Indirect ELISA (enzyme-linked immuno sorbent assay) detection kit for rabbit hemorrhagic disease wild virus antibody

Abstract

The invention discloses an indirect ELISA (enzyme-linked immuno sorbent assay) detection kit for a rabbit hemorrhagic disease wild virus antibody, and relates to the technical field of biological detection. The indirect ELISA detection kit for the rabbit hemorrhagic disease wild virus antibody comprises the following components: an elisa plate coated with an antigen, a washing and diluting solution, an elisa secondary antibody, a developing solution, a stopping solution, standard RHDV positive serum and standard RHDV negative serum, the antigen is a recombinant P16 protein; the amino acid sequence of the recombinant P16 protein is as shown in SEQ ID NO. 1. The rabbit hemorrhagic disease wild virus antibody indirect ELISA detection kit developed by the invention has very high accuracy, has no cross reaction with rabbit hemorrhagic disease inactivated vaccine immune serum and positive serum of common pathogens of rabbits, and can accurately detect rabbit hemorrhagic disease wild virus infection in an immune rabbit farm.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, in particular to an indirect ELISA detection kit for rabbit viral hemorrhagic disease wild-type virus antibodies. Background Art

[0002] Rabbit hemorrhagic disease, also known as rabbit plague, is an acute septicemic, highly contagious, fatal infectious disease of rabbits caused by rabbit hemorrhagic disease virus (RHDV), characterized by systemic parenchymal organ bleeding. It is defined as a Class B disease by the World Organization for Animal Health (OIE), and my country classifies it as a Class II disease.

[0003] Rabbit viral hemorrhagic disease only occurs in domestic rabbits. Rabbits used for fur are slightly more susceptible than rabbits used for leather, and Angora rabbits are the most susceptible. The disease develops rapidly and has a high mortality rate. It often presents an outbreak, with both the morbidity and mortality rates exceeding 95%. It is a devastating disease in domestic rabbits. The disease can occur all year round, but is more common in winter and spring in the north, which may be related to the reduced immunity caused by the cold winter and the monotonous feed.

[0004] The prevention of rabbit viral hemorrhagic disease mainly adopts comprehensive measures such as disinfection, isolation, blockade, harmless treatment, and immunization. Immunization is the most important measure. In the epidemic area, inactivated vaccines are regularly used for vaccination, twice a year, with a dose of 1mL, subcutaneous injection. The immune period can last for more than half a year. At present, there are two vaccines for preventing this disease in my country, namely, VP60 subunit vaccine with baculovirus vector and tissue inactivated vaccine, which have played a good role in clinical practice. However, there are great differences in biological activity and immunogenicity between the new variant subtype and the classical subtype. The protective effect of these two vaccines against the variant subtype is relatively low, and some rabbits in the rabbit group are often immune to poor effects for some reasons, and infection will also occur when wild toxins are present. Therefore, in order to eliminate the source of infection in the group, it is necessary to detect wild toxin infection antibodies in the immune rabbit group, exclude wild toxin infected rabbits from the immune rabbit farm, reduce the discharge of its pathogens and the harm as a source of infection, and play a more effective preventive role.

[0005] The existing methods for detecting rabbit viral hemorrhagic disease infection include two categories: molecular biological methods such as PCR and qPCR, and serological methods such as HA-HI and ELISA. Molecular biological methods have high requirements for experimental conditions and personnel, and have a small detection throughput, making it difficult to meet the needs of large-scale rapid detection. At the same time, the detection of pathogens is only applicable to the infection stage, and cannot be performed on recovered animals, making it difficult to investigate the true extent and scope of the infection. Serological methods such as HA-HI and ELISA can detect antibodies and can detect viral infections for a long time, but they all use VP60 protein as an antigen and can only detect VP60 antibodies. They cannot distinguish between immune antibodies and wild virus infection antibodies, and cannot detect wild virus infected rabbits from immunized rabbit farms. The present invention intends to develop a new ELISA method and kit for detecting wild virus antibodies of rabbit viral hemorrhagic disease based on the analysis of the specificity of non-structural proteins of rabbit viral hemorrhagic disease virus, thereby realizing high-throughput screening of wild virus infected rabbits in vaccine immunized rabbit farms. Summary of the invention

[0006] The purpose of the present invention is to provide an indirect ELISA detection kit for antibodies against wild-type rabbit viral hemorrhagic disease to solve the problems existing in the above-mentioned prior art. The indirect ELISA detection kit for antibodies against wild-type rabbit viral hemorrhagic disease developed by the present invention has high accuracy, has no cross-reaction with the immune serum of inactivated rabbit viral hemorrhagic disease vaccine and the positive serum of common pathogens of rabbits, and can accurately detect the infection of wild-type rabbit viral hemorrhagic disease in immunized rabbit farms.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an indirect ELISA detection kit for rabbit viral hemorrhagic disease wild-type virus antibodies, comprising the following components:

[0009] Antigen-coated ELISA plate, washing and diluent, enzyme-labeled secondary antibody, color development solution, stop solution, standard RHDV-positive serum and standard RHDV-negative serum;

[0010] The antigen is recombinant P16 protein;

[0011] The amino acid sequence of the recombinant P16 protein is shown in SEQ ID NO.1.

