Rabies virus G protein recombinant antigen, monoclonal antibody and preparation method and application thereof

Abstract

The present invention provides a rabies virus G protein recombinant antigen, a monoclonal antibody and a preparation method and application thereof. A competitive ELISA detection method is constructed by using prokaryotically expressed rabies virus G protein as a coating antigen and a screened blocking monoclonal antibody as an enzyme-labeled antibody, and is applied to the detection of rabies virus neutralizing antibodies. Compared with the existing RFFIT and FAVN detection methods, the competitive ELISA detection method of the present invention is safer, more convenient and more efficient, and has a detection effect close to that of RFFIT. In addition, the prokaryotically expressed recombinant protein used therein has lower cost and higher efficiency, and is easier to obtain and safer than whole virus antigens and eukaryotically expressed recombinant antigens.

Description

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a rabies virus G protein recombinant antigen, a monoclonal antibody and a preparation method and application thereof. Background Art

[0002] Rabies belongs to the genus Rhabdovirus of the family Rhabdoviridae. It is a zoonotic infectious disease caused by the rabies virus (RABV). According to the provisions of the "Law of the People's Republic of China on the Prevention and Control of Infectious Diseases", it is a Class B infectious disease. Many species of mammals are associated with the spread of rabies, and dogs are the main reservoir and transmission hosts of human rabies in developing countries. The most common way for humans to be infected with rabies is through bites, scratches, and licking of damaged skin or mucous membranes by dogs, cats, wild carnivores, and insectivorous and vampire bats infected with rabies virus. Rabies virus mainly invades the human body through damaged skin or mucous membranes, and ascends to the central nervous system through nerve endings. The clinical manifestations are mainly acute, progressive, and almost irreversible encephalomyelitis, with a mortality rate of 100%. Therefore, active and effective rabies vaccination and testing are critical.

[0003] The surface of rabies virus particles has an envelope, which contains nucleoproteins. The nucleocapsid is helically symmetrical and contains single-stranded RNA. The virus contains 5 major proteins (L, N, G, M1 and M2) and 2 microproteins (P40 and P43). Among them, N protein is the main nucleoprotein that constitutes the virus particles and is the main component that induces rabies cell immunity. It is often used in the diagnosis, classification and epidemiological research of rabies virus; G protein is a glycoprotein that constitutes the fiber on the surface of the virus. It has the characteristics of agglutinating red blood cells and is the structure that binds rabies virus to cell receptors. It plays a key role in the pathogenesis and immunity of rabies virus. The G protein of rabies virus is a trimer of about 67kD, encoding 524 amino acids, including a signal peptide composed of 19 amino acids. The G protein contains 3 virus neutralizing antigenic determinants, which mainly play a role when the virus adsorbs host cells, and accelerates the adsorption of RABV by interacting with cell surface receptors. It has been reported that there is a region in rabies virus G protein 109-203aa that can interact with acetylcholine receptors.

[0004] It is reported that more than 60,000 people die of rabies every year in the world, and as the number of pet dogs and cats continues to increase, the risk of infection caused by scratches or bites is also increasing. The effectiveness of rabies vaccines can be evaluated by detecting the level of RABV neutralizing antibodies in the serum of vaccinated subjects.

[0005] The rabies virus neutralizing antibody detection methods recommended by WHO and OIE mainly include the rapid fluorescent focus inhibition test (RFFIT) and the fluorescent antibody virus neutralization test (FAVN). It is generally believed that when the titer of neutralizing antibodies in the body is ≥0.5IU / mL, the effective protection level is reached. Among them, RFFIT is mainly used to detect serum antibody levels after human vaccination. It has been reported that RFFIT has a certain proportion of false positives when detecting antibodies in animal serum at a low level. In addition, these two methods are generally cumbersome to operate, the cost of detection is high, the requirements for laboratories and operators are high, and there are potential biosafety risks. Therefore, it is more urgent to develop a relatively safe and economical rabies virus neutralizing antibody detection method. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a rabies virus G protein recombinant antigen, monoclonal antibody and preparation method and application thereof. The present invention uses prokaryotically expressed rabies virus G protein as coating antigen and screened blocking monoclonal antibody as enzyme-labeled antibody to establish a competitive ELISA method, which can be applied to the detection of rabies virus neutralizing antibodies. Among them, the prokaryotic expressed protein has lower cost and higher efficiency, and is easier to obtain and safer than whole virus antigen and eukaryotic expressed recombinant antigen. The competitive ELISA method involved in the invention has low requirements on laboratories and personnel, can avoid potential biosafety risks of methods such as RFFIT and FVAN, is simple to operate, and the test results are easier to interpret. It has been verified that the detection effect of rabies virus neutralizing antibodies is very close to that of RFFIT, and has broad application prospects.

