Anti-idiotype antibody for ELISA (enzyme-linked immunosorbent assay) detection and application thereof in pharmacokinetic evaluation of anti-rabies virus monoclonal antibody

Abstract

The invention provides an anti-idiotype antibody for ELISA (enzyme-linked immunosorbent assay) detection and application of the anti-idiotype antibody in pharmacokinetic evaluation of an anti-rabies virus monoclonal antibody, and relates to the technical field of biological detection, the invention provides a pair of antibodies which are respectively a coated antibody 38H9G8C12 and a detection antibody 53C7B12C9, and the coated antibody contains a CDR (CDR region) as shown in SEQ ID NO.1-6. The detection antibody contains a CDR region of which the sequence is as shown in SEQ ID NO.7-12. By using the antibody pair provided by the invention, the R92 in an anti-rabies virus monoclonal antibody injection consisting of two monoclonal antibodies neutralizing different epitopes of the rabies virus can be specifically detected through a double-antibody sandwich method region, the detection sensitivity is high, and the result is stable and reliable.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to an anti-idiotypic antibody for ELISA detection and its application in the pharmacokinetic evaluation of anti-rabies virus monoclonal antibody. Background Art

[0002] Rabies is a global zoonotic disease caused by the rabies virus. For those who have been exposed to rabies, especially those who have been severely exposed, rabies vaccine and anti-rabies antibodies should be fully injected. Currently, the types of rabies vaccines mainly include new inactivated rabies vaccines, attenuated vaccines, nucleic acid vaccines, subunit vaccines, virus-like particle vaccines and oral vaccines.

[0003] Currently, there are two recombinant anti-rabies virus monoclonal antibodies on the market worldwide, namely SII RMab from the Serum Institute of India and ormutivimab injection from North my country Pharmaceutical Group. Since each monoclonal antibody has monospecificity, WHO recommends the use of a cocktail containing at least two monoclonal antibodies that bind to non-overlapping antigenic epitopes to limit the risk of failure due to lack of coverage of circulating RABV strains or sudden viral escape.

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

[0005] However, in the pharmacokinetic testing of this preparation, the use of inactivated rabies virus particles or rabies virus glycoproteins could not distinguish between the two monoclonal antibodies, so it was necessary to screen and express proteins that could specifically bind to R71 or R92, respectively.

[0006] Usually, when establishing pharmacokinetic detection methods for monoclonal antibodies, ELISA becomes 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 requires two strains of specific antibodies against the drug as coating antibodies and detection antibodies respectively. The binding site of the detection antibody and the target protein needs to be different from the binding site of the target protein and the coating antibody, which will involve the problem of antibody pairing and needs further exploration. Summary of the invention

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

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

[0009] Specifically, the coating antibody 38H9G8C12 contains any one or more of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; or any one or more amino acid sequences having at least 85% sequence similarity with SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; and / or Contains any one or more of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, or any one or more amino acid sequences having at least 85% sequence similarity with SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6, respectively.

[0010] Further specifically, the coating antibody 38H9G8C12 contains HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively, and / or contains LCDR1, LCDR2 and LCDR3 whose sequences are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0011] SEQ ID NO.1: SYFMH; SEQ ID NO.2: YIDPYNDGTKYNEKFKG; SEQ ID NO.3: GGGYPPTGYFDV; SEQ ID NO.4:RASKSVSTSGYSYMH; SEQ ID NO.5: LASNLES; SEQ ID NO.6: QHSRELPLT.

[0012] Specifically, the detection antibody 53C7B12C9 contains any one or more of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9, or any one or more amino acid sequences having at least 85% sequence similarity with SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9, respectively; and / or Contains any one or more of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, or any one or more amino acid sequences having at least 85% sequence similarity with SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, respectively.

[0013] Further specifically, the detection antibody 53C7B12C9 contains HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9, and / or contains LCDR1, LCDR2 and LCDR3 whose sequences are shown in SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, respectively.

[0014] SEQ ID NO.7: SYTMS; SEQ ID NO.8: YISNGGGNTYYPDTLKG; SEQ ID NO.9: QNYGNSPYAMDY; SEQ ID NO.10: KSSQSLLYSSNQKNYLA; SEQ ID NO.11: WASTRES; SEQ ID NO. 12: QQYYSYPLT.

