (404) is preferably downstream of the test lines (402) and (403). However, in other embodiments, the control zone (404) may be located upstream of each or both of the test lines (402) and (403).
[084] In a preferred embodiment, the control line (404) includes an antibody or other recombinant protein that binds to a component of the elution medium or other composition to be used in the test. In embodiments where nucleic acids are the targets, the control line (404) preferably includes a nucleic acid, in addition to the labeled nucleic acid in use, as a binding partner for the target nucleic acid.
[085] Although only one test line is shown in the figures for each of the viral and bacterial markers, multiple test lines for either or both of the viral and bacterial markers can be used in the spirit of the invention. In some embodiments where there are multiple bacterial and/or viral targets, the presence of each target preferably corresponds to a separate test line (402) or (403). In other embodiments, both bacterial and viral markers are detected in a single test line. In these embodiments, the presence of both bacterial and viral markers in the same test line has different characteristics than the presence of either bacterial or viral markers alone. For example, the presence of both bacterial and viral markers in the same test line can be visually indicated by a different color than the presence of either bacterial marker or viral marker alone.
[086] Whole fresh blood samples from patients showing symptoms of viral infections (flu-like symptoms and fever > 100.5°F) were tested to determine which blood MxA levels can be detected with flow tests side described here. The lateral flow assays used in these experiments had a similar configuration as the device shown in Figure 4B described above, without a second test line for the presence of a bacterial marker. More specifically, test strips include a reagent zone upstream of a sample application zone. The reagent zone includes mobilized antibodies to MxA (Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan) labeled with colloidal gold. The test strips also include a test line in the detection zone. The test line included an immobilized antibody to MxA (Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan). The control line in the detection zone included rabbit anti-chicken antibody plus rabbit Ig (for an additional stabilizing effect), which binds to mobilized chicken IgY labeled with blue latex beads.
[087] Whole blood samples were collected with EDTA as an anticoagulant. In these tests, the amount of MxA protein in the blood samples was determined using an MxA Protein ELISA Test Kit (Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan). The blood was lysed 1:10 with the lysis solution provided in the kit, before being applied to the test strips. 100 μl of lysed blood was tested in the ELISA assay. 10 μl of lysed blood was used as a sample in the MxA lateral flow test.
[088] The lysed blood samples were applied to the test strip application area. The MxA antibodies labeled in the reagent were eluted by the sample transport liquid and transported into the blood samples. In the test line, the immobilized MxA antibody trapped any labeled MxA antibody in the MxA-bound reagent zone. This location of MxA with its labeled antibody gave a red visual indication on the test line if there was a sufficient concentration of MxA. TABLE 1


[089] Table 1 shows the standards of the MxA ELISA kit performed according to the test instructions. As shown in Table 1, an MxA concentration of 24 ng/ml produced a positive result in the lateral flow test. The standard kit was used to generate the standard curve from which MxA concentrations were determined.
[090] Table 2 shows the results of clinical samples of fresh whole blood from patients showing symptoms of viral infections (flu-like symptoms and fever > 100.5°F). TABLE 2

[091] The OD (optical density) values were used in combination with the standard curve of the kit standard in order to determine the concentration of MxA in the samples. The column concentration (ng/ml) was the dilution concentration with the lysing agent. The concentration x dilution (10x) in the column (ng/ml) was the actual concentration in the whole blood sample. As shown in the table, the lateral flow test produced a positive result for MxA in samples C and F, which had approximately 105 ng/ml MxA and approximately 22 ng/ml MxA, respectively, in the samples.
[092] Table 3 shows the results of total frozen samples from normal subjects from the Tennessee blood bank. None of the blood samples had any noticeable amounts of MxA, and they were all negative in the lateral flow test. TABLE 3

[093] Table 4 shows fresh frozen whole blood samples from BioReclamation (BioReclamation, Hicksville, NY) from patients showing visible symptoms of viral infections (flu-like symptoms and fever >100.5°F). None of these patients had ODs corresponding to MxA levels higher than approximately 8 ng/ml. These samples were negative in the lateral flow test. TABLE 4

[094] The results of these tests indicate that the lateral flow tests described herein can detect MxA levels of at least as low as approximately 20 ng/ml in a 10 μl sample (diluted 1:10).
[095] An example of a rapid screening test to distinguish viral and bacterial infections is shown in Figure 1. As discussed above, MxA is a diagnostic marker for viral infection, while CRP is a diagnostic marker for bacterial infection. In this example, the blue line ("control line" in Figure A-D) represents the control. The green line represents a C-reactive protein (CRP) level > 15 mg/L ("PCR test" in A-D of the figure). The red line represents an MxA level >20 ng/ml ("MxA test" in A-D of the figure). A positive result for the MxA protein with a negative result for the PCR protein indicates only a viral infection (Visual Test Result A). A positive (PCR) result with a negative result for the MxA protein indicates only a bacterial infection (Visual Test Result B). A positive result for both MxA and PCR indicates co-infection (infection with bacteria and viruses) (Visual C Test Result). No bacterial or viral infection is indicated by the negative result for both MxA and PCR (Visual Test Result D). While certain color lines are discussed in this example, other colors, or the same colors at different locations on the test strips to indicate viral or bacterial markers, are within the spirit of the present invention.
[096] When developing different colored lines is used, the lines may or may not be separated by a space. In the latter case, the markers are chosen so that the color seen when both markers are present is different from the colors seen when the individual markers are present. For example, the presence of a viral marker can be indicated by the red line; the presence of the bacterial marker by the blue line; and the presence of both by the purple line (red and blue combined).
[097] The use of two colors to distinguish acute and chronic infection is shown in Figure 2. In the first cassette, only IgM antibodies are present, which indicate an acute infection. On this cassette, the test line is red. On the second cassette, the test line is blue because the immunoglobulins are IgG. The third cassette shows an intermediate case, where IgM and IgG antibodies are both present. Consequently, the test line is purple. While this example is shown for testing IgM and IgG, the same concept is alternatively used with a single line that detects both viral and bacterial markers for infection.
[098] In another preferred embodiment, the test strip may also include a control section which indicates the functionality of the test strips. Figure 1 shows a control line. Figure 2 shows an example where there is a control section for all three cassettes. If present, the control section may be designed to signal the user that the device has operated. For example, the control section may contain a reagent (e.g., an antibody) that will bind to labeled reagents in the reagent zone. In a preferred embodiment, chicken anti-rabbit is used as the control line and chicken IgY conjugated to a marker, for example blue latex beads, is the control conjugate. Alternatively, the control section may contain an anhydrous reagent which, when wetted, produces a color change or color formation, for example anhydrous copper sulfate which turns blue when wetted with an aqueous sample. As a further alternative, the control section may contain immobilized viral and bacterial markers that will react with excess labeled reagent from the reagent zone. The control section can be placed upstream or downstream in the detection zone. A positive control indicator informs the user that the sample has permeated the required distance along the test device.
[099] Figure 3 compares two test strips, the "Adeno 1" and the "Adeno HS", which include the two control lines. On Adeno 1, the two control lines (upper line on each cassette) and the test line (lower line on each cassette) are red. On Adeno HS, the control line is blue and the test line is red. In embodiments where the control line is a different color than the test line, it is easier to distinguish between the two lines, and ensure that the test is working.
[0100] In some embodiments, the device and methods of the present invention include the zone of lysis to help differentiate between viral and bacterial infections. In these embodiments, the sample that has been collected is not lysed prior to collection and transfer to the sample analysis device. This decreases the number of steps needed to collect and prepare the sample for analysis. One situation where a lysis agent improves assay efficiency is when assaying for the presence of MxA. As discussed here, the presence of this protein can help distinguish between bacterial and viral infection in febrile children. In situ lysis using a combination of 1% to 6% weight/volume CHAPS and 0.5% to 2% weight/volume NP40 as the lysis agent improves detection of MxA in fresh or frozen whole blood.
[0101] In embodiments using a lysing agent, after loading the sample, the sample mobilized with the carrier liquid (buffer) will encounter the lysing agent. The lysing agent should preferably be preloaded onto the test strips and is eluted by the carrier liquid. In some embodiments the lysing agent has been dried on the test strip. Alternatively, the lysing agent can be pre-dried by freeze drying or lyophilization and then preloaded onto the test strip. In other embodiments, the lysis agent can be absorbed, adsorbed, soaked in, or entrapped on the test strip. The initially dried lysis agent is preferably located between the sample application zone and a reagent zone. In embodiments where the reagent zone is upstream of the sample application zone, the lysis zone is downstream of the sample application zone. The lysis agent is preferably soluble in the sample transport liquid, and the lysis agent is solubilized and activated upon contact with the sample transport liquid. The sample transport liquid then contains the lysing agent in both solution and suspension and the suspended sample components. Any components susceptible to lysis in the sample, which are then exposed in suspension to the lysing agent, are themselves lysed in situ. The running buffer then transports the analyte, including non-lysed components, to the detection zone.
[0102] The location where the lysing agent is preloaded and dried can be varied as needed. In order to maximize the time the sample has to interact with the lysing agent as well as to minimize the amount of lysing agent reaching the detection zone, the dry, absorbed, adsorbed, incorporated or trapped lysing agent can be located downstream. or even in the sample application area. Or, in order to minimize the distance the lysis product must travel before reaching the reactant zone, the dry lysis agent may be located closer to the reactant zone. In other embodiments, the lysis agent may be included in the running buffer.
[0103] The concentration of the preloaded lysing agent in a test strip is preferably between 0.001% and 5% weight/volume. The volume to be preloaded depends on where the lysis agent is preloaded. Appropriate ranges are 1 to 10 microliters when preloaded on the sample collection fleece (the sample application zone) or 5 to 50 microliters when preloaded on the absorbent pad or other locations within the test strip. Ideally, the pre-loaded amount should be approximately 3 microliters pre-loaded on the sampler fleece or approximately 10 microliters pre-loaded on the absorbent pad or other locations within the test strip.
