Anti-monkey high-mobility group protein B1 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 a monkey high-mobility group protein B1 monoclonal antibody, an enzyme-linked immunosorbent assay kit and its application. Named HMGB1-11, it 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 sequences are 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 sequences are shown in SEQ ID NO.9-SEQ ID NO.11. The monoclonal antibody of the present invention can specifically bind to monkey high-mobility group protein B1, and the monoclonal antibody can be paired with a monoclonal antibody after screening. Based on the monoclonal antibody combination, a monkey high-mobility group protein B1 enzyme-linked immunosorbent assay kit is constructed, which can detect the content of monkey high-mobility group protein B1 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 high-mobility group protein B1 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 most commonly used indicators are C-reactive protein (CRP), procalcitonin (PCT), serum amyloid A (SAA), interleukin-6 (IL-6), and high mobility group protein B1 (HMGB1).

[0003] Inducing inflammatory response Recent studies have found that once HMGB1 is secreted outside the cell, it can play an inflammatory role. It is now believed that HMGB1 is an important late inflammatory factor and has more important clinical significance than early rapid inflammatory factors such as TNF and IL-1. Mechanically damaged and necrotic cells can release nuclear HMGB1 to the extracellular space to induce inflammatory response. The co-culture of damaged cells with macrophages can cause the nuclear translocation of macrophage nuclear transcription factor-κB (NF-κB) and produce an inflammatory response similar to that caused by tissue necrosis; the co-culture of damaged cells from HMGB1 gene knockout mice with macrophages significantly reduced the intensity of the inflammatory response. This shows that the passive release of HMGB1 plays an important role in tissue necrosis inflammatory response. Histones in apoptotic cells are generally under-acetylated, so HMGB1 in apoptotic cells can be tightly bound to nucleosomes and will not be released, nor will it cause an inflammatory response; on the contrary, if the deacetylation in the cell is inhibited, an inflammatory response will occur. Endotoxin and a variety of inflammatory factors can induce the release of HMGB1 to mediate inflammatory response. A series of experimental studies have shown that HMGB1 can act as an endogenous pyrogen in the central nervous system and mediate the central inflammatory response. When cells are necrotic or damaged, HMGB1 in the nucleus can be released to the extracellular space, triggering mononuclear macrophages to secrete proinflammatory factors; and proinflammatory factors in turn promote the secretion of HMGB1, thus forming a positive feedback loop. In the later stages of the inflammatory response, this positive feedback effect plays a very important role in maintaining the inflammatory response.

[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 high-mobility group protein B1 enzyme-linked immunosorbent assay kit that can detect the content of monkey high-mobility group protein B1 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 high-mobility group protein B1 monoclonal antibody, an enzyme-linked immunosorbent assay kit and its application, which can specifically bind to monkey high-mobility group protein B1, and use the above monoclonal antibody to screen for paired monoclonal antibodies, and further construct a monkey high-mobility group protein B1 enzyme-linked immunosorbent assay kit based on the pair of monoclonal antibodies, which is used for the detection of the monkey high-mobility group protein B1 content in monkey serum, plasma or tissue fluid.

[0007] In the first aspect of the present invention, an anti-monkey high mobility group protein B1 monoclonal antibody is provided. Named as HMGB1-11, HMGB1-11 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.

[0008] Furthermore, the amino acid sequence of the heavy chain variable region of the HMGB1-11 is shown as SEQ ID NO.3, and the amino acid sequence of the light chain variable region of the HMGB1-11 is shown as SEQ ID NO.4.

[0009] Furthermore, the monoclonal antibody further comprises: 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.

[0010] In other embodiments, V H and / or V LThe 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.

[0011] The second aspect of the present invention provides a use of the anti-monkey high-mobility group protein B1 monoclonal antibody in the preparation of a monkey high-mobility group protein B1 detection reagent or kit.

[0012] The third aspect of the present invention provides a monkey high mobility group protein B1 enzyme-linked immunosorbent assay kit, The kit comprises two anti-monkey high mobility group protein B1 monoclonal antibodies HMGB1-5 and HMGB1-11, wherein the monoclonal antibody HMGB1-5 is used as a coating antibody, and the monoclonal antibody HMGB1-11 is used as a detection antibody; The HMGB1-5 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 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 complementary 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.

