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    • 专利摘要:
    Diagnostic procedure for Systemic Lupus Erythematosus (SLE). The present invention relates to a diagnostic method based on an interfacial process of molecular antigen-antibody recognition, namely Anti-RO52-Protein RO52, in a piezoelectric resonator for its application in the diagnosis of autoimmune diseases such as Systemic Lupus Erythematosus (LES). (Machine-translation by Google Translate, not legally binding)
    公开号:ES2644580A1
    申请号:ES201730972
    申请日:2017-07-26
    公开日:2017-11-29
    发明作者:Noelle MARINE DO NASCIMENTO；David GIMENEZ ROMERO；Sergi Beñat MORAIS EZQUERRO；Angel Maquieira Catala；Augusto Miguel JUSTE DOLZ；Elena GRAU GARCIA；Rosa Puchades Pla；Jose Andres ROMAN IBORRA
    申请人:Instituto De Investigacion Sanitaria La Fe - Fundacion Para La Investigacion Del Hospital Universitario Y Politecnico La Fe de la Comunidad Valenciana；Instituto De Investigacion Sanitaria La Fe - Fundacion Para La Investigacion Del Hospital Univ Y Pol；Universidad Politecnica de Valencia；Universitat de Valencia；
    IPC主号:
    专利说明:

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    Main limitation refers to the low sensitivity that limits the determination of extremely low concentrations of circulating serum antibodies. Therefore, the development of high sensitivity biosensors is of great interest to determine autoantibodies at very low concentrations.
    The microbalance technique of the quartz crystal (Quartz Crystal Microbalances, QCM) has been used extensively as a biosensor, thanks to its good performance, high sensitivity and low detection limits, offering excellent advantages over traditional detection techniques. In particular, the microbalance of the quartz crystal with dissipation (Quartz Crystal Microbalances with Dissipation, QCM-D) has great advantages in relation to sensitivity. In addition, QCM-D is attractive due to the sensor response in the form of variations in the dissipation factor (AD) that is related to viscoelasticity, which in turn is associated with structural changes of the attached film on the QCM transducer. D. This adds complementary information in relation to the structural interactions between probe and target, showing a high potential to diagnose and predict autoimmune diseases such as SLE. These biosensors operate on the principle that a change in mass, resulting from the interaction between the antibody and its respective antigen, can be determined directly.
    US5501986A describes a method for detecting an analyte (proteins, hormones, enzymes, antibodies, carbohydrates or nucleic acids) in a sample using QCM, measuring the change in resonance frequency and correlating the change in frequency with the amount of analyte In the sample. However, only the presence of an analyte is related to a single variable.
    The usual measurements of the piezoelectric experiments correspond to measurements of surface concentration and reaction time, without taking into account the viscoelastic properties of the adhered layer. Therefore, using this traditional measurement methodology, transition states cannot be detected during surface processes, since the monitored signal cannot be separated. To solve this problem, the simultaneous measurement of the resonance frequency (f) (adhered mass) and the dissipation factor (D) (viscoelastic properties) were introduced into the new piezoelectric sensors. With this, interfacial processes can be analyzed as a function of the nature of their surface. The big problem with this approach is that it does not introduce the measurement time in a direct way, so it cannot be measured in situ and also the time analysis of the monitored process becomes complex.
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    An example is WO2008114003A1, which describes a method and apparatus for detecting the presence and / or quantity of an oligomeric analyte (antibody) in a sample, by means of a piezoelectric biosensor in which the relationships between different variables such as the frequency of the resonance and dissipation factor. This invention is directed to the detection of certain diseases, in particular Alzheimer's.
    Recently, the use of a function (-df / dD) that allows the introduction of the time factor into the structural study of the interface monitored by QCM-D has been published in Biosensors & Bioelectronics 2017, 90, 166. However, this function has two clear limitations: its interpretation implies very specialized personnel and is not very sensitive, so it has little discrimination.