[0007] The technical solution of the present invention is:

[0008] A rabies virus G protein recombinant antigen, the amino acid sequence of the recombinant antigen is shown as SEQ ID No.1.

[0009] The nucleotide sequence of the gene encoding the recombinant antigen is shown in SEQ ID No.2.

[0010] A method for preparing a rabies virus G protein recombinant antigen, the specific operations are as follows:

[0011] (1) connecting the gene encoding the recombinant antigen to an expression vector to construct a recombinant plasmid containing the recombinant antigen gene;

[0012] (2) The recombinant plasmid is transformed into competent cells, induced for expression, and then purified to obtain the recombinant antigen.

[0013] In step (1), the expression vector is any one of pCold-TF, pET40b, and pSmart1.

[0014] In step (2), the competent cells are any one of BL21 (DE3), Rosetta, and Origami B (DE3).

[0015] A monoclonal antibody that specifically reacts with the recombinant antigen is specifically the variable region amino acid sequence of a mouse monoclonal antibody specific to rabies virus G protein, which has a blocking effect on rabies virus and can be used in competitive ELISA detection of rabies virus neutralizing antibodies.

[0016] The method for preparing the monoclonal antibody comprises the following specific steps:

[0017] S1) adding an adjuvant to the purified recombinant antigen, mixing, and immunizing female BALB / c mice;

[0018] S2) taking all spleen cells of the immunized mice and fusing them with SP2 / 0 myeloma cells in the logarithmic growth phase, screening and culturing them to obtain hybridoma cells;

[0019] S3) The hybridoma cells obtained by screening are expanded and cultured, and injected intraperitoneally into female BALB / c mice, and the ascites is collected and purified after centrifugation to obtain monoclonal antibodies.

[0020] In step (S1), the adjuvant is any one of MF59 aqueous adjuvant, Freund's complete adjuvant, and aluminum hydroxide adjuvant;

[0021] The volume ratio of the purified recombinant antigen to the adjuvant is 1:1.

[0022] A competitive ELISA detection method is constructed based on the monoclonal antibody. Specifically, the prokaryotic expressed rabies virus G protein is used as the coating antigen, and the screened blocking monoclonal antibody is used as the enzyme-labeled antibody to establish a competitive ELISA method that can be applied to the detection of rabies virus neutralizing antibodies.

[0023] The competitive ELISA method is used in detecting rabies virus neutralizing antibodies. The competitive ELISA method has low requirements on laboratories and personnel, can avoid potential biosafety risks of methods such as RFFIT and FVAN, is easy to operate, and the test results are easier to interpret. It has been verified that the detection effect of rabies virus neutralizing antibodies is very close to that of RFFIT, and has broad application prospects.

[0024] The beneficial effects of the present invention are:

[0025] (1) The present invention provides a rabies virus G protein recombinant antigen obtained by prokaryotic expression technology, which has a high expression level and biological activity.

