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Melamine hapten, antigen and preparation method and applications thereof

A technology of melamine and antigen is applied in the preparation method of peptide, chemical instrument and method, animal/human protein, etc., to achieve the effect of simple and feasible preparation method, low cost and high yield

Inactive Publication Date: 2011-05-25
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Defects in the protein content testing methods of the food and feed industry, plus melamine as a white crystalline powder, has no smell and taste, and it is not easy to be found after being mixed with melamine in food and feed. For this reason, melamine is often used by unscrupulous businessmen as Food additives to improve the protein content index in food testing, so melamine is also known as "protein essence"

Method used

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  • Melamine hapten, antigen and preparation method and applications thereof
  • Melamine hapten, antigen and preparation method and applications thereof
  • Melamine hapten, antigen and preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, preparation and identification of melamine hapten

[0034] 1. Preparation of melamine hapten

[0035] 1. Weigh 120 mg of 4,6-diamino-2-hydroxyl-1,3,5-triazine (purchased from Acros Organics, CAS No.: 3397-62-4), 100 μL of ethyl 4-bromobutyrate, Put 100 mg of anhydrous sodium carbonate in a 50 mL flask, add 20 mL of acetone, reflux at 70°C for 16 hours, and evaporate the solvent to dryness under reduced pressure.

[0036] 2. The residue was dissolved in 15 mL of methanol-potassium hydroxide mixture (15%, 88 mg of potassium hydroxide dissolved in 5 mL of methanol), refluxed at 80° C. for 1 hour, and the solvent was evaporated to dryness under reduced pressure.

[0037] 3. Add 8 mL of distilled water to the residue (all dissolved), acidify with 2M HCl to pH 3 (white precipitate appears).

[0038] 4. Collect the precipitate by centrifugation and dry at 50°C. 143 mg of the reaction product (melamine hapten) was obtained with a yield of 68%.

[0039] 2. Iden...

Embodiment 2

[0044] Embodiment 2, preparation and identification of melamine artificial antigen

[0045] 1. Synthesis of melamine artificial antigen

[0046] 1. Dissolve 10 mg of melamine hapten in 1 mL of dioxane, stir to dissolve completely; then add 50 μL of anhydrous tri-n-butylamine, cool the mixture in an ice bath at 4°C, and stir for 15 minutes. Add 25 μL redistilled isobutyl chloroformate and stir at about 4°C for 30 minutes.

[0047] 2. Weigh 50 mg bovine serum albumin (BSA) and dissolve it in water, stir to dissolve it, and adjust the pH to 9.0 with 0.1 mol / L NaOH; add the hapten solution obtained in step 1 dropwise to the dissolved protein, and Stir gently at 4°C for 4 hours, maintaining the pH at 9.0 throughout.

[0048] 3. The mixture was dialyzed in 0.1 mol / L, pH 7.4 PBS at 4°C for 72 hours, and the dialysate was changed every 6 hours. Freeze-dried and stored at -20°C.

[0049] 2. Identification of melamine artificial antigen

[0050] According to the ratio of the hapten...

Embodiment 3

[0051]Embodiment 3, preparation and specific identification of antiserum

[0052] 1. Preparation of melamine antiserum

[0053] Adopt 8 7-week-old, 2-3 kilograms of body weight, healthy New Zealand white rabbits as immunization animals; the melamine artificial antigen prepared in embodiment 2 is immunogen; immunization dose: basic immunization is 1.0 mg / kg, booster immunization dose is 1.5mg / kg. The specific steps of immunization are as follows: Dilute an appropriate amount of immunogen with a diluent, add an equal volume of Freund’s complete adjuvant, fully emulsify, and inject into the rabbit’s back at multiple points for the first immunization; 2 weeks later, take the antigen and add Freund’s incomplete adjuvant After emulsifying the agent, inject it subcutaneously at multiple points on the back, and boost the immunization once every 2 weeks.

[0054] From the third booster immunization, blood was collected from the rabbit’s ear vein on the 7th day after each immunization...

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Abstract

The invention discloses a melamine hapten, a melamine antigen and a preparation method and applications thereof. The melamine hapten provided in the invention is a compound shown in formula (I), the compound in the formula (I) is connected with carrier protein to obtain the melamine antigen which can be used for preparing melamine specific antibody. The invention has the advantages of being specific, sensitive, accurate, rapid, convenient, and cheap and the like. The preparation method in the invention features convenience and feasibility, lower cost and higher yield of hapten. The melamine artificial antigen can generate specific antibody aiming at the melamine through immune animals, is used for rapidly testing the melamine residue in foods, and has the advantages of simplicity, rapidness, large quantity of processing samples, high sensitivity, strong specificity and the like.

Description

technical field [0001] The invention relates to a melamine hapten, an antigen, a preparation method and application thereof. Background technique [0002] Melamine, molecular formula C 3 N 6 h 6 , referred to as triamine, is a triazine nitrogen-containing heterocyclic organic compound with a structure as shown in formula (II): [0003] Formula (II). [0004] Melamine is weakly alkaline and can form melamine salts with hydrochloric acid, sulfuric acid, nitric acid, acetic acid, oxalic acid, etc. Melamine is an important nitrogen-heterocyclic organic chemical raw material. It condenses with formaldehyde under neutral or slightly alkaline conditions to form various methylol melamines, but in slightly acidic environments (pH5.5-6.5) with methylol The derivatives undergo polycondensation reaction to form resin products. Melamine is a basic organic chemical intermediate product with a wide range of uses. The main use of melamine is as a raw material for the production of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D251/52G01N33/566C07K14/765C07K14/77G01N33/531C07K16/44C07K1/113
Inventor 沈建忠张素霞丁双阳史为民江海洋王战辉王亚辉
Owner CHINA AGRI UNIV
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