[0104] Selection of a specific lysis environment and agent will depend on viral and bacterial markers and assay. pH and ionic strength are key to the lysis environment. Depending on the pH set by the lysing agent, a pH below 4.0 tends to precipitate materials, especially proteins. Higher pH, above approximately 10.0, tends to lyse materials such as proteins and cell walls. Therefore, a pH of approximately 10.0 or above is preferred for many applications. Alternatively, lower pH may be preferred for nucleic acid targets.
[0105] Like the ionic strength established by the lysis agent, both high and low ionic strength can be used for lysis. For example, a lower (hypotonic) ionic strength tends to break down erythrocytes. For example, water itself can lyse erythrocytes. Higher ionic strength environments can be used to disrupt certain cell walls and membranes.
[0106] As for specific lysis agents, they can be grouped and selected based on their properties: salts, amphoteric and cationic agents, and ionic and non-ionic detergents. The salt, Ammonium Chloride (NH4Cl) lyses erythrocytes. Other salts, including, but not limited to, high concentrations of Sodium Chloride (NaCl) and Potassium Chloride (KCl), can disrupt certain cell walls and membranes. Other lysis agents are amphoteric agents including, but not limited to, Lyso PC, CHAPS, and Zwittergent. Alternatively, cationic agents including, but not limited to, C16 TAB and Benzalkonium Chloride can be used as a lysis agent. Both ionic and non-ionic detergents are often used to break down or lyse cell wall or cell membrane components such as lipoproteins and glycoproteins. Common ionic detergents include, but are not limited to, SDS, Cholate, and Deoxycholate. Ionic detergents are good solubilizing agents. Antibodies retain their activity in SDS at 0.1% or less. Common nonionic detergents include, but are not limited to, Octylglucoside, Digitonin, C12E8, Lubrol, Triton X-100, Noniodet P-40, Tween 20, and Tween 80. Nonionic and mildly ionic detergents are weaker denaturants and are often used to solubilize membrane proteins such as viral surface proteins. Additional lysing agents include, but are not limited to, urea and enzymes. Combinations of different lysis agents can be used to optimize the lysis environment.
[0107] Surfactants are generally wetting agents and decrease the surface tension of a liquid. This then allows for easier spreading by decreasing the interfacial tension between liquids. Therefore, surfactants can interfere with the natural binding of antigen and antibody or ligand and receptors. Concentrations are therefore experimentally chosen for each class of lysis agent. Once lysis occurs it is important that the desired binding reactions are not impaired. Generally, a lysing agent concentration of 0.001% is considered the lower limit, and the upper limit is approximately 1%. There is an additive or synergistic effect when combinations of lysis agents are used. This expands the working range of operating concentrations from approximately 0.001% to 1%. Finally, some undesired non-specific binding can be prevented at a Tween 20 concentration of 5%. In all cases, the total amount of lysis agent preloaded at all locations on an individual test strip must be sufficient to lyse barriers to immunodetection, allowing for practical test strip operation.
[0108] The lysis agent itself shall not interfere with any other assay detecting or indicator agents and therefore not interfere with any other assay interactions and reactions to an extent that would prevent the practical operability of the assay. A lysis agent must have sufficient shelf life to permit manufacture, distribution, and storage prior to use of a test strip in point-of-care testing.
[0109] In preferred embodiments where MxA is the viral marker, in situ lysis using a combination of 1% to 6% weight/volume of CHAPS and 0.5% to 2% weight/volume of NP40 as the lysis agent is preferably used. As a specific example, 2 microliters of 100 mM HEPES buffer (pH 8.0) containing 5% CHAPS and 2% NP-40 with 150 mM Sodium Chloride, 0.1% BSA, and 0.1% Sodium Azide (all weight/volume percentages) are dried in the lysis zone of the test strip.
[0110] In a preferred embodiment, as shown in Figures 5A through 5D, the sample is applied to the application zone (201) on a chromatographic test strip (200). The sample passes the lysis zone (250), where the lysing agent will preferably have been preloaded onto the test strips and is eluted by the carrier liquid. The lysing agent lyses any lysable components in the sample in situ.
[0111] The chromatographic test strip contains a sample application zone (201), the lysis zone (250) containing a lysing agent, and a reagent zone (260) containing at least one labeled binding partner that binds to a viral tag and at least one tagged binding partner that binds to a bacterial tag which are eluted by and then able to migrate with a sample of carrier liquid (e.g. a buffer solution). While the reagent zone (260) is shown downstream of the sample application zone in these figures, in alternative embodiments, the reagent zone (260) may be upstream of the sample application zone (see Figure 4B), provided that the reagents encounter the sample at some point after the sample reaches the lysis zone and is effectively lysed. The labeled viral binding partner is capable of specifically binding a viral or bacterial marker of interest to form a complex which in turn is capable of specifically binding another specific reagent or binding partner in the detection zone. Although not shown in these Figures, an absorbent pad, as well as other known lateral flow immunoassay components including, but not limited to, a waste zone, a carrier support, a housing, and an opening in the housing for reading the result, may optionally also be a component of the test strips (200) in these embodiments.
[0112] In a preferred embodiment, the lysis agent is located in the lysis zone (250) between the sample application zone (201) and the reagent zone (260). The lysis agent is preferably soluble or miscible in the sample transport liquid, and the lysis agent is solubilized and activated upon contact with the sample transport liquid. The sample transport liquid then contains the lysing agent in both solution and suspension and the suspended sample components. Any components susceptible to lysis in a sample, which are then exposed in suspension to the lysing agent, are themselves lysed in situ. The running buffer then transports the sample, including any of the non-lysed components, to the detection zone (205).
[0113] The lysis zone (250) is preferably located between the sample application zone (201) and the reagent zone (260), as shown in Figure 5A. In other embodiments, the lysis zone (250) overlaps the sample application zone (201), the reagent zone (260), or both the sample application zone (201) and the reagent zone. (260) as shown in Figures 5B, 5C, and 5D, respectively. Note that the figures are schematic, not drawn to scale. The amount of overlap between different zones (as shown in Figures 5B through 5D) can be highly variable.
[0114] The test strips (200) also include a detection zone (205) containing a first section for detecting at least one bacterial marker, e.g. a test line (203), including an immobilized specific binding partner , complementary to the bacterial complex formed by the bacterial marker and its labeled binding partner. Thus, at the test line (203), the binding partner detection zone traps the labeled bacterial binding partners of the reagent zone (260) along with their bound bacterial labels. This location of the bacterial markers with their labeled binding partners gives rise to an indication on the test line (203). In the test line (203), the presence of a bacterial marker is determined by qualitatively and/or quantitatively reading the test line (203) indication resulting from the accumulation of the labeled binding partners.
[0115] The detection zone (205) also includes a second section for detecting at least one viral marker, for example a test line (202), including an immobilized specific binding partner, complementary to the viral conjugate formed by the viral marker and its tagged binding partner. Thus, at the test line (202), the binding partner detection zone traps the labeled viral binding partners from the reagent zone (260) along with their bound viral markers. This location of the viral markers with their labeled binding partners gives rise to an indication at the test line (202). In the test line (202), the presence of a viral marker is determined by qualitatively and/or quantitatively reading the test line (202) indication resulting from the accumulation of the labeled binding partners. While the test line (203) is upstream of the test line (202) with respect to the flow direction (208) in the figures, in alternative embodiments, the test line (202) is upstream of the test line (203). In still other embodiments, test lines (202) and (203) are located at the same location on the test strips.
[0116] Optionally, the detection zone (205) can contain other test lines to detect other bacterial and/or viral markers, as well as a control line (204). The control line (204) indicates that the labeled specific binding partner has traveled the length of the assay, even though it may not have any tags attached, thus confirming the proper operation of the assay. As shown in Figures 5A through 5D, the control zone (204) is preferably downstream of the test lines (203) and (202). However, in other embodiments, the control zone (204) may be located upstream of each or both of the test lines (203) and (202).
[0117] In a preferred embodiment, the control line (204) includes an antibody or other recombinant protein that binds to a component of the elution medium or other composition to be used in the test. In embodiments where nucleic acids are the targets, the control line (204) preferably includes a nucleic acid, in addition to the labeled nucleic acid in use, as a binding partner for the target nucleic acid.
[0118] Although only one test line is shown in the figures, several test lines are in the spirit of the present invention. In some embodiments where multiple targets exist, the presence of each target preferably corresponds to a separate test line (202). In other embodiments where multiple targets exist, the presence of multiple targets may be indicated on the same test line such that the presence of more than one target has different characteristics than the presence of a single target. For example, the presence of multiple targets on the same test line can be visually indicated by a different color than the presence of each of the targets alone.
[0119] In other embodiments, it is possible to have one or more light lysis agents in the running buffer itself. In these embodiments, there is no adverse effect on the reagent zone which should be downstream and the sample may be upstream or downstream of the reagent zone. A lysis enzyme in the running buffer can "target" its substrate and cut it open to open the cell membrane or cell wall. As an example, penicillin can weed out or “punch a hole” in susceptible bacteria. In other embodiments, when the lysis agent is applied to the sampling material, then the reagent zone may be upstream of the sample application zone.
[0120] As an example, one or more lysing agents are dried in the sample application zone of a lateral flow strip. On a per strip basis, the lysis agent consists of approximately 2 microliters of 100 mM HEPES buffer (pH 8.0) containing 5% CHAPS and 2% NP-40 with 150 mM Sodium Chloride, 0.1 % BSA, and 0.1% Sodium Azide (all percentages by weight/volume). Up to 10 microliters of whole blood are then added to the application area of the sample to be lysed in situ. MxA protein is released from inside white blood cells to react with a monoclonal MxA antibody in a visual marker (colloidal gold or visible latex granules). This complex crosses with a working buffer containing Triton X-100 and is captured by MxA monoclonal antibodies immobilized on the test line of the nitrocellulose membrane. This connection on the test line gives a visible indication. SAMPLE ANALYSIS DEVICE WITH DOUBLE BIMODAL TEST STRIPS
[0121] MxA is a cell derivative of alpha/beta interferon that rises in the presence of viral infections but is not specific for a particular type of virus. The expression of MxA protein in peripheral blood is a sensitive and specific marker for viral infection.
