Anti-idiotype antibody for ELISA detection and its application in pharmacokinetic evaluation of anti-rabies virus monoclonal antibody

Abstract

The application provides an anti-idiotype antibody for ELISA detection and application thereof in evaluation of pharmacokinetics of anti-rabies virus monoclonal antibody, relates to the technical field of biological detection, and provides a pair of antibodies, namely a coating antibody 38H9G8C12 and a detection antibody 53C7B12C9. The coating antibody contains CDR regions with sequences shown in SEQ ID NO. 1-6. The detection antibody contains CDR regions with sequences shown in SEQ ID NO. 7-12. The antibody pair provided by the application can be used to specifically detect R92 in anti-rabies virus monoclonal antibody injection containing two strains of monoclonal antibodies with different epitopes of neutralizing rabies virus by a double antibody sandwich method, and the detection sensitivity is high, and the result is stable and reliable.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to an anti-idiotype antibody for ELISA detection and application of the anti-idiotype antibody in pharmacokinetic evaluation of anti-rabies virus monoclonal antibodies. BACKGROUND

[0002] Rabies is a global infectious disease caused by rabies virus. For rabies exposure, especially severe exposure, rabies vaccine and anti-rabies antibodies should be injected. Currently, rabies vaccines mainly include rabies inactivated vaccine, attenuated vaccine, nucleic acid vaccine, subunit vaccine, virus-like particle vaccine and oral vaccine.

[0003] Currently, there are two recombinant anti-rabies virus monoclonal antibodies on the market in the world, SII RMab of the Indian Serum Institute and ormutivimab injection of the North China Pharmaceutical Group. Since each monoclonal antibody has monospecificity, the WHO recommends using a cocktail containing at least two monoclonal antibodies that can bind to non-overlapping epitopes to limit the risk of failure due to lack of coverage of the prevailing RABV strain or the emergence of viral escape.

[0004] Chinese patent CN117771365A is a human anti-rabies virus monoclonal antibody preparation developed by the applicant before, which has a low dosage, high affinity and strong neutralizing activity. The preparation is composed of two strains of monoclonal antibodies R71 and R92 with different epitopes of neutralizing rabies virus, and the concentration is mixed, and the total protein concentration is 1 mg / mL. The drug specification is 1500 IU / mg / mL, and the minimum dosing dose in cynomolgus monkeys is 50 IU / kg.

[0005] However, in the pharmacokinetic detection of the preparation, neither inactivated rabies virus particles nor rabies virus glycoprotein can distinguish the two strains of monoclonal antibodies, so it is necessary to screen and express proteins that can specifically bind to R71 or R92.

[0006] Generally, when establishing a pharmacokinetic detection method, ELISA is the most commonly used detection method due to its high throughput and low cost. Double antibody sandwich ELISA has high sensitivity and high specificity, but it needs two specific antibodies against the drug as coating antibodies and detection antibodies, respectively. The binding site of the detection antibody to the target protein needs to be different from the binding site of the target protein to the coating antibody, which involves the problem of antibody pairing, and further exploration is needed. SUMMARY

[0007] In order to solve the above problems, the application provides an anti-idiotype antibody for ELISA detection and application thereof in pharmacokinetic evaluation of anti-rabies virus monoclonal antibody.

[0008] In one aspect, the application provides a pair of anti-idiotype antibodies for ELISA detection, wherein the anti-idiotype antibodies are a coating antibody 38H9G8C12 and a detection antibody 53C7B12C9.

[0009] Specifically, the coating antibody 38H9G8C12 contains any one or more of HCDR1, HCDR2 and HCDR3 having sequences as shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, or any one or more of the amino acid sequences having at least 85% sequence similarity with SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0010] and / or

[0011] contains any one or more of LCDR1, LCDR2 and LCDR3 having sequences as shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, or any one or more of the amino acid sequences having at least 85% sequence similarity with SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6, respectively.

[0012] Further specifically, the coating antibody 38H9G8C12 contains HCDR1, HCDR2 and HCDR3 having sequences as shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively, and / or contains LCDR1, LCDR2 and LCDR3 having sequences as shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

[0013] SEQ ID NO. 1: SYFMH;

[0014] SEQ ID NO. 2: YIDPYNDGTKYNEKFKG;

[0015] SEQ ID NO. 3: GGGYPPTGYFDV;

[0016] SEQ ID NO. 4: RASKSVSTSGYSYMH;

[0017] SEQ ID NO. 5: LASNLES;

[0018] SEQ ID NO. 6: QHSRELPLT.