[0015] Specifically, the coating antibody 38H9G8C12 contains a heavy chain variable region having a sequence as shown in SEQ ID NO.13, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO.13; and / or contains a light chain variable region as shown in SEQ ID NO.14, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO.14.

[0016] More specifically, the coating antibody 38H9G8C12 contains a heavy chain variable region whose sequence is shown in SEQ ID NO.13, and / or contains a light chain variable region whose sequence is shown in SEQ ID NO.14.

[0017] SEQ ID NO. 13: EVQLQQSGPELVKPGASVKMSCKASGYTFTSYFMHWVKQQPGQGLEWIGYIDPYNDGTKYNEKFKGKATLTSDKSSTTAYMELSNLTSEDSAVYYCARGGGYPPTGYFDVWGAGTTVTVSS.

[0018] SEQ ID NO. 14: DIVLTQSPASLGVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLDLK.

[0019] Specifically, the detection antibody 53C7B12C9 contains a heavy chain variable region as shown in SEQ ID NO.15, or any one or more amino acid sequences having at least 85% sequence similarity with SEQ ID NO.15, and / or contains a light chain variable region as shown in SEQ ID NO.16, or any one or more amino acid sequences having at least 85% sequence similarity with SEQ ID NO.16.

[0020] More specifically, 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.

[0021] SEQ ID NO. 15: EVKLVESGGGLVQPGGSLKLSCAASGFTFRSYTMSWVRQTPEKRLEWVAYISNGGGNTYYPDTLKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQNYGNSPYAMDYWGQGTSVTVSS.

[0022] SEQ ID NO. 16: DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK.

[0023] Specifically, the coated antibody 38H9G8C12 contains a heavy chain as 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 contains a light chain as shown in SEQID NO.18, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO.18.

[0024] In certain specific embodiments of the present invention, 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.

[0025] SEQ ID NO. 17: MEWSWIFLFLLSGTAGVHSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYFMHWVKQQPGQGLEWIGYIDPYNDGTKYNEKFKGKATLTSDKSSTTAYMELSNLTSEDSAVYYCARGGGYPPTGYFDVWGAGTTVTVSS.

[0026] SEQ ID NO. 18: METDTLLLWVLLLWVPGSTGDIVLTQSPASLGVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLDLK.

[0027] Specifically, the detection antibody 53C7B12C9 contains a heavy chain as shown in SEQ ID NO.19, or any one or more amino acid sequences having at least 85% sequence similarity with SEQ ID NO.19, and / or contains a light chain as shown in SEQID NO.20, or any one or more amino acid sequences having at least 85% sequence similarity with SEQ ID NO.20.

[0028] In certain embodiments of the present invention, 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.

[0029] SEQ ID NO. 19: MNFGLSLIFLVLVLKGVLCEVKLVESGGGLVQPGGSLKLSCAASGFTFRSYTMSWVRQTPEKRLEWVAYISNGGGNTYYPDTLKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQNYGNSPYAMDYWGQGTSVTVSS.

[0030] SEQ ID NO. 20: MDSQAQVLMLLLLWVSGTCGDIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK.

[0031] Specifically, the above antibodies are all monoclonal antibodies.

[0032] In another aspect, the present invention provides a nucleic acid molecule encoding any of the above antibodies.

[0033] In yet another aspect, the present invention provides a biological expression vector, characterized in that the biological expression vector comprises the above-mentioned nucleic acid molecule.

[0034] Specifically, the biological expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

[0035] More specifically, the prokaryotic expression vector includes but is not limited to: pET series vectors, pGEX series vectors, pBAD series vectors or pQE series vectors.

[0036] More specifically, the eukaryotic expression vector includes but is not limited to: CMV4 or pSV2.

[0037] In yet another aspect, the present invention provides a host cell comprising the above-mentioned nucleic acid molecule and / or biological expression vector.

[0038] More specifically, the host cell includes but is not limited to: Escherichia coli, Saccharomyces cerevisiae, Kluyveromyces marxianus, CHO cells, NS0 cells or H293T cells.