[0012] Furthermore, the preparation method of the antigen-coated ELISA plate comprises the following steps: adding a carbonate buffer solution containing the recombinant P16 protein to a 96-well microplate, coating at 37°C for 2 hours, then adding PBST containing 5% donkey serum, blocking at 37°C for 1 hour, and obtaining the antigen-coated ELISA plate.

[0013] Furthermore, in the carbonate buffer solution, the concentration of the recombinant P16 protein is 10 ng / mL.

[0014] Furthermore, the washing and diluting solution is PBST solution.

[0015] Furthermore, the enzyme-labeled secondary antibody is HRP-labeled goat anti-rabbit IgG.

[0016] Furthermore, the color developing solution is TMB solution.

[0017] Furthermore, the stop solution is a 10% sulfuric acid solution.

[0018] The present invention also provides a method for detecting rabbit viral hemorrhagic disease wild-type antibodies for non-disease diagnosis purposes, comprising the steps of using the above-mentioned rabbit viral hemorrhagic disease wild-type antibody indirect ELISA detection kit to detect rabbit viral hemorrhagic disease wild-type antibodies:

[0019] Add 100-fold diluted standard RHDV positive serum, standard RHDV negative serum or the sample to be tested to the antigen-coated ELISA plate, incubate at 37° C. for 1 hour, and wash the plate 5 times with PBST after incubation;

[0020] Add 100 μL of 2000-fold diluted enzyme-labeled secondary antibody to each reaction well and incubate at 37°C for 30 min. After incubation, wash the plate 5 times with PBST;

[0021] Add 100 μL of the color developing solution to each reaction well and react in the dark until the solution turns obviously blue;

[0022] After adding 50 μL of the stop solution to each reaction well to terminate the reaction, the OD was read using an ELISA reader. 450 Value, and calculate the S / P value:

[0023] S / P = OD of sample 450 Value / OD of positive control 450 value;

[0024] Determine the test results based on the S / P value:

[0025] When the OD of the standard RHDV positive serum 450 ≥1, and the OD of the standard RHDV-negative serum 450 When ≤0.4, the test result is valid;

[0026] When the S / P value was ≥0.8, it was judged as positive, otherwise it was negative.

[0027] The present invention discloses the following technical effects:

[0028] The present invention selects non-structural protein P16 as coating antigen based on the analysis of the specificity of non-structural protein of rabbit viral hemorrhagic disease virus, establishes an ELISA method for detecting rabbit viral hemorrhagic disease wild-type virus antibody, and develops an indirect ELISA detection kit for rabbit viral hemorrhagic disease wild-type virus antibody. The indirect ELISA detection kit for rabbit viral hemorrhagic disease wild-type virus antibody developed by the present invention has high accuracy, has no cross reaction with rabbit viral hemorrhagic disease inactivated vaccine immune serum and positive serum of common pathogens of rabbits, and can accurately detect rabbit viral hemorrhagic disease wild-type virus infection in immunized rabbit farms.

[0029] By using the indirect ELISA detection kit for antibodies to wild-type rabbit viral hemorrhagic disease provided by the present invention, rabbits infected with wild-type rabbits can be screened at a high throughput in a vaccine-immunized rabbit farm, and immunization and detection and elimination can be combined in the prevention and control of rabbit plague, which is beneficial to improving the efficiency and effect of prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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 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.

[0031] Figure 1 It is the BLAST-PSI alignment diagram of P16 protein;

[0032] Figure 2 This is the SDS identification image of the purified P16 protein. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Example 1

[0039] 1. Bioinformatics analysis of P16

[0040] The protein sequence of P16 was obtained from the NCBI website, and its full length and antigen epitope were analyzed by BLASTP ( Figure 1 ) to determine its specificity as an antigen.

[0041] 2. Preparation of recombinant P16 protein

[0042] According to the amino acid sequence of rabbit viral hemorrhagic disease P16 protein (SEQ ID NO.1), codon optimization was performed to obtain a gene sequence that can be efficiently expressed in Escherichia coli (SEQ ID NO.2). After gene synthesis, it was digested by BamH I and Xho I and inserted into the pET28a expression vector to construct the pET28a-P16 expression vector, and then the BL21 (DE3) Escherichia coli was transfected by chemical method to construct a recombinant expression strain. After cultivation, the expression of recombinant protein P16 was induced by IPTG, and affinity purification was performed by Ni column to prepare a high-purity recombinant P16 protein, which was identified by SDS-PAGE. The SDS identification results of the purified P16 protein are shown in Figure 2 .

