Anti-PLA2R fully human IgG monoclonal antibody and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an anti-PLA2R fully human IgG monoclonal antibody and application thereof. The invention provides a human-derived anti-alkali phospholipase A2 receptor antibody PLA2R PL82 screened by using a phage surface display technology, the subtype of the human-derived anti-alkali phospholipase A2 receptor antibody PLA2R PL82 is IgG4, and amino acid sequences of light chain and heavy chain variable regions of the human-derived anti-alkali phospholipase A2 receptor antibody PLA2R PL82 are respectively shown as SEQ ID NO: 6 and SEQ ID NO: 7. The antibody can specifically recognize an alkali phospholipase A2 receptor, the reactivity, the stability and the matrix effect all meet the performance use requirements of a kit, and the antibody plays an important role in alkali phospholipase A2 IgG project application as a quality control product.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the preparation and application of anti-PLA2R fully human IgG monoclonal antibodies. Background Art

[0002] Membranous nephropathy (MN), also known as membranous glomerulonephritis (MG), can be divided into primary membranous glomerulonephritis (PMN), also known as idiopathic membranous nephropathy (IMN), and secondary membranous glomerulonephritis (SMN).

[0003] PMN / IMN is treated as a separate disease, particularly when associated with nephrotic syndrome and hyperosmolar PMN. Currently, PMN / IMN is the only type of kidney disease that can be diagnosed by blood draw. It may be an autoimmune disease involving target antigens on glomerular epithelial cells (such as PLA2R, TSHD7A, and NELL1). Approximately one-third of patients experience spontaneous remission, while 20%-40% progress to end-stage renal failure. The remainder present with persistent proteinuria and stable renal function. Membranous nephropathy peaks in the 30s to 50s; it is rare in children (less than 5%), with a male-to-female prevalence of 2:1. Clinically, 60%-80% present with nephrotic syndrome (NS), while the remainder present with asymptomatic proteinuria (24-hour urine protein greater than 3.5 g and serum albumin less than 30 g / L).

[0004] PLA2R belongs to the mannose receptor family and is a type I transmembrane protein with a relative molecular mass of approximately 180–186 kDa. It consists of extracellular, transmembrane, and intracellular segments. It is primarily expressed in the cytoplasm and membrane of normal glomerular podocytes and is also expressed in human lung and placenta. In 2009, it was discovered to be the primary target antigen for IMN. Anti-PLA2R antibodies have a specificity of 99% and a sensitivity of 78% for diagnosing membranous nephropathy. Anti-PLA2R antibodies are detected in serum and immune complexes in 70% of adult IMN patients. THSD7A is positive in 5%–10% of anti-PLA2R antibody-negative patients; the two rarely coexist. Anti-PLA2R antibodies are predominantly of the IgG4 subtype, circulating antibodies targeting discontinuous epitopes.

[0005] In most patients, anti-PLA2R antibodies precede clinical disease activity and relapse, making them useful for diagnosing IMN, monitoring treatment response, and prognosticating disease. The temporal course of antibody titers can predict response to treatment and, consequently, the risk of worsening renal function. Therefore, anti-PLA2R antibody testing holds significant clinical significance. However, in vitro diagnostics (IVDs) primarily utilize plasma from positive patients, which presents numerous challenges, such as difficulty collecting and scaling up. The development of fully human anti-PLA2R IgG monoclonal antibodies through in vitro methods is urgently needed for use as quality controls and calibrators for use in test kits. Summary of the Invention

[0006] In view of this, the present invention provides a method for preparing a fully human anti-PLA2R IgG monoclonal antibody. Using this recombinant monoclonal antibody as a quality control and calibrator, the PLA2R antigen undergoes a significant enzyme-linked immunosorbent assay (ELISA) reaction, and the reactivity, stability, and matrix effect all meet the performance requirements of the kit. The preparation method is simple and can be mass-produced.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides an antibody, whose heavy chain CDR1 has:

