Pla2r igg4 antibodies and uses thereof

Abstract

The application provides a PLA2R monoclonal antibody and an ELISA kit comprising the antibody. Specifically, the application provides a monoclonal antibody which specifically binds to PLA2R, wherein the antibody only binds to non-reduced PLA2R antigen with a conformational epitope, does not bind to reduced PLA2R antigen, and does not bind to another antigen THSD7A of primary membranous nephropathy. An ELISA kit prepared based on the antibody can more efficiently and accurately detect primary membranous nephropathy than existing commercial kits.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and in particular relates to a PLA2R IgG4 antibody and application thereof. BACKGROUND

[0002] Primary membranous nephropathy (PMN) is a glomerular disease that often occurs in middle-aged and elderly men, and its clinical manifestation is nephrotic syndrome. In recent years, the incidence of PMN in China has shown a trend of rising year by year. A large number of clinical and experimental data show that PMN is an autoimmune disease mediated by phospholipase A2 receptor (PLA2R) antibody.

[0003] PLA2R is a member of the mannose receptor family, and PLA2R has conformation dependence as an antigen. Four conformational epitopes have been found in the 10 domains of PLA2R: CysR, CTLD1, CTLD7 and CTLD8.

[0004] In recent years, enzyme-linked immunosorbent assay (ELISA) has been widely used in the screening and diagnosis of PMN. The existing standardized ELISA kit for detecting PLA2R antibody is produced by Euroimmun Company. The diagnostic specificity of this kit is close to 100%, but the diagnostic sensitivity is limited, and there is a problem of diagnostic gray interval, that is, the sample with antibody concentration in the critical value range cannot be accurately judged.

[0005] In addition, the standard and positive control of PLA2R antibody are obtained by serum purification and concentration method of PMN patients. The serum purification and concentration method has many problems, such as large batch difference, complicated quality control, potential risk of contamination by infectious sources, etc.

[0006] Therefore, there is an urgent need in the art to develop a PLA2R antibody detection ELISA kit with better specificity and sensitivity, and at the same time, a batch of standard products (such as monoclonal antibodies recognizing PLA2R) can be produced. SUMMARY

[0007] The present application provides an antibody specifically binding to PLA2R, and an ELISA kit with high sensitivity, good specificity, good stability and small batch difference prepared using the antibody.

[0008] In a first aspect, the present application provides a PLA2R antibody binding to phospholipase A2 receptor (PLA2R), the PLA2R antibody having:

[0009] (a) a heavy chain variable region comprising the following three complementarity determining regions CDRs:

[0010] H-CDR1 as shown in SEQ ID NO: 3,

[0011] H-CDR2 as shown in SEQ ID NO: 4, and

[0012] H-CDR3 as shown in SEQ ID NO: 5; and

[0013] (b) a light chain variable region comprising the following three complementarity determining regions (CDRs):

[0014] L-CDR1 as shown in SEQ ID NO: 6,

[0015] L-CDR2 as shown in SEQ ID NO: 7, and

[0016] L-CDR3 as shown in SEQ ID NO: 8.

[0017] In another preferred embodiment, the complementarity determining regions are annotated using IMGT numbering.

[0018] In another preferred embodiment, the H-CDR1 comprises an amino acid sequence having one or more conservative amino acid mutations compared to SEQ ID NO: 3.

[0019] In another preferred embodiment, the H-CDR2 comprises an amino acid sequence having one or more conservative amino acid mutations compared to SEQ ID NO: 4.

[0020] In another preferred embodiment, the H-CDR3 comprises an amino acid sequence having one or more conservative amino acid mutations compared to SEQ ID NO: 5.

[0021] In another preferred embodiment, the L-CDR1 comprises an amino acid sequence having one or more conservative amino acid mutations compared to SEQ ID NO: 6.

[0022] In another preferred embodiment, the L-CDR2 comprises an amino acid sequence having one or more conservative amino acid mutations compared to SEQ ID NO: 7.

[0023] In another preferred embodiment, the L-CDR3 comprises an amino acid sequence having one or more conservative amino acid mutations compared to SEQ ID NO: 8.

[0024] In another preferred embodiment, the one or more is preferably 1-3, more preferably 1-2, most preferably 1.

[0025] In another preferred embodiment, the mutation is preferably a substitution, insertion or deletion of an amino acid.

[0026] In another preferred embodiment, the antibody has a heavy chain variable region comprising the sequence shown in SEQ ID NO: 1 :

[0027] VHSQVQLLESGPGLVKSSQTLSLTCTVSGGSIISGGSFWSWIRQHPGKGLEWIGHIHYSGSTHYNPSLKSRVNISVDTSKTQFFLKLSSVTAADTAVYYCARDTDAFDMWGQGTMVSVSS (SEQ ID NO: 1).

[0028] In another preferred embodiment, the antibody has a light chain variable region comprising the sequence set forth in SEQ ID NO: 2:

[0029] VHSDIQMTQSPLSLSASVGDRVTITCRASRIITSYLNWYQQRPGQAPKLLIFGTSTLQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYYCQQSYSSPRTFGQGTRVEIK (SEQ ID NO: 2).

[0030] In another preferred embodiment, the heavy chain variable region has a sequence that is at least 85% identical, preferably at least 90% identical, more preferably at least 95% or more, such as 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 1.

[0031] In another preferred embodiment, the light chain variable region has a sequence that is at least 85% identical, preferably at least 90% identical, more preferably at least 95% or more, such as 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 2.

[0032] In another preferred embodiment, the antibody specifically binds to a non-reduced form of PLA2R antigen, but not to a reduced form of PLA2R antigen.

[0033] In another preferred embodiment, the antibody has a heavy chain constant region and / or a light chain constant region.

[0034] In another preferred embodiment, the heavy chain constant region is a heavy chain constant region of a mammal (rodent or primate), including wild-type and mutant heavy chain constant regions.

[0035] In another preferred embodiment, the heavy chain constant region is a heavy chain constant region of human or murine origin.

[0036] In another preferred embodiment, the heavy chain constant region is derived from murine IgGl, murine IgG2a, murine IgG2b, or murine IgG3.

[0037] In another preferred embodiment, the heavy chain constant region is derived from human IgGl, human IgG2, human IgG3, or human IgG4.

[0038] In another preferred embodiment, the heavy chain constant region is derived from human IgG4.

[0039] In another preferred embodiment, the antibody has a heavy chain constant region comprising the sequence set forth in positions 121-450 of SEQ ID NO: 9.

