Rapid detection of aflatoxin (ELlSA method)

[Detection principle]

The known antigen is adsorbed on the surface of the solid carrier, the unadsorbed antigen is washed away, a certain amount of antibody and the mixture of the sample (containing the antigen) extract are added, and after competitive culture, the antigen-antibody complex is formed on the surface of the solid carrier

[Instruments and reagents]

Instruments: microplate, constant temperature incubator, microplate reader, vortex oscillator

Reagents:

(1) tetramethylbenzidine , 30% hydrogen peroxide , bovine serum albumin (BSA), Tween-20 and the like

(2) Antibody specific monoclonal antibody (or antiserum) against aflatoxin B1

(3) The conjugate of the coating antigen aflatoxin B1 and the carrier protein (BSA or polylysine, etc.

(4) Enzyme-labeled secondary antibody goat anti-mouse immunoglobulin G combined with horseradish peroxidase

(5) The buffer system coating buffer is a phosphate buffer of pH 9.

(6) The standard solution of aflatoxin B1 is prepared with methanol to prepare a 1mg/mL aflatoxin B1 stock solution, and stored in a refrigerator at -20℃.

(7) Substrate solution 10mg tetramethylbenzidine in 1mL dimethylformamide, take 75uL tetramethylbenzidine solution, add 10mL substrate buffer, add 10μL 30% hydrogen peroxide solution

[Operation method]

(1) Sample processing Weigh 10g of the crushed sample in an Erlenmeyer flask, use 50mL acetonitrile -water (50% + 50%, volume fraction), use 2mol/L carbonate buffer to adjust the pH to 8.

(2) Coating the enzyme-labeled microtiter plate with coating antigen (coating buffer diluted to 10μg/mL), 100ul per well at 4℃ overnight

(3) Wash the enzyme-labeled microplate with washing solution 3 times, 3 minutes each time, add 50uL series aflatoxin B1 standard solution and 50μL sample extract to each well, then add 50μL diluted antibody, and incubate at 37°C for 1.

(4) Wash the microtiter plate with enzyme-labeled washing solution 3 times, 3min each time, add 100uL enzyme-labeled secondary antibody to each well, and incubate at 37°C for 2h

(5) Wash the microtiter plate with washing solution 3 times, add 100uL substrate solution 372 to each well and incubate for 0.

[Result judgment]

Calculation:

Learning unit two-meter noodle products rapid detection

In the formula, m'——the amount of aflatoxin measured on the microtiter plate, ng (calculated according to the standard curve);

v ₁ ——The volume of the sample extraction solution, mL;

v ₂ ——The volume of the dripped sample solution, mL;

d——the total dilution factor of the sample solution;

m is the mass of the sample, g