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Toxoplasma gondii IgG antibody immunoblotting kit and preparation method thereof

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

Inactive Publication Date: 2011-12-21
厦门市湖里区妇幼保健院
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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 IgG antibody immunoblotting kit and preparation method thereof
  • Toxoplasma gondii IgG antibody immunoblotting kit and preparation method thereof
  • Toxoplasma gondii IgG antibody immunoblotting kit and preparation method thereof

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preparation example Construction

[0033] The preparation method of described toxoplasma gondii IgG 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 IgG detection line, human IgG antibody was coated on the control line, and dried.

[0038] 3) Preparation of anti-human IgG specific fragment γ chain monoclonal antibody

[0039] Balb / c mice were immunized with human IgG specific fragment γ chain as antigen, and hybridoma cell lines stably secreting anti-human IgG monoclonal antibody were screened by hybri...

Embodiment 1

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

[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. Add 1.5ml of the diluted sample to ...

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

Toxoplasma gondii IgG antibody immunoblotting kit and preparation method thereof relate to a kit. The kit is provided with a carrier plate, a nitrocellulose membrane, a toxoplasma IgG antibody detection line and a control line; the toxoplasma IgG antibody detection line and the control line are sequentially arranged on the nitrocellulose membrane; Toxoplasma recombinant antigen, coated with human IgG antibody at the control line; horseradish peroxidase-labeled anti-human γ-chain antibody. Preparation of Toxoplasma recombinant antigen; spotting of nitrocellulose membrane; preparation of anti-human IgG specific fragment γ chain monoclonal antibody; anti-human IgG specific fragment γ chain monoclonal antibody labeled with horseradish peroxidase; preparation of western blot Reagent test kit. When testing, the required sample volume is extremely small, no special equipment is required, and the results can be directly interpreted with the naked eye. The detection is simple and fast, with 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 detection of toxoplasma gondii IgG antibody 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 nor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/535
Inventor 李淑莲林志锋林惠玲张惠姗王容美张长弓
Owner 厦门市湖里区妇幼保健院
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