Preparation method of polyase marker antibody

A technology for labeling antibodies and polymerases, applied in the field of enzyme immunity, can solve the problems of many steps and complexity in the preparation process, and achieve the effects of simplified preparation process, high speed and high yield

Active Publication Date: 2008-05-14
武汉原谷生物科技有限责任公司
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Problems solved by technology

But whether it is Dako's patent (US005543332A 1996) or Chinese patent application number 03150895.2, its main problem comes from the need to use polymer carrier molecules-dextran and toxic and dangerous vinyl sulfone in the reagent preparation process, and the preparation The process has many steps and is complicated

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  • Preparation method of polyase marker antibody

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Embodiment 1

[0023] A method for preparing a polymerase-labeled antibody, comprising the steps of:

[0024] 1) Oxidation reaction: take 8mg horseradish peroxidase (HRP), add 8mmol NaIO 4 , reacted for 20 minutes; NaIO 4 The addition of (oxidizing agent) plays an oxidation role to obtain HRP containing aldehyde groups;

[0025] 2) polymerization to form polymerase (Poly-HRP): step 1) adding 2ml of mass concentration to the aldehyde-containing HRP obtained is the non-ionic surfactant TritonX-100 of 3%, and then 1M Na 2 CO 3 -NaHCO 3 Adjust the pH of the solution to 8.8-9.2 (optimally 9.0), then rotate and react overnight at 4°C, and react at room temperature for 1 hour the next day to obtain Poly-HRP; add non-ionic surfactant to form micellized coating;

[0026] 3) Purification: the polymerase obtained in step 2) was dialyzed in 10 mM phosphate physiological buffer solution (PBS) for 3 times to obtain a dialyzate;

[0027] 4) IgG connection: take out the dialyzate obtained in step 3) (t...

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Abstract

The invention belongs to the technical field of enzyme immunity, and in particular relates to a preparation method of a polymerase-labeled antibody. The preparation method of the polymerase-labeled antibody is characterized in that it comprises the steps of: in the presence of a nonionic surfactant, soluble horseradish peroxidase (HRP) is micellized, and the sugar molecules on the surface of the HRP are first oxidized with NaIO form an aldehyde group, and then use the amino group on HRP to undergo a condensation reaction with the aldehyde group to generate a polymerase (Poly-HRP), and then use the excess aldehyde group on the polymerase to combine with the amino group on IgG to generate soluble Poly-HRP-IgG , After reducing the generated unsaturated bond with NaBH4, a stable polymerase-labeled antibody (Poly-HRP-IgG) is generated. The preparation process of the method is simple, and the prepared polymerase-labeled antibody has faster color development speed, higher sensitivity and better storage stability when applied.

Description

technical field [0001] The invention belongs to the technical field of enzyme immunity, and in particular relates to a preparation method of a polymerase-labeled antibody (Poly-HRP-IgG). technical background [0002] Enzyme markers include enzyme-labeled antigen, enzyme-labeled antibody, and enzyme-labeled SPA. The quality of enzyme markers is directly related to the success of immunoenzyme technology, so it is called a key reagent. The most commonly used enzyme-labeled antibody is an enzyme-labeled antibody, which is formed by linking an enzyme with a specific antibody by an appropriate method. The quality of enzyme-labeled antibodies mainly depends on the enzymes and antibodies with good purity, strong activity and high affinity, followed by good preparation methods. At present, high-quality enzymes (such as horseradish peroxidase, referred to as HRP) have been commercially available in China. High-quality antibodies can be obtained by extraction and purification. Dako...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535
Inventor 宋晓华张薇范力仁
Owner 武汉原谷生物科技有限责任公司
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