[0026] (2) The present invention screened out a high-binding monoclonal antibody with blocking activity based on the rabies virus G protein recombinant antigen, used the blocking monoclonal antibody as an enzyme-labeled antibody, and used the rabies virus G protein recombinant antigen as a coating antigen to construct a competitive ELISA detection method for the detection of rabies virus neutralizing antibodies. Compared with the existing RFFIT and FAVN detection methods, the competitive ELISA detection method of the present invention is safer, more convenient, and more efficient, and has a detection effect close to that of RFFIT. In addition, the prokaryotically expressed recombinant protein used therein has lower cost and higher efficiency, and is easier to obtain and safer than whole virus antigens and eukaryotically expressed recombinant antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 Shown is a diagram of the identification results of the rabies virus G recombinant protein (RABV / G protein) of the present invention;

[0029] Figure 2 It shows the binding activity of the rabies virus G recombinant protein of the present invention with different polysera of mice immunized with dog vaccine. DETAILED DESCRIPTION

[0030] 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, rather than all of the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0031] The above technical solution is described in detail below in conjunction with specific embodiments.

[0032] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0033] Example 1 Preparation of recombinant antigens

[0034] This embodiment provides a rabies virus G protein recombinant antigen, whose amino acid sequence is shown in SEQ ID No.1, and the nucleotide sequence of the gene encoding the recombinant antigen is shown in SEQ ID No.2.

[0035] SEQ ID No. 1 is as follows:

[0036] DKLGPWSPIDLHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPY PDYHWLRTVKTTKESLVIISPSVTDLDPYDKSLHSRVFPGGNCSGITVSSTYCSTNHDYTIWMPENLRLGTSCDIFTHSRGKRASKGDKTCGFVDERGLYKSLKGACK LKLCGVLGLRLMDGTWVAMQTSDETKWCPPGQLVNLHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSV QTWNEIIPSKGCLRVGERCHPHVNGVFFNGIILGSDGHVLIPEMQSSLLQQHMELLESSVIPLMHPLADPSTVFKDGDEVEDFVEVHLPDVHKQVSGVDLGLPKWGK.

[0037] SEQ ID No. 2 is as follows:

[0038] GATAAGCTGGGCCCGTGGAGTCCGATTGATCTGCATCATCTGAGTTGTCCGAATAATCTGGTTGTGGAAGATGAAGGCTGCACCAATCTGAGCGGCTTTAGCTATATGGAACTGAAAGTTGGCTATATTAGTGCAATTAAGGTTAATGGCTTCACCTGTACCGGCGTGGTTACCGAAGCAGAAACCTATACCAATTTTGTTGGCTATGTTACCACCACCTTTAAACGCAAACATTTTCGCCCGACCCCGGATGCATGTCGCGCAGCTTATAATTGGAAAATGGCCGGTGACCCGCGTTATGAAGAAAGTCTGCATAATCCGTATCCGGATTATCATTGGCTGCGCACCGTTAAAACCACCAAAGAAAGCCTGGTTATTATTAGCCCGAGTGTTACCGATCTGGACCCTTATGATAAAAGCCTGCATAGCCGTGTTTTTCCGGGCGGTAATTGCAGCGGCATTACCGTGAGCAGCACCTATTGCAGTACCAATCATGATTATACCATTTGGATGCCGGAAAATCTGCGCCTGGGCACCAGCTGCGATATTTTTACCCATAGCCGTGGCAAACGTGCCAGCAAAGGCGATAAAACCTGCGGTTTTGTGGATGAACGCGGTCTGTATAAAAGTCTGAAAGGCGCATGCAAACTGAAACTGTGCGGTGTTCTGGGCCTGCGCCTGATGGATGGTACCTGGGTTGCCATGCAGACCAGTGATGAAACCAAATGGTGCCCGCCGGGTCAGCTGGTGAATCTGCATGATTTTCGTAGTGATGAAATTGAACATCTGGTTGTGGAGGAACTGGTGAAAAAACGTGAAGAATGTCTGGATGCCCTGGAAAGTATTATGACCACCAAAAGCGTGAGTTTTCGTCGTCTGAGTCATCTGCGTAAACTGGTTCCGGGTTTTGGTAAAGCCTATACCATTTTTAATAAGACCCTGATGGAAGCAGATGCACATTATAAAAGCGTTCAGACCTGGAATGAAATTATTCCGAGTAAAGGTTGCCTGCGTGTTGGCGAACGTTGCCATCCGCATGTGAATGGTGTTTTCTTTAATGGTATTATCCTGGGTAGCGATGGTCATGTTCTGATTCCGGAAATGCAGAGCAGTCTGCTGCAGCAGCATATGGAACTGCTGGAAAGCAGCGTGATTCCGCTGATGCATCCGCTGGCCGATCCGAGCACCGTGTTTAAAGATGGCGATGAAGTGGAAGATTTTGTTGAAGTTCATCTGCCGGATGTGCATAAACAGGTGAGCGGTGTTGATCTGGGTCTGCCGAAATGGGGCAAA.