[0019] In particular, the detection antibody 53C7B12C9 contains any one or more of HCDR1, HCDR2 and HCDR3 having the sequence as set forth in SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9, respectively, or any one or more of the amino acid sequences having at least 85% sequence similarity to SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9, respectively;

[0020] and / or

[0021] contains any one or more of LCDR1, LCDR2 and LCDR3 having the sequence as set forth in SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, respectively, or any one or more of the amino acid sequences having at least 85% sequence similarity to SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, respectively.

[0022] Further in particular, the detection antibody 53C7B12C9 contains HCDR1, HCDR2 and HCDR3 having the sequence as set forth in SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9, respectively, and / or contains LCDR1, LCDR2 and LCDR3 having the sequence as set forth in SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, respectively.

[0023] SEQ ID NO. 7: SYTMS;

[0024] SEQ ID NO. 8: YISNGGGNTYYPDTLKG;

[0025] SEQ ID NO. 9: QNYGNSPYAMDY;

[0026] SEQ ID NO. 10: KSSQSLLYSSNQKNYLA;

[0027] SEQ ID NO. 11: WASTRES;

[0028] SEQ ID NO. 12: QQYYSYPLT.

[0029] In particular, the detection antibody 53C7B12C9 contains any one or more of HCDR1, HCDR2 and HCDR3 having the sequence as set forth in SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9, respectively, or any one or more of the amino acid sequences having at least 85% sequence similarity to SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9, respectively;

[0030] and / or contains a heavy chain variable region of the sequence as shown in SEQ ID NO. 13, and / or contains a light chain variable region of the sequence as shown in SEQ ID NO. 14.

[0031] Further particularly, the coating antibody 38H9G8C12 contains a heavy chain variable region of the sequence as shown in SEQ ID NO. 13, and / or contains a light chain variable region of the sequence as shown in SEQ ID NO. 14.

[0032] SEQ ID NO. 13: EVQLQQSGPELVKPGASVKMSCKASGYTFTSYFMHWVKQQPGQGLEWIGYIDPYNDGTKYNEKFKGKATLTSDKSSTTAYMELSNLTSEDSAVYYCARGGGYPPTGYFDVWGAGTTVTVSS.

[0033] SEQ ID NO. 14: DIVLTQSPASLGVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLDLK.

[0034] Particularly, the detection antibody 53C7B12C9 contains a heavy chain variable region of the sequence as shown in SEQ ID NO. 15, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO. 15, and / or contains a light chain variable region of the sequence as shown in SEQ ID NO. 16, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO. 16.

[0035] Further particularly, the detection antibody 53C7B12C9 contains a heavy chain variable region of the sequence as shown in SEQ ID NO. 15, and / or contains a light chain variable region of the sequence as shown in SEQ ID NO. 16.

[0036] SEQ ID NO. 15: EVKLVESGGGLVQPGGSLKLSCAASGFTFRSYTMSWVRQTPEKRLEWVAYISNGGGNTYYPDTLKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQNYGNSPYAMDYWGQGTSVTVSS.

[0037] SEQ ID NO. 16: DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK.

[0038] In particular, the coating antibody 38H9G8C12 contains a heavy chain having the sequence shown in SEQ ID NO. 17, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO. 17, and / or a light chain having the sequence shown in SEQ ID NO. 18, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO. 18.

[0039] In particular, the coating antibody 38H9G8C12 contains a heavy chain having the sequence shown in SEQ ID NO. 17, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO. 17, and / or a light chain having the sequence shown in SEQ ID NO. 18, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO. 18.

[0040] SEQ ID NO. 17: MEWSWIFLFLLSGTAGVHSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYFMHWVKQQPGQGLEWIGYIDPYNDGTKYNEKFKGKATLTSDKSSTTAYMELSNLTSEDSAVYYCARGGGYPPTGYFDVWGAGTTVTVSS.

[0041] SEQ ID NO. 18: METDTLLLWVLLLWVPGSTGDIVLTQSPASLGVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLDLK.

[0042] In particular, the coating antibody 38H9G8C12 contains a heavy chain having the sequence shown in SEQ ID NO. 17, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO. 17, and / or a light chain having the sequence shown in SEQ ID NO. 18, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO. 18.

[0043] In some embodiments of the present application, the detection antibody 53C7B12C9 contains a heavy chain with a sequence as shown in SEQ ID NO. 19, and / or contains a light chain as shown in SEQ ID NO. 20.

