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Toxoplasma IgM antibody immunoblotting kit and preparation method thereof

A technology of immunoblotting and toxoplasma gondii, which is applied in the field of kits, can solve the problems of high price and achieve the effect of low cost, wide application, simple and fast detection

Inactive Publication Date: 2012-05-02
厦门市仙岳医院
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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 IgM antibody immunoblotting kit and preparation method thereof
  • Toxoplasma IgM antibody immunoblotting kit and preparation method thereof
  • Toxoplasma IgM antibody immunoblotting kit and preparation method thereof

Examples

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preparation example Construction

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

[0038] 3) Preparation of anti-human IgM specific fragment μ chain monoclonal antibody

[0039] Balb / c mice were immunized with human IgM-specific fragment μ chain as antigen, and hybridoma cell lines stably secreting anti-human IgM monoclonal antibody were sc...

Embodiment 1

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

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 which relates to a kit concretely relates to a toxoplasma IgM antibody immunoblotting kit and a preparation method thereof. The kit is characterized in that: the kit is provided with a vector plate, a nitrocellulose film, a toxoplasma IgM antibody detection line and a contrast line; the toxoplasma IgM antibody detection line and the contrast line are sequentially arranged on the nitrocellulose film; the toxoplasma IgM antibody detection line is coated with a toxoplasma recombinant antigen, and the contrast line is coated with a human IgM antibody; and an anti-human mum-chain antibody is labeled with horseradish peroxidase. The preparation method comprises the following steps: 1, preparing the toxoplasma antigen; 2, carrying out nitrocellulose film spotting; 3, preparing an anti-human IgM specific fragment mum-chain monoclonal antibody; 4, labeling the anti-human IgM specific fragment mum-chain monoclonal antibody with the horseradish peroxidase; and 5, preparing the immunoblotting kit. The kit of the invention, which allows the needed sample amount to be extremely small and no special instrument to be needed during detection and allows the result to be directly interpreted by naked eyes, has the advantages of simple and rapid 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 detection of toxoplasma gondii IgM 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 norm...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/535
Inventor 郭永炼章家新吴统健陈桂美曹凤玲张长弓
Owner 厦门市仙岳医院
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