Anti-monkey interleukin-6 monoclonal antibody, enzyme-linked immunosorbent assay kit and application thereof

Abstract

The present invention belongs to the field of biological detection technology, and specifically relates to an anti-monkey interleukin-6 monoclonal antibody, an enzyme-linked immunosorbent assay kit and its application. Named IL-6-12, IL-6-12 includes a heavy chain variable region and a light chain variable region; the heavy chain variable region has three complementary determining regions CDR-H1 to CDR-H3, and the amino acid sequence is shown in SEQ ID NO.6-SEQ ID NO.8; the light chain variable region has three complementary determining regions CDR-L1 to CDR-L3, and the amino acid sequence is shown in SEQ ID NO.9-SEQ ID NO.11. The monoclonal antibody of the present invention can specifically bind to monkey interleukin-6, and the above-mentioned monoclonal antibody is used to screen a pairable monoclonal antibody. Based on the combination of the monoclonal antibodies, a monkey interleukin-6 enzyme-linked immunosorbent assay kit is obtained, which can detect the content of monkey interleukin-6 in monkey serum, plasma or tissue fluid.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to an anti-monkey interleukin-6 monoclonal antibody, an enzyme-linked immunosorbent assay kit and applications thereof. Background Art

[0002] Infectious diseases are extremely common in clinical practice, and laboratory auxiliary examinations are crucial for the diagnosis and treatment of infectious diseases. The four most commonly used indicators are C-reactive protein (CRP), procalcitonin (PCT), serum amyloid A (SAA), and interleukin-6 (IL-6).

[0003] Interleukin-6 is a lymphokine secreted by activated inflammatory cells, which can promote the proliferation and differentiation of lymphocytes, as well as the proliferation and division of hematopoietic stem cells, and can play an immunomodulatory role. If the level of interleukin-6 in the body increases, it may be caused by a decrease in the body's immunity due to bacterial infection, viral infection, etc., or it may be caused by diseases such as systemic lupus erythematosus and rheumatoid arthritis.

[0004] Laboratory animals are the main experimental subjects for preclinical evaluation of drugs, and monkeys are an important species in preclinical research. The application of laboratory animals in the development of new drugs covers both preclinical and clinical research stages, especially in preclinical research. Different experimental methods are used to evaluate the pharmacological effects of drugs, study their mechanisms of action, and observe their toxic effects to prove the effectiveness and safety of drugs. Monkeys are the closest to humans in terms of kinship, with 75% to 98.5% homology with human genetic material. They are the animals with the highest homology with humans, and therefore are the ideal animals closest to humans in experiments.

[0005] Currently, there is no monkey interleukin-6 enzyme-linked immunosorbent assay kit that can detect the content of monkey interleukin-6 in monkey serum, plasma or tissue fluid. Summary of the invention

[0006] In order to solve the above problems, one of the objects of the present invention is to provide an anti-monkey interleukin-6 monoclonal antibody, an enzyme-linked immunosorbent assay kit and its application, which can specifically bind to monkey interleukin-6, and use the above monoclonal antibody to screen for paired monoclonal antibodies, and further construct a monkey interleukin-6 enzyme-linked immunosorbent assay kit based on the pair of monoclonal antibodies for the detection of the monkey interleukin-6 content in monkey serum, plasma or tissue fluid.

[0007] In a first aspect of the present invention, an anti-monkey interleukin-6 monoclonal antibody is provided.

[0008] Named IL-6-12, the IL-6-12 includes a heavy chain variable region and a light chain variable region;

[0009] The heavy chain variable region has three complementary determining regions, the amino acid sequence of CDR-H1 is shown in SEQ ID NO.6, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.7, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.8;

[0010] The light chain variable region has three complementary determining regions, the amino acid sequence of CDR-L1 is shown in SEQ ID NO.9, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.10, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.11.

