Anti-pla2r fully human igg monoclonal antibody and application thereof

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the preparation and application of anti-PLA2R fully human IgG monoclonal antibody. Background Technology

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

[0003] PMN / IMN is treated as an independent disease, especially PMN accompanied by nephrotic syndrome and hyperosmolarity. PMN / IMN is currently the only type of kidney disease that can be diagnosed by blood tests. It may be an autoimmune disease with target antigens on glomerular epithelial cells (PLA2R, TSHD7A, NELL1, etc.). About one-third of patients experience spontaneous remission, 20% to 40% of patients progress to end-stage renal failure, and the remaining patients present with persistent proteinuria and stable renal function. The peak age of onset for membranous nephropathy is 30 to 50 years; it is rare in children (incidence less than 5%), and the male-to-female ratio is 2:1. Its clinical characteristics are that 60% to 80% of cases present with nephrotic syndrome (NS), while the rest mainly present with asymptomatic proteinuria (24-hour urine protein quantification greater than 3.5 g, 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 includes extracellular, transmembrane, and intracellular segments and is mainly expressed in the cytoplasm and membrane of normal glomerular podocytes. It is also expressed in human lungs and the placenta. In 2009, it was discovered to be a major target antigen of membranous nephropathy (IMN). Anti-PLA2R antibodies have a specificity of up to 99% and a sensitivity of 78% in diagnosing membranous nephropathy. Anti-PLA2R antibodies are detected in the serum of 70% of adult IMN patients in neutralizing immune complexes. 5%-10% of patients who are negative for anti-PLA2R antibodies are positive for THSD7A. The two types of antibodies generally do not coexist. Anti-PLA2R antibodies are predominantly of the IgG4 subtype and are circulating antibodies with discontinuous epitopes.

[0005] In most patients, anti-PLA2R antibodies precede clinical disease activity and relapse, and can be used to diagnose IMN, monitor treatment response, and predict disease progression. The time progression of antibody titers can predict treatment response, thereby predicting the risk of renal function deterioration. Therefore, anti-PLA2R antibody detection has significant clinical implications. However, in the field of in vitro diagnostics, plasma from positive patients is mostly used, which presents numerous problems such as difficulty in collection and amplification. Therefore, the preparation of fully human anti-PLA2R IgG monoclonal antibodies in vitro as quality control and calibrators for reagent kit detection is urgently needed. Summary of the Invention

[0006] In view of this, the present invention provides a method for preparing a fully human IgG monoclonal antibody against PLA2R. Using this recombinant monoclonal antibody as a quality control and calibrator, the PLA2R antigen exhibits a significant enzyme-linked immunosorbent assay (ELISA), 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] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides an antibody whose heavy chain CDR1 has:

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

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

[0012] Its heavy chain CDR2 has:

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

[0014] (5) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (4), and whose function is the same as or similar to that of (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) An amino acid sequence as shown in SEQ ID NO: 3; or

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

[0020] Its light chain CDR1 has:

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

[0022] (11) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (10), and whose function is the same as or similar to that of (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 shown in (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 as shown in (13), and whose function is the same as or similar to that of (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) An amino acid sequence as shown in SEQ ID NO: 5; or

[0030] (17) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (16), and whose function is the same as or similar to that of (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) An amino acid sequence as shown in SEQ ID NO: 6; or

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

[0035] (21) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (19) or (20);

[0036] Its light chain variable region has:

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

[0038] (23) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (22), and whose function is the same as or similar to that of (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 antibody is human IgG4, and the light chain constant region is Lambda type.

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

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

[0043] (ii) Prepare products for detecting anti-PLA2R antibodies;

[0044] (iii) Prepare products for the prevention, diagnosis or treatment of membranous nephropathy.

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

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

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

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

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

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

[0051] (a) Construct an expression vector containing the above-mentioned nucleic acid molecules, transform the expression vector into a host, culture the host, separate and purify the resulting culture to obtain the antibody; or

[0052] (b) Transform the above expression vector into a host, culture the host, and isolate and purify the resulting culture to obtain the antibody; or

[0053] (c) Culturing the above-mentioned host, separating and purifying the obtained culture to obtain the antibody.

[0054] The present invention also provides quality control samples, calibrators or positive sample analogs, including the antibodies described above, and acceptable excipients or adjuvants.

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

[0056] The present invention also provides a diagnostic method, including diagnosis based on the above-described antibodies or the above-described detection products.

