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Dehydroepiandrosterone enzyme-linked immunosorbent assay kit development method

A technology of dehydroepiandrosterone and epiandrosterone enzyme, which is applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of difficult to produce specific antibodies and weak immunogenicity, and achieve stability and low toxicity , reduce toxicity, high stability effect

Inactive Publication Date: 2013-03-27
WUHAN CLOUD CLONE CORP
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, many small molecular haptens have weak immunogenicity, and it is difficult to produce specific antibodies through traditional immunization methods. They need to be coupled to macromolecular substances (carriers) to obtain immunogenicity, and then high-quality antibodies can be produced

Method used

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  • Dehydroepiandrosterone enzyme-linked immunosorbent assay kit development method
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  • Dehydroepiandrosterone enzyme-linked immunosorbent assay kit development method

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preparation example Construction

[0046] a. Preparation of DHEA whole antigen and coated antigen:

[0047] Prepare DHEA-BSA by mixed acid anhydride method. Take 25-75mg dehydroepiandrosterone and 25-75mg succinic anhydride (weight 1:1) to react overnight at room temperature, TLC thin layer detection reaction yield, silica gel column chromatography, petroleum Ether / acetone mixed solution gradient elution, collected in 50ml bottles, recovered solvent, TLC tracking detection, combined the same part, compared with TLC and dehydroepiandrosterone, and retained the reaction product as the modified hapten.

[0048] Take 5mg-20mg of the modified hapten, add 10-30ul chloromethyl isobutyl ester and 5-20ul tri-n-butylamine, react at 4°C for 2 hours to obtain liquid A. Dissolve 10-30 mg of BSA in 1-4 ml of carbonate buffer to obtain liquid B, which is cooled to 4°C. Add liquid A dropwise to liquid B, stir and react for 6h, then pass through Sephadex G-100 column, elute with PBS, collect protein fraction, and remove unreac...

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Abstract

The present invention discloses a dehydroepiandrosterone (DHEA) enzyme-linked immunosorbent assay kit development method, which comprises: adopting dehydroepiandrosterone and succinic anhydride as raw materials, and adopting an anhydride mixing method to respectively prepare dehydroepiandrosterone artificial antigen (DHEA-BSA and DHEA-OVA) and anti-dehydroepiandrosterone (DHEA) monoclonal antibody so as to prepare an enzyme-linked immunosorbent assay (ELISA) kit. Compared with the high performance liquid chromatography (HPLC) method, the method of the present invention has the following advantages that: operation steps are simple, a plurality of samples can be detected in one time, expensive instrument is not required, and detection sensitivity, the required time and other aspects are better than the HPLC method. Compared to the HPLC method, the ELISA method has the following characteristics that: uses of organic reagents such as n-hexane, acetonitrile, methanol and the like are not required, poison on experimenters and environment can be reduced, and an environmental protection characteristic is provided.

Description

technical field [0001] The technical invention is a method for developing a dehydroepiandrosterone enzyme-linked immunosorbent assay kit, and is designed in the field of biotechnology. Background technique [0002] Dehydroepiandrosterone (DHEA), a C19 steroid hormone, is mainly secreted by the zona reticularis of the adrenal cortex, and exists in circulating blood as DHEA and its storage sulfate form (DHEAS) (the latter is 100 times the former in the body Left and right), DHEA can be converted into other hormones such as estrogen, androgen, progesterone, cortisol and corticosterone, so it is known as a multidirectional "hormone buffer". Many studies have shown that DHEA has positive effects in promoting fat metabolism, anti-oxidation, improving immunity, enhancing cell activity, and preventing and treating senile dementia. After the body ages, the levels of DHEA and DHEAS in the body decrease significantly, resulting in a significant decrease in the content of various stero...

Claims

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Application Information

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IPC IPC(8): G01N33/74G01N33/531
Inventor 何峰容李华渊童晓玲杨娟章秀波汪景谢正红
Owner WUHAN CLOUD CLONE CORP
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