[0013] Furthermore, the amino acid sequence of the HMGB1-5 heavy chain variable region is shown as SEQ ID NO.1, and the amino acid sequence of the HMGB1-5 light chain variable region is shown as SEQ ID NO.2.

[0014] Furthermore, the monoclonal antibody further comprises: 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.

[0015] 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.

[0016] Furthermore, the monoclonal antibodies HMGB1-5 and HMGB1-11 are both obtained by immunizing monkeys with high mobility group protein B1 recombinant protein.

[0017] Furthermore, the monkey high-mobility group protein B1 recombinant protein is a segment of monkey high-mobility group protein B1 polypeptide selected and optimized using bioinformatics software, and the segment can be expressed in Escherichia coli according to prediction analysis.

[0018] The invention provides a monkey high-mobility group protein B1 recombinant protein, and realizes the efficient expression of the monkey high-mobility group protein B1 in Escherichia coli by optimizing antigen peptide sequence and expression vector.

[0019] Furthermore, the amino acid sequence of the monkey high mobility group protein B1 recombinant protein is shown in SEQ ID NO.5.

[0020] The fourth aspect of the present invention provides an application of the monkey high-mobility group protein B1 enzyme-linked immunosorbent assay kit, wherein the kit is used to detect the content of monkey high-mobility group protein B1 in monkey serum, plasma or tissue fluid.

[0021] The beneficial effects of the present invention are: (1) The monoclonal antibody of the present invention can specifically bind to monkey high mobility group protein B1 and has high specificity.

[0022] (2) The present invention screened out a monoclonal antibody that specifically binds to monkey high-mobility group protein B1 based on the monkey high-mobility group protein B1 recombinant protein, and used the above monoclonal antibody to screen a pairing monoclonal antibody, and further constructed a monkey high-mobility group protein B1 enzyme-linked immunosorbent assay kit based on the monoclonal antibody pair, which is used to detect the content of monkey high-mobility group protein B1 in monkey serum, plasma or tissue fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Shown is a graph showing the detection standard of the monkey high mobility group protein B1 ELISA of the present invention. DETAILED DESCRIPTION

[0024] 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.

[0025] 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.

[0026] The above technical solution is described in detail below in conjunction with specific embodiments.

[0027] Example 1

[0028] Preparation of recombinant antigens: This embodiment provides a monkey high mobility group protein B1 recombinant protein, the amino acid sequence of which is shown in SEQ ID NO.5: MGKGDPREARGKMSSYAFFVQTCREEHKEFHPDASVNFSEFSSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKGSFKDPNAPKRPPSAFFLFCSEYRPPGLSIGDVAKKLGEMWNNKEDRDKQPYEKKAAKLKEKPEKDIAAYRAKGKKSAAKKGVVKAEKSKKKKEEEEDEEDEEEEEEDEEDEDEEEDDDDE.

[0029] This embodiment provides a method for preparing a monkey high mobility group protein B1 recombinant protein, comprising the following steps: (1) Plasmid construction: The computational software was used to predict and select the main antigenic epitopes of monkey high-mobility group protein B1 for expression. The polypeptide segment can be expressed in the E. coli prokaryotic expression system. The gene was cloned into the pET28a vector via the E. coli expression system to obtain the recombinant plasmid pET28a-HMGB1 expressing monkey high-mobility group protein B1 protein; (2) Induced expression: The recombinant plasmid pET28a-HMGB1 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.

[0030] (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 recombinant protein, which is the monkey high-mobility group protein B1 recombinant protein, and the amino acid sequence is shown in SEQ ID NO.5: MGKGDPREARGKMSSYAFFVQTCREEHKEFHPDASVNFSEFSSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKGSFKDPNAPKRPPSAFFLFCSEYRPPGLSIGDVAKKLGEMWNNKEDRDKQPYEKKAAKLKEKPEKDIAAYRAKGKKSAAKKGVVKAEKSKKKKEEEEDEEDEEEEEEDEEDEDEEEDDDDE.

[0031] (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.

[0032] Example 2

[0033] Screening of monoclonal antibodies: This embodiment provides a method for obtaining monoclonal antibodies by screening based on the recombinant protein monkey high mobility group protein B1, and the specific operations are as follows: S1) Mouse immunization: 50 μg of monkey high-mobility group protein B1 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 high-mobility group protein B1 recombinant protein through the tail vein. After 3 days, the spleen of the mouse was collected for the preparation of hybridoma cells.