[0039] This embodiment provides a method for preparing a rabies virus G protein (RABV / G protein) recombinant antigen, comprising the following steps:

[0040] (1) Plasmid construction:

[0041] The G protein of rabies virus contains a signal peptide and a transmembrane region, which are not used for in vitro recombinant expression. The 28-458aa containing the main epitope of the G protein is predicted and selected for expression using computational software, as shown in SEQ ID No. 2. The gene is synthesized by Anhui General Biotechnology after codon optimization and cloned into the pCold-TF vector to obtain the recombinant plasmid pCold-TF-RABV / G expressing RABV / G;

[0042] (2) Induced expression:

[0043] The recombinant plasmid pCold-TF-RABV / G was transformed into BL21 (DE3) competent cells, and the transformed bacteria were spread on LB agar plates containing 100 μg / mL ampicillin and cultured at 37°C overnight. A single colony was picked and inoculated into 5 mL of LB medium containing 100 μg / mL ampicillin, and cultured at 37°C and 220 rpm overnight. 1% of the total volume of the culture medium was inoculated into LB medium containing 100 μg / mL ampicillin, and cultured at 37°C and 220 rpm for about 3 hours until OD600 was 0.5, cooled at 16°C for 1.5 hours, and IPTG was added at a final concentration of 0.1 mM. After induction at 16°C and 220 rpm for 16 hours, the bacteria were collected.

[0044] (3) Purification of recombinant protein:

[0045] Since the expressed recombinant protein carries a histidine tag, it is purified using GE's AKTA Start, HisTrap HP affinity chromatography columns and Superdex 200 molecular sieves to obtain the purified recombinant protein, which is the recombinant antigen, and the amino acid sequence is shown in SEQ ID No.1.

[0046] Specifically: A buffer is 50mM PB, 300mM NaCl, pH 8.0, B buffer is 50mM PB, 300mM NaCl, 0.5M imidazole, pH 8.0, the chromatography column is balanced with A buffer, then the fermented bacterial solution is centrifuged at 8000rpm for 10min, the precipitate is resuspended with A solution, ultrasonically broken in ice water for 30min, ultrasonicated for 5 seconds at intervals of 5 seconds, centrifuged at 12000rpm for 30min, the supernatant is filtered with a 0.22 micron filter, loaded, the chromatography column is washed with A buffer, and finally gradient eluted with B buffer, and the purification is observed by SDS-PAGE protein gel electrophoresis. Select the target protein collection peak, dialyze and change the solution to buffer A, then use the sample loop to load the sample onto Superdex 200 molecular sieve, collect the target protein peak and concentrate it, determine the protein concentration by Nanodrop, and pack it into 1ml / tube and store it at -20℃.

[0047] (4) Identify the purity of the recombinant protein by SDS-PAGE electrophoresis:

[0048] Protein sample pretreatment: add an equal volume of 2×SDS loading buffer to each sample, boil in a boiling water bath for 10 minutes, and centrifuge at 12000rpm for 3 minutes. Dilute 5× glycine buffer to the working concentration, add it to the electrophoresis tank to the appropriate liquid level, and gently pull the comb out of the solidified gel. Add protein marker and 10μl of treated protein sample to the sample well. Turn on the power supply, adjust the voltage to 80V constant voltage electrophoresis to the separation gel, and then change to 120V until bromophenol blue reaches the bottom of the gel. Cut the gel from the glass plate, place it in Coomassie brilliant blue dye, shake and dye for 4 hours, and then decolorize it with decolorizing solution until the bands are clear.