    Description of the invention
    The present invention solves the problems existing in the state of the art by means of a procedure for the diagnosis of autoimmune diseases in a human. This procedure is based on the measurement in a piezoelectric resonator of the relationship between different variables such as surface concentration and the quality factor, through the function dAr / dAQ, of an interfacial process of molecular recognition antigen-antibody for the screening of samples of autoimmune patients
    The dAr / dAQ function monitors the structural changes involved in the deposition of a reactive species on the surface of a piezoelectric crystal. Certain approaches are necessary for the clear interpretation of their meaning:
    The acoustic response of a piezoelectric sensor (of density "p0", thickness "h0" and modulus of elastic shear "p0") coated with a viscoelastic film (of density "p", thickness "h", sliding viscosity "%" and modulus of elastic shear "px") immersed in a Newtonian liquid (of sliding viscosity "%" and depth of viscous penetration "£ 3") is modeled by the following expressions:
    Ar
    poAo 1 2/2 2 ^ p0h0
    ^ + l PiW - 2hi
    d3
    F
    V ^ 3 J
    2 2 2 Ai + w h
    AQ "or
    2rfpo hoQo

    + 2h
    f 2
    S3 J
    PiW
    2 2 2 Ai + w h
    (one)
    where "Q" is the quality factor, "D" the dissipation factor and "f" the resonance frequency of the piezoelectric sensor (Figure 1).
    If we consider a pure elastic film (y ^ 0) and consequently:
    Aa / PoMo
    AT
    one
    2f 2npoK
    Y + Kpw
    m3
    AQ «-
    Dn
    And + 2h
    rd3Y V ^ 3 and
    w
    Me
    (3)
    (4)
    2 ^ fpo hoQo
    deriving both expressions as a function of the monitoring time (t) you get:
    dAT Ay] PoMo 1 d {h1p1w) w ^ cte dlPoMo w d {h1p1)
     dt  2fl 2Pho dt 2f2 2Pho
     dAQ  Do 2 f Y ^ 2 d f w ^ w ^ cte wD0
     dt  2rfPo hoQo VM3 and dt V M1 J 2rfPo hoQo
    -2
    dt
    C V
    VM3 and
    d_
    dt
    v my j
    (5)
    (6)
    5
    and dividing them ((dAT / dt) / (dAQ / dt)), the function dAT / dAQ (8) is obtained:
    dAT dAT _ dt
    Aa / PoMo QoM
    d (h1P1)
    dt
    dAQ dAQ
    dt
    2f Do 2 Y d
    dt
    f d)
    M and
    AaI PoMo Qod d (hjPj)
    2f Do2Y f h ^
    d
    M1 j
    (7)
    Aa / PoMo Qod32
    2f Do2Y32
    h
    d (P1), _ d (h1)
    fu + P1 fhd
    d
    h
    M1 J
    d
    VM1 j y
    h d‘e AJ PoMo QoM dP1
    “2 f ^
    d
    2f Do2Y
    M1y
    dat
    dAQ
    aa / PoMo Qo5l
    2f Do2Y
    dP
    image 1
    TO
    VM J
    (8)
    10
    Equation (8) shows how the dAT / dAQ function is directly proportional to the variation of the density of the attached film on the sensor surface with respect to the
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    Application no.
    inverse of its viscoelastic properties. In this way, if the film adhered to a piezoelectric surface is modified only by one process, the value of the dAr / dAQ function will remain constant with the transformation time, given that the function:
    In that case, each adhered species will modify in a regular way the viscoelastic properties of the film monitored by the piezoelectric sensor. However, if the monitored transformation corresponds to multiple parallel or consecutive processes, the dAr / dAQ function will vary with time, presenting maximum n-1, where n corresponds to the number of species constituted during the transformation process. Therefore, this function makes it possible to unambiguously characterize the transformation processes of the adhered films on piezoelectric materials, both for their temporal evolution and for the temporal values they present.
