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Toxoplasma gondii total antibody immune blotting kit and preparation method thereof

A technology for immunoblotting and toxoplasma gondii, applied in the field of kits, can solve problems such as high price and achieve the effects of low cost, high sensitivity and wide application

Inactive Publication Date: 2012-01-18
ZHONGSHAN HOSPITAL XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The commercially available Western-blot reagents are all imported reagents, which are expensive

Method used

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  • Toxoplasma gondii total antibody immune blotting kit and preparation method thereof
  • Toxoplasma gondii total antibody immune blotting kit and preparation method thereof
  • Toxoplasma gondii total antibody immune blotting kit and preparation method thereof

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preparation example Construction

[0033] The preparation method of described toxoplasma gondii total antibody immunoblotting kit comprises the following steps:

[0034] 1) Preparation of SAG1 (P30), SAG2 (P22), ROP2 and GRA7 Toxoplasma gondii recombinant antigens

[0035]Using gene cloning technology, PCR amplified the DNA encoding the T. gondii antigen, and inserted it into Escherichia coli to express it, and obtained the T. gondii recombinant antigens SAG1 (P30), SAG2 (P22), ROP2, and GRA7.

[0036] 2) Spotting on nitrocellulose membrane

[0037] SAG1 (P30), SAG2 (P22), ROP2 and GRA7 Toxoplasma recombinant antigens were coated on the nitrocellulose membrane total antibody detection line, human Ig antibody was coated on the control line, and dried.

[0038] 3) Preparation of anti-human Ig monoclonal antibody

[0039] Immunize Balb / c mice with human Ig as antigen, and obtain a hybridoma cell line that stably secretes anti-human Ig monoclonal antibody through hybridoma technology;

[0040] 4) Horseradish per...

Embodiment 1

[0065] Paste the nitrocellulose membrane on the surface of the carrier plate, and the total antibody detection line and control line of Toxoplasma gondii are arranged on the nitrocellulose membrane in turn; the recombinant antigens SAG1 (P30) and SAG2 (P22) of Toxoplasma gondii are coated at the total antibody detection line of Toxoplasma gondii ), ROP2 and GRA7, coated with human Ig antibody at the control line, cut into strips with a strip cutter, and assembled with enzyme-labeled anti-human Ig monoclonal antibody and its substrate to form a Toxoplasma gondii total antibody immunoblotting kit .

[0066] 1) The sample was diluted 1:50 with 0.01mol / LPBS buffer.

[0067] 2) Blocking: 5% skimmed milk powder (prepared with 0.01 mol / L PBST buffer) was used as the blocking solution, and incubated at room temperature (18-25° C.) on a rocking table for 30 min.

[0068] 3) Serum incubation: take out the required membrane strips, put them into the incubation tank, and number them. Ad...

Embodiment 2

[0076] Similar to Example 1, the difference is that the nitrocellulose membrane detection line only consists of SAG2 (P22), ROP2 and GRA7 Toxoplasma recombinant antigens, and does not contain SAG1 (P30) Toxoplasma recombinant antigens. Result judgment is identical with embodiment 1.

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Abstract

The invention discloses a toxoplasma gondii total antibody immune blotting kit and a preparation method thereof, relating to a kit. The kit comprises a carrier plate, a nitrocellulose membrane, a toxoplasma gondii total antibody detection line and a contrast line, wherein the toxoplasma gondii total antibody detection line and the contrast line are sequentially arranged on the nitrocellulose membrane; a toxoplasma gondii recombinant antigen is coated on the toxoplasma gondii total antibody detection line, a human Ig antibody is coated on the contrast line; and horse radish peroxidase labels the human Ig antibody. The toxoplasma gondii recombinant antigen is prepared, the nitrocellulose membrane is sampled, an anti-human Ig monoclonal antibody is prepared, the horse radish peroxidase of the anti-human Ig monoclonal antibody is labeled, and the immune blotting kit is prepared. During the detection, the required quantity of specimens is minimum, no special apparatuses are needed, naked eyes directly judge and read results; and the invention has the advantages of convenience and rapidness for detection, strong specificity, high sensitivity, accuracy and reliability, low cost and wide application.

Description

technical field [0001] The invention relates to a kit, in particular to an immunoblotting kit for detecting total antibodies to Toxoplasma gondii and a preparation method thereof. Background technique [0002] Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasmagondii that seriously endangers human health. The pathogen Toxoplasma can parasitize in the nucleated cells of humans and various animals. and animals are generally susceptible. The disease is widely distributed all over the world. According to statistics, about 1 billion people in the world are infected by Toxoplasma gondii ([1] Xi Linlin, Li Wei. Research progress on pathogenic mechanism, virulence and genotype of Toxoplasma gondii[J]. Chinese Journal of Pathogenic Biology , 2009, 4(11): 859-861.). Toxoplasmosis is widely distributed in my country, and there are reports of toxoplasmosis infection in all provinces, municipalities and autonomous regions of the country. The average infection rate of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531G01N33/535
Inventor 杨天赐张忠英刘莉莉林丽蓉张长弓
Owner ZHONGSHAN HOSPITAL XIAMEN UNIV