[0011] Furthermore, the amino acid sequence of the heavy chain variable region of the IL-6-12 is shown as SEQ ID NO.3, and the amino acid sequence of the light chain variable region of the IL-6-12 is shown as SEQ ID NO.4.

[0012] Furthermore, the monoclonal antibody further comprises:

[0013] An antibody having the same function obtained by replacing, deleting and / or adding one or more amino acids to the amino acid sequence of the monoclonal antibody; or a heavy chain variable region comprising an amino acid sequence having at least 80% homology with the heavy chain variable region; and a light chain variable region having an amino acid sequence having at least 80% homology with the light chain variable region; or an antibody obtained by connecting a tag to the N-terminus and / or C-terminus of the monoclonal antibody.

[0014] In other embodiments, V H and / or V L The amino acid sequence may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the above sequence. H and V L The region is highly (i.e. 80% or more) homologous to the V H and V L Antibodies to the regions can be obtained by mutagenizing (eg, site-directed mutagenesis or PCR-mediated mutagenesis) nucleic acid molecules encoding SEQ ID NOs. 6-11, and then testing the encoded altered antibodies for retained function using the functional assays described herein.

[0015] In the second aspect of the present invention, there is provided a use of the anti-monkey interleukin-6 monoclonal antibody in the preparation of a monkey interleukin-6 reactive protein detection reagent or kit.

[0016] In a third aspect of the present invention, a monkey interleukin-6 enzyme-linked immunosorbent assay kit is provided, the kit comprising two anti-monkey interleukin-6 monoclonal antibodies, IL-6-4 and IL-6-12, wherein the monoclonal antibody IL-6-4 is used as a coating antibody, and the monoclonal antibody IL-6-12 is used as a detection antibody;

[0017] The IL-6-4 includes a heavy chain variable region and a light chain variable region, the heavy chain variable region has three complementarity determining regions, the amino acid sequence of its CDR-H1 is shown in SEQ ID NO.12, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.13, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.14; the light chain variable region has three complementarity determining regions, the amino acid sequence of its CDR-L1 is shown in SEQ ID NO.15, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.16, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.17.

[0018] Furthermore, the amino acid sequence of the IL-6-4 heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the IL-6-4 light chain variable region is shown in SEQ ID NO.2.

[0019] Furthermore, the monoclonal antibody further comprises:

[0020] An antibody having the same function obtained by replacing, deleting and / or adding one or more amino acids to the amino acid sequence of the monoclonal antibody; or a heavy chain variable region comprising an amino acid sequence having at least 80% homology with the heavy chain variable region; and a light chain variable region having an amino acid sequence having at least 80% homology with the light chain variable region; or an antibody obtained by connecting a tag to the N-terminus and / or C-terminus of the monoclonal antibody.

[0021] In other embodiments, V H and / or V L The amino acid sequence may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the above sequence. H and V L The region is highly (i.e. 80% or more) homologous to the V H and V L Antibodies to the regions can be obtained by mutagenizing (eg, site-directed mutagenesis or PCR-mediated mutagenesis) nucleic acid molecules encoding SEQ ID NOs. 12-17, and then testing the encoded altered antibodies for retained function using the functional assays described herein.

[0022] Furthermore, the monoclonal antibodies IL-6-4 and IL-6-12 are both obtained by immunizing monkey interleukin-6 recombinant protein.

[0023] Furthermore, the amino acid sequence of the monkey interleukin-6 recombinant protein is shown in SEQ ID NO.5.

[0024] Furthermore, the monkey interleukin-6 recombinant protein is obtained by selecting and optimizing a segment of monkey interleukin-6 polypeptide using bioinformatics software, and the segment can be expressed in Escherichia coli according to prediction analysis.

[0025] The invention provides a monkey interleukin-6 protein recombinant protein, and realizes the efficient expression of monkey interleukin-6 in Escherichia coli by optimizing antigen peptide sequence and expression vector.