[0122] MxA inhibits the replication of a wide variety of viruses. MxA has a low basal concentration [less than 50 ng/ml] and rapid induction [1-2 hours]. It peaks at 4 pm and remains elevated in the presence of elevated interferon. MxA also has a long half-life [2.3 days] and constant titers in the presence of interferonemia. Viral infections raise MxA levels while having only a modest increase in CRP levels.
[0123] In a prospective clinical trial using ELISA (Towbin H et al. J Interferon Res 1992;12:67-74, incorporated herein by reference), the study included 87 normal healthy adults. MxA levels were measured to be < 5 ng/ml in 66% of adults, between 5-50 ng/ml in 29% of adults, and above 50 ng/ml in 5% of adults.
[0124] Another prospective clinical trial using ELISA (Chieux V et al., J Virol Methods 1998;70:183-191, incorporated herein by reference) involved 174 children. 45 of these children had acute fever (respiratory infection and/or gastroenteritis) and there were 30 age-matched controls. MxA values were 7 ng/ml ±7 ng/ml in the 30 age-matched controls. MxA values were 10 ng/ml ±6 ng/ml in 13 confirmed bacterial infections.
[0125] Another prospective clinical trial using ELISA involved 60 patients (Kawamura M et al. J Clin Lab Anal 2012;26:174-183, incorporated herein by reference). 42 of these patients had acute fever (respiratory infection and/or gastroenteritis) and there were 18 age-matched controls. The mean MxA value was 110.0 ng/ml in 31 confirmed viral infections. The mean MxA value was 10.6 ng/ml in 11 confirmed bacterial infections. The mean MxA value was 2.0 ng/ml in the 18 age-matched controls. The ELISA test had a sensitivity of 87.1% and a specificity of 90.9% for differentiating viral from bacterial infection, but only MxA values were tested to make this determination. Patients with viral infection were sharply distinguished from healthy controls with 100% sensitivity and specificity. A cutoff of 36.7 ng/ml MxA was used to determine viral infection by ELISA in this study.
[0126] Another prospective clinical trial using ELISA involved 174 children (Nakabayashi Met al., Pediatr Res 2006;60:770-774, incorporated herein by reference, data corrected to standard recalibrated ELISA). 122 of these children had acute fever (respiratory infection and/or gastroenteritis) and there were 52 age-matched controls. Mean MxA values were 123.7 ng/ml ± 83.0 ng/ml in 95 confirmed viral infections. Mean MxA values were 12.3 ng/ml ± 10.0 ng/ml in 27 confirmed bacterial infections. The mean MxA value was 14.5 ng/ml ± 11.0 in the 52 age-matched controls. The test showed a specificity of 92.6% and a positive odds ratio of 13.1 to accurately identify the viral infection. A cutoff of 36.7 ng/ml MxA was used to determine viral infection by ELISA in this study.
[0127] The cut-off point in the ELISA test is artificial and is chosen to discriminate between positive and negative. Therefore, it is preferable to routinely assign 10% CV from this cut-off point. At a treatment test point, 100% of people can clearly see the test line at >40 ng/ml, but some people may see a positive result at lower levels.
[0128] CRP becomes elevated in the presence of bacterial infections but is not specific to a specific type of bacteria. CRP is a non-specific indicator for the presence of acute inflammation and is elevated in the presence of bacterial infections. CRP is an acute phase protein synthesized by the liver. IL-6 is the primary mediator of CRP production. Bacterial infection is a potent stimulus of marked CRP elevation. Following antibiotic treatment, CRP levels drop rapidly. Bacterial infections dramatically raise CRP levels while MxA levels remain low. Bacterial infection is a potent CRP stimulus with marked elevation in serum CRP levels occurring within hours. CRP levels rise at 4-6 hours after stimulation and reach a peak after 36 hours. The serum concentration of CRP is normally less than 3 mg/l. With infection or severe inflammation, CRP can rise above 500 mg/L.
[0129] Pneumonia has elevated serum CRP levels (>10 mg/L). Serum CRP levels are typically greater than 100 mg/L for severe pneumonia. 32% of patients with pneumococcal bacteremia had a serum CRP of less than 60 mg/l. In viral infections, serum CRP usually does not rise above 10 mg/l. Invasive Adenovirus and Influenza can increase CRP up to 10-80 mg/l. Very rarely, CRP levels exceed 60 mg/l in viral infections.
[0130] The meta analysis of ten studies (Aouifi et al., Crit Care Med. 2000, 28:3171-6; Hatherill et al., Arch Dis Child 1999: 81:417-21; Muller et al., Crit Care Med. 2000, 28: 977-83; Penel et al., Rev Med Interne 2001:22: 706714; Rothenberger et al., Clin Chem Lab Med, 1999, 37: 275-9; Schwarz et al., Crit Care Med. 2000, 28: 1828-32; Selberg et al., Crit Care Med 2000, 28: 2793-8; Suprin et al., Intensive Care Med 2000, 26: 1232-8; Ugarte et al., Crit Care Med 1999, 27: 498-504; Viallon et al., Intensive Care Med 2000, 26: 1082-8, all incorporated herein by reference) which aimed for a single serum CRP value to be used as a cut-off point for bacterial disease resulted in a bimodal result. Three of the studies (Aouifi et al., Crit Care Med. 2000, 28:3171-6; Penel et al., Rev Med Interne 2001:22: 706714; Schwarz et al., Crit Care Med 2000, 28: 1828-32 ) recommended that the CPR cut-off value be set at 615 mg/L, while the other seven studies (Hatherill et al., Arch Dis Child 1999: 81: 417-21; Muller et al., Crit Care Med. 2000, 28: 977-83; Rothenberger et al., Clin Chem Lab Med, 1999, 37: 275-9; Selberg et al., Crit Care Med 2000, 28: 2793-8; Suprin et al., Intensive Care Med 2000, 26: 1232-8; Ugarte et al., Crit Care Med 1999, 27: 498-504; Viallon et al., Intensive Care Med 2000, 26: 1082-8) recommended a cutoff of 60-100 mg/L.
[0131] In isolation, neither MxA nor CRP alone are sensitive or specific in identifying both viral and bacterial infection. Low CRP threshold values show high sensitivity and low specificity for detecting bacterial infection. High CRP threshold values show low sensitivity and high specificity for detecting bacterial infection. MxA is specific for identifying viral infection, but not sensitive to bacterial infection. A multiplexed pattern of results including medical decision points reflected that threshold levels of low CRP, high CRP, and MxA together provide a sensitive and specific way to identify an immune response to a viral and/or bacterial infection.
[0132] In a preferred embodiment of a multiplexed lateral flow immunoassay, the fingerstick blood pattern of test results shows a positive result with a serum equivalence to a low threshold CRP level of approximately 10 mg/L , a serum equivalence to an elevated CRP threshold level of approximately 80 mg/L, and an MxA cutoff of approximately 40 ng/ml. These preferred values are shown in Table 5. TABLE 5

[0133] The specificity of the test is further enhanced by restricting the intended use. For example, in preferred embodiments, only certain ages of the patient population are tested (preferably one year of age or older) and/or patients with specific threshold conditions that can lead to confounding factors are preferably not data from this test.
[0134] A rapid, point-of-care MxA immunoassay was developed and compared with the MxA ELISA assay on 25 peripheral blood samples from patients with febrile respiratory disease, as shown in Table 6. Table 7 orders the same data from the for the highest amounts of MxA in the ELISA test.
[0135] The ELISA MxA cut-off value was 36.7 ng/ml (+/- 10%CV = 33 ng/ml at 40/5 ng/ml). Patient 19 tested positive on the MxA rapid test at the point of care, even though the ELISA results were much less than 40 ng/ml (15 ng/ml). However, the MxA immunoassay demonstrated 100% (9/9) sensitivity and 94% (15/16) specificity. TABLE 6
TABLE 7


[0136] Rapid immunoassays of low point-of-care CRP level and high CRP level were developed and compared with PCR ELISA on 25 peripheral blood samples from patients with a febrile respiratory illness. These patients are the same patients who were tested for MxA in Tables 6 and 7. These preferred values are shown in Table 8. TABLE 8


[0137] Patients numbers 6 and 8 showed a positive PCR result even though the ELISA results were less than 80 mg/L. Patient 23 showed a negative result even when the ELISA results were exactly 80 mg/L. Patient number 9 showed a negative low CRP test result despite ELISA results for that patient being above 10 mg/L. But overall, the CRP values correlated well with the PCR ELISA at both cut-off values.
[0138] Using an MxA and CRP ELISA, RPS analyzed 25 normal, healthy blood bank samples for the presence or absence of elevated MxA and CRP. The mean plasma concentration of CRP was shown to be 1.6 mg/l. CRP levels have been shown to range in the range from 0.1 to 3.7 mg/L. The results are shown in Table 9. TABLE 9


[0139] Dual bimodal test strips can be used to differentiate bacterial and viral infection in humans, but can also be used in veterinary applications for animals. Since PCR differs depending on the species, there are no common antibodies to PCR between species. Therefore, veterinary tests need to include PCR specific to the particular species being tested. MxA is well conserved across species, so it is possible to use human MxA in veterinary testing. However, MxA for a particular species could alternatively be used to try to further increase specificity. Veterinary tests using the dual bimodal test strips described herein can be developed for a specific species, including, but not limited to, cats, dogs, rabbits, pigs, sheep, horses, cows, monkeys, chimpanzees, baboons, and orangutans.
