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Protein A immobilized antibody-based micro-sphere immunoassay method
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An immobilized antibody and immunoassay technology, which is applied in the field of immunoassay, can solve the problems of inability to identify antigens, false positives, and inactivation of antibody molecules, and achieve the effects of improving detection results, increasing accuracy, and reducing non-specific adsorption
Active Publication Date: 2013-07-24
厦门圣科环保科技股份有限公司
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[0003] The main defect of the existing double-antibody sandwich method is that during the antibody coupling process, the antibody molecule is inactivated due to the lodging of the antibody, so that it cannot correctly recognize the antigen
The binding of protein A to the Fc end of the antibody is not selective, causing it to directly bind to the antibody labeled with the signal molecule FITC (fluorescein isothiocyanate), resulting in false positives
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Embodiment 1
[0024] 1) Treat the surface of the amino microbeads with 5% glutaraldehyde at 25° C. for 1 hour;
[0025] 2) Coupling with protein A by using the Schiff base reaction between aldehyde group and amino group, modifying protein A molecules on the surface of amino microbeads, the coupling buffer is PBS solution, the coupling temperature is 25 ° C, and the time is 3 h;
[0026] 3) Using sodium borohydride to reduce the carbon-nitrogen double bond formed between protein A and microbeads, the treatment condition is: put the amino microbeads into 6% sodium borohydride aqueous solution for 1 hour;
[0027] 4) Taking advantage of the characteristic of directional binding between protein A and the Fc end of the antibody, incubate the antibody and amino microbeads in phosphate buffer overnight at 4°C to obtain antibody-immobilized microbeads;
[0028] 5) Use DMP (dimethylpimelic acid imide-dihydrochloride) to fix the antibody-immobilized microbeads. The treatment method is to treat the mi...
Embodiment 2
[0036] 1) Take three groups of amino microbeads, and treat the surface of the amino microbeads with 5% glutaraldehyde at 25° C. for 1 hour;
[0037] 2) Coupling with protein A by using the Schiff base reaction between aldehyde group and amino group, modifying protein A molecules on the surface of amino microbeads, the coupling buffer is PBS solution, the coupling temperature is 25 ° C, and the time is 3 h;
[0038] 3) Using sodium borohydride to reduce the carbon-nitrogen double bond formed between protein A and microbeads, the treatment condition is: put the amino microbeads into 6% sodium borohydride aqueous solution for 1 hour;
[0039] 4) Taking advantage of the characteristic of directional binding between protein A and the Fc end of the antibody, incubate the antibody and amino microbeads in phosphate buffer overnight at 4°C to obtain antibody-immobilized microbeads;
[0040] 5) Use DMP (dimethylpimelic acid imide-dihydrochloride) to fix the antibody-immobilized microbea...
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Abstract
The invention provides a protein A immobilized antibody-based micro-sphere immunoassay method, relates to the immunoassay method, and provides the protein A immobilized antibody-based micro-sphere immunoassay method capable of realizing micro-sphere molecular aligned coupling on an amino micro-sphere, relating to the immunoassay method. The method comprises the following steps: treating the surface of the amino micro-sphere by a bridging reagent; coupling protein A onto the amino micro-sphere; incubating with AFP antibody 1 to obtain antibody immobilized micro-sphere; incubating the antibody immobilized micro-sphere with a human serum sample; incubating the antibody AFP combined antibody immobilized micro-sphere with AFP antibody 2; and incubating the antibody immobilized micro-sphere with secondary antibody which is labeled by FITC and can identify the AFP antibody 2, and detecting the fluorescent strength to obtain the AFP in the human serum sample.
Description
technical field [0001] The invention relates to an immunodetection method, in particular to a microbead immunoassay method based on protein A immobilized antibody. Background technique [0002] Protein A is the abbreviation of Staphylococcus protein A (SPA), which is a protein isolated from the cell wall of Staphylococcus aureus. Protein A mainly has five antibody-binding functional domains, and each domain consists of approximately 58 amino acid residues (1, Karen L.A, Julia D.B, Emily S.S. aureus IgG-binding proteins SpA and Sbi: Host specificity and mechanisms of immune complex formation, Molecular Immunology, 2008, 45: 1600-1611; 2. Feng Shaojiao, Liang Hao, Song Shuliang, Staphylococcus protein A activity mechanism and modern application, Chemistry of Life, 2008, Vol. 28 No. 6, 748-751). [0003] The main defect of the existing double-antibody sandwich method is that during the antibody coupling process, the antibody molecules are inactivated due to the lodging of the ...
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