Recombinant rabbit monoclonal antibody against BHV-1 and double antibody sandwich ELISA reagent and its application

Abstract

The present invention is applicable to the field of immunoassay, and provides a recombinant rabbit monoclonal antibody against BHV-1 and a double antibody sandwich ELISA reagent and application, wherein the recombinant rabbit monoclonal antibody against BHV-1 is any one of rabbit monoclonal antibody D1, rabbit monoclonal antibody D2 and rabbit monoclonal antibody D3; the amino acid sequence of rabbit monoclonal antibody D1 is shown in the sequence table SEQ ID NO.1; the amino acid sequence of rabbit monoclonal antibody D2 is shown in the sequence table SEQ ID NO.2; the amino acid sequence of rabbit monoclonal antibody D3 is shown in the sequence table SEQ ID NO.3. In addition, the double antibody sandwich ELISA reagent includes the above-mentioned rabbit monoclonal antibody D2 and rabbit monoclonal antibody D3, which can be used for rapid and accurate detection of BHV-1, has the characteristics of strong specificity and high sensitivity, and is more suitable for promotion in clinical detection.

Description

Technical Field

[0001] The invention belongs to the field of immune detection, and in particular relates to a recombinant rabbit monoclonal antibody against BHV-1 and a double antibody sandwich ELISA reagent and applications thereof. Background Art

[0002] Bovine herpesvirus 1 (BHV-1) is a highly contagious and latent virus that can cause a variety of clinical syndromes such as respiratory and reproductive tract diseases, which have a significant impact on the milk production of dairy cows, the fertility of bulls and the fattening rate of beef cattle. At the same time, as one of the important pathogens of bovine respiratory disease syndrome, it is the direct cause of cattle death. The virus is widely present in my country and has a high positive detection rate. With the continuous increase in the number of cattle in my country, this has had a serious impact on the economy of the cattle industry.

[0003] BHV-1 is a member of the alphaherpesvirus subfamily of the herpesviridae family. It is an enveloped double-stranded DNA virus. The gD protein is one of its main structural proteins and is essential for viral replication and infection. Many papers have shown that the gD protein can often induce higher neutralizing antibody titers than other proteins. Due to its immunodominance and sequence conservation, it is considered to be an ideal antigen for diagnostic testing and vaccine immune evaluation.

[0004] In view of the sequence and functional characteristics of gD protein, the development of gD-specific monoclonal antibodies based on phage display technology is extremely beneficial for BHV-1 detection and diagnosis and post-vaccine immunization evaluation. Phage technology is easy to operate and highly flexible, and is considered to be a fast and effective tool for discovering and developing monoclonal antibodies. At present, most antibodies in BHV-1 detection kits are mouse monoclonal antibodies. Although mouse monoclonal antibody technology is widely used, it has relatively weak affinity and low specificity, and is still insufficient in the field of immunological detection. Compared with mouse monoclonal antibodies, rabbit monoclonal antibodies have more advantages. Rabbit monoclonal antibodies can recognize more epitope antigenic determinants of immune antigens, with strong affinity, good specificity and high stability. Therefore, the development of a rabbit monoclonal antibody with high affinity and strong specificity is of great significance for the high-sensitivity detection of BHV-1.

[0005] In addition, the current diagnosis method of BHV-1 mainly uses virus isolation and identification, PCR and virus neutralization experiments, but they must be operated in a special laboratory, and the experimental cycle is long and the cost is high. Therefore, it is urgent to establish a method that is simple to operate, low-cost and conducive to large-scale detection of pathogens. The ELISA method is simple and fast to operate, can be used for large-scale detection, and has high sensitivity and good specificity of the test results. It is currently the most widely used method. Among them, the double antibody sandwich ELISA method has the advantages of high sensitivity and strong specificity through the dual screening of capture antibodies and detection antibodies, and is often used for accurate detection of viral serology. Summary of the invention

[0006] The purpose of the embodiments of the present invention is to provide a recombinant rabbit monoclonal antibody against BHV-1, aiming to solve the problems raised in the background technology.

[0007] In view of the above problems, an embodiment of the present invention is implemented as follows: a recombinant rabbit monoclonal antibody against BHV-1 is provided, wherein the recombinant rabbit monoclonal antibody is any one of rabbit monoclonal antibody D1, rabbit monoclonal antibody D2 and rabbit monoclonal antibody D3; the amino acid sequence of the rabbit monoclonal antibody D1 is shown in the sequence listing SEQ ID NO.1; the amino acid sequence of the rabbit monoclonal antibody D2 is shown in the sequence listing SEQ ID NO.2; the amino acid sequence of the rabbit monoclonal antibody D3 is shown in the sequence listing SEQ ID NO.3.

[0008] Preferably, the method for preparing the recombinant rabbit monoclonal antibody comprises the following steps:

[0009] The recombinant gD protein expressed in prokaryotes was used to construct a scFv phage display library specific for BHV-1 gD protein;

[0010] The amino acid sequence of the recombinant rabbit monoclonal antibody is obtained by screening the specific scFv phage display library;

[0011] According to the amino acid sequence of the recombinant rabbit monoclonal antibody, a recombinant expression vector is constructed, and cells are transfected for recombinant expression to obtain the recombinant rabbit monoclonal antibody.

[0012] Preferably, the method for obtaining the recombinant gD protein is: removing the transmembrane region and signal peptide of the gD protein, adding a 6×His tag to the C-terminus, constructing a pET32a-gD recombinant expression vector for prokaryotic expression, and obtaining the recombinant gD protein.

[0013] Preferably, the amino acid sequence of the gD protein is as shown in SEQ ID NO.4 in the sequence listing.

[0014] Another object of the embodiment of the present invention is to provide a use of a recombinant rabbit monoclonal antibody in preparing a BHV-1 detection kit.

