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Melamine hapten, artificial antibody, and preparation method and application thereof

A melamine and hapten technology, applied in the field of immunochemistry, can solve problems such as poor stability of carbon-nitrogen double bonds, many side reactions, and different products, and achieve the effect of increasing recognition sites and improving antigenicity

Active Publication Date: 2020-03-31
GUANGDONG DAYUAN OASIS FOOD SAFETY TECH CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, patent CN 101402683 B discloses a melamine hapten and a complete antigen and a preparation method thereof. This method adopts glutaraldehyde to directly couple with melamine. During the amine group reaction, the product is not single, there are many side reactions, and the carbon-nitrogen double bond generated has poor stability, which also affects the electron cloud density of melamine; a connecting arm of a melamine hapten disclosed in patent CN 101643453 B The strong electron group benzene ring is directly introduced, which has a great influence on the electron cloud density of the entire structure

Method used

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  • Melamine hapten, artificial antibody, and preparation method and application thereof
  • Melamine hapten, artificial antibody, and preparation method and application thereof
  • Melamine hapten, artificial antibody, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Melamine hapten and its preparation method

[0044] A melamine hapten whose structural formula is:

[0045]

[0046] The synthetic route of the hapten is:

[0047]

[0048] The preparation method is as follows:

[0049] Take 3.1g (21.3mmol) of 2-chloro-4,6-diamino-1,3,5-triazine (compound a) in a 50mL round bottom flask, and then add 27mL of ethanol, 3mL of water, 1.0g (8.5 mmol) 3,4-diaminobutyric acid (compound b) and 1.4 g (34.0 mmol) sodium hydroxide. React at 80°C for 20h. After the reaction, the solvent was removed under reduced pressure, 50 mL of purified water was added, and the mixture was extracted twice with dichloromethane. The resulting aqueous solution was adjusted to pH 4.5 with 1M dilute hydrochloric acid, filtered, and the solid was rinsed twice with 80% ethanol / water, and dried. 0.6g of melamine hapten is dried, and the mass spectrum is as follows figure 1 As shown, ESI-MS: 335[M-H] - .

Embodiment 2

[0050] Example 2: Melamine artificial antigen and its preparation method

[0051] A melamine artificial antigen whose structural formula is:

[0052]

[0053] Among them, Protein is the carrier protein, and the carrier protein is bovine serum albumin or hemocyanin;

[0054] The synthetic route of the artificial antigen is:

[0055]

[0056] 1. The synthesis method of the melamine artificial antigen with bovine serum albumin as the carrier protein is as follows:

[0057] (1) Take 10 mg of the melamine hapten prepared in Example 1, and dissolve it in 0.5 mL of dimethylformamide (DMF). After stirring well, add 10 mg of 1-(3-dimethylaminopropyl)-3-ethyl Ethylcarbodiimide (EDC) and 10mg N-hydroxysuccinimide (NHS), stirred at room temperature for 4h, then the hapten activated ester can be obtained;

[0058] (2) Weigh 50 mg of bovine serum albumin (BSA) and fully dissolve it in 5 mL of phosphate buffered saline (PBS) with a concentration of 0.01 mol / L to form a carrier protein solution, and ac...

Embodiment 3

[0066] Example 3: Application of melamine artificial antigen in the preparation of anti-melamine monoclonal antibodies

[0067] The preparation method of anti-melamine monoclonal antibody is as follows:

[0068] The melamine artificial antigen with hemocyanin as the carrier protein was used as the immunogen, and after emulsification with an equal volume of Freund's adjuvant, BALB / C mice were immunized. The immunization dose of each mouse was 80 μg, and the immunization interval was 2 weeks. After 3 immunizations, blood from the tail vein of the mouse was collected to detect serum titer. If the antibody titer does not meet the requirements, a booster immunization is required. After the antibody titer no longer rises, a subcutaneous booster immunization is performed with 100 μg of the whole antigen. After 5 days, mouse spleen cells are fused with SP20 cells (mouse myeloma cells). The fused cells were selected in HAT medium, and 5 days later, the complete medium was replaced with HAT...

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Abstract

The invention discloses a melamine hapten, an artificial antigen, and a preparation method and an application thereof, wherein the structure of the hapten is represented as the formula I. The hapten is prepared from 2-chloro-4,6-diamino-1,3,5-triazine and 3,4-diaminobutyric acid as raw materials. The artificial antigen is a conjugate of the hapten and a protein carrier and can be used for preparing a melamine monoclonal antibody, ELISA detection, and fluorescent quantitative immunochromatography detection and products thereof. When being used for ELISA detection, the artificial antigen has strong specificity. A fluorescent quantitative immunochromatography test strip can achieve detection of melamine rapidly and conveniently, sensitivity can reach up to 10 [mu]g / L. Through the novel haptenstructure, the fluorescent quantitative immunochromatography product can be finally prepared, so that the researching content of melamine is enriched, and certain positive effects on security detection of foods and pharmacokinetics researching of the melamine are achieved.

Description

Technical field [0001] The invention relates to the technical field of immunochemistry, in particular to a melamine hapten and an artificial antibody, and a preparation method and application thereof. Background technique [0002] Melamine, molecular formula C 3 N 6 H 6 , Is a triazine nitrogen-containing heterocyclic organic compound. The main purpose of melamine is as a raw material for the production of melamine formaldehyde resin, and it is also widely used in materials, electrical and pharmaceutical industries. [0003] Protein detection commonly used "Kjeldahl method" to estimate protein content by measuring nitrogen content, protein is mainly composed of amino acids, and its nitrogen content is usually not higher than 30%, while the nitrogen content of melamine is as high as 66%. Because of its high nitrogen content, it is often illegally added to food or feed by illegal manufacturers to improve the determination of nitrogen atom content in food protein. After being ingest...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D251/70C07K14/765C07K14/77C07K14/795C07K16/44G01N33/558G01N33/577G01N33/58
CPCC07D251/70C07K14/765C07K14/77C07K14/795C07K16/44G01N33/558G01N33/577G01N33/582
Inventor 李斌石松黄智永王莹莹
Owner GUANGDONG DAYUAN OASIS FOOD SAFETY TECH CO LTD
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