PLA2R IgG4 antibody and application thereof

Abstract

The invention provides a PLA2R monoclonal antibody and an ELISA (Enzyme-Linked Immunosorbent Assay) kit containing the PLA2R monoclonal antibody. Specifically, the invention provides a monoclonal antibody capable of being combined with PLA2R with high specificity, and the antibody is only combined with a non-reduced PLA2R antigen with conformational epitopes, is not combined with a reduced PLA2R antigen, and is not combined with another antigen THSD7A of primary membranous nephropathy. Compared with an existing commercial kit, the ELISA kit prepared based on the antibody can more efficiently and accurately detect the primary membranous nephropathy.

Description

Technical Field

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

[0002] Primary membranous nephropathy (PMN) is a glomerular disease that predominantly affects middle-aged and elderly men, with clinical manifestations of nephrotic syndrome. The incidence of PMN in my country has been increasing annually in recent years. Extensive clinical and experimental data indicate that PMN is an autoimmune disease mediated by antibodies against the phospholipase A2 receptor (PLA2R).

[0003] PLA2R is a member of the mannose receptor family. Its antigenicity is conformation-dependent. Four conformational epitopes have been identified within the 10 structural domains of PLA2R: CysR, CTLD1, CTLD7, and CTLD8.

[0004] In recent years, the enzyme-linked immunosorbent assay (ELISA) has been widely used for the screening and diagnosis of PMNs. The current standardized ELISA kit for detecting PLA2R antibodies is produced by Euromonitor. While the kit's diagnostic specificity is close to 100%, its sensitivity is limited, including a gray zone diagnostic range, meaning it cannot accurately detect samples with antibody concentrations within the critical range.

[0005] In addition, the PLA2R antibody standards and positive controls were obtained by purifying and concentrating serum from PMN patients. This method of serum purification and concentration has many problems, such as large batch-to-batch variability, cumbersome quality control, and the risk of potential contamination from infectious sources.

[0006] Therefore, there is an urgent need in the art to develop an ELISA kit for detecting PLA2R antibodies with better specificity and sensitivity, and at the same time, a standard product (such as a monoclonal antibody that recognizes PLA2R) that can be mass-produced. Summary of the Invention

[0007] The present invention provides an antibody that specifically binds to PLA2R, and an ELISA kit prepared using the antibody, which has high sensitivity, good specificity, good stability and small batch difference.

[0008] In a first aspect of the present invention, there is provided a phospholipase A2 receptor (PLA2R) antibody that binds to the PLA2R, wherein the PLA2R antibody has: (a) a heavy chain variable region, comprising the following three complementarity determining regions (CDRs): H-CDR1 shown in SEQ ID NO: 3, H-CDR2 shown in SEQ ID NO: 4, and H-CDR3 shown in SEQ ID NO: 5; and (b) a light chain variable region comprising the following three complementarity determining regions (CDRs): L-CDR1 shown in SEQ ID NO:6, L-CDR2 shown in SEQ ID NO:7, and L-CDR3 shown in SEQ ID NO:8.

[0009] In another preferred embodiment, the complementarity determining regions are annotated using the IMGT coding rules.

[0010] In another preferred embodiment, the H-CDR1 comprises an amino acid sequence having one or more conservative amino acid mutations compared with SEQ ID NO: 3.

[0011] 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.

[0012] In another preferred embodiment, the H-CDR3 comprises an amino acid sequence having one or more conservative amino acid mutations compared with SEQ ID NO:5.

[0013] In another preferred embodiment, the L-CDR1 comprises an amino acid sequence having one or more conservative amino acid mutations compared with SEQ ID NO: 6.

[0014] In another preferred embodiment, the L-CDR2 comprises an amino acid sequence having one or more conservative amino acid mutations compared with SEQ ID NO: 7.

[0015] In another preferred embodiment, the L-CDR3 comprises an amino acid sequence having one or more conservative amino acid mutations compared with SEQ ID NO: 8.

[0016] In another preferred embodiment, the one or more preferably refers to 1-3, more preferably 1-2, most preferably 1.

[0017] In another preferred embodiment, the mutation is preferably an amino acid substitution, insertion or deletion.

[0018] In another preferred embodiment, the antibody has a heavy chain variable region, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 1: VHSQVQLLESGPGLVKSSQTLSLTCTVSGGSIISGGSFWSWIRQHPGKGLEWIGHIHYSGSTHYNPSLKSRVNISVDTSKTQFFLKLSSVTAADTAVYYCARDTDAFDMWGQGTMVSVSS (SEQ ID NO: 1).

[0019] In another preferred embodiment, the antibody has a light chain variable region, and the light chain variable region comprises the sequence shown in SEQ ID NO: 2: VHSDIQMTQSPLSLSSASVGDRVTITCRASRIITSYLNWYQQRPGQAPKLLIFGTSTLQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYYCQQSYSSPRTFGQGTRVEIK (SEQ ID NO: 2).

[0020] In another preferred example, the heavy chain variable region has a sequence that is at least 85% identical to SEQ ID NO: 1, preferably at least 90% identical, more preferably at least 95% or higher, for example 95%, 96%, 97%, 98%, 99% identical.

[0021] In another preferred example, the light chain variable region has a sequence that is at least 85% identical to SEQ ID NO: 2, preferably at least 90% identical, more preferably at least 95% or higher, for example 95%, 96%, 97%, 98%, 99% identical.

[0022] In another preferred embodiment, the antibody specifically binds to the non-reduced PLA2R antigen and does not bind to the reduced PLA2R antigen.

[0023] In another preferred embodiment, the antibody has a heavy chain constant region and / or a light chain constant region.

[0024] 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.

[0025] In another preferred embodiment, the heavy chain constant region is a human or mouse heavy chain constant region.

