Recombinant Rabbit Monoclonal Antibody Against BHV-1, Double Antibody Sandwich ELISA Reagent and Application

Abstract

The present invention is applicable to the field of immunoassay, and provides a recombinant rabbit monoclonal antibody against BHV-1, a double-antibody sandwich ELISA reagent and applications thereof. Among them, 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 as shown in SEQ ID NO.1 in the sequence listing; the amino acid sequence of rabbit monoclonal antibody D2 is as shown in SEQ ID NO.2 in the sequence listing; the amino acid sequence of rabbit monoclonal antibody D3 is as shown in SEQ ID NO.3 in the sequence listing. In addition, the double-antibody sandwich ELISA reagent includes the above-mentioned rabbit monoclonal antibody D2 and rabbit monoclonal antibody D3, and can be used for rapid and accurate detection of BHV-1, with characteristics such as strong specificity and high sensitivity, and is more suitable for popularization in clinical detection.

Description

Technical Field

[0001] The present invention belongs to the field of immunoassay, and particularly relates to a recombinant rabbit monoclonal antibody against BHV-1, 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 various clinical syndromes such as respiratory and reproductive tract diseases, which have a great 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. This virus is widespread in China with a high positive detection rate. With the continuous increase in the cattle inventory in China, it has caused serious economic impacts on the cattle industry.

[0003] BHV-1 is a member of the Alphaherpesvirinae 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 also essential for virus replication and infection. Multiple literatures have shown that the gD protein can often induce higher neutralizing antibody titers compared with other proteins. Due to its immunodominant and sequence-conserved characteristics, it is considered an ideal antigen for diagnostic detection and vaccine immune evaluation.

[0004] In view of the sequence and functional characteristics of the gD protein, the development of specific monoclonal antibodies against gD based on phage display technology is extremely beneficial for the detection and diagnosis of BHV-1 and the evaluation after vaccination. The phage technology is easy to operate and highly flexible, and is considered a rapid and effective tool for discovering and developing monoclonal antibodies. At present, most of the antibodies in BHV-1 detection kits are murine monoclonal antibodies. Although murine monoclonal antibody technology is widely used, there are problems such as relatively weak affinity and poor specificity, and there are still deficiencies in the field of immunological detection. Compared with murine monoclonal antibodies, rabbit monoclonal antibodies have more advantages. Rabbit monoclonal antibodies can recognize more epitope antigen determinants of immunogens, and have 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 highly sensitive detection of BHV-1.

[0005] In addition, the current methods for diagnosing BHV-1 mainly utilize virus isolation and identification, PCR, and virus neutralization tests. However, all of them must be operated in specialized laboratories, and the experimental cycle is long and the cost is relatively high. Therefore, there is an urgent need to establish a method that is simple to operate, low in cost, and conducive to large-scale detection of pathogens. The ELISA method is simple and rapid to operate, can be used for large-scale detection, and has high sensitivity and good specificity in the test results. It is the most widely used method at present. Among them, the double antibody sandwich ELISA method has the advantages of high sensitivity and strong specificity through the double screening of capture antibodies and detection antibodies, and is commonly used for accurate virus serological detection. 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 response to the above problems, the embodiments of the present invention are 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 SEQ ID NO.1 in the sequence listing; the amino acid sequence of the rabbit monoclonal antibody D2 is shown in SEQ ID NO.2 in the sequence listing; the amino acid sequence of the rabbit monoclonal antibody D3 is shown in SEQ ID NO.3 in the sequence listing.

[0008] Preferably, the preparation method of the recombinant rabbit monoclonal antibody includes the following steps:

[0009] Construct a specific scFv phage display library against the BHV-1 gD protein using the recombinant gD protein expressed in prokaryotes;

[0010] Screen the amino acid sequence of the recombinant rabbit monoclonal antibody according to the specific scFv phage display library;

[0011] According to the amino acid sequence of the recombinant rabbit monoclonal antibody, construct a recombinant expression vector, and after transfection into cells, perform recombinant expression to obtain the recombinant rabbit monoclonal antibody.

