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    • 专利摘要:
    The present invention describes an artificial immunocomplex comprising the human beta-2 glycoprotein 1 (B2GP1) protein bound to a fragment of the constant region of the heavy chain of an immunoglobulin, its use as a caliber, a method for calibrating an analytical detection system of circulating immunocomplexes B2GP1-antiB2GP1 comprising performing a calibration curve with artificial immunocomplexes and a method to determine if a subject is at risk of suffering pathological manifestations associated with the antiphospholipid syndrome (APS). (Machine-translation by Google Translate, not legally binding)
    公开号:ES2727261A1
    申请号:ES201830367
    申请日:2018-04-12
    公开日:2019-10-15
    发明作者:Hernandez Antonio Serrano；Flores José Angel Martínez；Blanco Manuel Serrano
    申请人:Fundacion Investigacion Biomedica Hospital Universitario 12 Octubre；
    IPC主号:
    专利说明:

    [0001] ARTIFICIAL IMMUNOCOMPLEJOS AND ITS USE AS CALIBERS IN CIRCULATING IMMUNOCOMOBETIC DETECTION SYSTEMS B2GP1-aB2GP1
    [0002]
    [0003]
    [0004]
    [0005] TECHNICAL SECTOR
    [0006]
    [0007] The present invention falls within the technical sector of medicine, more specifically in the field of immunopathology.
    [0008]
    [0009] BACKGROUND OF THE INVENTION
    [0010]
    [0011] Antiphospholipid syndrome (APS) or Hughes syndrome is a multisystem autoimmune disorder characterized by the appearance of thrombosis and / or gestational morbidity in patients presenting with antiphospholipid antibodies (aPL) in the blood.
    [0012]
    [0013] APLs are a heterogeneous group of autoantibodies directed against phospholipids, phospholipid complexes associated with proteins or phospholipid binding proteins in isolation.
    [0014]
    [0015] Some of the associated manifestations of APS are: blood clots (thrombosis), spontaneous abortions, fetal death, premature delivery, stroke or transient ischemic accident.
    [0016]
    [0017] There are currently no specific diagnostic criteria for PHC, although the criteria for classifying patients for clinical trials are used to diagnose the disease. These classification criteria, currently in force, were established in Sydney in 2004 (Miyakis S et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006; 4: 295-306) . These criteria mark the practice in all clinical laboratories and are summarized in Table 1.
    [0018] Table 1: APS classification criteria.
    [0019]
    [0020]
    [0021]
    [0022] One or more abortions before week 10 of anti-beta2 glycoprotein (aB2GPI) gestation antibodies documented.
    [0023] One or more premature (<34 weeks)
    [0024] preclampsia or eclampsia Three or more spontaneous abortions
    [0025]
    [0026] Many patients with APS clinic remain undiagnosed when no consensus aPL that meets the laboratory criteria is detected.
    [0027]
    [0028] Currently the diagnosis of the disease in clinical laboratories is carried out by the detection of aPL by solid-phase techniques such as ELISA, Fluoroenzyme immunoassay or Multiplex technology, which detect anti-cardiolipin antibodies (aCL) or anti-beta-2 glycoprotein 1 (aB2GP1 or anti -B2GP1) in serum or plasma. To meet the criteria for laboratory classification as a subject with APS, antibodies must be maintained in the serum for at least 12 weeks.
    [0029]
    [0030] Despite the relationship between the presence of aPL and the disease, the value of the presence of aPL to predict events related to APS (for example, thrombosis or complications during pregnancy) is low. For this reason, new methods with better predictive value are necessary for the identification of subjects carrying aPL antibodies and, therefore, are at risk of suffering, for example, a thrombotic event. An effective system to identify these patients will allow it to be treated more quickly, minimizing the damage to the subject and its cost derived to the corresponding national health system.
    [0031]
    [0032] The detection of circulating immunocomplexes B2GP1-aB2GP1 is one of the identification systems of patients with a higher risk of developing thrombotic events within those who are carriers of aPL. These immunocomplexes have been described as risk biomarkers in the study of the pathogenesis of PHC (Serrano M et al. Beta2-Glycoprotein I / IgA Immune Complexes: A Marker to Predict Thrombosis After Renal Transplantation in Patients With Antiphospholipid Antibodies. Circulation 2017, 135 (20): 1922-34).
    [0033]
    [0034] These B2GP1-aB2GP1 circulating immune complex detection systems detect their concentration in the plasma or serum previously obtained from a subject. In order to determine its concentration, it is necessary to prepare a calibration curve (or calibration) based on known concentrations of the immune complexes (which function as calibrators or calibrators), to determine, from the equation of the calibration curve, to determine the concentration of the B2GP1-aB2GP1 immunocomplexes in the sample of the subject and conclude if said subject has an amount of immunocomplexes above the risk point that will determine if he or she is at risk of suffering any pathology related to APS.
    [0035]
    [0036] Current methods of determining circulating immunocomplexes in serum samples from patients use known concentrations of native B2GP1-aB2GP1 immunocomplexes as calibrators (Martinez-Flores JA et al. Detection of circulating immune complexes of human IgA and beta 2 glycoprotein I in patients with antiphospholipid syndrome symptomatology. J Immunol Methods 2015, 422: 51-8).
    [0037]
    [0038] The closest state of the art is the Martinez-Flores JA et al. 2015, which detects the circulating immunocomplexes B2GP1-aB2GP1 (IgA) in patients with symptoms associated with APS. To determine the concentration of the immunocomplexes, a preliminary calibration curve is performed with native immunocomplexes B2GP1-aB2GP1.