[0026] In another preferred embodiment, the heavy chain constant region is derived from mouse IgG1, mouse IgG2a, mouse IgG2b, or mouse IgG3.

[0027] In another preferred embodiment, the heavy chain constant region is derived from human IgG1, human IgG2, human IgG3, or human IgG4.

[0028] In another preferred embodiment, the heavy chain constant region is derived from human IgG4.

[0029] In another preferred embodiment, the antibody has a heavy chain constant region, and the heavy chain constant region comprises the sequence shown at positions 121-450 in SEQ ID NO:9.

[0030] In another preferred embodiment, the antibody has a light chain constant region.

[0031] 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.

[0032] In another preferred embodiment, the light chain constant region is a human or mouse light chain constant region.

[0033] In another preferred embodiment, the light chain constant region is derived from mouse IgG1, mouse IgG2a, mouse IgG2b, or mouse IgG3.

[0034] In another preferred embodiment, the light chain constant region is derived from human IgG1, human IgG2, human IgG3, or human IgG4.

[0035] In another preferred embodiment, the light chain constant region is derived from human kappa chain.

[0036] In another preferred embodiment, the antibody has a light chain constant region, and the light chain constant region comprises the sequence shown at positions 111-216 in SEQ ID NO:10.

[0037] In another preferred embodiment, the antibodies include: monoclonal antibodies, polyclonal antibodies, double-chain antibodies, single-chain antibodies (scFv), Fab, Fab', and F(ab')2 antibodies.

[0038] In another preferred embodiment, the antibody is a double-chain antibody.

[0039] In another preferred embodiment, the antibody is a monospecific antibody, a bispecific antibody, or a trispecific antibody.

[0040] In another preferred embodiment, the PLA2R antibody is an IgG4 antibody.

[0041] In another preferred embodiment, the IgG4 antibody comprises an Fc fragment.

[0042] In another preferred embodiment, the Fc fragment is glycosylated.

[0043] In another preferred embodiment, the hinge region of the IgG4 antibody has an S228P mutation.

[0044] In another preferred embodiment, the IgG4 antibody further contains: L234F, H268Q, A330S, P331S, L235E, L235A, F234A, G237A, D255A, or a combination thereof.

[0045] In another preferred embodiment, the antibody comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 9, and a light chain having an amino acid sequence as shown in SEQ ID NO: 10.

[0046] In another preferred example, the antibody has a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region comprises the sequence shown at positions 121-450 in SEQ ID NO:9, and the light chain constant region comprises the sequence shown at positions 111-216 in SEQ ID NO:10.

[0047] In a second aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has: (i) the first aspect of the invention or the antibody according to the first aspect of the invention; and (ii) optional signal peptide and / or tag sequence.

[0048] In another preferred embodiment, the recombinant protein specifically binds to PLA2R.

[0049] In another preferred embodiment, the tag sequence is selected from the following group: FLAG, Myc, and His tags.

[0050] In a third aspect of the present invention, an antibody conjugate is provided, comprising: (Z1) the antibody according to the first aspect or 2 of the present invention or the recombinant protein according to the second aspect of the present invention; and (Z2) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0051] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.

[0052] In the fourth aspect of the present invention, there is provided a use of the antibody described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, or the antibody conjugate described in the third aspect of the present invention for preparing a kit for detecting primary membranous nephropathy.

[0053] In another preferred embodiment, the antibody, the recombinant protein, or the antibody conjugate is used as a positive control reagent.

[0054] In another preferred embodiment, the kit is used to detect PLA2R IgG4 antibodies in a sample.

[0055] In another preferred embodiment, the kit comprises a secondary antibody, and the secondary antibody specifically binds to the antibody, the recombinant protein, or the antibody conjugate.

[0056] In a fifth aspect of the present invention, a kit for detecting primary membranous nephropathy is provided, the kit comprising: the antibody described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, or the antibody conjugate described in the third aspect of the present invention.

[0057] In another preferred embodiment, the kit is an ELISA kit.

[0058] In another preferred embodiment, the antibody, the recombinant protein, or the antibody conjugate is used as a positive control reagent.

[0059] In another preferred embodiment, the kit further comprises an instruction manual, which indicates that the kit is used to detect PLA2R IgG4 antibodies in a sample.

[0060] In another preferred embodiment, the sample includes: a serum sample or a urine sample.

[0061] In another preferred embodiment, the sample is a serum sample.

[0062] In another preferred embodiment, the kit comprises a secondary antibody, and the secondary antibody specifically binds to the antibody described in the first aspect of the present invention.

[0063] In another preferred embodiment, the secondary antibody is a polyclonal antibody or a monoclonal antibody.

[0064] In another preferred embodiment, the secondary antibody is a goat antibody, a rabbit antibody, or a combination thereof.

[0065] In another preferred embodiment, the secondary antibody comprises a detectable marker or enzyme In another preferred embodiment, the secondary antibody is an anti-IgG4 secondary antibody.

[0066] In another preferred embodiment, the secondary antibody is an anti-human IgG4 secondary antibody.

[0067] In another preferred embodiment, the anti-human IgG4 secondary antibody is labeled with a detectable marker.

[0068] In another preferred embodiment, the detectable marker includes peroxidase.

[0069] In another preferred embodiment, the ELISA kit further comprises other reagents required for ELISA detection, including: washing solution required for ELISA detection, analysis buffer, blocking solution, color development solution, color development stop solution, PLA2R protein, or a combination thereof.

[0070] In another preferred embodiment, the instructions indicate that the kit is used to detect primary membranous nephropathy.

[0071] In another preferred embodiment, the kit further comprises other diagnostic reagents for primary membranous nephropathy.

