RHDV2 antibody indirect ELISA detection kit and determination method

Abstract

The invention is applicable to the field of biological detection application, and provides an RHDV2 antibody indirect ELISA detection kit and a determination method, the RHDV2 antibody indirect ELISA detection kit comprises an antigen-coated ELISA plate, RHDV2 negative serum and RHDV2 positive serum; according to antigen epitope analysis, truncated prokaryotic expression of four segments of antigen epitopes, namely, RHDV2VP60-1, RHDV2VP60-2, RHDV2VP60-3 and RHDV2VP60-4, is designed, and a recombinant protein RHDV2VP60-2 with optimal biological activity and stability is screened from the four segments of antigen epitopes, namely, RHDV2VP60-1, RHDV2VP60-2, RHDV2VP60-3 and RHDV2VP60-4; a recombinant protein RHDV2VP60-2 is used as an antigen to establish an indirect ELISA antibody detection method, and reaction conditions are optimized. In conclusion, the detection kit disclosed by the invention is high in sensitivity and can be used for rapidly and accurately detecting the RHDV2.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to an RHDV2 antibody indirect ELISA detection kit and a determination method. Background Art

[0002] Rabbit hemorrhagic disease (RHD), caused by rabbit hemorrhagic disease virus (RHDV), poses a serious threat to the global rabbit industry due to its high lethality and acute infectious characteristics. Currently, there are two pathogenic pathogens of RHDV: RHDV1 and RHDV2. RHDV2 spreads rapidly and replaces the dominant position of RHDV1. However, the current commercial RHDV1 vaccine cannot form effective cross-protection against RHDV2. Therefore, rapid and accurate detection of RHDV2 has become the focus of disease prevention and control.

[0003] RHDV belongs to the Caliciviridae family and is a single-stranded positive-strand RNA virus without an envelope. The virus particles are spherical and icosahedral. VP60 protein is its main structural protein. It is not only structurally stable, but also closely related to the immunogenicity of the virus as a viral capsid protein. Therefore, most of the immunological research on RHDV is centered around VP60 protein.

[0004] ELISA is convenient and quick, and the test results are intuitive. It has been widely used in viral disease detection. There have been many studies on the establishment of RHDV1-related ELISA detection methods.

[0005] Paper: Establishment and application of double antibody sandwich ELISA method for identification and detection of rabbit hemorrhagic disease virus type 1 and type 2. Jiangsu Agricultural Science, 2023, 51(24): 161-167. A double antibody sandwich ELISA method for identification of type 1 and type 2 was established using RHDV broad-spectrum monoclonal antibodies and RHDV1 and RHDV2 specific monoclonal antibodies.

[0006] The patent with publication number CN116284264A discloses a polypeptide, a kit and a method for detecting type 1 RHDV antibodies, which have high specificity for type 1 RHDV antibodies, good safety and good antigenicity; the patent is used to detect RHDV type 1 virus antibodies, using a partial antigenic epitope with an amino acid sequence of the 43-57 region and a sequence of AENSSASVATAGIGG (SEQ ID NO.7).

[0007] However, there is no report on the establishment of an indirect ELISA detection method for rabbit hemorrhagic disease RHDV2 serum antibodies in the prior art. Therefore, there is an urgent need for a method and kit for detecting RHDV2 antibodies to provide technical support for preventing and controlling the spread of RHDV2. Summary of the invention

[0008] In view of this, the present invention proposes an indirect ELISA detection kit for RHDV2 antibodies. A prokaryotic expression system is used to induce the expression of recombinant truncated proteins RHDV2 VP60-1 (aa 1-312), VP60-2 (aa 1-276), VP60-3 (aa 27-312), and VP60-4 (aa 77-312). After sensitivity tests and stability tests, RHDV2 VP60-2 is screened out to establish an antibody indirect ELISA detection method, and a kit is developed, which can quickly and accurately detect RHDV2.

[0009] The technical solution of the present invention is achieved in this way:

[0010] The invention provides an indirect ELISA detection kit for RHDV2 antibodies, comprising 5 antigen-coated ELISA plates, 2 mL of RHDV2 negative serum, and 2 mL of RHDV2 positive serum; the detection kit also comprises 300 mL of sample diluent and 300 mL of 10× concentrated washing solution.

