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 B1 appears nearly in all cases together with Aflatoxin B2, G1 and G2 and it is the analyte with the highest toxic significance.Thin-layer chromatography has been a common method of aflatoxin detection, but sample preparation and samples analysis of this method is time-consuming and exhausting. The use of aflatoxin B1 ELISA kit to analyze aflatoxin B1 residue of samples is fast and accurate. Refer to the National Standard GB / T 5009.22-2003.

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 antigen. Free aflatoxin in sample solutions and the immobilized antigen compete for aflatoxin enzyme conjugate. Any unbound enzyme conjugate is then removed in a washing step. Substrate/chromogen is added to the wells, bound enzyme conjugate converts the chromogen into a blue product. The addition of the stop solution leads to a color change from blue to yellow. The measurement is made photometrically at 450 nm. The absorbance is inversely proportional to the aflatoxin concentration in the sample.

3.  Application

This test kit can be used to detect aflatoxin B1 in maize, rice, wheat, beans, peanuts, peanut butter, feed, edible oil and other samples qualitatively and quantitatively.

4.  Technical specifications

Sensitivity: 0.1 ppb

Detection limit:

Peanuts, peanut butter, cereals, pastries …………………………………………2ppb

Feed ………………………………………………………………………………...2.4ppb

Feed (recommended method) ……………………………………………………1.2ppb

Edible oil …………………………………………………………………………….1.5ppb

Soy sauce, vinegar, wine, dry miso, hot pot bottom material…………………….1ppb

Recovery rate:

Peanuts, maize and other cereal samples, cookies, edible oil, vinegar, wine… 90±15%

Honey cake, soy sauce, animal feed………………………………………………. 85±15%

Dry miso……………………………………………………………………………….. 80±10%

Pot bottom material…………………………………………………………………… 95±10%

Coefficient of variation:   <10%

5.  Components

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

2)  6 x Concentrated Aflatoxin B1 standards…………………………………...1ml each

0 ppb (zero standard), 1 ppb, 3 ppb, 9 ppb, 27 ppb, 81 ppb

Standard working concentration: 0ppb, 0.1ppb, 0.3ppb, 0.9ppb, 2.7ppb

3)  AFB1 Enzyme conjugate ................................................................................ 6ml

4)  AFB1 Antibody working solution………………………………………………….. 6ml

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

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

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

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

9)  AFB1 Concentrated sample diluent （2×）................................................. 40ml

6.  Materials required but not provided

1)  Equipment: microtiter plate spectrophotometer (450 nm/630nm), shaker, vortex shaker, centrifuge, water bath, balance: 0.01g quantity sensitive, rotary evaporator/ nitrogen-drying device, micropipettes: single-channel 20ml ~ 200ml, 200ml ~ 1000ml, multi-channel 250ml, gaduated pipettes: 10ml, ear wash ball, funnel, separatory funnel, beaker: 50 ml, polystyrene centrifuge tube: 10ml,50ml, quantitative analysis of filter paper

2)  Reagents: methanol（analysis pure）, deionized water, dichloromethane, petroleum ether/n-hexane

7.  Sample pre-treatment

Solution preparation before sample pre-treatment:

1)  sample diluent

(for sample: peanuts and peanut butter; Corn, wheat etc. ; Cookies, honey cake)

Use 1 part of AFB1 Concentrated sample diluent （2×）and dissolve with 1 parts of deionized water to obtain the ready to use sample diluent. Solution is stable for 1 months when stored at 4 °C.

2)  washing buffer

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

3)  sample extract buffer 1

Use 3 part of methanol and dissolve with 2 parts of deionized water to obtain the ready to use sample extract.

* Note: sample extract buffer 1 is recommended method suitable for peanuts, peanut butter; corn, wheat and other grains; cookies, honey cake; dry miso, hot pot bottom material and feed etc.

4)  sample extract buffer 2 (for feed sample method one)

Use 4 part of methanol and dissolve with 1 parts of deionized water to obtain the ready to use sample extract.

5)  sample extract buffer 3 (for edible oil sample)

Use 1 part of methanol and dissolve with 1 parts of deionized water to obtain the ready to use sample extract.

6)  6 Aflatoxin B1 standards

Take six 1.5ml centrifuge tubes, numbered from 1 to 6. Take six concentrations of standard (10 ×) (0ppb, 1ppb, 3ppb, 9ppb, 27ppb, 81ppb) 50μl ,add to the above six tubes, then each tube adding 450μl deionized water to dilute them by 1: 9, obtain 0ppb, 0.1ppb, 0.3ppb, 0.9ppb, 2.7ppb, 8.1ppb the standard solution(see the following table). Configured standard solution should be used in one week (4 ℃ storage)