Malachite green artificial antigen and antibody, and preparation method and application thereof

A malachite green and hapten technology, which is applied in chemical instruments and methods, cyanide reaction preparation, organic compound preparation, etc., can solve the problems of cumbersome operation, inapplicable rapid detection, expensive equipment, etc.

Active Publication Date: 2014-01-22
深圳市微宇藻业科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the detection of malachite green and cryptomalachite green residues is mainly performed by gas chromatography and high performance liquid chromatography. Although the instrument detection method has high accuracy, it is not suitable for on-site rapid detection due to expensive equipment and cumbersome operation.

Method used

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  • Malachite green artificial antigen and antibody, and preparation method and application thereof
  • Malachite green artificial antigen and antibody, and preparation method and application thereof
  • Malachite green artificial antigen and antibody, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] (a) 4-[bis(4-dimethylamino)phenyl]methylbenzoic acid

[0056]

[0057] Synthetic route of hapten (a):

[0058] Accurately weigh 1.8 g of methyl p-formylbenzoate and 3.7 g of zinc chloride into a 50 mL flask, then add 5.3 g of N,N-dimethylaniline, heat to 120 ° C, stir for 4 hours, and ethyl acetate Ester extraction and purification by silica gel column chromatography (ethyl acetate / n-hexane = 1:2) gave the target compound 4-[bis(4-dimethylamino)phenyl]methylbenzoic acid with a yield of 10%.

[0059] Example 2 Synthesis of malachite green hapten 5-{4-[bis(4-dimethylamino)phenyl]methyl}phenylvaleric acid

Embodiment 2

[0060] (b) 5-{4-[bis(4-dimethylamino)phenyl]methyl}phenylvaleric acid

[0061]

[0062] 1. Accurately weigh 10 g of p-hydroxybenzoic acid and dissolve in dichloromethane, add 7.8 ml of benzyl bromide and 9.3 ml of triethylamine at 0°C, stir at room temperature for 1-2 days, add saturated ammonium chloride at 0°C to quench The reaction was quenched, extracted with dichloromethane, concentrated, and purified on a silica gel column (ethyl acetate / n-hexane=1:3) to obtain the compound 4-hydroxymethylbenzoic acid benzyl ester;

[0063] 2. Dissolve 4.1g of the product in dichloromethane, add 7.9g of trichloroisocyanuric acid and 2.86g of NaHCO at 0°C 3 and 2,2,6,6-tetramethylpiperidine oxide, reacted for 5 minutes, purified by silica gel short column (ethyl acetate / n-hexane = 1:2), and the eluent was concentrated to obtain the compound 4-formylbenzoic acid Benzyl ester;

[0064] 3. Weigh 2.87g of wittig reagent and dissolve in tetrahydrofuran, slowly add 23.25ml of lithium hexam...

Embodiment 3

[0069] The hapten is coupled to ovalbumin (OVA) or hemocyanin (keyhole limpet hemocyanin, KLH) respectively by the activated ester method, the specific method is:

[0070] n=0, 4

[0071] Take 0.5 mmol of the above hapten, 0.625 mmol of N-hydroxysuccinimide (NHS) in a 25 ml round bottom flask and add 3 ml of anhydrous tetrahydrofuran, under N 2 Under protection, 4 ml of anhydrous tetrahydrofuran solution dissolved in 0.625 mmol N, N-dicyclohexylcarbodiimide DCC was added dropwise, stirred while dripping, stirred at room temperature for 4 h, purified by silica gel column to obtain hapten-activated ester .

[0072] The activated ester of the above hapten was dissolved in DMF to form solution A, and the carrier protein was dissolved in phosphate buffer solution to form solution B. Then solution A was slowly added to solution B under an ice bath. After stirring, react overnight at 4°C, and finally put the reaction solution into a dialysis bag, and dialyze in a PBS solution w...

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Abstract

The invention provides preparation of malachite green artificial antigen and antibody, and relates to preparation of artificial hapten, artificial antigen and antibody of a triphenylmethane chemical substance malachite green. By adopting the preparation method, the problems of complex process, high cost and low analysis speed of the traditional physical and chemical analysis method are overcome. A simple, convenient, quick, sensitive and accurate enzyme immunoassay is provided. The method comprises the following steps: taking 4-[bi(4-dimethylamino)phenyl]methyl benzoic acid and 5-{4-[bi-(4-dimethylamino)phenyl]methyl} phenyl valeric acid as haptens to be respectively connected with carrier proteins such as hemocyanin, ovalbumin and the like to synthetize artificial antigen; carrying out animal immunization, taking blood, separating out antiserum, and purifying, so as to prepare the antibody. The antibody is stable, and has good specificity and sensitivity, and a good application prospect, and can be applied to fast immunodetection of the malachite green.

Description

technical field [0001] The application of the present invention relates to the synthesis of malachite green artificial hapten with triphenylmethane structure, specific antigen and preparation of specific antibody, belonging to the technical field of immunochemistry and residual analysis of small molecule compounds. Background technique [0002] With the rapid development of the national economy and the continuous improvement of the living standards of urban and rural residents, people's dietary structure is undergoing profound changes. With its low fat, low cholesterol, high protein, rich nutrition, delicious taste and other advantages, aquatic products are becoming one of the foods favored by consumers. [0003] Compared with other meat products, microorganisms are more likely to multiply in aquatic products. We can easily find various pathogenic bacteria, viruses and parasites in the intestines, skin, and muscles of aquatic products. Farmers want to prevent and control aqu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07C227/10C07C229/52C07C229/42C07K14/77C07K14/795C07K16/06G01N33/53
Inventor 胡章立
Owner 深圳市微宇藻业科技有限公司
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