[0057] The present invention also provides methods for prevention or treatment, including prevention or treatment based on the above-mentioned antibodies or the above-mentioned detection products.

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

[0059] The anti-PLA2R fully human IgG monoclonal antibody provided by this invention has a purity >90%, a transient expression level of 100 mg / L, and exists in monomeric form. It has been identified that this monoclonal antibody can be used as a positive control. Its potency, stability, and matrix effect all meet the requirements for use, providing a reliable basis for clinical diagnosis and treatment. Attached Figure Description

[0060] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0061] Figure 1The SDS-PAGE diagram shows that "reduced" represents the reduced band (treated with reducing agent / DTT) and "non-reduced" represents the non-reduced band (not treated with reducing agent / DTT).

[0062] Figure 2 The HPLC analysis chromatogram shows that the blue peaks are markers, and their 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 Implementation

[0063] This invention discloses a method for preparing a fully human IgG monoclonal antibody against PLA2R and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0064] This invention provides a fully human IgG monoclonal antibody against PLA2R, the amino acid sequence of which is shown in SEQ ID NO: 6 and the amino acid sequence of which is shown in SEQ ID NO: 7.

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

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

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

[0070] This invention also provides a method for preparing a fully human IgG monoclonal antibody against PLA2R, comprising:

[0071] (1) Construct an expression vector containing the DNA molecule as described in claim 3 or 4;

[0072] (2) Transform the expression vector described in step (1) into host cells;

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

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

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

[0076] The sequence information involved in this 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…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0094] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0095] It should be understood that the order of steps or the sequence of actions is not important as long as this application remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0096] The use of any and all instances or exemplary language such as “e.g.” or “include” in this document is intended merely to better illustrate the application and does not constitute a limitation on the scope of the application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of this application.

[0097] Furthermore, the numerical ranges and parameters used to define this application are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

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

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

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

[0101] (1) Materials

[0102] Antigen, vector, and cell sources: PLA2R recombinant antigen was purchased from Beijing Yuanye Biotechnology; pComb3XSS phage display plasmid was obtained from Fenghui Biotechnology; helper phage M13KO7 was purchased from NEB; and HEK 293 F cells used for transient expression were purchased from Beijing Yiqiao Company.

[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 (Sangon Biotech, Shanghai). Using Oligo-dT primers, the extracted RNA was used as a template for first-strand synthesis using NEB's ProtoScript kit. ® The First Strand cDNA Synthesis Kit (Cat No. E6300S) was used to reverse transcribe cDNA, which was then used as a template to amplify the scFv heavy and light chain gene fragments. 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] The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 30 cycles; and a final extension at 72℃ for 10 min. The light and heavy chain variable regions, purified by gel excision, were ligated into T vectors and sent for assay to obtain the light and heavy chain variable region sequences. Then, using the SfiI restriction site, the two sequences were spliced ​​into the scFv gene fragment using OverLap PCR technology.

[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 2YT-AG medium (containing 2% glucose, 100 μg / mL ampicillin) was added and cultured at 37°C with shaking for 1 h. 10 µL of the bacterial culture was then inoculated into 2YT-AG medium and cultured overnight. Phage colony formation was observed, and antibody library titers were calculated. 10–20 positive colonies were randomly selected and sent to Sangon Biotech for sequencing and colony PCR identification to analyze antibody library diversity. The remaining bacterial culture was added to helper phage-M13K07, incubated at 37°C for 30 min, and then centrifuged to collect the bacterial cells. The cells were resuspended in 100 mL of 2YT medium (containing 1 μmol / L IPTG and 100 μg / mL Amp) and cultured overnight at 30°C in a shaker. The supernatant was collected by centrifugation, and a quarter volume of 20% PEG / 2.5 mol / L NaCl was added. The cells were incubated on ice for 50 min to precipitate, and the precipitate was collected by centrifugation. The precipitate was resuspended in PBS to obtain the ScFV phage library.

[0135] Example 2: Enrichment and Screening of Human Anti-PLA2R Phage Antibody Library

[0136] The PLA2R antigen was coated onto immunotubes 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, followed by washing 10 times with PBST. 1 mL of triethylamine dissociation buffer (100 mmol / L) was added, and the tubes were incubated at room temperature for 10 min. The solution was neutralized with 1 mol / L Tris-HCl (pH=6.4), and 50 mL of TG1 bacterial culture in logarithmic growth phase was added. After incubation at 37°C for 30 min, 10 μL of the culture was plated onto 2YT / GA plates and incubated overnight at 37°C. The yield was then measured. The remaining bacterial culture was expanded until the A600 of the culture reached approximately 0.5. Helper phage M13K07 was then added, and the subsequent process was the same as in Example 1. This screening process was repeated four times to enrich phages that bound to the PLA2R antigen.