[0011] The antigen is the recombinant protein RHDV2 VP60-2, and the amino acid sequence used by RHDV2 is the 43-57 region, and the sequence is TENASTSIATAGIGG (SEQ ID NO.1).

[0012] The determination method of the RHDV2 antibody indirect ELISA detection kit comprises the following steps:

[0013] Step 1: Design truncated prokaryotic expression of RHDV2 VP60-1 (aa 1~312), RHDV2 VP60-2 (aa 1~276), RHDV2 VP60-3 (aa 27~312), and RHDV2 VP60-4 (aa 77~312), express and purify the recombinant proteins, and screen out RHDV2 VP60-2 recombinant proteins with good activity and stability.

[0014] Step 2: Using RHDV2 VP60-2 recombinant protein as an antigen, adding the antigen to the coating solution, adding it to the ELISA reaction plate at a dose of 100 μL / well for reaction, washing with PBST after the reaction is completed, and patting and drying;

[0015] During the antigen coating process of ELISA plate, the antigen coating mass concentration was 5 mg / L; the blocking condition was 5% skim milk powder at 37°C for 1 hour; the dilution multiples of RHDV2 negative serum and RHDV2 positive serum were both 200 times; the first antibody was RHDV2 positive rabbit serum; the second antibody was goat anti-rabbit IgG-HRP, and the dilution multiple was 1:10000 times.

[0016] The RHDV2 antibody indirect ELISA detection kit of the present invention has the following beneficial effects compared with the prior art:

[0017] In order to establish an indirect ELISA method for detecting serum antibodies against rabbit hemorrhagic disease RHDV2, four truncated prokaryotic expressed RHDV2 VP60-1 (aa 1-312), RHDV2 VP60-2 (aa 1-276), RHDV2 VP60-3 (aa 27-312), and RHDV2 VP60-4 (aa 77-312) antigenic epitopes were designed according to antigenic epitope analysis. The recombinant protein RHDV2 VP60-2 (aa 1-276) with the best biological activity and stability was screened. The amino acid sequence used for RHDV2 was 43-57 region, and the sequence was TENASTSIATAGIGG (SEQ ID NO.1). An indirect ELISA antibody detection method was established using the recombinant protein RHDV2 VP60-2 as the antigen, the reaction conditions were optimized, and the sensitivity, specificity and repeatability were evaluated.

[0018] The test results showed that the method has good sensitivity, and the result was still positive when the positive serum was diluted to 12,800 times; the specificity test results showed that the positive serum antibodies of various rabbit susceptible pathogens were negative; the repeatability test results showed that the intra-batch and inter-batch coefficients of variation were 2.20% to 7.79% and 5.09% to 9.46%, respectively. The positive rates of using this method to detect clinical serum samples and comparing with the HI test were 73.91% and 67.39%, respectively, proving that the established method has good sensitivity.

[0019] The indirect ELISA established in this study provides technical support for RHDV2 epidemiological surveys and antibody monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1This is the SDS-PAGE analysis of the recombinant protein of the present invention; M: pre-stained protein molecular standard; 1 is the bacteria before induction; 2 is the bacteria after induction; 3 is the supernatant after ultrasonic disruption; 4 is the precipitate after ultrasonic disruption;

[0022] Figure 2 It is the Western blot identification of the recombinant protein of the present invention; M: pre-stained protein molecular standard; 1 is RHDV2VP60-1, 2 is RHDV2 VP60-2, 3 is RHDV2 VP60-3, 4 is RHDV2 VP60-4 recombinant protein detection;

[0023] Figure 3 For the recombinant protein reactivity analysis of the present invention;

[0024] Figure 4 It is the stability analysis of the recombinant protein of the present invention; M: pre-stained protein molecular standard; 1 is the swimming lane of the recombinant protein in a 37°C water bath for 0 hours, 2 is the swimming lane of the recombinant protein in a 37°C water bath for 4 hours, 3 is the swimming lane of the recombinant protein in a 37°C water bath for 8 hours, and 4 is the swimming lane of the recombinant protein in a 37°C water bath for 12 hours;