[0015] Another object of an embodiment of the present invention is to provide a double antibody sandwich ELISA reagent, which includes at least one of the above-mentioned recombinant rabbit monoclonal antibodies.

[0016] Preferably, the rabbit monoclonal antibody D2 and the rabbit monoclonal antibody D3 are included.

[0017] Preferably, the rabbit monoclonal antibody D2 is used as the detection antibody, and the rabbit monoclonal antibody D3 is used as the capture antibody.

[0018] Another object of an embodiment of the present invention is to provide a use of the above-mentioned double antibody sandwich ELISA reagent in preparing a kit for detecting BHV-1 by ELISA method.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] The recombinant rabbit monoclonal antibody provided in the embodiment of the present invention is a rabbit antibody, which has higher affinity and stronger specificity than mouse antibodies. In addition, the embodiment of the present invention uses rabbit monoclonal antibody D2 as a detection antibody and rabbit monoclonal antibody D3 as a capture antibody to establish a double antibody sandwich ELISA method for rapid detection of BHV-1. In the reaction, the antigen binds to different antigenic determinants of the two antibodies, which greatly improves the specificity of the detection and makes the detection result more reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the result diagram of antiserum titer test;

[0022] Figure 2 Figure 1 is the sequencing result of positive clones;

[0023] Figure 3 It is the sequence diagram of rabbit monoclonal antibodies D1, D2, and D3;

[0024] Figure 4 Schematic diagram of the construction of eukaryotic recombinant expression vector;

[0025] Figure 5 The result of enzyme digestion identification of the vector;

[0026] Figure 6 This is the SDS PAGE result diagram of the recombinant rabbit monoclonal antibody in the non-reducing state and under reducing conditions;

[0027] Figure 7 This is the Western Blot result of the recombinant rabbit monoclonal antibody under reducing conditions;

[0028] Figure 8 This is the result of affinity test between rabbit monoclonal antibodies D1, D2, D3 and recombinant gD protein;

[0029] Fig. 9 It is the P / N ratio diagram using different types of sealants;

[0030] Fig.10 It is the P / N ratio diagram of different incubation times;

[0031] Fig.11 It is the P / N ratio diagram of different color development time;

[0032] Fig.12 OD of negative and positive samples 450 Value detection result diagram;

[0033] Fig.13 This is a statistical analysis chart of the clinical sample test results;

[0034] Fig.14 This is a graph showing the specificity test results. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0036] The embodiments of the present invention utilize the characteristics of phage display technology, such as simple preparation process, short development cycle and low cost, to prepare rabbit monoclonal antibodies with strong specificity and high sensitivity, and at the same time develop a double antibody sandwich ELISA kit for detecting BHV-1 to replace the indirect ELISA method, thereby overcoming the disadvantages of low sensitivity and poor specificity of the indirect ELISA method, thereby facilitating the rapid and accurate detection of clinical samples of animal pathogens.

[0037] Specifically, in one embodiment of the present invention, three recombinant rabbit monoclonal antibodies against BHV-1 are provided, namely rabbit monoclonal antibody D1, rabbit monoclonal antibody D2 and rabbit monoclonal antibody D3;

[0038] Among them, the amino acid sequence of rabbit monoclonal antibody D1 is shown in SEQ ID NO.1 in the sequence listing, specifically: QS LEESGGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGIIGGSGSTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARVYDDYGDDYFSIWGPGTLVTVSSGGGGSGGGGSGGGGSAQVMTQTPSSKSVPVGDTVTINCLASEFLFNAVSWYQQKPGQPPKLLIYGASNLESGVPPRFSGSGSGTDYTLTIGGVQAEDVATYYCLGGCSGSADLTFGGGTELEIL;

[0039] The amino acid sequence of rabbit monoclonal antibody D2 is shown in SEQ ID NO.2 in the sequence listing, specifically: QSLEESG GRLVTPGTPLTLTCTVSGFSLSSYDMSWVRQAPGKGLEWIGIINSYGTTYYASWVNGRFTISKTSTTVDLKITSPTTEDTATYFCARAADANGWPFKLWGPGTLVTVSSGGGGSGGGGSGGGGSAQVMTQTPSSKSVPVGDTVTINCQSSQSVYNNNQLSWYQQKPGQPPKLLIYSSTLASGVPPRFSGSGSGTDYTLTIGGVQAEDVATYYCLGTYSGFGGGTELEIL;

[0040] The amino acid sequence of rabbit monoclonal antibody D3 is shown in SEQ ID NO.3 in the sequence listing, specifically: QSLEESG GRLVTPGTPLTLTCTVSGFSLSTYAMNWVRQAPGKGLEWIGIISSSGLKYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCAREYDSYGYAYDHGMDLWGPGTLVTVSSGGGGSGGGGSGGGGSAQVMTQTPSSKSVPVGDTVTINCQASQSISSYLSWYQQKPGQPPKLLIYKASTLASGVPPRFSGSGSGTDYTLTIGGVQAEDVATYYCQSNYHSSSSNYVHAFGGGTELEIL.

[0041] In a preferred embodiment of the present invention, the method for preparing the above-mentioned recombinant rabbit monoclonal antibody comprises the following steps:

[0042] S1. Construct a specific scFv phage display library for BHV-1 gD protein using prokaryotically expressed recombinant gD protein;

[0043] S2. obtaining the amino acid sequence of the recombinant rabbit monoclonal antibody by screening the specific scFv phage display library;

[0044] S3. According to the amino acid sequence of the recombinant rabbit monoclonal antibody, a recombinant expression vector is constructed, and cells are transfected for recombinant expression to obtain the recombinant rabbit monoclonal antibody.