[0026] In a fourth aspect, the present invention provides a use of the monkey interleukin-6 enzyme-linked immunosorbent assay kit, wherein the kit is used to detect the content of monkey interleukin-6 in monkey serum, plasma or tissue fluid.

[0027] The beneficial effects of the present invention are:

[0028] (1) The monoclonal antibody of the present invention can specifically bind to monkey interleukin-6 with high specificity.

[0029] (2) The present invention screened out a monoclonal antibody that specifically binds to monkey interleukin-6 based on monkey interleukin-6 recombinant protein, and used the above monoclonal antibody to screen a pairing monoclonal antibody, and further constructed a monkey interleukin-6 enzyme-linked immunosorbent assay kit based on the monoclonal antibody pair, which is used to detect the content of monkey interleukin-6 in monkey serum, plasma or tissue fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Shown is the standard curve graph of monkey interleukin-6 ELISA detection of the present invention. DETAILED DESCRIPTION

[0031] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0032] Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without making any creative work are within the scope of protection of the present invention.

[0033] The above technical solution is described in detail below in conjunction with specific embodiments.

[0034] Example 1

[0035] Preparation of recombinant antigens:

[0036] This embodiment provides a monkey interleukin-6 recombinant protein, whose amino acid sequence is shown in SEQ ID No. 5:

[0037] MNSFSTSAFGPVFESLGLLLPAAFPAVLPGEDSKNQPAPHSQPLTSSERIRSHIRYILDGISALRKETSERSNMCESSKRENNLNLPKMAEFRGCFQSGEQEDTCLVLLEFEVYLEYLQNRFESSEEQARTTSGSTKVLIQFLQKKAKNLDTPEPTTNASLLTKLQAQNQWLKNMTTHLILRSFKLFLQSNLRALRQM.

[0038] This embodiment provides a method for preparing monkey interleukin-6 recombinant protein, comprising the following steps:

[0039] (1) Plasmid construction: Computational software was used to predict and select the main antigenic epitopes of monkey interleukin-6 protein for expression. This polypeptide segment can be expressed in the Escherichia coli prokaryotic expression system. The gene was cloned into the pET28a vector via the E. coli expression system to obtain the recombinant plasmid pET28a-IL-6 expressing monkey interleukin-6 protein.

[0040] (2) Induced expression: The recombinant plasmid pET28a-IL-6 was transformed into BL21 (DE3) competent cells, and the transformed bacteria were spread on LB agar plates containing 50 μg / mL kanamycin and cultured at 37°C overnight. A single colony was picked and inoculated into 4 mL of LB medium containing 50 μg / mL kanamycin, and cultured at 37°C and 230 rpm overnight. 1% of the total volume of the culture medium was inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37°C and 220 rpm for about 3 hours until the OD600 reached 0.6. A final concentration of 0.1 mM IPTG was added, and the bacteria were collected after induction at 16°C and 180 rpm for 15 hours.

[0041] (3) Purification of recombinant protein: Since the expressed recombinant protein carries a histidine tag, it is initially purified using a protein purifier and a chromatography column, and then purified using ion exchange chromatography to obtain the monkey interleukin-6 recombinant protein, whose amino acid sequence is shown in SEQ ID No. 5:

[0042] MNSFSTSAFGPVFESLGLLLPAAFPAVLPGEDSKNQPAPHSQPLTSSERIRSHIRYILDGISALRKETSERSNMCESSKRENNLNLPKMAEFRGCFQSGEQEDTCLVLLEFEVYLEYLQNRFESSEEQARTTSGSTKVLIQFLQKKAKNLDTPEPTTNASLLTKLQAQNQWLKNMTTHLILRSFKLFLQSNLRALRQM.

[0043] (4) Identify the purity of the recombinant protein by SDS-PAGE electrophoresis: Protein sample pretreatment: add an equal volume of 2×SDS loading buffer to each sample, boil in a boiling water bath for 10 min, centrifuge at 12,000 rpm for 3 min, and add the supernatant to the sample loading well.