[0025] Figure 5 This is the RHDV2 VP60-2 ELISA sensitivity test result of the present invention. DETAILED DESCRIPTION

[0026] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] In the present invention, 2×Taq Plus MasterMixⅡ was purchased from Vazyme; Q5 high-fidelity DNA polymerase, restriction endonuclease, and T4 DNA ligase were all purchased from NEB; Gel Extraction Kit D2500 was purchased from Omega; Biospin plasmid DNA small-amount extraction kit was purchased from BioFlux; BCA protein concentration determination kit was purchased from Solarbio; soluble single-component TMB substrate solution was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; goat anti-rabbit IgG-HRP was purchased from Thermo Fisher; GLK-Gel Ni IDA affinity chromatography filler was purchased from Wuxi Jialeke Chromatography Technology Co., Ltd.; and human "O" type red blood cells were purchased from Shanghai Blood Biomedicine Co., Ltd.

[0028] coli DH5α and Rosetta competent cells, pET-28a, negative serum, and positive serum (RHDV1, RHDV2, rabbit rotavirus, rabbit Bordetella, rabbit Pasteurella, rabbit Escherichia coli, etc.) were all preserved by Shandong Binzhou Animal Husbandry and Veterinary Research Institute.

[0029] The sample diluent was phosphate buffered saline (PBST) containing 0.05% Tween-20, pH 7.4, and molar concentration of 0.01 mol / L.

[0030] The 10× concentrated washing solution is a phosphate buffer containing 0.5% Tween-20, a pH of 7.4, and a molar concentration of 0.1 mol / L.

[0031] The color developing solution is a mixture of tetramethylbenzidine (TMB) solution and citric acid-phosphate buffer solution, the volume ratio of the two is 1:1, the mass concentration of the tetramethylbenzidine solution is 0.2 mg / mL, and the citric acid-phosphate buffer solution contains urea hydrogen peroxide with a concentration of 0.5.

[0032] The stop solution is a hydrofluoric acid solution with a volume concentration of 0.25-0.31%.

[0033] The coating solution was carbonate buffer with a pH of 9.6 and a molar concentration of 0.05 mol / L.

[0034] Example 1 Expression of truncated RHDV2 VP60 protein

[0035] 1. Construction of prokaryotic expression vector of VP60 protein

[0036] Shanghai Jierui Bioengineering Co., Ltd. was commissioned to synthesize the RHDV2 VP60 gene, and specific primers were designed and commissioned to be synthesized by Shanghai Shenggong Co., Ltd. The primer sequences are shown in Table 1. The synthesized RHDV2 VP60 gene was used as a template, and the target gene was amplified according to the instructions for use of Q5 high-fidelity DNA polymerase. The gel-recovered PCR product and the prokaryotic expression vector pET-28a were double-digested with BamHⅠ and HindⅢ, and further purified and recovered. After ligation according to the instructions for use of T4 DNA ligase, the competent cells E. coli DH5α were transformed, and the recombinant plasmids were screened by culture on kanamycin-resistant agar plates, extracted, and sequenced to identify the recombinant plasmids.

[0037] Table 1 VP60 gene primer sequences

[0038]

[0039] The synthetic RHDV2 VP60 gene was used as a template and RHDV2 VP60 primers were used for PCR amplification. The products were subjected to 1% agarose gel electrophoresis, and the electrophoresis results showed clear bands of 936bp, 828bp, 854bp, and 704bp, which were consistent with the expected results. After double enzyme digestion, ligation, transformation, resistance screening, plasmid extraction and sequencing verification, positive recombinant plasmids pET-28a-VP60-1, pET-28a-VP60-2, pET-28a-VP60-3, and pET-28a-VP60-4 were obtained.