    [0039]
    [0040] Native immunocomplexes or natural immunocomplexes are those obtained from human subjects. The native immunocomplexes have the disadvantage of being labile and unstable, which makes their detection difficult, generates great variability between detection tests and their use is also limited to a few days or weeks. To increase the life of these calibres, it is necessary to freeze the native immune complexes, but these molecules are very sensitive to freeze-thaw cycles. In addition, because the number of analyzes for which the calibres can be used is limited and it is necessary to replenish new calibres periodically to be able to continue the analyzes to the patient samples, with the consequent element of uncertainty that generates the variability of the lots of sample sizes. Therefore, calibration curves for the detection of circulating immunocomplexes B2GP1-aB2GP1 prepared with known concentrations of native immunocomplexes are not suitable for establishing the true and reliable concentration of circulating immunocomplexes in the patient sample.
    [0041]
    [0042] The objective technical problem is the need for stable calibres and to prepare a calibration curve in a B2GP1-aB2GP1 circulating immunocomplex detection system.
    [0043]
    [0044] DESCRIPTION OF THE INVENTION
    [0045]
    [0046] The present invention relates to an artificial immunocomplex comprising a fragment of the human beta-2 glycoprotein 1 protein (B2GP1) linked to a fragment of the heavy chain constant region of an immunoglobulin (Fc).
    [0047]
    [0048] Immune or immunocomplex complexes (CIC) are macromolecules formed by subunits consisting of antigen (s) and antibody (s) linked together by low-energy chemical bonds (Van der Waals forces, hydrogen bonds or hydrophobic interactions).
    [0049]
    [0050] For the purposes of the present invention, several types of immunocomplexes are distinguished:
    [0051] 1) circulating immunocomplexes: they are immunocomplexes produced naturally by the organism of a subject, circulate in the blood in a soluble way and can be deposited in the inner wall of the blood vessels;
    [0052] 2) native (or natural) immune complexes: they are circulating immune complexes that have been isolated from blood or serum of a subject; and
    [0053] 3) artificial immunocomplexes (or artificial immune complexes): they have been produced in the laboratory. The sequence of these immunocomplexes does not necessarily correspond to that of the circulating immunocomplexes or that of the natives. Antigen-antibody binding does not take place through low energy chemical bonds but through peptide bonds or high energy covalent bonds between the two subunits of the immunocomplex. Artificial immunocomplexes are stable for several months at 4 ° C.
    [0054] Apolipoprotein H (Apo H), also called beta-2 glycoprotein 1 (B2GP1, reference UniProtKB P02749, Reference NCBI No.: NP_000033.2), is a plasma amino acid glycoprotein of 345 amino acids that binds to negatively charged substances such as heparin , phospholipids and dextran sulfate. This protein can bind phosphatidylserine and prevent the activation of blood coagulation by binding to phospholipids on the surface of damaged cells. Its sequence is encoded in the APOH gene.
    [0055]
    [0056] The artificial immunocomplexes of the invention are formed by two subunits: one subunit corresponds to the B2GP1 protein described above (antigen) and the other subunit corresponds to a fragment of the constant region of the heavy chain of an immunoglobulin (antibody). The fragment of the immunoglobulin heavy chain constant region of the artificial immunocomplex is linked by a peptide bond, a covalent bond, to the amino or carboxyl terminus of the B2GP1 protein. In a preferred embodiment, the fragment of the immunoglobulin heavy chain constant region binds to the carboxyl terminus of the B2GP1 protein. The union of the subunits of the immunocomplexes by peptide bonds or covalent bonds has the advantage that, being a high energy chemical bond, it is very stable to changes in ionic strength, pH or temperature. On the contrary, the antigen-antibody binding of natural immunocomplexes is performed by low-energy chemical bonds, such as hydrogen bonds, hydrophobic interactions and Van der Waals forces. These links are weak and the unions mediated by them are unstable.
    [0057]
    [0058] In one embodiment, the artificial immunocomplexes can have at least one amino acid linker between the B2GP1 protein sequence and the fragment of the constant region of the immunoglobulin heavy chain.
    [0059]
    [0060] In a preferred embodiment. The fragment of the constant region of the immunoglobulin heavy chain is selected from the IgM, IgG or IgA isotypes.
    [0061]
    [0062] The first artificial immunocomplex of the invention is characterized in that it comprises the sequence SEQ ID NO: 1. The sequence SEQ ID NO: 1 corresponds to the chimeric protein B2PG1-IgM, composed of the human B2PG1 sequence defined above in which the stop codon has replaced the amino acid lysine (K) and through a peptide bond the amino acids 240 to 590 of the chain heavy of the constant region of human immunoglobulin M (Accession No. NCBI AAS01769.1).
    [0063]
    [0064] The first artificial immunocomplex of the invention is considered to have at least 98%, 99% or 100% identity with the sequence SEQ ID NO: 1.
    [0065]
    [0066] The first artificial immunocomplex of the invention may be encoded in any DNA sequence whose translation results in SEQ ID NO: 1, or results in a protein whose amino acid sequence has at least 98%, 99% or 100% Identity with the sequence SEQ ID NO: 1. As an example, and without limiting the present invention, the nucleotide sequence of the first artificial immunocomplex comprises the sequence SEQ ID NO: 2.
    [0067]
    [0068] The second artificial immune complex of the invention is characterized in that it comprises the sequence SEQ ID NO: 3. The sequence SEQ ID NO: 3 corresponds to the chimeric protein B2PG1-IgG, composed of the human B2PG1 sequence defined above, in which the stop codon has been replaced by a linker peptide composed of the amino acids lysine (K), asparagine (D) and proline (P), linked by peptide bonding to amino acids 31 to 246 of the constant region of the human G immunoglobulin heavy chain (NCBI Accession No. AAW65947.1).
    [0069]
    [0070] The second artificial immune complex of the invention is considered to have at least 98%, 99% or 100% identity with the sequence SEQ ID NO: 3.
    [0071]
    [0072] The second artificial immunocomplex of the invention may be encoded in any DNA sequence whose translation results in the sequence SEQ ID NO: 3, or in the place of a protein whose amino acid sequence has at least 98%, 99% or 100 % identity with the sequence SEQ ID NO: 3. As an example, and without limiting the present invention, the nucleotide sequence of the second artificial immunocomplex comprises SEQ ID NO: 4.