[0072] In another preferred embodiment, the kit further comprises a therapeutic agent for primary membranous nephropathy.

[0073] In another preferred embodiment, the kit further comprises an instruction manual, which records the operating method and judgment criteria for diagnosing primary membranous nephropathy.

[0074] In another preferred embodiment, the judgment criteria include: if the antibody unit value of PLA2R IgG4 antibody in the sample is ≥17, the sample is judged to be positive for primary membranous nephropathy; if the antibody unit value of PLA2R IgG4 antibody in the sample is <17, the sample is judged to be negative for primary membranous nephropathy.

[0075] In another preferred embodiment, the unit of the antibody unit value X is U / mL.

[0076] In a sixth aspect of the present invention, a method for detecting PLA2R in a sample is provided, comprising the steps of: The sample is contacted with the antibody described in the first aspect of the present invention, and the formation of a "PLA2R-antibody" complex is observed. If a "PLA2R-antibody" complex is formed, it indicates that PLA2R is present in the sample; if no "PLA2R-antibody" complex is formed, it indicates that PLA2R is not present in the sample.

[0077] In a seventh aspect of the present invention, a method for detecting anti-PLA2R autoantibodies in a sample is provided, comprising the steps of: (a) In the test group, the sample was incubated with PLA2R to observe whether a "PLA2R-anti-PLA2R autoantibody" complex was formed; In a positive control group, the antibody of the first aspect of the present invention is incubated with PLA2R to observe whether a "PLA2R-positive antibody" complex is formed; (b) Comparing the test results of the test group with the test results of the positive control group to determine whether anti-PLA2R autoantibodies are present in the test sample.

[0078] In another preferred embodiment, in step (a), the method further comprises: in a blank control group, incubating PLA2R with a blank buffer under the same conditions, and obtaining blank experimental results; In another preferred embodiment, in the test group, the positive control group and the blank control group, except for the addition of sample, the addition of positive antibody, and the addition of neither sample nor positive antibody, other experimental conditions are the same.

[0079] In another preferred embodiment, the method is an ELISA method, which comprises the following steps: (S1) In the blank control group, blank buffer was co-incubated with PLA2R protein pre-coated in the detection wells of the ELISA plate; In the positive control group, under the same conditions, the antibody according to the first aspect of the present invention was co-incubated with the PLA2R protein pre-coated in the detection wells of the ELISA plate; In the experimental group, under the same conditions, the test samples were incubated with PLA2R protein pre-coated in the detection wells of the ELISA plate; (S2) 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 to the detection wells of the blank control group, the positive control group, and the experimental group, respectively, and continuing incubation; (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, indicating that the antibody unit value X of the PLA2R IgG4 antibody in the sample is 100×(Y3-Y1) / (Y2-Y1).

[0080] In another preferred embodiment, the anti-human IgG4 secondary antibody is linked to peroxidase.

[0081] In another preferred embodiment, the relative parameter is the absorbance at 450 nm after a color development reaction using peroxidase.

[0082] In another preferred embodiment, the unit of the antibody unit value X is U / mL.

[0083] In another preferred embodiment, step (S1) includes the following sub-steps: (S1-1) Coating the PLA2R protein on the detection wells of the ELISA plate; (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; (S1-3) In a blank control group, a blank buffer solution was co-incubated with the PLA2R protein pre-coated in the detection wells of an ELISA plate; in a positive control group, under the same conditions, the antibody described in the first aspect of the present invention was co-incubated with the PLA2R protein pre-coated in the detection wells of an ELISA plate; in an experimental group, under the same conditions, the test sample was co-incubated with the PLA2R protein pre-coated in the detection wells of an ELISA plate.

[0084] In another preferred embodiment, in step (S1-1), the coating refers to incubation at 4°C overnight.

[0085] In another preferred embodiment, in step (S1-2), the blocking comprises discarding the liquid in the detection well, washing with a blocking buffer, and adding a blocking buffer for blocking.

[0086] In another preferred embodiment, the blocking by adding blocking buffer means blocking on a shaker at room temperature for 60-120 minutes, preferably 90 minutes, after adding blocking buffer.

[0087] In another preferred embodiment, in step (S2), the washing refers to discarding the liquid in the detection well, adding a washing buffer solution and standing for washing.

[0088] In another preferred embodiment, the standing refers to standing at room temperature for 1 minute.

[0089] In another preferred embodiment, in step (S2), the incubation refers to incubation on a shaker at room temperature for 40-80 minutes, preferably 60 minutes.

[0090] In another preferred embodiment, in step (S3), the following sub-steps are further included: (S3-1) Discard the liquid in the detection well, add washing buffer, let it stand and then wash; (S3-2) Adding the substrate solution to the microplate and incubating the plate. After the incubation is complete, adding the stop solution; (S3-3) 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 are respectively measured. The antibody unit value X of the PLA2R IgG4 antibody in the sample is X = 100 × (A3 - A1) / (A2 - A1).

[0091] In step (S3-2), the static incubation refers to static incubation at room temperature for 6 minutes.

[0092] In another preferred embodiment, the room temperature is 18-28°C, preferably 25°C.

[0093] In another preferred embodiment, the washing refers to washing 2-4 times, preferably, washing 3 times.

[0094] In another preferred embodiment, the ELISA method comprises the following steps: (M1) PLA2R protein was coated on the detection wells of the ELISA plate; (M2) blocking the detection wells obtained in step (M1), thereby obtaining the PLA2R protein pre-coated in the detection wells of the ELISA plate; (M3) In the blank control group, blank buffer was co-incubated with the PLA2R protein pre-coated in the detection wells of the ELISA plate; in the positive control group, the antibody described in the first aspect of the present invention was co-incubated with the PLA2R protein pre-coated in the detection wells of the ELISA plate under the same conditions; in the experimental group, the test sample was co-incubated with the PLA2R protein pre-coated in the detection wells of the ELISA plate under the same conditions.