[0012] Preferably, the method for obtaining the recombinant gD protein is as follows: Remove the transmembrane region and signal peptide of the gD protein, add a 6×His tag at the C-terminus, construct a pET32a-gD recombinant expression vector for prokaryotic expression, and obtain the recombinant gD protein.

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

[0014] Another purpose of the embodiments of the present invention is to provide an application of the recombinant rabbit monoclonal antibody in the preparation of a BHV-1 detection kit.

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

[0016] Preferably, it comprises the rabbit monoclonal antibody D2 and the rabbit monoclonal antibody D3.

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

[0018] Another object of the embodiments of the present invention is to provide an application of the above double-antibody sandwich ELISA reagent in the preparation of 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 by the embodiments of the present invention is a rabbit-derived antibody, which has higher affinity and stronger specificity compared with murine antibodies. In addition, by using the rabbit monoclonal antibody D2 as the detection antibody and the rabbit monoclonal antibody D3 as the capture antibody in the embodiments of the present invention, 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, greatly improving the specificity of the detection and making the detection result more reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a graph of the detection result of the antiserum titer;

[0022] Figure 2 It is a graph of the sequencing result of the positive clone;

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

[0024] Figure 4 It is a schematic diagram of the construction of the eukaryotic recombinant expression vector;

[0025] Figure 5 It is the restriction enzyme digestion identification result of the vector;

[0026] Figure 6 It is a graph of the SDS PAGE results of the recombinant rabbit monoclonal antibody under non-reducing and reducing conditions;

[0027] Figure 7 It is a graph of the Western Blot results of the recombinant rabbit monoclonal antibody under reducing conditions;

[0028] Figure 8 It is a graph of the detection results of the affinity of the rabbit monoclonal antibodies D1, D2, and D3 with the recombinant gD protein;

[0029] Figure 9 P / N ratio graphs for different types of sealants

[0030] Figure 10 P / N ratio graphs for different incubation times

[0031] Figure 11 P / N ratio graphs for different chromogenic times

[0032] Figure 12 OD of negative and positive samples 450 value detection result graph

[0033] Figure 13 Data statistical analysis graph of clinical sample detection results

[0034] Figure 14 Specificity test result graph Detailed implementation manners

[0035] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below with reference to 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 used to limit the present invention.

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

[0037] Specifically, in an 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 Sequence Listing SEQ ID NO.1, specifically: QS LEESGGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGIIGGSGSTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARVYDDYGDDYFSIWGPGTLVTVSSGGGGSGGGGSGGGGSAQVMTQTPSSKSVPVGDTVTINCLASEFLFNAVSWYQQKPGQPPKLLIYGASNLESGVPPRFSGSGSGTDYTLTIGGVQAEDVATYYCLGGCSGSADLTFGGGTELEIL;

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

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

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

[0042] S1. Construct a specific scFv phage display library against BHV-1 gD protein using the recombinantly expressed gD protein in prokaryotes;

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

[0044] S3. Construct a recombinant expression vector according to the amino acid sequence of the recombinant rabbit monoclonal antibody, transfect cells and perform recombinant expression to obtain the recombinant rabbit monoclonal antibody.

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

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

[0047] Among them, the amino acid sequence of the gD protein refers to Gene Bank accession number 001847.1 (shown as SEQ ID NO.4 in the sequence listing), specifically: MLPTPAPRVTVYVDPPAYPMPRYNYTERWHTT GPIPSPFADGREQPVEVRYATSAAACDMLALIADPQVGRTLWEAVRRHARAYNATVIWYKIESGCARPLYYMEYTECEPRKHFGYCRYRTPPFWDSFLAGFAYPTDDELGLIMAAPARLVEGQYRRALYIDGTVAYTDFMVSLPAGDCWFSKLGAARGYTFGACFPARDYEQKKVLRLTYLTQYYPQEAHKAIVDYWFMRHGGVVPPYFEESKGYEPPPAADGGSPAPPGDDEAREDEGETEDGAAGREGNGGPPGPEGDGESQTPEANGGAEGEPKPGPSPDADRPEGWPSLEAITHPPPAPATPAAPDAVPVSYTRRRGAGPLPRKPKKLPAFGNVNYSALPGHHHHHH*.

