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A sheep echinococcosis antibody ELISA detection kit and its preparation method and application

A detection kit and technology for hydatid, applied in the field of sheep hydatid antibody ELISA detection kit and preparation thereof, can solve the problem that the sensitivity cannot meet the clinical immune effect evaluation requirements, the detection kit is not available, and the scale is unfavorable. To avoid complex purification steps and interference of impurity proteins, improve binding efficiency, and reduce inspection costs

Active Publication Date: 2022-02-22
CHONGQING AULEON BIOLOGICALS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the preparation of recombinant antigens in this method requires fermentation of recombinant engineering bacteria, bacterial disruption, inclusion body washing, inclusion body denaturation, dialysis, filtration, affinity chromatography, thrombin hydrolysis, polyacrylamide gel electrophoresis identification, antigen concentration determination, etc. A series of cumbersome operation steps and complex production process; in addition, this method requires the use of various equipment such as bacterial culture equipment, ultrasonic breaker, dialysis equipment, protein purifier, protein electrophoresis instrument, etc., which is not conducive to large-scale production; the antigen produced by this method Contains a small amount of Escherichia coli impurity protein, and when it is coated with an enzyme-plated plate for detection, there are often false positives, resulting in a certain amount of misjudgment in the result judgment
At home and abroad, a large number of detection methods have been studied with cystic fluid antigens, lysed antigens, and recombinant antigens. Either the sensitivity cannot meet the clinical immune effect evaluation requirements, or the specificity is not good (cross-reaction with antibodies to other sheep parasitic diseases). None of them are ideal, so far, there is still no commercialized relevant detection kit available at home and abroad

Method used

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  • A sheep echinococcosis antibody ELISA detection kit and its preparation method and application
  • A sheep echinococcosis antibody ELISA detection kit and its preparation method and application
  • A sheep echinococcosis antibody ELISA detection kit and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0045] In the present embodiment, ELISA detection kit for echinococcosis antibody includes: ELISA plate (2×96 wells), diluent (2 bottles, 30mL / bottle); washing solution (1 bottle, 30mL / bottle); blocking solution (1 bottle, 15mL / bottle); positive control serum (1 bottle, 50 μL / bottle); negative control serum (1 bottle, 50 μL / bottle); enzyme-labeled secondary antibody (2 bottles, 200 μL / bottle); substrate color development Solution: chromogenic solution A (1 bottle, 30mL / bottle), chromogenic solution B (1 bottle, 30mL / bottle); stop solution (1 bottle, 15mL / bottle); and serum dilution plate (2 pieces).

[0046] The source gene sequence of the antigenic epitope polypeptide is the complete sequence of Echinococcus granulosus EG95, the complete gene sequence of which is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6. Carry out genetic engineering prokaryotic expression to this sequence (results such as figure 1 shown) and Western Blot verification (result...

Embodiment 2

[0057] In this embodiment, the sheep echinococcosis antibody ELISA detection kit, in addition to using a polypeptide synthesizer to synthesize the amino acid sequence shown in SEQ ID NO: 2, after purification to obtain the EG95-PT-2 polypeptide antigen, is replaced by using a polypeptide synthesizer to synthesize The amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4, after purification, the EG95-PT-1, EG95-PT-3 or EG95-PT-4 polypeptide antigens are obtained in sequence, respectively prepared Except for the EG95-PT-1, EG95-PT-3 or EG95-PT-4 polypeptide antigen coated microtiter plate, the rest are the same as in Example 1.

Embodiment 3

[0059] The sheep Echinococcus antibody ELISA detection kit in Example 1 and Example 2 was used to detect 3 parts of goat positive sera (SP1, SP2, SP3), 3 parts of goat negative sera (SN1, SN2, SN3), and sheep positive sera Echinococcus ovis EG95 antibody in 2 (MP1, MP2) and 2 sheep negative sera (MN1, MN2).

[0060] The specific operation steps are:

[0061]1) Take EG95-PT-1, EG95-PT-2, EG95-PT-3 or EG95-PT-4 polypeptide antigen-coated microtiter plates respectively, and add 100 μL each of the diluted serum to be tested and the control to the In the wells of the antigen-coated microplate plate, make 1 well of the serum to be tested, and set 2 wells for the negative control and positive control, 100 μL in each well, gently shake the serum to be tested in the wells (do not overflow), and incubate at 37°C 60 minutes;

[0062] 2) Remove the solution in the wells of the plate, add 200 μL of diluted washing solution to each well, let it stand for 3 minutes, pour it out, then pat d...

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Abstract

The invention discloses an ELISA detection kit for sheep echinococcosis antibody and its preparation method and application, belonging to the technical field of biological detection. Sheep Echinococcus Antibody ELISA Kit includes EG95 Polypeptide Antigen Coated Microtiter Plate, Diluent, Washing Solution, Blocking Solution, Positive Control Solution, Negative Control Solution, Enzyme Conjugate, Substrate Chromogenic Solution, Stop Solution and Serum Dilution plate. The sheep echinococcosis antibody ELISA detection kit of the present invention adopts the Echinococcus granulosus EG95 protein B cell epitope-specific polypeptide antigen as the coating antigen, and has a simple and convenient production method and high antigen purity. It avoids the complex purification steps and impurity protein interference produced by E. coli fermentation, and is suitable for the detection of a large number of serum samples. It has the advantages of economy and convenience in animal disease detection and epidemiological investigation, and the inspection cost is low, which is conducive to popularization and application. It has broad application prospects in the detection of sheep hydatid (hydatid) EG95 protein antibody.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a sheep echinococcosis antibody ELISA detection kit and a preparation method and application thereof. Background technique [0002] Echinococcosis, also known as echinococcosis, is a serious zoonotic parasitic disease mainly caused by the middle-stage larva of Echinococcus granulosus (Echinococcus granulosus) infecting the intermediate host. Echinococcus granulosus parasitizes in the small intestines of dogs, foxes, wolves and other animals, and Echinococcus parasitizes in sheep, goats, cattle (yaks), pigs, camels and other animals as well as in the liver, lungs and other organs of humans. The disease seriously threatens human health and animal husbandry development. [0003] The EG95 protein is the natural antigen of Echinococcus granulosus Hexa, the most effective vaccine molecule, and the protective antigen component of the genetically engineered subu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/543G01N33/535
CPCG01N33/6893G01N33/543G01N33/535G01N2333/43543
Inventor 冉智光赵扬扬吕航赖茂林伏刚王盼举古静殷远滔高洋
Owner CHONGQING AULEON BIOLOGICALS
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