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Blocking ELISA (enzyme-linked immunosorbent assay) kit for detection of fowl adenovirus I type-4 antibody and application thereof

A technology of poultry adenovirus and kit, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of inaccurate results, inconvenient detection of a large number of samples, time-consuming and laborious separation and identification, etc., and achieve good repeatability, simple and fast operation, good specific effect

Active Publication Date: 2019-01-04
北京市动物疫病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the traditional laboratory detection methods for FAdV have certain defects, such as virus isolation and identification, neutralization test, agar diffusion test and other methods are time-consuming and laborious, and the results are not accurate enough. PCR, LAMP, etc. have high requirements for kit equipment and are inconvenient. For the detection of a large number of samples, the ELISA method has the advantages of simplicity, rapidity, and strong specificity, so it can be used as an effective method for the detection of FAdV antigens and antibodies

Method used

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  • Blocking ELISA (enzyme-linked immunosorbent assay) kit for detection of fowl adenovirus I type-4 antibody and application thereof
  • Blocking ELISA (enzyme-linked immunosorbent assay) kit for detection of fowl adenovirus I type-4 antibody and application thereof
  • Blocking ELISA (enzyme-linked immunosorbent assay) kit for detection of fowl adenovirus I type-4 antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Expression, purification and identification of FAdV-4 Hexon protein

[0056] 1. Construction of FAdV-4 Hexon protein expression vector:

[0057] According to the gene sequence of SDSX1 strain registered in GenBank (GenBank: KU845700), a pair of specific primers were designed using Primer5.0 software, and 5'linker and 3'linker were introduced into the primer sequence to facilitate the subsequent recombination reaction with the expression vector. The primer sequences are as follows: upstream primer P1: 5'-GGGTCCAGCGGCGCTGGATCCATGGCGGCCCTCACGCCCGA-3' (SEQ ID NO: 1); downstream primer P2: 5'-CGCTCCACTGCCTCCTGCAGCTTACACGGCGTTGCCTGTGG-3' (SEQ ID NO: 2). The size of the expected amplified fragment is about 2856bp, wherein the nucleotide sequence of the hexon gene is shown in SEQ ID NO:3. Recover the hexon gene target fragment (amplification product) by gel, and recombine the hexon target gene with the pQE1 (Cat No. / ID: 32932, QIAGEN) vector in a certain ratio. Recom...

Embodiment 2

[0064] Example 2 Preparation and identification of hybridoma cell lines and the monoclonal antibodies they secrete

[0065] 1. Antigen Immunization and Fusion

[0066] The prepared renatured and purified Hexon protein was emulsified in an amount of 50 μg with an equal amount of Freund's adjuvant, and then subcutaneously injected into multiple points to immunize several BALB / c mice. The adjuvant for the first immunization was complete Freund's adjuvant, and the adjuvant for subsequent immunization was incomplete Freund's adjuvant. The immunization interval was 2 weeks, and the serum titer was determined by direct ELISA after 3 immunizations. Screen the mice with high serum titer and low cross-reaction, and perform cell fusion according to conventional methods, and screen hybridoma cell lines secreting specific monoclonal antibodies.

[0067] 2. Screening of monoclonal antibodies

[0068] Screening antigens are inactivated avian-derived viruses such as avian influenza virus, ...

Embodiment 3

[0075] The establishment of embodiment 3 blocking ELISA method

[0076] 1. Preparation of sample coating solution, diluent, washing solution and stop solution

[0077] The coating solution is 0.05M carbonate buffer, and its formula is: Na 2 CO 3 1.59g, NaHCO 3 2.93g, add distilled water to make up to 1000mL, pH is 9.6.

[0078] The sample diluent was washing solution containing 1% bovine serum albumin.

[0079] The washing solution is 0.01M phosphate buffer solution with a pH of 7.4 containing 0.1% Tween-20, and its formula is: NaCl: 8.5g, NaH 2 PO 4 2H 2 O: 0.356g, Na 2 HPO 4 12H 2 O: 2.772g, after mixing it, dissolve it with distilled water and set the volume to 1000mL, then add 0.5mL Tween-20 to it to obtain the above washing solution.

[0080] The stop solution is a solution containing 0.5M sulfuric acid: dilute 21.8mL of concentrated sulfuric acid to 800mL to obtain the above 0.5M sulfuric acid solution.

[0081] 2. Determination of the optimum coating concen...

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Abstract

The invention discloses a hybridoma strain prepared with fowl adenovirus I type-4 Hexon protein, a monoclonal antibody secreted by the hybridoma strain, and a blocking ELISA (enzyme-linked immunosorbent assay) kit for detection of fowl adenovirus I type-4 antibody. The kit herein includes a coated plate, a horse radish peroxidase-marked fowl adenovirus type-4 monoclonal antibody, a cleaning solution, serum diluent, FAdV-4 standard positive serum, FAdV-4 standard negative serum, a substrate rendering liquid, and a stop solution, wherein the hybridoma strain is collected under CGMCC No. 16203; the fowl adenovirus type-4 monoclonal antibody is secreted by the hybridoma strain collected under CGMCC No. 16203; the coated plate employs Escherichia coli system expressed recombinant fowl adenovirus type-4 Hexon protein as a coating antigen. The kit herein employs the blocking ELISA as a principle to detect fowl adenovirus I type-4 antibody in a fowl serum sample, has the advantages of high sensitivity and specificity, good repeatability, high detection speed, good detection convenience, good convenience of standardization and the like, and is very convenient to popularize and apply in basic-level farming units.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a blocking ELISA kit for detecting group I poultry adenovirus antibodies and a detection method and application thereof. Background technique [0002] Since June 2015, diseases characterized by pericardial effusion and liver hypertrophy have broken out in commercial chicken flocks in Shandong, Anhui, Hebei, Henan, Liaoning and other provinces. According to the lesions, it is tentatively called chicken pericardial effusion-hepatitis syndrome. The disease mostly occurs in broilers aged about 20 to 30 days. The peak period of death is 4 to 8 days after the onset, the course of disease is 8 to 15 days, and the mortality rate reaches 20% to 30%. At the same time, 20-70-day-old and 200-300-day-old laying hens also occurred, but the mortality rate was lower than that of broiler chickens. The pathogen of the disease was identified as fowl adenovirus 4 (Fowl adenovirus 4, FA...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/531G01N33/535
CPCG01N33/531G01N33/535G01N33/56983
Inventor 王林冯小宇孙丹韦海涛宋彦军袁万哲赵荣茂
Owner 北京市动物疫病预防控制中心
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