[0048] S12. Animal immunization: Mix the recombinant gD protein with complete Freund's adjuvant at a mass ratio of 1:1 and emulsify it, and immunize multiple New Zealand white rabbits by subcutaneous injection method. After an interval of two weeks, perform the second, third, and fourth immunizations with the above recombinant gD protein and incomplete Freund's adjuvant.

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

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

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

[0052] S21. Construction of a scFv (Single-chain fragment variable) phage display library of gD protein: The immunized rabbits in the eighth week were deeply anesthetized and sacrificed. The spleen tissue RNA was extracted using the RNA Easy Fast Total RNA Extraction Kit for Animal Tissues / Cells, and a reverse transcription experiment was carried out to obtain cDNA. Specific primers were used for PCR to amplify VH and VL. The 350bp fragment was recovered by gel extraction to obtain the first fragment. VH and VL were ligated into ScFv using overlap extension PCR. Then, after digestion with SfiI enzyme, it was ligated into the pComb3XSS vector to obtain the recombinant phagemid. It was electrotransformed into ER2738 super-competent cells to form the phage scFv library bacteria. Then, the wild-type helper phage M13KO7 was added to infect the host bacteria, and the host bacteria were amplified overnight. The supernatant was obtained by precipitating the bacteria with PEG-NaCl, and thus the primary phage scFv display library was obtained.

[0053] S22. Enrichment and screening of positive clones: Use three rounds of solid-phase panning to screen a recombinant gD protein phage scFv display library with good enrichment and specific monoclonal aggregation. In a laminar flow hood, take a sterile ELISA-specific microtiter plate and coat it with the BHV-1 gD protein prepared by prokaryotic expression as an antigen. Dilute the antigen with sterile PBS to a concentration of 25 μg / mL. Add 100 μL of the diluted antigen solution to each well of the microtiter plate and incubate overnight at 4°C. Use the same antigen in the second round of panning and screening, but reduce the coating concentration to 15 μg / mL. In the third round of panning and screening, reduce the coating concentration of the antigen to 5 μg / mL. The next day, wash the above microtiter plate with a plate washer, carefully pat the plate on clean paper to remove the PBST washing solution, and use a sterile PBS buffer containing 3% BSA as a blocking solution. Add 200 μL to each well and incubate statically in a 37°C incubator for 2 h. After the blocking is completed, wash the microtiter plate, carefully pat to remove the PBST washing solution, and set aside for use. Take 3 mL of the primary antibody library phage solution prepared above and dilute it 5 times with PBS containing 1% BSA (use the primary antibody library phage solution in the second round of panning and screening, and so on). Let it stand at room temperature for 10 min. Add 150 μL to each well of the blocked microtiter plate and incubate at 37°C for 2 h. After the incubation is completed, wash the microtiter plate 3 times, 3 min each time, and pat to remove the PBST washing solution. Add 100 μL of a 0.2 M Gly-HCl elution solution with a pH of 2.2 to each well of the above microtiter plate, rotate at 37°C and 80 rpm for 8 min, then pipette up and down repeatedly to mix evenly. Carefully aspirate the elution solution into a clean 15 mL centrifuge tube, and immediately add an equal volume of Tris-HCl with a pH of 7.4 to neutralize the phage elution solution. Set aside 1 mL of the eluted phage for storage, which is used for library capacity determination and colony PCR to identify the insertion rate of the target gene.

[0054] In the embodiment of the present invention, by using rabbits as immunized animals, since rabbits have large spleens and high gene diversity, antibodies with a wider epitope coverage can be obtained.