[0009] (1) the amino acid sequence shown in SEQ ID NO: 1; or

[0010] (2) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or

[0011] (3) an amino acid sequence that is at least 80%, 83%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 98% or 99% homologous to the amino acid sequence of (1) or (2);

[0012] Its heavy chain CDR2 has:

[0013] (4) the amino acid sequence shown in SEQ ID NO: 2; or

[0014] (5) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (4), and having the same or similar function as (4); or

[0015] (6) an amino acid sequence that is at least 80%, 83%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 98% or 99% homologous to the amino acid sequence shown in (4) or (5);

[0016] Its heavy chain CDR3 has:

[0017] (7) the amino acid sequence shown in SEQ ID NO: 3; or

[0018] (8) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (7), and having the same or similar function as (7); or

[0019] (9) an amino acid sequence that is at least 80%, 83%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 98% or 99% homologous to the amino acid sequence of (7) or (8);

[0020] Its light chain CDR1 has:

[0021] (10), the amino acid sequence shown in SEQ ID NO: 4; or

[0022] (11) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (10), and having the same or similar function as (10); or

[0023] (12) an amino acid sequence that is at least 80%, 83%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 98% or 99% homologous to the amino acid sequence of (10) or (11);

[0024] Its light chain CDR2 has:

[0025] (13), amino acid sequence EDH; or

[0026] (14) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (13), and having the same or similar function as (13); or

[0027] (15) an amino acid sequence that is at least 80%, 83%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 98% or 99% homologous to the amino acid sequence shown in (13) or (14);

[0028] Its light chain CDR3 has:

[0029] (16), the amino acid sequence shown in SEQ ID NO: 5; or

[0030] (17) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (16), and having the same or similar function as (16); or

[0031] (18) An amino acid sequence that is at least 80%, 83%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 98% or 99% homologous to the amino acid sequence shown in (16) or (17).

[0032] In some specific embodiments of the present invention, the heavy chain variable region of the above-mentioned antibody has:

[0033] (19), the amino acid sequence shown in SEQ ID NO: 6; or

[0034] (20) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (19), and having the same or similar function as (19); or

[0035] (21) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (19) or (20);

[0036] Its light chain variable region has:

[0037] (22), the amino acid sequence shown in SEQ ID NO: 7; or

[0038] (23) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (22), and having the same or similar function as (22); or

[0039] (24) An amino acid sequence that is at least 80%, 83%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 98% or 99% homologous to the amino acid sequence shown in (22) or (23).

[0040] In some specific embodiments of the present invention, the heavy chain constant region of the above-mentioned antibody is human IgG4, and the light chain constant region is Lambda type.

[0041] The present invention also provides the use of the above-mentioned antibody in any of the following items:

[0042] (i) Preparation of quality control products, calibrators or positive sample analogs for diagnostic reagents;

[0043] (ii) preparing a product for detecting anti-PLA2R antibodies;

[0044] (iii) preparing a product for preventing, diagnosing or treating membranous nephropathy.

[0045] In some specific embodiments of the present invention, the diagnostic reagent used above is a reagent for diagnosing membranous nephropathy.

[0046] In some specific embodiments of the present invention, the membranous nephropathy in the above application may be primary membranous glomerulonephritis or secondary membranous glomerulonephritis.

[0047] The present invention also provides a nucleic acid molecule encoding the above antibody.

[0048] The present invention also provides an expression vector containing the above nucleic acid molecule.

[0049] The present invention also provides a host containing the above nucleic acid molecule or the above expression vector.

[0050] The present invention also provides a method for preparing the above-mentioned antibody, comprising:

[0051] (a) constructing an expression vector containing the above-mentioned nucleic acid molecule, transforming the expression vector into a host, culturing the host, and isolating and purifying the obtained culture to obtain the antibody; or

[0052] (b) transforming the above expression vector into a host, culturing the host, and isolating and purifying the obtained culture to obtain the antibody; or

[0053] (c) culturing the host and isolating and purifying the obtained culture to obtain the antibody.