[0044] Example 2

[0045] Screening of monoclonal antibodies:

[0046] This embodiment provides a method for obtaining monoclonal antibodies based on the recombinant protein monkey interleukin-6 screening, and the specific operations are as follows:

[0047] S1) Mouse immunization 50 μg of monkey interleukin-6 recombinant protein was mixed with an equal volume of Freund's complete adjuvant to obtain a mixture, which was then injected subcutaneously at multiple points into female BALB / c mice for 6 weeks. At week 0 and week 2, Freund's incomplete adjuvant was mixed with the recombinant protein in equal proportions and emulsified according to the above method, and then immunized again. At week 6, mouse serum was collected to test the antibody titer. The mouse with the highest titer was selected and boosted with 20 μg of monkey interleukin-6 recombinant protein through the tail vein. After 3 days, the spleen of the mouse was taken for the preparation of hybridoma cells.

[0048] S2) Screening of hybridoma cell lines:

[0049] All spleen cells of immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and placed in HAT medium for screening and culture. When the fused cells grew to 1 / 2 of the bottom of the well, positive clones were screened by indirect ELISA of monkey interleukin-6 protein, and the positive rate was 100% through 2-3 consecutive limiting dilution cloning methods, and the cell lines were expanded and frozen; the method of screening positive clones by indirect ELISA is as follows: first, the recombinant protein was coated in the microplate: the coating buffer was carbonate buffer (1.59g sodium carbonate, 2.93g sodium bicarbonate, fixed to 1L pure water), the coating concentration was 1μg / mL, 50μl / well, 4℃ overnight; 3% sucrose, 1% BSA blocking, 150μL per well, 37°C for 2 hours, and the plate was washed once with washing buffer PBST (PBS containing 0.05% Tween-20), patted dry; 50μL cell culture supernatant was added, and the reaction was 37℃ for 30min. Shake out the liquid in the wells, wash the plate 4 times with PBST washing solution, pat dry, add 50 μL / well of HRP-labeled goat anti-mouse (diluted 1:5000 in PBS), react at 37℃ for 30 min, wash the plate 4 times again, pat dry, add 50 μL / well of TMB color developing solution, develop at room temperature for 10 min, finally add 0.5 M sulfuric acid as a terminator to terminate the reaction, and measure the OD450nm value with an enzyme reader.

[0050] S3) Preparation of monoclonal antibody ascites:

[0051] After the selected monoclonal cell lines were expanded and cultured, 0.2 mL (containing 2.5×106 cells) pre-treated with Freund's incomplete adjuvant was injected into the peritoneum of female BALB / c mice. About 10 days later, when the abdomen of the mice was obviously swollen, ascites was collected using a sterile syringe needle. The collected ascites was centrifuged at 3000r / min for 10 minutes to collect the middle layer.

[0052] S4) Purification of monoclonal antibodies:

[0053] Centrifuge the ascites at 12000r / min for 5 minutes, take 5mL of the supernatant, add 20ml of acetate buffer (68mmol / L, pH 4.5), mix well, slowly add 50μL of octanoic acid, stir while adding, continue stirring for 30 minutes after adding, centrifuge at 12000r / min for 30 minutes at 2-8℃, and take the supernatant. Filter the supernatant with absorbent cotton, add saturated ammonium sulfate at a ratio of 50% (V / V) of the final volume, stir while adding, continue stirring for 30 minutes after adding, let it precipitate at 2-8℃ for 4-5 hours, centrifuge at 12000r / min for 30 minutes at 2-8℃, and take the precipitate. The precipitate was resuspended with 3 ml Tris-HCl (10 mmol / L, pH 9.0), placed in a dialysis bag (MW: 8000-14000), and dialyzed in 2 L Tris-HCl (10 mmol / L, pH 9.0) solution at 2-8°C for 14 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 r / min for 5 minutes. The supernatant was the purified monoclonal antibody. The concentration of the purified monoclonal antibody was measured with an ultra-micro spectrophotometer and stored at -20°C to avoid repeated freezing and thawing.