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

[0138] Single colonies from four rounds of enrichment were randomly selected and placed into 96-well plates, for a total of six plates. 400 μL of 2YT / GA medium (2% glucose, 50 μg / mL Amp) was added to each well, and the plates were incubated overnight at 37°C. The next day, 5 μL of the overnight culture was transferred to a 96-well plate containing 100 μL of medium for activation. The plates were incubated at 37°C for 2–3 h. When the OD value was approximately 2, 20 μL of a mixture of 2YT+ helper phage (MOI=5) was added to each well, and the plates were incubated statically at 37°C for 30 min. After 1.5 h of shaking culture, 300 μL of 2YT-AK (100 μg / mL Amp, 50 μg / mL Kan+) was added to each well, and the plates were incubated overnight for 16–18 h. The next day, the supernatant was collected by centrifugation for detection.

[0139] Example 4: Detection of bacteriophage monoclonal antibodies

[0140] PLA2R antigen was coated onto an ELISA plate at a concentration of 0.5 μg / mL and incubated overnight at 4°C. The plate was then blocked with 4% skim milk and incubated at 37°C for 1 h. A single phage clone was added and incubated at 37°C for 30 min. HRP-M13 (1 / 5K dilution) was added and incubated at 37°C for 30 min. The plate was then developed with chromogenic buffer, and the reaction was terminated with 2 mol / L H2SO4. The absorbance was measured at 450 nm using an ELISA 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 antibody

[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 constructed into laboratory vectors IgG-PCMV3 and LC-PCMV3 (containing constant region sequences) via HindIII / XbaI. After the recombinant plasmids were transformed into competent DH5α cells, positive clones were selected for sequencing and plasmid extraction.

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

[0146] The HEK 293 F cell density was adjusted to 2×10⁻⁶ cells the day before transfection. 6 On the day of transfection, cells were diluted to 3 × 10⁶ cells / mL using HEK 293F cell culture medium. 6 Cells / mL. The extracted plasmids were diluted with liposomes according to the instructions of the liposome transfection reagent, and transfected into cells at a ratio of 1.5 μg:1 mL. The PL82 and PL61 antibody plasmids contained both light and heavy chain plasmids, with an HC:LC ratio of 1:1. After 24 h, 2.5% feed was added, and the cells were cultured until cell viability decreased to approximately 70%–75%, at which point the supernatant was harvested.

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

[0148] The supernatants harvested from PL82 and PL61 antibodies were filtered through a 0.45 μm filter membrane. The chromatography column packed with Protein A packing material was equilibrated with ultrapure water and buffer (0.02 mol / L PBS, pH 7.4). The filtered antibody supernatant was loaded onto the column 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 the expression levels of PL82 and PL61 antibodies after purification were 100 mg / L and 94 mg / L, respectively. The protein concentration was adjusted to 5 mg / mL using a NanoDrop One spectrophotometer. SDS-PAGE ( Figure 1 The results showed that the molecular weight of the heavy chain was approximately 50 kD, and the molecular weight of the light chain was approximately 23 kD, consistent with expectations; HPLC detection ( Figure 2 The results show that the green peak represents PL82IgG4, which mainly exists in monomeric form (around 150 KD) with a small number of aggregate peaks, and the antibody purity is over 98%. PL61IgG4 exists in monomeric form, with the antibody elution 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, 100, 500, 1000, and 2000 times, and their titers were detected using the Antu Anti-Alkaline Phospholipase A2 Antibody-IgG Kit according to the product instructions. The detection results of the prepared human antibodies PL82 and PL61 are shown in Table 1. The average detection concentrations of PL82 IgG4 and PL61 IgG4 were 18.7 × 10⁻⁶. 4 AU / mL and 10.9×10 4 Both AU / mL met the reactivity requirements for positive control samples, but PL82 antibody showed 70% higher reactivity than PL61 antibody and was therefore preferred.

[0152] Table 1: Results of antibody reactivity testing for PL82 IgG4 and PL61 IgG4

[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 two levels, low and high (target values ​​of 15±5 AU / mL and 95±5 AU / mL, respectively). After lyophilization, the titer changes after reconstitution and 37°C accelerated heating for 7 and 14 days were evaluated. The stability data are shown in Table 2. The results show that the activity values ​​of PL82 IgG4 and PL61 IgG4 after 37°C accelerated heating for 7 and 14 days were within ±5%, which meets the requirements for use as quality control and calibrators.