[0054] The present invention also provides a quality control product, a calibrator or a positive sample analog, comprising the above-mentioned antibody, and an acceptable excipient or auxiliary agent.

[0055] The present invention also provides a detection product, comprising the above-mentioned antibody and acceptable adjuvants or auxiliary agents.

[0056] The present invention also provides a diagnostic method, comprising performing diagnosis based on the above-mentioned antibody or the above-mentioned detection product.

[0057] The present invention also provides a method for prevention or treatment, comprising prevention or treatment based on the above-mentioned antibody or the above-mentioned detection product.

[0058] The present invention has the following beneficial effects:

[0059] The anti-PLA2R fully human IgG monoclonal antibody provided by the present invention has a purity of >90%, a transient expression level of 100 mg / L, and exists in monomeric form. After identification, this monoclonal antibody can be used as a positive quality control product. Its potency, stability, and matrix effect all meet the requirements of use, providing a reliable basis for clinical diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0061] Figure 1: shows an SDS-PAGE image, wherein "reduced" represents a reduced band (treated with reducing agent / DTT), and "non-reduced" represents a non-reduced band (not treated with reducing agent / DTT);

[0062] Figure 2 Figure 2 shows the HPLC analysis graph, in which the blue peak is the marker, and the sizes from left to right are 1340 kDa, 670 kDa, 300 kDa, 150 kDa, 45 kDa, 17 kDa, and 1 kDa, the green peak is the target protein PL82 IgG4 antibody peak, and the red peak is the target protein PL61 IgG4 antibody peak. DETAILED DESCRIPTION

[0063] The present invention discloses methods for preparing fully human anti-PLA2R IgG monoclonal antibodies and their applications. Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications obvious to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art can modify or appropriately alter and combine the methods and applications described herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0064] The present invention provides an anti-PLA2R fully human IgG monoclonal antibody, the amino acid sequence of the heavy chain variable region of which is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of which is shown in SEQ ID NO: 7.

[0065] The anti-PLA2R fully human IgG monoclonal antibody of the present invention further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is of human IgG4 subtype, and the light chain constant region is of Lambda type.

[0066] The present invention also provides a DNA molecule encoding the anti-FluB fully human monoclonal antibody. The present invention also provides an expression vector containing a DNA molecule encoding the anti-PLA2R fully human IgG monoclonal antibody.

[0067] In some specific embodiments, the backbone vector of the expression vector of the present invention is pCMV3.

[0068] The present invention also provides a recombinant host containing the expression vector.

[0069] In some specific embodiments, the host cell is a HEK 293F cell.

[0070] The present invention also provides a method for preparing an anti-PLA2R fully human IgG monoclonal antibody, comprising:

[0071] (1) Constructing an expression vector containing the DNA molecule according to claim 3 or 4;

[0072] (2) Transforming the expression vector described in step (1) into a host cell;

[0073] (3) culturing the host cells obtained in step (2);

[0074] (4) Isolate and purify to obtain the monoclonal antibody.

[0075] The present invention also provides the use of the anti-PLA2R fully human IgG monoclonal antibody in in vitro diagnostic reagents.

[0076] The sequence information involved in the present invention is as follows:

[0077] PL82 heavy chain CDR amino acid sequence:

[0078] GFIFSSYA (SEQ ID NO: 1).......___ISDDSGK (SEQ ID NO: 2).......ARDNGYSYAYDVFGV............. (SEQ ID NO: 3);

[0079] PL82 light chain CDR amino acid sequence:

[0080] SGSIATNY (SEQ ID NO: 4).............___EDH...HSYADGDYV (SEQ ID NO: 5).....................;

[0081] PL82 heavy chain variable region amino acid sequence:

