Aflatoxin M1 Immunoaffinity Column

Operation Manual for Total Aflatoxin M1 Immunoaffinity Column

[Summary]

Aflatoxin is a secondary metabolite produced by fungi such as Aspergillus flavus and Aspergillus parasiticus. It is classified as a Class 1 carcinogen by the World Health Organization's cancer research agency and is also a highly toxic substance that is widely present in various foods. Grains such as corn, peanuts, rice, wheat, and their by-products are the main sources of aflatoxin. At the same time, aflatoxin B1 in feed is metabolized by animals to produce aflatoxin M1, which still has strong toxicity and carcinogenicity, and is widely present in foods such as milk, blood, and tissues. Aflatoxin, as one of the three major causes of liver cancer and the main cause of diseases such as gastric cancer and colon cancer, poses a great threat to human health. Countries around the world have strict regulations on aflatoxins in food and feed. The national standard for dairy safety, which came into effect on June 1, 2010, specifies the detection of aflatoxin M1 in milk and dairy products. The main sample pretreatment method used is the immunoaffinity column purification method.

【 Scope and Performance 】

Our company's fungal toxin immunoassay column series products are tested in the form of a high-performance immunoaffinity column, designed for cleaning (purifying) or concentrating samples before performing HPLC (high-performance liquid chromatography), GC-MS (gas chromatography-mass spectrometry), ELISA (enzyme-linked immunosorbent assay).

Used for qualitative and quantitative detection of aflatoxin M1 in samples such as milk, formula, fermented milk, cheese, cream, and sample pretreatment.

Column capacity: ≥ 100ng

Recovery rate: ≥ 90%

【 Experimental Principle 】

The determination of this product is based on antigen antibody reaction. The monoclonal antibody of aflatoxin M1 is connected to the gel in the column. The aflatoxin M1 in the sample is extracted, filtered and diluted, and then the sample extract is slowly passed through the aflatoxin M1 immunoaffinity chromatography column. In the immunoaffinity column, the toxin is combined with the antibody, and then the immunoaffinity column is washed to remove other substances that are not combined. Wash aflatoxin M1 with methanol and inject it into the analytical instrument for detection.

【 Packaging Composition 】

Aflatoxin M1 standard (optional)

25 pieces/box, 3mL column tube/piece

1 copy of product manual

Equipment and reagents needed but not provided

equipment

High speed homogenizer or tissue homogenizer

Crusher

200mL/50mL measuring cylinder

Balance: Sensitivity 0.1g

Acidimeter

Separation funnel

Micro pipette: single channel 100mL~1000mL

Groove filter paper (folded rapid qualitative or quantitative filter paper)

50mL funnel

Rotary evaporator

Disposable glass test tube

Pump flow control rack (including air pump, glass injectors of different volumes, etc.)

Beakers, volumetric flasks, conical flasks, etc

Reagent:

Methanol (chromatographic grade)

Acetonitrile (chromatographic grade)

Petroleum ether

Distilled water or deionized water

Weigh 2g of solution (0.5mol/L) and dissolve it in 100mL of water

Sample processing steps

（1） Milk

Heat 100mL of milk in a water bath to 40 ° C;

Centrifuge at 4000r/min for 15 minutes, collect 50mL of clear liquid for later use

（2） Milk powder and powdered infant formula food

Weigh 10g of milk powder sample, add 100mL of 50 ° C water in small amounts multiple times, stir to fully dissolve the milk powder

Centrifuge at 6000r/min for 15 minutes, collect 50mL of clear liquid for later use

Weigh 60 g of the mixed sample and adjust the pH to 7.4 using a 0.5 mol/L * solution indicated by an acidity meter;

Use a high-speed homogenizer to homogenize at 9500 rpm for 5 minutes

Heat the water bath to 40 ° C;

Centrifuge at 4000r/min for 15 minutes, collect 50mL of clear liquid for later use

（4） Cheese

Weigh 5g of the sample that has been finely cut and sieved through a 10 mesh circular sieve, and place it in a 50 mL centrifuge tube. Add 2 mL of water and 30 mL of methanol;

Use a high-speed homogenizer to homogenize at 9500r/min for 5 minutes;

Ultrasonic extraction for 30 minutes;

Centrifuge at 6000r/min for 15 minutes, collect the supernatant and transfer it into a 250 mL separatory funnel.

Add 30 mL of petroleum ether (4.3) to the separating funnel and shake for 2 minutes;

After stratification, transfer the lower layer to a 50 mL beaker and discard the petroleum ether layer.

Repeat extraction with petroleum ether twice;.

Transfer the lower solution to a 100 mL round bottom flask and concentrate under reduced pressure to about 2 mL. Pour the concentrate into a centrifuge tube and wash the flask twice with 5 mL of acetonitrile water solution (1+4). Pour the washing solution into a 50 mL centrifuge tube;

Dilute with water to about 50 mL, centrifuge at 6000 rpm for 5 minutes, and purify the supernatant.

（5） Cream

Weigh 5g of the sample and place it in a 50mL beaker;

Dissolve it in 20 mL of petroleum ether and transfer it to a 250 mL stoppered conical flask.

Add 20 mL of water and 30 mL of methanol, shake for 30 minutes, and transfer all the liquid to a separatory funnel;

After stratification, transfer all the lower solution to a 100 mL round bottom flask, concentrate under reduced pressure to about 5 mL in a rotary evaporator, dilute with water to about 50 mL for purification treatment.

Purification steps of affinity column

Connect the immunoaffinity column to a 50.0mL glass syringe. Accurately transfer 50.0mL of clear solution and inject it into a glass syringe;

Connect the air pressure pump to the glass syringe and adjust the pressure to slowly pass the solution through the immunoaffinity column at a flow rate of about 2-3mL/min until 2-3mL of air passes through the column;

Replace the 10ml glass syringe and connect it to the affinity column. Add 10ml of water to the syringe and adjust the pressure to clean the affinity column at a flow rate of approximately 6mL/min.

Accurately add 4mL of chromatographic grade acetonitrile for elution, with a flow rate of 1 mL/min to 2 mL/min. Collect all the elution solution in a glass test tube for detection.

【 Result judgment 】

After purification by an immunoaffinity column, the collected acetonitrile eluent can be directly detected using a fluorescence meter or HPLC, or detected using thin-layer chromatography or enzyme-linked immunosorbent assay kits. The aflatoxin content in the collected 4ml eluent is equivalent to the aflatoxin M1 content in 50ml milk sample, fermented milk sample, or 5g milk powder sample, cheese sample, and cream sample.

If other solvents are required for HPLC detection, the acetonitrile eluent can be dried with nitrogen and then dissolved again

【 Precautions 】

Aflatoxin is extremely harmful and should be operated with gloves.

It is recommended to soak the used glass containers and aflatoxin solution in a 5% concentration sodium hypochlorite solution overnight.

Do not use immune affinity columns that have exceeded their expiration date.

【 Storage conditions and shelf life 】

Storage conditions: The affinity column should be stored at 2-8 ℃ and not frozen.

Shelf life: The product is valid for 12 months.