    [0073]
    [0074] The third artificial immunocomplex of the invention is characterized in that it comprises the sequence SEQ ID NO: 5. The sequence SEQ ID NO: 5 corresponds to the chimeric protein B2PG1-IgA, composed of the human B2PG1 sequence defined above in which the stop codon has been replaced by a linker peptide composed of the amino acids lysine (K) and asparagine (D), linked by peptide bond to amino acids 117 to 352 of the heavy chain constant region of immunoglobulin A (Accession No. NCBI AAC82528.1).
    [0075]
    [0076] The third artificial immune complex of the invention is considered to have at least 98%, 99% or 100% identity with the sequence SEQ ID NO: 5.
    [0077]
    [0078] The third artificial immunocomplex of the invention may be encoded in any DNA sequence whose translation results in SEQ ID NO: 5, or results in a protein whose amino acid sequence has at least 98%, 99% or 100% Identity with the sequence SEQ ID NO: 5. As an example, and without limiting the present invention, the nucleotide sequence of the third artificial immunocomplex comprises SEQ ID NO: 6.
    [0079]
    [0080] In a preferred embodiment, the artificial immunocomplex comprising the human beta-2 glycoprotein 1 (B2GP1) protein bound to a fragment of the constant region of the heavy chain of an immunoglobulin is characterized in that the sequence of said immunocomplex comprises a sequence identified by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.
    [0081]
    [0082] The object of the present invention is a method for calibrating an analytical system of detection of immune complexes B2GP1-aB2GP1 comprising:
    [0083] a) dilute an artificial immunocomplex to at least three different known concentrations;
    [0084] b) determine the optical density of each concentration of the previous stage; c) perform a calibration curve that relates each concentration of stage a) to its respective optical density obtained in stage b); d) obtain a mathematical function that defines the calibration curve of stage c).
    [0085]
    [0086] The calibration curve is performed with at least three known concentrations of the same type of artificial immunocomplexes (B2GP1-IgG, B2GP1-IgM or B2GP1-IgA) in step a). The curve is performed with at least 3, at least 4, at least 5, at least 6 or at least 7 known concentrations of the same type of artificial immunocomplexes.
    [0087]
    [0088] The calibrators or calibrators are known concentrations of certain molecules that serve to establish a standard curve (or calibration curve) that relates a quantifiable physical property (eg concentration) and a response or physical property thereof (eg optical density). In a preferred embodiment, the calibration curve it relates the optical density (OD) of the average of several measurements of the same caliber to the known concentration of said caliber.
    [0089]
    [0090] The optical density value is proportional to the concentration of the substance of interest in a sample (artificial immunocomplexes), which allows to perform the calibration curve that relates the optical density and the concentration of the immunocomplexes. In a preferred embodiment, the optical density of a sample is detected an absorbance at 450 nm.
    [0091]
    [0092] The detection of the optical density can be carried out by any method known in the state of the art, such as colorimetry or turbidity. Analytical immunocomplete detection systems refer to any detection system and quantify proteins, such as spectrometry, immunoassays or immunoblotting. An analytical detection system is ELISA ( Enzyme-Linked ImmunoSorbent Assay) which, for example, may be based on alkaline phosphatase or peroxidase.
    [0093]
    [0094] The mathematical function, function of the curve or curve equation is a quadratic equation of type y = ax bx2 c, in which the variable "y" is the known concentration of artificial immunocomplexes in a caliber sample, the variable "x" it is the optical density value for said sample and the values a, b and c are the coefficients of the calibration curve. The a, b and c values are specific to each calibration curve and can be determined manually or automatically using software installed in the analytical detection system.
    [0095]
    [0096] For the purposes of the present invention, the concentration of immunocomplexes in a sample is represented in units per milliliter (U / mL).
    [0097]
    [0098] Another embodiment of the present invention is a method for determining the concentration of a circulating immunocomplex B2PG1-aB2GP1 in a sample obtained from a subject comprising:
    [0099] a) dilute an artificial immunocomplex to at least three different concentrations; b) determine the optical density of each concentration of immunocomplexes of the previous stage;
    [0100] c) perform a calibration curve that relates each concentration of stage a) to its respective optical density obtained in stage b); d) obtain a mathematical function that defines the calibration curve of the stage c);
    [0101] e) measure the optical density of a biological sample obtained from a subject; and f) determine the concentration of circulating immune complexes B2GP1-aB2GP1 in the sample from the function obtained in step d).
    [0102]
    [0103] In a preferred embodiment, the subject's biological sample is serum or plasma. In a more preferred embodiment, the subject's biological sample is serum. Whey is a liquid without cells that is obtained from the blood after the coagulation process. Serum differs from plasma primarily because it lacks fibrinogen and platelets.
    [0104]
    [0105] For the determination of the concentration of a circulating immunocomplex in a sample of a subject, a calibration curve must be performed previously, as indicated above, that relates the known concentration of immune complexes to a quantifiable physical characteristic, such as optical density.
    [0106]
    [0107] The concentration of circulating immunocomplexes in step d) is determined by replacing in the equation obtained in step d) the variable "x" with the value of the optical density obtained in step e).
    [0108]
    [0109] Another embodiment of the present invention is directed to a method for determining if a subject is at risk of suffering manifestations associated with the antiphospholipid syndrome (APS). This method comprises determining the concentration of circulating immunocomplexes B2GP1-aB2GP1 in a sample of a subject according to any of the methods defined above, in which a concentration value of circulating immunocomplexes B2GP1-aB2GP1 equal to or greater than 20.5 U / mL is indicative that the subject is at high risk of suffering pathological manifestations associated with PHC. In another embodiment, a value below 20.5 U / mL of circulating immunocomplexes is considered to be indicative that the subject has a low risk of suffering manifestations associated with PHC, a risk that is equal to that of the healthy population, a population carrying aPL antibodies or a carrier population of a2GP1 antibodies that do not form circulating immune complexes.