[0054] S5) Identification of binding activity of monoclonal antibodies:

[0055] The indirect ELISA method was used to coat monkey interleukin-6 recombinant protein and control carrier protein (monkey PCT recombinant protein), respectively. The purified antibodies were tested by ELISA to identify the affinity of the monoclonal antibody. The antibody was diluted to 1 μg / ml. The results are shown in Table 1. "Ctrl" in Table 1 represents the negative control monkey PCT mouse monoclonal antibody.

[0056] Table 1 Binding activity identification of monoclonal antibodies

[0057]

[0058] As can be seen from Table 1, the binding activity of the 18 selected monkey interleukin-6 monoclonal antibodies to monkey interleukin-6 protein and control carrier protein was detected by indirect ELISA. The data showed that the monoclonal antibodies could specifically bind to monkey interleukin-6 protein.

[0059] Example 3

[0060] Screening and application of paired monoclonal antibodies:

[0061] 1. HRP labeling of antibodies:

[0062] The screened monoclonal antibodies were labeled with HRP according to the classic sodium periodate method. Finally, the labeled antibodies were dialyzed overnight in 0.01M PBS, pH 7.4 buffer, glycerol was added at a volume of 1:1, and the antibodies were stored in separate devices at -20°C.

[0063] 2. Establishment of double antibody sandwich immunoassay method:

[0064] The purified monoclonal antibody was diluted to a concentration of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL with coating buffer (1.59 g sodium carbonate and 2.93 g sodium bicarbonate, fixed to 1 L pure water, pH 9.6), and added to the ELISA plate at 50 μL / well for overnight coating at 4°C. The coating solution was discarded the next day, and the plate was washed once with washing solution (PBST, PBS containing 0.05% Tween-20), patted dry, and blocked with 3% sucrose and 1% BSA at 150 μL / well. Incubated at 37°C for 2 h, the blocking solution was discarded, and patted dry. The monkey interleukin-6 recombinant protein of the test sample and the monkey PCT protein of the control sample were diluted with PBS at 2μg / mL, 1μg / mL, and 0.5μg / mL, and then added to the ELISA plate at 50μL / well, incubated at 37°C for 35min, washed 4 times with PBST washing solution, patted dry, and enzyme-labeled monoclonal antibody diluted to 500, 1000, and 2000 times with PBS was added at 50μL / well, incubated at 37°C for 35min, washed 4 times, patted dry, and TMB color developing solution was added at 50μL / well, and color was developed at room temperature for 10min. Finally, 0.5M sulfuric acid was added at 50μL / well to terminate the reaction, and the OD450nm value was measured with an ELISA reader.

[0065] Then, the most suitable reaction conditions were determined by adjusting the dilution of the coating antibody and HRP antibody.

[0066] Specifically: monoclonal antibody was coated in a microplate (coating buffer carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate fixed to 1 L pure water), the coating concentration gradient was 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 50 μL / well, 4°C overnight; the plate was washed once with washing solution (PBST, PBS containing 0.05% Tween-20), patted dry, blocked with 3% sucrose, 1% BSA, 150 μL per well, 37°C for 2 hours, patted dry and stored for later use; monkey interleukin-6 recombinant protein was diluted to a concentration of 10 ng / ml with PBS, 50 μL was added to the microplate coated with monoclonal antibody, and PCT protein was diluted to 10 ng / ml as a negative control, and reacted at 37°C for 30 min. The liquid in the wells was shaken off, the plate was washed 4 times with PBST washing solution, and patted dry; the HRP-labeled monoclonal antibody was diluted with PBS to a concentration of 1μg / mL, 2μg / mL, and 4μg / mL, and 50μL / well was added, reacted at 37℃ for 30min, and the plate was washed 4 times again, and after patting dry, 50μL / well of TMB colorimetric solution was added to develop at room temperature for 10min, and finally the stop solution was added to terminate the reaction, and the OD450nm value was measured with an enzyme reader. The coating conditions with the most obvious positive and negative differences and the optimal reaction conditions for the HRP-labeled antibody were selected.