[0095] (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 to the detection wells of the blank control group, the positive control group, and the experimental group, respectively, and continuing incubation; (M5) washing the detection wells obtained in step (M4), adding the substrate solution to the microplate and incubating it statically, and adding the stop solution after the incubation is completed; (M6) 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. The antibody unit value X of the PLA2R IgG4 antibody in the sample is X = 100 × (A3 - A1) / (A2 - A1).

[0096] In another preferred embodiment, the unit of the antibody unit value X is U / mL.

[0097] In another preferred embodiment, the method is a method for non-diagnostic and non-therapeutic purposes.

[0098] 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.

[0099] In another preferred embodiment, if A1>0.100 or A2<0.400, it is determined that retesting is required.

[0100] In an eighth aspect of the present invention, a polynucleotide is provided, wherein the polynucleotide encodes the antibody according to the first aspect of the present invention or the recombinant protein according to the second aspect of the present invention.

[0101] In the ninth aspect of the present invention, a vector is provided, wherein the vector comprises the polynucleotide according to the eighth aspect of the present invention.

[0102] In another preferred embodiment, the vector comprises a eukaryotic cell expression vector or a prokaryotic cell expression vector.

[0103] In another preferred embodiment, the vector is a plasmid.

[0104] In the tenth aspect of the present invention, a host cell is provided, wherein the host cell comprises the vector according to the ninth aspect of the present invention or the polynucleotide according to the eighth aspect of the present invention is integrated into its genome.

[0105] In another preferred embodiment, the host cell comprises a eukaryotic cell or a prokaryotic cell.

[0106] In another preferred embodiment, the host cell is HEK293T cell.

[0107] In the eleventh aspect of the present invention, there is provided a use of the antibody described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, or the antibody conjugate described in the third aspect of the present invention for preparing a detection reagent, a detection plate, or a kit for detecting PLA2R.

[0108] In another preferred embodiment, the detection includes qualitative detection or quantitative detection.

[0109] In the twelfth aspect of the present invention, a detection reagent is provided, which comprises the antibody described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, or the antibody conjugate described in the third aspect of the present invention.

[0110] In another preferred embodiment, the detection reagent further comprises a carrier or excipient acceptable in detection.

[0111] In the thirteenth aspect of the present invention, a detection plate is provided, which comprises the antibody described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, or the antibody conjugate described in the third aspect of the present invention.

[0112] In a fourteenth aspect of the present invention, a device for detecting PLA2R IgG4 antibodies is provided, the device comprising the following modules: (N1) a sample incubation module, wherein the sample incubation module is configured to: in a blank control group, incubate a blank buffer solution with the PLA2R protein pre-coated in the detection wells of an ELISA plate; in a positive control group, incubate the antibody described in the first aspect of the present invention with the PLA2R protein pre-coated in the detection wells of an ELISA plate under the same conditions; and in an experimental group, incubate the sample to be tested with the PLA2R protein pre-coated in the detection wells of an ELISA plate under the same conditions; (N2) a secondary antibody incubation module, wherein the secondary antibody incubation module is configured to: wash the detection wells of the blank control group, the positive control group, and the experimental group, respectively, and add anti-human IgG4 secondary antibody to the detection wells of the blank control group, the positive control group, and the experimental group, respectively, and continue incubation; (N3) an analysis and calculation module, wherein the analysis and calculation module is configured to: wash and detect a relative parameter Y1 of the PLA2R protein-PLA2R IgG4 complex in the blank control group, a relative parameter Y2 of the PLA2R protein-PLA2R IgG4 complex in the positive control group, and a 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 is 100×(Y3-Y1) / (Y2-Y1).

[0113] In another preferred embodiment, the anti-human IgG4 secondary antibody is linked to peroxidase.

[0114] In another preferred embodiment, the relative parameter is the absorbance at 450 nm after a color development reaction using peroxidase.

[0115] In another preferred embodiment, the unit of the antibody unit value X is U / mL.

[0116] In another preferred embodiment, if Y1>0.100 or Y2<0.400, it is determined that retesting is required.

[0117] In another preferred embodiment, the device further comprises a result output module, which is configured to: if the antibody unit value X of the PLA2R IgG4 antibody in the sample is ≥17, then 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, then the sample is determined to be negative for primary membranous nephropathy.

[0118] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 Flow cytometric gating of PLA2R antibody-secreting cells is shown.

[0120] Figure 2 Shown is the agarose gel electrophoresis of amplified base fragments of the VDJ region of a single PLA2R antibody-secreting cell.

[0121] Figure 3 The CDR sequences of the variable region of the heavy chain of Abl 1 are shown with different conventions (underlined in red).

[0122] Figure 4 The CDR sequences of the light chain variable region of Abl 1 are shown with different conventions (red underlined).

[0123] Figure 5 WB detection showed that the recombinant PLA2R monoclonal antibody (Ab11) had complete light and heavy chains after reduction denaturation.

[0124] Figure 6 The PLA2R monoclonal antibody screening is shown, in which Ab11 is the PLA2R monoclonal antibody with the highest affinity.

[0125] Figure 7 Specific detection of a recombinant PLA2R monoclonal antibody (Abl 1) is shown. DETAILED DESCRIPTION

[0126] After extensive and in-depth research, the inventors analyzed specific B cells that recognize PLA2R in the peripheral blood of PMN patients, resulting in the first identification of a monoclonal antibody that specifically recognizes PLA2R from PMN peripheral blood. The inventors also provided an IgG4 subclass monoclonal antibody of this monoclonal antibody. This antibody binds only to the non-reduced PLA2R antigen with a conformational epitope, not to the reduced PLA2R antigen, and certainly not to THSD7A, another antigen implicated in primary membranous nephropathy. An ELISA kit prepared using this antibody as a standard exhibits superior specificity for detecting primary membranous nephropathy, with minimal intra- and inter-assay variability. This led to the completion of the present invention.