[0045] In practical applications, the above step S1 may specifically include the following steps:

[0046] S11. Antigen preparation: Analyze the amino acid sequence of gD protein, remove the transmembrane region and signal peptide of gD protein, add 6×His tag at the C-terminus, construct pET32a-gD recombinant expression vector for prokaryotic expression, obtain recombinant gD protein, and use the recombinant gD protein as the antigen for immunizing rabbits.

[0047] The amino acid sequence of the gD protein is shown in Gene Bank Accession No. 001847.1 (as shown in the sequence table SEQ ID NO. 4), specifically: MLPTPAPRVTVYVDPPAYPMPRYNYTERWHTT GPIPSPFADGREQPVEVRYATSAAACDMLALIADPQVGRTLWEAVRRHARAYNATVIWYKIESGCARPLYYMEYTECEPRKHFGYCRYRTPPFWDSFLAGFAYPTDDELGLIMAAPARLVEGQYRRALYIDGTVAYTDFMVSLPAGDCWFSKLGAARGYTFGACFPARDYEQKKVL RLTYLTQYYPQEAHKAIVDYWFMRHGGVVPPYFEESKGYEPPPAADGGSPAPPGDDEAREDEGETEDGAAGREGNNGGPPGPEGDGESQTPEANGGAEGEPKPGPSPDADRPEGWPSLEAITHPPPAPATPAAPDAVPVSYTRRRGAGPLPRKPKKLPAFGNVNYSALPGHHHHHH*.

[0048] S12. Animal immunization: The recombinant gD protein was mixed and emulsified with complete Freund's adjuvant at a mass ratio of 1:1, and multiple New Zealand white rabbits were immunized by subcutaneous injection. After an interval of two weeks, the recombinant gD protein was mixed with incomplete Freund's adjuvant for the second, third and fourth immunizations.

[0049] S13. Evaluation of immune effect: Blood was collected after the fourth immunization and serum titer was determined by gradient dilution using ELISA method.

[0050] In the embodiment of the present invention, rabbits are used as immunized animals. Since rabbits have large spleens and high genetic diversity, antibodies with greater epitope coverage can be obtained.

[0051] The above step S2 may specifically include the following steps:

[0052] Construction of scFv (Single-chain fragment variable, scFv) phage display library of S21 and gD proteins: The immunized rabbits in the eighth week were deeply anesthetized and killed, and the spleen tissue RNA was extracted using the RNA Easy Fast Animal Tissue / Cell Total RNA Extraction Kit, and a reverse transcription experiment was performed to obtain cDNA. PCR was performed using specific primers to amplify VH and VL, and a 350bp fragment was recovered by gel recovery to obtain the first fragment. VH and VL were connected to ScFv using overlap extension PCR, and then after SfiI digestion, they were connected to the pComb3XSS vector to obtain recombinant phage plasmids, which were electroporated into ER2738 super competent cells to form phage scFv library bacteria, and then wild-type helper phage M13KO7 was added to infect the host bacteria, and the host bacteria were amplified overnight. The bacteria were precipitated with PEG-NaCl to obtain the supernatant, and the primary phage scFv display library was obtained.

[0053] S22. Enrichment and screening of positive clones: Three rounds of solid phase panning were used to screen the recombinant gD protein phage scFv display library with good enrichment and specific monoclonal aggregation. In the clean bench, a sterile ELISA plate was coated with the BHV-1gD protein prepared by prokaryotic expression as the antigen, and the antigen was diluted with sterile PBS to a concentration of 25μg / mL. 100μL of diluted antigen solution was added to each well of the plate and incubated at 4°C overnight. The same antigen was used in the second round of panning, but the coating concentration was reduced to 15μg / mL. In the third round of panning, the coating concentration of the antigen was reduced to 5μg / mL. The next day, take the above-mentioned ELISA plate and wash it with a plate washer, carefully tap the plate on a clean paper, remove the PBST washing solution, use PBS sterile buffer containing 3% BSA as a blocking solution, add 200 μL to each well and incubate it in a 37°C constant temperature incubator for 2 hours. After the blocking is completed, wash the ELISA plate and carefully tap off the PBST washing solution for use. Take 3mL of the above-prepared primary antibody library phage solution and dilute it 5 times with 1% BSA PBS (use the primary antibody library phage solution in the second round of panning, and so on), and let it stand at room temperature for 10 minutes. Add 150 μL to each well of the blocked ELISA plate, incubate it at 37°C for 2 hours, wash the ELISA plate 3 times after the incubation is completed, 3 minutes each time, and tap off the PBST washing solution. Add 100 μL of 0.2M GLy-HCl eluent at pH=2.2 to each well of the above ELISA plate, rotate at 37°C, 80 rpm for 8 min, and then repeatedly blow and mix. Carefully pipette the eluent into a clean 15 mL centrifuge tube, and immediately add an equal volume of Tris-HCl at pH=7.4 to neutralize the phage eluent. Set aside 1 mL of the eluted phage for storage capacity determination and bacterial solution PCR to identify the insertion rate of the target gene.

[0054] In the embodiment of the present invention, rabbits are used as immunized animals. Since rabbits have large spleens and high genetic diversity, antibodies with greater epitope coverage can be obtained.