[0040] In another preferred embodiment, the antibody has a light chain constant region.

[0041] In another preferred embodiment, the light chain constant region is a light chain constant region of a mammal (rodent or primate), including wild-type and mutant light chain constant regions.

[0042] In another preferred embodiment, the light chain constant region is a light chain constant region of human or murine origin.

[0043] In another preferred embodiment, the light chain constant region is derived from murine IgGl, murine IgG2a, murine IgG2b, or murine IgG3.

[0044] In another preferred embodiment, the light chain constant region is derived from human IgGl, human IgG2, human IgG3, or human IgG4.

[0045] In another preferred embodiment, the light chain constant region is derived from human kappa chain.

[0046] In another preferred embodiment, the antibody has a light chain constant region comprising the sequence set forth in positions 111-216 of SEQ ID NO: 10.

[0047] In another preferred embodiment, the antibody includes a monoclonal antibody, a polyclonal antibody, a diabody, a single chain antibody (scFv), a Fab, a Fab', a F(ab')2 antibody.

[0048] In another preferred embodiment, the antibody is a diabody.

[0049] In another preferred embodiment, the antibody is a monospecific antibody, a bispecific antibody, or a trispecific antibody.

[0050] In another preferred embodiment, the PLA2R antibody is an IgG4 type antibody.

[0051] In another preferred embodiment, the IgG4 type antibody comprises an Fc fragment.

[0052] In another preferred embodiment, the Fc fragment has a glycosylation modification.

[0053] In another preferred embodiment, the IgG4-type antibody further comprises a S228P mutation in the hinge region.

[0054] In another preferred embodiment, the IgG4-type antibody further comprises L234F, H268Q, A330S, P331S, L235E, L235A, F234A, G237A, D255A, or a combination thereof.

[0055] In another preferred embodiment, the antibody comprises a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 9, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 10.

[0056] In another preferred embodiment, the antibody has a heavy chain constant region comprising the sequence set forth in positions 121-450 of SEQ ID NO: 9, and / or a light chain constant region comprising the sequence set forth in positions 111-216 of SEQ ID NO: 10.

[0057] In a second aspect of the present application, there is provided a recombinant protein, said recombinant protein having:

[0058] (i) the antibody of the first aspect of the present application or the first aspect of the present application; and

[0059] (ii) an optional signal peptide and / or a tag sequence.

[0060] In another preferred embodiment, the recombinant protein specifically binds to PLA2R.

[0061] In another preferred embodiment, the tag sequence is selected from the group consisting of FLAG, Myc, His tag.

[0062] In a third aspect of the present application, there is provided an antibody conjugate, said antibody conjugate comprising:

[0063] (Z1) the antibody of the first aspect or 2 of the present application or the recombinant protein of the second aspect of the present application; and

[0064] (Z2) a conjugating moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0065] In another preferred embodiment, the conjugate is selected from the group consisting of a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agent, or an enzyme capable of generating a detectable product, a radionuclide, a biological toxin, a cytokine (such as IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle / nanorod, a viral particle, a liposome, a nanomagnetic particle, a prodrug-activating enzyme (e.g., DT-diaphorase (DTD) or benzpyrene hydroxylase-like protein (BPHL)), a chemotherapeutic agent (e.g., cisplatin), or any form of nanoparticle, etc.

[0066] In a fourth aspect of the present application, there is provided use of the antibody of the first aspect of the present application, the recombinant protein of the second aspect of the present application, or the antibody conjugate of the third aspect of the present application, in the preparation of a kit for detecting primary membranous nephropathy.

[0067] In another preferred embodiment, the antibody, the recombinant protein, or the antibody conjugate is used as a positive control reagent.

[0068] In another preferred embodiment, the kit is used for detecting PLA2R IgG4 antibody in a sample.

[0069] In another preferred embodiment, the kit comprises a secondary antibody that specifically binds to the antibody, the recombinant protein, or the antibody conjugate.

[0070] In a fifth aspect of the present application, there is provided a kit for detecting primary membranous nephropathy, the kit comprising: the antibody of the first aspect of the present application, the recombinant protein of the second aspect of the present application, or the antibody conjugate of the third aspect of the present application.

[0071] In another preferred embodiment, the kit is an ELISA kit.

[0072] In another preferred embodiment, the antibody, the recombinant protein, or the antibody conjugate is used as a positive control reagent.

[0073] In another preferred embodiment, the kit further comprises an instruction manual indicating that the kit is used for detecting PLA2R IgG4 antibody in a sample.

[0074] In another preferred embodiment, the sample comprises: a serum sample, or a urine sample.

[0075] In another preferred embodiment, the sample is a serum sample.

[0076] In another preferred embodiment, the kit comprises a secondary antibody that specifically binds to the antibody of the first aspect of the present application.

[0077] In another preferred embodiment, the secondary antibody is a polyclonal or monoclonal antibody.

[0078] In another preferred embodiment, the secondary antibody is a goat antibody, a rabbit antibody, or a combination thereof.

[0079] In another preferred embodiment, the secondary antibody comprises a detectable label or an enzyme

[0080] In another preferred embodiment, the secondary antibody is an anti-IgG4 secondary antibody.

[0081] In another preferred embodiment, the secondary antibody is an anti-human IgG4 secondary antibody.

[0082] In another preferred embodiment, the anti-human IgG4 secondary antibody is labeled with a detectable label.

[0083] In another preferred embodiment, the detectable label comprises a peroxidase.

[0084] In another preferred embodiment, the ELISA kit further comprises other reagents required for the ELISA detection, including: wash solution, assay buffer, blocking solution, color developing solution, color developing termination solution, PLA2R protein, or a combination thereof.

[0085] In another preferred embodiment, the instruction manual indicates that the kit is used for detecting primary membranous nephropathy.

[0086] In another preferred embodiment, the kit further comprises other diagnostic reagents for primary membranous nephropathy.

[0087] In another preferred embodiment, the kit further comprises therapeutic reagents for primary membranous nephropathy.

[0088] In another preferred embodiment, the kit further comprises an instruction manual, which records the operation method and determination criteria for diagnosing primary membranous nephropathy.

[0089] In another preferred embodiment, the determination criteria comprises: if the antibody unit value of PLA2R IgG4 antibody in the sample is ≥ 17, the sample is determined to be positive for primary membranous nephropathy; if the antibody unit value of PLA2R IgG4 antibody in the sample is < 17, the sample is determined to be negative for primary membranous nephropathy.