[0049] like Figure 1 As shown in the figure, "M" represents a protein marker, and "1" represents that lane 1 is the purified RABV / G protein. It can be seen from SDS-PAGE that the recombinant RABV / G protein has a very high purity, which is greater than 90%.

[0050] ELISA to identify the activity of recombinant protein

[0051] The purified recombinant RABV / G protein was coated (1 μg / mL), and its reaction with the serum of mice immunized with rabies vaccine was identified by indirect ELISA method, and blank mouse serum was used as negative control. The recombinant protein was first coated in the microplate: the coating buffer was carbonate buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, fixed to 1L pure water), the coating concentration was 1 μg / mL, 50 μl / well, 4°C overnight; 1% BSA was blocked, 100 μl 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; the polyclonal antibody serum was diluted with PBS in a gradient of 1000 times, 10000 times, and 100000 times, and 50 μL was added to the microplate coated with antigen, and the rabies virus N protein mouse monoclonal antibody was used as a negative control, and the reaction was carried out at 37°C for 30 minutes. 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 secondary antibody (diluted 1:5000 in PBS), react at 37°C for 30 min, wash the plate 4 times, 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 to terminate the reaction, and measure the OD450nm value with an enzyme reader.

[0052] like Figure 2 As shown, "vaccine immune 1" represents the serum of mice immunized with vaccine 1#, "vaccine immune 2" represents the serum of mice immunized with vaccine 2#, and "control serum" represents the negative control blank mouse serum. It can be seen from the figure that the recombinant RABV / G protein has a good reaction with the polyclonal antibody serum immunized with canine rabies vaccine, and it is still positive when the serum is diluted 10,000 times, while the negative control serum does not react, indicating that the prokaryotic expressed G protein has good biological activity.

[0053] There are many ways to obtain recombinant proteins, and the differences lie in the expression system, expression plasmid or gene, expression vector, transfection or induction expression conditions, etc. The present invention only lists one embodiment of preparing recombinant protein, but different expression vectors, strains or induction conditions based on this gene can achieve the purpose. There are many specific implementation methods, which are not described here one by one.

[0054] Example 2 Screening of monoclonal antibodies

[0055] This embodiment provides a method for obtaining monoclonal antibodies based on the recombinant antigen screening, and the specific operations are as follows:

[0056] S1) Mouse immunization

[0057] 200 μL of the mixture obtained by mixing the commercially available canine rabies inactivated vaccine (PV / BHK-21 strain) with an equal volume of MF59 adjuvant was injected intramuscularly into 6-week-old female BALB / c mice, and then immunized again in the 2nd and 4th weeks according to the above method. At the 5th week, the mouse serum was collected to detect the antibody titer using the recombinantly expressed G protein. The mouse with the highest titer was selected and boosted with 20 μg of recombinant rabies virus G protein through the tail vein. Three days later, the mouse spleen was collected for the preparation of hybridoma cells;

[0058] S2) Screening of hybridoma cell lines

[0059] 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, clones positive for rabies virus G protein were screened by indirect ELISA, and then positive cells were cloned to a monoclonal state by limiting dilution, and the cell lines were expanded and frozen;

[0060] S3) Screening of positive clones by indirect ELISA:

[0061] RABV / G recombinant protein and RABV / N recombinant protein (expressed by pCold vector, containing histidine tag) were coated in microplates, the coating buffer was carbonate buffer (1.59g sodium carbonate, 2.93g sodium bicarbonate, fixed to 1L pure water), the coating concentration was 1μg / mL, and the plate was blocked at 4°C overnight; 1% gelatin was blocked, 150μL per well, blocked at 37°C for 2 hours, washed once with detergent, and patted dry; 50μL of cell culture supernatant was added, and reacted at 37°C for 30min. The liquid in the well was thrown out, the plate was washed 4 times with PBST washing solution, and 50μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 with PBS) was added after patting dry, reacted at 37°C for 30min, and the plate was washed 4 times again, 50μL / well of TMB color development solution was added after patting dry, and color was developed at room temperature for 10min, and finally 0.5M sulfuric acid was added to terminate the reaction, and the OD450nm value was measured with an enzyme marker. Positive cell lines that react only with RABV / G recombinant protein but not with control antigen were selected for subsequent experiments.