[0055] The above step S3 may specifically include the following steps:

[0056] S31, eukaryotic expression and purification of monoclonal antibody: The heavy chain variable region and light chain variable region in the sequences of the above rabbit monoclonal antibody D1, rabbit monoclonal antibody D2, and rabbit monoclonal antibody D3 are connected in series with the rabbit heavy chain constant and light chain constant regions, respectively, and the kozak sequence (GCCGCCACC, as shown in the sequence table SEQ ID NO.5) is added before the start codon, and the human serum albumin sequence MKWVTFISLLFLFASAYS (as shown in the sequence table SEQ ID NO.6, see GeneBank accession number: NP_000468) is added after the start codon, and Xbal is added to the 5' end of the sequence, and the EcoRV restriction site is added to the 3' end, and inserted into the pcDNA3.4 empty vector to construct a eukaryotic recombinant expression vector, which is sent to Nanjing GenScript Biotechnology Co., Ltd. to optimize the codon preference, GC content, mRNA secondary structure, and repeat sequence region using CHO cells as the host, and gene synthesis is performed at Nanjing GenScript. The light chain and heavy chain are combined and co-transfected into mammalian cells for expression purification, transfection and purification, and the method is as follows:

[0057] Take CHO-K1 cells with good growth status and cell viability greater than 90% and centrifuge them. Take a cell volume of 750 μL and the number of cells is 1×10 7 , after mixing, centrifuge at 650rpm for 5min, discard the supernatant, add 800μL of high-glucose DMEM medium to resuspend, add 50ug of heavy chain and light chain plasmids respectively, add to the pre-cooled electrode cup and mix slowly, use 4mm Bole electric shock cup for electrotransformation, the electrotransformation conditions are 300v, 900uF, after electrotransformation, absorb the liquid in the electric shock cup and put it into 15mL high-glucose DMEM medium for culture, screen with 800μg / mL G418, collect the supernatant, centrifuge at 8000rpm for 5min. Use 20mL PBS, 1mL / min flow rate to balance the chromatography column, load at 1mL / min flow rate, elute nonspecific proteins with 20mL PBS, 1mL / min flow rate, elute with glycine buffer (pH=3.0), flow rate 1mL / min, collect in separate tubes, about 3mL per tube. Collect 2 tubes in total, add 100 μL of neutralizing solution before elution to ensure that the eluted protein is stored at a neutral pH, collect the purified antibodies, and then store them in aliquots at -20°C.

[0058] S32, affinity test of rabbit monoclonal antibodies D1, D2, D3 and recombinant gD protein: dilute the recombinant gD protein to 0.5μg / ml with coating solution, add 100μL / well, and incubate at 2-8℃ overnight; wash the plate 3 times with washing solution, pat dry, add 300μL / well blocking solution, and block at room temperature for 1 hour; wash the plate 3 times with washing solution, pat dry for use; dilute rabbit monoclonal antibodies D1, D2, and D3 into 7 concentration gradients starting from 4000ng / ml by 5 times, Add 100 μL / well to the microplate; wash the plate 3 times with washing solution and pat dry; dilute the enzyme-linked antibody to the working concentration, mix well, incubate 100 μL well at room temperature for 1 hour; wash the plate 3 times with washing solution and pat dry; use an 8-channel gun to add 200 μL / well of color development solution and place it at room temperature away from light for 15 minutes; use a gun to add 100 μL / well of stop solution to terminate the reaction; use an enzyme reader to measure the 450nm signal value, perform statistical analysis on the data, and calculate the EC50 value.

[0059] In the above embodiments of the present invention, the phage display technology used to screen antibody sequences is simple and easy to operate, does not require the culture and screening of hybridoma cells and the use of expensive equipment and instruments, and allows the production and screening of antibodies that bind to target antigens with high specificity in a relatively short period of time. The prepared rabbit monoclonal antibodies have stronger specificity and higher affinity than mouse monoclonal antibodies, and have higher sensitivity and accuracy in the application of BHV-1 immunological diagnostic detection.

[0060] In addition, the method for preparing the gD recombinant rabbit monoclonal antibody provided in the embodiment of the present invention is based on phage display technology to screen the antibody sequence, and then prepare a recombinant expression vector, and then transfect the cell to obtain the antibody by recombinant expression. Compared with traditional hybridoma cells, the advantages are: higher preparation efficiency, more convenient operation, no need to sacrifice a large number of animals, higher storage stability of the recombinant antibody expression vector, and it is easy to scale up production, and a large number of antibodies can be prepared.

[0061] In addition, since the domestic immunoassay kits for BHV-1gD are mainly indirect ELISA and indirect immunofluorescence, the sensitivity and specificity are insufficient. Most of the currently recognized kits are produced by the American IDEX Biotech Company. Due to the high price and long delivery cycle of foreign kits, it is urgent to develop a new BHV-1 sensitive detection kit to replace foreign products. Moreover, most of the antibodies in the current BHV-1 pathogen immune diagnosis kits are mouse monoclonal antibodies, which have relatively weak affinity and low specificity, and are still insufficient in the field of immunological detection.

[0062] In order to solve the above technical problems, in another embodiment of the present invention, a double antibody sandwich ELISA reagent is provided, which includes at least one of the above recombinant rabbit monoclonal antibodies.

[0063] In a preferred embodiment of the present invention, the double antibody sandwich ELISA reagent comprises the rabbit monoclonal antibody D2 and the rabbit monoclonal antibody D3. It should be noted that the double antibody sandwich ELISA reagent may also include other reagents required for ELISA detection.

[0064] In a preferred embodiment of the present invention, the above-mentioned rabbit monoclonal antibody D2 is used as the detection antibody, and the above-mentioned rabbit monoclonal antibody D3 is used as the capture antibody.

[0065] In practical applications, the composition of the double antibody sandwich ELISA reagent can be screened by the following test methods, as follows:

[0066] 1. Screening of the best antibody pairing combination: (1) The three rabbit monoclonal antibodies D1, D2, and D3 were used as capture antibodies, diluted to a concentration of 2 μg / mL using coating solution, 100 μL was added to each well, and coated at 4°C overnight;

[0067] (2) The ELISA plate was washed three times with 0.1% PBST and patted dry, and 200 μL of 5% skim milk powder was added and blocked at 37°C for 2 h;

[0068] (3) After blocking, wash three times and pat dry. Add 5 μg / mL and 10 μg / mL recombinant gD protein and BHV-1 virus solution diluted with PBS to the ELISA plate, add 100 μL to each well, and incubate at 37°C for 1 h.