[0140] The strip with MxA and low CRP could be made with any configuration, for example the configurations shown in Figures 4A and 4B, or Figures 5A through 5D, where MxA is the viral marker being detected and relatively low levels of CRP is the bacterial marker being detected. In other embodiments, the MxA test line and the PCR test line may overlap, or be in the same location as the test strips. In these embodiments, the presence of PCR and MxA in the same test line has different characteristics than the presence of either bacterial or viral markers alone. For example, the presence of low CRP and MxA in the same test line can be visually indicated by a different color than the presence of MxA or low CRP alone. In these embodiments, a positive result for MxA would give a different color or indication than a positive result for low CRP, so that the person reading the assay can distinguish between a completely negative result, a positive result for MxA, a positive result for low CRP, and a positive result for both MxA and low CRP. For example, a positive result for MxA can result in a red test line, and a positive result for low PCR can result in a blue test line. Thus, when a sample is positive for both low PCR and MxA, the line is visibly purple.
[0141] Some embodiments for lateral flow assay devices that detect high levels of CRP are shown in Figures 6A-6B and 7A-7D. These configurations are similar to the configurations shown in Figures 4A-4B and 5A-5D, without a test line for a viral marker, and the same numerical references are used for the same strip components (600), (700).
[0142] Figures 6A and 6B show a chromatographic test strip (600) with a test line (623) that detects the presence of a bacterial marker, such as high levels of CRP. The sample is applied to the application zone (401) of the chromatographic test strip (600). As shown in Figure 6A, the sample then passes a reagent zone (660) containing at least one labeled bacterial binding partner and which is eluted by and then capable of migrating with the sample transport liquid (e.g. a buffer solution). Alternatively, as shown in Figure 6B, the reagent zone (660) is located upstream of a sample application zone (401) as the labeled binding partners in the reagent zone are eluted by the sample carrying liquid and traveling for the sample. The labeled bacterial binding partner is capable of specifically binding a bacterial marker of interest, for example high levels of PCR, to form a complex which in turn is capable of specifically binding another specific reagent or binding partner. in the detection zone. Although not shown in these Figures, an absorbent pad, as well as other known lateral flow immunoassay components including, but not limited to, a waste zone, a carrier support, a housing, and an opening in the housing for reading the result, may optionally also be a component of the test strips (600) in these embodiments.
[0143] The test strips (600) also include a detection zone (605) containing a section for detecting a bacterial marker, for example a test line (623), including an immobilized specific binding partner, complementary to the complex of the bacterial reagent formed by the bacterial tag and its labeled binding partner. Thus, at the test line (623), the binding partner detection zone traps the labeled bacterial binding partners of the reagent zone (660) along with their bound bacterial labels. This location of the bacterial marker with its labeled binding partners gives rise to an indication on the test line (623). At test line (623), the presence of the bacterial marker is determined by qualitatively and/or quantitatively reading the test line (623) indication resulting from accumulation of the labeled binding partners.
[0144] Optionally, the detection zone (605) can contain other test lines to detect other bacterial and/or viral markers, as well as a control line (404). The control line (404) indicates that the specific binding partner labeled has traveled throughout the assay, even though it may not have any labels attached, thus confirming the proper operation of the assay. As shown in Figures 6A through 6B, the control zone (404) is preferably downstream of the test lines (623). However, in other embodiments, the control zone (404) may be located upstream of the test line (623).
[0145] In a preferred embodiment, the control line (404) includes an antibody or other recombinant protein that binds to a component of the elution medium or other composition to be used in the test. In embodiments where nucleic acids are the targets, the control line (404) preferably includes a nucleic acid, in addition to the labeled nucleic acid in use, as a binding partner for the target nucleic acid.
[0146] In other preferred embodiments for testing a bacterial marker, such as PCR levels, as shown in Figures 7A through 7D, the sample passes the lysis zone (250), where a lysis agent will preferably have been pre- loaded onto the test strips and is eluted by the liquid carrier. The lysing agent lyses any lysable components in the sample in situ.
[0147] The chromatographic test strip (700) contains a sample application zone (201), the lysis zone (250) containing a lysis agent, and a reagent zone (760) containing at least one binding partner label that binds to a bacterial marker, e.g. high levels of PCR, which is eluted in and then able to migrate with the carrier liquid sample (e.g. a buffer solution). While the reagent zone (760) is shown downstream of the sample application zone in these figures, in alternative embodiments, the reagent zone (760) may be upstream of the sample application zone (see Figure 6B), provided that the reagents encounter the sample at some point after the sample reaches the lysis zone and is effectively lysed. The labeled binding partner is capable of specifically binding a bacterial marker of interest, e.g. high levels of PCR, to form a complex which in turn is capable of specifically binding another specific reagent or binding partner. in the detection zone. Although not shown in these Figures, an absorbent pad, as well as other known lateral flow immunoassay components including, but not limited to, a waste zone, a carrier support, a housing, and an opening in the housing for reading the result, may optionally also be a component of the test strips (700) in these embodiments.
[0148] In a preferred embodiment, the lysis agent is located in the lysis zone (250) between the sample application zone (201) and the reagent zone (760). The lysis agent is preferably soluble or miscible in the sample transport liquid, and the lysis agent is solubilized and activated upon contact with the sample transport liquid. The sample transport liquid then contains the lysing agent in both solution and suspension and suspended sample components. Any components susceptible to lysis in a sample, which are then exposed in suspension to the lysing agent, are themselves lysed in situ. The running buffer then transports the sample, including any of the non-lysed components, to the detection zone (705).
[0149] The lysis zone (250) is preferably located between the sample application zone (201) and the reagent zone (760), as shown in Figure 7A. In other embodiments, the lysis zone (250) overlaps the sample application zone (201), the reagent zone (760), or both the sample application zones (201) and the reagent zone. (260) as shown in Figures 7B, 7C, and 7D, respectively. Note that the figures are schematic, not drawn to scale. The amount of overlap between different zones (as shown in Figures 7B through 7D) can be highly variable.
[0150] The test strips (700) also include a detection zone (705) containing a first section for detecting at least one bacterial marker, for example a test line (723), including an immobilized specific binding partner, for example, a binding partner specific for a high level of PCR, complementary to the bacterial conjugate formed by the bacterial tag and its labeled binding partner. Thus, at the test line (723), the binding partner detection zone traps the labeled bacterial binding partners from the reagent zone (760) along with their bound bacterial labels. This location of the bacterial markers with their labeled binding partners gives rise to an indication on the test line (723). At test line 723, the presence of a bacterial marker is determined by qualitatively and/or quantitatively reading the test line indication (723) resulting from accumulation of the labeled binding partners.
[0151] Optionally, the detection zone (705) can contain other test lines to detect other bacterial and/or viral markers, as well as a control line (204). The control line (204) indicates that the labeled specific binding partner has traveled the length of the assay, even though it may not have any tags attached, thus confirming the proper operation of the assay. As shown in Figures 7A through 7D, the control zone (204) is preferably downstream of the test lines (723). However, in other embodiments, the control zone (204) may be located upstream of the test line (723).
[0152] In a preferred embodiment, the control line (204) includes an antibody or other recombinant protein that binds to a component of the elution medium or other composition to be used in the test. In embodiments where nucleic acids are the targets, the control line (204) preferably includes a nucleic acid, in addition to the labeled nucleic acid in use, as a binding partner for the target nucleic acid.
[0153] A preferred configuration of a bimodal dual sample analysis device test strip is shown in Figures 8A through 8C. The sample analyzing device or test card (800) includes a two-sided confineable housing (835), (836), (837) and a column or hinge portion (831). In a preferred embodiment, the test card (800) is approximately 11.5 cm long (L) x 7 cm wide (W) when the two sides (836), (837) are closed. However, any size test card (800) can be used to accommodate all components. Inside the first side (836) of the housing (835), there are two test strips (815), (825), each including a receiving pad (845), a bypass zone (850), a transfer pad (855) and a detection zone (805). The first side (836) also includes an absorbent pad (840) and preferably a waste pad (860). The first test strip (815) preferably includes a detection zone (805) with an MxA test line (802), a low PCR test line (803) and a control line (804). The second test strip (825) preferably includes a detection zone (805) with an elevated PCR test line (823) and a control line (804). All test lines are visible through windows (865) on the second side (837) of housing (835) when housing (835) is closed. The absorbent pad (840) is preferably a single pad to which the working buffer is added to initiate lateral flow. Similarly, the waste pad 860 is preferably a single pad that collects excess working buffer at the end of the test. However, in other embodiments, each strip could have an absorbent pad (840) and/or waste pad (860).
[0154] The second side (837) of the housing (835) includes three separate sections (838), (839) and (870). The middle portion, a sample compressor or flap (870), preferably includes two conjugated zones (872), (874), each including a labeled binding marker for at least one analyte, and a labeled control. A window (843) is placed in the lower portion (838) of the second side (837) of the wrapper so that the pad can be added to the absorbent pad (840) when the wrapper (835) is closed. Viewing windows (865) for detection zones (805) are in the upper portion (839) of the second side (837) of the housing (835).
[0155] The upper portion (839) and the lower portion (838) of the second side (837) of the housing (835) each preferably also include at least one button, handle or projection (875) corresponding to one or more holes (895) such that the lower and upper portions (838), (839) can be easily attached to the first side (836) of the housing (835). In a preferred embodiment, there are two handles (875) on the lower portion (838) that correspond to two holes (895) flanking the absorbent pad (840) on the first side (836) of the housing (835) and two handles (875). ) in the upper portion (839) which correspond to two holes (895) flanking the waste pad (860) on the first side (836) of the housing (835). In other embodiments, the holes (895) are on the second side (837) of the housing (835) and the handles (875) are on the first side (836) of the housing (835). In still other embodiments, other reversible attachment mechanisms could be used to secure the upper portion (838) and/or lower portion (839) of the second side (837) of the housing (835) to the first side (836) of the housing. (835). In other embodiments, the upper and lower sections (838), (839) are permanently closed, for example using an adhesive, before use.
[0156] The tab (870), also known as a sample compressor, on the second side (837) of the housing includes two torque zones (872), (874) and two sample application zones (873), (876). ), and can be easily opened and closed. The flap (870) also preferably includes at least one button, handle or projection (875) corresponding to one or more holes (895) such that the flap (870) is easily closed correctly on the first side (836) of the housing. (835) after the sample has been added to the sample application zones (873), (876). In other embodiments, the holes (895) are on the second side (837) of the housing (835) and the handles (875) are on the first side (836) of the housing (835). In still other embodiments, other reversible attachment mechanisms could be used to secure the tab (870) to the first side (836) of the housing (835).