    Therefore, a first aspect of the invention is a method for the diagnosis of autoimmune diseases in a human comprising two stages:
    (a) incubating a sample obtained from said human in a piezoelectric resonator comprising a protein antigen immobilized on the surface of the resonator and determining the value of the dAr / dAQ function described in function (8), and
    (b) make the diagnosis of autoimmune diseases by determining the maximum value of dAr / dAQ, the diagnosis being positive when the maximum is greater than a cut-off value of 600 ng / cm2.
    In another aspect of the invention, autoimmune diseases for which this procedure is suitable include Sjogren's Syndrome (SS), Systemic Lupus Erythematosus (SLE) and Neonatal Lupus Erythematosus (LEN).
    In another aspect of the invention, the protein antigen used for the diagnosis of the aforementioned diseases is the Ro protein antigen (anti-Ro). Specifically, the protein antigen is Ro52 since the conformational changes implied by its molecular recognition are very different between patients and healthy subjects.
    Systemic lupus erythematosus (SLE) is the disease that is most associated with anti-Ro, with a reported average positivity of 27.7%, which increases up to 45 to 50% when
    dp-
    image2
    It will be constant.
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    It is analyzed by ELISA. In the case of a late onset LE (greater than 50 years) the positivity is 92%.
    In addition, anti-Ro is an important marker of disease in neonatal lupus erythematosus (LEN), since more than 90% of mothers and their children express it, although the prevalence of LEN in anti-Ro mothers is 1% . Anti-Ro is also present in 70 to 88% of patients with primary Sjogren's Syndrome (SS) and 10 to 50% of secondary cases.
    In another aspect of the invention, the sample that is incubated in step (a) of the procedure is obtained from the blood serum of the patient for whom the diagnosis of the aforementioned autoimmune diseases is intended.
    Brief description of the figures
    Figure 1. Representation of the geometry of a piezoelectric crystal covered by a viscoelastic film (general representation of a concept known in the state of the art).
    Figure 2. Graphical representation of the dAr / dAQ function against time during the interaction of the Ro52 protein with autoantibodies from autoimmune anti-Ro + patients (a) and healthy individuals (b).
    Description of realization modes
    Example 1. Study of the anti-Ro52 antibody / Ro52 protein interaction.
    A chemical immobilization of specific autoantigens in the piezoelectric resonator and the determination of circulating autoantibodies were performed.
    The Ro52 protein was immobilized by covalent anchoring, forming a self-assembled monolayer. The monolayer was created by submerging the piezoelectric surface for 16 h in a 10 mM mercaptopropionic acid solution and subsequent activation by 10 mM EDC / NHS for 60 min. Then, the surface was treated with 5 mM carbohydrazide for 60 min. Then, 100 pL of Ro52 protelna (33 mg / L) was dispensed on the sensor surface, incubating for 60 min. Finally, free active waste is
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    blocked by treating the sensor surface with an aqueous solution containing 0.05% EDTA, BSA and Tween 20, pH 8.5.
    The antibodies were purified from the serum of anti-Ro + patients.
    Subsequently, the variation of the surface concentration and the quality factor during the selective interaction of autoantigens with specific autoantibodies present in a PBS1x solution were recorded, using a piezoelectric sensor.
    Subsequently, the function dAr / dAQ was calculated, where T ”is the surface concentration and“ Q ”the quality factor, during the molecular recognition process studied (Figure 2a). Figure 2a shows the characteristic fingerprint of the recognition process molecular, presenting a maximum of 3,000 ng / cm2 at 1,000 seconds, thus, according to the above described, this function shows that the monitored process corresponds to a cooperative bond.The antibodies bind the antigen by means of a specific interaction epltopo-paratopo, to later be recognized the Fc fragment of these antibodies through the PRY-SPRY domain of the Ro52 protein.This experiment demonstrates the use of the function to monitor transition states and molecular recognition patterns, establishing pathways of reaction of the process studied.
    Example 2. Study of the application of the dAr / dAQ function in healthy patients and autoimmune anti-Ro + patients.