[0044] SEQ ID NO. 19: MNFGLSLIFLVLVLKGVLCEVKLVESGGGLVQPGGSLKLSCAASGFTFRSYTMSWVRQTPEKRLEWVAYISNGGGNTYYPDTLKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQNYGNSPYAMDYWGQGTSVTVSS.

[0045] SEQ ID NO. 20: MDSQAQVLMLLLLWVSGTCGDIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK.

[0046] Specifically, each of the above-mentioned antibodies is a monoclonal antibody.

[0047] In another aspect, the present application provides a nucleic acid molecule encoding any of the above-mentioned antibodies.

[0048] In still another aspect, the present application provides a biological expression vector, characterized in that the biological expression vector comprises the above-mentioned nucleic acid molecule.

[0049] Specifically, the biological expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

[0050] Further specifically, the prokaryotic expression vector includes but is not limited to: pET series vectors, pGEX series vectors, pBAD series vectors, or pQE series vectors.

[0051] Further specifically, the eukaryotic expression vector includes but is not limited to: CMV4 or pSV2.

[0052] In yet another aspect, the present application provides a host cell comprising the above-mentioned nucleic acid molecule and / or biological expression vector.

[0053] Further specifically, the host cell includes but is not limited to: Escherichia coli, Saccharomyces cerevisiae, Kluyveromyces marxianus, CHO cells, NS0 cells, or H293T cells.

[0054] In another aspect, the present application provides use of the above antibody in R92 pharmacokinetic evaluation.

[0055] In another aspect, the present application provides use of the above antibody in preparation of a kit for detecting R92.

[0056] In another aspect, the present application provides a kit comprising the above antibody.

[0057] Specifically, the kit further comprises other reagents required for detecting R92.

[0058] Further specifically, the reagents include, but are not limited to, washing solution, blocking solution, coating solution or buffer solution.

[0059] In another aspect, the present application provides a method for detecting R92 pharmacokinetics by ELISA double antibody sandwich method, which uses 38H9G8C12 as a coating antibody and / or 53C7B12C9 as a detection antibody.

[0060] Compared with the prior art, the present application has the following advantages:

[0061] Specifically bind to R92, and almost completely identical to the variable region sequence outside, and simultaneously exist in the sample and bind to R71.

[0062] When the ELISA double antibody sandwich method is used for detection by using the antibody pair provided by the present application, the standard curve range is 10-130 ng / mL, the minimum dilution multiple is 5, the lower limit of quantification is 10 ng / mL, and the method has good selectivity, precision and accuracy, stability, parallelism, specificity, dilution linearity, no obvious Hook effect, and can be used for determining the content of R92 in serum. DETAILED DESCRIPTION

[0063] The present application will be further described in detail below in combination with specific examples, and the following examples are not used for limiting the present application, but for illustrating the present application. Unless otherwise specified, the experimental methods used in the following examples are generally performed according to conventional conditions. Unless otherwise specified, the materials, reagents and the like used in the following examples can be obtained from commercial channels.

[0064] The experimental reagents are purchased from the sources shown in Table 1 below:

[0065] Table 1

[0066] name factory Item number Pepsin Sigma-Aldrich P6887-250mg IdeS Genscript Biotech Co., Ltd. NA Protein A Resin Genscript Biotech Co., Ltd. NA 1M or 0.1M sodium citrate, pH 3.5 Self-configured NA 3.3M Tris Self-configured NA 1*PBS pH 7.4 Self-configured NA Freund's adjuvant, complete SIGMA-ALDRICH F5881 Freund's adjuvant, incomplete SIGMA-ALDRICH F5506 Imject Alum Adjuvant Thermo Fisher 77161 Mihen GenScript NA BALB / c mice Beijing Vital River Laboratory Animal Technology Co., Ltd. 2021-09-03 / 2021-09-17 Coating fluid Self-configured NA Wash Self-configured NA Blocking solution / diluent Self-configured NA Colorimetric solution A Self-configured NA Colorimetric solution B Self-configured NA Stop solution (1M HCl) Self-configured NA Peroxidase-AffiniPure Goat Anti-Mouse IgG, FcγFragment Specific (min X Hu,Bov,Hrs Sr Prot)-HRP(M6) Jackson 115-035-071 Buffer A: 100×HT Sigma H: H9636T:T1895 Buffer B: Aminopterin (100×A) Sigma A3411 Buffer C: 100×IL-6 GenScript Z02767 FBS (Fetal Bovine Serum) Wisent 086-150 DMEM basal culture medium Gibco / HyClone C1199550BT / SH30243 2×HT as a substitute for feeder cell culture medium (Buffer A, fetal bovine serum, Buffer C, DMEM). GenScript NA 2×A (Buffer B, DMEM) GenScript NA SP2 / 0 SGST NA Electrofusion buffer GenScript NA CD138 Positive Selection Kit STEMCELL 18957 1×HT medium (10%FBS+89%DMEM+1%100×HT) NA NA [TMB Substrate (A: C6H8O7, CH3COONa; B: TMB)] A: Sinopharm Chemical Reagent Co.LtdB: Suhou Yacoo Science Co. Ltd 1000711810018718S0025 Stop solution (1M HCl, self-prepared) NA NA 1×HT as a substitute for feeder cell culture medium (Buffer A, fetal bovine serum, Buffer C, DMEM). NA NA Trypan blue staining solution Gibco K940-100ML FEM Gibco C12338018 DMEM cytiva / Shanghai Yuanpei SH30243.01 / L110KJ FBS (Fetal Bovine Serum) Wisent 086-150 Binding buffer (1×PBS, pH 7.4) HyClone Phosphate Buffered Saline & 500 mL (1×PBS) SH30256.01 Elution buffer 1 (0.1 M Citric acid buffer, pH 3.0) KPL NA Elution buffer 2 (0.1 M Glycine, pH 2.5) KPL NA Neutralizing buffer (0.1 M Tris pH 9.0) KPL NA Dialysis buffer (1×PBS, pH 7.4) Shengwo B548117-0500 Secondary Antibody: Streptavidin-HRP GenScript M00091 Pierce High Sensitivity Streptavidin - HRP Thermo Fisher Scientific 21130

[0067] Example 1 Preparation and screening of monoclonal antibody

[0068] 1. Preparation of immunogen

[0069] 1.1 Mix the antibody with IDES industrial enzyme at a ratio of 1 mg / 10 μg, react at 37°C. Take 3 μg at 2, 4, 6, 8 hours, respectively.

[0070] 1.2 Perform reducing electrophoresis and non-reducing electrophoresis on the above samples, and use total molecular electrophoresis as a control.

[0071] 1.3 According to the results of the gel run, determine the appropriate enzyme digestion conditions, and further separate a large number of enzyme digestion fragments. After digestion, dialyze with 1x PBS. After dialysis, take samples and perform electrophoresis. According to the electrophoresis results, if the entire molecule still exists in the digestion product, remove it by passing through a protein A affinity column.

[0072] 1.4 After SDS-PAGE identification, if the purity reaches >85%, it can be used for the next step of the experiment.

[0073] 2. Animal immunization and serum detection

[0074] 2.1 Immunization

[0075] 2.1.1 Preparation of immunogen: prepare the antigen and adjuvant at a volume ratio of 1:1;

[0076] 2.1.2 Animal immunization: after disinfecting the immunization site with 75% alcohol, immunize the animals by intraperitoneal injection or subcutaneous injection. Immunize on days 0, 14, 21, 28, and 35, with an immunization dose of 25-50 μg of protein per animal.

[0077] 2.2 Serum collection

[0078] 2.2.1 Collection of animal blood: collect blood from the animals after immunization by submandibular vein, 100 μL per animal.

[0079] 2.2.2 Separate serum, serum volume requirement: ≥30 μL per animal.

[0080] 2.3 Serum detection

[0081] 2.3.1 Dilute the antigen to the same concentration with the coating solution, mix well, and add to the designed plate strip, 100 μL per well;

[0082] 2.3.2 After coating, place in a 4°C refrigerator overnight for 16 h;

[0083] 2.3.3 Discard the coating solution, wash the plate once, and pat dry the enzyme-labeled plate;

[0084] 2.3.4 Add 150 μL of blocking solution to each well, and incubate in a 37°C constant temperature incubator for 1 h;

[0085] 2.3.5 Remove the enzyme-labeled plate, discard the blocking solution;

[0086] 2.3.6 Serum sample addition step: pre-immune serum detection 1:1000 dilution per well, total 2 wells, average value, 100 µL / well; post-immune serum 2 wells, 1:100 or 1:1000 dilution respectively, 100 µL / well, 2-fold gradient dilution to the 10th well, the remaining 2 wells added diluent as blank control, the data of the final control well is represented as the average value of the two blank wells, 100 µL / well;

[0087] 2.3.7 Incubate at 37°C for 1 h, remove the enzyme-labeled plate, discard the inner liquid, and wash the plate 3 times;

[0088] 2.3.8 Add 100 µL of diluted enzyme-labeled secondary antibody to each well and incubate in a 37°C incubator for 0.5 h;

[0089] 2.3.9 Remove the enzyme-labeled plate, discard the inner liquid, and wash the plate 3 times, then add 100 µL of color developing solution, react at 25°C for 15 min; add 50 µL of 1M HCl to stop the reaction; and read OD450 nm on the enzyme-labeled instrument.