[0157] The conjugate zones (872), (874) and the sample application zones (873), (876) preferably overlap. In preferred embodiments, the conjugate zones (872), (874) are colored due to dyes in the sample conjugates and control conjugates, and the sample is placed directly on the colored portion of the tab (870). In a preferred embodiment, the conjugate zone (872) that is used for the first test strip (815) contains an MxA binding partner that is labeled with a red dye, a low PCR binding partner that is labeled with a black dye, and a control binding partner that is labeled with a blue dye. In this embodiment, the conjugate zone 872 appears purple. The other conjugate zone (874) contains a high PCR binding partner that is labeled with a black dye and a control binding partner that is labeled with a blue dye. In this embodiment, the conjugate zone 874 appears bluish.
[0158] The bypass zone (850) preferably includes a slit or barrier that interrupts the lateral flow, bypassing the operating plug to the flap (870) that includes the conjugate zones (872), (874) and the zones of sample application (873), (876).
[0159] In actuation, the upper and lower portions (838), (839) of the second side (837) of the housing (835) are kept closed before use by attaching the handles (875) to the holes (895). The sample analyzing device, or test card (800) is preferably placed on a flat surface. If the tab (870) is not already open, the user opens it to access the sample application zones (873), (876). A blood sample to be tested is taken from the patient. The sample can be taken by any procedure known in the art. In a preferred embodiment, a 5 µl sample of blood is added to each of the sample application zones (873), (876) and then the flap (870) is closed. Each of the 5 μl samples is preferably collected independently of each other. Blood samples are preferably added directly to the device (800), without any pre-treatment.
[0160] To ensure that the sample compressor or flap (870) has been properly closed pressure is preferably applied to the housing (835) above the handles (875) to keep the handles (875) closed. The top of the tab (870) needs to be aligned with the top of the rest of the second side (837) of the housing (835) for the test to work properly. The working buffer is added to the absorbent pad (840), which initiates lateral flow (885). In preferred embodiments, the working buffer includes one or more lysing agents, for example detergents, to lyse the blood sample and expose intracellular MxA in the sample. When the working plug reaches the bypass zone (850) it is bypassed to the flap (870). Travels through the conjugate zones (872), (874), collecting any complexes formed between the MxA and MxA binding partner in the sample, the low PCR binding partner and low levels of PCR in the sample, the Elevated PCR and high levels of PCR in the sample as well as the control conjugate.
[0161] Since the conjugate zones (872), (874) form a bridge over the bypass zone (850) on the lateral flow test strips (815), (825), the operating buffer, which contains Now the sample, conjugate, and the complexes described above then travel to the transfer pad (855), and to detection zones (805) on each of the test strips (815), (825). If MxA is present in the sample, the MxA test line (802) in the first test line (815) will be red. If a low threshold PCR level is present in the sample, the low PCR test line (803) in the first test line (815) will be black. If a high threshold level of CRP is present in the sample, the high CRP test line (823) in the first test line (825) will be black. If the test is operated correctly, the control lines (804) on both the first test strip (815) and the second test strip (825) will be blue. In preferred embodiments, detection levels are 40 ng/ml for MxA, 10 mg/l for low CRP on the first test strip (815) and 80 mg/l for high CRP on the second test strip (825). Test results should be visible after approximately 5-20 minutes, preferably within approximately 10 minutes.
[0162] Since the control link partner is on the sample compressor or tab (870) and not on any of the test strips (815), (825) there is true procedural control for this configuration. If the flap (870) is not closed properly, nothing will appear in the detection zone (805), indicating that the test has been improperly operated.
[0163] Figures 9A through 9F show test results using the device (800) shown in Figures 8A through 8C, with two test strips (815), (825) side by side, where a first test strip (815) tests for the presence of both MxA and low levels of CRP and the second test strip (825) tests for high levels of CRP.
[0164] Figure 9A shows a negative result on the MxA test line (802) and a negative result on the low PCR test line (803) on the first test line (815), as well as a negative result on the low PCR test line (803) in the first test line (815). high PCR test (823) on the second test strip (825). More specifically, the only visible lines in the detection zone (805) of the lateral flow assay (800) are the two blue control lines (804). This result indicates that the sample is negative for viral and bacterial infection.
[0165] Figures 9B and 9C are positive for viral infection. In Figure 9B, the presence of two blue control lines (804) and one red MxA line (802) indicates a viral infection. In Figure 8C, the presence of two blue control lines (804) and one red MxA line (802) indicates a viral infection. Since there is also a black low CRP line (803) in Figure 9C, there is a possibility of bacterial co-infection, although there is an absence of a high CRP line (823).
[0166] Figures 9D and 9E are positive for viral infection. In Figure 9D, the presence of two blue control lines (804) and a low black PCR line (803) indicates a bacterial infection. In Figure 9E, the presence of two blue control lines (804), a black low CRP line (803), and a black high CRP line (823) also indicates a bacterial infection. The MxA line is absent in both Figures 9D and 9E, indicating an absence of a viral infection.
[0167] Figure 9F indicates co-infection (bacterial and viral infection). The presence of two blue control lines (804), a black MxA line (802), a black low CRP line (803), and a black high CRP line (823) indicates the presence of viral and bacterial infection.
[0168] Another preferred configuration for a dual bimodal test strip sample analysis device (1000) is shown in Figures 10A through 10C. This configuration is similar to the configuration (800) shown in Figures 8A through 8C, but the sample application zones (1073), (1076) are located on each of the test strips (1015), (1025), downstream of the bypass zone (850). The sample analyzing device or test card (1000) includes a pluggable housing (835) with two sides (836), (837) and a column or hinge portion (831). In a preferred embodiment, the test card (1000) is approximately 11.5 cm long (L) x 7 cm wide (W) when the two sides (836), (837) are closed. However, any size test card (1000) can be used to accommodate all components. Inside the first side (836) of the housing (835), there are two test strips (1015), (1025), each including a receiving pad (845), a bypass zone (850), a transfer pad (1055) and a detection zone (805). The first side (836) also includes an absorbent pad (840) and preferably a waste pad (860). The first test strip (1015) preferably includes a detection zone (805) with an MxA test line (802), a low PCR test line (803) and a control line (804). The second test strip (1025) preferably includes a detection zone (805) with an elevated PCR test line (823) and a control line (804). All test lines are visible through windows (865) on the second side (837) of housing (835) when housing (835) is closed. The absorbent pad (840) is preferably a single pad to which the working buffer is added to initiate lateral flow. Similarly, the waste pad 860 is preferably a single pad that collects excess working buffer at the end of the test. However, in other embodiments, each strip could have an absorbent pad (840) and/or waste pad (860).
[0169] The second side (837) of the housing (835) includes three separate sections (838), (839) and (1070). The middle portion, or flap (1070), also known as a sample compressor, preferably includes two conjugate zones (872), (874), each including a labeled binding marker for at least one analyte, and a control marked. A window (843) is located in the lower portion (838) of the second side (837) of the housing such that the plug can be added when the housing (835) is closed. Viewing windows (865) for detection zones (805) are in the upper portion (839) of the second side (837) of the housing (835).
[0170] The upper portion (839) and the lower portion (838) of the second side (837) of the housing (835) each preferably also include at least one button, handle or projection (875) corresponding to one or more holes (895) so that the lower and upper portions (838), (839) can be easily attached to the first side (836) of the housing (835). In a preferred embodiment, there are two handles (875) on the lower portion (838) that correspond to two holes (895) flanking the absorbent pad (840) on the first side (836) of the housing (835) and two handles (875). ) in the upper portion (839) which correspond to two holes (895) flanking the waste pad (860) on the first side (836) of the housing (835). In other embodiments, the holes (895) are on the second side (837) of the housing (835) and the handles (875) are on the first side (836) of the housing (835). In still other embodiments, other reversible attachment mechanisms could be used to secure the upper portion (838) and/or lower portion (839) of the second side (837) of the housing (835) to the first side (836) of the housing. (835). In other embodiments, the upper and lower sections (838), (839) are permanently closed, for example using an adhesive, before use.
[0171] The flap (1070) on the second side (837) of the housing includes two torque zones (872), (874) and can be easily opened and closed. The flap (1070) also preferably includes at least one button, handle or projection (875) which corresponds to one or more holes (895) such that the flap (1070) is easily closed correctly on the first side (836) of the housing (835). ) after the sample has been added to the sample application zones (1073), (1076) on the test strips (1015), (1025). In other embodiments, the holes (895) are on the second side (837) of the housing (835) and the handles (875) are on the first side (836) of the housing (835). In still other embodiments, other reversible attachment mechanisms could be used to secure the tab (1070) to the first side (836) of the housing (835).
[0172] In preferred embodiments, the conjugate zones (872), (874) are colored due to dyes in the sample conjugates and control conjugates. In a preferred embodiment, the conjugate zone (872) that is used for the first test strip (1015) contains an MxA binding partner that is labeled with a red dye, a low PCR binding partner that is labeled with a black dye, and a control binding partner that is labeled with a blue dye. In this embodiment, the conjugate zone 872 appears purple. The other conjugate zone (874) contains a high PCR binding partner that is labeled with a black dye and a control binding partner that is labeled with a blue dye. In this embodiment, the conjugate zone 874 appears bluish.
[0173] The bypass zone (850), which preferably includes a slit or barrier, interrupts the lateral flow, bypassing the operating plug to the flap (1070) that includes the conjugate zones (872), (874).