[0069] (4) Wash three times and pat dry, add 100 μL of 1 μg / mL HRP-labeled detection antibody to each well, and incubate at 37°C for 1 h;

[0070] (5) Wash three times and pat dry, add 100 μL of TMB colorimetric solution to each well, and incubate at 37°C in the dark for 15 min;

[0071] (6) Add 50 μL of sulfuric acid to each well to terminate the reaction, read the OD450 absorbance using a fluorescent microplate reader, and use PBS as a negative control to determine the best paired antibody combination.

[0072] 2. Screening of optimal working concentrations of capture antibody and detection antibody: The capture antibody with different concentrations (1, 2, 4, 8 μg / mL) and the detection antibody with different dilutions (1:100, 1:200, 1:400, 1:800) were reacted, and the optimal working concentrations of capture antibody and detection antibody were determined by the chessboard method.

[0073] 3. Determination of the best blocking agent type: According to the best conditions screened above, with other conditions unchanged, the blocking agents were set to 3 types: 5% skimmed milk powder, 3% BSA, and 5% BSA. The best blocking agent was determined by the P / N ratio.

[0074] 4. Determination of the optimal incubation time of the antigen: According to the optimal conditions screened above, with other conditions unchanged, the antigen incubation time was set to 30, 60, 120, and 150 minutes. The optimal incubation time of the antigen was determined by the P / N ratio.

[0075] 5. Determination of the optimal color development time: According to the optimal conditions screened above, keep other conditions unchanged, set the incubation time of the detection antibody to 5, 10, 15, and 20 minutes, and determine the optimal color development time by the P / N ratio.

[0076] 6. Determination of negative and positive thresholds: 30 BHV-1 negative and 30 positive clinical samples were tested using the optimized double antibody sandwich ELISA method. The critical value was determined by measuring the OD450 value on an ELISA instrument and the model was evaluated.

[0077] 7. Specificity test: The specificity of ELISA comes from the specific binding of antigen and antibody. In order to verify whether the detection method has cross-reaction to other cattle-related viruses except BHV-1, BCV, BRSV, BRV, BVDV, and BHV-1 positive samples are used to verify whether the detection method is specific.

[0078] 8. Intra-batch and inter-batch repeat tests: The accuracy of the established double antibody sandwich ELISA method was evaluated by intra-batch and inter-batch variability. The established ELISA method was used to perform 4 intra-batch and 4 inter-batch positive sample tests, and the coefficient of variation was calculated to evaluate its accuracy.

[0079] In the above embodiment of the present invention, by using rabbit monoclonal antibody D2 as the detection antibody and rabbit monoclonal antibody D3 as the capture antibody, a double antibody sandwich ELISA method for rapid detection of BHV-1 can be established. In the reaction, the antigen binds to different antigenic determinants of the two antibodies, which greatly improves the specificity of the detection and makes the detection result more reliable. At the same time, the setting of negative control and positive control ensures the accuracy of each test repetition. The double antibody sandwich ELISA method can ensure that the operation steps are the same each time when detecting, and the sample detection is in the same reaction system, which can minimize the error in the operation process. In addition, the operation steps of the double antibody sandwich ELISA method are simple, time-consuming, and the professional requirements for the test personnel are relatively low. The reagents used for the double antibody sandwich ELISA method are very common and the reagents are cheap, which makes the double antibody sandwich ELISA method universal and can be used for the detection of large quantities of samples. Compared with traditional methods, it is more suitable for promotion in clinical detection.

[0080] It should be noted that the experimental methods in the examples of the present invention that do not specify specific experimental conditions are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the reagent manufacturer. The reagents, raw materials and equipment used in the present invention can all be obtained commercially.

[0081] The following embodiments are specific implementations and test cases of the present invention in practical applications, but are not limited thereto.

[0082] Example 1: This example provides a method for constructing a specific scFv phage display library for BHV-1gD protein, which specifically includes the following steps:

[0083] 1. Antigen preparation: The amino acid sequence of the gD protein was analyzed, the transmembrane region and signal peptide of the gD protein were removed, a 6×His tag was added to the C-terminus, and a pET32a-gD recombinant expression vector was constructed for prokaryotic expression. Specifically, the gene sequence of the recombinant gD protein was optimized by an E. coli host and then constructed into a pET-32a(+) vector, and induced to express in BL21(DE3). After that, the His tag carried by the recombinant target protein was used for affinity chromatography purification to obtain a soluble recombinant gD protein with biological activity. The recombinant gD protein was used as an antigen for immunizing rabbits. The above-mentioned vector construction, protein expression and purification methods are conventional operations in the art and will not be repeated here.

[0084] 2. Animal immunization: Dilute the endotoxin-free recombinant gD protein with PBS and mix it with Freund's complete adjuvant in a 1:1 ratio. The total amount of the initial immunization protein is 1 mg, and the total amount of the immunization adjuvant is 1 mL. Multiple injections are made subcutaneously on the abdomen and back of New Zealand white rabbits, and a negative control group is set up at the same time. During immunization, use an adjuvant emulsifier to stir and emulsify in a 2mL endotoxin-free ep tube for 10 minutes, and swing forward from top to bottom as a whole until a uniform and stable white milky suspension is formed. Uneven emulsification will reduce the immunization effect. Ten days after the first immunization, the second and third immunizations were carried out. The dose of the immunization protein was halved compared to the first. The difference was that it was mixed with Freund's incomplete adjuvant in a 1:1 ratio during emulsification. The fourth immunization operation was the same as the first immunization adjuvant dose and immunization adjuvant as a means of enhancing immunity. Immunization was performed once every two weeks, for a total of four times.