[0090] In another preferred embodiment, the unit of the antibody unit value X is U / mL.

[0091] In a sixth aspect of the present application, a method for detecting PLA2R in a sample is provided, comprising the steps of:

[0092] The sample is contacted with the antibody of the first aspect of the application, and it is observed whether a "PLA2R-antibody" complex is formed, wherein if the "PLA2R-antibody" complex is formed, it indicates that PLA2R exists in the sample; if the "PLA2R-antibody" complex is not formed, it suggests that PLA2R does not exist in the sample.

[0093] In the seventh aspect of the application, a method for detecting anti-PLA2R autoantibody in a sample is provided, comprising the steps of:

[0094] (a) in a test group, incubating the sample with PLA2R, and observing whether a "PLA2R-anti-PLA2R autoantibody" complex is formed;

[0095] and in a positive control group, incubating the antibody of the first aspect of the application with PLA2R, and observing whether a "PLA2R-positive antibody" complex is formed;

[0096] (b) comparing the test results of the test group with the test results of the positive control group, so as to determine whether anti-PLA2R autoantibody exists in the sample.

[0097] In another preferred embodiment, in step (a), further comprising: in a blank control group, incubating PLA2R and a blank buffer under the same conditions, and obtaining a blank experimental result;

[0098] In another preferred embodiment, in the test group, the positive control group and the blank control group, the experimental conditions are the same except that the sample is added, the positive antibody is added, and neither the sample nor the positive antibody is added.

[0099] In another preferred embodiment, the method is an ELISA method, and the ELISA method comprises the following steps:

[0100] (S1) in a blank control group, co-incubating a blank buffer with PLA2R protein pre-coated in a detection hole of an ELISA plate;

[0101] In a positive control group, co-incubating the antibody of the first aspect of the application with PLA2R protein pre-coated in a detection hole of an ELISA plate under the same conditions;

[0102] In an experimental group, co-incubating a sample to be tested with PLA2R protein pre-coated in a detection hole of an ELISA plate under the same conditions;

[0103] (S2) washing the detection holes of the blank control group, the positive control group, and the experimental group respectively, and adding anti-human IgG4 secondary antibody to continue incubation in the detection holes of the blank control group, the positive control group, and the experimental group respectively;

[0104] (S3) washing and detecting the relative parameter Y1 of the PLA2R protein-PLA2R IgG4 complex in the blank control group, the relative parameter Y2 of the PLA2R protein-PLA2R IgG4 complex in the positive control group, and the relative parameter Y3 of the PLA2R protein-PLA2R IgG4 complex in the experimental group, thereby indicating the antibody unit value X of the PLA2R IgG4 antibody in the sample, X = 100x (Y3-Y1) / (Y2-Y1).

[0105] In another preferred embodiment, the anti-human IgG4 secondary antibody is linked to peroxidase.

[0106] In another preferred embodiment, the relative parameter is the absorbance at 450 nm after color development with peroxidase.

[0107] In another preferred embodiment, the unit of the antibody unit value X is U / mL.

[0108] In another preferred embodiment, step (S1) comprises the following sub-steps:

[0109] (S1-1) coating the PLA2R protein in the detection wells of the ELISA plate;

[0110] (S1-2) blocking the detection wells obtained in step (S1-1), thereby obtaining the PLA2R protein pre-coated in the detection wells of the ELISA plate;

[0111] (S1-3) in the blank control group, co-incubating the blank buffer with the PLA2R protein pre-coated in the detection wells of the ELISA plate; in the positive control group, co-incubating the antibody of the first aspect of the application with the PLA2R protein pre-coated in the detection wells of the ELISA plate under the same conditions; in the experimental group, co-incubating the sample to be tested with the PLA2R protein pre-coated in the detection wells of the ELISA plate under the same conditions.

[0112] In another preferred embodiment, in step (S1-1), the coating refers to overnight incubation at 4°C.

[0113] In another preferred embodiment, in step (S1-2), the blocking comprises discarding the liquid in the detection wells, washing with blocking buffer, and adding blocking buffer for blocking.

[0114] In another preferred embodiment, the addition of blocking buffer for blocking refers to the addition of blocking buffer, followed by incubation at room temperature on a shaking table for 60-120 minutes, preferably 90 minutes.

[0115] In another preferred embodiment, in step (S2), the washing refers to discarding the liquid in the detection wells, adding washing buffer, and then washing after standing.

[0116] In another preferred embodiment, the standing at room temperature is for 1 minute.

[0117] In another preferred embodiment, in step (S2), the incubation at room temperature on a shaker is for 40-80 minutes, preferably 60 minutes.

[0118] In another preferred embodiment, in step (S3), the following sub-steps are further included:

[0119] (S3-1) Discard the liquid in the detection wells, add a washing buffer, and then wash after standing;

[0120] (S3-2) Add a substrate solution to the microplate wells and incubate after standing, and then add a termination solution after incubation;

[0121] (S3-3) Respectively detect the absorbance value A1 at 450 nm in the detection wells of the blank control group, the absorbance value A2 at 450 nm in the detection wells of the positive control group, and the absorbance value A3 at 450 nm in the detection wells of the experimental group, and then the antibody unit value X of the PLA2R IgG4 antibody in the sample = 100 x (A3-A1) / (A2-A1).

[0122] In step (S3-2), the incubation after standing at room temperature is for 6 minutes.

[0123] In another preferred embodiment, the room temperature is 18-28°C, preferably 25°C.

[0124] In another preferred embodiment, the washing is for 2-4 times, preferably 3 times.

[0125] In another preferred embodiment, the ELISA method includes the following steps:

[0126] (M1) Coating the PLA2R protein in the detection wells of the ELISA plate;

[0127] (M2) Blocking the detection wells obtained in step (M1), thereby obtaining the PLA2R protein pre-coated in the detection wells of the ELISA plate;

[0128] (M3) In the blank control group, incubating the blank buffer with the PLA2R protein pre-coated in the detection wells of the ELISA plate under the same conditions; in the positive control group, incubating the antibody of the first aspect of the application with the PLA2R protein pre-coated in the detection wells of the ELISA plate under the same conditions; and in the experimental group, incubating the sample to be tested with the PLA2R protein pre-coated in the detection wells of the ELISA plate under the same conditions.