[0090] 2.3.10 Results: the serum titer of the immune group after serum detection is >1:512,000, and 1:8,000 OD450>1.0, meeting the fusion standard.

[0091] 3. Hybridoma fusion

[0092] 3.1 Prepare a single cell suspension of myeloma cells (SP2 / 0).

[0093] 3.2 Extract the spleen or lymph node tissue and grind for homogenization to produce a single cell suspension.

[0094] 3.3 Mix the SP2 / 0 and spleen or lymph node cells obtained in the above steps, and use electrofusion technology to fuse with SP2 / 0 mouse myeloma cells. After fusion is complete, resuspend the fused cells in DMEM / 10% FBS medium containing hybridoma cell selection agents thymidine, hypoxanthine and aminopterin, and inoculate the cells into a 96-well plate.

[0095] 3.4 Incubate the 96-well plate in a 5.5% CO2 incubator at 37°C.

[0096] 3.5 After 5-8 days, count the fusion rate and screen positive clones.

[0097] 3.6 ELISA screening: Indirect ELISA was used to assess the binding ability of antibodies in supernatant to X. Sample preparation: The culture medium in the culture plate was pipetted into the ELISA plate coated with antigen, enzyme-labeled antibody reaction was added, and TMB color developing solution was added for color development after reading termination. Whole-page ELISA screening and confirmation screening binding were performed, and positive qualified clones were selected for further advancement.

[0098] 3.7 Cell limited dilution subcloning: Observe the cell state, and mix the cells in the hole with a pipette. Mix 20 μL of cell suspension with 20 μL of trypan blue. Take 20 μL of the mixture and put it into a cell counting plate, and use Count-star to count. Each clone is subcloned by limited dilution method, cultured in 1xHT medium, and plated into a 96-well plate at a density of 2-5 cells / well. Each plate is labeled with order number, clone ID, and operation date. After the 96-well plate is cultured at 37°C and 5.5% CO2 incubator for 1 week, the supernatant of the clone is taken for ELISA binding as described above to evaluate the presence of antibodies against x.

[0099] Subclone as above until a positive monoclonal is obtained.

[0100] 3.8 Subclass detection

[0101] 3.8.1 Coat the goat anti-mouse Ig on the 96-well plate at a concentration of 0.5 μg / mL, 100 μL / well. Seal the plate and incubate.

[0102] 3.8.2 Wash the plate and add 150 μL of blocking buffer to each well. Seal the plate and incubate. Add samples and dilute with dilution buffer, 9 wells per sample, 100 μL / well, seal the culture plate. Wash the plate 3 times.

[0103] 3.8.3 IgG2c secondary antibody dilution ratio is 1:2K, other secondary antibody dilution ratio is 1:500, add to the plate at 100 μL / well. Seal the plate and incubate.

[0104] 3.8.4 After washing 3 times, discard the solution. After wiping with a paper towel, add 100 μL of TMB substrate to each well and incubate at 25°C.

[0105] 3.8.5 Add 50 μL of stop buffer to each well to terminate the reaction. Read the readings of the enzyme-labeled plate on the enzyme-labeled instrument at OD450 nm.

[0106] 3.10 Results

[0107] 3.10.1 Parental clone screening results: Select the clones in Table 2 in bold and subclone them to the fixed strain.

[0108] Table 2

[0109]

[0110]

[0111] 3.10.2 The results of the screening of the stable clones are shown in Table 3: the stable clones 43D2F10E8 / 43D2F10C2 were tested negative, the other clones showed a decrease in OD value on the master test original C1480GH240-1A:R92 with increasing dilution gradient.

