A monoclonal antibody specifically binding to brucellin, a detection kit and application thereof

Abstract

The application provides a monoclonal antibody specifically combined with brucellin, a detection kit and application thereof, and belongs to the technical field of monoclonal antibodies. The application immunizes animals with brucellin as an antigen to screen a hybridoma with a high antibody titer, and the monoclonal antibody generated by the hybridoma is specifically combined with brucellin. The application also provides a competitive ELISA kit, in which brucellin is used as an antigen, and the monoclonal antibody specifically combined with brucellin is used as a competitive antibody. Compared with a commercial kit, the kit has the characteristics of high detection sensitivity and good repeatability, is not limited by the species of a sample, can be applied to the prevention and control of brucellosis in the breeding industry of cattle, sheep and pigs, meets the urgent needs of high-precision, high-efficiency and low-cost identification of detection technology, realizes precise epidemiological detection of brucellosis across species, and promotes the prevention and control of brucellosis to a new stage of precise identification.

Description

Technical Field

[0001] This invention belongs to the field of monoclonal antibody technology, specifically relating to a monoclonal antibody that specifically binds to brucellin, its detection kit, and its application. Background Technology

[0002] Brucellosis, also known as brucellosis, is caused by Brucella or Brucella bacteria (Brucella). Brucella Brucella is a globally distributed zoonotic infectious disease characterized by fever, inflammation, and abortion, caused by Brucella bacteria. Currently, detection of Brucella primarily relies on serological methods, including the Rose Bengal Plate Test (RBT), Standard Agglutination Test (SAT), Complement Fixation Test (CFT), Colloidal Gold Immunochromatography (GICA), Enzyme-Linked Immunosorbent Assay (ELISA), and Fluorescence Polarization Assay (FPA). These techniques often use Brucella lipopolysaccharide (LPS) as an antigen, including FPA and indirect ELISA. However, a major problem is that Brucella LPS cross-reacts with various Gram-negative bacteria, such as Yersinia enterocolitica, leading to false positives. As LPS is a common antigen of smooth Brucella, detection methods based on LPS cannot distinguish between natural Brucella infection and vaccine immunization, nor can they detect rough Brucella, limiting their application in disease control tracing and epidemiological investigations.

[0003] Brucella is an allergen used to diagnose brucellosis in humans and animals. It is detected through skin tests to determine the presence of delayed-type hypersensitivity to Brucella, thus aiding in the assessment of infection or immune status. However, there are currently no products based on brucellin for detecting Brucella, and there are also few reports of monoclonal antibodies against brucellin. Summary of the Invention

[0004] The purpose of this invention is to provide a monoclonal antibody that has good binding affinity for brucellosis.

[0005] This invention provides a monoclonal antibody that specifically binds to brucellin, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region; The heavy chain variable region includes heavy chain complementarity-determining regions CDR1, CDR2 and CDR3, and the amino acid sequences of the heavy chain complementarity-determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID NO:5~SEQ ID NO:7; The light chain variable region includes light chain complementarity-determining regions CDR1, CDR2 and CDR3, and the amino acid sequences of the light chain complementarity-determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID NO:8~SEQ ID NO:10.

[0006] Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1; The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 3.

[0007] The present invention provides a nucleic acid molecule encoding the monoclonal antibody.

[0008] Preferably, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region is shown in SEQ ID NO: 2; The nucleotide sequence of the nucleic acid molecule encoding the variable region of the light chain is shown in SEQ ID NO: 4.

[0009] The present invention provides a derivative product comprising the nucleic acid molecule, including at least one of the following: gene expression cassette, recombinant vector, recombinant microbial strain, and recombinant cell line.

[0010] This invention provides the use of the monoclonal antibody, or the monoclonal antibody prepared from the nucleic acid molecule or the derivative thereof, in the preparation of products for detecting Brucella or diagnosing brucellosis.

[0011] Preferably, the product includes detection reagents and / or detection kits.

[0012] Preferably, the product is prepared based on the following detection technologies: colloidal gold immunochromatography, enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, and fluorescence immunoassay.

[0013] This invention provides a competitive ELISA kit, comprising the monoclonal antibody, a ELISA plate coated with brucellin, washing concentrate, enzyme-labeled secondary antibody, substrate, and stop solution.

[0014] Preferably, the concentration of brucellin in the enzyme-labeled plate coated with brucellin is 0.2 μg / mL; The working solution of the monoclonal antibody is a 1% BSA solution containing a monoclonal antibody diluted 1:9000.