[0129] (M4) washing the detection wells of the blank control group, the positive control group, and the experimental group respectively, and adding anti-human IgG4 secondary antibody into the detection wells of the blank control group, the positive control group, and the experimental group respectively for further incubation;

[0130] (M5) washing the detection wells obtained in step (M4), adding substrate solution into the microwell plate and incubating, and adding stop solution after the incubation is completed;

[0131] (M6) detecting the absorbance value A1 at 450 nm in the detection well of the blank control group, the absorbance value A2 at 450 nm in the detection well of the positive control group, and the absorbance value A3 at 450 nm in the detection well of the experimental group respectively, and then the antibody unit value X of the PLA2R IgG4 antibody in the sample = 100 x (A3-A1) / (A2-A1).

[0132] In another preferred embodiment, the unit of the antibody unit value X is U / mL.

[0133] In another preferred embodiment, the method is a method for non-diagnostic and non-therapeutic purposes.

[0134] In another preferred embodiment, if the antibody unit value X of the PLA2R IgG4 antibody in the sample is ≥ 17, the sample is determined to be positive for primary membranous nephropathy; if the antibody unit value X of the PLA2R IgG4 antibody in the sample is < 17, the sample is determined to be negative for primary membranous nephropathy.

[0135] In another preferred embodiment, if A1 > 0.100 or A2 < 0.400, it is determined that re-detection is needed.

[0136] In an eighth aspect of the present application, a polynucleotide encoding the antibody of the first aspect of the present application or the recombinant protein of the second aspect of the present application is provided.

[0137] In a ninth aspect of the present application, a vector comprising the polynucleotide of the eighth aspect of the present application is provided.

[0138] In another preferred embodiment, the vector comprises a eukaryotic cell expression vector or a prokaryotic cell expression vector.

[0139] In another preferred embodiment, the vector is a plasmid.

[0140] In a tenth aspect of the present application, a host cell comprising the vector of the ninth aspect of the present application or the polynucleotide of the eighth aspect of the present application integrated into the genome is provided.

[0141] In another preferred embodiment, the host cell comprises a eukaryotic cell or a prokaryotic cell.

[0142] In another preferred embodiment, the host cell is a HEK293T cell.

[0143] In a twelfth aspect of the present application, there is provided a detection reagent comprising the antibody of the first aspect of the present application, the recombinant protein of the second aspect of the present application, or the antibody conjugate of the third aspect of the present application.

[0144] In another preferred embodiment, the detection comprises qualitative detection or quantitative detection.

[0145] In a twelfth aspect of the present application, there is provided a detection reagent comprising the antibody of the first aspect of the present application, the recombinant protein of the second aspect of the present application, or the antibody conjugate of the third aspect of the present application.

[0146] In another preferred embodiment, the detection reagent further comprises a pharmaceutically acceptable carrier or excipient.

[0147] In a thirteenth aspect of the present application, there is provided a detection plate comprising the antibody of the first aspect of the present application, the recombinant protein of the second aspect of the present application, or the antibody conjugate of the third aspect of the present application.

[0148] In a fourteenth aspect of the present application, there is provided a device for detecting PLA2R IgG4 antibody, the device comprising the following modules:

[0149] (N1) a sample incubation module configured to: in a blank control group, co-incubate a blank buffer with a PLA2R protein pre-coated in a detection well of an ELISA plate; in a positive control group, co-incubate the antibody of the first aspect of the present application with the PLA2R protein pre-coated in a detection well of an ELISA plate under the same conditions; and in an experimental group, co-incubate a sample to be tested with the PLA2R protein pre-coated in a detection well of an ELISA plate under the same conditions;

[0150] (N2) a secondary antibody incubation module configured to: wash the detection wells of the blank control group, the positive control group, and the experimental group, respectively, and add an anti-human IgG4 secondary antibody to continue incubation in the detection wells of the blank control group, the positive control group, and the experimental group, respectively;

[0151] (N3) an analysis calculation module configured to: wash and detect the relative parameter Y1 of the PLA2R protein-PLA2R IgG4 complex in the blank control group, the relative parameter Y2 of the PLA2R protein-PLA2R IgG4 complex in the positive control group, and the relative parameter Y3 of the PLA2R protein-PLA2R IgG4 complex in the experimental group, and then the antibody unit value X of the PLA2R IgG4 antibody in the sample = 100x (Y3-Y1) / (Y2-Y1).

[0152] In another preferred embodiment, the anti-human IgG4 secondary antibody is linked to peroxidase.

[0153] In another preferred embodiment, the relative parameter is the absorbance at 450 nm after color development with peroxidase.

[0154] In another preferred embodiment, the unit of the antibody unit value X is U / mL.

[0155] In another preferred embodiment, if Y1>0.100 or Y2<0.400, it is determined that re-detection is needed.

[0156] In another preferred embodiment, the device further comprises a result output module configured to: if the antibody unit value X of the PLA2R IgG4 antibody in the sample is ≥17, it is determined that the sample is positive for primary membranous nephropathy; and if the antibody unit value X of the PLA2R IgG4 antibody in the sample is <17, it is determined that the sample is negative for primary membranous nephropathy.

[0157] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features specifically described below (e.g., in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0158] Figure 1 Flow cytometry gating of PLA2R antibody-secreting cells is shown.

[0159] Figure 2 Amplification of single PLA2R antibody-secreting cell VDJ region base fragment agarose gel electrophoresis is shown.

[0160] Figure 3 CDR sequences of the Ab11 heavy chain variable region marked with different rules (marked with red underlines) are shown.

[0161] Figure 4 CDR sequences of the Ab11 light chain variable region marked with different rules (marked with red underlines) are shown.

[0162] Figure 5 WB shows that the recombinant PLA2R monoclonal antibody (Ab11) detects the intact light chain and heavy chain after reduction and denaturation.

[0163] Figure 6 PLA2R monoclonal antibody screening is shown, in which Ab11 is the highest affinity PLA2R monoclonal antibody.

[0164] Figure 7 Specificity detection of recombinant PLA2R monoclonal antibody (Ab11) is shown. DETAILED DESCRIPTION

[0165] The present inventors, through extensive and in-depth research, first obtained a monoclonal antibody capable of specifically recognizing PLA2R from the peripheral blood of PMN patients by analyzing the specific B cells recognizing PLA2R in the peripheral blood of PMN patients, and provided an IgG4 subtype monoclonal antibody of the monoclonal antibody. The antibody only binds to non-reduced PLA2R antigen with conformational epitopes, does not bind to reduced PLA2R antigen, and does not bind to another antigen THSD7A of primary membranous nephropathy. The ELISA kit prepared based on the antibody as a standard is more specific for the detection of primary membranous nephropathy, and the intra-batch difference and inter-batch difference are both small. On this basis, the present application is completed.