    To demonstrate the good use of the dAr / dAQ function for the discrimination of patients with autoimmune diseases, the molecular recognition of anti-Ro52 antibodies from autoimmune anti-Ro + patients and healthy people was studied. As Figure 2 shows, the antibodies of both populations show a similar recognition behavior when it is represented against time.
    The pattern shown is a peak-shaped function that has a maximum of about 1,000 seconds. Therefore, both processes have the same cooperative reaction mechanism. However, antibodies from anti-Ro + patients have a maximum close to 3,000 ng / cm2.
    This characteristic pattern remains unchanged when solutions containing anti-Ro52 antibodies from a population of 130 autoimmune anti-Ro + patients are analyzed.
    In contrast, when the serum of healthy patients is analyzed, the antibody recognition pattern has a maximum of 500 ng / cm2.
    Therefore, the dAr / dAQ function allows unequivocally discriminating anti-Ro + patients, by establishing a cut-off value of 600ng / cm2, identifying characteristic recognition patterns.
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    1. Procedure for the diagnosis of autoimmune diseases in a human, characterized in that it comprises:
    (a) incubating a sample obtained from said human in a piezoelectric resonator comprising a protein antigen immobilized on the surface of the resonator and determining the value of the formula
    give
    dAQ
    Nickname QpSi
    2f D02 ^ 23
    dPi
    image3

    W j
    with respect to time, where T "is the surface concentration and" Q "the quality factor," A "is the area of the resonator," p0 "is the density", "D0" the dissipation factor, f the frequency of
    resonance and "/ j0" the elastic shear module of the piezoelectric sensor, "p" is the density "and" p1 "is the elastic shear module of a viscoelastic film that covers the sensor," sliding viscosity and "S3" depth of viscous penetration of a Newtonian liquid in which the film is immersed, and
    (b) make the diagnosis of autoimmune diseases by determining the maximum value of dAr / dAQ, the diagnosis being positive when the maximum is greater than a cut-off value of 600 ng / cm2.
    2. Method according to claim 1, characterized in that the autoimmune diseases are selected from the group consisting of Sjogren's Syndrome (SS), Systematic Lupus Erythematosus (SLE) and Neonatal Lupus Erythematosus (LEN).
    3. Method according to claim 1 or 2, characterized in that the protein antigen is Ro (anti-Ro).
    4. Method according to claim 1 or 2, characterized in that the protein antigen is Ro52 (anti-Ro52).
    5. Method according to any of claims 1 to 4, characterized in that said sample is blood serum.
    权利要求:
    Claims (1)
    [1]

    image 1
    FIG. one
    image2
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    同族专利:
    公开号 | 公开日
    EP3640609A1|2020-04-22|
    ES2644580B2|2018-06-04|
    WO2019020847A1|2019-01-31|
    EP3640609A4|2020-08-05|
    US20210364513A1|2021-11-25|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO1996035103A1|1995-05-04|1996-11-07|Michael Rodahl|A piezoelectric crystal microbalance device|
    WO2008114003A1|2007-03-16|2008-09-25|Inverness Medical Switzerland Gmbh|Improvements in or relating to detection and/or characterisation of oligomers|
    US5501986A|1988-04-06|1996-03-26|E. I. Du Pont De Nemours And Company|Piezoelectric specific binding assay with mass amplified reagents|
    法律状态:
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    优先权:
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    ES201730972A|ES2644580B2|2017-07-26|2017-07-26|PROCEDURE OF DIAGNOSIS OF LUPUS ERITEMATOSO SISTÉMICO |ES201730972A| ES2644580B2|2017-07-26|2017-07-26|PROCEDURE OF DIAGNOSIS OF LUPUS ERITEMATOSO SISTÉMICO |
    PCT/ES2018/070467| WO2019020847A1|2017-07-26|2018-06-29|Method for the diagnosis of systemic lupus erythematosus |
    EP18838102.4A| EP3640609A4|2017-07-26|2018-06-29|Method for the diagnosis of systemic lupus erythematosus |
    US16/634,266| US20210364513A1|2017-07-26|2018-06-29|Method for the diagnosis of systemic lupus erythematosus |
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