[0067] Through the above-mentioned coating and HRP labeling of monoclonal antibodies, the best pairing combination was selected based on the detection of positive and negative samples. The final determination was: the concentration of coated monoclonal antibody IL-6-4 was 1μg / mL, and the concentration of HRP-labeled monoclonal antibody IL-6-12 was 3μg / mL.

[0068] 3. After sequencing:

[0069] IL-6-4 monoclonal antibody, including heavy and light chains:

[0070] The amino acid sequence of the IL-6-4 heavy chain variable region is shown in SEQ ID NO.1, specifically as follows: EVQLQESGPGLVKPSQTLSLTCTVSGTGSRTDYTAWSWIRQPPGKGLEWMGVIRRFLSTYYSPSLKSRTSISWDTSKNQFSLQLSSVTPEDTAVYYCTTNFDRVRSGTDGYWGQGTQVTVSS, the complementary determining region CDR-H1 amino acid sequence GTGSRTDYTA (SEQ ID NO.12), the complementary determining region CDR-H2 amino acid sequence IRRFLST (SEQ ID NO.13), and the complementary determining region CDR-H3 amino acid sequence TTNFDRVRSGTDGY (SEQ ID NO.14).

[0071] The amino acid sequence of the IL-6-4 light chain variable region is shown in SEQ ID NO.2, specifically as follows: QAVLTQPPLVSGTPGQTVTISCAGAQQGYTDYNYVSWYQQLPGTAPKLLIYKVTTRASGIPSRFSGSKSGNTASLTISGLQSEDEADYYCAQTDNRFSSVFGRGTHLTVL, the complementary determining region CDR-L1 amino acid sequence QQGYTDYNY (SEQ IDNO.15), the complementary determining region CDR-L2 amino acid sequence YKVTT (SEQ ID NO.16), and the complementary determining region CDR-L3 amino acid sequence AQTDNRFSSV (SEQ ID NO.17).

[0072] IL-6-12 monoclonal antibody, including heavy and light chains:

[0073] The amino acid sequence of the IL-6-12 heavy chain variable region is shown in SEQ ID NO.3, specifically as follows: QVTLKESGPTLVKPTQTLTLTCSFSGFRRSTDVDGVGWVRQPPGKALEWLAFISSGRYTYYSPSLESRLTITKDTSKNQVVLTMTNMDPVDTATYYCANLTDYRRTNSGYTWGQGTLVT, the complementary determining region CDR-H1 amino acid sequence GFRRSTDVDG (SEQ ID NO.6), the complementary determining region CDR-H2 amino acid sequence ISSGRYT (SEQ ID NO.7), and the complementary determining region CDR-H3 amino acid sequence ANLTDYRRTNSGYT (SEQ ID NO.8).

[0074] The amino acid sequence of the light chain variable region of the IL-6-12 is shown in SEQ ID NO.4, specifically as follows: DIQMTQSPSSLSAFVGDGVTMTCWASQTRFRYLNWYHQRPGEAPELLVFAASNLQIGVPSRFRGSGSETYFTLTINSLQPEDSGTYFCQGYTRSRWTFGGGTKLEI, the complementary determining region CDR-L1 amino acid sequence QTRFRY (SEQ ID NO.9), the complementary determining region CDR-L2 amino acid sequence FAASN (SEQ ID NO.10), and the complementary determining region CDR-L3 amino acid sequence QGYTRSRWT (SEQ ID NO.11).