[0166] TERMS

[0167] For easier understanding of the present application, certain technical and scientific terms are defined in detail below. Unless otherwise defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Before describing the present application, it should be understood that the present application is not limited to the particular methodology and experimental conditions described, as such methodology and conditions can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present application will only be limited by the appended claims.

[0168] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0169] As used herein, the term "and / or" refers to any and all possible combinations of one or more of the associated listed items.

[0170] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance can occur, but does not have to occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of a particular sequence can have but does not have to have, can be 1, 2 or 3.

[0171] The term "sequence identity" as used herein refers to the extent to which two nucleic acid or two amino acid sequences are identical, when optimally aligned and compared. The sequence identity between a sequence as described herein and a sequence having identity thereto can be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0172] As used herein, the term "antibody" or "immunoglobulin" is a heterotetramer formed from two light (L) and two heavy (H) chains. The N-terminal of each heavy chain is a variable region (VH) connected to a heavy chain constant region. The N-terminal of each light chain is a variable region (VL) connected to a light chain constant region.

[0173] As used herein, the term "variable" refers to the fact that certain portions of the antibody vary among antibodies, varying the antigen combining site and conferring specificity to, and affinity for, the particular antigen. The variable regions of the antibody include the Complementarity Determining Regions (CDRs) or hypervariable regions and the more conserved framework regions (FRs). The primary sequence of the heavy and light chain variable regions are each composed of four FR sequences, which are interspersed with three CDR sequences (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The sequence and spatial conformation of the variable regions of the heavy and light chains determine the specificity of an antibody for its epitope. The constant regions of the antibodies are not directly involved in binding to an antigen but can influence the ability of the antibody to capture and detect.

[0174] The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant regions. and Depending on the amino acid sequences of the constant regions, immunoglobulins can be assigned to different classes, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes) e.g., human IgG includes IgG1, IgG2, IgG3, IgG4 subtypes, and mouse IgG includes IgG1, IgG2a, IgG2b subtypes. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally (see, e.g., Basic and Clinical Immunology, 8th Ed., Daniel P. Stites, Temple R. Nowell, Richard E. Gallin, Harry R. Malech, eds., Appleton & Lange, 1994, Fig. 2-8).

[0175] As used herein, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present. Monoclonal antibodies are highly specific, being directed against a single antigenic determinant (epitope). The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0176] The present application also includes monoclonal antibodies having the corresponding amino acid sequences of the PLA2R monoclonal antibodies, monoclonal antibodies having the variable region chains of the PLA2R monoclonal antibodies, and other protein or protein conjugate and fusion expression products of these chains. In particular, the present application includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a light chain and a heavy chain containing a variable region (complementarity determining region, CDR) as long as the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the variable region of the light chain and the heavy chain of the present application.

[0177] As known by those skilled in the art, antibody conjugate and fusion expression products include: fluorescent or luminescent markers, enzymes capable of producing detectable products, gold nanoparticles / nanorods and other molecules useful for detection, conjugated to the PLA2R antibody.

[0178] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment of an antibody" include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a scFv fragment consisting of the VH and VL domains of a single arm of an antibody.

[0179] The present application includes not only intact monoclonal antibodies, but also antibody fragments having binding activity, such as Fab or (Fab')2 fragments; antibody heavy chains; antibody light chains or scFv.

[0180] The terms "specifically binds", "selectively binds", "selectively binds" and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen.

[0181] As used herein, the term "antigenic determinant" refers to a discontinuous, three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present application.

[0182] The present application includes not only whole antibodies, but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Therefore, the present application also includes fragments, derivatives, and analogs of the antibodies.

[0183] In the present application, the antibody can be monospecific, bispecific, trispecific, or more multispecific.

[0184] As used herein, the term "heavy chain variable region" is used interchangeably with "VH". The term "light chain variable region" is used interchangeably with "VL".

[0185] As used herein, "complementarity determining region" (CDR) is a region in an antibody that binds to an antigen. CDRs can be defined using various notations, such as Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al., J Mol Biol 196:901-17, 1987), IMGT (Lefranc et al., Dev Comp Immunol 27:55-77, 2003), and AbM (Martin and Thornton, J Biomol Biol 263:800-15, 1996). Unless otherwise specified in the specification, the terms "CDR," "H-CDR1," "H-CDR2," "H-CDR3," "L-CDR1," "L-CDR2," and "L-CDR3" as used in the present application include CDRs defined by the coding rules known to those skilled in the art by any of the methods described above, Kabat, Chothia, IMGT, or AbM. The correspondence between numbering systems, including, for example, Kabat numbering and IMGT unique numbering system, is known to those skilled in the art.

[0186] In the present application, the 3 CDRs of VH (H-CDR1, H-CDR2, and H-CDR3) and the 3 CDRs of VL (L-CDR1, L-CDR2, and L-CDR3) can be determined by Kabat rules, IMGT rules, Contact rules, AbM rules, Chothia rules, etc. Representative CDRs of the antibodies of the present application determined using different rules can be found in Figure 3 and Figure 4It is understood that the antibodies of the present application include antibodies defined by any of the six CDRs determined by any of the rules, and that the sequence of the FR regions outside the CDRs can vary to some extent, provided that the binding properties are the same or similar to those of the preferred antibody of the application (Ab 11), including binding to the same epitope; binding to non-reduced but not reduced PLA2R antigen with conformational epitopes.

[0187] "Fragment", "derivative" and "analogue" of an antibody refer to a polypeptide that substantially retains the same biological function or activity of an antibody of the present application. A polypeptide fragment, derivative or analogue of the present application can be (i) a polypeptide having one or more conservative or non-conservative amino acid substitutions, preferably conservative amino acid substitutions, and such substituted amino acid residues can or can not be encoded by the genetic code, or (ii) a polypeptide having a substituent group at one or more amino acid residues, or (iii) a polypeptide formed by conjugation of the mature polypeptide to another compound, such as a compound for chemiluminescence, e.g. acridinium ester, or (iv) a polypeptide formed by fusion of additional amino acid sequences to the polypeptide sequence, such as a leader sequence or a secretion sequence or a tag protein sequence for purification or detection of the polypeptide or other fusion protein sequences. These fragments, derivatives and analogues are within the scope of those skilled in the art.