[0127] the term In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can change. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0128] Unless defined otherwise, 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 invention belongs.

[0129] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0130] As used herein, the term "optionally" or "optionally" means that the event or situation described subsequently may occur but need not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence may have but need not have, and may have 1, 2, or 3.

[0131] As used herein, "sequence identity" refers to the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between a sequence described herein and a sequence to which it is identical may 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%, and 100%.

[0132] As used herein, the term "antibody" or "immunoglobulin" is a heterotetramer formed by two light chains (L) and two heavy chains (H). The N-terminus of each heavy chain is a variable region (VH), connected to the heavy chain constant region. The N-terminus of each light chain is a variable region (VL), connected to the light chain constant region.

[0133] As used herein, the term "variable" refers to an antibody in which the variable region is different in a specific sequence, forming the affinity and specificity of a particular antibody for binding to a specific antigen. The antibody variable region includes a complementary determining region (CDR) or a hypervariable region and a framework region (FR) whose sequence is relatively conservative. The primary sequence of the heavy chain and light chain variable regions is composed of four sections of FR sequences and three sections of CDR sequences arranged at intervals (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, 647-669 pages (1991)). The sequence and spatial structure conformation of the heavy chain and light chain variable regions determine the specific binding of the antibody to the antigen epitope. The antibody constant region does not directly participate in the binding of the antibody to the antigen, but has an impact on the performance of the capture antibody and the detection antibody.

[0134] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified according to the amino acid sequence of their constant regions into and Based on the amino acid sequence of their heavy chain constant regions, immunoglobulins can be classified into five main classes: IgA, IgD, IgE, IgG, and IgM, as well as antibody subtypes (isotypes). For example, human IgG includes IgG1, IgG2, IgG3, and IgG4 subtypes, while mouse IgG includes IgG1, IgG2a, and IgG2b subtypes. The subunit structures and three-dimensional configurations of different immunoglobulin classes are well known in the art.

[0135] As used herein, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical. Monoclonal antibodies are highly specific for 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 any particular method for production of the antibody.

[0136] The present invention 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 proteins or protein conjugates and fusion expression products having these chains. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having light and heavy chains containing variable regions (complementarity determining regions, CDRs), as long as the variable regions are identical to or at least 90% homologous to the variable regions of the light and heavy chains of the present invention, preferably at least 95% homologous.

[0137] As known to those skilled in the art, antibody conjugates and fusion expression products include: conjugates formed by combining fluorescent or luminescent markers, enzymes capable of producing detectable products, gold nanoparticles / nanorods and other detectable molecules with the PLA2R antibody.

[0138] 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 fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by 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 bond at the hinge region; and (iii) a scFv fragment consisting of the VH and VL domains of a single arm of an antibody.

[0139] The present invention includes not only complete monoclonal antibodies, but also antibody fragments with binding activity, such as Fab or (Fab')2 fragments; antibody heavy chains; antibody light chains or scFv.

[0140] The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen.

[0141] As used herein, the term "antigenic determinant" refers to a discrete three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present invention.

[0142] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0143] In the present invention, the antibodies can be monospecific, bispecific, trispecific, or more multispecific.

[0144] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably. The terms "light chain variable region" and "VL" are used interchangeably.

[0145] As used herein, "complementarity determining region" (CDR) is the region of an antibody that binds to an antigen. CDRs can be defined using various notations, such as Kabat (Kabat et al., Immunologically Significant Protein Sequences, 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 Bmol 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 herein include CDRs defined by any of the methods described above, as well as by numbering rules known to those skilled in the art, such as Kabat, Chothia, IMGT, or AbM. The correspondence between numbering systems, including, for example, the Kabat numbering system and the IMGT unique numbering system, is well known to those skilled in the art.

[0146] In the present invention, the three CDRs of VH (H-CDR1, H-CDR2 and H-CDR3) and the three 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 invention determined using different rules can be found in Figure 3 and Figure 4 It should be understood that the antibodies of the present invention include antibodies defined by the six CDRs determined by any rule, and the sequences of the FR regions outside the CDRs may vary to a certain extent, as long as the same or similar binding properties as the preferred antibody of the present invention (Ab11) are retained, including: binding to the same epitope; binding only to the non-reduced PLA2R antigen having a conformational epitope but not binding to the reduced PLA2R antigen.

[0147] "Fragments," "derivatives," and "analogs" of antibodies refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent group in one or more amino acid residues, or (iii) polypeptides formed by coupling a mature polypeptide with another compound (such as a chemiluminescent compound, e.g., an acridinium ester), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence, or a tag protein sequence or other fusion protein sequence used to purify or detect the polypeptide). These fragments, derivatives, and analogs are well known to those skilled in the art.

[0148] The antibodies of the present invention are those having PLA2R binding activity, for example, Figure 3 and Figure 4 The polypeptide of the CDR region. The term also includes variant forms of polypeptides comprising the aforementioned CDR region that have the same function as the antibody of the present invention. These variant forms include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids with similar or similar properties generally does not change the function of the protein. For another example, adding one or several amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0149] As used herein, "positive control reagent" and "standard" are used interchangeably and both refer to the positive control in the detection of autoantibodies against phospholipase A2 receptor (PLA2R) in primary membranous nephropathy. In the present invention, the positive control reagent is PLA2R IgG4 antibody.

