Anti-monkey high mobility group protein B1 monoclonal antibody, enzyme-linked immunosorbent assay kit and its application

Abstract

The present invention belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody against monkey high-mobility group protein B1, 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 complementarity-determining regions CDR-H1 to CDR-H3, and the amino acid sequences are shown in SEQ ID NO.6-SEQ ID NO.8 in sequence; the light-chain variable region has three complementarity-determining regions CDR-L1 to CDR-L3, and the amino acid sequences are shown in SEQ ID NO.9-SEQ ID NO.11 in sequence. The monoclonal antibody of the present invention can specifically bind to monkey high-mobility group protein B1. A paired monoclonal antibody can be screened for this monoclonal antibody. Based on this pair of monoclonal antibody combinations, an enzyme-linked immunosorbent assay kit for monkey high-mobility group protein B1 can be constructed, and the content of monkey high-mobility group protein B1 in monkey serum, plasma or tissue fluid can be detected.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody against monkey high mobility group protein B1, an enzyme-linked immunosorbent assay kit and their applications. Background Art

[0002] Infectious diseases are extremely common clinically, and laboratory tests 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 In recent years, studies have found that once HMGB1 is secreted extracellularly, it can exert a pro-inflammatory effect. It is now believed that HMGB1 is an important late pro-inflammatory factor and has more important clinical significance than early-onset inflammatory factors such as TNF and IL-1. Mechanically damaged and necrotic cells can release nuclear HMGB1 extracellularly to induce an inflammatory response. Co-culturing damaged cells with macrophages can cause nuclear translocation of nuclear transcription factor-κB (NF-κB) in macrophages and produce an inflammatory response similar to that caused by tissue necrosis; co-culturing damaged cells of HMGB1 gene knockout mice with macrophages results in a significant reduction in the intensity of the inflammatory response. This indicates that the passive release of HMGB1 plays an important role in tissue necrotic inflammatory responses. Histones in apoptotic cells are generally underacetylated, so HMGB1 in apoptotic cells can bind tightly to nucleosomes, will not be released, and will not cause an inflammatory response; on the contrary, if intracellular deacetylation is inhibited, an inflammatory response will occur. Endotoxin and various inflammatory factors can induce the release of HMGB1 to mediate an inflammatory response. A series of experimental studies suggest that HMGB1 can act as an endogenous pyrogen in the central nervous system and mediate central inflammatory responses. When cells are necrotic or damaged, nuclear HMGB1 can be released extracellularly, triggering the secretion of pro-inflammatory factors by monocytes and macrophages; and the pro-inflammatory factors in turn promote the secretion of HMGB1, thus forming a positive feedback loop. In the later stage 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 for preclinical research. The application of laboratory animals in new drug research and development covers the preclinical research and clinical research stages, especially more in preclinical research. The pharmacological effects of drugs are evaluated through different experimental methods, their mechanisms of action are studied, and their toxic effects are observed to prove the effectiveness and safety of drugs. Monkeys are the closest to humans in terms of genetic relationship, with 75% - 98.5% homology with human genetic material. They are the animals with the highest homology with humans and are therefore the most ideal animals closest to humans in experiments.

[0005] Currently, there is no ELISA kit for detecting high mobility group protein B1 (HMGB1) in monkeys that can detect the content of HMGB1 in monkey serum, plasma or tissue fluid. SUMMARY OF THE INVENTION

[0006] To solve the above problems, one of the objectives of the present invention is to provide a monoclonal antibody against monkey high mobility group protein B1, an ELISA kit and its application, which can specifically bind to monkey high mobility group protein B1, and use the above monoclonal antibody to screen paired monoclonal antibodies, and further construct an ELISA kit for detecting monkey high mobility group protein B1 based on the combination of the pair of monoclonal antibodies, for detecting the content of monkey high mobility group protein B1 in monkey serum, plasma or tissue fluid.

[0007] In the first aspect of the present invention, a monoclonal antibody against monkey high mobility group protein B1 is provided.

[0008] It is named HMGB1-11, and the HMGB1-11 includes a heavy chain variable region and a light chain variable region.

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

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

[0011] Furthermore, the amino acid sequence of the heavy chain variable region of HMGB1-11 is as shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region of HMGB1-11 is as shown in SEQ ID NO.4.