[0085] 3. Evaluation of immune effect: In the eighth week after immunization, the titer of antiserum of three immunized rabbits was determined by indirect ELISA to evaluate the immune effect. The specific steps are as follows:

[0086] (1) The recombinant gD protein was diluted with coating solution to a concentration of 1 μg / mL and coated on a polystyrene microplate at 4°C overnight. A blank control and a negative control group were set up with 3 replicates in each group.

[0087] (2) Pour off the coating solution, wash with PBST three times for 2 minutes each time, add 300 μL of 5% skim milk powder diluted with PBS and incubate in a 37°C constant temperature incubator for 2 h;

[0088] (3) After blocking, discard the blocking solution from the ELISA plate, wash the plate in the same manner as in step (2), and pat dry;

[0089] (4) The serum to be tested is diluted with antibody diluent in a gradient manner, with a starting concentration of 1:100. Add 20 μL of serum to 2 mL of antibody diluent to prepare a starting concentration of 1:100. Pipette 500 μL of diluted serum from the starting concentration of 1:100 and mix it evenly with 500 μL of PBS solution, and so on. Dilute in a gradient manner in this way so that the dilution multiple of each well is twice that of the previous well. Add pre-immune rabbit serum to the negative control well. Incubate in a 37°C constant temperature incubator for 1 hour;

[0090] (5) Wash the plate as in step (2) and pat dry. Dilute the HRP-labeled goat anti-rabbit secondary antibody 5000 times using antibody diluent, add 100 μL per well to the ELISA plate, and incubate in a 37°C constant temperature incubator for 0.5 h;

[0091] (6) Wash the plate as in step (2), pat dry, add 100 μL of single-component TMB to each well, and incubate in a 37°C constant temperature incubator in the dark for 15 min;

[0092] (7) Add 50 μL of 2 M sulfuric acid to each well as a color stop solution, and read the OD450 value with a microplate reader. The maximum dilution of a positive reaction is the titer of the sample to be tested;

[0093] (8) Calculate the absorbance ratio of the antibody to be tested and the negative control (Positive / negative, P / N). When P (positive) / N (negative) is greater than 2.1, it is positive; when P / N is less than 2.1, it is negative. The positive result with the largest dilution multiple is taken as the measured antibody titer.

[0094] The results of the above antiserum titer test are shown in Figure 1 The serum antibody titers of three positive rabbits immunized with gD recombinant protein were determined ( Figure 1 A, B, and C) all reached 4.1×10 5 , indicating that the immune effect is good.

[0095] 4. Using phage display technology, we constructed 2.1×10 13 pfu / mL of the scFv (Single-chain fragment variable, scFv) phage display library of the gD protein of the specific immune library: the eighth week of immunization rabbits were deeply anesthetized and killed, the spleen tissue RNA was extracted using the RNA Easy Fast Animal Tissue / Cell Total RNA Extraction Kit, and a reverse transcription experiment was performed to obtain cDNA, and PCR was performed using specific primers to amplify VH and VL. The 350bp fragment was recovered by gel recovery to obtain the first fragment, and VH and VL were connected to ScFv using overlap extension PCR, and then after SfiI digestion, they were connected to the pComb3XSS vector to obtain the recombinant phage plasmid, which was electroporated into ER2738 super competent cells to form a phage scFv library bacteria, and then the wild-type helper phage M13KO7 was added to infect the host bacteria, the host bacteria were amplified overnight, and the bacteria were precipitated with PEG-NaCl to obtain the supernatant, and the primary phage scFv display library was obtained.

[0096] Example 2: This example provides a method for enriching and screening positive clones, as follows:

[0097] Three rounds of solid phase panning were used to screen the recombinant gD protein phage scFv display library with good enrichment and specific monoclonal aggregation. In the clean bench, a sterile ELISA special enzyme plate was coated with the recombinant gD protein prepared by prokaryotic expression as the antigen, and the antigen was diluted with sterile PBS to a concentration of 25μg / mL. After adding 100μL of diluted antigen solution to each well of the enzyme plate, it was incubated at 4°C overnight. The same antigen was used in the second round of panning, but the coating concentration was reduced to 15μg / mL. In the third round of panning, the coating concentration of the antigen was reduced to 5μg / mL. The next day, the above-mentioned enzyme plate was washed with a plate washer, and the plate was carefully tapped on a clean paper, the PBST washing solution was removed, and a sterile PBS buffer containing 3% BSA was used as the blocking solution. After adding 200μL to each well, it was incubated in a 37°C constant temperature incubator for 2h. After the blocking was completed, the enzyme plate was washed and the PBST washing solution was carefully tapped for use. Take 3mL of the primary antibody library phage solution prepared above and dilute it 5 times with 1% BSA PBS (use the primary antibody library phage solution in the second round of panning, and so on), and let it stand at room temperature for 10min. Add 150μL to each well of the sealed ELISA plate, incubate at 37℃ for 2h, wash the ELISA plate 3 times after incubation, 3min each time, and tap off the PBST washing solution. Add 100μL of 0.2M GLy-HCl eluent with pH=2.2 to each well of the above ELISA plate, rotate at 37℃, 80rpm for 8min, and then repeatedly blow and mix, carefully draw the eluent into a clean 15mL centrifuge tube, and immediately add an equal volume of Tris-HCl with pH=7.4 to neutralize the phage eluent. Set aside 1mL of the eluted phage for storage and use in library capacity determination and bacterial liquid PCR to identify the insertion rate of the target gene.

[0098] The above panning results are shown in Table 1. After three rounds of panning, the positive rate measured in the first round of panning reached 88.23%, and the positive rates measured in the second and third rounds of panning both reached 100%, and the enrichment ratio was greater than 10. After three rounds of panning, a phage antibody library with good enrichment and specific monoclonal aggregation has been screened.