[0112] Table 3

[0113]

[0114]

[0115] 4. Antibody production

[0116] 4.1 Cultivation

[0117] The selected cell lines were expanded from 24-well plates to 10 cm cell culture dishes with complete medium (DMEM + 10% FBS); the cell status was observed and when the cell confluence in the dishes was more than 80%, the supernatant was discarded and the cells were suspended and transferred to a flask with FEM medium; the cells were inoculated at a density of 0.5-0.7 x 10 5 cells / mL (half the volume); the flask was shaken to check for cell contamination and FEM was added to the flask to the final volume; the cells in the flask were observed and checked for proliferation after several days of cultivation; when the viability was less than 20%, the sample was collected for downstream purification.

[0118] 4.2 Purification

[0119] The Protein A magnetic beads were taken out of the refrigerator. The supernatant was added to the magnetic beads and incubated at room temperature for 2 h. After incubation, the magnetic beads were equilibrated with 10 CV (column volume) of binding buffer. The magnetic beads were washed with elution buffer 1. The elution buffer was collected. The eluted antibody was neutralized by adding 1 / 10 volume of neutralization buffer. The antibody was filtered with a 0.22 pm filter. The antibody concentration was measured using a Nanodrop 2000.

[0120] 5. Detection of the binding ability of the purified antibody to the antigen

[0121] 5.1 The antigen was diluted with coating solution and mixed, then added to the designed strip, 100 pL / well;

[0122] 5.2 After coating, it was placed in a 4°C refrigerator overnight for 16 h;

[0123] 5.3 The coating solution was discarded, the plate was washed once, and the ELISA plate was tapped dry;

[0124] 5.4 Add 150 μL blocking solution to each well and incubate in a 37°C incubator for 1 h;

[0125] 5.5 Take out the enzyme-labeled plate and discard the blocking solution;

[0126] 5.6 The first well of the QC antibody to be tested has a concentration of 1.0 μg / mL, and is diluted by 3 times to the 10th well. The remaining 2 wells are added with diluent as blank controls. The final control well data is represented by the average of the two wells, 100 μL / well;

[0127] 5.7 Incubate at 37°C for 1 h, take out the enzyme-labeled plate, discard the inner liquid, and wash the plate 3 times;

[0128] 5.8 Add 100 μL of diluted enzyme-labeled secondary antibody to each well and incubate in a 37°C incubator;

[0129] 5.9 Take out the enzyme-labeled plate, discard the inner liquid, wash the plate 3 times, and add 100 μL of color developing solution after drying. React at 25°C for 15 min; add 50 μL of 1M HCl to terminate the reaction; and read OD 450 nm on the enzyme-labeled instrument.

[0130] Example 2: ELISA Pairing Detection Report of Purified Antibody

[0131] The reagents used in this example are shown in Table 4:

[0132] Table 4

[0133] Coating antigen Unlabeled antibody Coating concentration 2.0 μg / mL Coating solution Phosphate buffer Secondary antibody Streptavidin-HRP

[0134] 1. Dilute the unlabeled antibody to 2 μg / mL with the coating solution, mix well, and add to the designed plate strip, 100 μL / well;

[0135] 2. After coating, place in a 4°C refrigerator overnight for 16 h;

[0136] 3. Discard the coating solution, wash the plate once, and dry the enzyme-labeled plate;

[0137] 4. Add 150 μL of blocking solution to each well and incubate in a 37°C incubator for 1 h;

[0138] 5. Take out the enzyme-labeled plate and discard the blocking solution;

[0139] 6. The sandwich antibody R92 detection concentrations are 100 ng / mL, 10 ng / mL, 1 ng / mL, and 0 ng / mL; 100 μL / well;

[0140] 7. Incubate at 37°C for 1 h, take out the enzyme-labeled plate, discard the inner liquid, and wash the plate 3 times;

[0141] 8. Add biotin-labeled detection antibody, 1 μg / mL, 100 μL / well;

[0142] 9. Incubate at 37°C for 1 h, remove the enzyme-labeled plate, discard the liquid, and wash the plate 3 times;

[0143] 10. Add 100 μL of diluted enzyme-labeled secondary antibody to each well and incubate in a 37°C incubator;

[0144] 11. Remove the enzyme-labeled plate, discard the liquid, wash the plate 3 times, pat dry, add 100 μL of color developing solution, and react at 25°C for 15 min; add 50 μL of 1M HCl to stop the reaction; and measure the OD nm reading with an enzyme-labeled instrument. 450

[0145] 12. The results are shown in Table 5. When 38H9G8C12 is used as the coating antibody and 53C7B12C9 is used as the detection antibody, the signal is the strongest.