[0012] Furthermore, the monoclonal antibody further includes:

[0013] An antibody with the same function obtained by substituting, deleting and / or adding one or more amino acids to the amino acid sequence of the monoclonal antibody; or including a heavy chain variable region with an amino acid sequence having at least 80% homology with the heavy chain variable region; and a light chain variable region with 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 some other embodiments, VH and / or V L The amino acid sequence may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the above sequence. The V H and V L regions with a high degree (i.e., 80% or higher) of homology to the above sequence H and V L regions of the antibody can be obtained as follows: mutagenize (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) the nucleic acid molecule encoding SEQ ID NOs. 6-11, and then detect the retained function of the encoded altered antibody with the functional assays described herein.

[0015] The second aspect of the present invention provides the use of the anti-monkey high mobility group protein B1 monoclonal antibody described above in the preparation of a reagent or kit for detecting monkey high mobility group protein B1.

[0016] The third aspect of the present invention provides an enzyme-linked immunosorbent assay kit for monkey high mobility group protein B1,

[0017] The kit includes two anti-monkey high mobility group protein B1 monoclonal antibodies, HMGB1-5 and HMGB1-11, wherein the monoclonal antibody HMGB1-5 is used as the coating antibody and the monoclonal antibody HMGB1-11 is used as the detection antibody;

[0018] The HMGB1-5 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 CDR-H1 is as shown in SEQ ID NO. 12, the amino acid sequence of CDR-H2 is as shown in SEQ ID NO. 13, and the amino acid sequence of CDR-H3 is as shown in SEQ ID NO. 14; the light chain variable region has three complementarity-determining regions. The amino acid sequence of CDR-L1 is as shown in SEQ ID NO. 15, the amino acid sequence of CDR-L2 is as shown in SEQ ID NO. 16, and the amino acid sequence of CDR-L3 is as shown in SEQ ID NO. 17.

[0019] Furthermore, the amino acid sequence of the heavy chain variable region of HMGB1-5 is as shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region of HMGB1-5 is as shown in SEQ ID NO. 2.

[0020] Furthermore, the monoclonal antibody further includes:

[0021] An antibody with the same function obtained by substituting, deleting, and / or adding one or more amino acids to the amino acid sequence of the monoclonal antibody; or comprising a heavy chain variable region having an amino acid sequence with at least 80% homology to the heavy chain variable region described above; and a light chain variable region having an amino acid sequence with at least 80% homology to the light chain variable region described above; or an antibody obtained by linking a tag to the N-terminus and / or C-terminus of the monoclonal antibody.

[0022] In some 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. Having V with the above sequence H and V L regions highly (i.e., 80% or higher) homologous V H and V L region antibodies can be obtained as follows: mutagenize (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) the nucleic acid molecule encoding SEQ ID NOs. 12-17, and then detect the retained function of the encoded altered antibody using the functional assays described herein.

[0023] Furthermore, both the monoclonal antibodies HMGB1-5 and HMGB1-11 are obtained by immunizing monkeys with the recombinant high mobility group box protein B1.

[0024] Furthermore, the recombinant high mobility group box protein B1 of monkeys is obtained by using bioinformatics software to optimize and select a polypeptide of high mobility group box protein B1 of monkeys, and this polypeptide is predicted to be able to be expressed in Escherichia coli.

[0025] The present invention provides a recombinant high mobility group box protein B1 of monkeys, which realizes the efficient expression of high mobility group box protein B1 in Escherichia coli by optimizing the antigen peptide sequence and the expression vector.

[0026] Furthermore, the amino acid sequence of the recombinant high mobility group box protein B1 of monkeys is as shown in SEQ ID NO.5.

[0027] The fourth aspect of the present invention provides an application of the above-mentioned enzyme-linked immunosorbent assay kit for high mobility group box protein B1 of monkeys, and uses the kit to detect the content of high mobility group box protein B1 in monkey serum, plasma or tissue fluid.

[0028] The beneficial effects of the present invention are:

[0029] (1) The monoclonal antibody of the present invention can specifically bind to high mobility group box protein B1 of monkeys, and has high specificity.