[0099] Table 1

[0100]

[0101] 96 colonies were randomly selected and positive clones with a P / N value greater than 3.5 were screened using the phage-ELISA method. A total of 38 positive clones were sent to the company for sequencing (see the results). Figure 2 ). After analyzing the sequencing results, three scFv gene sequences with high abundance and consistent with the variable region of rabbit antibodies were obtained, which were named rabbit monoclonal antibodies D1, D2, and D3 (sequences are as follows Figure 3 (as shown in A, B, and C).

[0102] Example 3: This example provides a method for expressing and purifying the above-mentioned recombinant rabbit monoclonal antibody in a mammalian system, as follows:

[0103] The heavy chain variable region and light chain variable region in the sequences of the above rabbit monoclonal antibody D1, rabbit monoclonal antibody D2, and rabbit monoclonal antibody D3 were connected in series with the heavy chain constant region and light chain constant region of rabbit IgG, respectively. The kozak sequence (GCCGCCACC) was added before the start codon, and the human serum albumin sequence MKWVTFISLLFLFASAYS (see GeneBank accession number: NP_000468) was added after the start codon. Xbal was added to the 5' end of the sequence and EcoRV restriction site was added to the 3' end. The sequence was inserted into the pcDNA3.4 empty vector to construct a eukaryotic recombinant expression vector (such as Figure 4 and Figure 5 As shown, Figure 4 A, B, and C correspond to rabbit monoclonal antibodies D1, D2, and D3, respectively; Figure 5 A is the restriction enzyme digestion verification result of rabbit monoclonal antibody D1, M: DNA Marker, 1: single digestion with D1H EcoRV, 2: double digestion with D1H EcoRV and Xbal, 3: single digestion with D1L EcoRV, 4: double digestion with D1L EcoRV and Xbal; Figure 5 B is the result of rabbit monoclonal antibody D2 digestion verification, M: DNA Marker, 1: D2HEcoRV single digestion, 2: D2H EcoRV and Xbal double digestion, 3: D2L EcoRV single digestion, 4: D2LEcoRV and Xbal double digestion; Figure 5 C is the result of rabbit monoclonal antibody D3 restriction enzyme digestion verification, M: DNA Marker, 1: D3H EcoRV single restriction enzyme digestion, 2: D3H EcoRV and Xbal double restriction enzyme digestion, 3: D3L EcoRV single restriction enzyme digestion, 4: D3L EcoRV and Xbal double restriction enzyme digestion).

[0104] The above eukaryotic recombinant expression vector was sent to Nanjing GenScript Biotechnology Co., Ltd. to optimize the codon preference, GC content, mRNA secondary structure, and repeat sequence region using CHO cells as the host, and gene synthesis was performed at Nanjing GenScript. The light chain and heavy chain were combined and co-transfected into mammalian cells for expression purification, transfection and purification, and the method was as follows:

[0105] Centrifuge cells with good growth status and cell viability greater than 90% to obtain a cell volume of 750 μL and a cell number of 1×10 7, after mixing, centrifuge at 650rpm for 5min, discard the supernatant, add 800μL of high-glucose DMEM medium to resuspend, add 50ug of heavy chain and light chain plasmids respectively, add to the pre-cooled electrode cup and mix slowly, use 4mm Bole electric shock cup for electrotransformation, the electrotransformation conditions are 300v, 900uF, after electrotransformation, absorb the liquid in the electric shock cup and put it into 15mL high-glucose DMEM medium for culture, screen with 800μg / mL G418, collect the supernatant, centrifuge at 8000rpm for 5min. Use 20mL PBS, 1mL / min flow rate to balance the chromatography column, load at 1mL / min flow rate, elute nonspecific proteins with 20mL PBS, 1mL / min flow rate, elute with glycine buffer (pH=3.0), flow rate 1mL / min, collect in separate tubes, about 3mL per tube. A total of 2 tubes were collected, and 100 μL of neutralizing solution was added before elution to ensure that the eluted protein was stored at a neutral pH. The purified recombinant rabbit monoclonal antibody was collected and then stored in aliquots at -20°C.

[0106] The SDS PAGE results of the purified recombinant rabbit monoclonal antibody in non-reducing state and reducing condition are as follows: Figure 6 The Western Blot results under reducing conditions are shown in Figure 7 As shown; Figure 6 and Figure 7 1, 2, and 3 correspond to the experimental results of rabbit monoclonal antibodies D1, D2, and D3, respectively.

[0107] Example 4: This example provides a method for detecting the affinity between rabbit monoclonal antibodies D1, D2, D3 and recombinant gD protein, as follows:

[0108] Dilute the recombinant gD protein to 0.5 μg / ml with coating solution, add 100 μL / well, and incubate at 2-8°C overnight; wash the plate 3 times with washing solution, pat dry, add 300 μL / well blocking solution, and block at room temperature for 1 hour; wash the plate 3 times with washing solution, pat dry for use; dilute rabbit monoclonal antibodies D1, D2, and D3 into 7 concentration gradients starting from 4000 ng / ml by 5 times, and add 100 μL / well to the microplate; wash the plate 3 times with washing solution, pat dry; dilute the enzyme-linked antibody to the working concentration, mix well, incubate at room temperature for 1 hour in 100 μL well; wash the plate 3 times with washing solution, pat dry; add the color developing solution at 200 μL / well using an 8-channel shotgun, and place it at room temperature away from light for 15 minutes; use a shotgun to add the stop solution at 100 μL / well to terminate the reaction; use an enzyme reader to measure the 450nm signal value, perform statistical analysis on the data, and calculate the EC50 value.

[0109] The above test results are as follows Figure 8 As shown, at this time, the EC50 values ​​of D1, D2 and D3 were 31.12, 24.99 and 33.47, respectively, all of which had good binding activity.