[0039] In yet another aspect, the present invention provides use of the above antibody in the pharmacokinetics evaluation of R92.

[0040] In yet another aspect, the present invention provides use of the above antibody in preparing a kit for detecting R92.

[0041] In yet another aspect, the present invention provides a kit comprising the above-mentioned antibody.

[0042] Specifically, the kit also includes other reagents required for detecting R92.

[0043] More specifically, the reagents include but are not limited to: a washing solution, a blocking solution, a coating solution or a buffer solution.

[0044] In yet another aspect, the present invention provides a method for detecting the pharmacokinetics of R92 by an ELISA double antibody sandwich method, wherein 38H9G8C12 is used as a coating antibody and / or 53C7B12C9 is used as a detection antibody.

[0045] Compared with the prior art, the present invention has the following advantages: It can specifically bind to R92, is almost identical to the variable region sequence, and simultaneously binds to R71 in the sample.

[0046] When the antibody pair provided by the present invention is used for ELISA double antibody sandwich method detection, the standard curve range is 10-130 ng / mL, the minimum dilution factor is 5, the lower limit of quantification is 10 ng / mL, it has good selectivity, precision and accuracy, stability, parallelism, specificity, dilution linearity, and no obvious Hook effect, and can be used to determine the content of R92 in serum. DETAILED DESCRIPTION

[0047] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0048] The sources of experimental reagents are shown in Table 1 below: Table 1 name factory Part 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 Weitonglihua Laboratory Animal Technology Co., Ltd. 2021-09-03 / 2021-09-17 Coating fluid Self-configured NA Lotion Self-configured NA Blocking buffer / dilution buffer 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 medium Gibco / HyClone C1199550BT / SH30243 2×HT replacement 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 <![CDATA[TMB Substrate(A: C 6 H 8 THE 7 、CH 3 COONa;B: TMB)]]> A: Sinopharm Chemical Reagent Co.LtdB: Suhou Yacoo Science Co. Ltd 1000711810018718S0025 Stop solution (1M HCl, self-prepared) NA NA 1×HT replacement 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, pH7.4) HyClone Phosphate Buffered Saline&500 mL(1×PBS) SH30256.01 Elution buffer 1 (0.1 M Citric acid buffer, pH 3.0) GenScript Biopharmaceuticals NA Elution buffer 2 (0.1 M Glycine, pH 2.5) GenScript Biopharmaceuticals NA Neutralizing buffer (0.1 M Tris pH 9.0) GenScript Biopharmaceuticals NA Dialysis buffer(1×PBS, pH 7.4) Bioengineering B548117-0500 Secondary Antibody: Streptavidin-HRP GenScript M00091 Pierce High Sensitivity Streptavidin - HRP Thermo Fisher Scientific 21130 Example 1 Preparation and screening of monoclonal antibodies 1. Immunogen Preparation 1.1 Mix the antibody and IDES industrial enzyme at a ratio of 1 mg / 10 μg and react at 37°C. Take 3 μg at 2, 4, 6, and 8 hours respectively.

[0049] 1.2 Perform reduction electrophoresis and non-reduction electrophoresis on the above samples, and use total molecule electrophoresis as a control.

[0050] 1.3 According to the results of the gel run, determine the appropriate enzyme digestion conditions and further segment a large number of enzyme digestion fragments. After digestion, dialyze with 1× PBS. After dialysis, take samples and perform electrophoresis. According to the electrophoresis results, if the whole molecule still exists in the digestion product, remove it by passing it through a protein A affinity column.

[0051] 1.4 After SDS-PAGE identification, the purity reaches >85% and can be used for the next experiment.

[0052] 2. Animal immunization and serum testing 2.1 Immunity 2.1.1 Preparation of immunogens: Prepare the antigen and adjuvant in a volume ratio of 1:1; 2.1.2 Animal immunization: After disinfecting the immunization site with 75% alcohol, the animals were immunized by intraperitoneal injection or subcutaneous injection. The immunization was carried out on days 0, 14, 21, 28, and 35, with an immunization dose of 25-50 μg protein per animal.