[0075] 4. Linear detection based on double antibody sandwich ELISA detection method:

[0076] Referring to the above detection steps, the monkey interleukin-6 fixed value standard protein was first diluted with PBS buffer solution. The concentrations after dilution were 500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL, 32.3pg / mL, 16.2pg / mL, 8.1pg / mL, and 0pg / mL, respectively. 50μL of the diluted standard protein of different concentrations was taken for detection. Three parallel experiments were performed for each concentration to prepare a standard curve. The standard curve is as follows: Figure 1 As shown: According to the four-parameter fitting results, the linearity reaches 0.999.

[0077] 5. Sensitivity test based on double antibody sandwich ELISA test method:

[0078] Referring to the detection steps of Example 4, the present invention was used to first dilute the monkey interleukin-6 fixed value standard protein with a PBS buffer solution, and the concentrations after dilution were 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.3 pg / mL, 15.6 pg / mL, 7.8 pg / mL, and 0 pg / mL, respectively. 50 μL of the diluted standard protein of different concentrations and 20 PBS buffers were taken for detection, and a sensitivity test was performed. The standard curve and PBS buffer values ​​are shown in Table 2. The sensitivity of the double antibody sandwich ELISA detection method was calculated to be 3.2 pg / mL according to the formula "mean + 2SD".

[0079] Table 2: Sensitivity test data

[0080]

[0081] 6. Identification of monkey serum samples based on double antibody sandwich ELISA detection method:

[0082] Referring to the above detection steps, 16 uninfected monkey sera and 16 infected monkey sera were measured and identified based on the double antibody sandwich ELISA detection method, and the OD values ​​were measured as shown in Table 3:

[0083] Table 3 Results of the double antibody sandwich ELISA test on 16 non-infected monkey sera and 16 infected monkey sera

[0084]

[0085] From the test results, it can be seen that there are significant differences between infected and uninfected.

[0086] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. An anti-monkey interleukin-6 monoclonal antibody, characterized in that: Named IL-6-12, the IL-6-12 includes a heavy chain variable region and a light chain variable region; The heavy chain variable region has three complementary determining regions, the amino acid sequence of CDR-H1 is shown in SEQ ID NO.6, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.7, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.8; The light chain variable region has three complementary determining regions, the amino acid sequence of CDR-L1 is shown in SEQ ID NO.9, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.10, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

11.

2. The anti-monkey interleukin-6 monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the IL-6-12 heavy chain variable region is shown in SEQ ID NO.3, and the amino acid sequence of the IL-6-12 light chain variable region is shown in SEQ ID NO.

4.

3. A monkey interleukin-6 enzyme-linked immunosorbent assay kit, characterized in that: The kit comprises two anti-monkey interleukin-6 monoclonal antibodies, IL-6-12 and IL-6-4, as claimed in claim 1 or 2, wherein the monoclonal antibody IL-6-4 is used as a coating antibody, and the monoclonal antibody IL-6-12 is used as a detection antibody; The IL-6-4 includes a heavy chain variable region and a light chain variable region, the heavy chain variable region has three complementarity determining regions, the amino acid sequence of its CDR-H1 is shown in SEQ ID NO.12, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.13, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.14; the light chain variable region has three complementarity determining regions, the amino acid sequence of its CDR-L1 is shown in SEQ ID NO.15, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.16, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

17.

4. The monkey interleukin-6 enzyme-linked immunosorbent assay kit according to claim 3, characterized in that: The amino acid sequence of the IL-6-4 heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the IL-6-4 light chain variable region is shown in SEQ ID NO.

2.

5. The monkey interleukin-6 enzyme-linked immunosorbent assay kit according to claim 3, characterized in that: The monoclonal antibodies IL-6-4 and IL-6-12 are both obtained by immunizing monkey interleukin-6 recombinant protein.

6. The monkey interleukin-6 enzyme-linked immunosorbent assay kit according to claim 5, characterized in that: The amino acid sequence of the monkey interleukin-6 recombinant protein is shown in SEQ ID NO.5.

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