[0082] QVQLQQSGGGLVQPGKSLRLSCAASGFIFSSYAMHWVRQAPGKGLEWVAAISDDGSGKFYADFVKGRFATSRDNSKNTLYLQMNSLRAEDTAIYYCARDNGYSYAYDVFGVWGQGTMVTVSS (SEQ ID NO: 6);

[0083] PL82 light chain variable region amino acid sequence:

[0084] NFMLTQPHSVSGSPGETVTISCTASSGSIATNYVQWFQQRPGSAPTPMIFEDHKRASGVPDRFSASIDYPSNSATFTISGLQTEDEAAYFCHSYADGDYVFGTGTKVTVL (SEQ ID NO: 7);

[0085] PL61 heavy chain CDR amino acid sequence:

[0086] GYFFPSYA (SEQ ID NO: 8).......___VKGDKDDT (SEQ ID NO: 9).......AREDSSGWYVGSFFDS............ (SEQ ID NO: 10);

[0087] PL61 light chain CDR amino acid sequence:

[0088] SSNIRVNT (SEQ ID NO: 11).............___NDN...ASWDDSLNGWV (SEQ ID NO: 12).............;

[0089] PL61 heavy chain variable region amino acid sequence:

[0090] QVQLVQSGAEVKKPGASVKVSCKASGYFFPSYALHWVRQAPGQGLEWMGMVKGDKDDTKYSQKFQGRVTISRDTSANTGYMELSGLRAEDTAVYYCAREDSSGWYVGSFFDSWGQGTLVTVSS (SEQ ID NO: 13);

[0091] PL61 light chain variable region amino acid sequence:

[0092] NFLMLTHPPSASGTPGQRVTISSCSGSSSNIRVNTVNWYQHLPGTAPKLLIYNDNERPSGVPDRVSGSKSGTSGSLAISGLQSEDEADYYCASWDDSLNGWVFGGGTKLTVL (SEQ ID NO: 14).

[0093] It should be understood that the expression "one or more of" includes individually each of the items recited after the expression and various combinations of two or more of the recited items, unless otherwise apparent from the context and usage. The expression "and / or" in conjunction with three or more recited items should be understood to have the same meaning, unless otherwise apparent from the context.

[0094] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0095] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application remains operable. Additionally, two or more steps or actions may be performed simultaneously.

[0096] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and does not limit the scope of the present application. No language in this specification should be construed as indicating any non-claimed element is essential to the practice of the present application.

[0097] In addition, the numerical ranges and parameters used to define this application are approximate values. The relevant numerical values in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, amounts, values, and percentages used in this disclosure are modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0098] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0099] The present invention will be further described below with reference to the embodiments.

[0100] Example 1: Construction of a human anti-PLA2R phage antibody library

[0101] (1) Materials

[0102] Sources of antigens, vectors, and cells: PLA2R recombinant antigens were purchased from Beijing Yuanye Biotechnology; the pComb3XSS phage display plasmid was from Fenghui Biotechnology, the helper phage M13KO7 was purchased from NEB, and the HEK 293 F cells used for transient expression were purchased from Beijing Sino-Bio.

[0103] (2) Primer synthesis and gene acquisition

[0104] Lymphocytes were isolated from anticoagulated blood of respiratory patients using human peripheral blood lymphocyte separation medium (Solarbio, Beijing). Total cellular RNA was extracted using an RNA extraction kit (Shengang, Shanghai). The extracted RNA was used as a template using the first-strand synthesis kit (ProtoScript PCR) from NEB using oligo-dT primers. ® cDNA was generated by reverse transcription using the First Strand cDNA Synthesis Kit (Cat No. E6300S), and used as template to amplify the scFv heavy and light chain gene fragments, respectively. The primer sequences used are as follows.