[0015] This invention provides a monoclonal antibody that specifically binds to brucellosis. The monoclonal antibody includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes heavy chain complementarity-determining regions (CDR1, CDR2, and CDR3), and the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO:5~SEQ ID NO:7. The light chain variable region also includes light chain complementarity-determining regions (CDR1, CDR2, and CDR3), and the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO:8~SEQ ID NO:10. This monoclonal antibody has the characteristic of specifically binding to brucellosis and exhibits a high antibody titer. When a sample is detected by competitive ELISA, a positive serum concentration of 3.125 IU / mL is still considered positive. In contrast, commercially available competitive ELISA kits can only detect positive serum at a minimum concentration of 12.5 IU / mL. Therefore, the competitive ELISA kit of this invention has significantly higher sensitivity than commercially available competitive ELISA kits. It is evident that the monoclonal antibody exhibits high affinity for brucellin, providing a new method for the detection of brucellin and the qualitative or quantitative detection of Brucella, and offering a new avenue for the development of diagnostic products for brucellosis. Attached Figure Description

[0016] Figure 1 The SDS-PAGE results of the brucellin prepared in this invention are shown; where M: protein molecular weight standard; 1: brucellin; Figure 2 The results of SDS-PAGE analysis of purified brucellin monoclonal antibody specifically binding to it are shown; where M: protein molecular weight standard; 1: purified brucellin monoclonal antibody. Figure 3 Standard curve for competing ELISA kits for quantitative detection of Brucella antibodies. Detailed Implementation

[0017] This invention provides a monoclonal antibody that specifically binds to brucellin, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region includes heavy chain complementarity-determining regions CDR1, CDR2, and CDR3. The amino acid sequence of heavy chain complementarity-determining region CDR1 is shown in SEQ ID NO:5 (GYTFSDYY), the amino acid sequence of CDR2 is shown in SEQ ID NO:6 (INPNNGGT), and the amino acid sequence of CDR3 is shown in SEQ ID NO:7 (ARNSWYFDV). The light chain variable region includes light chain complementarity-determining regions CDR1, CDR2, and CDR3. The amino acid sequence of light chain complementarity-determining region CDR1 is shown in SEQ ID NO:8 (QSLLHSDGQTY), the amino acid sequence of CDR2 is shown in LVS, and the amino acid sequence of CDR3 is shown in SEQ ID NO:9 (WQGTHFPLT). The heavy chain complementarity-determining region and the light chain complementarity-determining region are CDR regions defined based on the International Immunogenetic Information System (IMGT).

[0018] In this invention, the preferred amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1 (EVQLQQSGPELVRPGASVKISCKASGYTFSDYYMNWVKQSHGKSLEWIGDINPNNGGTRNNQKFKGKATLTVDRSSSTAYMELRSLTSEDSAIYYCARNSWYFDVWGTGTTVTVSS). The preferred amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 3 (DVVMTQTPLTLSVTIGQPASISCKSTQSLLHSDGQTYLHWLLQRPGQSPKRLIYLVSKVDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPLTFGAGTKLELK).

[0019] The present invention provides a nucleic acid molecule encoding the monoclonal antibody.

[0020] In this invention, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region is preferably as shown in SEQ ID NO: 2 (GAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAGGCCTGGGGC). TTCAGTGAAGATATCCTGTAAGGCTTCTGGATACACGTTTCTCTGACTACTACATGAACTGGGTGAAAACAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATCAATCCTAACAATGGTGGTACCAGGAATAACCAGAAGTTCAAGGGCA AGGCCACATTGACTGTAGACAGGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAATCTATTACTGTGCAAGAAACTCCTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA). The nucleotide sequence of the nucleic acid molecule encoding the light chain variable region is shown in SEQ ID NO: 4 (GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAACTCAGAGCCTCTTACATAGTGATGGACAGACATATTTGCATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAAGTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAGATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACACTTTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA).

[0021] In this invention, the nucleic acid molecule also includes a nucleotide sequence that has been optimized according to host codon preference during in vitro recombinant expression.

[0022] The present invention provides a derivative product comprising the nucleic acid molecule, including at least one of the following: gene expression cassette, recombinant vector, recombinant microbial strain, and recombinant cell line.