[0053] 2.2 Serum collection 2.2.1 Animal blood collection: Blood was collected from immunized animals through the submandibular vein, 100 µL / animal.

[0054] 2.2.2Separate serum. Serum volume requirement: ≥30µL / mouse.

[0055] 2.3 Serological testing 2.3.1 Dilute the antigen to the same concentration with coating solution, mix well and add to the designed strips, 100µL / well; 2.3.2 After packaging, place in a 4℃ refrigerator overnight for 16 hours; 2.3.3 Discard the coating solution, wash the plate once, and pat the plate dry; 2.3.4 Add 150 µL of blocking solution to each well and incubate in a 37°C incubator for 1 hour; 2.3.5 Take out the ELISA plate and discard the blocking solution; 2.3.6 Serum sample addition steps: Before immunization, serum was diluted 1:1000 in each well, for a total of 2 wells, and the average value was taken, 100µL / well; after immunization, serum in the 2 wells was diluted 1:100 or 1:1000, 100µL / well, and 2-fold gradient dilution was performed to the 10th well. The diluent was added to the remaining 2 wells as blank controls. The data of the final control wells was reflected as the average value of the two blank wells, 100µL / well; 2.3.7 Incubate at 37℃ for 1 hour, remove the ELISA plate, discard the inner solution, and wash the plate three times; 2.3.8 Add 100 µL of diluted enzyme-labeled secondary antibody to each well and incubate at 37°C for 0.5 h; 2.3.9 Take out the ELISA plate, discard the internal solution, wash the plate three times, pat dry, add 100 μL of colorimetric solution, react at 25°C for 15 min; add 50 μL of 1M HCl to terminate the reaction; read the OD450 nm on a ELISA reader.

[0056] 2.3.10 Results: After testing the sera of the immunized group, the serum titer was >1:512,000, 1:8,000 and OD450>1.0, meeting the fusion standard.

[0057] 3. Hybridoma Fusion 3.1 Prepare myeloma cell (SP2 / 0) single cell suspension.

[0058] 3.2 Spleen or lymph node tissue was extracted and ground and homogenized to produce a single cell suspension.

[0059] 3.3 The SP2 / 0 obtained in the above steps was mixed with spleen cells or lymph node cells, and fused with SP2 / 0 mouse myeloma cells using electrofusion technology. After the fusion was completed, the fused cells were resuspended in DMEM / 10% FBS medium containing hybridoma cell selection agents thymidine, hypoxanthine and aminopterin, and the cells were seeded into 96-well plates.

[0060] 3.4 Incubate the 96-well plate at 37°C in 5.5% CO 2 Culture in an incubator.

[0061] 3.5 After 5-8 days, count the fusion rate and screen positive clones.

[0062] 3.6 ELISA screening: Indirect ELISA is used to evaluate the ability of antibodies in the supernatant to bind to X. Sample preparation: Use a pipette to aspirate the culture medium in the culture plate to the ELISA plate coated with the antigen, add the enzyme-labeled antibody to react, and finally add TMB colorimetric solution to develop the color. After the reading is terminated, perform full-plate ELISA screening and confirmation screening, and select positive clones that meet the conditions to continue.

[0063] 3.7 Cell limited dilution subcloning: Observe the cell status and gently pipette the cells in the well to mix. Mix 20μL of cell suspension with 20μL of trypan blue. Take 20µL of the mixture and put it into a cell counting plate and count it using Count-star. Each clone is subcloned by limiting dilution, cultured in 1×HT medium, and plated into a 96-well plate at a density of 2-5 cells / well. Each plate is marked with the order number, clone ID and operation date. Incubate the 96-well plate at 37°C and 5.5% CO 2 After 1 week of incubation in the incubator, the clone supernatant was taken for the above-mentioned ELISA combination to evaluate the presence of antibodies against x.

[0064] The subcloning process was the same as above until a positive single clone was obtained.

[0065] 3.8 Subclass Detection 3.8.1 Coat goat anti-mouse Ig at a concentration of 0.5 µg / mL on a 96-well plate, 100 µL / well. Seal the plate and incubate.

