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Nonstructural protein antibody ELISA (enzyme-linked immunosorbent assay) Kit for porcine foot-and-mouth disease virus

A porcine foot-and-mouth disease virus and non-structural protein technology, applied in the biological field, can solve the problems of uncertain animals, non-existence, and inability to be used as detection indicators, and achieve the effects of reducing losses, strong specificity, and good market prospects

Active Publication Date: 2017-12-26
CHINA ANIMAL HUSBANDRY IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

2C antibody fades faster than 3ABC, and 2C antibody can be detected in immunized cattle, so it cannot be used as a detection indicator
Some researchers have pointed out that if there are at least two non-structural protein antibodies (except 3D) in the serum, it can be concluded that the animal has been infected with FMDV, but the absence of one or all non-structural protein antibodies in the serum cannot be sure that the animal has been infected with FMDV. Never been infected with FMDV

Method used

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  • Nonstructural protein antibody ELISA (enzyme-linked immunosorbent assay) Kit for porcine foot-and-mouth disease virus
  • Nonstructural protein antibody ELISA (enzyme-linked immunosorbent assay) Kit for porcine foot-and-mouth disease virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1, preparation of coated antigen by ELISA kit for nonstructural protein antibody of porcine foot-and-mouth disease virus

[0050] The present invention uses bioinformatics methods to accurately analyze the epitopes of non-structural proteins, and screens out peptides suitable for ELISA detection from the main epitopes of 2B, 3A, 3B, and 3C proteins, that is, the non-structural protein of foot-and-mouth disease virus. Protein 2B, 3A, 3B, 3C epitope polypeptides, the sequences of which are shown in Sequence 1, Sequence 2, Sequence 3 and Sequence 4 in the sequence listing. The polypeptide composition is used as the coating source of the kit of the present invention to prepare an ELISA kit for nonstructural protein antibody of porcine foot-and-mouth disease virus.

[0051] The coated antigen of the present invention can be prepared using an Applied Biosystem automatic polypeptide synthesizer (model 433A). Using the Merrifield solid-phase synthesis method, Fmoc (...

Embodiment 2

[0083] Embodiment 2, composition of porcine foot-and-mouth disease virus nonstructural protein antibody ELISA kit

[0084] 1. Evaluation of combined B cell epitopes

[0085] In order to improve the positive detection rate of the detection method, the single peptide sequence 1 (Seq1), sequence 2 (Seq2), sequence 3 (Seq3 ), sequence 4 (Seq4) and the mixed peptide (MIX) mixed with 4 peptides were combined as the coating antigen (1.0 μg / ml / peptide), and 27 porcine foot-and-mouth disease type O virus-infected sera were tested according to the conventional indirect ELISA method. detection. The results show( figure 1 ), the positive detection rates of Sequence 1, Sequence 2, Sequence 3, Sequence 4 and mixed peptides to 27 infection sera were 81.5%, 88.9%, 92.3%, 85.2% and 100%, respectively. The results showed that the combined B cell expression The mixed peptides at the same position can detect all the infected serum, and the positive detection rate is significantly higher than t...

Embodiment 3

[0099] Embodiment 3, the detection method of porcine foot-and-mouth disease virus nonstructural protein antibody ELISA kit

[0100] 1.1 Sample preparation Take animal whole blood. After the blood coagulates, centrifuge at 3500r / min for 10 minutes to collect the supernatant. You can also collect blood. After coagulation, the serum will naturally precipitate. The serum should be clear and free of hemolysis.

[0101] 1.2 Washing solution preparation Before use, return the 20-fold concentrated washing solution to room temperature. If there is precipitation, dissolve it in a 37°C water bath, and then dilute it 20 times with deionized water for later use.

[0102] 1.3 Sample dilution Dilute the serum to be tested in the serum dilution plate at a ratio of 1:20. The negative and positive control serum has been diluted and can be used directly.

[0103] 1.4 Operation steps

[0104] 1.4.1 Adding samples Take the antigen-coated plate, add the diluted serum to be tested, negative control...

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Abstract

The invention discloses a nonstructural protein antibody ELISA (enzyme-linked immunosorbent assay) kit for a porcine foot-and-mouth disease virus. The kit comprises a foot-and-mouth disease virus nonstructural protein epitope polypeptide coated enzyme-linked reaction plate and an enzyme labeled antibody; foot-and-mouth disease virus nonstructural protein epitope polypeptides are a polypeptide shown as sequence 1, a polypeptide shown as sequence 2, a polypeptide shown as sequence 3 and a polypeptide shown as sequence 4 in a sequence table. According to the kit, a chemically synthesized nonstructural protein antigen peptide is used for coating the reaction plate, antigen dosage is small, sensitivity and specificity are high, and the presence of foot-and-mouth disease virus infection can be effectively detected. The kit provided by the invention has good specificity, sensitivity and high efficiency, and has good market prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, the invention relates to an ELISA kit for nonstructural protein antibody of foot-and-mouth disease virus, in particular to an ELISA kit for nonstructural protein antibody of porcine foot-and-mouth disease virus. Background technique [0002] Foot-and-mouth disease (FMD) is a severe infectious disease caused by foot-and-mouth disease virus (FMDV) infection in cloven-hoofed animals. The fatality rate of foot and mouth disease is very low, but the infection rate is high. The main symptoms of the disease are: blisters in the oral mucosa, tongue, lips, nose mirror, hoof forks, nipples and other parts, ulceration to form rotten spots, sick animals lying on the ground with hoof pain, and severe hoof shell edge cracking or shelling. The symptoms of different animals are slightly different. Pregnant cows may abort, which leads to reduced fertility; pigs have broken hooves as the main sympt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/09G01N33/68G01N33/569G01N33/545
CPCC07K14/005C12N2770/32122G01N33/545G01N33/56983G01N33/6854G01N2469/20
Inventor 张蕾董春娜肖进李静巴利民张欣王楠宋芳栗利芳张艳宾王振豹齐鹏赵洪涛张君
Owner CHINA ANIMAL HUSBANDRY IND
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