[0023] In this invention, the gene expression cassette preferably includes an enhancer, a promoter, a nucleic acid molecule, and a terminator. This invention does not impose any special limitations on the types of enhancers, promoters, and terminators; any types of enhancers, promoters, and terminators well-known in the art may be used.

[0024] In this invention, the recombinant vector is preferably a backbone vector in which the nucleic acid molecule is embedded. This invention does not have any particular limitation on the type of backbone vector; any backbone vector well-known in the art can be used, such as prokaryotic expression vectors or eukaryotic expression vectors. The recombinant microbial strain preferably includes recombinant bacterial strains, recombinant fungal strains, and recombinant viral strains. The host bacteria of the recombinant bacterial strain preferably include *Escherichia coli* (…). E. coli Bacillus subtilis ( Bacillus subtilis ), Lactococcus lactis ( Lactococcus lactis ), Corynebacterium glutamicum ( Corynebacterium glutamicum ) or cyanobacteria (Syntropha) Synechococcus The host microorganism of the recombinant eukaryotic strain preferably includes yeast, such as Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae Pichia pastoris () Komagataella pastoris Kluyveromycin (lactic acid yeast) Kluyveromyces lactis ), Yarrowia lipolytica ( Yarrowia lipolytica ) and Hansenula polymorpha ( Ogataea polymorpha The host cells of the recombinant cell line preferably include insect cells or mammalian cells, such as fall armyworm cells or CHO cell series.

[0025] This invention provides the use of the monoclonal antibody, or the monoclonal antibody prepared from the nucleic acid molecule or the derivative thereof, in the preparation of products for detecting Brucella or diagnosing brucellosis.

[0026] In this invention, the detection of Brucella preferably includes detecting whether a sample is infected with Brucella under in vitro conditions, such as testing the safety of food or meat. The detection of Brucella achieves the purpose of pathogen detection by detecting Brucella-specific antibodies in the sample. The diagnosis of brucellosis preferably achieves the diagnosis of the disease by detecting anti-brucellosis antibodies in the serum.

[0027] In this invention, the product preferably comprises detection reagents and / or detection kits. The product is preferably prepared based on the following detection technologies: colloidal gold immunochromatography, enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, and fluorescence immunoassay. The product preferably uses a competitive method to detect Brucella-specific antibodies in the sample.

[0028] This invention provides a competitive ELISA kit, comprising the monoclonal antibody, a ELISA plate coated with brucellin, washing concentrate, enzyme-labeled secondary antibody, substrate, and stop solution.

[0029] In this invention, the preferred concentration of brucellin in the ELISA plate coated with brucellin is 0.2 μg / mL. The preferred coating solution for brucellin is a carbonate-bicarbonate buffer. The preferred method for preparing the ELISA plate coated with brucellin is to add a brucellin solution containing the coating concentration to the ELISA plate, treat at 4°C for 10-14 h, discard the solution, wash 1-2 times, add blocking buffer, treat at 37°C for 2 h, discard the solution again, wash 1-2 times, and then dry and vacuum package. The preferred blocking buffer is a 5% BSA PBST solution.

[0030] In this invention, the working solution of the monoclonal antibody is preferably a 1% BSA solution containing a monoclonal antibody diluted 1:9000, or it can be a PBST solution containing a monoclonal antibody diluted 1:9000. The monoclonal antibody is preferably obtained by immunizing mice with hybridoma cells producing the monoclonal antibody, collecting the ascites fluid of the mice, and then purifying it. The purification method preferably includes purification using a Protein G column. The purity of the monoclonal antibody is preferably above 90%, the concentration is 3.4 mg / mL, and it is stored at -80℃.

[0031] In this invention, the washing concentrate is preferably a PBST solution, diluted 20 times before use. The enzyme-labeled secondary antibody is preferably HRP-labeled goat anti-mouse IgG, purchased from Acinetobacter Biotechnology Co., Ltd. (catalog number: A21010). The substrate is selected based on the type of enzyme and is suitable for the enzymatic hydrolysis. The stop solution is preferably a strong acid solution or a strong alkali solution. In this embodiment of the invention, both the substrate and the stop solution were purchased from Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.

[0032] In this invention, the kit preferably further includes a positive control serum and a negative control serum. The positive control serum is serum from sheep naturally infected with Brucella, and contains high levels of antibodies as detected by a commercially available Brucella antibody detection ELISA kit. The negative control serum is serum from healthy sheep that have not been infected with Brucella and have not been vaccinated against Brucella, and shows a negative result in the red agglutination test.

