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Pig cytomegalovirus antibody indirect blocking enzyme-linked immuno sorbent assay (ELISA) detection kit and detection method thereof

A technology of cytomegalovirus and detection kit, which is applied in the field of porcine cytomegalovirus antibody indirect blocking ELISA detection kit, which can solve the problems of difficulty in large-scale application and promotion, impossibility of large-scale application, high detection cost, etc., and achieve high sensitivity , simple operation, broad market prospects

Active Publication Date: 2012-08-01
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Assaf and Tjima respectively used the whole virus as the coated antigen to establish the immunofluorescence detection technology of PCMV antibody and serological detection methods such as enzyme-linked immunosorbent assay. However, the established method has certain limitations and it is difficult to apply it on a large scale
At present, the PCMV PCR diagnostic method established at home and abroad cannot be applied on a large scale in production due to the high detection cost.

Method used

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  • Pig cytomegalovirus antibody indirect blocking enzyme-linked immuno sorbent assay (ELISA) detection kit and detection method thereof
  • Pig cytomegalovirus antibody indirect blocking enzyme-linked immuno sorbent assay (ELISA) detection kit and detection method thereof
  • Pig cytomegalovirus antibody indirect blocking enzyme-linked immuno sorbent assay (ELISA) detection kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1. Solution preparation

[0047] (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.

[0048] (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.

[0049] (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 : 10000 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.

[0050] (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

[0084] 1. Kit detection operating procedures

[0085] (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.

[0086] (2) Set blocking antibody control wells on the antigen-coated plate, add diluted blocking antibody, 100 μl per well.

[0087] (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.

[0088] (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.

[0089] (5) Add the diluted blocking antibody to the test sample well, 100 μl per well, package it in a sealed bag and incubate at 37°C for 1 hour (repeat step 4).

[0090] (5) Add enzyme conjugate working solution (preparation method in Example 1), 100 μl per well, package in ...

Embodiment 3

[0098] 1. Construction of porcine cytomegalovirus envelope glycoprotein B (gB) epitope region gene expression vector:

[0099] (1) PCMV gB gene antigen epitope analysis: PCMV gB gene (GenBank: FJ595497.1) protein signal peptide, hydrophobicity, transmembrane region, secondary structure analysis by DNAstar and online bioinformatics software, gB protein antigen For epitope prediction, select a 270bp (nucleotide sequence, see SEQ ID NO: 1) dominant antigen epitope region encoding 90 amino acids as the target gene for codon optimization, and synthesize it in Invitrogen.

[0100] (2) TA cloning of the main epitope region of the gB gene: Synthetic primers were designed according to the main epitope region of the gB gene, and the dominant epitope region of the gB gene was amplified by PCR using the synthetic dominant epitope region gene fragment as a template.

[0101] Primers were designed as follows:

[0102] Upstream primer: 5'-GAATTCATGAGTTCTTCGGGAACTG-3' (see SEQ ID NO: 2)

[...

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Abstract

The invention discloses a pig cytomegalovirus antibody indirect blocking enzyme-linked immuno sorbent assay (ELISA) detection kit, wherein an antibody detection board, enzyme combination working solution, a blocking antibody, sample diluent, cleaning solution, developing solution A, developing solution B and stop solution are arranged in the kit. A detection board of the kit is a detachable 96-hole elisa plate wrapped by pig cytomegalovirus g B dominant antigen epitope area protein recombinant antigens, enzyme combination working solution is goat anti-rabbit antibody marked by horse radish peroxidase, and the blocking antibody is a rabbit anti-pig cytomegalovirus g B dominant antigen epitope area protein polyclonal antibody. The pig cytomegalovirus antibody indirect blocking ELISA detection kit has the advantages of being strong in specificity, high in sensitivity, easy to operate and suitable for clinical large-scale popularization and application and has wide market prospect.

Description

technical field [0001] The invention relates to a kit for detecting animal infectious diseases and a detection method thereof, in particular to a kit for detecting porcine cytomegalovirus antibody indirect blocking ELISA, which belongs to the field of veterinary biological products. Background technique [0002] Porcine cytomegalovirus (PCMV) is a virus that causes porcine inclusion body rhinitis or porcine cytomegalic inclusion disease. Porcine cytomegalovirus, also known as porcine herpesvirus type 2, belongs to the family Herpesviridae, subfamily Betaherpesviridae, and the genus Cytomegalovirus. The diameter of the virus particle is 150-200nm, with an electron-dense core, and its periphery is composed of capsid protein The icosahedral nucleocapsid, the genome is double-stranded DNA. It is difficult to culture PCMV. Only pig lung macrophages aged 3 to 5 weeks are highly sensitive. Giant cells appear 3 to 14 days after inoculation, forming nuclear inclusion bodies and occa...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/531
Inventor 徐志文朱玲刘骁郭万柱
Owner SICHUAN AGRI UNIV
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