[0188] An antibody of the present application refers to a polypeptide having PLA2R binding activity, e.g. comprising Figure 3 and Figure 4 the CDR regions described. The term also includes variants of polypeptides comprising the foregoing CDR regions that have the same function as the antibodies of the present application. These variants include, but are not limited to, deletion, insertion and / or substitution of one or more (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or more (typically 20 or fewer, preferably 10 or fewer, more preferably 5 or fewer) amino acids at the C-terminus and / or the N-terminus. For example, in the art, substitution with similar or identical amino acids typically does not change the function of the protein. Also, for example, addition of one or more amino acids at the C-terminus and / or the N-terminus typically does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present application.

[0189] As used herein, "positive control reagent" and "standard" are used interchangeably and refer to a positive control in the detection of autoantibodies to phospholipase A2 receptor (PLA2R) in primary membranous nephropathy, and in the present application, the positive control reagent is a PLA2R IgG4 antibody.

[0190] As used herein, the PLA2R IgG4 antibody refers to an antibody against PLA2R IgG4, i.e. an anti-PLA2R IgG4 antibody; similarly, the PLA2R antibody refers to an antibody against PLA2R, i.e. an anti-PLA2R antibody.

[0191] Primary membranous nephropathy

[0192] Primary membranous nephropathy (PMN) is a common cause of nephrotic syndrome in adults, which belongs to the autoimmune glomerular disease. Its characteristic is diffuse deposition of immune complexes on the epithelial side of the glomerular basement membrane, accompanied by diffuse thickening of the basement membrane.

[0193] In terms of pathogenesis, recent studies have shown that about 70%-80% of patients with primary membranous nephropathy have anti-phospholipase A2 receptor (PLA2R) autoantibodies in their bodies. The autoantibodies bind to the PLA2R antigen on the surface of podocytes, form in situ immune complexes, activate the complement system, and cause glomerular damage.

[0194] Epidemiologically, primary membranous nephropathy can occur at any age, with the majority of middle-aged and elderly people, and the incidence of men is higher than that of women. In different regions, the incidence rate is different, and the overall trend is rising.

[0195] The main clinical symptoms are nephrotic syndrome, i.e. massive proteinuria (urinary protein quantification > 3.5g / d), hypoproteinemia (plasma albumin < 30g / L), edema and hyperlipidemia. Some patients may also have microscopic hematuria, but gross hematuria is rare.

[0196] Diagnosis mainly relies on renal biopsy pathological examination. Under light microscope, diffuse thickening of glomerular basement membrane can be seen, and immunofluorescence shows that IgG and C3 are deposited along the glomerular capillary wall in granular form. Under electron microscope, subepithelial electron-dense material deposition can be seen. At the same time, detection of serum anti-PLA2R antibody and other autoantibodies is helpful for diagnosis and disease assessment.

[0197] A large number of clinical and experimental data show that PMN is an autoimmune disease mediated by autoantigen PLA2R and corresponding autoantibody. PLA2R antibody as a biomarker of primary membranous nephropathy plays an important role in early diagnosis, disease monitoring and prognosis evaluation of the disease.

[0198] Phospholipase A2 receptor (PLA2R)

[0199] Phospholipase A2 receptor (PLA2R) is a cell surface receptor and a transmembrane glycoprotein member of the mammalian mannose receptor family. It is a protein with a size of 180kDa, which is specifically expressed in human kidney podocytes.

[0200] PLA2R is the pathogenic antigen of primary membranous nephropathy. PLA2R as an antigen has conformation dependence. PLA2R has 10 different extracellular domains, from N-terminal to C-terminal, including cysteine-rich region domain (CysR), fibronectin type II domain (FnII), and 8 consecutive C-type lectin-like domains (CTLD1-CTLD8). The protein domains of PLA2R are mostly extracellular, and there are also some transmembrane domains and intracellular domains. Four conformational epitopes have been found in the 10 domains of PLA2R: CysR, CTLD1, CTLD7, and CTLD8.

[0201] However, the detection of PLA2R antibody still needs to be optimized. For example, the standard and positive control of PLA2R antibody are obtained by serum purification and concentration method. The serum purification and concentration method has problems such as large batch difference, complicated quality control, potential risk of contamination by infectious sources, etc. For another example, the antibodies in the peripheral blood of PMN patients that recognize PLA2R are mainly of IgG4 subtype, while the most commonly used Euroimmun kit in clinical practice detects IgG antibodies (including IgG4 and other IgG subtypes), which may recognize other IgG subtypes that are not specifically bound, resulting in a decrease in specificity.

[0202] Specifically, compared with the standard obtained by serum purification and concentration, the recombinant monoclonal antibody as a standard has the advantages of batch stability, high specificity, and large-scale production. Therefore, a series of monoclonal antibodies capable of recognizing PLA2R are obtained by analyzing specific B cells in the peripheral blood of PMN patients that recognize PLA2R, and a strain capable of being used as a standard for PLA2R-IgG4 antibody detection ELISA kit is screened. Further, an ELISA kit for detecting the content of PLA2R-IgG4 in PMN is established with IgG4 as the target subtype.

[0203] The main advantages of the present application include:

[0204] (a) The recombinant PLA2R monoclonal antibody prepared by the present application has high specificity and high affinity, and only binds to PLA2R protein with conformational epitopes.

[0205] (b) The present application uses anti-IgG4 enzyme-labeled antibody instead of anti-IgG enzyme-labeled antibody in similar products on the market, effectively reduces the background signal caused by non-specific binding of other IgG1 / 2 / 3 subtype antibodies with high proportion in serum, improves the detection specificity, and enhances the detection accuracy of low concentration of PLA2R antibody.

[0206] (c) The ELISA kit of the present application has higher sensitivity and stronger specificity than other similar products on the market for samples with gray zone of PLA2R antibody titer confirmed by pathological biopsy of primary membranous nephropathy, which can meet the needs of early screening and individualized treatment of primary membranous nephropathy.

[0207] (d) The recombinant PLA2R monoclonal antibody of the present application is easy to prepare by recombination, which is beneficial to ensure that there is no bacteria, virus or other infectious sources in the production process, and has higher safety and reliability.

[0208] (e) The positive control recombinant PLA2R monoclonal antibody of the present application has a standard synthesis process and strict quality control, which avoids batch differences in traditional biological source antibodies, ensures the stability of the potency of the standard product, and improves the repeatability and reliability of the experimental results.

[0209] (g) The ELISA kit of the present application is more objective than the existing PLA2R antibody IIFT detection, which avoids errors caused by subjective judgment.