[0033] This invention does not impose any special limitations on the method of using the kit; any well-known competitive ELISA detection kit method can be used. The detection results of the kit are considered reliable when the negative control OD... 450nm Value > 0.5 and positive control serum inhibition rate (PI) p The experiment is considered successful when the positive control serum inhibition rate (PIP) is greater than 60%. The positive control serum inhibition rate (PIP) is calculated according to Formula I: Positive control serum inhibition rate (PIP) = (Negative control OD) 450nm Value - Positive control OD 450nmValue) / Negative control OD 450nm Value × 100% Formula I.

[0034] If the test is successful, a sample with a PI value > 50% is considered positive for Brucella antibodies; a sample with a PI value ≤ 50% is considered negative for Brucella antibodies. The PI value of the sample is calculated according to Formula II: Inhibition rate (PIs) of the test sample = (OD of the negative control) 450nm Value - OD of the sample to be tested 450nm Value) / Negative control OD 450nm Value × 100% Formula II.

[0035] In this invention, the kit uses brucellin as the detection antigen and the monoclonal antibody as the competing antibody. It detects the level of Brucella-specific antibodies using a competitive method, thus achieving quantitative detection of Brucella. Because brucellin has good specificity, cross-reactions between brucellin and pathogens such as Yersinia enterocolitica O9 are avoided, ensuring the detection specificity of the kit. The kit is not limited to the species of the sample being tested, using the same enzyme-labeled antibody for all samples. There is no need to prepare specific enzyme-labeled secondary antibodies for samples from different species, exhibiting good universality. This competitive ELISA kit can be applied to the prevention and control of brucellosis in livestock such as cattle, sheep, and pigs, meeting the urgent need for high-precision, high-efficiency, and low-cost identification of brucellosis, achieving precise cross-species epidemiological detection of brucellosis, and propelling brucellosis prevention and control into a new stage of precise identification.

[0036] The following detailed description, in conjunction with embodiments, illustrates a monoclonal antibody that specifically binds to brucellin, its detection kit, and its applications, but these should not be construed as limiting the scope of protection of this invention.

[0037] Example 1 Preparation method of brucellin Brucella 104M strain (GenBank accessions: NZ_CP009625.1 and NZ_CP009626.1) was preserved in our laboratory. TSB was added to centrifuge tubes, with a TSB blank control included. Bacterial powder was collected and incubated at 37°C to prepare a concentrated bacterial suspension. The supernatant was then collected by autoclaving and centrifugation. The protein was dissolved by saturated ammonium sulfate precipitation, followed by repeated precipitation with trichloroacetic acid to obtain the target protein. Finally, the protein was dialyzed against PBS and filtered to remove bacteria, yielding brucellin. Brucella was detected by gel electrophoresis, and the results are shown below. Figure 1 As shown, the desired strip size is obtained.

[0038] Example 2 Preparation method of monoclonal antibodies that specifically bind to brucellin (1) Mouse immunization: Seven 6-8 week old BALB / c mice were selected and immunized with 50 μg / mouse by emulsifying a mixture of brucellin purified in Example 1 and Freund's complete adjuvant at a 1:1 ratio. Two weeks later, the mice were immunized again, followed by a third immunization two weeks later, and a fourth booster immunization one week later. Serum was collected from the tail vein of the mice, and the antibody titer was measured. Indirect ELISA was performed using brucellin purified in Example 1 as the coating antigen and goat anti-mouse IgG (H+L) as the secondary antibody. The OD was measured by an enzyme-linked immunosorbent assay (ELISA) reader. 450nm Value, serum OD of the test 450nm / Negative control OD 450nm When (S / N) ≥ 2.1, the maximum dilution factor is the antibody titer. Mice with high antibody titers are selected to prepare monoclonal antibodies.

[0039] (2) Hybridoma cell line screening: Spleen cells from immunized mice were isolated and fused with SP2 / 0 cells at a ratio of 5:1. Positive clones were detected after 7-10 days. After two rounds of subclonal screening, positive clones that stably secreted monoclonal antibodies against brucellosis were obtained. These clones were then passaged and expanded, and promptly cryopreserved in liquid nitrogen. A hybridoma cell line secreting anti-brucellosis monoclonal antibodies was successfully obtained and named 4B3. Cell supernatant was collected for monoclonal antibody subtype identification using mouse monoclonal antibody Ig class / subclass. Expanded cells were collected to prepare ascites fluid.