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

[0056] S31. Eukaryotic expression and purification of monoclonal antibodies: The heavy chain variable region and the light chain variable region in the sequences of the above-mentioned rabbit monoclonal antibodies D1, D2, and D3 were respectively concatenated with the rabbit heavy chain constant region and the light chain constant region. A kozak sequence (GCCGCCACC, as shown in SEQ ID NO.5 of the sequence listing) was added before the start codon, and the human serum albumin sequence MKWVTFISLLFLFASAYS (as shown in SEQ ID NO.6 of the sequence listing, see GeneBank accession number: NP_000468 for details) was added after the start codon. An Xbal site was added to the 5' end of the sequence, and an EcoRV restriction site was added to the 3' end. The resulting sequence was inserted into the pcDNA3.4 empty vector to construct a eukaryotic recombinant expression vector, which was sent to Nanjing Genscript Biotech Co., Ltd. for codon preference, GC content, mRNA secondary structure, and repeat sequence region optimization using CHO cells as the host, and gene synthesis was carried out at Nanjing Genscript. After combining the light chain and the heavy chain, they were co-transfected into mammalian cells for expression and purification. The transfection and purification methods are as follows:

[0057] Centrifuge CHO-K1 cells with good growth status and a cell viability greater than 90%. Take 750 μL of cell volume and 1×10 7 , mix well and centrifuge at 650 rpm for 5 min. Discard the supernatant, resuspend the cells in 800 μL of high-glucose DMEM medium. Add 50 μg of each plasmid of the heavy chain and the light chain, slowly mix in a pre-cooled electrode cup, and perform electroporation using a 4 mm Bole electroporation cup. The electroporation conditions are 300 v and 900 uF. After electroporation, aspirate the liquid in the electroporation cup and culture it in 15 mL of high-glucose DMEM medium. Screen with 800 μg / mL of G418, collect the supernatant, and centrifuge at 8000 rpm for 5 min. Equilibrate the chromatography column with 20 mL of PBS at a flow rate of 1 mL / min, load the sample at a flow rate of 1 mL / min, elute non-specific proteins with 20 mL of PBS at a flow rate of 1 mL / min, and elute with glycine buffer (pH = 3.0) at a flow rate of 1 mL / min. Collect the eluate in fractions, about 3 mL per tube. A total of 2 tubes are collected. Add 100 μL of neutralizing solution before elution to ensure that the eluted protein is stored at neutral pH. Collect the purified antibody and then aliquot and store it 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 above-mentioned double-antibody sandwich ELISA reagent includes the above-mentioned rabbit monoclonal antibody D2 and rabbit monoclonal antibody D3. It should be noted that the above-mentioned 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 serves as the detection antibody, and the above-mentioned rabbit monoclonal antibody D3 serves as the capture antibody.

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

[0066] 1. Screening of the best antibody pairing combination: (1) Dilute the above-mentioned 3 rabbit monoclonal antibodies D1, D2, and D3 as capture antibodies to a concentration of 2 μg / mL with coating buffer, add 100 μL to each well, and coat overnight at 4°C;

[0067] (2) Wash the enzyme-linked immunosorbent assay (ELISA) plate 3 times with 0.1% PBST and pat dry, add 200 μL of 5% skim milk powder, and block at 37°C for 2 h;

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

[0069] (4) Wash 3 times and pat dry. Add 100 μL of HRP-labeled detection antibody with a concentration of 1 μg / mL to each well, and incubate at 37°C for 1 h;

[0070] (5) Wash 3 times and pat dry. Add 100 μL of TMB chromogenic solution to each well, and incubate in the dark at 37°C for 15 min;

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

[0072] 2. Screening of the optimal working concentrations of the capture antibody and the detection antibody: React with different concentrations of the capture antibody (1, 2, 4, 8 μg / mL) and different dilutions of the detection antibody (1:100, 1:200, 1:400, 1:800) combinations, and use the checkerboard method to determine the optimal working concentrations of the capture antibody and the detection antibody.

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

[0074] 4. Determination of the best antigen incubation time: According to the best conditions screened above, with other conditions remaining unchanged, the antigen incubation times were set to four time points: 30, 60, 120, and 150 min. The best antigen incubation time was determined by the P / N ratio.

[0075] 5. Determination of the best color development time: According to the best conditions screened above, with other conditions remaining unchanged, the incubation times of the detection antibody were set to four time points: 5, 10, 15, and 20 min. The best color development time was determined by the P / N ratio.

[0076] 6. Determination of the negative and positive thresholds: The optimized double antibody sandwich ELISA method was used to detect 30 BHV-1 negative and 30 positive clinical samples. The critical value was determined by measuring the OD450 value on an enzyme-linked immunosorbent assay (ELISA) reader and the model was evaluated.

