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Indirect ELISA (Enzyme-linked Immunosorbent Assay) kit for detecting serum type 4 aviadenovirus antibody and detection method of kit

The technology of a poultry adenovirus and kit, applied in the field of biotechnology detection, can solve the problems such as the evaluation of the immune effect that cannot be specifically detected by the serum type 4 FAdV antibody, the economic loss of the domestic poultry industry, and the rapid diagnosis of unfavorable diseases. Reproducibility, high specificity, good effect of specificity

Inactive Publication Date: 2017-05-31
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, domestic reports of FAdV-4 infection have been increasing, causing huge economic losses to the domestic poultry industry
[0004] The diagnostic methods of FAdV-4 mainly include virus isolation and identification, agar diffusion test, enzyme-linked immunosorbent assay, etc. The results of virus isolation and identification are accurate and reliable, but the detection cycle is long and the operation is cumbersome, which is not conducive to the rapid diagnosis of the disease
[0005] At present, the only commercialized ELISA kit for general antibody detection of group I FAdV in the domestic market is expensive because it is an imported product, and cannot be used for specific detection of serum type 4 FAdV antibody and FAdV-4 vaccine immunization Effect evaluation

Method used

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  • Indirect ELISA (Enzyme-linked Immunosorbent Assay) kit for detecting serum type 4 aviadenovirus antibody and detection method of kit
  • Indirect ELISA (Enzyme-linked Immunosorbent Assay) kit for detecting serum type 4 aviadenovirus antibody and detection method of kit
  • Indirect ELISA (Enzyme-linked Immunosorbent Assay) kit for detecting serum type 4 aviadenovirus antibody and detection method of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1 is used for the expression of the fiber-2 protein of coating antigen

[0069] 1. According to the fiber-2 gene sequence of FAdV-4JSJ13 strain, design specific primers with EcoRV and HindIII restriction endonuclease sites. Upstream primer: 5'-CCG GATATC ATGCTCCGGGCCCCTAAAAGAAG-3'(EcoR V), downstream primer: 5'-CCG AAGCTT TTACGGGAGGGAGGCCGCTGG-3' (Hind III). Specific primers were used to amplify the open reading frame (ORF) of the fiber-2 gene with a length of 1440 bp.

[0070] 2. The genome of JSJ13 strain was extracted by DNAVzol method, and the fiber-2 gene fragment was amplified using its genome as a template. The PCR reaction system is as follows:

[0071]

[0072] The PCR reaction procedure of Prime STAR high-fidelity enzyme is as follows:

[0073]

[0074] 3. After gel recovery and purification of the target fragment, use EcoR V and Hind III together with the pET-32a vector for double enzyme digestion. The enzyme digestion reaction syste...

Embodiment 2

[0081] Embodiment 2 is used for the purification of the fiber-2 protein of coating antigen

[0082] Discard the lysed bacterial supernatant of the recombinant strain, and keep the bacterial pellet. Follow the instructions of the His-tag inclusion body protein purification kit. After purification, the flow-through and eluate were collected for SDS-PAGE inspection. Test results such as image 3 As shown, by using a high concentration of imidazole to elute the nickel column, fiber-2 protein with higher purity was obtained.

Embodiment 3

[0083] Example 3 Specific identification and renaturation of the fiber-2 protein used to coat the antigen

[0084] 1. Pipette 25 μL of the purified protein into a new centrifuge tube, add 5 μL of 6×Protein LoadingBuffer and mix well. The mixed sample was placed in a water bath at 100°C for 5 minutes and in an ice bath for 2 minutes.

[0085] 2. After preparing the gel, add the processed samples to the sample wells repeatedly and perform vertical electrophoresis for 90 minutes. After taking out the gel, cut out the gel with the molecular weight of the entire protein, and cut out two PVDF membranes with the same area and soak them in methanol for activation. 70V constant voltage transfer for 60min. After the transfer was completed, the PVDF membrane was soaked in 5% skim milk and blocked for 1 hour at 37°C. After blocking, the PVDF membrane was washed in PBST for 3 times, 5 min each time. Two identical PVDF membranes were soaked in 1:1000 diluted His-tag monoclonal antibody ...

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Abstract

The invention discloses an indirect ELISA kit for detecting a serum type 4 aviadenovirus antibody and a detection method of the kit. The ELISA kit comprises an ELISA reaction plate, recombinant FAdV-4 fiber-2 protein, a horseradish peroxidase conjugated rabbit anti-chicken IgG antibody, coating liquid, confining liquid, a cleaning solution, a substrate developing solution and a stop buffer. The kit and detection method disclosed by the invention are applicable to rapid detection of serum type 4 aviadenovirus and have the characteristics of simplicity in operation, high specificity, high sensitivity and low cost.

Description

technical field [0001] The invention relates to the field of biotechnology detection, in particular to an indirect ELISA kit for detecting serum type 4 avian adenovirus (FAdV-4) antibody and a preparation method thereof. Background technique [0002] Fowl adenovirus (FAdV) belongs to the family Adenoviridae and the genus Aviadenovirus. The virion has no envelope, has a diameter of about 70-90nm, and is icosahedrally symmetrical, and is composed of capsid, fiber, nucleic acid core and related proteins. According to the different group-specific antigens, avian adenoviruses can be divided into 3 groups (groups I-III). Group I poultry adenoviruses include adenoviruses isolated from chickens, turkeys, geese and other birds, which contain 5 genotypes (A-E) and 12 serotypes (1-11, 8a, 8b). Among the encoded proteins of FAdV, the amino acid composition of the F2 fiber-2 protein is not conserved, and the surface has subgroup and type-specific antigenic epitopes; at the same time, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 张国中赵静何梓榕阮思凡
Owner CHINA AGRI UNIV
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