[0040] Table 1 Comparison of ELISA results for multiple monoclonal antibodies against brucellosis strains

[0041] Table 1 shows that the reactivity of monoclonal antibodies secreted by each hybridoma cell line was compared using the ELISA method during the screening process of hybridoma cell lines. Among them, 4B3 was significantly better than other hybridoma cells in the same batch.

[0042] (3) Six- to eight-week-old BALB / c mice were sensitized by intraperitoneal injection of 500 μL Freund's complete adjuvant one week before hybridoma cell injection. One week later, hybridoma cells were injected intraperitoneally at a cell count of 5.0 × 10⁻⁶. 5 Each mouse was examined. Peritoneal fluid was collected after approximately 7-14 days when the peritoneum of the mice became distended. The ascites was collected after centrifugation at 4000 rpm for 15 min at 4℃. The intermediate layer was then purified using a Protein G column to obtain a monoclonal antibody specifically binding to brucellosis. The results were analyzed by SDS-PAGE electrophoresis. Figure 1 The monoclonal antibody has a purity of up to 90%, a concentration of 3.4 mg / mL, and is stored at -80℃.

[0043] Example 3 Determination of the light and heavy chain variable region sequences of brucellin-specific monoclonal antibodies The hybridoma cell line obtained in Example 2 was expanded to 1.0 × 10⁻⁶ cells / year. 6 In the above steps, RNA was extracted from hybridoma cells and its concentration was determined. Single-stranded cDNA was obtained using a reverse transcription kit and used as a template to amplify the variable region gene of the monoclonal antibody light and heavy chains.

[0044] The PCR primer sequences are as follows: Heavy chain upstream primer: 5'-GAGGTCCAGCTGCAACAATCTG-3' (SEQ ID NO: 10); Heavy chain downstream primer: 5'-TGAGGAGACGGTGACCGTG-3' (SEQ ID NO: 11); Light chain upstream primer: 5'-GATGTTGTGATGACCCAGACTCC-3' (SEQ ID NO: 12); Light chain downstream primer: 5'-TTTCAGCTCCAGCTTGGTCCC-3' (SEQ ID NO: 13). The PCR reaction procedure is as follows: The process involved denaturation at 98℃ for 10 seconds, annealing at 55℃ for 15 seconds, and extension at 72℃ for 5 seconds, for a total of 35 cycles, followed by a final extension at 72℃ for 5 minutes.

[0045] After amplification, the target fragment was subjected to agarose gel electrophoresis and recovered. It was then cloned into the puc19 vector and transformed into competent trans5α cells. The transformed bacterial culture was plated onto ampicillin-resistant LB medium and incubated overnight at 37°C. Single colonies were picked and amplified in LB liquid medium, followed by colony PCR detection. The colonies were then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing and analysis to obtain the variable region sequences of the light and heavy chains of the monoclonal antibody. Simultaneously, the CDR regions of the heavy and light chains were delineated using the International Immunogenetic Information System (IMGT).

[0046] The amino acid sequence of the heavy chain variable region of the brucellin-specific monoclonal antibody obtained by sequencing is listed in the sequence listing SEQ ID NO: 1: EVQLQQSGPELVRPGASVKISCKAS GYTFSDYY MNWVKQ SHGKSLEWIGD INPNNGGT RNNQKFKGKATLTVDRSSSTAYMELRSLTSEDSAIYYC ARNSWYFDV WGTGTTVTVSS.

[0047] The underlined portions are, in order, the amino acid sequences of the heavy chain complementarity-determining regions CDR1, CDR2, and CDR3, namely, amino acids 26 to 33 in SEQ ID NO: 1, amino acids 51 to 58 in SEQ ID NO: 1, and amino acids 97 to 105 in SEQ ID NO: 1.

[0048] The gene encoding the variable region of the heavy chain of the above-mentioned brucellin-specific monoclonal antibody is SEQ ID NO: 2: GAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAGGCCTGGGGCT TCAGTGAAGATATCCTGTAAGGCTTCTGGATACACGTTTCTCTGACTACTACATGAACTGGGTGAAAACAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATCAATCCTAACAATGGTGGTACCAGGAATAACCAGAAGTTCAAGGGC AAGGCCACATTGACTGTAGACAGGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAATCTATTACTGTGCAAGAAACTCCTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA.

