Solution for aflatoxin M1 detection in milk and dairy products

First, the background introduction

Aflatoxin M1 belongs to one of the structurally similar compounds of aflatoxin, and has the highest probability of aflatoxin in foods and feeds in hot and humid regions. The physicochemical properties are fairly stable and are not destroyed by pasteurization. When a mammal receives a feed or food contaminated with aflatoxin B1, it is converted to aflatoxin M1 by hydroxylation. Aflatoxin M1 is mainly caused by carcinogenicity and mutagenicity, and has a destructive effect on human and animal liver tissues, which can lead to liver cancer and even death.

At present, the detection methods of Aflatoxin M1 include high performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay. Liquid chromatography can accurately detect the small amount of aflatoxin in dairy products. The aflatoxin M1 ELISA kit allows rapid and accurate analysis of aflatoxin M1 residues in the sample.

As one of the major suppliers of mycotoxin detection technology and product services, Pribolab integrates the original Beijing Tellabs testing application laboratory to focus on the services of mycotoxin testing products and technical solutions. Quickly propose a solution. A complete set of solutions has been established for the problem of excessive aflatoxin M1 in milk and its products for several years.

Second, the reference standard : according to the national standard "GB 5413.37-2010" national food safety standards for the determination of aflatoxin M1 in milk and dairy products.

(1) Immunoaffinity column-high performance liquid chromatography : in line with GB 5413.37-2010 national standard method

1 .   Required equipment and supplies

High performance liquid chromatography

Column: Mycotoxin-specific chromatography column (PRC-18, Pribolab).

Immunoaffinity column: PriboFast® aflatoxin M 1 immunoaffinity column ( IAC-012-3, Pribolab, 25 pcs/box , fast column type, suitable for viscous samples: such as raw milk, yogurt, etc., effective Solve the problem of easy column plugging ) .

High-speed homogenizer: stainless steel is corrosion-resistant and has a rotational speed of over 22,000 rpm (EQ-WR-1L, Pribolab).

Glass fiber filter paper: PriboFast® glass fiber filter paper (GMF/A-110 , Pribolab )

PriboFast® eight-stage pump flow operator ( Pribolab for controlling the flow rate of the immunoaffinity column )

Standard : Aflatoxin M 1 (solid standard MSS1007 100ug or 0.5μg/ml Aflatoxin M 1 liquid standard , Pribolab )

2 .   Sample preparation: Prepbang offers different treatment options for raw milk, cheese, cream, etc. (For details, please contact Pribolab China, Puri State has developed an optimized pretreatment solution for multiple milk bases)

3. Immunoaffinity column purification : PriboFast® aflatoxin M 1 immunoaffinity column ( IAC-012-3, Pribolab)

Enrichment: the extract passes through the immunoaffinity column at a flow rate of 2-3 mL/min until 2-3 mL of air passes through the column;

Washing: 10 mL of 0.1% Tween/PBS wash, wash the column twice with 10 mL of water, and pass 2-3 mL of air through the column.

Elution: Elution was carried out by adding 1.0 mL of chromatographic grade methanol, incubating for more than 30 seconds, and passing the column at a flow rate of 1-2 mL/min, and collecting all the eluents for detection.

4. High performance liquid chromatography analysis

Column: C18, 25 cm long, 4.6 mm internal diameter;

Mobile phase: 25% aqueous acetonitrile (10.2.1);

Flow rate: 1 mL/min.

(two) enzyme-linked immunosorbent assay

Method principle

Aflatoxin M1 antigen was pre-coated on a microplate using direct competition enzyme-linked immunosorbent assay , and a sample (or aflatoxin M1 standard solution) and horseradish peroxidase-labeled aflatoxin M1 antibody were added. The antigen in the sample or standard solution competes with the antigen pre-coated on the plate well to bind to the enzyme-labeled antibody. Unbound enzyme-labeled antibodies are removed upon washing. Add TMB coloring solution and read the absorbance. The absorbance of the sample is inversely related to the amount of aflatoxin M1 contained therein , and the content of aflatoxin M 1 in the sample can be obtained by comparison with a standard curve .

2. Reagents and materials: ELISA kit (PriboFast R aflatoxin M1   Kit EKT-012 , Pribolab )

3. Pre-treatment of the sample: milk: centrifuge to remove the lower layer for testing. Milk powder: reconstituted and centrifuged to remove the lower layer for testing.

4. Detection steps

4.1 The immune reaction was carried out for 30 min; the plate was washed 5 times; the color reaction was carried out for 30 min; the reaction was terminated, and the OD value was read by the microplate reader.

5. Expression of analysis results

The results were interpreted using a microplate reader.

(3) Rapid detection method of immune gold standard

Method principle

The aflatoxin M1 (AFM1) rapid detection strip uses the principle of competitive inhibition immunochromatography. The aflatoxin M1 in the sample binds to the colloidal gold-labeled specific anti-aflatoxin M1 monoclonal antibody in the enzyme-labeled well. The antibody and NC membrane were tested for binding to the AFM1-BSA conjugate on the line. If the content of aflatoxin M1 in the sample is greater than 0.5 ug/L, the test line does not show color, and the result is positive; otherwise, the test line is red, and the result is negative.

2. Reagents and materials

Colloidal gold test strip: Pribo Strip TM aflatoxin M1 immunogold standard rapid test strip ( PR-01 2 , Pribolab )

3. Analysis steps: detection after reconstitution of milk powder; direct detection of liquid milk. Pipette the milk droplets into the well and read the results for 8-15 minutes. In the case of C-line color development: T-line color development is negative or deeper than C-line. The T line is lighter than the C line, or no coloration is a positive result.