    [0110]
    [0111] The determination of the concentration of immunocomplexes in patient samples makes it possible to identify subjects with antiphospholipid antibodies (aPL) that have high or low risk of suffering a thrombotic event or other manifestations. Pathological associated with PHC. The risk is determined by the presence of immunocomplexes above a numerical value corresponding to a cut-off point determined by ROC curve analysis.
    [0112]
    [0113] A person is at risk of developing a manifestation associated with PHC when the probability of suffering the associated pathology is at least double the risk of the general population. For example, people who have a concentration of circulating immunocomplexes B2GP1-aB2GP1 (IgA or IgG isotypes) equal to or greater than 20.5 U / mL have a risk of developing an APS-associated manifestation of at least 3%, compared to the general population that has a risk of 0.6% (five times higher). Studies have been described in which the risk of immunocomplex carriers is greater than 30% (Serrano etal., 2017).
    [0114]
    [0115] In a preferred embodiment, the subject at risk of suffering manifestations associated with the antiphospholipid syndrome (APS) is a carrier of antiphospholipid antibodies (aPL). A carrier subject of aPL is a person who has been detected at least one aPL of any target: lupus anticoagulant (aAL), anticardiolipin (aCL) or anti-beta2 glycoprotein (aB2GP1), of any isotype (IgG, IgM or IgA). A subject carrying aPL antibodies may manifest signs and symptoms of any of the pathological manifestations associated with APS or be an asymptomatic carrier.
    [0116]
    [0117] In a preferred embodiment, the method determines if the subject is at risk of suffering from antiphospholipid syndrome or any of its associated pathological manifestations, such as blood clots (thrombosis), spontaneous abortions, fetal death, premature delivery, stroke or transient ischemic accident.
    [0118]
    [0119] Thrombosis or a thrombotic event is any incident in which a thrombus (clot) develops in an artery. It can manifest as angina pectoris, ischemic stroke or peripheral arterial disease. The definition of thrombotic events excludes minor thrombosis such as thrombophlebitis and hemorrhoids.
    [0120]
    [0121] Another embodiment of the invention relates to the use of any of the artificial immunocomplexes of the invention B2GP1-IgG, B2GP1-IgM or B2GP1-IgGA as a caliber in a circulating immunocomplex detection system comprising at least one fragment of the bound B2GP1 protein to a fragment of aB2GP1 antibody (B2GP1-aB2GP1).
    [0122]
    [0123] A stable and reliable calibration system allows the detection of circulating immunocomplexes of a subject allows to determine more precisely the concentration of said immunocomplexes and favors that a patient at risk of suffering from antiphospholipid syndrome or any of its associated manifestations, be treated more quickly minimizing damage to the patient.
    [0124]
    [0125] Artificial immunocomplexes have the same anti-B2GP1 antigen recognition properties as native aBGP1 antibodies or anti-B2GP1 monoclonal antibodies. In addition, they are more than 100 times more stable than natural immunocomplexes, which allows them to be used as calibrators in circulating immunocomplex detection systems for weeks thanks to their stability and reproducibility between trials.
    [0126]
    [0127] The advantages of artificial immunocomplexes and their use as gauges in B2GP1-aB2GP1 circulating immunocomplete detection systems are:
    [0128] - Stable for several months at 4 ° C;
    [0129] - Reproducibility between trials (little inter-trial and intra-trial variability); - Ease of obtaining through standard molecular biology techniques;
    [0130] - Antigen recognition by anti-B2GP1 monoclonal antibodies identical to recognition by native immunocomplexes.
    [0131]
    [0132] DESCRIPTION OF THE FIGURES
    [0133]
    [0134] Figure 1: shows the detection of artificial complexes by ELISA developed with peroxidase (A, B) or alkaline phosphatase (C). The dilutions used were: 1: 1 (dilution -); 1: 2; 1: 4; 1: 8; 1:16; 1:32; 1:64 (Dilution); white (C-). A : B2GP1-IgM, B : B2GP1-IgG, C : B2GP1-IgA.
    [0135]
    [0136] Figure 2: Inter-assay variability of 10 trials with 8 dilutions of the B2GP1-IgM construct. The concentration of artificial immunocomplex is represented on the X axis and on the Y axis the optical density value measured at 450 nm. A : medium, B : medium.
    [0137] Figure 3: Inter-assay variability of 10 trials with 8 dilutions of the B2GP1-IgG construct. The concentration of artificial immunocomplex is represented on the X axis and on the Y axis the optical density value measured at 450 nm. A : medium, B : medium.
    [0138]
    [0139] Figure 4: Inter-assay variability of 10 trials with 8 dilutions of the B2GP1-IgA construct. The concentration of artificial immunocomplex is represented on the X axis and on the Y axis the optical density value measured at 450 nm. A : medium, B : medium.
    [0140]
    [0141] Figure 5: Calibration curve and corresponding equation for the B2GP1-IgM ( A ), B2GP1-IgG ( B ) and B2GP1-IgA ( C ), calculated with the average values for each point of the 10 experiments referred to in Figures 2 to 4 respectively. To the right of each curve is represented the equation of the curve, the coefficient of determination (R2) and the P value of the study for each caliber.
    [0142]
    [0143] Figure 6 Comparison between B2GP1 immunocomplex calibration systems. A (white), BH serial dilutions 1, 1: 2, 1: 4, 1: 8, 1:16, 1:32 and 1:64 in PBS respectively of concentrations of artificial immunocomplexes B2GP1-IgA (in duplicate, samples a and b ) and three samples of native B2GP1-IgA immune complexes (aB2GP1) obtained from three patients (samples # 1, # 2 and # 3).
    [0144]
    [0145] EXAMPLES
    [0146]
    [0147] With the intention of showing the present invention in an illustrative way, although in no way limiting, the following examples are provided.