[0049] The amino acid sequence of the light chain variable region of the brucellin-specific monoclonal antibody obtained by sequencing is listed in the sequence listing SEQ ID NO: 3: DVVMTQTPLTLSVTIGQPASISCKST QSLLHSDGQTY LHWLLQRPGQSPKRLIY LVS KVDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPLT FGAGTKLELK.

[0050] The underlined portions are, in order, the amino acid sequences of the light chain complementarity-determining regions CDR1, CDR2, and CDR3, namely, amino acids 27 to 37 in SEQ ID NO: 3, amino acids 55 to 57 in SEQ ID NO: 3, and amino acids 94 to 102 in SEQ ID NO: 3.

[0051] The gene encoding the variable region of the light chain of the above-mentioned brucellin-specific monoclonal antibody is SEQ ID NO: 4: GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGAC AACCAGCCTCCATCTCTTGCAAGTCAACTCAGAGCCTCTTACATAGTGATGGACAGACATATTTGCATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAAGTGGACTCTGGAGTCCCTG ACAGGTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAGATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTGCTGGCAAGGTACACACTTTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA.

[0052] Example 4 Preparation method of a competitive ELISA kit for quantitative detection of Brucella antibodies I. Competitive ELISA Kit for Quantitative Detection of Brucella Antibodies The Brucella antibody quantitative detection competitive ELISA kit is constructed using brucellin as the coating antigen and the specific monoclonal antibody sequenced in Example 3 as the competitive antibody.

[0053] The competitive ELISA kit for quantitative detection of Brucella antibodies of the present invention comprises: (1) Two microplates coated with 0.2 μg / mL brucellin, 96 wells each, are stored at 2-8℃; (2) One bottle of 20× washing concentrate, 40 mL / bottle; (3) One tube each of positive control serum and negative control serum, 0.5 mL / tube, stored at 2-8℃; (4) One bottle of working solution for specific binding brucellin monoclonal antibody, 15 mL / bottle, stored at 2-8℃; (5) One bottle of enzyme-labeled antibody (HRP-labeled goat anti-mouse IgG), 20 mL / bottle, stored at 2-8℃; (6) One bottle each of substrate solution A and substrate solution B, 12 mL / bottle, stored at 2-8℃; (7) One bottle of stop solution, 12 mL / bottle, stored at 2-8℃; (8) Two sheets of sealing film; (9) One copy of the instruction manual.

[0054] The kit can be stored at 2-8℃ for 12 months.

[0055] The method for coating brucellin onto an ELISA plate is as follows: Take out the brucin purified in Example 1, which was frozen at -80℃, and after thawing, dilute it to 0.2 μg / mL with carbonate-bicarbonate buffer. Add 100 μL / well to the microplate and coat overnight at 4℃. Discard the liquid in the microplate, wash three times with washing buffer, and then block with 5% BSA at 37℃ for 2 h, 100 μL / well. Discard the liquid, gently pat dry, and finally seal the microplate in a packaging bag for storage.

[0056] The positive control serum was serum from sheep naturally infected with Brucella, and it was found to contain high levels of antibodies using the Qingdao Lijian Brucellosis Antibody Detection Competitive ELISA Kit (catalog number: BRU-C2P).

[0057] The negative control serum was serum from healthy sheep that were not infected with Brucella and were not vaccinated against Brucella, and the blood agglutination test was negative.

[0058] The substrate solution A and substrate solution B, the stop solution, and the 20× wash concentrate in the competitive ELISA kit for detecting Brucella antibodies of the present invention were all purchased from Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences.

[0059] II. Instructions for using the Brucella antibody quantitative detection competitive ELISA kit The optimal reaction conditions were determined using the checkerboard titration method (Table 2). The optimal coating concentration for brucellin was 0.2 μg / mL, overnight coating at 4°C; blocking with 5% BSA; the optimal serum dilution was 1:4, the optimal monoclonal antibody dilution was 1:9000, and the incubation conditions were 37°C for 30 min; the optimal goat anti-mouse IgG (H+L) dilution was 1:10000, and the incubation conditions were 37°C for 30 min; the substrate solution color development conditions were 37°C in the dark for 10 min; and the OD titration was performed within 5 min after adding the stop solution. 450nm Testing.