[0174] In operation, the upper and lower portions (838), (839) of the second side (837) of the housing (835) are kept closed before use by attaching the handles (875) to the holes (895). The sample analyzing device, or test card (1000) is preferably placed on a flat surface. If the tab (1070) is not already open, the user opens it to access the sample application zones (1073), (1076). Sample application zones (1073), (1076) may be located on any portion of the transfer pad (1055). A blood sample to be tested is taken from the patient. The sample can be taken by any procedure known in the art. In a preferred embodiment, a 5 µl sample of blood is added to each of the sample application zones (1073), (1076) and then the flap (1070) is closed. Each of the 5 μl samples is preferably collected independently of each other. Blood is preferably directly added to the device (1000), without any pre-treatment. In preferred embodiments, an arrow (1002) or other indication (shown in Figure 10A), for example the words "add sample here", shows the user where to place the sample on the test strips (1015), (1025).
[0175] To ensure that the flap (1070) has been properly closed, pressure is preferably applied to the housing (835) above the handles (875) to close the handles (875). The top of the tab (1070) needs to be aligned with the top of the rest of the second side (837) of the housing (835) for the test to work properly. The working buffer is added to the absorbent pad (840), which initiates lateral flow (885). In preferred embodiments, the working buffer includes one or more lysing agents, for example detergents, to lyse the blood sample and expose intracellular MxA in the sample. When the working plug reaches the bypass zone (850) it is bypassed to the flap (1070). It travels through the conjugate zones (872), (874), collecting MxA binding partners, low PCR binding partners, and high PCR binding partners, as well as the control conjugate.
[0176] Since the conjugate zones (872), (874) bridge the bypass zone (850) on the side flow test strips (1015), (1025), the operating buffer, which contains now conjugated, then travels to the transfer pad (1055), which includes sample application zones (1073), (1076), and to detection zones (805) on each of the test strips (1015), (1025). If MxA is present in the sample, the MxA test line (802) in the first test line (1015) will be red. If a low threshold PCR level is present in the sample, the low PCR test line (803) in the first test line (1015) will be black. If a high threshold level of PCR is present in the sample, the high PCR test line (823) in the second test line (1025) will be black. In preferred embodiments, detection levels are 40 ng/ml for MxA, 10 mg/l for low CRP on the first test strip (1015) and 80 mg/l for high CRP on the second test strip (1025). Test results should be visible after approximately 5-20 minutes, preferably within approximately 10 minutes. If the test was correctly operated, the control lines (804) on both the first test strip (815) and the second test strip (825) will be blue.
[0177] Since the control link partner is on the tab (1070) and not on any of the test strips (1015), (1025) there is true procedural control for this configuration. If the flap (1070) is not properly closed, nothing will appear in the detection zone (805), indicating that the test has been improperly operated.
[0178] In an alternative embodiment, the sample application zones (1073), (1076) are located on the receiving pad (845), before the bypass zone (850). In this embodiment, the working plug travels through the sample application zones (1073), (1076), and then diverts to the flap (1070).
[0179] In preferred embodiments of the configurations shown in Figures 8A through 8C and 10A through 10C, more than approximately 1.2 ml of working buffer is placed in the absorbent pad (840). If less than 1.0 ml is added in embodiments where the bypass zone (850) is a slit, the plug will stagnate in the slit because the slit holds approximately 1.0 ml.
[0180] As shown in Figure 11, in a preferred embodiment, a kit (1100) includes the sample analyzing device (800), (1000), a lancet (1102), one or more pipettes (1101), and an operating plug (1103). The lancet (1102) is used to puncture the skin and one or more pipettes (1101) are used to draw blood from the puncture site. In a preferred embodiment, 5 µl of blood is transferred from a first pipette (1101) to the first conjugate zone (872) and another 5 µl of blood is transferred from a second pipette (1101) and added to the second conjugate zone (1101). conjugate (874). The flap (870) is closed, and the working plug (1103) is added to the absorbent pad (840), as described in the description of Figs. 8A up to 8C and 10A up to 10C.
[0181] The bypass zone (850) preferably includes at least one feature that interrupts the flow in the plane in which the flow is taking place. The bypass zone may include a barrier, a crack, a ditch, or any combination of these features. The barrier is preferably an impermeable membrane (or substantially impermeable membrane) which may be made of any material that prevents the flow of liquid from continuing to flow in the same plane. Some barrier materials include, but are not limited to, inert materials, semipermeable materials, plastics, hydrocarbons, metal, hydrophobic materials, Sephadex, Sepharose, cellulose acetate, a hygroscopic material (e.g. CaCl2, CaSO4 or silica gel) , or hydrogels. A crack or trench is any break in the plane of the side flow test strip that extends deep enough to stop the flow. In a preferred embodiment, the slot preferably has a depth of approximately 0.1 mm.
[0182] Bypass zone (850) in Figures 8A through 8C and 10A through 10C slows or completely stops flow until the compressor/sample flap (870), (1070) is brought into contact with the rest of the device, and creates a bridge along which fluid can flow. The sample compressor (870), (1070) acts as a bridge and redirects the flow to a different plane. The flow is diverted to the sample compressor (870), (1070). This increases the collection of reagents in the sample compressor (870), (1070). For example, in embodiments where the conjugate is in the sample compressor (870), (1070), conjugate collection increases in devices with a bypass zone (850). In embodiments where both the sample application zone (873), (876), (1073), (1076) and the conjugate are in the sample compressor (870), (1070), the sample and the conjugate both meet the operating buffer when they are bypassed to the sample compressor (870), (1070), and a full sandwich or sandwich (depending on where the second binding partner for the analyte is located in the sample analysis device) is formed before the working buffer is diverted back to the test strip if analyte is present in the sample. Embodiments with a bypass zone (850) and sample compressor (870), (1070) increase speed, allow for better interactions between conjugate and sample, and allow for more sensitivity because more conjugate is placed in the fluid. In these embodiments, all of the fluid preferentially interacts with the conjugate. This is a significant improvement over non-redirection compressor embodiments, where approximately 2030% of the fluid interacts with the couple.
[0183] Another preferred configuration for a dual bimodal test strip sample analysis device (1200) is shown in Figure 12. This configuration is similar to the configurations (800), (1000) shown in Figures 8A through 8C and Figures 10A and 10C, without a second section (837) of the housing (1235) or a bypass zone (850). Instead, all test components lie in the same plane and flow proceeds laterally from the absorbent pad (840) to the waste pad (860). Note that this embodiment may also include a housing with a window to facilitate application of the tampon to the absorbent pad (840), to a window located above each sample application zone (1273), (1276) for applying the sample to the device (1200), and viewing windows for the detection zone (805). In a preferred embodiment, the sample analyzing device (1200) is approximately 11.5 cm long (L) x 7 cm wide (W). However, any size test card (1200) can be used to accommodate all components. There are two test strips (1215), (1225), each including a receiving pad (845), a conjugate zone (1272), (1274), a transfer pad (1240) containing a sample application zone (1273), (1276), a detection zone (805) and a waste pad (860). The device (1200) also preferably includes an absorbent pad (840) and a waste pad (860). While the conjugate zones (1272), (1274) are shown upstream of the sample application zone (1273), (1276) in this figure, in other embodiments, one or both of the conjugate zones (1272), (1274 ) are located downstream of the sample application zone (1273), (1276). The detection zone (805) of the first test strip (1215) preferably includes an MxA test line (802), a low PCR (803) and a control line (804). The detection zone (805) on the second test strip (1225) also preferably includes an elevated PCR test line (823) and the control line (804). The absorbent pad (840) is preferably a single pad to which the working buffer is added to initiate lateral flow. Similarly, the waste pad 860 is preferably a single pad that collects excess working buffer at the end of the test. However, in other embodiments, each strip could have a separate absorbent pad (840) and/or waste pad (860).
[0184] In preferred embodiments, the conjugate zones (1272), (1274) are colored due to dyes in the sample conjugates and control conjugates. In a preferred embodiment, the conjugate zone (1272) that is used for the first test strip (1215) contains an MxA binding partner that is labeled with a red dye, a low PCR binding partner that is labeled with a black dye, and a control binding partner that is labeled with a blue dye. In this embodiment, the conjugate zone (1272) appears purple. The other conjugate zone (1274) contains a high PCR binding partner that is labeled with a black dye and a control binding partner that is labeled with a blue dye. In this embodiment, the conjugate zone (1274) appears bluish.
[0185] In the operation, a blood sample to be tested is taken from the patient. The sample can be taken by any procedure known in the art. In a preferred embodiment, a 5 µl blood sample is added to each of the sample application zones (1273), (1276). Each of the 5 μl samples is preferably collected independently of each other. In preferred embodiments, an arrow (1002) or other indication (shown in Figure 10A), for example the words "add sample here", shows the user where to place the sample on the test strips (1215), (1225).
[0186] Blood is preferably directly added to the device (1200) without any pre-treatment. The working buffer is added to the absorbent pad (840), which initiates lateral flow (1285). In preferred embodiments, the working buffer includes one or more lysing agents, e.g., detergents, to lyse the blood sample and expose intracellular MxA in the sample. It travels through the conjugate zones (1272), (1274), collecting the MxA binding partners, the low PCR binding partners, and the high PCR binding partners, as well as the control conjugate.
[0187] The running buffer, which now contains conjugate, then moves to the transfer pad (1255), which includes the sample application zones (1273), (1276), and detection zones (805) on each of the test strips (1215), (1225). If MxA is present in the sample, the MxA test line (802) in the first test line (1215) will be red. If a low threshold PCR level is present in the sample, the low PCR test line (803) in the first test line (1215) will be black. If a high threshold level of PCR is present in the sample, the high PCR test line (823) in the second test line (1225) will be black. In preferred embodiments, detection levels are 40 ng/ml for MxA, 10 mg/l for low CRP on the first test strip (1215) and 80 mg/l for high CRP on the second test strip (1225). Test results should be visible after approximately 5-20 minutes, preferably within approximately 10 minutes. If the test was correctly operated, the control lines (804) on both the first test strip (1215) and the second test strip (1225) will be blue.
[0188] In an alternative embodiment, the sample application zones (1273), (1276) are located upstream of the conjugate zones (1272), (1274). In this embodiment, the working buffer travels through the sample application zones (1273), (1276), and then diverts to the conjugate zones (1272), (1274). In yet another embodiment, the mating zones (1272), (1274) overlap a sample application zone (1273), (1276). In yet another embodiment, the mating zones (1272), (1274), and/or the sample application zone (1273), (1276) may be located on the receiving pad (845).
