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Indirect blocking ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit of antibody of porcine torque teno virus type II

A detection kit, a technology for parvoviruses, which are applied in measurement devices, instruments, scientific instruments, etc., can solve problems such as harm and impact on pig production, and achieve broad market prospects, high sensitivity, and strong specificity.

Active Publication Date: 2012-07-18
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Porcine leukovirus is often co-infected with porcine circovirus and blue ear virus, which has brought serious impact and harm to pig production

Method used

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  • Indirect blocking ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit of antibody of porcine torque teno virus type II
  • Indirect blocking ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit of antibody of porcine torque teno virus type II
  • Indirect blocking ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit of antibody of porcine torque teno virus type II

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] 1. Solution preparation

[0039] (1) Preparation of 0.05mol / L carbonate coating buffer: Na 2 CO 3 1.59g, NaHCO 3 2.93g, add distilled water to 1000mL, adjust pH to 9.6.

[0040] (2) Preparation of blocking solution: BSA 30g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, NaCl 8.0g, KCl 0.2g add distilled water to 1000mL, set at -20°C.

[0041] (3) Preparation of enzyme conjugate working solution: commercialized horseradish peroxide-labeled goat anti-rabbit antibody (Aimeijie Technology Co., Ltd., USA), directly added to the enzyme-labeled secondary antibody diluent according to the test results for 1 : 12000 diluted solution. Enzyme-labeled secondary antibody diluent: KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, NaCl 29g, KCl 0.2g, PEG6000 40g, add ddH 2 O was adjusted to 1000ml, and 0.01% to 0.05% thimerosal was added to adjust the pH to 7.4.

[0042] (4) Preparation of sample diluent: KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9g, NaCl 29g, KCl 0.2g, PEG6000...

Embodiment 2

[0076] 1. Kit detection operating procedures

[0077] (1) Preparation procedure before the test: add ddH to 10× washing buffer (preparation method in Example 1) 2 Dilute O into 1×washing working solution; dilute the blocking antibody with sample diluent at 1:1600; dilute the sample to be tested at 1:5.

[0078] (2) Set blocking antibody control wells on the antigen-coated plate, add diluted blocking antibody, 100 μl per well.

[0079] (3) Add the sample to be tested into the test sample well, 100 μl per well, package it in a sealed bag and incubate at 37°C for 1.5h.

[0080] (4) Pour off the liquid in each well and wash with 1×washing working solution, 300 μl per well, wash 4 times, 2 min each time.

[0081] (5) Add the diluted blocking antibody to the test sample wells, 100 μl per well, package them in sealed bags and incubate at 37°C for 1 hour, (repeat step 4).

[0082] (5) Add enzyme conjugate working solution (preparation method in Example 1), 100 μl per well, package ...

Embodiment 3

[0090] 1. The construction of the gene expression vector for the dominant antigenic epitope region of porcine leocirculation virus type II ORF1 gene:

[0091] (1) Analysis of the dominant antigenic epitopes of the porcine leocirculation virus type II ORF1 gene: DNAstar and online bioinformatics software were used to analyze the protein signal peptide, hydrophobicity, and transmembrane Region and secondary structure analysis, prediction of ORF1 gene epitope, selection of a length of 696bp (nucleotide sequence see SEQ ID NO: 1) encoding 232 amino acid dominant epitope region as the target gene for codon optimization, And synthesized in Treasure Biological Company.

[0092] (2) TA cloning of the main epitope region of the ORF1 gene: Synthetic primers were designed according to the main epitope region of the ORF1 gene, and the gene fragment of the dominant epitope region synthesized was used as a template to amplify the dominant epitope region of the ORF1 gene by PCR.

[0093] Pr...

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Abstract

The invention discloses an indirect blocking ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit of an antibody of porcine torque teno virus type II, which comprises an antibody detecting board, an enzyme conjugate working solution, a sample diluent, a coloration solution A, a coloration solution B, a termination solution, a blocking antibody and a washing buffer solution, wherein the blocking antibody is a polyclonal antibody which is obtained in rabbits after purified TTV2 (Torque Teno Virus type II) -ORF1 (Open Reading Frame1) gene antigen epitope area protein immunizes the rabbits. The kit has the beneficial effects of strong specificity and simpleness in operation, is suitable for large-scale clinical popularization and application, and has a good market prospect.

Description

technical field [0001] The invention relates to a virus antibody detection method for pigs, in particular to an indirect ELISA detection method for porcine leukovirus type II (TTV2) antibody. Background technique [0002] Pig farming is an important part of my country's animal husbandry and breeding industry, and occupies a very important position in my country's agricultural economy. The development of international multilateral trade such as the import and export of animal husbandry products in my country will bring huge social, economic and human life and property losses to countries and regions. Porcine circovirus, porcine circovirus and PRRS virus are often co-infected, which has brought serious impact and harm to pig production. The prevention and control of porcine fine ring virus directly affects the development of my country's pig industry, and is the focus of my country's animal husbandry production. [0003] In December 1997, Japanese scholar Nishizawa et al. use...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543G01N33/531
Inventor 徐志文朱玲李凇刘骁郭万柱廖珊
Owner SICHUAN AGRI UNIV
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