[0040] 2. Expression and purification of recombinant proteins

[0041] The correctly identified recombinant plasmid was transformed into E. coli Rosetta competent cells, cultured on kanamycin-resistant agar plates, and a single colony was inoculated into kanamycin-resistant LB medium. The culture was shaken at 37°C overnight, and the culture was expanded at a ratio of 1:100. 600nm When the value was 0.6, IPTG with a final concentration of 0.1 mmol / L was added to induce expression. After 4 hours, the bacterial solution was centrifuged at 8000g for 5 minutes. The precipitated bacteria were ultrasonically broken and then protein purified. The purified recombinant protein was concentrated by ultrafiltration and identified by Western blot.

[0042] The supernatant and precipitate of the pre-induction bacteria, post-induction bacteria, and ultrasonically disrupted bacteria after induction were taken for SDS-PAGE identification. The results showed that the truncated RHDV2 VP60-1, RHDV2 VP60-2, RHDV2VP60-3, and RHDV2 VP60-4 recombinant proteins were successfully expressed and were all inclusion bodies. The sizes of the recombinant proteins were approximately 45kDa, 40kDa, 35kDa, and 30kDa, respectively. Figure 1 shown.

[0043] The recombinant protein was transferred to a PVDF membrane after SDS-PAGE electrophoresis, incubated with RHDV2 positive rabbit serum as the primary antibody and goat anti-rabbit IgG-HRP as the secondary antibody, and the results were observed after color development. It can be seen that the truncated recombinant protein produced a good antigen-antibody binding reaction with the positive serum. The results are shown in Figure 2 shown.

[0044] RHDV2 VP60-1(aa 1~312):

[0045] MEGKARATPQGETAGTATTASVPGTTTDGMDPGVVATTSVVTTENASTSIATA

[0046] GIGGPPQQMDQQETWRTNFYYNDVFTWSVADAPGNILYTVQHSPQNNPFTAV

[0047] LSQMYAGWAGGMQFRFIVAGSGVFGGRLVAAVIPPGIEIGPGLEVRQFPHVVI

[0048] DARSLEPVTITMPDLRPNMYHPTGNPGLVPTLVLSVYNNLINPFGGSTSAIQV

[0049] TVETRPSEDFEFVMIRAPSSKTVDSISPADLLTTPVLTGVGTDNRWNGEIVGLQPVPGGFSTCNRHWNLNGSTYGWSSPRFAAIDHDRGNASFPGSSSSNV(SEQ ID NO.8)

[0050] RHDV2 VP60-2(aa 1~276):

[0051] MEGKARATPQGETAGTATTASVPGTTTDGMDPGVVATTSVVTTENASTSIATA

[0052] GIGGPPQQMDQQETWRTNFYYNDVFTWSVADAPGNILYTVQHSPQNNPFTAV

[0053] LSQMYAGWAGGMQFRFIVAGSGVFGGRLVAAVIPPGIEIGPGLEVRQFPHVVI

[0054] DARSLEPVTITMPDLRPNMYHPTGNPGLVPTLVLSVYNNLINPFGGSTSAIQV

[0055] TVETRPSEDFEFVMIRAPSSKTVDSISPADLLTTPVLTGVGTDNRWNGEIVGLQPVPGGFSTCNR(SEQID NO.9)

[0056] RHDV2 VP60-3(aa 27~312):

[0057] TDGMDPGVVATTSVVTTENASTSIATAGIGGPPQQMDQQETWRTNFYYNDVF

[0058] TWSVADAPGNILYTVQHSPQNNPFTAVLSQMYAGWAGGMQFRFIVAGSGVFG

[0059] GRLVAAVIPPGIEIGPGLEVRQFPHVVIDARSLEPVTITMPDLRPNMYHPTGNP

[0060] GLVPTLVLSVYNNLINPFGGSTSAIQVTVETRPSEDFEFVMIRAPSSKTVDSISP

[0061] ADLLTTPVLTGVGTDNRWNGEIVGLQPVPGGFSTCNRHWNLNGSTYGWSSPRFAAIDHDRGNASFPGSSSSNV(SEQ ID NO.10)

[0062] RHDV2 VP60-4 (aa 77~312):