    [0148]
    [0149] Example 1: Obtaining artificial immunocomplexes
    [0150]
    [0151] Fusion proteins, chimeric proteins or artificial immune complexes were generated by fusing the B2GP1 protein sequence to a fragment of the heavy chain constant region of IgM, IgG or IgA immunoglobulins.
    [0152]
    [0153] The sequences coding for the B2GP1-IgM (SEQ ID NO: 1), B2GP1-IgG (SEQ ID NO: 3) and B2GP1-IgA (SEQ ID NO: 5) constructs were amplified by PCR (30 cycles with a temperature of priming of 60 ° C 1 min and an expansion time of one minute at 72 ° C). Then the amplified sequences are introduced into plasmid pLVX-IRES-tdTomato (Clonteck, Takara Bio USA, Inc., Mountain View, CA 94043, USA). Three plasmids were obtained, in which each one contains respectively the B2GP1-Mu-C2C3C4 nucleotide sequences (B2GP1 fused to the constant domains 2, 3 and 4 of the IgM heavy chain, SEQ ID NO: 2), B2GP1- FcIgG (SEQ ID NO: 4) and B2GP1-FcIgA (SEQ ID NO: 6). Each of the plasmids was introduced into the "Stellar competent E.Coli cells" (Clontech, Takara Bio USA, Inc.) bacteria by thermal shock according to the manufacturer's protocol. Positive colonies were selected by antibiotic selection and grown in for 24-48 hours at 37 ° C. Plasmid DNA from the transforming colonies was extracted and purified by a purification kit, according to the manufacturer's instructions (Clontech, Takara Bio USA, Inc.).
    [0154]
    [0155] Next, human HEK-293 cells (embryonic kidney) were transfected with Lipofectamine 2000 according to manufacturer specifications (Thermofisher Scientific).
    [0156]
    [0157] During the culture the fusion protein encoded in the respective gene construct is expressed. The expressed proteins are secreted in the cell culture supernatant. Once 500 ml of supernatant was accumulated, the fusion protein was purified by affinity columns.
    [0158]
    [0159] This procedure was performed for each of the three constructions. Once purified, fusion proteins (or chimeric proteins or artificial immune complexes B2GP1-IgM (SEQ ID NO: 1), B2GP1-IgG (SEQ ID NO: 3) and B2GP1-IgA (SEQ ID NO: 5) were obtained.
    [0160]
    [0161] Figure 1 shows the detection of the constructs by ELISA revealed with the substrate peroxidase (A, B) or alkaline phosphatase (C). The experiment was performed in quadruplicate using several dilutions of the purified fusion protein. The dilutions used were: 1: 1 (dilution -); 1: 2; 1: 4; 1: 8; 1:16; 1:32; 1:64 (Dilution); white (C-). A: B2GP1-IgM, B: B2GP1-IgG, C: B2GP1-IgA.
    [0162]
    [0163] The fusion zone between the B2GP1 protein and the corresponding immunoglobulin heavy chain fragment is essential for the fusion protein to be stable and, therefore, can be used as a gauge in circulating immunocomplex detection systems.
    [0164] The amino acids selected for the fusion of the immunoglobulin heavy chain fragment in each isotype, have the objective that the generated chimeric molecule conserves the physiological folding of the two fused subunits. The maintenance of the structure is key in the design of artificial immunocomplexes. The selected area of the heavy chain of each immunoglobulin that binds to the sequence of B2GP1 is critical for the fusion product to maintain the three-dimensional structure of both subunits (B2GP1 and heavy chain fragment of the corresponding immunoglobulin) and therefore , its antigenic characteristics.
    [0165]
    [0166] It has been found that, if the binding between B2GP1 and the IgG heavy chain takes place, for example, at the beginning of the hinge region of the heavy chain, the resulting protein has the appropriate size, but this construct is not recognized by anti-antibodies. -B2GP1 H219 (Mabtech, Stockholm, Sweden) nor by the human anti-IgG 1 polyclonal antibodies (Sigma, Jackson and Inova).
    [0167]
    [0168] On the contrary, these anti-B2GP1 H219 antibodies (Mabtech, Stockholm, Sweden) and the three human anti-IgG 1 polyclonal antibodies (Sigma, Jackson and Inova) perfectly recognize their target molecule in the native form (B2GP1 or IgG1, respectively) ).
    [0169]
    [0170] In the case where the fusion between B2GP1 and IgG takes place excluding 21 amino acids from the hinge region of IgG1 from the heavy chain, it has been found that the resulting fusion product can be recognized by the anti-B2GP1 H219 monoclonal antibody and by the three polyclonal anti-IgG 1 antibodies mentioned above.
    [0171]
    [0172] With these experiments it is demonstrated that fusions between B2GP1 and fragments of the immunoglobulin heavy chain must be performed at certain positions in the sequence. From the data obtained with B2GP1-IgG, homologous regions in the IgM and IgA heavy chain sequences were identified.
    [0173] Example 2: Calibration of circulating immunocomplex detection systems with artificial immunocomplexes.
    [0174]
    [0175] Next, it was determined whether the constructions can be used in the calibration of circulating immune complex detection systems.
    [0176]
    [0177] The detection of immunocomplexes was carried out as described in following the protocols written in Martínez-Flores et al. 2015
    [0178]
    [0179] A stock solution of each fusion protein (caliber) was prepared at 400 U / ml by diluting in PBS. Next, 0.1% Proclin was added to each stock solution (preservative for antibody solutions). From the stock solution of each construct, dilutions were prepared in PBS with known concentration values to be used as calibres: 1.5 U / mL, 3.1 U / mL, 6.3 U / mL, 12.5 U / mL, 25 U / mL, 25 U / mL, 50 U / mL, 100 U / mL and 200 U / mL.
    [0180]
    [0181] Ten independent tests (calibration curves) were performed for each concentration (caliber) of each of the artificial immunocomplexes in triplicate. The coefficients of variation were always less than 5%, for all points of all experiments, indicating that the intra-test variability of triplicates paca each dilution is very low.