[0105] Heavy chain upstream primer:

[0106] VHF1: CAGGTBCAGCTGGTRCAGTC (SEQ ID NO: 15);

[0107] VHF2: CAGGTCAACTTAAGGGAGTCTGG (SEQ ID NO: 16);

[0108] VHF3: CAGGGTGCAGCTGGTGGAGTCTGG (SEQ ID NO: 17);

[0109] VHF4: CAGGGTGCAGCTGCAGGAGTCGGG (SEQ ID NO: 18);

[0110] VHF5: CAGGTGCAGCTGTTGCAGTCTGC (SEQ ID NO: 19);

[0111] VHF6: CAGGTACAGCTGCAGCAGTCAGG (SEQ ID NO: 20);

[0112] VHF7: CAGGTCCAGCTKGTGCARTC (SEQ ID NO: 21);

[0113] VHF8: CAGGTCCAGCTGGTGCAGTC (SEQ ID NO: 22);

[0114] VHF9: CAGGTACAGCTGGTGGAGTC (SEQ ID NO: 23);

[0115] Heavy chain downstream primer:

[0116] VHR1: GCCAGAACCACCTCCCGCTGA (SEQ ID NO: 24);

[0117] VHR2:GCCAGAACCACCTCCGCCTGA(SEQ ID NO: 24)

[0118] VHR3:GCCAGAACCACCTCCGCCTGA(SEQ ID NO: 24)

[0119] VHR4:GCCAGAACCACCTCCGCCTGAA(SEQ ID NO: 25)

[0120] Enter the lambda system:

[0121] λ1F:CAGTCTGTGTTGACGCAGCCGCC(SEQ ID NO: 26)

[0122] λ2F:CAGTCTGCCCTGACTCAGCCTGC(SEQ ID NO: 27)

[0123] λ3F:TCCTATGTGCTGACTCAGCCACC(SEQ ID NO: 28)

[0124] λ4F:TCTTCTGAGCTGACTCAGGACCC(SEQ ID NO: 29)

[0125] λ5F:CACGTTATACTGACTCAACCGCC(SEQ ID NO: 30)

[0126] λ6F:CAGGCTGTGCTCACTCAGCCGTC(SEQ ID NO: 31)

[0127] λ7F:AATTTTATGCTGACTCAGCCCCA(SEQ ID NO: 32)

[0128] Enter the lambda cover:

[0129] λ1R:TAGGACGGTGACCTTGGTCCC(SEQ ID NO: 33)

[0130] λ2R:TAGGACGGTCAGCTTGGTCCC(SEQ ID NO: 34)

[0131] λ3R:TAAAACGGTGAGCTGGGTCCC(SEQ ID NO: 35).

[0132] PCR reaction conditions were: 95°C pre-denaturation for 5 minutes; 30 cycles of 95°C denaturation for 30 seconds, 56°C annealing for 30 seconds, and 72°C extension for 30 seconds; followed by an additional extension at 72°C for 10 minutes. The light and heavy chain variable regions, purified from gels, were ligated to T-vectors and sent for testing to obtain the light and heavy chain variable region sequences. Subsequently, the two regions were spliced into the scFv gene fragment using OverLap PCR using the SfiI restriction site.

[0133] (3) Construction of ScFV phage library

[0134] The scFv gene fragment was ligated to the phage vector pComb3XSS via the SfiI restriction site. The ligation product was electroporated into E. coli TG1 competent cells and incubated with shaking at 37°C for 1 hour in 2YT-AG medium (containing 2% glucose and 100 μg / mL Amp). 10 μL of the bacterial suspension was inoculated into 2YT-AG medium and cultured overnight. Phage colony formation was observed, and the antibody library titer was calculated. 10–20 positive colonies were randomly selected and sent to Sangon Technology Co., Ltd. for sequencing and identification by colony PCR to analyze the diversity of the antibody library. Helper phage-M13K07 was added to the remaining bacterial liquid, and the cells were incubated at 37°C for 30 min. The cells were collected by centrifugation and resuspended in 100 mL of 2YT medium (containing 1 μmol / L IPTG and 100 μg / mL Amp). The cells were cultured on a shaking platform at 30°C overnight. The supernatant was collected by centrifugation, and one-quarter volume of 20% PEG / 2.5 mol / L NaCl was added. The cells were incubated on ice for 50 min to precipitate the phages. The precipitate was collected by centrifugation and resuspended in PBS to obtain the ScFV phage library.