[0034] S2) Screening of hybridoma cell lines: All spleen cells of immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and then 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 high-mobility group protein B1 protein, and the positive rate was 100% through 2-3 consecutive limiting dilution cloning methods, and the cell lines were expanded and frozen; among them, 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, made up to 1L pure water), the coating concentration was 1μg / mL, 50μl / well, 4°C 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), and patted dry; 50μL cell culture supernatant was added, and the reaction was 37°C 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.

[0035] S3) Preparation of monoclonal antibody ascites: 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.

[0036] S4) Purification of monoclonal antibodies: 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.

[0037] S5) Identification of binding activity of monoclonal antibodies: The indirect ELISA method was used to coat the monkey high-mobility group protein B1 recombinant protein and the control carrier protein (monkey SAA recombinant protein), respectively, and the purified antibodies were tested by ELISA to identify the affinity of the monoclonal antibody. The antibody was diluted to 1μg / ml. The results are shown in Table 1. "Ctrl" in Table 1 represents the negative control monkey SAA mouse monoclonal antibody.

[0038] Table 1 Binding activity identification of monoclonal antibodies

[0039] As can be seen from Table 1, the 22 selected monkey high-mobility group protein B1 monoclonal antibodies were tested for their binding activity with monkey high-mobility group protein B1 protein and control carrier protein by indirect ELISA. The data showed that the monoclonal antibodies could specifically bind to monkey high-mobility group protein B1 protein.

[0040] Example 3

[0041] Screening and application of paired monoclonal antibodies: 1. HRP labeling of antibodies: 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.

[0042] 2. Establishment of double antibody sandwich immunoassay method: 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 high-mobility group protein B1 recombinant protein and the control monkey SAA protein 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 antibodies diluted to 500, 1000, and 2000 times with PBS were 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.

[0043] Then, the most suitable reaction conditions were determined by adjusting the dilution of the coating antibody and HRP antibody.

[0044] 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 high-mobility group protein B1 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 SAA protein was diluted to 10 ng / ml as a negative control, and reacted at 37°C for 30 minutes. 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.

[0045] Through the above-mentioned monoclonal antibody coating and HRP labeling, the best pairing combination was selected based on the detection of positive and negative samples, and the most obvious difference in positive and negative detection values ​​was selected. Finally, it was determined that the concentration of coated monoclonal antibody HMGB1-5# was 1μg / mL, and the concentration of HRP labeled monoclonal antibody HMGB1-11# was 2μg / mL.

[0046] 3. After sequencing: HMGB1-5# monoclonal antibody, including heavy chain and light chain: The amino acid sequence of the HMGB1-5# heavy chain variable region is shown in SEQ ID NO.1, specifically as follows: EVQLQQSGAELVKPGASVKLSCTASGSNITGSYMHWVKQRPEQGLEWIGRINTRNFSTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCAGEAYWGQGTLVTVSA, the complementary determining region CDR-H1 amino acid sequence GSNITGSY (SEQ ID NO.12), the complementary determining region CDR-H2 amino acid sequence INTRNFST (SEQ ID NO.13), the complementary determining region CDR-H3 amino acid sequence AGEAY (SEQ ID NO.14); The amino acid sequence of the HMGB1-5# light chain variable region is shown in SEQ ID NO.2, specifically as follows: DIVLTQSPASLAVSLGQRATISCKASQGFDRTGDDYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDTLNIHPVEEEDAATYYCQNDNTAPRTFGSGTKLEIK, the complementary determining region CDR-L1 amino acid sequence is QGFDRTGDDY (SEQ ID NO.15), the complementary determining region CDR-L2 amino acid sequence is YAASN (SEQ ID NO.16), and the complementary determining region CDR-L3 amino acid sequence is QNDNTAPRT (SEQ ID NO.17).