    [0182]
    [0183] The inter-assay variability for the 10 8-point trials (dilutions of each construct) was also very low. The variability of the measurements of each artificial immunocomplex is shown in Figures 2-4 ( Figure 2 : B2GP1-IgM, Figure 3 : B2GP1-IgG, Figure 4 : B2GP1-IgA) in which graph A shows the mean and the Graph B shows the median of the optical density (OD) values at 450 nm for each concentration. The determinations were performed automatically in a Triturus® autoanalyzer (Grifols, Barcelona, Spain).
    [0184]
    [0185] To determine the strength and reproducibility of the trials, the intraclass correlation coefficients of the data of the 10 calibration curves were analyzed for each artificial immunocomplex (Figures 2, 3 and 4). The intraclass correlation coefficient for the B2GP1-IgM fusion protein is 0.985 (95% CI: 0.964-0.997; p <0.0001), for the B2GP1-IgG fusion protein is 0.991 (95% CI: 0.978- 0.998; p <0.0001) and for the fusion protein with IgA 0.988 (95% Confidence interval 0.970 0.997; p <0.0001). These data indicate that the calibration curves performed using the artificial immunocomplexes of the invention have a very low variability. Statistical analysis was performed with the MedCalc V. 14.12 program (MedCalc Software Bvba, Oostende, Belgium).
    [0186]
    [0187] Figure 5 shows the calibration curves of the mean values of the 10 tests for each of the artificial immunocomplexes (CA). To the right of each curve the function (or equation) of the corresponding curve is indicated, its coefficient of determination (R2) and the P-value of each test, which expresses the probability that the results obtained are due to chance. Values of p <0.01 are indicative that the results are not due to chance. The formulas are obtained automatically from the autoanalyzer software. The functions of the calibration curves correspond to an equation type y = ax bx2 c (U / mL = aOD bOD2 c). By substituting the values of optical density of a patient's sample in the calibration curve, the concentration of circulating immune complexes is obtained.
    [0188]
    [0189] Example 3. Comparison between calibration systems.
    [0190]
    [0191] In order to show the differences between the native B2GP1-aB2GP1 and the artificial B2GP1-Ig calibres, an experiment was conducted in which the two calibration systems were compared.
    [0192]
    [0193] For this, serial dilutions 1, 1: 2, 1: 4, 1: 8, 1:16, 1:32 and 1:64 were made in PBS of known concentrations of three samples of native B2GP1-IgA immunocomplexes (aB2GP1) obtained from three patients (samples # 1, # 2 and # 3) and another of a sample of artificial immunocomplexes B2GP1-IgA. The test with the artificial immunocomplex was performed in duplicate (a, b).
    [0194]
    [0195] Patient samples were obtained from the Immunology Service of the Hospital 12 de Octubre from samples submitted for a health examination. Only the leftover serum from the samples was used anonymously as tests.
    [0196]
    [0197] The concentrations of immunocomplexes used as calibres were those indicated in Table 2 .
    [0198] Table 2: Immunocomplex gauges
    [0199]
    [0200] B2GP1-IgA Patient # 1 Patient # 2 Patient # 3 Dilution (U / mL) (U / mL) (U / mL) / mL) A White 0 0 0 0
    [0201]
    [0202]
    [0203]
    [0204]
    [0205]
    [0206]
    [0207]
    [0208]
    [0209] Immunocomplex levels were determined following the protocol described in Martínez-Flores et al. (2015), in which instead of sera the calipers were added: native immunocomplexes (patients # 1, # 2 and # 3) and artificial immunocomplexes (B2GP1-IgA).
    [0210]
    [0211] Figure 6 shows the result of the experiment in which the differences in the concentration of immunocomplexes in relation to the intensity of color in each well are observed. The rows AH correspond to the concentration of immunocomplexes indicated in Table 1. It is observed that the artificial immunocomplexes B2GP1-IgA (in duplicate, samples a and b) can be used as gauges in the determination of circulating immunocomplexes in the same way as up to Date the native B2GP1-IgA immunocomplexes (aB2GP1) were used. This experiment was repeated with the artificial immunocomplexes B2GP1-IgG and B2GP1-IgM, obtaining the same result as with B2GP1-IgA.
    [0212]
    [0213] The function to calculate the units of each patient from the optical density of the test based on the reference values of the calibration curve is performed automatically by the autoanalyzer software. The function of the curve is calculated by the autoanalyzer for each calibration test independently.
    [0214]
    [0215] Example 4: Detection of circulating serum immune complexes
    [0216]
    [0217] In order to determine whether artificial immunocomplexes can be used as gauges in B2GP1-aB2GP1 circulating immune complex detection systems, A capture ELISA was performed in which the presence of circulating immunocomplexes in the patients' serum was detected.
    [0218]
    [0219] 86 consecutive samples were obtained from the 12 de Octubre Hospital serrate (Spain) and the concentration of circulating immunocomplexes was determined as described in Martínez-Flores et al. (2015), using 200, 100, 50, 25, 12.5, 6.3, 3.1 and 1.5 concentrations of the artificial immunocomplexes B2GP1-IgM, B2GP1-IgG and B2GP1-IgA as calibers.
    [0220]
    [0221] 96-well plates Nunc maxisorp ™ (A / S Nunc, Kamstrup, Roskilde, Denmark) were covered with 100 pl per well of 2 pg / ml solution of human anti-B2GP1 monoclonal antibody H219 (Mabtech AB, Nacka Strand, Sweden) in PBS at pH 7.4. The plates were incubated for 16 hours at 4 ° C and then washed three times with PBS-Tween-200.05% (PBS-T). The wells were subsequently blocked for 1 hour at room temperature with 0.1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) in PBS.