[0077] 7. Specificity test: The specificity of ELISA comes from the specific binding of antigen and antibody. To verify whether the detection method has cross-reactivity with other bovine-related viruses except BHV-1, positive samples of BCV, BRSV, BRV, BVDV, and BHV-1 were used to verify the specificity of the method.

[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 detect positive samples four times within a batch and four times between batches, and the coefficient of variation was calculated to evaluate its accuracy.

[0079] In the above embodiments 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, greatly improving the specificity of the detection and making the detection results more reliable. At the same time, the setting of negative and positive controls ensures the accuracy of each test repetition. When using the double-antibody sandwich ELISA method, each operation step can be guaranteed to be the same, 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 is short, and the professional requirements for experimental personnel are relatively low. The reagents used in the double-antibody sandwich ELISA method are very common and the reagent prices are cheap, making the double-antibody sandwich ELISA method universal and applicable to the detection of a large number of samples. Compared with traditional methods, it is more suitable for popularization in clinical detection.

[0080] It should be noted that for the experimental methods without specific experimental conditions indicated in the embodiments of the present invention, they 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 and instruments 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 actual applications, but are not limited thereto.

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

[0083] 1. Antigen preparation: Analyze the amino acid sequence of the above gD protein, remove the transmembrane region and signal peptide of the gD protein, add a 6×His tag at the C-terminus, and construct a pET32a-gD recombinant expression vector for prokaryotic expression; specifically, the gene sequence of the recombinant gD protein is optimized by an Escherichia coli host and then constructed into the pET-32a(+) vector, induced to express in BL21(DE3), and then affinity purified through the His tag carried by the recombinant target protein to obtain a soluble recombinant gD protein with biological activity. The recombinant gD protein is used as the antigen for immunizing rabbits; the above-mentioned vector construction, protein expression, and purification methods are all conventional operations in the art and will not be elaborated here.

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

[0085] 3. Evaluation of immunization effect: Indirect ELISA was used to measure the antibody titers of the antiserum of three immunized rabbits eight weeks after immunization to evaluate the immunization effect. The specific steps are as follows:

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

[0087] (2) The coating buffer was poured out, and the plate was washed 3 times with PBST for 2 minutes each time. Then, 300 μL of 5% skim milk powder diluted with PBS was added and incubated at 37°C in a constant temperature incubator for 2 hours;

[0088] (3) After the blocking of the enzyme-labeled plate, the blocking solution was discarded. After washing the plate as in step (2), it was patted dry;

[0089] (4) The serum to be tested was serially diluted with antibody diluent, starting from a concentration of 1:100. 20 μL of serum was added to 2 mL of antibody diluent to prepare a starting concentration of 1:100. 500 μL of the diluted serum at the starting concentration of 1:100 was mixed evenly with 500 μL of PBS solution, and so on, for serial dilution in this way, so that the dilution factor of each well was 2 times that of the previous well. The negative control well was added with pre-immunization rabbit serum. Incubate at 37°C in a constant temperature incubator for 1 hour;

[0090] (5) Wash the plate as in step (2) and pat it dry. The HRP-labeled goat anti-rabbit secondary antibody was diluted 5000 times with antibody diluent, and 100 μL was added to each well of the enzyme-labeled plate and incubated at 37°C in a constant temperature incubator for 0.5 hour;

[0091] (6) Wash the plate as in step (2) and pat it dry. Single-component TMB was added at 100 μL per well and incubated at 37°C in a constant temperature incubator in the dark for 15 minutes;

[0092] (7) Add 50 μL of 2 M sulfuric acid to each well as the color development termination solution, and read the OD450 value with an enzyme-linked immunosorbent assay (ELISA) reader. The maximum dilution of the positive reaction is the titer of the test sample.

[0093] (8) Calculate the absorbance ratio of the antibody to be detected to 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. Take the positive result with the largest dilution factor as the measured antibody titer.