[0066] 3.8.2 Wash the plate and add 150μL of blocking buffer to each well. Seal the plate and incubate. Add the sample and dilute with dilution buffer, 9 wells per sample, 100μL / well, seal the plate. Wash the plate 3 times.

[0067] 3.8.3 The dilution ratio of IgG2c secondary antibody is 1:2K, and the dilution ratio of other secondary antibodies is 1:500. Add 100µL / well to the plate. Seal the plate and incubate.

[0068] 3.8.4 After washing 3 times, discard the solution. After drying with a paper towel, add 100 µL of TMB substrate to each well and incubate at 25°C.

[0069] 3.8.5 Add 50 µL of stop buffer to each well to terminate the reaction. Read the plate at OD450nm on a microplate reader.

[0070] 3.10 Results 3.10.1 Mother clone screening results: Select the bold clones in Table 2 and subclone them into fixed strains.

[0071] Table 2

[0072]

[0073] 3.10.2 The results of screening of fixed clones are shown in Table 3: the titer test of the fixed clone 43D2F10E8 / 43D2F10C2 was negative, and the OD values ​​of other clones on the main detection source C1480GH240-1A:R92 decreased with the increase of the dilution gradient.

[0074] Table 3

[0075]

[0076] 4. Antibody Production 4.1 Cultivation The screened cell lines were cultured and expanded from 24-well plates to 10 cm cell culture dishes, and complete culture medium (DMEM + 10% FBS) was added. The cell status was observed. When the cell confluence in the dish reached more than 80%, the supernatant was discarded, and the cells were suspended in FEM culture medium and transferred to a shaking flask. The culture medium was adjusted to 0.5-0.7×10 5 Inoculate at a density of 100 cells / mL (half the volume); shake the flask to check if the cells are contaminated, and add FEM to the flask to the final volume; culture for several days, observe the cells in the flask, and check if the cells proliferate; when the activity rate is lower than 20%, collect samples and send them to downstream purification.

[0077] 4.2 Purification Remove the ProteinA magnetic beads from the refrigerator. Add the supernatant to the magnetic beads and incubate at room temperature for 2 hours. After incubation, equilibrate the magnetic beads with 10CV (column volume) of binding buffer. Wash the magnetic beads with elution buffer 1. Collect the elution buffer. Neutralize the eluted antibody by adding 1 / 10 volume of neutralization buffer. Filter the antibody through a 0.22µm filter membrane. Measure the antibody concentration using Nanodrop 2000.

[0078] 5. Detection of purified antibody and antigen binding ability 5.1 Dilute the antigen with coating solution, mix well and add to the designed strips, 100 µL / well; 5.2 After packaging, place in a 4℃ refrigerator overnight for 16 hours; 5.3 Discard the coating solution, wash the plate once, and pat the ELISA plate dry; 5.4 Add 150 µL of blocking solution to each well and incubate in a 37°C incubator for 1 h; 5.5 Take out the ELISA plate and discard the blocking solution; 5.6 The concentration of the first well of the QC test antibody is 1.0 μg / mL, and it is diluted 3 times to the 10th well. The diluent is added to the remaining 2 wells as a blank control. The final control well data is the average of the two wells, 100 μL / well; 5.7 Incubate at 37℃ for 1 h, remove the ELISA plate, discard the inner solution, and wash the plate three times; 5.8 Add 100 µL of diluted enzyme-labeled secondary antibody to each well and incubate in a 37°C incubator; 5.9 Take out the ELISA plate, discard the inner solution, wash the plate 3 times, pat dry, add 100 μL of colorimetric solution, react at 25℃ for 15 min; add 50 μL of 1M HCl to terminate the reaction; 450 nm readings.