[0062] S4) Preparation of monoclonal antibody ascites

[0063] After the selected monoclonal cell lines were expanded and cultured, 0.2 mL (containing 2.5×10 6 Cells) were pretreated with Freund's incomplete adjuvant in female BALB / c mice. After about 10 days, 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.

[0064] S5) Affinity chromatography purification of monoclonal antibodies

[0065] The ascites was centrifuged at 12000r / min for 5 minutes, the supernatant was diluted 10 times with binding buffer (20mM PBS, 150mM NaCl, pH7.4), filtered through a 0.22μm filter, and the filtered sample was pumped into the Protein G (Cytiva) purification column equilibrated with binding buffer at a low speed through a peristaltic pump, connected to an AKTA purifier, washed with binding buffer for 5-10 column volumes until the UV absorption peak was washed flat, and then eluted with elution buffer (0.1M glycine, pH 2.7), and the elution peak was collected. The collected sample was adjusted to neutral with 1M Tris-HCl, pH 9, and placed in a dialysis bag (MW: 8000-14000), and dialyzed in 0.01M PBS pH7.4 solution at 2-8°C for 14 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000r / min for 5 minutes. The supernatant was the purified monoclonal antibody.

[0066] S6) Identification of binding activity of monoclonal antibodies

[0067] Referring to the aforementioned indirect ELISA method, the monoclonal antibody was graded diluted at concentrations of 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, and 1 ng / mL. The rabies virus N protein mouse monoclonal antibody was used as a negative control to determine the binding activity of the screened monoclonal antibody, and the monoclonal antibody with high binding activity was selected for subsequent blocking screening.

[0068] Example 3 Screening and application of blocking monoclonal antibodies

[0069] 1. Screening of blocking monoclonal antibodies

[0070] Antibody HRP labeling: The screened monoclonal antibodies were labeled according to the G-Biosciences HOOK TM The labeling operation was performed according to the instructions of H RP PLUS Labeling Kit (Cat#: 786-313). Finally, the labeled antibody was dialyzed overnight in 0.01 M PBS, pH 7.4 buffer, glycerol was added at a volume of 1:1, and the cells were stored at -20°C.

[0071] Specifically: dilute the antibody to be labeled with coupling buffer to a final concentration of 1-2 mg / mL, then add the diluted antibody solution to the tube containing HRP (provided with the kit), mix it by pipetting, and incubate it at room temperature for 1 hour. Mix it regularly during the incubation period. Add 50uL of stop solution, mix it for 15 minutes to terminate the labeling reaction, dialyze it in PBS buffer overnight, and add an equal volume of glycerol for freezing.

[0072] Dilute the antigen to 1μg / ml with coating solution, add 100μL to each well of the ELISA plate, and incubate at 4℃ overnight. Discard the coating solution in the well, add 200μL of blocking solution to each well, incubate at 37℃ for 2h, and discard the blocking solution in the well. Set the gradient of 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32 for positive serum (4.5IU / mL) and negative serum, and dilute the HRP-labeled monoclonal antibody to the same working concentration. Add 50μL of the serum to be tested and 50μL of the same volume of enzyme-labeled monoclonal antibody working solution to each well, incubate at 37℃ for 1h, discard the solution in the well, and wash the plate 5 times with washing solution. Add TMB color substrate to each well and color for 10min at room temperature in the dark. Add 100μL of stop solution to each well to stop the reaction, and measure the OD450nm value on the ELISA reader within 10min. Calculate the inhibition rate, serum inhibition rate (I%) = (1-S / N) × 100%. Select the antibody with the highest inhibition rate (i.e., blocking monoclonal antibody) for subsequent optimization and testing of the competitive ELISA system.