[0110] Example 5: This example provides a method for optimizing the composition of double antibody sandwich ELISA reagents and detection conditions, as follows:

[0111] 1. Screening of the best antibody pairing combination: (1) The three rabbit monoclonal antibodies D1, D2, and D3 were used as capture antibodies, diluted to a concentration of 2 μg / mL using coating solution, 100 μL was added to each well, and coated at 4°C overnight;

[0112] (2) The ELISA plate was washed three times with 0.1% PBST and patted dry, and 200 μL of 5% skim milk powder was added and blocked at 37°C for 2 h;

[0113] (3) After blocking, wash three times and pat dry. Add 5 μg / mL and 10 μg / mL recombinant gD protein and BHV-1 virus solution diluted with PBS to the ELISA plate, add 100 μL to each well, and incubate at 37°C for 1 h.

[0114] (4) Wash three times and pat dry, add 100 μL of 1 μg / mL HRP-labeled detection antibody to each well, and incubate at 37°C for 1 h;

[0115] (5) Wash three times and pat dry, add 100 μL of TMB colorimetric solution to each well, and incubate at 37°C in the dark for 15 min;

[0116] (6) Add 50 μL of sulfuric acid to each well to terminate the reaction, read the OD450 absorbance using a fluorescent microplate reader, and use PBS as a negative control to determine the best paired antibody combination.

[0117] The three purified monoclonal antibodies were paired one by one with the HRP-labeled monoclonal antibodies to ensure that the antibody coating concentration and the antigen concentration remained unchanged. The results are shown in Table 2. Table 2 shows that the antibodies were successfully paired and a pair of optimal combinations were screened out. When the capture antibody was D3 and the detection antibody was D2, the combination was the best.

[0118] Table 2

[0119]

[0120] 2. Screening of optimal working concentrations of capture antibody and detection antibody: The capture antibody with different concentrations (1, 2, 4, 8 μg / mL) and the detection antibody with different dilutions (1:100, 1:200, 1:400, 1:800) were reacted, and the optimal working concentrations of capture antibody and detection antibody were determined by the chessboard method.

[0121] The above screening results are shown in Table 3. The optimal coating concentration of the capture antibody can be determined to be 4 μg / mL by the chessboard method, and the optimal dilution of the detection antibody is 1:400.

[0122] Table 3

[0123]

[0124] 3. Determination of the best blocking agent: According to the best conditions screened above, with other conditions unchanged, the blocking agents were set to 5% skim milk powder, 3% BSA, and 5% BSA, a total of 3 types. The best blocking agent was determined by the P / N ratio. The results are as follows: Fig. 9 As shown, optimal conditions were obtained when blocking with 3% BSA.

[0125] 4. Determination of the optimal incubation time of antigen: According to the optimal conditions screened above, with other conditions unchanged, the antigen incubation time was set to 30, 60, 120, and 150 minutes. The optimal incubation time of antigen was determined by the P / N ratio. The results are as follows: Fig.10 As shown, the optimal condition was when the antigen incubation time was 90 min.

[0126] 5. Determination of the optimal color development time: According to the optimal conditions screened above, keep other conditions unchanged, set the incubation time of the detection antibody to 5, 10, 15, and 20 minutes, and determine the optimal color development time by the P / N ratio. The results are as follows: Fig.11 The results show that the best condition is when the color development time is 15 minutes.

[0127] 6. Determination of negative and positive thresholds: 30 BHV-1 negative and 30 positive clinical samples were tested using the optimized double antibody sandwich ELISA method. The critical value was determined by measuring the OD450 value on an ELISA instrument and the model was evaluated.

[0128] 60 clinical samples were tested and the results of statistical analysis were obtained (see Fig.12 , Fig.13 ) showed that the yin-yang critical value was 0.8525 and the AUC value was 0.968, indicating that the established model had good classification performance and high accuracy in disease detection.

[0129] 7. Specificity test: The specificity of ELISA comes from the specific binding of antigen and antibody. In order to verify whether the detection method responds to other cattle-related viruses besides BHV-1, BCV, BRSV, BRV, BVDV, and BHV-1 are used to verify whether the detection method is specific.

[0130] The specificity test was performed using the ELISA experimental conditions established above. The results are as follows Fig.14As shown, it was found that this method had no cross-reaction with BCV, BRSV, BRV, BVDV, BAstV, and BNoV, and the values ​​were significantly different, indicating that the established method has high specificity.

[0131] 8. Intra-batch and inter-batch repeat tests: The accuracy of the established double antibody sandwich ELISA method was evaluated by intra-batch and inter-batch variability. The established ELISA method was used to perform 4 intra-batch and 4 inter-batch positive sample tests, and the coefficient of variation was calculated to evaluate its accuracy.

[0132] In order to detect whether the established double antibody sandwich ELISA method has good stability, 4 positive clinical samples were selected for determination in the intra-batch and inter-batch experiments respectively. The results (see Table 4 and Table 5) showed that the coefficient of variation within and between batches did not exceed 5%, which indicated that the established double antibody sandwich ELISA method had good stability.

[0133] Table 4

[0134]

[0135] Table 5

[0136]

[0137] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A recombinant rabbit monoclonal antibody against BHV-1, characterized in that: The recombinant rabbit monoclonal antibody is rabbit monoclonal antibody D1; the amino acid sequence of the rabbit monoclonal antibody D1 is shown in the sequence table SEQ ID NO.

1.

2. Use of the recombinant rabbit monoclonal antibody as claimed in claim 1 in the preparation of a BHV-1 detection kit.

3. Double antibody sandwich ELISA reagent, characterized in that: Comprising the recombinant rabbit monoclonal antibody as claimed in claim 1.

4. Use of the double antibody sandwich ELISA reagent as claimed in claim 3 in the preparation of a kit for detecting BHV-1 by ELISA method.

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