ELISA (Enzyme-linked Immuno Sorbent Assay) antigen detection kit of animal echinococcosis granulosa

A technology for echinococcosis and antigen detection, which is applied in the field of genetic engineering technology and diagnostic reagents, can solve the problems of complex components, instability, and limited sources, and achieve strong specificity, good kit stability, and high sensitivity Effect

Inactive Publication Date: 2018-07-24
QINGHAI ACAD OF ANIMAL SCI & VETERINARY MEDICINE
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0003] Currently, hydatid cyst fluid is commonly used in the serological diagnosis of echinococcosis. Due to its complex composition, it has serious cross-reacti

Method used

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Examples

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Embodiment 1

[0016] Example 1: Preparation of an animal echinococcosis ELISA antigen detection kit

[0017] An ELISA antigen detection kit for animal echinococcosis, comprising a monoclonal antibody against EgAgB8 / 2 protein pre-coated microtiter plate, sample diluent, positive control solution, negative control solution, enzyme conjugate, and washing solution , substrate chromogenic solution and stop solution.

[0018] The preparation method of the animal echinococcosis ELISA antigen detection kit is as follows:

[0019] 1. Preparation of monoclonal antibody against EgAgB8 / 2 protein: first use RT-PCR to amplify EgAgB8 / 2 gene cDNA, clone EgAgB8 / 2 gene, construct prokaryotic expression vector pET-EgAgB8 / 2, and in Escherichia coli BL21 (DE3 ), use IPTG to induce the expression of the prokaryotic expression vector, collect the cells by centrifugation, dissolve them in bacterial lysate, and break them up by ultrasonication, collect the supernatant by centrifugation, purify them with Ni-NTA gel...

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Abstract

The invention discloses an ELISA (Enzyme-linked Immuno Sorbent Assay) antigen detection kit of animal echinococcosis granulosa. The detection kit is prepared from an anti-EgAgB8/2 protein monoclonal antibody pre-coated ELISA plate, a sample diluting solution, a positive control solution, a negative control solution, an enzyme conjugate, a washing solution, a substrate color developing solution anda stopping solution. An anti-EgAgB8/2 protein monoclonal antibody is prepared through the following method: firstly, amplifying EgAgB8/2 gene cDNA (complementary Deoxyribonucleic Acid) by utilizing RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and cloning an EgAgB8/2 gene; constructing a prokaryotic expression vector pET/EgAgB8/2 and expressing in escherichia coli BL21(DE3); inducing the expression of the prokaryotic expression vector by utilizing IPTG (isopropyl-beta-d-thiogalactoside); centrifuging and collecting thalli; after dissolving with a bacterium lysis solution and carrying out ultrasonic crushing; centrifuging and collecting supernatant; purifying by utilizing Ni-NTA glue; immunizing a BALB/c mouse by utilizing the purified EgAgB8/2 protein, so as to obtain a hybridoma cell line capable of stably secreting the anti-EgAgB8/2 protein monoclonal antibody; selecting the excellent monoclonal antibody to produce an antibody and purifying. The kit disclosed by the invention can be used for qualitative detection of an animal echinococcosis granulosa antigen and has the advantages of stable antibody source, good specificity, high sensitivity and good repeatability.

Description

technical field [0001] The invention belongs to the field of genetic engineering technology and diagnostic reagents, in particular to an ELISA antigen detection kit for animal Echinococcus granulosus. Background technique [0002] Echinococcosis, also known as echinococcosis, is a multiple zoonotic parasitic disease with a worldwide distribution. It is the most serious parasitic disease that endangers the development of western my country and people's health. The adults of Echinococcus parasitize in the upper small intestines of carnivorous animals such as dogs, foxes, wolves, etc., and are fixed in the crypts at the base of the intestinal villi by means of apical hooks and suckers. Intermediate hosts are artiodactyls such as sheep, cattle, camels, deer, mice, certain rodents, etc., and primates (including humans). Animals such as dogs, foxes and wolves are the main transmitters of echinococcosis. There are 5 kinds of Echinococcus that are prevalent in the world, and 3 of ...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 朵红付永何轶群沈秀英郭志宏彭毛李伟
Owner QINGHAI ACAD OF ANIMAL SCI & VETERINARY MEDICINE
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