[0063] VFTWSVADAPGNILYTVQHSPQNNPFTAVLSQMYAGWAGGMQFRFIVAGSGV

[0064] FGGRLVAAVIPPGIEIGPGLEVRQFPHVVIDARSLEPVTITMPDLRPNMYHPTG

[0065] NPGLVPTLVLSVYNNLINPFGGSTSAIQVTVETRPSEDFEFVMIRAPSSKTVDSI

[0066] SPADLLTTPVLTGVGTDNRWNGEIVGLQPVPGGFSTCNRHWNLNGSTYGWSSPRFAAIDHDRGNASFPGSSSSNV(SEQ ID NO.11)

[0067] Example 2 Detection antigen screening

[0068] 1. Reactivity test

[0069] The recombinant protein was used as the antigen, and the mass concentration of the antigen coating was 5 mg / L. Five RHDV2 negative and positive sera were selected and diluted 1:200 for indirect ELISA detection. The D 450nm The recombinant protein with better reaction activity was selected based on the comparison results.

[0070] The results showed that the recombinant proteins RHDV2 VP60-1 and RHDV2 VP60-2 had good reactivity with positive serum, while the recombinant proteins RHDV2 VP60-3 and RHDV2 VP60-4 had relatively poor reactivity. Figure 3 shown.

[0071] 2. Comparison of recombinant protein stability

[0072] In order to screen out recombinant proteins with good stability during the experiment, the purified proteins were placed in a 37°C water bath, and samples were taken at 0h, 4h, 8h, and 12h to select recombinant proteins with good stability.

[0073] Since RHDV2 VP60-4 is very easy to precipitate during protein concentration, RHDV2 VP60-1, RHDV2VP60-2, and RHDV2 VP60-3 were selected for stability test. The purified protein was placed in a 37°C water bath, and samples were taken at 0h, 4h, 8h, and 12h for SDS-PAGE analysis.

[0074] Results Figure 4 , showing that both RHDV2 VP60-1 and RHDV2 VP60-2 have good thermal stability. However, since RHDV2VP60-1 is easily precipitated during the concentration process and after freezing at -80°C, the recombinant protein RHDV2 VP60-2 was selected as the antigen to establish the indirect ELISA method. The amino acid sequence of RHDV2 used was the 43-57 region, and the sequence was TENASTSIATAGIGG (SEQ ID NO.1).

[0075] Example 3 Establishment of indirect ELISA detection method

[0076] The antigen-coated ELISA plate is prepared by the following method: adding the antigen to carbonate buffer (pH 9.6), adding it to the ELISA plate at a dose of 100 μL / well for coating, reacting under certain conditions, washing with PBST after the reaction is completed, patting dry, drying, and storing it in a packaging bag containing a desiccant for future use.

[0077] 1. Optimization of indirect ELISA reaction conditions

[0078] Optimization of indirect ELISA reaction conditions: Optimize the antigen coating concentration, serum dilution multiple, blocking conditions and secondary antibody dilution to measure D 450nm The maximum P / N ratio was selected as the optimal reaction condition.

[0079] The checkerboard method was used to determine the antigen coating concentration. The purified recombinant protein RHDV2 VP60-2 was diluted to 6 gradients of 10 mg / L, 5 mg / L, 2.5 mg / L, 1.25 mg / L, 0.625 mg / L, and 0.3125 mg / L using carbonate buffer (pH 9.6). The remaining steps were performed using conventional ELISA experimental methods. The color development was stopped immediately after the color development was completed. 450nm The optimal coating concentration of antigen was obtained when the P / N ratio was the largest. The results are shown in Table 2.

[0080] Table 2 Antigen coating concentration

[0081] 10mg / L 5mg / L 2.5mg / L 1.25mg / L 0.625mg / L 0.3125mg / L P / N Ratio 6.3463 6.7851 6.1734 5.4592 4.9716 4.7095

[0082] The results showed that the P / N ratio was the highest when the antigen coating concentration was 5 mg / L.

[0083] The chessboard method was used to determine the serum dilution multiple. The negative and positive sera were diluted with PBST at 1:50, 1:100, 1:200, and 1:400, respectively. 100 μL was added to each well. Two replicates were set for each dilution gradient. The remaining steps were performed using conventional ELISA experimental methods. The color development was stopped immediately after the color development was completed. 450nm The optimal serum dilution factor was the one with the largest P / N ratio. The results are shown in Table 3.