[0079] Example 2 Purified Antibody ELISA Paired Test Report The reagents used in this example are shown in Table 4: Table 4 Packing original Unlabeled antibody Coating concentration 2.0 μg / mL Coating fluid Phosphate buffered saline Secondary Antibodies Streptavidin-HRP 1. Dilute the unlabeled antibody to 2 μg / mL with coating solution, mix well and add to the designed strips, 100 μL / well; 2. After coating, place in a 4℃ refrigerator overnight for 16 hours; 3. Discard the coating solution, wash the plate once, and pat the ELISA plate dry; 4. Add 150 µL of blocking solution to each well and incubate in a 37°C incubator for 1 hour; 5. Take out the ELISA plate and discard the blocking solution; 6. The detection concentration of sandwich antibody drug R92 is 100ng / mL, 10ng / mL, 1ng / mL, 0ng / mL; 100µL / well; 7. Incubate at 37℃ for 1 h, remove the ELISA plate, discard the inner solution, and wash the plate 3 times; 8. Add biotin-labeled detection antibody, 1 μg / mL, 100 μL / well; 9. Incubate at 37℃ for 1 h, remove the ELISA plate, discard the inner solution, and wash the plate 3 times; 10. Add 100 µL of diluted enzyme-labeled secondary antibody to each well and incubate in a 37°C incubator; 11. Take out the ELISA plate, discard the inner solution, wash the plate 3 times, pat dry, add 100 μL of colorimetric solution, react at 25℃ for 15 min; add 50 μL of 1M HCl to terminate the reaction; 450 nm readings.

[0080] 12. The results are shown in Table 5. When 38H9G8C12 was used as the coating antibody and 53C7B12C9 was used as the detection antibody, the signal was the strongest.

[0081] Table 5

[0082] Example 3 Sensitivity of purified antibody ELISA paired detection 1. Dilute 38H9G8C12 to 2.0 μg / mL with coating solution, mix well and add to the designed strips, 100 μL / well; 2. After coating, place in a 4℃ refrigerator overnight for 16 hours; 3. Discard the coating solution, wash the plate once, and pat the ELISA plate dry; 4. Add 150 µL of blocking solution to each well and incubate in a 37°C incubator for 1 h; 5. Take out the ELISA plate and discard the blocking solution; 6. The detection concentration of sandwich antibody drug is 512ng / mL in the first well, and then diluted 2-fold to 0.5ng / mL, 100 µL / well; 7. Incubate at 37℃ for 1 h, remove the ELISA plate, discard the inner solution, and wash the plate 3 times; 8. Add biotin-labeled antibody 53C7B12C9, 1.0 μg / mL, 100 μL / well; 9. Incubate at 37℃ for 1 h, remove the ELISA plate, discard the inner solution, and wash the plate 3 times; 10. Add 100 µL of diluted enzyme-labeled secondary antibody to each well and incubate in a 37°C incubator; 11. Take out the ELISA plate, discard the internal solution, wash the plate 3 times, pat dry and add 100 μL of colorimetric solution, react at 25°C for 15 min; add 50 μL of 1M HCl to terminate the reaction; read the OD450 nm on a ELISA reader.

[0083] 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 was determined by adding two times the standard deviation (SD) to the average OD 450 nm value of 24 zero standard replicates and calculating the corresponding concentration. The LOD of this method was 0.179 ng / ml.

[0084] If the effect of serum on the sample is not taken into account, the sensitivity on the plate can theoretically reach about 0.895 ng / ml after the sample is diluted 5 times, which is much lower than the target concentration of 2 ng / ml. This pair of antibodies can be used to establish a pharmacokinetic (PK) ELISA detection method for recombinant human anti-rabies virus monoclonal antibody R92.

[0085] Table 6

[0086] Table 7

[0087] Experimental Example 1 Methodological Investigation The test steps are as follows: (1) Coating: Add 100 μL / well of the diluted coating antibody 38H9G8C12 to the ELISA plate, seal the plate, and incubate overnight at 2-8°C; (2) Washing: Wash the plate once with washing solution, 300 μL / well / time; (3) Blocking: Add 200 μL / well of blocking solution, incubate at 25°C 750rpm for about 1 hour; (4) Washing: Wash the plate once with washing solution, 300 μL / well / time; (5) Add standard: Take the finished product of anti-rabies virus monoclonal antibody, of which R92 concentration is 0.5mg / ml; (6) Add sample: Add the diluted sample to the corresponding well, 100μL / well, incubate at 25℃750rpm for about 2h; (7) Wash the plate: Wash the plate 6 times with the washing solution, 300μL / well / time; (8) Add detection antibody working solution: Add 100μL / well of the diluted biotin-labeled detection antibody 53C7B12C9 to the ELISA plate, incubate at 25℃750rpm for about 1h; (9) Add secondary antibody working solution: Add 100μL / well of the diluted HRP-labeled Streptavidin HRP to the ELISA plate, incubate at 25℃ Incubate at 750 rpm for about 1 h; (10) Wash: Wash 6 times with washing solution, 300 μL / well / time; (11) Color development: Add 100 μL / well of TMB color development solution, incubate at room temperature in the dark for about 10 min, depending on the color development; (12) Stop: Add 50 μL / well of stop solution, read the OD value at 450 nm and save the result.

