1.  General

Aflatoxins are secondary metabolites of the fungi species Aspergillus flavus, parasiticus and nomius.  Aflatoxins belong to the strongest natural occurring carcinogenic substances, commonly found in corn, peanuts, cotton seed, human blood and animal feed. Aflatoxin M1 is the hydroxylated metabolite of aflatoxin B1, also a strong carcinogen. Milk and its products are one of the food susceptible to aflatoxin M1 contamination. The methods to test Aflatoxin M1 residue is high-performance liquid chromatography (HPLC), thin layer chromatography (TLC) etc. The use of aflatoxin M1 ELISA kit to analyze aflatoxin M1 residue of samples is fast and accurate.

This kit is a new generation of fungal toxins detection products in application of ELISA technology and the operation time is shorter than 60min, to minimize operating errors and work intensity.

2.  Principle

The test is based on a direct competitive enzyme immunoassay method. The microtiter wells are coated with aflatoxin M1 antigen. Free aflatoxin M1 in sample solutions and the immobilized antigen compete for aflatoxin enzyme conjugate.

While aflatoxin M1 antibody and HRP secondary antibody (HRP material) combined, via the TMB substrate show color, sample absorbance values ??of aflatoxin M1 has a negative correlation with its content, compared with the standard curve and then multiplied by the corresponding the dilution factor, can draw aflatoxin M1 content in samples.

3.  Application

This test kit can be used to detect aflatoxin M1 in Liquid milk, fermented milk, milk powder, cream, condensed milk, cheese etc. samples qualitatively and quantitatively.

4.  Technical specifications

Sensitivity:  0.05 ppb

Detection limit:

Liquid milk ……………………………………………………………………………0.1ppb

Milk powder, fermented milk, light cream, condensed milk, cheese………… 0.15ppb

Recovery rate: ………………………………………………………………………… 90±15%

Coefficient of variation:   <10%

5.  Components

1)  Micro-well strips: 12 strips with 8 removable wells each

2)  1 x High concentrated Aflatoxin M1 standard (810ppb)……………………….1ml

(Remark: work standard 0 ppb (zero standard), 0.05 ppb, 0.15 ppb, 0.45 ppb, 1.35 ppb)

3)  AEM1 Enzyme conjugate ..............................................................................6ml

4)  AFM1 Antibody working solution ...................................................................6ml

5)  Substrate A......................................................................................................6ml

6)  Substrate B......................................................................................................6ml

7)  Stop solution................................................................................................... 6ml

8)  Concentrated washing buffer（10×）...........................................................40ml

9)  Concentrated standard diluent……………………………………………………12ml

10) Concentrated sample diluent..........................................................................40ml

6.  Materials required but not provided

1)  Equipment: microtiter plate spectrophotometer (450 nm/630nm), shaker, centrifuge (or funnel), balance: 0.01g quantity sensitive, micropipettes: single-channel 20ml ~ 200ml, 200ml ~ 1000ml, multi-channel 250ml, gaduated pipettes: 25ml, ear wash bath, filter paper (optional), triangle bottle: 100 ml, polystyrene centrifuge tube: 50ml

2)  Reagents: acetonitrile (analysis pure), deionized water

7.  Sample pre-treatment

Solution preparation before sample pre-treatment:

1)  standard diluent

Use 1 part of concentrated standard diluent and dissolve with 3 parts of distilled water to obtain the ready to use sample diluent. Solution is stable for 1 months when stored at 4 °C.

2)  sample diluent 1

Use 1 part of concentrated sample diluent and dissolve with 7 parts of distilled water to obtain the ready to use sample diluent. Solution is stable for 1 month when stored at 4 °C.

3)  sample diluent 2

Use 1 part of concentrated sample diluent and dissolve with 3 parts of distilled water to obtain the ready to use sample diluent. Solution is stable for 1 month when stored at 4 °C.

4)  sample extract buffer

Use 84 part of acetonitrile and dissolve with 16 parts of distilled water to obtain the ready to use milk powder sample extraction. Solution is stable for 1 month when stored at room temperature.

5)  washing buffer

Use 1 part of concentrated washing buffer （10×）and dissolve with 9 parts of distilled water to obtain the ready to use washing buffer. Solution is stable for 1 month when stored at 4 °C.

6） 5 Aflatoxin M1 standards

Take six 1.5ml centrifuge tubes, numbered from 1 to 6, add 995μl, 400μl, 400μl, 400μl, 400μl, 400μl standard diluent separately to prepare standard liquid as per the following table. For example, add 5μl high concentrated Aflatoxin M1 standard (810ppb) to No.1 tube, mix evenly with 995μl standard diluent to obtain 4.05ppb  Aflatoxin M1 standard. Take 200μl ready to use 4.05ppb to No.2 tube, mix evenly with 400μl standard diluent to obtain 1.35ppb Aflatoxin M1 standard. Take 200μl ready to use 1.35ppb to No.3 tube, mix evenly with 400μl standard diluent to obtain 0.45ppb Aflatoxin M1 standard.  Accordingly to obtain 1.35ppb、0.45ppb、0.15ppb、0.05ppb、0ppb standard . (see following table) . The standard is currently in use in match. Configured standard solution should be used in one week (4 ℃ storage)