[0030] (2)Based on the recombinant protein of monkey high-mobility group protein B1, the present invention screened out monoclonal antibodies specifically binding to monkey high-mobility group protein B1, and used the above monoclonal antibodies to screen paired monoclonal antibodies. Further, an enzyme-linked immunosorbent assay kit for monkey high-mobility group protein B1 was constructed based on the pair of monoclonal antibody combinations, which is used for detecting the content of monkey high-mobility group protein B1 in monkey serum, plasma or tissue fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The attached figure shows the standard curve diagram of the ELISA detection of monkey high-mobility group protein B1 described in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0033] All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope protected by the present invention.

[0034] The above technical solutions will be described in detail below in conjunction with specific embodiments.

[0035] Example 1

[0036] Preparation of recombinant antigen:

[0037] This example provides a recombinant protein of monkey high-mobility group protein B1, and its amino acid sequence is shown in SEQ ID NO.5:

[0038] MGKGDPREARGKMSSYAFFVQTCREEHKEFHPDASVNFSEFSSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKGSFKDPNAPKRPPSAFFLFCSEYRPPGLSIGDVAKKLGEMWNNKEDRDKQPYEKKAAKLKEKPEKDIAAYRAKGKKSAAKKGVVKAEKSKKKKEEEEDEEDEEEEEDEEDEDEEEDDDDE.

[0039] This example provides a preparation method of a recombinant protein of monkey high-mobility group protein B1, including the following steps:

[0040] (1)Plasmid construction: Computational software was used to predict and select the sequence containing the major antigenic epitopes of monkey high mobility group protein B1 for expression. This polypeptide segment can be expressed in the E. coli prokaryotic expression system. The gene was cloned into the pET28a vector through the E. coli expression system to obtain the recombinant plasmid pET28a-HMGB1 expressing monkey high mobility group protein B1;

[0041] (2)Induced expression: The recombinant plasmid pET28a-HMGB1 was transformed into BL21(DE3) competent cells. The transformed bacteria were spread on an LB agar plate containing 50 μg / mL kanamycin and cultured overnight at 37 °C. A single colony was picked and inoculated into 4 mL of LB medium containing 50 μg / mL kanamycin, and cultured overnight at 37 °C with shaking at 230 rpm. It was inoculated into LB medium containing 50 μg / mL kanamycin according to 1% of the total volume of the medium, and cultured with shaking at 37 °C and 220 rpm for about 3 hours until the OD600 reached 0.6. IPTG with a final concentration of 0.1 mM was added, and the cells were induced at 16 °C and 180 rpm for 15 hours, and then the cells were collected.

[0042] (3)Purification of the recombinant protein: Since the expressed recombinant protein carried a histidine tag, it was initially crudely purified using a protein purifier and a chromatography column, and then the purified recombinant protein, namely the recombinant monkey high mobility group protein B1, was obtained by ion exchange chromatography. The amino acid sequence is shown in SEQ ID NO.5:

[0043] MGKGDPREARGKMSSYAFFVQTCREEHKEFHPDASVNFSEFSSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKGSFKDPNAPKRPPSAFFLFCSEYRPPGLSIGDVAKKLGEMWNNKEDRDKQPYEKKAAKLKEKPEKDIAAYRAKGKKSAAKKGVVKAEKSKKKKEEEEDEEDEEEEEDEEDEDEEEDDDDE.

[0044] (4)Identifying the purity of the recombinant protein by SDS-PAGE electrophoresis: Pretreatment of the protein sample: Samples were reserved and an equal volume of 2×SDS loading buffer was added, then placed in a boiling water bath for 10 min, centrifuged at 12000 rpm for 3 min, and the supernatant was taken and added to the loading well.

[0045] Example 2

[0046] Screening of monoclonal antibodies:

[0047] This example provides a method for screening monoclonal antibodies based on the recombinant protein monkey high mobility group protein B1. The specific operation is as follows:

[0048] S1) Mouse immunization: Mix 50 μg of recombinant simian high-mobility group protein B1 with an equal volume of Freund's complete adjuvant to obtain a completely emulsified mixture. Subcutaneously inject the mixture at multiple points to immunize 6-week-old female BALB / c mice. At week 0 and week 2, emulsify an equal proportion of Freund's incomplete adjuvant and recombinant protein according to the above method and immunize again. At week 6, collect mouse serum to detect the antibody titer. Select the mouse with the highest titer and boost immunize 20 μg of recombinant simian high-mobility group protein B1 through the tail vein. Three days later, take the mouse spleen for the preparation of hybridoma cells.