[0088] The test used cynomolgus monkey serum as the experimental system, and the standard curve, precision and accuracy, selectivity, specificity, dilution linearity, hook effect, stability, and parallelism of the detection method were fully validated. The validation results are shown in Table 8: Table 8

[0089] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A pair of anti-idiotypic antibodies for ELISA detection, characterized in that The idiotype antibody is used to specifically detect R92 in the anti-rabies virus monoclonal antibody injection, and the anti-idiotype antibody includes the coating antibody 38H9G8C12; The coating antibody 38H9G8C12 contains any one or more of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; or any one or more amino acid sequences having at least 85% sequence similarity with SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; and / or Contains any one or more of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, or any one or more amino acid sequences having at least 85% sequence similarity with SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6, respectively.

2. A pair of anti-idiotypic antibodies for ELISA detection, characterized in that The idiotype antibody is used to specifically detect R92 in the anti-rabies virus monoclonal antibody injection, and the anti-idiotype antibody includes the detection antibody 53C7B12C9; The detection antibody 53C7B12C9 contains any one or more of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9, or any one or more amino acid sequences having at least 85% sequence similarity with SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 respectively; and / or Contains any one or more of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, or any one or more amino acid sequences having at least 85% sequence similarity with SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, respectively.

3. The anti-idiotypic antibody according to claim 1, characterized in that The coated antibody 38H9G8C12 contains a heavy chain variable region having a sequence as shown in SEQ ID NO.13, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO.13; and / or contains a light chain variable region as shown in SEQ ID NO.14, or any one or more amino acid sequences having at least 85% sequence similarity to SEQ ID NO.

14.

4. The anti-idiotypic antibody according to claim 2, characterized in that The detection antibody 53C7B12C9 contains a heavy chain variable region 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 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.

5. The anti-idiotypic antibody according to claim 3, characterized in that The coating antibody 38H9G8C12 contains a heavy chain as 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 contains a light chain as 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.

6. The anti-idiotypic antibody according to claim 4, characterized in that The detection antibody 53C7B12C9 contains a heavy chain as shown in SEQ ID NO.19, or any one or more amino acid sequences having at least 85% sequence similarity with SEQ ID NO.19, and / or contains a light chain as shown in SEQ ID NO.20, or any one or more amino acid sequences having at least 85% sequence similarity with SEQ ID NO.

20.

7. The anti-idiotypic antibody according to any one of claims 1 to 6, characterized in that The antibody is a monoclonal antibody.

8. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-idiotypic antibody according to any one of claims 1-6.

9. A biological expression vector, characterized in that: The biological expression vector comprises the nucleic acid molecule according to claim 8.

10. A host cell, characterized in that The host cell comprises the nucleic acid molecule according to claim 8 and / or the biological expression vector according to claim 9.

11. Use of the anti-idiotypic antibody according to any one of claims 1 to 6 in the pharmacokinetics evaluation of R92 in anti-rabies virus monoclonal antibody injection.

12. Use of the anti-idiotypic antibody according to any one of claims 1 to 6 in the preparation of a kit for detecting R92 in an anti-rabies virus monoclonal antibody injection.

13. A kit, characterized in that: The kit comprises the anti-idiotypic antibody according to any one of claims 1-6.

14. A method for detecting the pharmacokinetics of R92 by ELISA double antibody sandwich method, characterized in that: The method uses 38H9G8C12 as a coating antibody and / or 53C7B12C9 as a detection antibody.

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