[0084] Table 3 Serum dilution multiples

[0085] 1∶50 1∶100 1∶200 1∶400 P / N Ratio 6.2979 6.5577 7.1698 6.8516

[0086] The results showed that the P / N ratio was the highest when the serum dilution was 200 times.

[0087] 5% BSA, 5% skim milk powder, and 1% gelatin were selected as blocking solutions, and the remaining steps were performed using conventional ELISA experimental methods. The color development was stopped immediately after the color development was completed. 450nm The best blocking solution was obtained when the P / N ratio was the largest. The results are shown in Table 4.

[0088] Table 4 Blocking solution

[0089] 5% BSA 5% skim milk powder 1% Gelatin P / N Ratio 3.7575 4.2879 3.8231

[0090] The results showed that the P / N ratio was the highest when 5% skimmed milk powder was selected as the blocking solution.

[0091] The blocking temperature and time were set at 37°C for 0.5 h, 37°C for 1 h, 37°C for 1.5 h, 37°C for 2 h, 37°C for 4 h, and 4°C for 4 h. The remaining steps were performed using conventional ELISA experimental methods. The color development was terminated immediately after the color development was completed. 450nmThe optimum sealing temperature and time are as follows:

[0092] Table 5 Sealing temperature and time

[0093] 37℃0.5h 37℃1h 37℃1.5h 37℃2h 37℃4h 4℃4h P / N Ratio 5.0205 6.0719 5.7815 5.4834 5.0203 5.5335

[0094] The results showed that the P / N ratio was the highest when the blocking condition was 37°C for 1h.

[0095] The HRP-labeled goat anti-rabbit IgG secondary antibody was diluted to 1:5000, 1:10000, 1:15000, and 1:20000. The remaining steps were performed using conventional ELISA experimental methods. The color development was stopped immediately after the color development was completed. 450nm When the P / N ratio is the largest, the optimal dilution of the secondary antibody is determined. The results are shown in Table 6.

[0096] Table 6 HRP labeled goat anti-rabbit IgG secondary antibody dilution

[0097] 1∶5000 1∶10000 1∶15000 1∶20000 P / N Ratio 6.4879 6.6529 6.5671 6.1558

[0098] The results showed that when goat anti-rabbit IgG-HRP was diluted at 1:10000, the P / N ratio was the highest.

[0099] In order to further verify the optimization effect of the indirect ELISA reaction conditions, the present invention also designed comparative examples (see Table 7, wherein √ represents the conditions selected for use in each comparative example).

[0100] Table 7 Optimization of indirect ELISA reaction conditions

[0101]

[0102] The results showed that when the indirect ELISA reaction conditions: antigen coating mass concentration of 5 mg / L, serum dilution of 200 times, 5% skim milk powder, blocking at 37°C for 1h, and goat anti-rabbit IgG-HRP diluted at 1:10000 were all met, the P / N ratio was the highest. The lack of any one of the conditions would affect the final P / N ratio.

[0103] 2. Determination of critical value

[0104] The indirect ELISA method initially established according to the above optimized conditions was used to test 18 randomly selected rabbit negative serum samples, with 2 replicates for each sample. 450nm Calculate the mean, standard deviation and critical value. According to statistical principles, critical value = mean + 3 × standard deviation.

[0105] The results show that the mean value is 0.1561, the standard deviation is 0.0276, and the calculated critical value is 0.2389.

[0106] 3. Sensitivity test

[0107] According to the initially established indirect ELISA detection method, the recombinant protein RHDV2 VP60-2 was coated, and the positive serum was diluted at 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800, respectively. Each dilution gradient was repeated 2 times to determine the sensitivity of the detection method.

[0108] The results showed that it was still positive when diluted to 1:12800, which was higher than the critical value of 0.2389, indicating that the detection method had good sensitivity. Figure 5 shown.

[0109] 4. Specificity test

[0110] According to the initially established indirect ELISA detection method, RHDV2-negative, RHDV2-positive, rabbit rotavirus-positive, rabbit Bordetella-positive, Pasteurella-positive, and Escherichia coli-positive sera were tested to measure D 450nm value to determine the specificity of the detection method.