[0135] Example 2: Enrichment and screening of human anti-PLA2R phage antibody library

[0136] Immunotubes were coated with PLA2R antigen at a concentration of 20 μg / mL and incubated overnight at 4°C. The tubes were then blocked with 5% skim milk at 37°C for 2 h. After washing twice with PBST, 1 mL of the ScFV phage library prepared in Example 1 was added. The tubes were incubated with shaking at room temperature for 2 h, washed 10 times with PBST, and 1 mL of triethylamine dissociation buffer (100 mmol / L) was added. The tubes were incubated at room temperature for 10 min, neutralized with 1 mol / L Tris-HCl (pH 6.4), and 50 mL of TG1 bacteria in the logarithmic growth phase was added. After incubation at 37°C for 30 min, 10 μL of the solution was plated on 2YT / GA plates and incubated overnight at 37°C for assay yield. The remaining culture was expanded until the A600 of the solution was approximately 0.5. Helper phage M13K07 was added. The subsequent process was consistent with Example 1. Four rounds of screening were repeated to enrich for phages that bound to the PLA2R antigen.

[0137] Example 3: Induced expression of positive phage clones

[0138] Single colonies after four rounds of enrichment were randomly selected and plated in 96-well plates (a total of six plates). 400 μL of 2YT / GA medium (2% glucose, 50 μg / mL Amp) was added to each well and cultured overnight at 37°C. The next day, 5 μL of the overnight culture was transferred to a 96-well plate containing 100 μL of culture medium for activation. The cells were incubated at 37°C for 2–3 hours. When the OD value was approximately 2, 20 μL of a mixture of 2YT and helper phage (MOI = 5) was added to each well and incubated at 37°C for 30 minutes. After incubation on a shaker for 1.5 hours, 300 μL of 2YT-AK (100 μg / mL Amp, 50 μg / mL Kan) was added to each well and cultured overnight for 16–18 hours. The next day, the supernatant was centrifuged and collected for analysis.

[0139] Example 4: Phage monoclonal detection

[0140] PLA2R antigen was coated onto an ELISA plate at 0.5 μg / mL overnight at 4°C. The plate was blocked with 4% skim milk for 1 hour at 37°C. A phage monoclonal clone was added and incubated at 37°C for 30 minutes. HRP-M13 (1 / 5K dilution) was added and incubated at 37°C for 30 minutes. The plate was developed with a colorimetric solution, and the reaction was terminated with 2 mol / L H2SO4. The absorbance was measured at 450 nm using a microplate reader. Two highly reactive clones were sent for sequencing.

[0141] Example 5: Nucleic acid sequence analysis of variable region genes and construction of full-length IgG antibodies

[0142] Antibody 1-PL82: Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 6; its light chain variable region has the amino acid sequence shown in SEQ ID NO: 7.

[0143] Antibody 2-PL61: its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 13; its light chain variable region has the amino acid sequence shown in SEQ ID NO: 14.

[0144] Using two selected positive monoclonal clones as templates, the variable region gene sequences were amplified by PCR and then constructed into the laboratory vectors IgG-PCMV3 and LC-PCMV3 (containing the constant region sequences) using HindIII / XbaI. The recombinant plasmids were transformed into competent DH5α cells, and positive clones were selected for sequencing and plasmid extraction.

[0145] Example 6: Transient expression of full-length IgG antibody

[0146] One day before transfection, adjust the density of HEK 293 F cells to 2 × 10 6 On the day of transfection, cells were diluted to 3×10 cells / mL using HEK 293F cell culture medium. 6 The extracted plasmids were diluted with lipofectamine according to the instructions for Sino-Bio transfection reagent and transfected into cells at a ratio of 1.5 μg:1 mL. PL82 and PL61 antibody plasmids contain light and heavy chain plasmids in a 1:1 ratio of HC:LC. After 24 h, 2.5% feed was added and the cells were cultured until cell viability dropped to approximately 70%-75%, and the supernatant was harvested.

