Rapid detection of aflatoxin (immunoaffinity column method)

Aflatoxin is the most toxic mycotoxin discovered so far, and it is a highly toxic substance

Aflatoxins are a group of compounds with similar chemical structures.

The relative molecular mass of aflatoxin is 312~346, it is hardly soluble in water, easily soluble in organic solvents such as oil, methanol, acetone and chloroform, but insoluble in petroleum spirit, hexane and acetone

Aflatoxins are often found in soil, animals and plants, and various nuts, especially peanuts and walnuts.

Aflatoxin is a highly toxic substance, and its toxicity is equivalent to 10 times that of potassium cyanide and 68 times that of arsenic

According to reports from the World Health Organization, aflatoxin content of 30-50μg/kg is low toxicity, 50-100μg/kg is toxic, 100-1000μg/kg is highly toxic, and more than 1000μg/kg is extremely toxic

The relevant laws of the US federal government stipulate that the aflatoxin content in human consumption food and dairy cattle feed (referring to the total amount of aflatoxin B1 + aflatoxin B2 + aflatoxin G1 + aflatoxin G2) shall not exceed 15 μg/kg for human consumption.

[Detection principle]

Using a monoclonal immunoaffinity column as a separation method, a large dose of aflatoxin monoclonal antibody is solidified on a water-insoluble carrier.

[Instruments and reagents]

(1) Instrument 4 series of special fluorescence analyzer for mycotoxins, 1.

(2) Reagent 0.

[Operation method]

(1) Sample preparation: Weigh 25g of finely ground sample (measured directly for liquid samples), 5g of sodium chloride, place them in a stirring cup, add 125mL of methanol-water solution, close the lid, stir at high speed for 1min, filter, and take the filtrate for later use

(2) Detection: Pipette 20mL of filtrate, add 20mL of water, and filter with glass fiber filter paper; take 10mL of filtrate and pass through the affinity column at a flow rate of 1 to 2 drops/s until air enters the affinity column

(3) Pass 10 mL of water through the affinity column at a flow rate of 2 drops/s, and repeat it once until the air enters the affinity column

(4) Wash the affinity column with 10 mL of chromatographically pure methanol at a flow rate of 1 to 2 drops/s, collect all sample eluent in a test tube, add 1.

[Result judgment]

Measure with a fluorometer: place the prepared sample liquid test tube in a calibrated fluorometer, and read after 60 seconds.

Calculation formula:

F = KP

In the formula, F——fluorescence intensity;

K——Fluorescence absorption coefficient;

P——concentration of tested substance, g/100mL;

Measuring range: 0~300μg/kg

[Precautions]

(1) Adding bromine solution to the eluent for derivatization can improve the sensitivity of the determination
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(2) When the fluorometer is zeroed, put pure water in the test tube and calibrate the fluorometer to read 0
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(3) The immunoaffinity column overcomes the use of highly toxic mycotoxins as calibration standards during the operation of thin-layer chromatography and the use of a variety of toxic and odorous organic solvents during sample pretreatment, which can poison operators and pollute the environment.
The disadvantages
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