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Hepatitis C antibody polymer enzyme marker, and preparation and application thereof

A technology of polymer enzymes and markers, applied in the field of enzyme immunity, can solve problems such as irritation to the eyes, respiratory system and skin, undisclosed application methods, strong toxicity, etc., to achieve improved sensitivity and stability, high sensitivity, and good specificity Effect

Active Publication Date: 2017-08-11
山东莱博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the vinyl sulfone used in these two patents is highly toxic and irritates the eyes, respiratory system and skin
Chinese patent (patent application No. 200710168647.5) discloses a method for preparing a polymerase-labeled antibody, the key technology of which is to micellize soluble horseradish peroxidase (HRP) in the presence of a nonionic surfactant 4 The sugar molecules that decorate the surface of HRP are first oxidized to aldehyde groups, and then the amino groups on HRP are condensed with the aldehyde groups to form a polymer (Poly-HRP), but the specific application method is not disclosed
After searching, the antibody was modified with sulfhydryl group, and the modified antibody was exposed to hydroxylamine and then glued with the polymer enzyme marker (Poly-HRP) and purified to prepare a literature and product of a hepatitis C antibody polymer enzyme marker Not yet reported

Method used

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  • Hepatitis C antibody polymer enzyme marker, and preparation and application thereof
  • Hepatitis C antibody polymer enzyme marker, and preparation and application thereof
  • Hepatitis C antibody polymer enzyme marker, and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The steps of polymerase labeling of hepatitis C antibody are as follows:

[0042] Step 1: Activation of polymers: Dextran T500 is used as a polymer carrier and activated by hydrazine hydrochloride

[0043] 1) Weigh out 25mg of Dextran T500 and dissolve in 0.5ml 1×PBS (0.1M PBS, pH7.2);

[0044] 2) Add 0.5ml 0.1M NaIO 4 , and react in the dark for 2 hours at room temperature;

[0045] 3) Use PD-10 desalting column to desalt with 0.1M PBS (pH7.2), collect the eluate containing dextran, add 0.6g hydrazine hydrochloride, and use 2M NaHCO 3 Adjust the pH to 9.0, and gently stir the reaction at room temperature for 2 hours;

[0046] 4) Add 5 mg of trimethylamine borane compound (TMAB), and react at room temperature for 2 hours;

[0047] 5) Use a PD-10 desalting column to desalt with 0.1M PBS (pH7.2), collect the liquid from the first peak, and store it at 2-8°C for later use;

[0048] The second step: the cross-linking of the polymer and horseradish peroxidase (HRP), tha...

Embodiment 2

[0060] 1. The preparation and use of the multimer enzyme stabilizer used in supporting the hepatitis C antibody multimer enzyme marker of the present invention:

[0061] For the long-term effective preservation of the prepared multimer enzyme marker of the hepatitis C antibody, BSA can be added to the prepared polymer enzyme marker of the hepatitis C antibody (to make the final concentration 10mg / ml), and kathon (final Concentration 10ul / ml), add iodoacetamide (final concentration is 1 / 10,000), add 1 / 3 volume of glycerol.

[0062] The prepared multimer enzyme marker can be stable for at least two years after adding the enzyme stabilizer. Related monitoring results can be seen in Example 3.

[0063] 2. The preparation method of the diluent used for supporting the hepatitis C antibody multimer enzyme marker of the present invention is as follows:

[0064] In 0.1M PBS (pH7.2), add 20% neonatal bovine serum by volume ratio, Procin300 of 5 / 10,000, by weight volume ratio, 1% BSA, ...

Embodiment 3

[0067] The hepatitis C antibody multimer enzyme marker prepared in Example 1 has higher sensitivity than the enzyme marker of the hepatitis C antibody labeled by the common enzyme labeling method (sodium periodate method), and its use and verification methods are as follows:

[0068] Carry out 1:3000 dilution of the multimer enzyme marker of the hepatitis C antibody labeled by Example 1 without adding the enzyme stabilizer with the enzyme diluent in Example 2, and add the enzyme stabilizer in Example 2. The polymer enzyme marker of the antibody was diluted 1:2000, compared with the hepatitis C antibody enzyme marker labeled by the common enzyme labeling method (sodium periodate method) in the enzyme-linked immunoassay kit, and the OD value was recorded , calculate P mean / N mean, the results are shown in Table 1.

[0069] A. Ordinary enzyme labeling, the enzyme diluent used in the original kit, diluted 1:500

[0070] B. The multimer enzyme marker marked by the method of the pr...

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Abstract

The invention discloses a hepatitis C antibody polymer enzyme marker. The Hepatitis C antibody polymer enzyme marker is prepared according to the following steps: with glucosan T500 as a polymer carrier, activating with hydrazine hydrochloride and then crosslinking with HRP, thereby acquiring polymer enzyme marker (Poly-HRP); modifying Hepatitis C antibody with sulfydryl and then cross-linking the Hepatitis C antibody modified with sulfydryl with the polymer enzyme marker (Poly-HRP). The invention also discloses a polymer enzyme stabilizer and a diluent which are used together with the hepatitis C antibody polymer enzyme marker. A test proves that the hepatitis C antibody polymer enzyme marker disclosed by the invention can be stably stored for two years after the polymer enzyme stabilizer is added, and meanwhile, after the hepatitis C antibody polymer enzyme marker is diluted with the diluent, the sensitivity of the diluted hepatitis C antibody polymer enzyme marker used for enzyme-linked immunosorbent assay and chemiluminescence immunodetection is greatly higher than that of a enzyme marker for a traditional sodium periodate method or glutaraldehyde method.

Description

technical field [0001] The invention belongs to the technical field of enzyme immunity, and in particular relates to a hepatitis C antibody multimer enzyme marker and its preparation method and application. [0002] technical background [0003] Detection of hepatitis C virus (Hepatitis C virus, HCV) core antigen can directly reflect the infection status of HCV and shorten the detection window period of HCV infection. However, after HCV infects the human body, the content of its core antigen in the blood is very low, and a highly sensitive method is required for detection. [0004] In enzyme immunoassay detection, antigen or antibody enzyme markers play an important role in the sensitivity and specificity of immunoassay, and are key reagents in enzyme immunoassay detection. Horseradish Peroxidase (HRP) has high specific activity, stability, small molecular weight, easy preparation of pure enzyme, and is now the most commonly used raw material for the preparation of protease ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535
CPCG01N33/535
Inventor 盖中涛欧兰香汪运山王露楠朱之炜江长林丁兴龙
Owner 山东莱博生物科技有限公司
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