[0043] SEQ ID NO.1:

[0044] MAAMSRLTGMTTAILPEKKPLDFFLDLRDKTPPCCIRATGRLAWPVFPGQNGKEGPLETCNKCGKWLNGFGNFGLEDLGDVCLCSIAQQKHKFGPVCLCNRVYIHDCGRWRRRSRFLKHYKALNKVIPCAYQFDESFPTPIF.

[0045] SEQ ID NO.2:

[0046] ATGGCTGCTATGTCTCGTCTGACCGGTATGACCACCGCTATCCTGCCGGAAAAAAAACCGCTGGACTTCTTCCTGGACCTGCGTGACAAAACCCCGCCGTGCTGCATCCGTGCTACCGGTCGTCTGGCTTGGCCGGTTTTCCCGGGTCAGAACGGTAAAGAAGGTCCGCTGGAAACCTGCAACAAATGCGGTAAATGGCTGAACGGTTTCGGTAAC TTCGGTCTGGAAGACCTGGGTGACGTTTGCCTGTGCTCTATCGCTCAGCAGAAACACAAATTCGGTCCGGTTTGCCTGTGCAACCGTGTTTACATCCACGACTGCGGTCGTTGGCGTCGTCGTTCTCGTTTCCTGAAACACTACAAAGCTCTGAACAAAGTTATCCCGTGCGCTTACCAGTTCGACGAATCTTTCCCGACCCCGATCTTCGAATAA.

[0047] Example 2

[0048] Rabbit virus hemorrhagic disease wild-type virus antibody indirect ELISA detection kit, including the following components:

[0049] ELISA plate: ELISA plate coated with antigen (recombinant P16 protein prepared in Example 1);

[0050] Washing and dilution solution: PBST solution;

[0051] Enzyme-labeled secondary antibody: HRP-labeled goat anti-rabbit IgG;

[0052] Color developing solution: TMB solution;

[0053] Stop solution: 10% (V / V) sulfuric acid solution;

[0054] Standard RHDV-positive serum;

[0055] Standard RHDV negative serum.

[0056] The preparation method of the ELISA plate coated with antigen is as follows:

[0057] Add 100 μL of carbonate buffer solution containing 10 ng / mL recombinant P16 protein to a 96-well microplate, place it in a 37°C incubator for 2 hours for coating, then add 200 μL of PBST containing 5% (V / V) donkey serum, block it at 37°C for 1 hour, and obtain an antigen-coated ELISA plate.

[0058] Example 3

[0059] The rabbit viral hemorrhagic disease wild virus antibody indirect ELISA detection kit of Example 2 was used to detect the positive and negative serum of rabbits (15 wild virus positive sera, 42 immune positive sera and RHDV negative sera) and positive sera of common infectious diseases (pasteurellosis, myxomatosis, Clostridium perfringens disease, Escherichia coli disease, salmonellosis), and the specific method is as follows:

[0060] (1) Add 100-fold diluted standard RHDV positive serum, standard RHDV negative serum or serum to be tested to the antigen-coated ELISA plate, incubate at 37°C for 1 hour, and wash the plate 5 times with PBST after incubation;

[0061] (2) Add 100 μL of 2000-fold diluted enzyme-labeled secondary antibody (HRP-labeled goat anti-rabbit IgG) to each reaction well and incubate at 37°C for 30 min. After incubation, wash the plate 5 times with PBST.

[0062] (3) Add 100 μL of colorimetric solution (TMB solution) to each reaction well and react in the dark until the solution turns a distinct blue color;

[0063] (4) Add 50 μL of stop solution (10% sulfuric acid solution) to each reaction well to terminate the reaction and read the OD value using an ELISA reader. 450 value, and calculate the S / P value: S / P = OD of the sample 450 Value / OD of positive control 450 value.

[0064] (5) Result judgment criteria

[0065] When the OD of the standard RHDV positive serum 450 ≥1, and the OD of the standard RHDV-negative serum 450 When ≤0.4, the test result is valid;

[0066] When the S / P value of the serum sample is ≥0.8, the serum sample is determined to be positive for rabbit hemorrhagic disease virus antibody; otherwise, it is negative.

[0067] (6) Determination of test results

[0068] The test results of the negative and positive sera of rabbits and the positive sera of common infectious diseases are shown in Table 1. The results show that the S / P values ​​of immune positive sera, RHDV negative sera and other positive sera are all less than 0.8, and only the S / P value of wild virus positive serum is greater than 0.8, indicating the accuracy of the test.

