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Kit for detecting capripoxvirus serum antibody based on synthetic peptide

A technology of sheep pox virus and serum antibody, which is applied in the direction of biological testing, material inspection products, etc., can solve the problems of low sensitivity and specificity, and achieve the effect of easy operation and reliable technical support

Active Publication Date: 2013-03-20
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a kit for detecting goatpox virus serum antibody based on goatpox virus synthetic peptide antigen coated ELISA plate, which can detect goatpox virus serum antibody quickly, specifically and accurately , to provide a reference for the clinical diagnosis and prevention of sheeppox virus, to overcome the shortcomings of existing detection methods such as low specificity and low sensitivity

Method used

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  • Kit for detecting capripoxvirus serum antibody based on synthetic peptide
  • Kit for detecting capripoxvirus serum antibody based on synthetic peptide
  • Kit for detecting capripoxvirus serum antibody based on synthetic peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] 1. Use DNAstar software to design two short peptides for the main immune protein P32 of sheep pox virus, and use conventional synthetic peptide methods to synthesize peptides. The sequence of the peptides is:

[0024] A: PELKSDNDIFYKKVDTVKDFKNSDVNFFLKDKKDDI

[0025] B: YKLEKELKLNRQVLNDSSKYILHNTKYLSKKRANEMKN

[0026] 2. Dilute the two synthesized short peptides with 0.5M PBS buffer to 6 μg / ml, then mix A and B in equal volumes, add 50 μL of the mixture of A and B to each well of the ELISA plate, overnight at 4°C, and A is 0.15 μg / well, and B is 0.15 μg / well.

[0027] 3. Discard the liquid in the wells, add 220 μL 1×PBST (phosphate Tween) washing solution to each well, wash 3 times, and spin dry for the last time. Afterwards, 50 μL of blocking solution (1% gelatin) was added to each well, incubated at 37°C for 45 minutes, washed 3 times with PBST, and dried by spinning for the last time.

[0028]

[0029] 4. Dilute the negative and positive serum with PBS to 5 dilutio...

Embodiment 2

[0045] 1-6 steps are the same as in Example 1.

[0046] 7. Preparation of Sample 2 (Goat Pox Positive Serum, Goat Pox Negative Serum)

[0047] For the 5 serum samples from 5 antibody-positive sheep from different sources and 2 serum samples from 2 antibody-negative sheep from different sources, take 5 μL for each, and then add 95 μL PBS respectively, and the total volume of each diluted serum is 100 μL (1 :20 dilution), and stored at -20°C for later use.

[0048] 8. Perform an intra-batch repeatability test on the detection reagents prepared in the same batch, and do 5 wells for each serum sample to repeat the intra-batch repeat test.

[0049] 8.1 Add 50 μL each of the positive and negative serum samples prepared in step 7 to the ELISA plate, repeat for 5 wells, incubate at 37°C for 45 minutes, wash the plate 3 times with PBST, and spin dry the last time (repeat within the batch).

[0050] 8.2 Add 50 μL 1:400 diluted rabbit anti-goat enzyme-labeled secondary antibody to each...

Embodiment 3

[0057] 1-6 steps are the same as in Example 1.

[0058] 7. Preparation of sample 3 (sheep pox vaccine immune serum).

[0059] Purchasing commercially available sheep pox vaccine, immunizing 5 goats according to the vaccine instruction manual, collecting the whole blood of the immunized goat two months after immunization, and separating the serum; at the same time collecting the whole blood of a goat that has not received any vaccine immunization, and separating the serum, all The prepared samples were stored at -20°C for later use.

[0060] 8. The prepared detection reagent is used to detect the immune sheep serum antibody.

[0061] Take 1 part of 5μL sheeppox immune serum and 1 part of 5μL sheeppox non-immune serum from each isolated goat, add 95μL PBS respectively, each with a total volume of 100μL (1:20 dilution), detect and analyze according to the prepared ELISA reagent.

[0062] 8.1 Add 50 μL each of the prepared 5 sheeppox immunized and 1 non-immune serum samples to t...

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Abstract

The invention discloses a kit for detecting a capripoxvirus serum antibody based on a synthetic peptide. A preparation method of the kit comprises the following steps of 1, designing a short-peptide aiming at a capripoxvirus main immunogene P32, 2, synthesizing a peptide fragment by a conventional peptide synthesis technology, 3, preparing the kit for detecting a capripoxvirus serum antibody based on a synthetic peptide, and 4, optimizing conditions and finishing the preparation of the kit for detecting a capripoxvirus serum antibody. A coating antigen adopted by the kit is an external synthetic immunoactive peptide fragment, reduces the danger of totvirus use and prevents virus diffusion and escape. The synthetic peptide adopted by the kit has high purity, has immunological characteristics similar to capripoxvirus particles, and can replace capripoxvirus particles and be used as an antigen for detection and thus the kit realizes sensitive, specific, safe and reliable detection of a capripoxvirus serum antibody.

Description

technical field [0001] The invention relates to a kit for detecting serum antibody of sheeppox virus, in particular to a kit for detecting serum antibody of sheeppox virus prepared on the basis of coating an ELISA plate with a synthetic peptide antigen. Background technique [0002] Capripox (Capripox) is caused by Capripoxvirus (CPV), which can infect goats, sheep and cattle and cause goatpox (variolacaprin, Goatpox), sheep pox (variolacaprin, Sheeppox) and cattle pimple skin disease (Lumpy shindisease) An acute, febrile, contact infectious disease. The main manifestations are fever, papules and herpes on the skin or mucous membranes of hairless or less hairy parts. Goat pox is the most serious of all animal pox diseases, which has a high fatality rate and can cause huge economic losses, seriously affecting the development of international trade and sheep farming. Among them, goatpox is widely prevalent in Africa, Turkey, India, and some Asian countries, and it is also sp...

Claims

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Application Information

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IPC IPC(8): G01N33/68
Inventor 刘湘涛田宏吴锦艳陈妍尚佑军尹双辉王光祥靳野张克山杨顺利刘永杰
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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