Enzyme-labeled coupled secondary antibody and preparation method thereof

A technology of coupling and enzyme labeling, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of insufficient antigen sensitivity, loose chain polymer-enzyme-antibody polymer structure, large volume, etc., to achieve good specificity, The effect of high sensitivity and stable structure

Pending Publication Date: 2021-04-13
苏州百道医疗科技有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, this method also has certain shortcomings. The overall structure of the chain polymer-enzyme-antibody polymer is relatively loose and the volume is large. It has certain resistance when entering the nuclear membrane, and is not sensitive enough to detect some intranuclear antigens.

Method used

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  • Enzyme-labeled coupled secondary antibody and preparation method thereof
  • Enzyme-labeled coupled secondary antibody and preparation method thereof
  • Enzyme-labeled coupled secondary antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] In this example, the enzyme-labeled goat anti-mouse antibody was prepared according to the following method:

[0045] (1) Modification of antibody; the modification method is as follows:

[0046] ① Dissolve goat anti-mouse IgG in 0.1M sodium phosphate, 0.15M NaCl, pH 7.5 phosphate buffer solution at room temperature to a concentration of 5mg / ml.

[0047] ② will 12-NHS ester was dissolved in DMSO at room temperature to prepare a reaction modification solution with a concentration of 10 mg / ml. CAUTION Use a fume hood to handle organic solvents.

[0048] ③According to goat anti-mouse IgG and The molar ratio of 12-NHS ester was 1:10 for antibody modification, and the reaction was carried out at room temperature for 30 minutes.

[0049] ④ After the reaction, replace the buffer of the above reaction system with a phosphate buffer solution of 0.1M sodium phosphate, 0.15M NaCl, pH 5.5 with a 50k ultrafiltration tube.

[0050] ⑤ Using a 5mL desalting column, purify by dia...

Embodiment 2

[0090] In this example, the enzyme-labeled goat anti-rabbit antibody was prepared according to the following method:

[0091] (1) Modification of antibody; the modification method is as follows:

[0092] ① Dissolve goat anti-rabbit IgG in 0.1M sodium phosphate, 0.15M NaCl, pH 7.5 phosphate buffer solution at room temperature to a concentration of 10mg / ml.

[0093] ② will 12-NHS ester was dissolved in DMSO at room temperature to prepare a reaction modification solution with a concentration of 10 mg / ml. Caution Use a fume hood to handle organic solvents.

[0094] ③According to goat anti-mouse IgG and The molar ratio of 12-NHS ester was 1:10 for antibody modification, and the reaction was carried out at room temperature for 30 minutes.

[0095] ④ After the reaction, replace the buffer solution of the above reaction system with 0.1M sodium phosphate, 0.15M NaCl, pH5.5 phosphate buffer solution with a 50k ultrafiltration tube to obtain a solution of phthalimide oxygen-modifie...

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Abstract

The invention relates to an enzyme-labelled coupled secondary antibody. The enzyme-labelled coupled secondary antibody is formed by coupling and connecting a modified antibody and a modified labelled enzyme through an oxime bond, wherein the modified antibody is formed by connecting amino of an antibody lysine residue with an amino oxygen group in a modified manner, and the modified labelled enzyme is formed by connecting amino of a lysine residue of an enzyme molecule with aryl aldehyde in a modified manner. The enzyme-labeled coupled secondary antibody is stable in structure, moderate in size, high in sensitivity, good in specificity, free of non-specific staining due to interference of biotin existing in organism tissue, moderate in structure and size, especially suitable for detection of some intracellular antigens, and very high in sensitivity.

Description

technical field [0001] The invention relates to the technical field of enzyme-labeled antibodies, in particular to an enzyme-labeled secondary antibody and a preparation method thereof. Background technique [0002] Immunoassay is a method based on the principle of "antigen-antibody specific binding" to detect proteins in biological samples and then perform qualitative and quantitative analysis. It has the advantages of high sensitivity and specificity. Enzyme-labeled secondary antibodies are widely used in immunoassays, mainly including immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), western blotting (WB), etc. [0003] (1) There are mainly three types of enzyme-labeled secondary antibodies currently on the market: 1) Antibody-enzyme direct labeling method, that is, a single secondary antibody and HRP enzyme are directly conjugated and labeled with a small molecule reagent. Since labeled enzymes are usually bulky, and the surface area and functional ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535
CPCG01N33/535
Inventor 吴纯宋砚明刘杨闵豆
Owner 苏州百道医疗科技有限公司
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