[0049] S2) Screening of hybridoma cell lines:

[0050] After fusing all spleen cells of immunized mice with SP2 / 0 myeloma cells in the logarithmic growth phase, culture them in HAT medium for screening. When the fused cells grow to 1 / 2 of the well bottom, screen for positive clones by the indirect ELISA method of simian high-mobility group protein B1, and achieve a 100% positive rate through 2 - 3 consecutive limited dilution cloning methods. Expand the culture and cryopreservation of the cell line; among them, the method for screening positive clones by the indirect ELISA method is as follows: First, coat the recombinant protein in a microplate: The coating buffer is carbonate buffer (prepared by dissolving 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate in 1 L of pure water), the coating concentration is 1 μg / mL, 50 μL per well, overnight at 4°C; Block with 3% sucrose and 1% BSA, 150 μL per well, 2 hours at 37°C, wash the plate once with washing solution PBST (PBS containing 0.05% Tween-20), pat dry; Add 50 μL of cell culture supernatant and react at 37°C for 30 min. Discard the liquid in the well, wash the plate 4 times with PBST washing solution, pat dry, then add 50 μL of HRP-labeled goat anti-mouse (diluted 1:5000 with PBS) per well, react at 37°C for 30 min, wash the plate 4 times again, pat dry, add 50 μL of TMB chromogenic solution per well and develop color at room temperature for 10 min, and finally add 0.5 M sulfuric acid as a terminator to terminate the reaction. Measure the OD450nm value with an enzyme-linked immunosorbent assay (ELISA) reader.

[0051] S3) Preparation of monoclonal antibody ascites:

[0052] After expanding the culture of the screened monoclonal cell line, intraperitoneally inject 0.2 mL (containing 2.5×106 cells) of female BALB / c mice pretreated with Freund's incomplete adjuvant. Approximately 10 days later, when the mouse abdomen is significantly swollen, collect ascites using a sterile syringe needle. Centrifuge the collected ascites at 3000 r / min for 10 minutes and collect the middle layer.

[0053] S4) Purification of monoclonal antibody:

[0054] Centrifuge the ascites at 12,000 r / min for 5 minutes, take 5 mL of the supernatant, add 20 mL of acetate buffer (68 mmol / L, pH 4.5), mix well, then slowly add 50 μL of n-octanoic acid while stirring, continue to stir for 30 minutes after addition, centrifuge at 12,000 r / min for 30 minutes at 2 - 8 °C, and take the supernatant. Filter the supernatant through absorbent cotton, add saturated ammonium sulfate at a ratio of 50% (V / V) of the final volume while stirring, continue to stir for 30 minutes after addition, precipitate at 2 - 8 °C for 4 - 5 hours, centrifuge at 12,000 r / min for 30 minutes at 2 - 8 °C, and take the precipitate. Resuspend the precipitate with 3 mL of Tris-HCl (10 mmol / L, pH 9.0), load it into a dialysis bag (MW: 8000 - 14000), and dialyze in 2 L of Tris-HCl (10 mmol / L, pH 9.0) solution at 2 - 8 °C for 14 hours. Transfer the liquid in the dialysis bag to a centrifuge tube, centrifuge at 12,000 r / min for 5 minutes, and the supernatant is the purified monoclonal antibody. Determine the concentration of the purified monoclonal antibody using an ultra-micro spectrophotometer and store it at -20 °C, avoiding repeated freezing and thawing.

[0055] S5) Identification of the binding activity of monoclonal antibodies:

[0056] Using the indirect ELISA method, coat the recombinant protein of monkey high-mobility group protein B1 and the control vector protein (recombinant protein of monkey SAA) respectively, perform ELISA detection on the purified antibody to identify the affinity of the monoclonal antibody. Dilute the antibody to 1 μg / ml, and the results are shown in Table 1. In Table 1, "Ctrl" represents the negative control monkey SAA mouse monoclonal antibody.

[0057] Table 1 Identification of the binding activity of monoclonal antibodies

[0058]

[0059] As can be seen from Table 1, for the 22 strains of monoclonal antibodies against monkey high-mobility group protein B1 screened, the binding activities of the monoclonal antibodies to monkey high-mobility group protein B1 protein and the control vector protein were detected by indirect ELISA. The data showed that the monoclonal antibodies could specifically bind to monkey high-mobility group protein B1 protein.