[0094] The results of the above antiserum titer detection are shown in Figure 1 , and the serum antibody titers of the 3 positive rabbits immunized with the gD recombinant protein ( Figure 1 A, B, and C in 5 ) all reached 4.1×10

[0095] 4. Using phage display technology, construct a single-chain fragment variable (scFv) phage display library of specific immunogenic gD protein at 2.1×10 13 pfu / mL: Deeply anesthetize and sacrifice the immunized rabbits in the eighth week, extract spleen tissue RNA using the RNA Easy Fast Total RNA Extraction Kit for Animal Tissues / Cells, and perform reverse transcription experiments to obtain cDNA. Use specific primers for PCR to amplify VH and VL. After gel extraction of the 350 bp fragment, obtain the first fragment. Use overlap extension PCR to ligate VH and VL into ScFv, then digest with SfiI enzyme and ligate to the pComb3XSS vector to obtain the recombinant phagemid. Electroporate it into ER2738 supercompetent cells to form the phage scFv library bacteria. Then add the wild-type helper phage M13KO7 to infect the host bacteria, amplify the host bacteria overnight, and precipitate the bacteria with PEG-NaCl to obtain the supernatant, thus obtaining the primary phage scFv display library.

[0096] Example 2: This example provides a method for enrichment and screening of positive clones, which is as follows:

[0097] A recombinant gD protein phage scFv display library with good enrichment and specific monoclonal aggregation was screened by three rounds of solid-phase panning. In a laminar flow hood, a sterile ELISA-specific microtiter plate was coated with the recombinant gD protein prepared by prokaryotic expression as an antigen. The antigen was diluted with sterile PBS to a concentration of 25 μg / mL. 100 μL of the diluted antigen solution was added to each well of the microtiter plate and incubated overnight at 4°C. The same antigen was used in the second round of panning, but the coating concentration was reduced to 15 μg / mL. In the third round of panning, the coating concentration of the antigen was reduced to 5 μg / mL. The next day, the above microtiter plate was washed with a plate washer, carefully patted on a clean paper to remove the PBST washing solution, and a sterile PBS buffer containing 3% BSA was used as a blocking solution. 200 μL was added to each well and incubated statically in a 37°C incubator for 2 h. After the blocking was completed, the microtiter plate was washed, and the PBST washing solution was carefully patted off for standby. 3 mL of the primary antibody library phage solution prepared above was diluted 5-fold with PBS containing 1% BSA (the primary antibody library phage solution was used in the second round of panning, and so on), and left to stand at room temperature for 10 min. 150 μL was added to each well of the blocked microtiter plate and incubated at 37°C for 2 h. After the incubation was completed, the microtiter plate was washed 3 times, 3 min each time, and the PBST washing solution was patted off. 100 μL of a 0.2 M Gly-HCl elution solution with a pH of 2.2 was added to each well of the above microtiter plate, rotated at 37°C and 80 rpm for 8 min, then pipetted up and down repeatedly to mix evenly. The eluate was carefully aspirated into a clean 15 mL centrifuge tube, and an equal volume of Tris-HCl with a pH of 7.4 was immediately added to neutralize the phage eluate. 1 mL of the eluted phage was set aside for storage, for library capacity determination and bacterial liquid PCR to identify the insertion rate of the target gene.

[0098] The 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 3 rounds of panning, a phage antibody library with good enrichment and specific monoclonal aggregation was 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 by the phage-ELISA method. A total of 38 positive clones were sent to the company for sequencing (the results are shown in Figure 2 ). By analyzing the sequencing results, three scFv gene sequences with relatively high abundances and conforming to the variable regions of rabbit antibodies were obtained, named rabbit monoclonal antibodies D1, D2, and D3 respectively (the sequences are shown as A, B, and C in Figure 3 ).