    [0222]
    [0223] The plates were washed three times with PBS-T and then 100 pL of the test sera diluted in PBS 1: 100 in PBS were added. They were incubated 2 hours at room temperature, washed with PBS-T three times and the presence of each isotype of aB2GP1 antibody was detected with 100 pL of goat anti-IgG, anti-IgM or human anti-IgA antibody (Jackson ImmunoResearch Laboratories , Inc., West Grove, PA, USA) conjugated with horseradish peroxidase, diluted 1: 1000 in PBS. The plates were incubated 30 minutes with the human anti-Ig antibody at room temperature and washed 3 times with PBS-T.
    [0224]
    [0225] The reaction was developed with a solution of tetramethylbenzidine (TMB) for 30 minutes and finally stopped with H2SO41N. The optical density (OD) of each well was read with a 450 nm filter (reference filter, 620 nm) and the absorbance of the wells containing the blank was subtracted. The OD values obtained for each sample were used to calculate the amount of each type of immunocomplexes according to the calibration values previously obtained. The entire procedure was performed at Triturus Analyzer (Diagnostics Grifols S.A., Barcelona, Spain).
    [0226]
    [0227] Table 3 shows the OD values of the calibration curves for each artificial immunocomplex concentration. The values in the table correspond to the value recorded by the autoanalyzer minus the value of the target in each case.
    [0228] Table 3. Optical density (OD) of the gauges of each immunocomplex
    [0229]
    [0230] Concentration
    [0231] DO B2GP1-IgM DO B2GP1-IgG DO B2GP1-IgA (U / mL)
    [0232] 0 (blanc 0.066 0.047 0.043
    [0233]
    [0234]
    [0235]
    [0236]
    [0237] 12.5 0.363 0.309 0.490
    [0238]
    [0239]
    [0240] 1.5
    [0241]
    [0242] Table 4 shows the concentration for the samples based on their OD at 450 nm, calculated from the function of the calibration curve obtained from the results of Table 3 for each of the artificial immunocomplexes. For a subject to be considered at risk it is necessary that the concentration value of immunocomplexes at least one of the analyzed isotypes (IgM, IgG or IgA) is greater than 20.5 U / mL.
    [0243]
    [0244] Table 4: Determination of circulating immune complexes in patient sera
    [0245] OD 450 nm Concentration
    [0246]
    [0247]
    [0248] Sample B2GP1-aB2GP1 B2GP1-aB2GP1
    [0249] IgM IgG IgA IgM IgG IgA
    [0250] 1 0.097 0.061 0.019 5.1 2.9 <2
    [0251] 2 0.183 0.091 0.063 6.3 4.3 2.5
    [0252] 3 0.378 0.116 0.155 10 5.6 4.9
    [0253] 4 0.043 0.065 0.159 4.5 3.1 5
    [0254] 5 0.085 0.1 0.079 5 4.8 2.9
    [0255] 6 0.056 0.063 0.049 4.6 3 2.1
    [0256] 7 0.144 0.09 0.136 5.8 4.3 4.4
    [0257] 8 0.086 0.427 0.08 5 22.5 2.9
    [0258] 9 0.075 0.074 0.075 4.9 3.5 2.8
    [0259] 10 1,952 0.156 0.467 85.3 7.6 16.1
    [0260] 11 0.105 0.125 0.118 5.2 6 3.9
    [0261] 12 0.071 0.059 0.083 4.8 2.8 3
    [0262]
    [0263] , 218 0.082 0.058 6.9 3.9 2.4 Negative, 121 0.102 0.024 5.4 4.9 <2 Negative, 027 0.076 0.098 4.3 3.6 3.3 Negative, 049 0.065 0.036 4.6 3, 1 <2 Negative, 259 0.082 0.043 7.6 3.9 2 Negative, 095 0.061 0.099 5.1 2.9 3.4 Negative, 142 0.061 0.022 5.7 2.9 <2 Negative, 064 0.133 0.162 4.7 6.9 5.1 Negative, 341 0.094 0.18 9.2 4.5 5.6 Negative, 056 0.088 0.059 4.6 4.2 2.4 Negative, 066 0.145 0.123 4.8 7 4 Negative, 387 0.177 0.177 10.2 8.7 5.5 Negative, 06 0.04 0.021 4.7 <2 <2 Negative, 032 0.123 0.02 4.4 5.9 <2 Negative, 072 0.11 0.037 4.8 5 , 3 <2 Negative, 44 0.057 0.026 11.4 2.7 <2 Negative, 041 0.056 0.072 4.5 2.6 2.7 Negative, 029 0.071 0.017 4.3 3.4 <2 Negative, 225 0.045 0.074 7 2.1 2.7 Negative 0.1 0.063 0.093 5.2 3 3.2 Negative, 245 0.072 0.12 7.4 3.4 3.9 Negative, 24 0.057 0.027 7.3 2.7 <2 Negative, 125 0.064 0.099 5.5 3 3.4 Negative, 114 0.123 0.15 5.4 5.9 4.7 Negative, 051 0.11 0.116 4.6 5.3 3.8 Negative, 072 0.107 0.111 4.8 5.1 4.2 Negative, 031 0.137 0.046 4.4 6.6 2.1 Negative, 043 0.015 0.017 4.5 <2 <2 Negative, 073 0.055 0.057 4.8 2.6 2.3 Negative, 047 0.036 0.237 4.5 <2 7.4 Negative, 035 0.024 0.027 4.4 <2 <2 Negative, 145 0.048 0.582 5.8 2.2 21.4 Positive IgA , 052 0.065 0.051 4.6 3.1 2.2 Negative, 64 0.094 0.069 16.9 4.5 2.6 Negative, 08 0.067 0.048 4.9 3 , 2 2.1 Negative, 568 0.053 0.099 14.8 2.5 3.4 Negative, 256 0.043 0.036 7.6 2 <2 Negative, 076 0.281 0.05 4.9 14.2 2.2 Negative, 044 0.058 0.035 4.5 2.7 <2 Negative, 035 0.077 0.022 4.4 3.6 <2 Negative 3.7 0.124 0.872 264.6 6 37.6 Positive IgM, IgA , 049 0.036 0.167 4.6 <2 5, 2 Negative, 57 0.076 0.082 14.8 3.6 2.9 Negative, 227 0.105 0.062 7.1 5 2.5 Negative, 217 0.024 0.062 6.9 <2 2.5 Negative, 05 0.055 0.024 4.6 2, 6 <2 Negative, 043 0.062 0.029 4.5 2.9 <2 Negative, 059 0.041 0.024 4.7 <2 <2 Negative 62 0.22 0.046 0.022 6.9 2.2 <2 Negative