[0156] Table 2: Results of stability evaluation of PL82 IgG4 and PL61 IgG4 antibodies

[0157]

[0158] Example 10: Evaluation of the matrix effect of human anti-PLA2R full-length IgG antibody

[0159] The reagents in the Antu Anti-Alkaline Phospholipase A2 Antibody-IgG Kit were used for antibody and clinical sample evaluation after being accelerated at 37 degrees Celsius for 10 days. Samples prepared with PL82 IgG4 and PL61 IgG4 antibodies at different levels were tested using both the Antu Anti-Alkaline Phospholipase A2 Antibody-IgG Kit (after 10 days of accelerated heating) and the Antu Anti-Alkaline Phospholipase A2 Antibody-IgG Kit (stored at 4 degrees Celsius). The changes in luminescence values ​​of the tested samples detected by the accelerated heating kits at 4 degrees Celsius and 37 degrees Celsius were compared. The results are shown in Table 3.

[0160] Data shows that the luminescence value of PL61 IgG4 antibody detected on the thermally accelerated reagent kit increased by about 50% compared to 4 degrees, which is inconsistent with the variation of clinical samples and indicates a matrix effect. On the other hand, the luminescence value of PL82 IgG4 antibody detected on the thermally accelerated reagent kit decreased by 10% to 20% compared to 4 degrees, which is consistent with the variation of clinical samples and does not show a matrix effect. It meets the performance requirements of quality control and calibrators, and antibody PL82 IgG4 is the preferred choice.

[0161] Table 3: Evaluation results of matrix effects of PL82 IgG4 and PL61 IgG4 antibodies

[0162]

[0163] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An anti-PLA2R antibody, characterized in that: The amino acid sequence of its heavy chain CDR1 is shown in SEQ ID NO: 1; The amino acid sequence of its heavy chain CDR2 is shown in SEQ ID NO: 2; The amino acid sequence of its heavy chain CDR3 is shown in SEQ ID NO: 3; The amino acid sequence of its light chain CDR1 is shown in SEQ ID NO: 4; Its light chain CDR2 amino acid sequence is shown in EDH. The amino acid sequence of its light chain CDR3 is shown in SEQ ID NO:

5.

2. The anti-PLA2R antibody as described in claim 1, characterized in that: Its heavy chain variable region has: (1) An amino acid sequence as shown in SEQ ID NO: 6; or (2) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (1), and whose function is the same as or similar to that of (1); or (3) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (1) or (2); Its light chain variable region has: (4) An amino acid sequence as shown in SEQ ID NO: 7; or (5) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (4), and whose function is the same as or similar to that of (4); or (6) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (4) or (5).

3. The anti-PLA2R antibody as described in claim 1 or 2, characterized in that, Its heavy chain constant region is human IgG4, and its light chain constant region is Lambda type.

4. The use of the anti-PLA2R antibody as described in any one of claims 1 to 3 in the preparation of quality control materials, calibrators, or positive sample analogs for diagnostic reagents for idiopathic membranous nephropathy.

5. A nucleic acid molecule encoding the anti-PLA2R antibody according to any one of claims 1 to 3.

6. An expression carrier, characterized in that, It contains the nucleic acid molecule as described in claim 5.

7. A host, excluding totipotent animal or plant cells, characterized in that, Contains the nucleic acid molecule of claim 5 or the expression vector of claim 6.

8. The method for preparing the anti-PLA2R antibody according to any one of claims 1 to 3, characterized in that, include: (a) Constructing an expression vector containing the nucleic acid molecule of claim 5, transforming the expression vector into a host, culturing the host, separating and purifying the resulting culture to obtain the anti-PLA2R antibody; or (b) Transforming the host with the expression vector of claim 6, culturing the host, separating and purifying the resulting culture to obtain the anti-PLA2R antibody; or (c) Cultivate the host according to claim 7, separate and purify the obtained culture to obtain the anti-PLA2R antibody.

9. Quality control samples, calibrators, or positive sample analogues, characterized in that, It includes the anti-PLA2R antibody as described in any one of claims 1 to 3, and acceptable excipients or adjuvants.

10. The product being tested, characterized in that, It includes the anti-PLA2R antibody as described in any one of claims 1 to 3, and acceptable excipients or adjuvants.

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