[0060] Table 2 Optimization of Competitive ELISA Conditions for Brucellosis Antibody Detection

[0061] The method of using the competitive ELISA kit for quantitative detection of Brucella antibodies of the present invention is as follows: (1) Preparation before use: Before use, the solution should be placed at room temperature for 15-30 minutes to reach room temperature before use. After the 20× washing concentrate has returned to room temperature (20-25℃), shake it to dissolve the crystals (it can be heated in a 37℃ water bath for 5-10 minutes). Then dilute it with deionized water at a ratio of 1:20 and mix thoroughly.

[0062] (2) Sample addition: Dilute the serum to be tested 1:2 with 1× washing buffer. Add the diluted serum to be tested, positive control serum and negative control serum to the ELISA plate, 50 μL / well, with 2 wells for each control serum. Dilute the monoclonal antibody 1:4500 and add it to the ELISA plate, 50 μL / well, and incubate at 37℃ for 30 min. If the antigen-coated plate is not used up after opening, the enzyme-labeled strips should be stored in a sealed bag.

[0063] (3) Incubation: Seal the ELISA plate with sealing film and incubate at 37°C for 30 min.

[0064] (4) Washing the plate: Take out the microplate and shake it dry. Wash it 3 times with 1× washing solution and pat it dry on absorbent paper.

[0065] (5) Add enzyme-labeled antibody: Add enzyme-labeled secondary antibody to the ELISA reaction wells, 100 μL / well, and incubate at 37℃ for 30 min.

[0066] (6) Washing the plate: Take out the microplate and shake it dry. Wash it 3 times with 1× washing solution and pat it dry on absorbent paper.

[0067] (7) Color development: Mix substrate solution A and substrate solution B at a volume ratio of 1:1 and immediately add 100 μL / well to the ELISA reaction plate. Let stand at 37°C in the dark for 10 min.

[0068] (8) Termination: Add 100 µL of stop solution per well to terminate the reaction, and measure the OD using a microplate reader within 5 min. 450nm value.

[0069] (9) Result interpretation: Calculate the OD of positive control serum, negative control serum and test sample respectively. 450nm The average value was calculated, and the inhibition rate (PI%) of the positive control serum and the sample to be tested was calculated.

[0070] Positive control serum inhibition rate (PI) P = (Negative control OD) 450nm Value - Positive control OD 450nm Value) / Negative control OD 450nm Value × 100%; Inhibition rate (PIs) of the test sample = (OD of the negative control) 450nm Value - OD of the sample to be tested 450nmValue) / Negative control OD 450nm Value × 100%.

[0071] When negative control OD 450nm Value > 0.5 and positive control serum inhibition rate PI P The experiment is valid when the percentage is >60%.

[0072] If the test is successful, the sample is positive for Brucella antibodies when the PIs value of the sample is >50%; the sample is negative for Brucella antibodies when the PIs value of the sample is ≤50%.

[0073] Example 5 Performance test of the Brucella antibody quantitative detection competitive ELISA kit of the present invention I. Sensitivity test of the Brucella antibody quantitative detection competitive ELISA kit of the present invention Standard positive serum was diluted to 200 IU / mL, and then serially diluted 2-fold to 100 IU / mL, 50 IU / mL, 25 IU / mL, 12.5 IU / mL, 6.25 IU / mL, 3.125 IU / mL, 1.563 IU / mL, 0.781 IU / mL, and 0.391 IU / mL. Different concentrations of standard positive serum were detected using the competitive ELISA kit of this invention, and a standard curve was plotted. The results are shown in [Figure number missing]. Figure 3 .

[0074] Figure 3 This indicates that a positive result can still be detected even when the serum concentration is 3.125 IU / mL, demonstrating that the competitive ELISA kit of this invention has high sensitivity to Brucella antibodies.

[0075] By detecting the above-mentioned standard positive serum at different concentrations using a commercially available competitive ELISA kit, it was found that the lowest detection concentration for positive serum was 12.5 IU / mL. The competitive ELISA kit of the present invention has significantly higher sensitivity than the commercially available competitive ELISA kit.

[0076] II. Repeatability Experiments of the Brucella Antibody Quantitative Detection Competitive ELISA Kit of the Present Invention Twenty positive serum samples and twenty negative serum samples were tested three times using a kit constructed from the same batch, and the results were identical. Tests were also performed on the same serum samples using kits from different batches, and the results were identical. Furthermore, tests were conducted on the same serum samples using the same batch of kits by different technicians, and the results were also identical. This competing ELISA kit of the present invention exhibits excellent reproducibility.