[0210] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples without specific conditions are usually carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0211] Example 1: Preparation and identification of PLA2R antibody.

[0212] This example provides a preparation method of the PLA2R antibody of the present application, which comprises the following steps:

[0213] (s1) PLA2R protein fluorescein labeling. Specifically, APC or PE fluorescein is coupled to PLA2R protein to prepare PLA2R-APC and PLA2R-PE, and the coupled protein concentration is determined and stored in the dark.

[0214] (s2) Cell Staining: PBMCs were isolated from peripheral anti-coagulated blood of a patient with primary membranous nephropathy who had not been treated with immunosuppressive agents, and resuspended in FACS buffer. The PBMCs were labeled with CD19 magnetic bead antibodies, and the magnetic bead-positive PBMCs were selected for all cells positive for the CD19 surface marker. The CD19-positive cells were then subjected to CD20, CD27, human IgG, and live cell fluorescent staining for 30 minutes. Then, PLA2R-APC and PLA2R-PE were added in equal proportions and stained for 30 minutes. Subsequently, FACS buffer was added to wash the cells, which were centrifuged at 400g for 10 minutes at 4°C, and the process was repeated three times.

[0215] (s3) Preparation of capture cell buffer: 145ul of RNase free water, 1.5ul of Tris-HCl buffer (pH 8.0) with a concentration of 1M, and 3.62ul of RNasin were mixed to prepare a total volume of 150ul of capture cell buffer. 10ul of the capture cell buffer was added to each well of a 96-well plate.

[0216] (s4) Flow sorting of PLA2R antibody-secreting cells: After removing dead cells and adherent cells, the cell population of CD20+CD27+human IgG+was sorted for a cell population positive for both PLA2R-APC and PLA2R-PE (as shown in Figure 1 ). The single cells were sorted into a 96-well plate containing the capture cell buffer. After sorting, the 96-well plate was immediately sealed and frozen on dry ice, and then stored at -80°C.

[0217] (s5) Single cell amplification: The antigen recognition region sequences of the light chain and heavy chain of the BCR (antibody) of the single target cell were amplified using a one-step method, and purified by agarose gel electrophoresis.

[0218] The results are shown in Figure 2 , where the single cells corresponding to the first lane, the second lane, and the fourth lane were successfully amplified for the heavy chain ( Figure 2 , left) and the light chain ( Figure 2 , right), and were used to synthesize Ab11, Ab12, and Ab13 antibodies, respectively; while the single cell corresponding to the third lane was successfully amplified for the heavy chain but had no band for the light chain.

[0219] The purified DNA fragments were subjected to TA cloning and sequencing, and the translated sequences (amino acid sequences) after sequencing are shown in Table 1.

[0220] Table 1 Antibody sequences

[0221]

[0222] According to IMGT encoding rules, the complementarity determining region sequences of the heavy chain variable region of the Ab11 antibody are as follows:

[0223] H-CDR1: GGSIISGGSF (SEQ ID NO: 3);

[0224] H-CDR2: IHYSGST (SEQ ID NO: 4);

[0225] H-CDR3: ARDTDAFDM (SEQ ID NO: 5); and

[0226] The complementarity determining region sequences of the light chain variable region of the Ab11 antibody are as follows:

[0227] L-CDR1: RIITSY (SEQ ID NO: 6);

[0228] L-CDR2: GTS (SEQ ID NO: 7);

[0229] L-CDR3: QQSYSSPRT (SEQ ID NO: 8).

[0230] The complementarity determining regions of the heavy chain variable region of the Ab11 antibody marked according to other encoding rules are as shown in Table 2, and the complementarity determining regions of the light chain variable region of the Ab11 antibody marked according to other encoding rules are as shown in Table 3. Figure 3 Figure 4

[0231] Finally, the sequencing results were analyzed, the constant region sequence of IgG4 was integrated on the variable region sequence of Ab11 (the heavy chain constant region was derived from human IgG4, and the light chain constant region was derived from human kappa chain), and was cloned into a pcDNA3.1 plasmid. The plasmid containing the antibody light chain sequence and the plasmid containing the heavy chain sequence were co-transfected into HEK293T cells, the supernatant was collected, and the concentrated purified solution was the recombinant monoclonal antibody solution.

[0232] The recombinant PLA2R monoclonal antibody was detected by WB, and the results are shown in Table 4. Figure 5

[0233] The heavy chain sequence of the obtained recombinant PLA2R monoclonal antibody Ab11 is as follows: VHSQVQLLESGPGLVKSSQTLSLTCTVSGGSIISGGSFWSWIRQHPGKGLEWIGHIHYSGSTHYNPSLKSRVNISVDTSKTQFFLKLSSVTAADTAVYYCARDTDAFDMWGQGTMVSVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK ​​​DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9, wherein the underlined sequence is the heavy chain constant region);

[0234] The light chain sequence is: VH SDIQMTQSPLSLSASVGDRVTITCRASRIITSYLNWYQQRPGQAPKLLIFGTSTLQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYYCQQSYSSPRTFGQGTRVEIK RTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGE (SEQ ID NO: 10, wherein the underlined sequence is).

[0235] The affinities of Ab11, Ab12, and Ab13 were explored, respectively, and the results are shown in Table 1. Figure 6 The results show that the affinity of Ab11 is significantly higher than that of the other two antibodies.

[0236] The EC 50 value of Ab11 is 0.68 ug / mL; the EC 50 value of Ab12 is 1.73 ug / mL; and the EC 50 value of Ab13 is 4.9 ug / mL.

[0237] The antigen specificity of the recombinant PLA2R monoclonal antibody Ab11 was further characterized, and the results are shown in Table 2. Figure 7 The results show that the Ab11 only binds to the non-reduced PLA2R antigen with a conformational epitope, and does not bind to the reduced PLA2R antigen; the Ab11 does not recognize other antigens of PMN, such as THSD7A.

[0238] Example 2: Preparation of an ELISA kit based on the recombinant PLA2R antibody Ab11.

[0239] This example provides a method for preparing an ELISA kit based on a recombinant PLA2R antibody, which specifically includes the following steps:

[0240] Coating: Prepare a sterile microplate, dilute the 1 mg / mL PLA2R protein solution to 1 ug / mL with 1x PBS buffer, take 100 ul and add it to the microplate, and incubate overnight at 4 degrees.

[0241] Blocking: Discard the liquid in the microplate, prepare the blocking buffer (mix 4% skimmed milk and 0.05% Tween-20 in PBS buffer), wash the plate 3 times, then add 300 ul of blocking buffer to each microplate, and block on a shaker at room temperature for 90 minutes.