[0060] Example 3

[0061] Screening and application of paired monoclonal antibodies:

[0062] 1. HRP labeling of antibodies:

[0063] Perform HRP labeling operation on the screened monoclonal antibodies according to the classical sodium periodate method. Finally, dialyze the labeled antibodies overnight in a buffer of 0.01 M PBS, pH 7.4, add glycerol at a volume ratio of 1:1, and store them in aliquots at -20 °C.

[0064] 2. Establishment of double-antibody sandwich immunoassay method:

[0065] The purified monoclonal antibodies were respectively diluted to concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL with coating buffer (1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L of pure water, pH 9.6), and added to the enzyme-linked immunosorbent assay (ELISA) plate at 50 μL / well. Coating was carried out overnight at 4°C. The next day, the coating solution was discarded, and the plate was washed once with washing solution (PBST, PBS containing 0.05% Tween-20), patted dry, blocked with 3% sucrose and 1% BSA at 150 μL / well, incubated at 37°C for 2 h, the blocking solution was discarded, and the plate was patted dry. The recombinant protein of monkey high-mobility group protein B1 in the sample to be tested and the control sample of monkey SAA protein were diluted with PBS to 2 μg / mL, 1 μg / mL, and 0.5 μg / mL, then added to the ELISA plate at 50 μL / well, incubated at 37°C for 35 min, washed 4 times with PBST washing solution, patted dry, added enzyme-labeled monoclonal antibodies diluted 500-, 1000-, and 2000-fold with PBS at 50 μL / well, incubated at 37°C for 35 min, washed 4 times again, patted dry, then added 50 μL of TMB chromogenic solution per well, developed color at room temperature for 10 min, and finally added 50 μL of 0.5 M sulfuric acid per well to terminate the reaction. The OD450nm value was measured with an enzyme-linked immunosorbent assay (ELISA) reader.

[0066] Furthermore, by adjusting the dilution ratios of the coating antibody and HRP antibody, the most suitable reaction conditions were determined.

[0067] Specifically: Monoclonal antibodies were coated in the microplate (coating buffer carbonate buffer: 1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate made up to 1 L of pure water), the coating concentration gradients were 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, 50 μL / well, overnight at 4°C; the plate was washed once with washing solution (PBST, PBS containing 0.05% Tween-20), patted dry, blocked with 3% sucrose and 1% BSA, 150 μL per well, at 37°C for 2 h, patted dry and stored dry for later use; the recombinant protein of monkey high-mobility group protein B1 was diluted with PBS to a concentration of 10 ng / ml, 50 μL was added to the microplate coated with monoclonal antibodies, and at the same time, SAA protein was diluted to 10 ng / ml as a negative control, reacted at 37°C for 30 min. The liquid in the wells was flicked out, the plate was washed 4 times with PBST washing solution, patted dry; the HRP-labeled monoclonal antibody was diluted with PBS to concentrations of 1 μg / mL, 2 μg / mL, and 4 μg / mL, added at 50 μL / well, reacted at 37°C for 30 min, washed 4 times again, patted dry, then added 50 μL of TMB chromogenic solution per well, developed color at room temperature for 10 min, and finally added the termination solution to terminate the reaction. The OD450nm value was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The coating conditions with the most obvious difference between negative and positive results and the optimal reaction conditions of the HRP-labeled antibody were selected.

[0068] By separately coating the monoclonal antibody and labeling it with HRP as described above, and based on the detection of positive and negative samples, the optimal paired combination was selected as the one with the most obvious difference in the detection values of positive and negative samples. Finally, it was determined that: the coated monoclonal antibody HMGB1-5# at a concentration of 1 μg / mL, and the HRP-labeled monoclonal antibody HMGB1-11# at a concentration of 2 μg / mL.