[0189] In preferred embodiments of the configurations shown in Figures 4A through 8C, 10A through 10C and 12 the control is anti-rabbit chicken and the control conjugate is blue latex granules coupled to chicken IgY. In other preferred embodiments, there is at least one lysing agent, preferably a detergent, in the running buffer. SIMULTANEOUS DETECTION OF INTRACELLULAR AND EXTRACELLULAR PROTEINS
[0190] There are extracellular analytes and there are intracellular analytes. The detection of each is often a separate event. The intracellular analyte must be lysed out of the cells so that the internalized analyte is externalized and available for testing.
[0191] The methods disclosed herein simultaneously detect at least one extracellular analyte and at least one intracellular analyte. An "intracellular" target or analyte, as described in this embodiment, is an analyte that is inside the cell and does not touch anything inside the cell (such as surface proteins, the cell wall, or internal surfaces). An "extracellular" target or analyte, as described in this embodiment, is a completely extracellular analyte, which does not contact anything outside the cell. For example, the extracellular analyte is in plasma, which does not contain cells. The cell can be completely removed, and the extracellular analyte can still be collected.
[0192] In contrast, viral particles, while outside the cell, are attached to the cell wall. It is well known in the art to mix an antibody cocktail to detect an intracellular bound fraction and a surface bound fraction. But, the methods described here are different, and detect a lysed, intracellular portion and a dissociated serum protein.
[0193] In a preferred embodiment, the extracellular analyte is C-reactive protein and the intracellular analyte is MxA protein. This is a serological test for the detection of PCR and MxA antigens. MxA is an intracellular protein within white blood cells. CRP is an extracellular protein found in whole blood, plasma and serum.
[0194] In a preferred method, glass fibers such as Whatman GD filters physically entrap the erythrocytes. Specific erythrocyte binding lectins and/or antibodies can be added to physically bind to erythrocytes in the glass fiber matrix. Leukocyte lysis solutions, which lyse leukocytes to release intracellular MxA, can be incorporated into glass fiber filters.
[0195] In some embodiments, the whole blood sample is added to the glass filter. Liquid blood dissolves the embedded lysing agents, which lyse leukocytes. Lateral flow immunochromatography is initiated by the addition of working buffer. The running buffer then loads the appropriate antibody conjugates that bind to extracellular PCR and the newly released intracellular MxA. The entire complex moves into the detection zone where immobilized specific antibodies capture the respective complexes to form the sandwich. Different test lines are formed with MxA and PCR. This test line can be visual, fluorescent, phosphorescent, chemiluminescent, paramagnetic, or any combination thereof. Different dyes can be incorporated to distinguish the MxA and PCR test lines. Any of the lateral flow assays and configurations described herein can be used to simultaneously detect MxA and PCR or other intracellular and extracellular analytes. MXA AND PCR AGGLUTINATION
[0196] Some embodiments include a simple agglutination test for MxA and blood PCR. As an analogy, the present inventors believe that cement in a brick wall is holding the bricks apart rather than holding them together. The reasoning is that if the cement is removed, the bricks clump together and fall together in a heap. Therefore, if the cement is "inactivated" or removed, the individual bricks coalesce together.
[0197] Gold conjugates and latex granules are colloidal particles that repel each other and therefore are gathered in suspension. If the repulsive force is removed, or the individual colloidal particles are crosslinked together, they clump together and the clumped particles can be visualized. Crosslinking to overcome this natural repulsion is achieved by the presence of the analyte antigen in question.
[0198] In a preferred embodiment, the monoclonal MxA KM 1124 and/or KM 1135 is conjugated to red colloidal gold particles. Anti-PCR monoclonal antibodies are conjugated to appropriately sized green latex beads. In the presence of MxA, the monoclonal antibody KM 1124 and/or KM 1135 binds and, since there is a multiplicity of epitopes recognized by KM 1124 and/or KM 1135, a natural cross-linking of the colloidal gold takes place and one sees the agglomeration of colloidal red gold particles. This process is antigen-dependent and quite rapid, usually occurring within a minute or two. The same phenomenon occurs with green colloidal latex beads coated with suitable monoclonal antibodies in the presence of a PCR analyte.
[0199] Agglomeration of red gold particles means that at least a threshold amount of MxA is in the sample, indicating a viral infection. Clustering of green beads means that at least a threshold amount of PCR is in the sample, indicating bacterial infection. The agglomeration of the red and green particles either signifies a co-infection or is indicative of an indeterminate outcome. The absence of any agglutination indicates that the sample is negative for viral and bacterial infections.
[0200] In a preferred embodiment, the threshold concentration of C-reactive protein is equal to or greater than approximately 6-15 mg/L of C-reactive protein and the threshold concentration of MxA protein is equal to or greater than an equivalent of serum of approximately 15-250 ng/ml. Since MxA is an intracellular biomarker, the blood sample is preferentially lysed during the assay to lyse white blood cells and externalize the MxA antigen. In other embodiments, the sample is lysed prior to performing the assay. Any immunoassay format known in the art can be used for the agglutination assay. Reagents are added, then the user waits to see if the reagents agglutinate in the presence of the sample. NANOPARTICLES IN VIRUS DIAGNOSIS
[0201] MxA levels are elevated in some but not all viral infections. Some exceptions to elevated MxA levels during infection are Hepatitis B and these chronic viral infections, including possibly HIV and Hepatitis C. Sialic acid is in most viruses. But, sialic acid is not in some viruses, such as the Cox virus.
[0202] The combined MxA-PCR detector devices described above are preferentially used in people with fever to distinguish between viral and bacterial infections. The sialic acid nanoparticle test is preferably used in all other suspected viral infections (whether or not accompanied by fever), including chronic viral infections. However, in some embodiments, sialic acid replaces MxA in the methods and devices described herein.
[0203] In another preferred embodiment, a test includes MxA, PCR, and a nanoparticle homologue sialic acid.
[0204] In a preferred embodiment, the test line is a nanoparticle specific for a given virus. Some examples include, but are not limited to, avian influenza, and viruses that cause chronic infection, such as HIV or hepatitis C. The conjugate is either a) a virus-specific nanomycelle with a dye inside or b) a homologous nanomycelle of sialic acid with a dye inside.
[0205] For example, Nanoviricides, Inc. (West Haven, Connecticut) is a Nanoparticle specific to Avian Influenza. These nanoparticles can be used in the Test Line and a homologous sialic acid nanomicelle as the conjugate to detect Avian Influenza.
[0206] Another embodiment includes a broad spectrum viral detector. In this embodiment, the test line is a sialic acid homologous nanoparticle that captures all viruses and the conjugate is a sialic acid nanomicel with dye inside.
[0207] In preferred embodiments, the test line is made of nanoparticles, not the nanomicelles that contain the nanoparticles. The conjugate, on the other hand, is preferably a nanomicel (analogous to a soap bubble) which carries the dyes within the micelle. The lipid portion of the micelle is responsible for the "slime" property of the micelle.
[0208] Embodiments of sialic acid may utilize any of the test strip configurations described herein, or known in the art, including those shown in Figures 4-8 and 10-12, or other assay configurations known in the art.
[0209] Accordingly, it is to be understood that the embodiments of the invention described herein are merely illustrative of the application of the principles of the invention. Reference herein to details of the illustrated embodiments is not intended to limit the scope of the claims, which themselves express those features considered essential to the invention.

权利要求:
Claims (24)
[0001]
1. METHOD TO DETERMINE IF AN INFECTION IS BACTERIAL AND/OR VIRAL, characterized by comprising the steps of: a) collecting a sample; b) transferring the sample to a sample analyzing device (800, 1000) comprising: i) a sample compressor (870, 1070) comprising: A) a first reagent zone comprising at least one first protein-specific reagent C-reactive, so that when the sample contacts the first reagent, a first labeled complex is formed if a low level of C-reactive protein is present in the sample, and at least a second reagent specific for MxA, so that, when the sample contacts the second reagent, a second labeled complex is formed if MxA is present in the sample; and B) a second reagent zone comprising at least a third reagent specific for C-reactive protein, wherein the third reagent detects only a level of C-reactive protein, which is higher than the level of C-reactive protein detected by the first reagent, so such that when the sample contacts the third reagent, a labeled third complex is formed if the level of C-reactive protein is present in the sample; ii) a first flow side chromatographic test strip (815, 1015) comprising: A) a first detection zone comprising a first binding partner which binds the first labeled complex; and a second binding partner which binds the second labeled complex; and B) a first bypass zone (850) located upstream of the first detection zone on the lateral flow chromatographic test strip, wherein the first bypass zone interrupts lateral flow on the first lateral flow chromatographic test strip; and iii) a second lateral flow chromatographic test strip (825, 1225) parallel in a lateral flow direction to a first lateral flow chromatographic test strip (815), comprising: A) a second detection zone comprising a third binding partner that binds the third labeled complex; and B) a second bypass zone located upstream of the first detection zone on the lateral flow chromatographic test strip, wherein the bypass zone interrupts the lateral flow on the second lateral flow chromatographic test strip; iv) a first sample application zone (873, 1073), where the sample is placed in the sample analyzing device, wherein the first sample application zone is placed at the selected location of the group consisting of: i) the first side flow chromatographic test strip upstream of the detection zone and ii) in the first reagent zone of the sample compressor; and v) a second sample application zone (876, 1076) where the sample is placed in the sample analyzing device, wherein the second sample application zone is placed at a location selected from the group consisting of: i) the second side flow chromatographic test strip upstream of the detection zone and ii) in the second reagent zone of the sample compressor; wherein the sample compressor is in a different plane than the lateral flow chromatographic test strip and a second lateral flow chromatographic test strip; wherein the sample compressor's first reagent zone bridges the first bypass zone and the sample compressor's second reagent zone bridges the second bypass zone, bypassing the flow to the sample compressor and returning the flow to the first chromatographic test strip and the second chromatographic test strips at the end of the first bypass zone and the second bypass zone; and c) analyzing the sample for the presence of the level of C-reactive protein, MxA, and the highest level of C-reactive protein.