[0111] The results showed that except for the RHDV2 positive serum, the others were all negative with a value less than 0.2389, indicating that the established indirect ELISA had good specificity. The results are shown in Table 8.

[0112] Table 8 Specificity test results

[0113]

[0114] 5. Repeatability test

[0115] Repeat within the batch: The same batch of purified RHDV2 VP60-2 recombinant protein was coated on the ELISA plate at different times, and 5 positive and 5 negative sera were tested, with 4 replicates for each sample. 450nm The coefficient of variation (CV) was calculated from the values: CV = (standard deviation / mean value) × 100%.

[0116] The purified RHDV2 VP60-2 recombinant protein from different batches was coated on the ELISA plate at different times, and 5 positive and 5 negative sera were tested, with 4 replicates for each sample. 450nm The coefficient of variation (CV) was calculated from the values.

[0117] The results showed that the coefficients of variation of intra-batch and inter-batch repeats were 2.20%-7.79% and 5.09%-9.46%, respectively, both less than 10%, indicating that the detection method has good repeatability.

[0118] 6. Compliance test

[0119] The established indirect ELISA method and HI method were used for detection, and the positive rates were 73.91% (34 / 46) and 67.39% (31 / 46), respectively. This indicates that the established detection method has better sensitivity than HI, and the results are shown in Table 9.

[0120] Table 9 Clinical sample test results

[0121] Detection Methods Number of samples (pieces) Positive number (copies) Positive rate (%) Indirect ELISA 46 34 73.91％ HI 46 31 67.39％

[0122] Example 4 Clinical sample detection

[0123] The indirect ELISA method established by the present invention was used to detect 46 serum samples from rabbit farms in various regions, and 4 hemagglutination unit (4HAU) antigen suspension and "O" type red blood cell suspension were prepared with reference to "Diagnostic Technology for Rabbit Hemorrhagic Disease" (NY-T 572-2023). The HI method was used for parallel detection to compare the positive rates.

[0124] The results showed that the positive rate of the indirect ELISA method established by the present invention was 73.91%; the positive rate of the HI method was 67.39%.

[0125] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An indirect ELISA detection kit for RHDV2 antibodies, characterized in that: The method comprises 5 antigen-coated ELISA plates, 2 mL of RHDV2-negative serum, 2 mL of RHDV2-positive serum, 300 mL of sample diluent, 300 mL of 10× concentrated washing solution, and 300 mL of coating solution.

2. The RHDV2 antibody indirect ELISA detection kit as described in claim 1, characterized in that: The antigen is the recombinant protein RHDV2 VP60-2.

3. The RHDV2 antibody indirect ELISA detection kit as described in claim 2, characterized in that: The amino acid sequence used by RHDV2 is the 43-57 region, and the sequence is TENASTSIATAGIGG (SEQ ID NO. 1).

4. A method for determining the RHDV2 antibody indirect ELISA detection kit as claimed in claim 3, characterized in that: The steps include: Step 1: Design truncated prokaryotic expression of RHDV2 VP60-1 (SEQ ID NO.8), RHDV2 VP60-2 (SEQ ID NO.9), RHDV2 VP60-3 (SEQ ID NO.10), and RHDV2 VP60-4 (SEQ ID NO.11), express and purify the recombinant proteins, and screen out the RHDV2 VP60-2 recombinant proteins with good activity and stability; Step 2: Using the RHDV2 VP60-2 recombinant protein as an antigen, adding the antigen to the coating solution, adding it to the ELISA plate at a dose of 100 μL / well for reaction, washing with PBST after the reaction is completed, and drying.

5. The assay method of the kit as claimed in claim 4, characterized in that: During the antigen coating process of the ELISA plate, the antigen coating mass concentration was 5 mg / L; the blocking condition was 5% skim milk powder at 37°C for 1 h; The dilution multiples of RHDV2-negative serum and RHDV2-positive serum were both 200 times; the secondary antibody was goat anti-rabbit IgG-HRP, and the dilution multiple was 1:10000 times.

Images