[0150] As used herein, the PLA2R IgG4 antibody refers to an antibody against PLA2R IgG4, ie, an anti-PLA2R IgG4 antibody; similarly, the PLA2R antibody refers to an antibody against PLA2R, ie, an anti-PLA2R antibody.

[0151] Primary membranous nephropathy Primary membranous nephropathy (PMN) is a common cause of nephrotic syndrome in adults and is an autoimmune glomerular disease characterized by diffuse deposition of immune complexes on the epithelial side of the glomerular basement membrane, accompanied by diffuse thickening of the basement membrane.

[0152] In terms of pathogenesis, recent studies have shown that approximately 70%-80% of patients with primary membranous nephropathy have autoantibodies against phospholipase A2 receptor (PLA2R). These autoantibodies bind to the PLA2R antigen on the surface of podocytes to form in situ immune complexes, activate the complement system, and lead to glomerular damage.

[0153] Epidemiologically, primary membranous nephropathy can occur at any age, but is more common in middle-aged and elderly people, with a higher incidence in men than in women. While the incidence varies across regions, it is generally on an upward trend.

[0154] The main clinical symptoms are nephrotic syndrome, characterized by heavy proteinuria (urine protein >3.5 g / day), hypoproteinemia (plasma albumin <30 g / L), edema, and hyperlipidemia. Some patients may also experience microscopic hematuria, but gross hematuria is rare.

[0155] Diagnosis primarily relies on renal biopsy and pathological examination. Light microscopy reveals diffuse thickening of the glomerular basement membrane, immunofluorescence reveals granular deposition of IgG and C3 along the glomerular capillary walls, and electron microscopy reveals subepithelial electron-dense deposits. Serum detection of autoantibodies, such as anti-PLA2R antibodies, also aids in diagnosis and assessment.

[0156] Extensive clinical and experimental data indicate that PMN is an autoimmune disease mediated by the autoantigen PLA2R and corresponding autoantibodies. As a biomarker for primary membranous nephropathy, PLA2R antibodies play an important role in early diagnosis, disease monitoring, and prognosis assessment.

[0157] Phospholipase A2 receptor (PLA2R) The phospholipase A2 receptor (PLA2R) is a cell surface receptor and a transmembrane glycoprotein member of the mammalian mannose receptor family. It is a 180 kDa protein specifically expressed in human kidney podocytes.

[0158] PLA2R is the pathogenic antigen of primary membranous nephropathy. Its antigenicity is conformation-dependent. PLA2R possesses 10 distinct extracellular domains, consisting, from N-terminus to C-terminus, of a cysteine-rich region (CysR), a fibronectin type II domain (FnII), and eight consecutive C-type lectin-like domains (CTLD1-CTLD8). The majority of PLA2R protein domains are located extracellularly, with some transmembrane and intracellular domains also present. Four conformational epitopes have been identified within the 10 PLA2R domains: CysR, CTLD1, CTLD7, and CTLD8.

[0159] However, the detection of PLA2R antibodies still needs to be optimized. For example, the PLA2R antibody standards and positive controls are obtained by purifying and concentrating the serum of PMN patients. This method of serum purification and concentration is subject to problems such as large batch-to-batch variability, cumbersome quality control, and the risk of potential contamination from infectious sources. For another example, the antibodies that recognize PLA2R in the peripheral blood of PMN patients are mainly of the IgG4 subtype. However, the most commonly used European test kit in clinical practice detects IgG antibodies (including IgG4 and other IgG subtypes). As a result, this test method may recognize other IgG subtypes that bind nonspecifically, resulting in decreased specificity.

[0160] Specifically, compared to standards obtained through serum purification and concentration, recombinant monoclonal antibodies offer advantages such as batch stability, high specificity, and scalable production. Therefore, this application identified a series of monoclonal antibodies capable of recognizing PLA2R by analyzing PLA2R-specific B cells in the peripheral blood of PMN patients. A single monoclonal antibody was screened for use in an ELISA kit for detecting PLA2R-IgG4 antibodies. Furthermore, an ELISA kit was developed for detecting PLA2R-IgG4 levels in PMNs, targeting IgG4 as the target subtype.

[0161] The main advantages of the present invention include: (a) The recombinant PLA2R monoclonal antibody prepared in the present invention has high specificity and high affinity, and only binds to the PLA2R protein with a conformational epitope.

[0162] (b) The present invention uses anti-IgG4 enzyme-labeled antibodies to replace the anti-IgG enzyme-labeled antibodies of similar products on the market, effectively reducing the background signal caused by nonspecific binding of other IgG1 / 2 / 3 subtype antibodies that account for a high proportion in serum, improving the detection specificity, and enhancing the detection accuracy of low-concentration PLA2R antibodies.

[0163] (c) Compared with other similar products on the market, the ELISA kit of the present invention has higher sensitivity and stronger specificity for samples with pathologically confirmed primary membranous nephropathy and PLA2R antibody titers in the gray zone, which can meet the needs of early screening and personalized treatment of primary membranous nephropathy.

[0164] (d) The recombinant PLA2R monoclonal antibody of the present invention is easy to prepare by recombinant methods, which helps to ensure that there are no bacteria, viruses or other infectious sources during the production process, and has higher safety and reliability.

[0165] (e) The positive control recombinant PLA2R monoclonal antibody of the present invention has a standard synthesis process and strict quality control, which avoids the batch differences in traditional biologically derived antibodies, ensures the potency stability of the standard, and improves the repeatability and reliability of the experimental results.

[0166] (g) Compared with the existing PLA2R antibody IIFT test, the ELISA kit of the present invention is more objective and avoids errors caused by subjective judgment.

[0167] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and parts by weight.

[0168] Example 1: Preparation and identification of PLA2R antibodies.