[0146] Table 5

[0147]

[0148] Example 3 Sensitivity of ELISA Pairing Detection of Purified Antibody

[0149] 1. Dilute 38H9G8C12 with the coating solution to 2.0 μg / mL, mix well, and add to the designed plate strip, 100 μL / well;

[0150] 2. After coating, place in a 4°C refrigerator overnight for 16 h;

[0151] 3. Discard the coating solution, wash the plate once, and pat dry the enzyme-labeled plate;

[0152] 4. Add 150 μL of blocking solution to each well and incubate in a 37°C incubator for 1 h;

[0153] 5. Remove the enzyme-labeled plate, discard the blocking solution;

[0154] 6. The sandwich antibody detection concentration is 512 ng / mL in the first well, and is diluted by 2-fold gradient to 0.5 ng / mL, 100 μL / well;

[0155] 7. Incubate at 37°C for 1 h, remove the enzyme-labeled plate, discard the liquid, and wash the plate 3 times;

[0156] 8. Add biotin-labeled antibody 53C7B12C9, 1.0 μg / mL, 100 μL / well;

[0157] 9. Incubate at 37°C for 1 h, remove the enzyme-labeled plate, discard the liquid, and wash the plate 3 times;

[0158] ​10. Add 100 μL of diluted enzyme-labeled secondary antibody to each well and incubate in a 37°C incubator;

[0159] 11. Take out the enzyme-labeled plate, discard the inner liquid, wash the plate 3 times, pat dry, add 100 μL of color developing solution, react for 15 min at 25°C, add 50 μL of 1M HCl to terminate the reaction, and read OD450 nm on an enzyme-labeled instrument.

[0160] 12. Experimental results: The preliminary experimental results are shown in Tables 6 and 7, which can meet the sensitivity requirements of ELISA detection. The LOD is determined by adding two standard deviations (SD) to the average OD 450 nm value of 24 zero standard repeats and calculating the corresponding concentration, and the LOD of this method is 0.179 ng / ml.

[0161] If the influence of serum on the sample is not considered, after 5-fold dilution of the sample, the sensitivity on the well plate can theoretically reach about 0.895 ng / ml, which is much lower than the target concentration of 2 ng / ml, and the antibody can be used for the establishment of a pharmacokinetic (PK) ELISA detection method for recombinant human anti-rabies virus monoclonal antibody R92.

[0162] Table 6

[0163]

[0164] Table 7

[0165]

[0166] Methodology investigation of experimental example 1

[0167] The test steps are as follows: (1) coating: add the diluted coating antibody 38H9G8C12 100 μL / well to the enzyme-labeled plate, seal the plate, and incubate at 2-8°C overnight; (2) washing the plate: wash the plate once with 300 μL / well / time; (3) blocking: add blocking solution 200 μL / well, incubate at 25°C 750 rpm for about 1 h; (4) washing the plate: wash the plate once with 300 μL / well / time; (5) adding standard: take the anti-rabies virus monoclonal antibody product, wherein the concentration of R92 is 0.5 mg / ml; (6) adding sample: add the diluted sample to the corresponding well, 100 μL / well, incubate at 25°C 750 rpm for about 2 h; (7) washing the plate: wash the plate 6 times with 300 μL / well / time; (8) adding detection antibody working solution: add the diluted biotin-labeled detection antibody 53C7B12C9 100 μL / well to the enzyme-labeled plate, incubate at 25°C 750 rpm for about 1 h; (9) adding secondary antibody working solution: add the diluted HRP-labeled Streptavidin HRP 100 μL / well to the enzyme-labeled plate, incubate at 25°C 750 rpm for about 1 h; (10) washing the plate: wash the plate 6 times with 300 μL / well / time; (11) color development: add TMB color developing liquid 100 μL / well, incubate at room temperature for about 10 min, and determine according to the color development; (12) termination: add termination solution 50 μL / well, read OD value at 450 nm and save the results.

[0168] The test uses cynomolgus monkey serum as the experimental system, and the methodological full verification of the standard curve, precision and accuracy, selectivity, specificity, dilution linearity, hook effect, stability, and parallelism of the detection method is carried out. The verification results are shown in Table 8.

[0169] Table 8

[0170]

[0171] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A pair of anti-idiotypic antibodies for use in an ELISA assay, characterized in that, The unique antibody is used for specifically detecting R92 in the anti-rabies virus monoclonal antibody injection, the anti-unique antibody includes a coating antibody 38H9G8C12, a light chain sequence of the R92 antibody is as shown in SEQ ID NO: 21, and a heavy chain sequence of the R92 antibody is as shown in SEQ ID NO: 22; The coating antibody 38H9G8C12 sequentially contains HCDR1, HCDR2 and HCDR3 with sequences as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; and sequentially contains LCDR1, LCDR2 and LCDR3 with sequences as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.

