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Application of 1- amino-2-naphthol as hapten and corresponding artificial antigen and antibody

A technology of artificial antigen and hapten, which is applied in the field of food safety to achieve the effect of improving detection efficiency, reducing detection cost and shortening detection time

Inactive Publication Date: 2014-07-30
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still few immunoassay reagents that can simultaneously detect multiple red azo dyes.

Method used

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  • Application of 1- amino-2-naphthol as hapten and corresponding artificial antigen and antibody
  • Application of 1- amino-2-naphthol as hapten and corresponding artificial antigen and antibody
  • Application of 1- amino-2-naphthol as hapten and corresponding artificial antigen and antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Synthesis of β-naphthol-derived dye artificial antigen

[0026] Experiment 1:

[0027] (a) Put 40mmol of 1-amino-2-naphthol in 3ml of N,N-dimethylformamide, stir and dissolve to obtain solution A; dissolve 50mg of carrier protein in 2mL of dihydrogen phosphate and hydrogen phosphate In the buffer solution of composition, obtain solution B, dihydrogen phosphate and hydrogen phosphate buffer solution preparation method are that 0.2 g potassium dihydrogen phosphate, 0.2 g potassium chloride, 1.15 g disodium hydrogen phosphate and 8.0 g sodium chloride are dissolved in 1000 mL of water, pH 7.2;

[0028] (b) Slowly add the above solution A into solution B, and stir thoroughly to obtain solution C;

[0029] (c) Add 20 μL of glutaraldehyde aqueous solution with a mass fraction of 25% to solution C dropwise, react at room temperature for 4 hours, and centrifuge at 4000 rpm for 5 minutes;

[0030] (d) Place the liquid obtained in step (c) in a dialysis bag, and dia...

Embodiment 2

[0043] Embodiment two: the preparation of monoclonal antibody:

[0044] (a) Immunize 5 Balb / C mice with the artificial antigen with the structure of formula I as the immunogen. The immunization dose is 100-300 μg / mouse. The immunization method is as follows: fully emulsify the immunogen with the same amount of complete Freund’s adjuvant, Inject subcutaneously at multiple points on the back of the neck, at intervals of 2-3 weeks, emulsify the immunogen with the same amount of Freund's incomplete adjuvant, and then boost the immunization once, a total of 6 booster immunizations;

[0045] (b) Seven days after the last immunization, the mouse with the highest titer of the above serum was selected and sacrificed by cervical dislocation. Under sterile conditions, the spleen was removed, splenocytes were separated, and mixed with mouse myeloma SP2 / 0 cells at a ratio of 10:1. Carry out fusion, use β-naphthol as a standard inhibitor to screen positive hybridoma cells, and then obtain a...

Embodiment 3

[0047] Embodiment three: performance measurement of monoclonal antibody:

[0048] (1) The lowest detection limit (LOD value) and half inhibitory amount (IC) of the antiserum 50 ) detection

[0049] The working concentration of the aforementioned β-naphthol monoclonal antibody and the artificial antigen with the structure of formula 4 was determined by square array titration. Different concentrations of β-naphthol standard substances were used as experimental solutions, and the concentrations were as follows: 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 40 (unit: ng / mL), and 6 groups of parallel experiments (n =6)

[0050] Indirect competitive enzyme-linked immunosorbent assay (ELISA): the working concentration of β-naphthol artificial antigen prepared in the above-mentioned Example 4 was used to coat the microtiter plate, overnight at 4°C or 2 hours at 37°C. Then, shake off the solution in the plate and wash it with PBST for 3-5 times, each time for 3 minutes. Add blocking solution and...

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Abstract

The invention discloses an application of a known compound 1-amino-2-naphthol as beta-naphthol source dye hapten. An artificial antigen and a monoclonal antibody of the corresponding beta-naphtho source dye are prepared; the monoclonal antibody has good immune detection performance for various beta-naphthol source dyes according to experimental data.

Description

technical field [0001] The invention relates to the field of food safety, in particular to the structure and preparation method of a red azo dye family-specific artificial antigen and a broad-spectrum specific monoclonal antibody with β-naphthol as the structural nucleus. Background technique [0002] Azo red dyes are a kind of red dyes containing azo bonds synthesized by chemical methods, mainly used in industry and printing and dyeing industry. Among all red dyes, many varieties are made by coupling β-naphthol with different compounds containing aniline structure as the core structure. Common varieties include Acid Orange No. 2, Sudan Red No. 1, and Sudan Red 2 No., Sudan Red No. 3, Sudan Red No. 4, Parabit Red, Sudan Red G, Sudan Red B, etc. It has been proven that among these red azo dyes, some species (such as Sudan Red No. 1) can cause cancer in humans and animals, so they are listed as Class 3 carcinogens by the World Cancer Research Organization. At the same time, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/77C07K14/765C07K1/107C07K16/44G01N33/577
CPCC07K14/765C07K14/77C07K16/44C07K19/00G01N33/577
Inventor 王建平王萍张会彩刘静
Owner HEBEI AGRICULTURAL UNIV.
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