[0242] Sample dilution: take the serum sample to be tested, mix well, and dilute 1:100 with reaction buffer.

[0243] Sample incubation: take 100ul of positive control reagent, negative control reagent and diluted serum sample to be tested, and add them to the microplate for incubation at room temperature for 90 minutes.

[0244] Plate washing: discard the liquid in the micro-wells, add 300ul of washing buffer to each well, stand at room temperature for 1 minute, then discard the liquid in the micro-wells, and repeat for 3 times.

[0245] Incubation of enzyme-labeled antibody: take 100ul of peroxidase-labeled anti-human IgG4 secondary antibody and add it to the microplate for incubation at room temperature for 60 minutes.

[0246] Plate washing: discard the liquid in the micro-wells, add 300ul of elution buffer to each well, stand at room temperature for 1 minute, then discard the liquid in the micro-wells, and wash for 3 times.

[0247] Incubation of substrate solution: take 100ul of substrate solution and add it to the microplate for incubation at room temperature for 6 minutes.

[0248] Reaction termination by termination solution: take 100ul of termination solution and add it to the microplate.

[0249] Reading by enzyme-labeled instrument: colorimetric at 450nm, and record the reading.

[0250] Result calculation: calculate the PLA2R IgG4 antibody unit value (U / mL) according to the formula: PLA2R IgG4 antibody unit value (U / mL) = 100 x (A450 待测样本 -A450 阴性对照 ) / (A450 阳性对照 -A450 阴性对照 ).

[0251] Result determination: unit value < 17 is negative, unit value ≥ 17 is positive, and the higher the unit value, the higher the concentration of PLA2R IgG4 antibody in the sample to be tested.

[0252] Quality control: each test result must meet OD450 阳性对照 ≥ 0.400, OD450 阴性对照 ≤ 0.100, otherwise the result is invalid.

[0253] In the present embodiment, the reaction buffer is 1x PBS containing 4% skimmed milk powder and 0.05% Tween 20; the washing buffer is 1x PBS containing 0.05% Tween 20; the positive control is recombinant PLA2R monoclonal antibody (Ab11) diluted in the reaction buffer; the negative control is 1x PBS containing 4% skimmed milk powder and 0.05% Tween 20; the peroxidase-labeled anti-human IgG4 secondary antibody is a commercial antibody (Invitrogen A10654) diluted 1:5000 in the reaction buffer; the substrate solution is TMB color developing liquid; and the termination solution is TMB color developing termination liquid.

[0254] Example 3: Detection performance of the kit of the present application.

[0255] The present embodiment provides the sensitivity, specificity, and stability of the ELISA kit of the present application in detecting primary membranous nephropathy.

[0256] The results are shown in Table 2.

[0257] The results show that for primary membranous nephropathy with a titer of ≥20 RU / mL as indicated by the OMRON kit, the detection rate of the kit of the present application is 100%; for primary membranous nephropathy with a titer of <20 RU / mL as indicated by the OMRON kit, the OMRON kit judges as negative or suspicious, but the detection rate of the kit of the present application is 91%, confirming that the kit of the present application has higher detection sensitivity and basically no or small gray interval in detection.

[0258] In addition, the kit of the present application has high specificity for primary membranous nephropathy, and in the existing samples, it will not misdiagnose healthy people and non-primary membranous nephropathy. However, the OMRON kit has false positives in diabetic nephropathy.

[0259] Table 2 Sensitivity and specificity of the ELISA kit of the present application

[0260]

[0261] The ELISA kit of the present application was used to continuously detect 3 serum samples with different PLA2R antibody titers for 5 days, 3 times a day, and the results showed that the intra-batch CV% of the PLA2R IgG4 antibody unit value was 1.53%, and the inter-batch CV% was 12%, both of which were within the allowable error range; the consistency of positive results of qualitative detection was 100%, and the consistency of negative results was 100%, indicating that the qualitative ELISA kit has stable repeatability.

[0262] In summary, the results of the present embodiment show that the kit of the present application exhibits excellent stability and sensitivity in the detection of primary membranous nephropathy, has good specificity, and has a very low probability of false positives, and has important significance for early diagnosis of primary membranous disease and selection of accurate treatment methods.

[0263] All documents referred to in this disclosure are incorporated herein by reference as if each individual document were incorporated by reference. In addition, it is to be understood that various alterations and modifications can be made to the present application upon reading and understanding the above lecture of the present application, and these equivalent forms also fall within the scope of the appended claims of the present application.

Claims

1. A PLA2R antibody that binds to phospholipase A2 receptor (PLA2R), characterized in that, The PLA2R antibody has: (a) a heavy chain variable region comprising the following three complementarity determining regions CDRs: H-CDR1 set forth in SEQ ID NO: 3, H-CDR2 set forth in SEQ ID NO: 4, and H-CDR3 set forth in SEQ ID NO: 5; and (b) a light chain variable region comprising the following three complementarity determining regions CDRs: L-CDR1 set forth in SEQ ID NO: 6, L-CDR2 set forth in SEQ ID NO: 7, and L-CDR3 set forth in SEQ ID NO:

8.

2. The antibody of claim 1, wherein The PLA2R antibody is an IgG4 type antibody.

3. A recombinant protein, characterized in that, The recombinant protein has: (i) the antibody of claim 1 or claim 2; and (ii) optionally a signal peptide and / or a tag sequence.

4. An antibody conjugate characterized in that, The antibody conjugate comprises: (Z1) the antibody of claim 1 or 2 or the recombinant protein of claim 3; and (Z2) a conjugating moiety selected from the group consisting of a detectable label or a radionuclide.

5. Use of the antibody according to claim 2, the recombinant protein according to claim 3 or the antibody conjugate according to claim 4, characterized in that, A kit for preparing a reagent for detecting primary membranous nephropathy.

6. A kit for detecting primary membranous nephropathy, characterized by, The kit comprises the antibody of claim 2, the recombinant protein of claim 3, or the antibody conjugate of claim 4.

7. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1-6. The polynucleotide encodes the antibody of claim 1 or the recombinant protein of claim 3.

8. A vector, characterized in that, The vector comprises the polynucleotide of claim 7.

9. A host cell, characterized in that, The host cell comprises the vector of claim 8 or the polynucleotide of claim 7 integrated in the genome. The host cell comprises the vector of claim 8 or the polynucleotide of claim 7 integrated in the genome.

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