[0169] This embodiment provides a method for preparing the PLA2R antibody of the present invention, comprising the following steps: (s1) Fluorescent labeling of PLA2R protein. Specifically, APC or PE fluorescein was conjugated to PLA2R protein to prepare PLA2R-APC and PLA2R-PE, respectively. The concentration of the conjugated protein was determined and the conjugated protein was stored in the dark.

[0170] (s2) Cell staining: Peripheral anticoagulated blood was collected from patients with primary membranous nephropathy who had not been treated with immunosuppressants and had been confirmed by biopsy. PBMCs were isolated and resuspended in FACS buffer. PBMCs were labeled with CD19 magnetic beads and all cells positive for the CD19 surface marker in the PBMCs were positively selected by magnetic beads. CD19-positive cells were then stained for CD20, CD27, human IgG, and live cell fluorescence for 30 minutes. PLA2R-APC and PLA2R-PE were then added in equal proportions and stained for 30 minutes. The cells were then washed with FACS buffer and centrifuged at 400 g for 10 minutes at 4°C. This was repeated three times.

[0171] (s3) Prepare capture cell buffer: Mix 145 μl of RNase-free water, 1.5 μl of 1 M Tris-HCl buffer (pH 8.0), and 3.62 μl of RNasin to make a total volume of 150 μl capture cell buffer. Add 10 μl of capture cell buffer to each well of a 96-well plate.

[0172] (s4) Flow cytometry sorting of PLA2R antibody secreting cells: After removing dead cells and adherent cells, the cell population with positive PLA2R-APC and PLA2R-PE double staining was sorted from the CD20+CD27+ human IgG+ cell population (e.g. Figure 1 Single cells were sorted into a 96-well plate containing capture cell buffer. After sorting, the 96-well plate was immediately sealed and placed on dry ice for quick freezing, and then stored at -80°C.

[0173] (s5) Single-cell amplification: A one-step method is used to amplify the antigen recognition region sequences of the light and heavy chains of the BCR (antibody) of a single target cell and purify them by agarose gel electrophoresis.

[0174] The results are as follows Figure 2 As shown, in the single cells corresponding to the first lane, the second lane, and the fourth lane, the heavy chain ( Figure 2 , left) and light chain ( Figure 2 , right) were successfully amplified and used to synthesize Ab11, Ab12, and Ab13 antibodies, respectively; while the single cell corresponding to the third lane successfully amplified the heavy chain, but had no light chain band.

[0175] The purified DNA fragment was subjected to TA cloning sequencing, and the sequence (amino acid sequence) after sequencing was shown in Table 1.

[0176] Table 1 Antibody sequences According to the IMGT coding rules, the complementarity determining region sequence of the heavy chain variable region of Ab11 antibody is as follows: H-CDR1:GGSIISGGSF (SEQ ID NO:3); H-CDR2: IHYSGST (SEQ ID NO:4); H-CDR3: ARDTDAFDM (SEQ ID NO: 5); and The complementarity determining region sequence of the light chain variable region of Ab11 antibody is shown below: L-CDR1: RIITSY (SEQ ID NO:6); L-CDR2: GTS (SEQ ID NO: 7); L-CDR3: QQSYSSPRT (SEQ ID NO:8).

[0177] The complementarity determining regions of the heavy chain variable region of Ab11 antibody labeled according to other coding rules are as follows: Figure 3 As shown, the complementary determining regions of the light chain variable region of Ab11 antibody marked according to other coding rules are as follows Figure 4 shown.

[0178] Finally, the sequencing results were analyzed, and the IgG4 constant region sequence (the heavy chain constant region was derived from human IgG4, and the light chain constant region was derived from human kappa chain) was integrated into the variable region sequence of Ab11 and cloned into the pcDNA3.1 plasmid. The plasmid containing the antibody light chain sequence and the heavy chain sequence were co-transfected into HEK293T cells, and the supernatant was collected, purified, and concentrated to obtain the recombinant monoclonal antibody solution.

[0179] The recombinant PLA2R monoclonal antibody was detected by WB. Figure 5 As shown, the results showed that Ab11 had complete heavy and light chains after reduction denaturation.

[0180] The heavy chain sequence of the obtained recombinant PLA2R monoclonal antibody Ab11 is: VHSQVQLLESGPGLVKSSQTLSLTCTVSGGSIISGGSFWSWIRQHPGKGLEWIGHIHYSGSTHYNPSLKSRVNISVDTSKTQFFLKLSSVTAADTAVYYCARDTDAFDMWGQGTMVSVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9, wherein the underlined sequence is the heavy chain constant region); The light chain sequence is: VHSDIQMTQSPLSLSASVGDRVTITCRASRIITSYLNWYQQRPGQAPKLLIFGTSTLQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYYCQQSYSSPRTFGQGTRVEIK RTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGE (SEQ ID NO: 10, wherein the underlined sequence is ).

[0181] The affinity of Ab11, Ab12, and Ab13 were investigated respectively, and the results were as follows: Figure 6 The results showed that the affinity of Ab11 was significantly higher than that of the other two antibodies.

[0182] EC of Ab11 50 The value is 0.68 ug / mL; the EC of Ab12 50 The value was 1.73 ug / mL; the EC of Ab13 50 The value is 4.9 ug / mL.

[0183] The antigen specificity of the recombinant PLA2R monoclonal antibody Ab11 was further characterized. Figure 7 The results showed that the Ab11 only bound to the non-reduced PLA2R antigen with the conformational epitope, but not to the reduced PLA2R antigen; the Ab11 did not recognize other PMN antigens, such as THSD7A.

[0184] Example 2: Preparation of an ELISA kit based on the recombinant PLA2R antibody Ab11.