[0073] The full-length amino acid sequence of the heavy chain (H chain) variable region of the obtained blocking monoclonal antibody is shown in SEQ ID No:3, and the nucleotide sequence is shown in SEQ ID No.4.

[0074] SEQ ID No.3 is as follows:

[0075] EVQLVESGAEVVKPGASVKMSCKASGYPFTSYNIHWIKQTPGQGLEW IGAIYPGNGDTSYTQKFKVKATLTSDKSSSTAYMQLSSLTSEDSAVYFCAR YGNYPSYAMDYWGQGTSVTVSS.

[0076] The three CDRs sequences of the heavy chain (H chain) are as follows:

[0077] CDR-H1 amino acid sequence: GYPFTSYN (SEQ ID No. 7);

[0078] CDR-H2 amino acid sequence: IYPGNGDT (SEQ ID No. 8);

[0079] CDR-H3 amino acid sequence: ARYGNYPSYAMDY (SEQ ID No. 9).

[0080] SEQ ID No. 4 is as follows:

[0081] Gaggtgcagctggtggagtctggggctgaggtggtgaaacctggggcctcagtgaagatgtcctgcaaggcttctggctacccatttaccagttacaatattcactggataaagcagacacctggacagggcctggaatggattggagctatttatccaggaaatggtgatacttcctaca ctcagaagttcaaagtcaaggccacattgacttcagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaggactctgcggtctatttctgtgcaagatacggtaactatccttcctatgctatggactactggggtcaaggaacctcagtcaccgtctcctcag.

[0082] The full-length amino acid sequence of the light chain (L chain) variable region of the obtained blocking monoclonal antibody is shown in SEQ ID No.5, and the nucleotide sequence is shown in SEQ ID No.6.

[0083] SEQ ID No.5 is as follows:

[0084] DIQMIQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQYKPGQP PRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSRKV PSTFGGGTKLELK.

[0085] The three CDRs sequences of the light chain (L chain) are as follows:

[0086] CDR-L1 amino acid sequence: QSVDYDGDSY (SEQ ID No. 10);

[0087] CDR-L2 amino acid sequence: LVS;

[0088] CDR-L3 amino acid sequence: QQSRKVPST (SEQ ID No.11).

[0089] SEQ ID No.6 is as follows:

[0090] gacatccagatgattcagtctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgttgattatgatggtgatagttatatgaactggtaccaatacaaaccaggacagccacccagactcctcatctatcttgtatc caacctagaatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggatgctgcaacctattactgtcaacaaagtaggaaggttccttcgacattcggtggaggcaccaagctggagctgaaa.

[0091] 2. Optimization and application of competitive ELISA detection system

[0092] Determination of antigen coating concentration and enzyme-labeled monoclonal antibody dilution multiple: The prokaryotic expressed pCold-RABV / G antigen was diluted with coating solution to 0.5μg / ml, 1μg / ml, 2μg / ml, and 4μg / ml, and then added to a 96-well ELISA plate at 100μl / well, respectively. The plate was coated at 4℃ overnight, and the coating solution in the wells was discarded. 150μL of blocking solution was added to each well, and the plate was incubated at 37℃ for 2h, and the blocking solution in the wells was discarded. The enzyme-labeled monoclonal antibody was diluted with a secondary antibody protective agent (HRP protective agent, provided by Beijing Meikowande Biological) at 1:200, 1:400, 1:800, 1:1600, and 1:3200, respectively. The positive serum and negative serum were mixed with the 6 dilutions of enzyme-labeled monoclonal antibodies, respectively, and incubated at 37°C for 1h. The solution in the wells was discarded, and the plate was washed 5 times with a washing solution, 300μL / well. The color substrate was added to each well, and the color was developed at room temperature in the dark for 10min. 100μL of the stop solution was added to each well to stop the reaction, and the OD450nm value was measured on the microplate reader within 10min. The antigen coating concentration and enzyme marker concentration corresponding to the well with the smallest positive value / negative value (P / N value) were the optimal antigen coating concentration and enzyme marker working concentration.