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

[0103] The heavy-chain variable region and the light-chain variable region in the sequences of the above-mentioned rabbit monoclonal antibodies D1, D2, and D3 are respectively concatenated with the constant regions of rabbit IgG heavy chain and light chain. A kozak sequence (GCCGCCACC) is added before the start codon, and the human serum albumin sequence MKWVTFISLLFLFASAYS (see GeneBank accession number: NP_000468 for details) is added after the start codon. An Xbal site is added to the 5' end of the sequence, and an EcoRV restriction site is added to the 3' end. It is inserted into the pcDNA3.4 empty vector to construct a eukaryotic recombinant expression vector (as shown in Figure 4 and Figure 5 ; in Figure 4 A, B, and C respectively correspond to rabbit monoclonal antibodies D1, D2, and D3; Figure 5 In Figure 5 A is the restriction enzyme digestion verification result of rabbit monoclonal antibody D1. M: DNA Marker, 1: D1H EcoRV single digestion, 2: D1H EcoRV and Xbal double digestion, 3: D1L EcoRV single digestion, 4: D1L EcoRV and Xbal double digestion; Figure 5 In

[0104] The above-mentioned eukaryotic recombinant expression vector is sent to Nanjing Genscript Biotech Co., Ltd. to optimize the codon preference, GC content, mRNA secondary structure, and repetitive sequence region with CHO cells as the host, and gene synthesis is carried out at Nanjing Genscript. After combining the light chain and the heavy chain, they are co-transfected into mammalian cells for expression and purification. The methods of transfection and purification are as follows:

[0105] Centrifuge cells with good growth status and a cell viability greater than 90%. Take 750 μL of cell volume and the number is 1×10 7, Mix well and centrifuge at 650 rpm for 5 min. Discard the supernatant, add 800 μL of high-glucose DMEM medium to resuspend, add 50 μg of plasmid for heavy chain and light chain respectively, add to a pre-chilled electrode cup and mix gently. Use a 4 mm Bole electroporation cup for electroporation. The electroporation conditions are 300 v and 900 uF. After electroporation, aspirate the liquid in the electroporation cup and place it in 15 mL of high-glucose DMEM medium for culture. Screen with 800 μg / mL of G418, collect the supernatant, and centrifuge at 8000 rpm for 5 min. Equilibrate the chromatography column with 20 mL of PBS at a flow rate of 1 mL / min, load the sample at a flow rate of 1 mL / min, elute non-specific proteins with 20 mL of PBS at a flow rate of 1 mL / min, elute with glycine buffer (pH = 3.0) at a flow rate of 1 mL / min, collect in fractions, about 3 mL per tube. A total of 2 tubes are collected. Add 100 μL of neutralizing solution in advance before elution to ensure that the eluted protein is stored at neutral pH. Collect the purified recombinant rabbit monoclonal antibody, and then aliquot and store at -20 °C.

[0106] The SDS-PAGE results of the above purified recombinant rabbit monoclonal antibody under non-reducing and reducing conditions are as Figure 6 shown, and the Western Blot results under reducing conditions are as Figure 7 shown; Figure 6 and Figure 7 1, 2, and 3 in

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

[0108] Dilute the recombinant gD protein to 0.5 μg / ml with coating buffer, add 100 μL / well, and incubate overnight at 2 - 8 °C; Wash the plate 3 times with washing buffer, pat dry, add 300 μL / well of blocking solution, and block at room temperature for 1 hour; Wash the plate 3 times with washing buffer, pat dry and set aside; Dilute rabbit monoclonal antibodies D1, D2, D3 from an initial concentration of 4000 ng / ml in 5-fold serial dilutions for 7 concentration gradients, and add 100 μL / well to the microplate; Wash the plate 3 times with washing buffer, pat dry; Dilute the enzyme-linked antibody to the working concentration, mix well, add 100 μL / well, and incubate at room temperature for 1 hour; Wash the plate 3 times with washing buffer, pat dry; Use an 8-channel pipette to add 200 μL / well of chromogenic solution, and place in the dark at room temperature for 15 minutes; Use a pipette to add 100 μL / well of stop solution to terminate the reaction; Measure the signal value at 450 nm with an enzyme-linked immunosorbent assay reader, perform statistical analysis on the data, and calculate the EC50 value.

[0109] The above detection results are as Figure 8 shown. At this time, the EC50 values of D1, D2, and D3 are 31.12, 24.99, and 33.47 respectively, all having good binding activities.

