A Japanese blood fluke ovum antibody magnetic particle EILSA detecting method

A technology of magnetic particles and schistosomiasis, which is applied in the direction of measuring devices, analytical materials, color/spectral characteristics measurement, etc., can solve the problems of increasing operational complexity, inability to detect antibodies, and inability to use universally, so as to save detection time, good repeatability, The effect of easy operation

Inactive Publication Date: 2009-09-16
北京倍爱康生物技术有限公司
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

However, this method also has some disadvantages; ①This method can only detect a single subclass of antibodies
For example, kits for detecting IgG antibodies cannot detect antibodies such as IgM and IgA
Because only IgM antibodies may exist in the early infection stage of schistosomiasis, there is a certain phenomenon of missed diagnosis; Can target humans or animals, not universal
③ Serum samples to be tested often need to be diluted in advance, which increases the complexity of the operation

Method used

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  • A Japanese blood fluke ovum antibody magnetic particle EILSA detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Sj-SEA is coupled with surface amino (COOH-) magnetic particles to prepare a magnetic separation reagent

[0031] Take 100 mg of magnetic particles containing carboxyl (COOH-) active groups on the surface and wash them three times with 0.1 M MES (2-[N-morpholino] ethanesulfonic acid), 10 ml of pH 4.5-5 solution. The magnetic particles were resuspended in 1ml of this solution, and 2mg of Sj-SEA was added and mixed evenly. Add 100μl 10mg / ml EDC solution, mix well and react at room temperature for 2h. After washing the magnetic beads three times with 10 ml of 0.01 M PBS pH7.4 solution containing 1% bovine serum albumin (BSA), the solution was used to prepare a 2.5 mg / ml magnetic separation reagent working solution.

Embodiment 2

[0032] Example 2: Sj-SEA coupled alkaline phosphatase (ALP), preparation of enzyme-labeled reagents

[0033] Take 5 mg of SJ-SEA antigen, concentrate it to 5 mg / ml, add 10 μl of 13.76 mg / mL activator 2-Iminothiolane HCl (2IT) solution, place at room temperature for 20 minutes, add glycine to terminate the activation reaction, and place at room temperature for 5 minutes. Use Sephadex G25 column to desalt and collect protein elution peaks.

[0034] Take 5 mg of ALP solution, add 50 μl of 6.69 mg / mL Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) solution, leave it at room temperature for 30 minutes, add glycine to terminate the activation reaction, and leave it at room temperature for 5 minutes. Use Sephadex G25 column to desalt and collect protein elution peaks.

[0035] The activated Sj-SEA was mixed with the activated ALP, left at room temperature for 10 hours, then separated and purified using a Supperdex200 gel chromatography column, and the second and thi...

Embodiment 3

[0038] Example 3: Schistosoma japonicum egg antibody magnetic particle separation enzyme immunoassay

[0039] Materials and Instruments

[0040] (1) Magnetic separation reagent: see the preparation of the above-mentioned magnetic separation reagent;

[0041] (2) Enzyme-labeled reagent: see the preparation of the above-mentioned enzyme-labeled reagent;

[0042] (3) Washing solution: 500mL (produced by Beijing Beiaikang Biotechnology Co., Ltd.);

[0043] (4) Substrate solution (PMP solution): 300mL (produced by Beijing Bei Aikang Biotechnology Co., Ltd.);

[0044] (5) Stop solution: 600mL (produced by Beijing Bei Aikang Biotechnology Co., Ltd.);

[0045] (6) National reference product of Schistosoma japonicum: 15 positive reference products (P1-P15), 15 negative reference products (N1-N15), 1 sensitivity reference product (P16), 1 precision reference product (P17), Purchased from China Institute for the Control of Pharmaceutical and Biological Products.

[0046] (7) Serum s...

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Abstract

The invention provides a Japanese blood fluke ovum antibody magnetic particle EILSA detecting method, which belongs to verminosis immunodiagnosis technology. The invention adopts the immunodetection theory of antigen sandwich assay method, which uses the surface of magnetic microsphere which is connected with the Japanese blood fluke ovum antigen and has the diameter of 0.1 to 5.0 micrometer as the solid phase reagent; the Japanese blood fluke dissoluble ovum antigen labeled by biological enzyme is used as enzyme reagent. The magnetic separation reagent and the enzyme reagent are respectively combined with the Japanese blood fluke ovum antibody in the sample to form the solid phase- antigen- antibody- enzyme antigen sandwiched immunocomposite. The invention is characterized in that first, magnetic particles are used for replacing traditional enzyme plate ELISA as the solid phase carriers so that the immune reaction is carried on in the condition of liquid phase and the reaction if more abundant and quick; compared with the traditional ELISA method, the method is characterized by the quick detecting speed, the high specificity and the good repetitiveness; second, due to the adoption of bi-antigen sandwich method theory, one kit can be used for the detection of both people and animal Japanese blood flukes.

Description

technical field [0001] The invention belongs to the technical field of immunological detection and analysis, relates to an immunological detection and analysis technology of zoonotic infectious diseases, and in particular provides a magnetic particle separation enzyme-linked immunological detection method for rapid detection of Schistosoma japonicum egg antibodies, which is suitable for human and animal Schistosoma japonicum Qualitative detection of egg antibodies. Background technique [0002] At present, schistosomiasis is still prevalent in the middle and lower reaches of the Yangtze River in my country. There are 810,000 schistosomiasis patients and 60,000 schistosomiasis cattle. 60 million people are exposed to the risk of infection, and acute infections continue to occur. Although my country has achieved great success in controlling schistosomiasis by using simultaneous chemotherapy for humans and animals, schistosomiasis is still an important public health problem in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N21/31
Inventor 陈立杰黄进仝文斌姜昌富姚蓝雷焱朱世伟
Owner 北京倍爱康生物技术有限公司
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