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Melamine hapten, artificial antibody, preparation method and application thereof

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

Active Publication Date: 2021-03-09
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, preparation method and application thereof
  • Melamine hapten, artificial antibody, preparation method and application thereof
  • Melamine hapten, artificial antibody, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: Melamine hapten and preparation method thereof

[0044] A kind of melamine hapten, its structural formula is:

[0045]

[0046] The synthetic route of this hapten is:

[0047]

[0048] The preparation method is as follows:

[0049] Take 3.1g (21.3mmol) 2-chloro-4,6-diamino-1,3,5-triazine (compound a) in a 50mL round bottom flask, then add 27mL ethanol, 3mL water, 1.0g (8.5 mmol) 3,4-diaminobutyric acid (compound b) and 1.4 g (34.0 mmol) sodium hydroxide. Reaction at 80°C for 20h. After the reaction was completed, remove the solvent under reduced pressure, add 50 mL of purified water, extract twice with dichloromethane, adjust the pH value of the resulting aqueous solution to 4.5 with 1M dilute hydrochloric acid, filter, rinse the solid twice with 80% ethanol / water, and dry Dried melamine hapten 0.6g, mass spectrogram as figure 1 Shown, ESI-MS: 335[M-H] -.

Embodiment 2

[0050] Embodiment 2: Melamine artificial antigen and preparation method thereof

[0051] A kind of melamine artificial antigen, its structural formula is:

[0052]

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

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

[0055]

[0056] 1, carrier protein is the synthetic method of the melamine artificial antigen of bovine serum albumin as follows:

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

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

Embodiment 3

[0066] Embodiment 3: the application of melamine artificial antigen in the preparation of anti-melamine monoclonal antibody

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

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

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Abstract

The invention discloses a melamine hapten and an artificial antigen and a preparation method and application thereof. The structure of the hapten is shown in formula I: the hapten is composed of 2-chloro-4,6-diamino-1,3,5- It is prepared from triazine and 3,4-diaminobutyric acid. The artificial antigen is a conjugate of the above-mentioned hapten and a protein carrier, and the artificial antigen can be used in the preparation of melamine monoclonal antibody, ELISA detection, fluorescence quantitative immunochromatography detection and its products, and it is used for ELISA detection specificity Strong, the fluorescent quantitative immunochromatography test strip prepared by it can quickly and conveniently realize the detection of melamine, and the sensitivity is as high as 10 μg / L; through the new hapten structure, a quantifiable fluorescent immunochromatographic product is finally prepared, It enriches the research content of melamine, and has a certain positive effect on food safety monitoring and melamine pharmacokinetics research.

Description

technical field [0001] The invention relates to the technical field of immunochemistry, in particular to a melamine hapten and an artificial antibody as well as 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 most important use 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 is 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 unscrupulous manufacturers to improve the determination of nitrogen atom content in foo...

Claims

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Application Information

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Patent Type & Authority Patents(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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