[0069] 3. After sequencing:

[0070] The monoclonal antibody HMGB1-5# includes a heavy chain and a light chain:

[0071] The amino acid sequence of the variable region of the heavy chain of HMGB1-5# is shown in SEQ ID NO.1, specifically as follows: EVQLQQSGAELVKPGASVKLSCTASGSNITGSYMHWVKQRPEQGLEWIGRINTRNFSTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCAGEAYWGQGTLVTVSA, the amino acid sequence of complementary determining region CDR-H1 is GSNITGSY (SEQ ID NO.12), the amino acid sequence of complementary determining region CDR-H2 is INTRNFST (SEQ ID NO.13), and the amino acid sequence of complementary determining region CDR-H3 is AGEAY (SEQ ID NO.14);

[0072] The amino acid sequence of the variable region of the light chain of HMGB1-5# is shown in SEQ ID NO.2, specifically as follows: DIVLTQSPASLAVSLGQRATISCKASQGFDRTGDDYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDTLNIHPVEEEDAATYYCQNDNTAPRTFGSGTKLEIK, the amino acid sequence of complementary determining region CDR-L1 is QGFDRTGDDY (SEQ ID NO.15), the amino acid sequence of complementary determining region CDR-L2 is YAASN (SEQ ID NO.16), and the amino acid sequence of complementary determining region CDR-L3 is QNDNTAPRT (SEQ ID NO.17).

[0073] The monoclonal antibody HMGB1-11# includes a heavy chain and a light chain:

[0074] The amino acid sequence of the heavy chain variable region of HMGB1-11# is shown in SEQ ID NO.3, specifically as follows: QVQLQQSGPELVRPGTSVKISCKTSGNVFLDDTMNWVKQRPGQGLEWIGQISNASFNYYYNEMFKDKATLTVDTSSSTAYIHLSSLTSEDTAVYFCARERRLMNYWGQGTSVTVSS. The amino acid sequence of complementary determining region CDR-H1 is GNVFLDDT (SEQ ID NO.6), the amino acid sequence of complementary determining region CDR-H2 is ISNASFNY (SEQ ID NO.7), and the amino acid sequence of complementary determining region CDR-H3 is ARERRLMNY (SEQ ID NO.8).

[0075] The amino acid sequence of the light chain variable region of HMGB1-11# is shown in SEQ ID NO.4, specifically as follows: DIQMTQTTSSLSASLGDRVTVSCSASRRDNTYLNWYQQKPDGTVKFLIYYASSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQTYVPLPSTFGGGTKLEFK. The amino acid sequence of complementary determining region CDR-L1 is RRDNTY (SEQ ID NO.9), the amino acid sequence of complementary determining region CDR-L2 is YYASS (SEQ ID NO.10), and the amino acid sequence of complementary determining region CDR-L3 is QTYVPLPST (SEQ ID NO.11).

[0076] 4. Linear detection based on the double antibody sandwich ELISA detection method:

[0077] Referring to the above detection steps, first, the monkey high mobility group protein B1 value standard protein was serially diluted with PBS buffer solution, and the diluted concentrations were 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.3 pg / mL, and 0 ng / mL respectively. 50 μL of the diluted standard protein with different concentrations was taken for detection, and three parallel experiments were performed for each concentration to prepare a standard curve. The standard curve is as Figure 1 shown: Calculated according to the four-parameter fitting result, the linearity reached 0.999.

[0078] 5. Sensitivity detection based on the double antibody sandwich ELISA detection method:

[0079] Referring to the detection steps of Example 4, using this invention patent, first, the high-mobility group protein B1 quantitative standard protein of monkeys was serially diluted with PBS buffer solution, and the concentrations after dilution were 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.3 pg / mL, and 0 ng / mL respectively. 50 μL of the diluted standard protein with different concentrations and 20 PBS buffer solutions were taken for detection to conduct a sensitivity test. The standard curve and the measured values of the PBS buffer solution are shown in Table 2. According to the formula "average value + 2SD", the sensitivity of the double-antibody sandwich ELISA detection method was calculated to be 12 pg / mL.

[0080] Table 2 Sensitivity test data

[0081]

[0082] 6. Identification of monkey serum samples based on the double-antibody sandwich ELISA detection method:

[0083] Referring to the above detection steps, 16 non-infected monkey sera and 16 infected monkey sera were measured and identified based on the double-antibody sandwich ELISA detection method, and the measured OD values are shown in Table 3:

[0084] Table 3 Results of measuring 16 non-infected monkey sera and 16 infected monkey sera based on the double-antibody sandwich ELISA detection method

[0085]

[0086] It can be seen from the detection results that there are significant differences between the infected and non-infected.

[0087] The above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to 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 HMGB1-11 and HMGB1-5 according to claim 1 or 2, 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.

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