[0147] Example 7: Purification of human anti-PLA2R full-length IgG antibody

[0148] The supernatant of PL82 and PL61 antibodies was filtered through a 0.45 μm filter membrane. Ultrapure water and buffer (0.02 mol / L PBS, pH 7.4) were used to equilibrate a chromatography column filled with Protein A. The filtered antibody supernatant was loaded and equilibrated again. Finally, the target protein antibody was eluted with dissociation buffer (0.2 mol / L Gly + 1.5 mol / L NaCl, pH 2.7). The dissociation peak was collected and the protein content was detected using a NanoDrop One spectrophotometer. The protein expression level was detected by SDS-PAGE and SEC-HPLC.

[0149] The results showed that after purification, the expression levels of PL82 and PL61 antibodies were 100 mg / L and 94 mg / L, respectively. The protein concentration was adjusted to 5 mg / mL using a spectrophotometer NanoDrop One. SDS-PAGE ( Figure 1 ) showed that the molecular weight of the heavy chain was about 50 kD, and the molecular weight of the light chain was about 23 kD, which was consistent with expectations; HPLC detection ( Figure 2 ) showed that the green peak was PL82IgG4, which existed mainly in monomeric form (around 150 KD) with a small amount of aggregate peaks. The antibody purity reached more than 98%. PL61IgG4 existed in monomeric form, with an antibody peak time later than PL82 and a smaller molecular weight than PL82.

[0150] Example 8: Titer Detection of Human Anti-PLA2R Full-Length IgG Antibody

[0151] The PL82 and PL61 IgG4 antibodies were diluted 50 times, 100 times, 500 times, 1000 times and 2000 times, and the titer was detected using the Antu anti-alkaline phospholipase A2 antibody IgG kit according to the product instructions. The test results of the prepared PL82 and PL61 human antibodies are shown in Table 1. The average detection concentrations of PL82 IgG4 and PL61 IgG4 were 18.7×10 4 AU / mL and 10.9×10 4 AU / mL, both meet the reactivity requirements for positive quality control products, but the reactivity of PL82 antibody is 70% higher than that of PL61 antibody, so it is preferred.

[0152] Table 1: PL82 IgG4 and PL61 IgG4 antibody reactivity test results

[0153]

[0154] Example 9: Stability Evaluation of Human Anti-PLA2R Full-Length IgG Antibody

[0155] The antibody titers (AU / mL) of PL82 IgG4 and PL61 IgG4 were adjusted to low and high levels (target values were 15±5 AU / mL and 95±5 AU / mL, respectively). After lyophilization, the titer fluctuations after reconstitution were assessed by heat-accelerated assays at 37°C for 7 and 14 days. The stability data are shown in Table 2. The results showed that the activity values of PL82 IgG4 and PL61 IgG4 were within ±5% after heat-accelerated assays at 37°C for 7 and 14 days, meeting the requirements for use as quality control and calibrators.

[0156] Table 2: Stability evaluation results of PL82 IgG4 and PL61 IgG4 antibodies

[0157]

[0158] Example 10: Evaluation of Matrix Effects of Human Anti-PLA2R Full-Length IgG Antibody

[0159] The reagents in the Antu Anti-Alkaline Phospholipase A2 Antibody IgG Kit were heat-accelerated at 37°C for 10 days before use in antibody and clinical sample evaluation. PL82 IgG4 and PL61 IgG4 antibodies were formulated into samples at varying concentrations. Samples formulated with recombinant antibodies PL82 and PL61, as well as random clinical samples, were tested using the Antu Anti-Alkaline Phospholipase A2 Antibody IgG Kit after 10 days of heat acceleration and the Antu Anti-Alkaline Phospholipase A2 Antibody IgG Kit stored at 4°C. The luminescence value variations of the samples tested using the 4°C and 37°C heat-accelerated kits were compared. The results are shown in Table 3.