[0185] This example provides a method for preparing an ELISA kit based on a recombinant PLA2R antibody, specifically comprising the following steps: Coating: Prepare a sterile microplate. Dilute 1 mg / mL PLA2R protein solution to 1 μg / mL with 1× PBS buffer. Add 100 μL of the solution to the microwells and incubate overnight at 4°C.

[0186] Blocking: Discard the liquid in the microwells, prepare blocking buffer (4% skim milk and 0.05% Tween-20 mixed in PBS buffer), wash the plate three times, add 300ul of blocking buffer to each microwell, and block on a shaker at room temperature for 90 minutes.

[0187] Sample dilution: Take the serum sample to be tested, mix well and dilute it 1:100 with reaction buffer.

[0188] Sample incubation: 100ul of each of the positive control reagent, negative control reagent, and diluted serum sample to be tested were added to the microplate and incubated on a shaker at room temperature for 90 minutes.

[0189] Wash the plate: discard the liquid in the microwells, add 300ul washing buffer to each well, let it stand at room temperature for 1 minute, then discard the liquid in the microwells, and repeat 3 times.

[0190] Enzyme-labeled antibody incubation: 100 μl of peroxidase-labeled anti-human IgG4 secondary antibody was added to the microplate and incubated on a shaker at room temperature for 60 minutes.

[0191] Wash the plate: discard the liquid in the microwells, add 300ul elution buffer to each well, let it stand at room temperature for 1 minute, then discard the liquid in the microwells and wash 3 times.

[0192] Substrate solution incubation: Add 100ul of substrate solution to the microplate and incubate at room temperature for 6 minutes.

[0193] Stop the reaction with stop solution: add 100ul of stop solution into the microplate.

[0194] Microplate reader reading: 450nm colorimetric, record the reading.

[0195] Result calculation: PLA2R IgG4 antibody unit value (U / mL) = 100 × (A450 待测样本 -A450 阴性对照 ) / (A450 阳性对照 -A450 阴性对照 ).

[0196] Result determination: A unit value of <17 is negative, a unit value of ≥17 is positive, and a higher unit value indicates a higher concentration of PLA2R IgG4 antibodies in the tested sample.

[0197] Quality control: Each test result must meet the OD450 阳性对照 ≥0.400, OD450 阴性对照 ≤0.100, otherwise the result is invalid.

[0198] In this example, the reaction buffer was 1×PBS containing 4% skim milk powder and 0.05% Tween 20; the wash buffer was 1×PBS containing 0.05% Tween 20; the positive control was recombinant PLA2R monoclonal antibody (Ab11) diluted in reaction buffer; the negative control was 1×PBS containing 4% skim milk powder and 0.05% Tween 20; the peroxidase-labeled anti-human IgG4 secondary antibody was a commercial antibody (Invitrogen A10654) diluted 1:5000 in reaction buffer; the substrate solution was TMB colorimetric solution; and the stop solution was TMB colorimetric stop solution.

[0199] Example 3: Detection performance of the kit of the present invention.

[0200] This example provides the sensitivity, specificity, and stability of the ELISA kit of the present invention for detecting primary membranous nephropathy.

[0201] The results are shown in Table 2.

[0202] The results showed that for primary membranous nephropathy with a titer ≥20RU / mL indicated by the Oumun test kit, the detection rate of the test kit of the present invention was 100%; for primary membranous nephropathy with a titer <20RU / mL indicated by the Oumun test kit, the Oumun test kit was judged as negative or suspected, but the detection rate of the test kit of the present invention was 91%, confirming that the test kit of the present invention has higher detection sensitivity, and there is basically no grayscale interval for detection or the grayscale interval is small.

[0203] In addition, the kit of the present invention has high specificity for primary membranous nephropathy and will not misdiagnose healthy individuals or non-primary membranous nephropathy in existing samples. However, the Omen kit has false positives in diabetic nephropathy.

[0204] Table 2 Sensitivity and specificity of the ELISA kit of the present invention The ELISA kit of the present invention was used to continuously test three serum samples with different PLA2R antibody titers, three times a day for five consecutive days. The results showed that the intra-assay CV% of the PLA2R IgG4 antibody unit value was 1.53%, and the inter-assay CV% was 12%, both within the allowable error range. The consistency of the positive results of the qualitative test was 100%, and the consistency of the negative results was 100%, indicating that the qualitative ELISA kit has stable repeatability.

[0205] In summary, the results of this example show that the kit of the present invention exhibits excellent stability and sensitivity in the detection of primary membranous nephropathy, good specificity, and an extremely low probability of false positives, which is of great significance for the early diagnosis of primary membranous diseases and the selection of accurate treatment methods.

[0206] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

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

8.

2. The antibody according to claim 1, wherein The PLA2R antibody is an IgG4 antibody.

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

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

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: Used for preparing a kit for detecting primary membranous nephropathy.

6. A kit for detecting primary membranous nephropathy, characterized in that: The kit comprises: the antibody according to claim 2, the recombinant protein according to claim 3, or the antibody conjugate according to claim 4.

7. A method for detecting anti-PLA2R autoantibodies in a sample, characterized in that: Including steps: (a) In the test group, the sample was incubated with PLA2R to observe whether a "PLA2R-anti-PLA2R autoantibody" complex was formed; In the positive control group, the positive antibody according to claim 2 is incubated with PLA2R to observe whether a "PLA2R-positive antibody" complex is formed; (b) Comparing the test results of the test group with the test results of the positive control group to determine whether anti-PLA2R autoantibodies are present in the test sample.

8. A polynucleotide, characterized in that The polynucleotide encodes the antibody according to claim 1 or the recombinant protein according to claim 3.

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

10. A host cell, characterized in that The host cell comprises the vector according to claim 9 or the polynucleotide according to claim 8 is integrated into its genome.

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