[0069] Table 1 Test results of positive and negative serum of rabbits and positive serum of common infectious diseases

[0070]

[0071] Example 4

[0072] Detection of wild-type rabbit hemorrhagic disease virus infection in meat rabbit farms:

[0073] In a meat rabbit farm where immunization with rabbit plague VP60 subunit vaccine was carried out, blood was collected from the ear vein of 10 sick rabbits with rabbit viral hemorrhagic disease symptoms and 5 healthy rabbits, and the serum was separated by centrifugation. A blind test for rabbit viral hemorrhagic disease wild-type virus antibodies was performed according to the method of Example 3. The results are shown in Table 2.

[0074] Table 2 Detection results of wild-type rabbit hemorrhagic disease virus infection in meat rabbit breeding farms

[0075]

[0076] Example 5

[0077] Detection of wild-type rabbit hemorrhagic disease virus infection in Rex rabbit farms:

[0078] At a Rex rabbit farm where rabbits were immunized with inactivated rabbit hemorrhagic disease virus vaccine, blood was collected from the ear veins of 5 sick rabbits with symptoms of viral hemorrhagic disease and 20 healthy rabbits. The serum was separated by centrifugation, and a blind test for rabbit viral hemorrhagic disease wild virus antibodies was performed according to the method of Example 3. The results are shown in Table 3.

[0079] Table 3 Detection results of wild-type rabbit hemorrhagic disease virus infection in Rex rabbit breeding farms

[0080]

[0081] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An indirect ELISA kit for detecting antibodies against wild-type rabbit viral hemorrhagic disease, characterized in that: Includes the following components: Antigen-coated ELISA plate, washing and diluent, enzyme-labeled secondary antibody, color development solution, stop solution, standard RHDV-positive serum and standard RHDV-negative serum; The antigen is recombinant P16 protein; The amino acid sequence of the recombinant P16 protein is shown in SEQ ID NO.

1.

2. The indirect ELISA detection kit for rabbit viral hemorrhagic disease wild-type virus antibodies according to claim 1, characterized in that: The preparation method of the antigen-coated ELISA plate comprises the following steps: adding a carbonate buffer solution containing the recombinant P16 protein to a 96-well microplate, coating at 37°C for 2 hours, then adding PBST containing 5% donkey serum, blocking at 37°C for 1 hour, and obtaining the antigen-coated ELISA plate.

3. According to claim 2, indirect ELISA detection kit for rabbit viral hemorrhagic disease wild-type virus antibodies, characterized in that: In the carbonate buffer solution, the concentration of the recombinant P16 protein is 10 ng / mL.

4. The indirect ELISA detection kit for rabbit viral hemorrhagic disease wild-type virus antibody according to claim 1, characterized in that: The washing and diluting solution is PBST solution.

5. The indirect ELISA detection kit for rabbit viral hemorrhagic disease wild-type virus antibody according to claim 1, characterized in that: The enzyme-labeled secondary antibody is HRP-labeled goat anti-rabbit IgG.

6. The indirect ELISA detection kit for rabbit viral hemorrhagic disease wild-type virus antibodies according to claim 1, characterized in that: The color developing solution is TMB solution.

7. The indirect ELISA detection kit for rabbit viral hemorrhagic disease wild-type virus antibodies according to claim 1, characterized in that: The stop solution is 10% sulfuric acid solution.

8. A method for detecting antibodies to rabbit viral hemorrhagic disease for non-disease diagnosis purposes, characterized in that: The method comprises the steps of using the indirect ELISA detection kit for rabbit viral hemorrhagic disease wild-type virus antibody according to any one of claims 1 to 7 to detect rabbit viral hemorrhagic disease wild-type virus antibody: Add 100-fold diluted standard RHDV positive serum, standard RHDV negative serum or the sample to be tested to the antigen-coated ELISA plate, incubate at 37° C. for 1 hour, and wash the plate 5 times with PBST after incubation; Add 100 μL of 2000-fold diluted enzyme-labeled secondary antibody to each reaction well and incubate at 37°C for 30 min. After incubation, wash the plate 5 times with PBST; Add 100 μL of the color developing solution to each reaction well and react in the dark until the solution turns obviously blue; After adding 50 μL of the stop solution to each reaction well to terminate the reaction, the OD was read using an ELISA reader. 450 Value, and calculate the S / P value: S / P = OD of sample 450 Value / OD of positive control 450 value; Determine the test results based on the S / P value: When the OD of the standard RHDV positive serum 450 ≥1, and the OD of the standard RHDV-negative serum 450 When ≤0.4, the test result is valid; When the S / P value was ≥0.8, it was judged as positive, otherwise it was negative.

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