[0002]
2. METHOD according to claim 1, characterized in that the sample analysis device further comprises a first control connection partner located in each of the first reagent zone and the second reagent zone of the sample compressor and a second sampling partner. control binding immobilized in a control zone of each of the first lateral flow chromatographic test strips and second lateral flow chromatographic test strips, wherein the first control binding partner is a binding partner for the second partner control link.
[0003]
3. METHOD, according to any one of claims 1 or 2, characterized in that it comprises the step of: a) collecting a sample; b) transferring the sample to a sample analysis device comprising: i) a first lateral flow chromatographic test strip comprising: a first reagent zone comprising at least a first reagent specific to a first level of C-reactive protein in such a way that when the sample contacts the first reagent, a labeled first complex is formed if a low level of C-reactive protein is present in the sample; a second reagent zone comprising at least one second reagent specific for MxA such that, when the sample contacts the second reagent, a second labeled complex is formed if MxA is present in the sample; and a first detection zone comprising a first binding partner which binds the first labeled complex; and a second binding partner which binds the second labeled complex; and ii) a second lateral flow chromatographic test strip parallel in a lateral flow direction to a first lateral flow chromatographic test strip, comprising: at least a third reagent zone comprising: at least a third reagent specific for a second C-reactive protein level, where the third reagent detects only a level of C-reactive protein, which is higher than the level of C-reactive protein detected by the first reagent, so that when the sample contacts the third reagent, it forms if a third labeled complex if high level of C-reactive protein is present in the sample; and a detection zone comprising a third binding partner that binds the third labeled complex; and c) analyzing the sample for the presence of low level of C-reactive protein, MxA, and high level of C-reactive protein.
[0004]
4. METHOD according to any one of claims 1 to 3, characterized in that a concentration threshold to obtain a positive result for the low level of C-reactive protein in the detection zone of the first lateral flow chromatographic test strip is equal at or greater than a serum equivalent of 6-15 mg/l of C-reactive protein, and preferably equal to or greater than a serum equivalent of approximately 10 mg/l of C-reactive protein.
[0005]
5. METHOD according to any one of claims 1 to 4, characterized in that a concentration threshold for obtaining a positive result for the high level of C-reactive protein in the detection zone of the first lateral flow chromatographic test strip is equal at or greater than a serum equivalent of 60-100 mg/l, and preferably equal to or greater than a serum equivalent of approximately 80 mg/l.
[0006]
6. METHOD according to any one of claims 1 to 5, characterized in that a concentration threshold for obtaining a positive result for MxA in the detection zone of the first lateral flow chromatographic test strip is equal to or greater than 15- 250 ng/ml, and preferably equal to or greater than approximately 40 ng/l.
[0007]
7. METHOD, according to any one of claims 1 or 3, characterized in that step c) comprises the sub-steps of: i) eluting the sample in the sample analyzing device; and ii) visually determining a result for the detection zone.
[0008]
METHOD according to either of claims 1 or 3, characterized in that the presence of MxA is indicated by a first test line (202) located in the detection zone of the first lateral flow chromatographic test strip and the presence of the low C-reactive protein level is indicated by a second test line (203) located in the detection zone of the first zone of the lateral flow chromatographic test strip.
[0009]
9. METHOD, according to claim 8, characterized in that the first test line displays a first color when positive and the second test line displays a second color different from the first color when positive, and wherein optionally both the first test line and the second test line are placed in the same space on the sample analyzing device so that a third color is formed when both the first test line and the second test line are positive.
[0010]
METHOD according to claim 8, characterized in that the first test line is spatially separated from the second test line on the first lateral flow chromatographic test strip.
[0011]
11. METHOD according to any one of claims 1 or 3, characterized in that the sample is a blood sample or the sample contains leukocytes.
[0012]
12. SIDE FLOW DEVICE FOR DETECTION OF AN ANALYTE IN A SAMPLE, characterized in that it comprises: a) a sample compressor (870, 1070) comprising: i) a first reagent zone comprising at least one first reagent specific for a C-reactive protein, so that when the sample contacts the first reagent, a first labeled complex is formed if a low level of C-reactive protein is present in the sample, and at least a second reagent specific for MxA so that, when the sample contacts the second reagent, a second labeled complex is formed if MxA is present in the sample; and ii) a second reagent zone comprising at least a third reagent specific for C-reactive protein, which detects only a level of C-reactive protein, which is higher than the level of C-reactive protein detected by the second reagent, such that, when the sample contacts the third reagent, a third labeled complex is formed if high level of C-reactive protein is present in the sample; b) a first lateral flow chromatographic test strip (815, 1215) comprising: i) a first detection zone comprising a first binding partner which binds the first labeled complex; and a second binding partner which binds the second labeled complex; and ii) a first bypass zone (850) located upstream of the first detection zone on the lateral flow chromatographic test strip, wherein the first bypass zone interrupts lateral flow on the first lateral flow chromatographic test strip; and c) a second lateral flow chromatographic test strip (825, 1225) parallel in the lateral flow direction to the first lateral flow chromatographic test strip, comprising: i) a second detection zone comprising a third binding partner that binds to the third marked complex; and ii) a second bypass zone located upstream of the first detection zone on the lateral flow chromatographic test strip, wherein the second bypass zone interrupts lateral flow on the second lateral flow chromatographic test strip; and d) a first sample application zone (1273) where the sample is placed in the sample analyzing device, wherein the first sample application zone is placed at the location selected from the group consisting of: i) the first test strip side flow chromatography upstream of the detection zone and ii) in the first reagent zone of the sample compressor; e) a second sample application zone (1276) where the sample is placed in the sample analyzing device, where the second sample application zone is placed at a location selected from the group consisting of: i) the second test strip side flow chromatography upstream of the detection zone and ii) in the second reagent zone of the sample compressor; wherein the sample compressor is in a different plane than the lateral flow chromatographic test strip and a second lateral flow chromatographic test strip; wherein the sample compressor's first reagent zone bridges the first bypass zone and the sample compressor's second reagent zone bridges the second bypass zone, bypassing the flow to the sample compressor and returning the flow to the first chromatographic test strip and the second chromatographic test strips at the end of the first bypass zone and the second bypass zone.
[0013]
DEVICE according to claim 12, characterized in that the sample analysis device further comprises a first control link partner located in each of the first reagent zones and the second reagent zone of the sample compressor and a second partner. control binding element immobilized in a control zone (204) of each of the first lateral flow chromatographic test strip and the second lateral flow chromatographic test strip, wherein the first control binding partner is a binding partner to the second control binding partner.
[0014]
DEVICE according to claim 12, characterized in that it comprises: a) a first lateral flow chromatographic test strip comprising: a first reagent zone comprising at least one first reagent specific for C-reactive protein such that, when when the sample contacts the first reagent, a first labeled complex is formed if a low level of C-reactive protein is present in the sample; a second reagent zone comprising at least one second reagent specific for MxA such that, when the sample contacts the second reagent, a second labeled complex is formed if MxA is present in the sample; and a first detection zone comprising a first binding partner which binds the first labeled complex; and a second binding partner which binds the second labeled complex; and b) a second lateral flow chromatographic test strip parallel in the lateral flow direction to the first lateral flow chromatographic test strip, comprising: at least a third reagent zone comprising at least one third reagent specific for C-reactive protein, wherein the third reagent only detects a level of C-reactive protein, which is higher than the level of C-reactive protein detected by the first reagent, so that when the sample contacts the third reagent, a labeled third complex is formed if the high level of C-reactive protein is present in the sample; and a second detection zone comprising a third binding partner that binds the third labeled complex.
[0015]
DEVICE according to either of claims 12 or 14, characterized in that the first detection zone of the first lateral flow chromatographic test strip comprises the first test line for detecting a positive result for MxA in the sample and a second line of test. test to detect a positive result for the low level of C-reactive protein in the sample.
[0016]
16. DEVICE according to claim 15, characterized in that the first test line displays a first color when positive and the second test line displays a second color different from the first color when positive; and optionally both the first test line and the second test line are placed in the same space on the first lateral flow chromatographic test strip so that a third color is formed when both the first test line and the second test line are test are positive.
[0017]
DEVICE according to claim 15, characterized in that the first test line is spatially separated from the second test line on the first lateral flow chromatographic test strip.
[0018]
18. DEVICE according to either of claims 12 or 14, characterized in that the first detection zone and the second detection zone each comprise a control line which is visible to the naked eye when the device is in operation.
[0019]
DEVICE according to either of claims 12 or 14, characterized in that the first lateral flow chromatographic test strip further comprises a first sample application zone upstream of the first reagent zone, the second reagent zone, and the first detection zone is downstream of the first reagent zone and the second reagent zone.
[0020]
DEVICE according to either of claims 12 or 14, characterized in that the second lateral flow chromatographic test strip further comprises a second sample application zone upstream of the third reagent zone, and the second detection zone, and the second detection zone is downstream of the third reagent zone.
[0021]
DEVICE according to either of claims 12 or 14, characterized in that the first lateral flow chromatographic test strip further comprises a lysis zone comprising at least one lysing agent, wherein the lysing agent contacts the sample in the first side flow chromatographic test strip.
[0022]
DEVICE according to either of claims 12 or 14, characterized in that the second lateral flow chromatographic test strip further comprises a lysis zone comprising at least one lysing agent, wherein the lysing agent contacts the sample in the second. side flow chromatographic test strip.
[0023]
DEVICE according to either of claims 12 or 14, characterized in that the first lateral flow chromatographic test strip further comprises a first sample application zone downstream of the first reagent zone and the second reagent zone upstream. of the first detection zone.
[0024]
DEVICE according to either of claims 12 or 14, characterized in that the second lateral flow chromatographic test strip further comprises a second sample application zone downstream of the third reagent zone and upstream of the second detection zone.

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