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 direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of being unsuitable for on-site monitoring and screening of a large number of samples, expensive equipment, and cumbersome detection process
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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 the ELISA 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;
[0108] (8) The concent...
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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