[0077] III. Specificity Experiment of the Competitive ELISA Kit for Quantitative Detection of Brucella Antibodies in this Invention To evaluate the specificity of the Brucella antibody competitive ELISA kit constructed in this invention, cross-reactivity tests were performed on the brucellin, Yersinia enterocolitica O9, and Salmonella O157. The results are shown in Table 3. The monoclonal antibody 4B3 prepared in this invention showed no cross-reactivity with Salmonella O157 and Yersinia enterocolitica O9, but exhibited good specificity with brucellin.

[0078] Table 3 Results of Monoclonal Antibody 4B3 Cross-Reaction ELISA

[0079] IV. Shelf life of the Brucella antibody quantitative detection competitive ELISA kit of the present invention For competing ELISA kits assembled in the same batch, 10 positive serum samples and 10 negative serum samples were tested every 1 week.

[0080] The results show that the shelf life of the kit of the present invention is 12 months.

[0081] V. Results of the Brucella antibody quantitative detection competitive ELISA kit of the present invention in detecting serum from different species The present invention was used to detect antibodies in infected sera from known species and immune sera from known vaccine strains, and the results showed that all samples were positive (Table 4). The competitive ELISA of the present invention can detect antibodies in infected sera from different species and immune sera from different vaccine strains, and has broad applicability.

[0082] Table 4. Suitability analysis of the Brucella competitive ELISA antibody detection kit

[0083] VI. Clinical Detection Experiment of the Brucella Antibody Quantitative Detection Competitive ELISA Kit of the Present Invention The Brucella competitive ELISA kit of the present invention, the Brucella red agglutination test, and the commercial competitive ELISA kit were used to simultaneously detect 356 clinical serum samples, and the consistency rate of the results was verified (Table 5).

[0084] Table 5. Concordance rates between the Brucella competitive ELISA antibody detection kit and the Rose Bengal agglutination assay and commercially available competitive ELISA kits.

[0085] Table 5 shows that the Brucella antibody quantitative detection competitive ELISA kit of the present invention achieves a 100% positive concordance rate, a 94.41% negative concordance rate, and an overall concordance rate of 97.75% with the Rose Bengal agglutination assay; and a 100% positive concordance rate, a 95.74% negative concordance rate, and an overall concordance rate of 98.31% with commercially available competitive ELISA kits. The competitive ELISA kit of the present invention exhibits good concordance rates and is suitable for a wide range of applications in detecting Brucella antibodies.

[0086] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody that specifically binds to brucellin, characterized in that, The monoclonal antibody includes a heavy chain variable region and a light chain variable region; The heavy chain variable region includes heavy chain complementarity-determining regions CDR1, CDR2 and CDR3, and the amino acid sequences of the heavy chain complementarity-determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID NO:5~SEQ ID NO:7; The light chain variable region includes light chain complementarity-determining regions CDR1, CDR2 and CDR3, and the amino acid sequences of the light chain complementarity-determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID NO:8~SEQ ID NO:

10.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1; The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:

3.

3. A nucleic acid molecule encoding the monoclonal antibody of claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region is shown in SEQ ID NO: 2; The nucleotide sequence of the nucleic acid molecule encoding the variable region of the light chain is shown in SEQ ID NO:

4.

5. A derivative product comprising the nucleic acid molecule of claim 3 or 4, characterized in that, It includes at least one of the following: gene expression cassette, recombinant vector, recombinant microbial strain, and recombinant cell line.

6. The use of the monoclonal antibody of claim 1 or 2, the monoclonal antibody prepared from the nucleic acid molecule of claim 3 or 4, or the derivative of claim 5, in the preparation of products for detecting Brucella or diagnosing brucellosis.

7. The application according to claim 6, characterized in that, The products include testing reagents and / or testing kits.

8. The application according to claim 6 or 7, characterized in that, The product is prepared based on the following detection technologies: colloidal gold immunochromatography, enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, and fluorescence immunoassay.

9. A competitive ELISA kit, characterized in that, It includes the monoclonal antibody as described in claim 1 or 2, an enzyme-labeled plate coated with brucellin, a washing concentrate, an enzyme-labeled secondary antibody, a substrate, and a stop solution.

10. The competitive ELISA kit according to claim 9, characterized in that, The concentration of brucellin in the enzyme-labeled plate coated with brucellin was 0.2 μg / mL. The working solution of the monoclonal antibody is a 1% BSA solution containing a monoclonal antibody diluted 1:9000.

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