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Enzyme-linked immunosorbent assay kit for detecting Sudan red and paranitroaniline red medicaments and application thereof

An enzyme-linked immunosorbent reagent, Sudan red technology, applied in the detection of Sudan red and para red drug residues in chili sauce, feed samples, and chili oil, can solve the problem of expensive equipment, unsuitable for on-site monitoring and screening of a large number of samples , The detection process is cumbersome and other problems

Active Publication Date: 2012-07-04
BEIJING WANGER BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, most domestic and foreign detection methods for Sudan Red in food are detected by liquid chromatography or liquid phase coupled with mass spectrometry. Large Sample Screening

Method used

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  • Enzyme-linked immunosorbent assay kit for detecting Sudan red and paranitroaniline red medicaments and application thereof
  • Enzyme-linked immunosorbent assay kit for detecting Sudan red and paranitroaniline red medicaments and application thereof
  • Enzyme-linked immunosorbent assay kit for detecting Sudan red and paranitroaniline red medicaments and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] The preparation of embodiment 1 kit components

[0063] 1. Synthesis of Immunogen

[0064] The immunogen was obtained by coupling the Sudan red hapten with bovine serum albumin by a diazotization method.

[0065] Immunogen preparation process:

[0066] 1) Take 25 mg of p-diaminobenzidine, add 4.5 ml of 0.2 mol / L hydrochloric acid to dissolve, and bathe in ice;

[0067] 2) Dissolve 10-20 mg of sodium nitrite in 0.5 ml of water and add it to (1), stir and react on ice for 0.5-1 hour, take a part for the next reaction, and store the rest at -20°C;

[0068] 3) Dissolve 20-100mg of BSA in PH9.0, 5ml boric acid buffer to prepare a BSA solution;

[0069] 4) Dissolve 5-20mg of β-naphthol with pH9.0 and 5ml of boric acid buffer to prepare a β-naphthol solution;

[0070] 5) Mix the BSA solution and the β-naphthol solution and put it on ice, add 1-3ml diazotized p-diaminobenzidine solution, and react in the dark for 12-24h;

[0071] 6) Dialyze with pH7.4, 0.01mol / L phosphate ...

Embodiment 2

[0099] Embodiment 2 detects the formation of the ELISA kit of Sudan Red

[0100] Set up an enzyme-linked immunosorbent assay kit for detecting Sudan Red to include the following components:

[0101] (1) A microtiter plate coated with Sudan red-coupled antigen;

[0102] (2) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0103] (3) Sudan red monoclonal antibody working solution;

[0104] (4) 6 bottles of Sudan red standard solution, the concentrations are 0μg / L, 0.5μg / L, 1.5μg / L, 4.5μg / L, 13.5μg / L, 40.5μg / L;

[0105] (5) The substrate chromogenic solution is composed of A liquid and B liquid, the substrate chromogenic liquid A liquid is carbamide peroxide, and the substrate chromogenic liquid B liquid tetramethylbenzidine;

[0106] (6) The stop solution is 2mol / L sulfuric acid;

[0107] (7) The concentrated washing solution has a pH value of 7.2 to 7.6, a phosphate buffer solution containing 1.0% to 3.0% Tween-20, and 0.02 to 0.04‰ thimerosal preservative...

Embodiment 3

[0109] The detection of Sudan Red in the sample of embodiment 3

[0110] 1. Sample pretreatment

[0111] a) chili paste

[0112] Weigh 3±0.05g of homogeneous sample into a centrifuge tube, add 6ml of acetonitrile, vortex for 30s, and centrifuge at 4000rpm for 3min at room temperature; pipette 2ml of supernatant and dry it with nitrogen at 50°C; add 1ml of 2mol / L hydroxide Sodium solution, vortex for 30s; add 2ml of n-hexane and vortex for 10s to dissolve the residue; centrifuge at 4000rpm for 3min at room temperature; pipette 1ml of supernatant and dry it with nitrogen at 50°C; add 0.5ml of DMF to fully dissolve the residue; take 50μl and add Mix 950 μl complex solution; take 50 μl for analysis.

[0113] b) Chilli oil, feed samples

[0114] Weigh 1±0.05g sample into a centrifuge tube, add 2ml 2mol / L sodium hydroxide, add 8ml acetonitrile, vortex for 30s, and centrifuge at 3800rpm for 3min; absorb 1ml supernatant, add 1.5ml water, mix well, and put on C18 column Purify (con...

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Abstract

The invention provides an enzyme-linked immunosorbent assay kit for detecting Sudan red and paranitroaniline red medicaments. The enzyme-linked immunosorbent assay kit comprises an enzyme-linked immunosorbent assay plate coated with a coating antigen, an enzyme label, a Sudan red specific antibody working solution (occurring when the antigen is coated on the enzyme-linked immunosorbent assay plate and the enzyme label is of an enzyme-labeled anti-antibody or the anti-antibody is coated on the enzyme-linked immunosorbent assay plate and the enzyme label is of an enzyme-labeled antigen), a Sudan red standard product solution, a substrate color development solution, a stop solution, a concentrated cleaning solution and a concentrated complex solution. The invention further discloses a method for detecting Sudan red and paranitroaniline red by applying the enzyme-linked immunosorbent assay kit, and the method comprises the following steps: firstly performing pretreatment on a sample, then detecting through the kit and finally analyzing a detection result. The enzyme-linked immunosorbent assay kit provided by the invention can be used for detecting the content of the Sudan red and the paranitroaniline red in the samples, such as chili sauce, chili oil, feed and the like, has the advantages of simplicity and convenience in operation, low cost, high sensitivity and capability of performing site monitoring, and is suitable for screening a large number of the samples.

Description

technical field [0001] The invention relates to an enzyme-linked immunosorbent detection technology, in particular to an enzyme-linked immunosorbent assay kit for detecting Sudan red and para-red drugs, which is especially suitable for Sudan red and para-red drugs in chili sauce, chili oil, and feed samples residual detection. technical background [0002] Sudan red (Sudan I) belongs to one of artificially synthesized azo chemical dyes, and is mainly used for dyeing industrial products such as engine oil, wax and shoe polish. Foreign animal experiments have shown that Sudan red dye can cause cancer in mice, and its metabolite aniline can directly act on human liver cells, thereby causing toxic liver disease. Long-term intake of aniline can cause damage to the human nervous system. For this reason, the International Cancer Research The agency lists it as the third class of carcinogens. Although such substances lack sufficient evidence to directly cause human cancer, their p...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/543G01N33/535
Inventor 汪善良冯静扶胜余厚美段盈盈何丽霞
Owner BEIJING WANGER BIOTECH
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