[0110] Example 5: This example provides an optimization method for the composition of a double-antibody sandwich ELISA reagent and detection conditions, which is as follows:

[0111] 1. Screening of the best antibody pairing combination: (1) Dilute the above 3 rabbit monoclonal antibodies D1, D2, and D3 as capture antibodies to a concentration of 2 μg / mL with coating buffer, add 100 μL to each well, and coat overnight at 4°C;

[0112] (2) Wash the enzyme-linked immunosorbent assay (ELISA) plate 3 times with 0.1% PBST and pat dry, add 200 μL of 5% skim milk powder, and block at 37°C for 2 h;

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

[0114] (4) Wash 3 times and pat dry, add 100 μL of HRP-labeled detection antibody at a concentration of 1 μg / mL to each well, and incubate at 37°C for 1 h;

[0115] (5) Wash 3 times and pat dry, add 100 μL of TMB chromogenic solution to each well, and incubate in the dark at 37°C for 15 min;

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

[0117] Pair the 3 purified monoclonal antibodies with the HRP-labeled monoclonal antibody one by one, ensuring that the antibody coating concentration and antigen concentration remain unchanged. The results are shown in Table 2. Table 2 shows that the antibodies are successfully paired and a pair of the best combination is screened out. When the capture antibody is D3 and the detection antibody is D2, the combination is the best.

[0118] Table 2

[0119]

[0120] 2. Screening of the best working concentrations of the capture antibody and the detection antibody: React with different concentrations of the capture antibody (1, 2, 4, 8 μg / mL) and different dilutions of the detection antibody (1:100, 1:200, 1:400, 1:800) combinations, and use the checkerboard method to determine the best working concentrations of the capture antibody and the detection antibody.

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

[0122] Table 3

[0123]

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

[0125] 4. Determination of the best antigen incubation time: According to the best conditions screened above, with other conditions unchanged, the antigen incubation times were set to 30, 60, 120, and 150 min. By the P / N ratio, the best antigen incubation time was determined. The results are as Figure 10 shown. The conditions are optimal when the antigen incubation time is 90 min.

[0126] 5. Determination of the best color development time: According to the best conditions screened above, with other conditions unchanged, the incubation times of the detection antibody were set to 5, 10, 15, and 20 min. By the P / N ratio, the best color development time was determined. The results are as Figure 11 shown. The conditions are optimal when the color development time is 15 min.

[0127] 6. Determination of the negative and positive thresholds: The optimized double-antibody sandwich ELISA method was used to detect 30 BHV-1 negative and 30 positive clinical samples. The critical value was determined by measuring the OD450 value on an enzyme-linked immunosorbent assay (ELISA) reader and the model was evaluated.

[0128] Sixty clinical samples were detected. According to the results of data statistical analysis (see Figure 12 , Figure 13 ), it is shown that the positive and negative critical value is 0.8525 and the AUC value is 0.968, indicating that the established model has 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. To verify whether the detection method reacts to other bovine-related viruses except BHV-1, BCV, BRSV, BRV, BVDV, and BHV-1 were used to verify the specificity of the detection method.

[0130] The specificity experiment was verified using the above-established ELISA experimental conditions. The results are as Figure 14As shown, it was found that the method had no cross-reaction with BCV, BRSV, BRV, BVDV, BAstV, and BNoV, and the values differed significantly, indicating that the established method had high specificity.

[0131] 8. Intra-batch and inter-batch repeated experiments: The accuracy of the established double-antibody sandwich ELISA method was evaluated through intra-batch and inter-batch variability. The established ELISA method was used to detect positive samples 4 times within a batch and 4 times between batches, and the coefficient of variation was calculated to evaluate its accuracy.

[0132] To detect whether the established double-antibody sandwich ELISA method had good stability, 4 positive clinical samples were selected for determination in intra-batch and inter-batch experiments respectively. The results (see Tables 4 and 5) showed that the coefficients of variation within and between batches were both not more than 5%, indicating that the established double-antibody sandwich ELISA method had good stability.

[0133] Table 4

[0134]

[0135] Table 5

[0136]

[0137] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these 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 appended claims.

Claims

1. A recombinant rabbit monoclonal antibody against BHV-1, characterized in that: The recombinant rabbit monoclonal antibody is rabbit monoclonal antibody D3; the amino acid sequence of the rabbit monoclonal antibody D3 is shown in the sequence table SEQ ID NO.

3.

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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