[0047] HMGB1-11# monoclonal antibody, including heavy chain and light chain: The amino acid sequence of the HMGB1-11# heavy chain variable region is shown in SEQ ID NO.3, specifically as follows: QVQLQQSGPELVRPGTSVKISCKTSGNVFLDDTMNWVKQRPGQGLEWIGQISNASFNYYYNEMFKDKATLTVDTSSSTAYIHLSSLTSEDTAVYFCARERRLMNYWGQGTSVTVSS, the complementary determining region CDR-H1 amino acid sequence is GNVFLDDT (SEQ ID NO.6), the complementary determining region CDR-H2 amino acid sequence is ISNASFNY (SEQ ID NO.7), and the complementary determining region CDR-H3 amino acid sequence isARERRLMNY (SEQ ID NO.8); The amino acid sequence of the light chain variable region of the HMGB1-11# is shown in SEQ ID NO.4, specifically as follows: DIQMTQTTSSLSASLGDRVTVSCSASRRDNTYLNWYQQKPDGTVKFLIYYASSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQTYVPLPSTFGGGTKLEFK, the complementary determining region CDR-L1 amino acid sequence RRDNTY (SEQ ID NO.9), the complementary determining region CDR-L2 amino acid sequence YYASS (SEQ ID NO.10), and the complementary determining region CDR-L3 amino acid sequence QTYVPLPST (SEQ ID NO.11).

[0048] 4. Linear detection based on double antibody sandwich ELISA detection method: Referring to the above detection steps, the monkey high-mobility group protein B1 fixed value standard protein was first diluted with PBS buffer solution. The concentrations after dilution were 2000pg / mL, 1000pg / mL, 500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL, 31.3pg / mL, and 0ng / 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.

[0049] 5. Sensitivity test based on double antibody sandwich ELISA test method: Referring to the detection steps of Example 4, the present invention was used to first perform gradient dilution of the monkey high mobility group protein B1 standard protein with PBS buffer solution, and the concentrations after dilution were 2000pg / mL, 1000pg / mL, 500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL, 31.3pg / mL, and 0ng / mL, respectively. 50μL of the diluted standard protein of different concentrations and 20 PBS buffer solutions were taken for detection, and a sensitivity test was performed. The standard curve and PBS buffer solution values ​​are shown in Table 2. The sensitivity of the double antibody sandwich ELISA detection method was calculated to be 12pg / mL according to the formula "mean value + 2SD".

[0050] Table 2 Sensitivity test data

[0051] 6. Identification of monkey serum samples based on double antibody sandwich ELISA detection method: 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: Table 3 Results of the double antibody sandwich ELISA test on 16 non-infected monkey sera and 16 infected monkey sera

[0052] From the test results, it can be seen that there are significant differences between infected and uninfected.

[0053] 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 high mobility group protein B1 monoclonal antibody, characterized in that: Named as HMGB1-11, HMGB1-11 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 high mobility group protein B1 monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the HMGB1-11 heavy chain variable region is shown in SEQ ID NO.3, and the amino acid sequence of the HMGB1-11 light chain variable region is shown in SEQ ID NO.

4.

3. Use of the anti-monkey high-mobility group protein B1 monoclonal antibody according to claim 1 or 2 in the preparation of a monkey high-mobility group protein B1 detection reagent or kit.

4. A monkey high mobility group protein B1 enzyme-linked immunosorbent assay kit, characterized in that: The kit comprises two anti-monkey high mobility group protein B1 monoclonal antibodies HMGB1-5 and HMGB1-11, wherein the monoclonal antibody HMGB1-5 is used as a coating antibody, and the monoclonal antibody HMGB1-11 is used as a detection antibody; The HMGB1-5 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 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 complementary 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.

5. The monkey high mobility group protein B1 ELISA kit according to claim 4, characterized in that: The amino acid sequence of the HMGB1-5 heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the HMGB1-5 light chain variable region is shown in SEQ ID NO.

2.

6. The monkey high mobility group protein B1 ELISA kit according to claim 4, characterized in that: The monoclonal anti-HMGB1-5 and anti-HMGB1-11 are both obtained by immunizing monkey high mobility group protein B1 recombinant protein.

7. The monkey high mobility group protein B1 ELISA kit according to claim 6, characterized in that: The amino acid sequence of the monkey high-mobility group protein B1 recombinant protein is shown in SEQ ID NO.

5.

8. An application of the monkey high mobility group protein B1 enzyme-linked immunosorbent assay kit according to any one of claims 4 to 7, characterized in that: The kit is used to detect the content of monkey high-mobility group protein B1 in monkey serum, plasma or tissue fluid.

Images