6.

2. A pair of anti-idiotypic antibodies for use in an ELISA assay, characterized in that, The unique antibody is used for specifically detecting R92 in the anti-rabies virus monoclonal antibody injection, the anti-unique antibody includes a detection antibody 53C7B12C9, a light chain sequence of the R92 antibody is as shown in SEQ ID NO: 21, and a heavy chain sequence of the R92 antibody is as shown in SEQ ID NO: 22; The detection antibody 53C7B12C9 sequentially contains HCDR1, HCDR2 and HCDR3 with sequences as shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9; and sequentially contains LCDR1, LCDR2 and LCDR3 with sequences as shown in SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.

12.

3. The anti-idiotype antibody of claim 1, wherein the anti-idiotype antibody is an anti-idiotype antibody to an antibody that binds to a target antigen. The coating antibody 38H9G8C12 contains a heavy chain variable region with a sequence as shown in SEQ ID NO.13, and / or contains a light chain variable region with a sequence as shown in SEQ ID NO.

14.

4. The idiotype antibody of claim 1, wherein, The coating antibody 38H9G8C12 contains a heavy chain variable region with at least 85% sequence similarity with SEQ ID NO.13, and / or contains a light chain variable region with at least 85% sequence similarity with SEQ ID NO.

14.

5. The anti-idiotype antibody of claim 2, wherein the anti-idiotype antibody is an anti-idiotype antibody to an antibody that binds to a target antigen. The detection antibody 53C7B12C9 contains a heavy chain variable region with a sequence as shown in SEQ ID NO.15, and / or contains a light chain variable region with a sequence as shown in SEQ ID NO.

16.

6. The anti-idiotype antibody of claim 2, wherein, The detection antibody 53C7B12C9 contains a heavy chain variable region with at least 85% sequence similarity with SEQ ID NO.15, and / or contains a light chain variable region with at least 85% sequence similarity with SEQ ID NO.

16.

7. The anti-idiotype antibody of claim 3, wherein the anti-idiotype antibody is an anti-idiotype antibody to an antibody that binds to a human PD-1. The coating antibody 38H9G8C12 contains a heavy chain with a sequence as shown in SEQ ID NO.17, and / or contains a light chain with a sequence as shown in SEQ ID NO.

18.

8. The anti-idiotype antibody of claim 3, wherein the anti-idiotype antibody is an anti-idiotype antibody to an antibody that binds to a target antigen. The coating antibody 38H9G8C12 contains a heavy chain with at least 85% sequence similarity with SEQ ID NO.17, and / or contains a light chain with at least 85% sequence similarity with SEQ ID NO.

18.

9. The anti-idiotype antibody of claim 5, wherein, The detection antibody 53C7B12C9 contains a heavy chain with a sequence as shown in SEQ ID NO. 19, and / or contains a light chain with a sequence as shown in SEQ ID NO.

20.

10. The anti-idiotype antibody of claim 5, wherein The detection antibody 53C7B12C9 contains a heavy chain with at least 85% sequence similarity to SEQ ID NO. 19, and / or contains a light chain with at least 85% sequence similarity to SEQ ID NO.

20.

11. The anti-idiotype antibody according to any one of claims 1 to 10, characterized in that, The antibody is a monoclonal antibody.

12. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-idiotype antibody according to any one of claims 1-10.

13. A biological expression vector, characterized by, The biological expression vector comprises the nucleic acid molecule according to claim 12.

14. A host cell, characterized in that, The host cell comprises the nucleic acid molecule according to claim 12 and / or the biological expression vector according to claim 13.

15. Use of the anti-idiotype antibody according to any one of claims 1-10 in the evaluation of the pharmacokinetics of R92 in an anti-rabies virus monoclonal antibody injection.

16. Use of the anti-idiotype antibody according to any one of claims 1-10 in the preparation of a kit for detecting R92 in an anti-rabies virus monoclonal antibody injection.

17. A kit comprising, The kit comprises the anti-idiotype antibody according to any one of claims 1-10.

18. A method of detecting R92 pharmacokinetics by an ELISA double antibody sandwich method, characterized by, The method uses 38H9G8C12 according to claim 1 as the coating antibody and / or 53C7B12C9 according to claim 2 as the detection antibody.

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