[0160] The data showed that the luminescence value of the PL61 IgG4 antibody detected on the heat acceleration kit increased by about 50% compared with 4 degrees, which was inconsistent with the variation range of clinical samples and showed a matrix effect; while the luminescence value of the PL82 IgG4 antibody detected on the heat acceleration kit decreased by 10%~20% compared with 4 degrees, which was consistent with the variation range of clinical samples and showed no matrix effect. The performance requirements of quality control products and calibrators were met, and the antibody PL82 IgG4 was preferred.

[0161] Table 3: Matrix effect evaluation results of PL82 IgG4 and PL61 IgG4 antibodies

[0162]

[0163] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An antibody, characterized in that: Its heavy chain CDR1 has: (1) the amino acid sequence shown in SEQ ID NO: 1; or (2) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or (3) an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (1) or (2); Its heavy chain CDR2 has: (4) the amino acid sequence shown in SEQ ID NO: 2; or (5) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (4), and having the same or similar function as (4); or (6) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (4) or (5); Its heavy chain CDR3 has: (7) the amino acid sequence shown in SEQ ID NO: 3; or (8) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (7), and having the same or similar function as (7); or (9) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (7) or (8); Its light chain CDR1 has: (10), the amino acid sequence shown in SEQ ID NO: 4; or (11) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (10), and having the same or similar function as (10); or (12) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (10) or (11); Its light chain CDR2 has: (13), amino acid sequence EDH; or (14) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (13), and having the same or similar function as (13); or (15) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (13) or (14); Its light chain CDR3 has: (16), the amino acid sequence shown in SEQ ID NO: 5; or (17) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (16), and having the same or similar function as (16); or (18) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (16) or (17).

2. The antibody according to claim 1, wherein: Its heavy chain variable region has: (19), the amino acid sequence shown in SEQ ID NO: 6; or (20) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (19), and having the same or similar function as (19); or (21) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (19) or (20); Its light chain variable region has: (22), the amino acid sequence shown in SEQ ID NO: 7; or (23) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (22), and having the same or similar function as (22); or (24) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (22) or (23).

3. The antibody according to claim 1 or 2, wherein Its heavy chain constant region is human IgG4, and its light chain constant region is Lambda type.

4. Use of the antibody according to any one of claims 1 to 3 in any of the following: (i) Preparation of quality control products, calibrators or positive sample analogs for diagnostic reagents; (ii) preparing a product for detecting anti-PLA2R antibodies; (iii) preparing a product for preventing, diagnosing or treating membranous nephropathy.

5. A nucleic acid molecule encoding the antibody according to any one of claims 1 to 3.

6. An expression vector, characterized in that Containing the nucleic acid molecule according to claim 5.

7. A host, excluding totipotent animal cells or plant cells, characterized in that: Containing the nucleic acid molecule according to claim 5 or the expression vector according to claim 6.

8. The method for preparing an antibody according to any one of claims 1 to 3, wherein: include: (a) constructing an expression vector containing the nucleic acid molecule of claim 5, transforming the expression vector into a host, culturing the host, and isolating and purifying the obtained culture to obtain the antibody; or (b) transforming the expression vector according to claim 6 into a host, culturing the host, and isolating and purifying the obtained culture to obtain the antibody; or (c) culturing the host according to claim 7, and isolating and purifying the obtained culture to obtain the antibody.

9. A quality control product, calibrator or positive sample analog, characterized in that: The invention comprises the antibody according to any one of claims 1 to 3, and an acceptable adjuvant or auxiliary agent.

10. The detection product is characterized in that, The invention comprises the antibody according to any one of claims 1 to 3, and an acceptable adjuvant or auxiliary agent.

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