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.

2. Test principle

This test kit is based on the competitive enzyme immunoassay for the detection of AFLATOXINS in the sample. The coupling antigens are pre-coated on the micro-well stripes. The AFLATOXINS in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti-AFLATOXINS antibodies. After the addition of the enzyme conjugate, the TMB substrate is added for coloration. The optical density (OD) value of the sample has a negative correlation with the AFLATOXINS in it. This value is compared to the standard curve and the AFLATOXINS concentration is subsequently obtained.

3. Application

This test kit can be used to detect total aflatoxins in maize, corn, feed, edible oil and other samples qualitatively and quantitatively .

4. Reagents provided

Each kit contains sufficient materials for 96 measurements (including standard analyses). Each test kit contains:

1 x Microtiter plate (12 strips with 8 removable wells each) coated with capture antigen

6 x standards, 1 ml each: 0 ppb (zero standard), 0.1 ppb, 0.3 ppb, 0.9 ppb, 2.7 ppb, 8.1 ppb

1 x High concentration standard, 1ml     100ppb

1 x Enzyme conjugate......................................................... 10 ml

1 x Antibody working solution.............................................6 ml

1 x Substrate A.........................................................................10ml

1 x Substrate B............................................................ ......10ml

1 x Stop solution......................................................................6ml

1 x Concentrated washing buffer.......................................40ml

5. Reagents required but not provided

5.1. Equipment:

− microtiter plate spectrophotometer (450 nm/630nm)

− shaker

− vortex Shaker

− centrifuge

− water bath

− blance: 0.01g quantity sensitive

− gaduated pipettes: 10ml

−rubber suction bulb

− polystyrene centrifuge tube: 10ml,50ml

− filter funnel

− Rotary evaporator/nitrogen evaporator

− Glass Centrifuge Tube:10ml

− Variable 20 μl - 200 μl und 200 - 1000 μl micropipettes

− filter

5.2. Reagents:

− methanol

− distilled or deionized water

− petroleum ether /n-hexane

6. Solutions

a. washing buffer

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

b. 60/40 (v/v) MeOH/Water

Add 600 mL MeOH to 400 mL distilled water.

c. 70/30 (v/v) MeOH/Water

Add 700 mL MeOH to 300 mL distilled water.

7. Samples Preparation

7.1 Sample Preparation of peanut

− weigh 10 g of ground sample into a suitable container and add 30 ml of 60/40 (v/v) MeOH/Water

− shake vigorously for five minutes (manually or with shaker)

− filter the extract through filter or centrifuge at 3500r/min for 5 minutes.

− dilute 1 ml of the obtained filtrate with 5 ml of distilled water.

− use 50 μl of the diluted filtrate for test

Dilution factor: 18

7.2 Preparation of animal feed and corn sample

− weigh 3 g of ground sample into a suitable container and add 15 ml of 70/30 (v/v) MeOH/Water.

− shake vigorously for 10 minutes (manually or with shaker)

− filter the extract through filter or centrifuge at 3500r/min for 5 minutes.

− dilute 1 ml of the obtained filtrate or suppernant with 6 ml of distilled water.

− use 50 μl of the diluted filtrate for test

Dilution factor: 35

7.3 Preparation of edible oil

− weigh 5 g of edible oil sample into a beaker

− Transfer the sample into a 125ml separating funnel with 10ml petroleum ether /n-hexane

− Add 15ml 60/40 (v/v) MeOH/Water, cover tightly and shake for 5min. Put it aside for layering and then release the lower methanol extract

− dilute 1 ml of the obtained extract with 5 ml of distilled water/ deionized water.

− use 50 μl of the diluted extract for test

Dilution factor: 18

8. Warnings and precautions for the users

a.The standard solutions contain aflatoxin B1, particular care should be taken. Avoid contact of the reagent with the skin (use gloves).

b. Decontamination of glassware and aflatoxin B1 solutions is best carried out using a sodium hypochlorite (bleach) solution (10 % (v/v)), adjust solution with HCl to pH 7) overnight.

e. The stop solution contains 2 M sulfuric acid.