    [0264] 63 0.054 0.057 0.046 4.6 2.7 2.1 Negative
    [0265] 64 0.125 0.063 0.544 5.5 3 19.6 Negative
    [0266] 65 0.086 0.23 0.021 5 11.4 <2 Negative
    [0267] 66 0.125 0.041 0.093 5.5 <2 3.2 Negative
    [0268] 67 0.087 0.067 0.107 5 3.2 3.6 Negative
    [0269] 68 0.042 0.059 0.048 4.5 2.8 2.1 Negative
    [0270] 69 0.164 0.038 0.123 6.1 <2 4 Negative
    [0271] 70 0.073 0.105 0.07 4.8 5 2.6 Negative
    [0272] 71 0.018 0.05 0.027 4.2 2.3 <2 Negative
    [0273] 72 0.089 0.045 0.029 5 2.1 <2 Negative
    [0274] 73 0.207 0.059 0.036 6.7 2.8 <2 Negative
    [0275] 74 0.08 0.058 0.06 4.9 2.7 2.4 Negative
    [0276] 75 0.145 0.033 0.041 5.8 <2 2 Negative
    [0277] 76 0.011 0.054 0.021 4.2 2.5 <2 Negative
    [0278] 77 0.063 0.051 0.076 4.7 2.4 2.8 Negative
    [0279] 78 0.128 0.06 0.025 5.5 2.8 <2 Negative
    [0280] 79 0.12 0.109 0.367 5.4 5.2 12 Negative
    [0281] 80 0.092 0.082 0.07 5.1 3.9 2.6 Negative
    [0282] 81 0.091 0.084 0.097 5.1 4 3.3 Negative
    [0283] 82 0.077 0.048 0.075 4.9 2.2 2.8 Negative
    [0284] 83 0.615 0.214 0.004 16.1 10.6 <2 Negative
    [0285] 84 0.049 0.099 0.069 4.6 4.7 2.6 Negative
    [0286] 85 0.177 0.076 0.036 6.3 3.6 Negative
    [0287]
    [0288]
    [0289] This experiment concludes that the calibration curve prepared with artificial immunocomplexes B2GP1-IgM, B2GP1-IgG and B2GP1-IgA serves to perform calibration curves with high reproducibility for use in the detection of circulating immunocomplexes of patients.
    [0290]
    [0291] SEQUENCE LIST
    [0292]
    [0293] The following sequences are described as part of the invention:
    [0294]
    [0295] - SEQ ID NO: 1 Protein sequence of the artificial immunocomplex B2GP1-IgM. - SEQ ID NO: 2 Nucleotide sequence of the artificial immunocomplex B2GP1-IgM. - SEQ ID NO: 3 Protein sequence of the artificial immunocomplex B2GP1-IgG. - SEQ ID NO: 4 Nucleotide sequence of the artificial immunocomplex B2GP1-IgG. - SEQ ID NO: 5 Protein sequence of the artificial immunocomplex B2GP1-IgA. - SEQ ID NO: 6 Nucleotide sequence of the artificial immunocomplex B2GP1-IgA.
    权利要求:
    Claims (6)
    [1]
    1. An artificial immunocomplex comprising human beta-2 glycoprotein 1 (B2GP1) protein bound to a fragment of the heavy chain constant region of an immunoglobulin, characterized in that the sequence of said immunocomplex comprises a sequence identified by SEQ ID NO : 1, SEQ ID NO: 3 or SEQ ID NO: 5.
    [2]
    2. Method for calibrating an analytical system of detection of circulating immunocomplexes B2GP1-aB2GP1 comprising:
    a) diluting the artificial immunocomplex of claim 1 to at least two distinct known concentrations;
    b) determine the optical density of each concentration of the previous stage;
    c) perform a calibration curve that relates each concentration of stage a) to its respective optical density obtained in stage b); d) obtain a mathematical function that defines the calibration curve of stage c).
    [3]
    3. Method for determining the concentration of a circulating immunocomplex B2GP1-aB2GP1 in a sample, comprising:
    a) diluting the artificial immunocomplex of claim 1 to at least two different concentrations;
    b) determine the optical density of each concentration of the previous stage;
    c) perform a calibration curve that relates each concentration of stage a) to its respective optical density obtained in stage b); d) obtain a mathematical function that defines the calibration curve of stage c);
    e) measure the optical density of a biological sample obtained from a subject,
    f) determine the concentration of B2GP1-aB2GP1 circulating immune complexes in the sample from the function obtained in step d).
    [4]
    4. Method according to claim 3, wherein the biological sample of the subject is serum or plasma.
    [5]
    5. Method for determining whether a subject is at risk of suffering pathological manifestations associated with the antiphospholipid syndrome (APS), which comprises determining the concentration of circulating immunocomplexes in a sample of a patient according to the method of claims 3 or 4, in The fact that a concentration value equal to or greater than 20.5 U / mL is indicative that the subject is at high risk of suffering pathological manifestations associated with PHC.
    [6]
    6. Use of any of the artificial immunocomplexes of claim 1 as a gauge in a B2GP1-aB2GP1 circulating immunocomplex detection system.
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    ES2565543T3|2005-01-24|2016-04-05|Board Of Regents, The University Of Texas System|Fc fusion constructs to phosphatidylserine binding and its therapeutic use|
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