[0093] Determination of serum sample dilution multiples: dilute 4.5IU / mL positive serum, 0.5IU / mL positive serum and negative serum at 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64 and 1:128 respectively, perform cELISA test, calculate P / N value (positive value / negative value), and select the serum dilution multiple corresponding to the hole with the smallest P / N value as the optimal dilution multiple of the serum to be tested. The level of serum neutralizing antibodies after rabies vaccine immunization is an indicator for determining the degree of vaccine immunity. The WHO Rabies Expert Committee believes that serum with a neutralizing antibody level equal to or higher than 0.5IU / ml can be effectively protected, that is, serum with an antibody titer greater than or equal to 0.5IU / mL is considered positive, otherwise it is negative.

[0094] Through the optimization of the antigen coating concentration, the test serum dilution multiple and the enzyme-labeled monoclonal antibody dilution multiple, it was determined that 1 μg / ml antigen was used for coating, 1:1 dilution of the test serum and 800-fold dilution of the enzyme-labeled monoclonal antibody were the best detection conditions.

[0095] 3. Determination of critical value of competitive ELISA detection method

[0096] 200 sera with known background were randomly selected for cELISA testing, including 150 negative sera and 50 positive sera. Two wells were set for each control serum. The S / N value was calculated and the data were subjected to ROC curve analysis.

[0097] By testing known background serum and using statistical software to analyze and generate the ROC curve, the critical point of this detection method was determined to be 0.6, based on the CUT-OFF point corresponding to the maximum Youden index. That is, when S / N≤0.6, the result was judged to be positive (S represents sample, N represents negative control), indicating that the RABV neutralizing antibody value of the sample to be tested is ≥0.5IU / mL; when S / N>0.6, the result was judged to be negative, indicating that the RABV neutralizing antibody value of the sample to be tested is <0.5IU / mL.

[0098] 4. Sensitivity and compliance rate of competitive ELISA detection method

[0099] The rabies positive reference serum was diluted at 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128, for a total of 8 samples, and cELISA detection was performed. The results are shown in Table 1. The detection sensitivity of the competitive ELISA detection method of the present invention for positive reference serum is 1:32, and the positive can still be detected after the reference serum is diluted 32 times.

[0100] Table 1 - Sensitivity test results of competitive ELISA detection method (S / N value)

[0101] serum 1:1 1:2 1:4 1:8 1:16 1:32 1:64 1:128 NC PC S / N 0.11 0.18 0.27 0.38 0.46 0.58 0.71 0.72 2.11 0.23

[0102] 100 serum samples were randomly selected and tested by the competitive ELISA method of the present invention and RFFIT, and the coincidence rates of the two were compared. The results are shown in Table 2. The test results show that the sensitivity is 95%, the specificity is 98.3%, and the total coincidence rate is 97%, which is greater than 95%, indicating that the accuracy of the detection method of the present invention is high and can be applied to the detection of clinical samples.

[0103] Table 2-Competitive ELISA and rapid fluorescence focus inhibition test coincidence rate

[0104]

[0105] 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. A rabies virus G protein monoclonal antibody, characterized in that: The antibody comprises a VH domain and a VL domain; The VH domain includes CDR-H1 of the amino acid sequence shown in SEQ ID No.7, CDR-H2 of the amino acid sequence shown in SEQ ID No.8, and CDR-H3 of the amino acid sequence shown in SEQ ID No.9; The VL domain includes CDR-L1 with the amino acid sequence shown in SEQ ID No.10, CDR-L2 with the amino acid sequence of LVS and CDR-L3 with the amino acid sequence shown in SEQ ID No.

11.

2. The rabies virus G protein monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the VH domain is shown in SEQ ID No.3, and the nucleotide sequence is shown in SEQ ID No.

4.

3. The rabies virus G protein monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the VL domain is shown in SEQ ID No.5, and the nucleotide sequence is shown in SEQ ID No.

6.

4. A competitive ELISA detection method constructed based on the monoclonal antibody described in any one of claims 1 to 3.

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