Description

1. Principle

Total Aflatoxins ELISA Test Kit is a competitive enzyme immunoassay for the quantitative analysis of Aflatoxin B1,B2,G1,G2,M1,M2 in cereal grains and feed , etc.

An antigen with a affinity for Aflatoxins is coated onto microwells. Standard or sample is added to the appropriate well and if Aflatoxins is present it will compete with the coated antigen to bind to Aflatoxins antibody. After the addition of the enzyme conjugate, the TMB substrate is added for coloration. The resulting color intensity, has an inverse relationship with the target concentration in the sample. The levels of Total Aflatoxins in the sample are determined by comparison of the color intensity of unknown samples with the standard curve, which is made using the Aflatoxins standards provided in the kit.

2. Technical specifications

Sensitivity : 0.025ppb

Detect time: Approx. 75 min

Detection limit:
Grains...........................2.0 ppb
Feed.............................1.0 ppb
Oil................................ 2.0 ppb
Milk..............................0.5 ppb

Recovery rate:
Grains,Milk,Oil................85 ±10%
Feed..................................75 ±10%

Cross-reaction rate:
Aflatoxin B1........................100 %
Aflatoxin M1........................90%
Aflatoxin B2.........................65%
Aflatoxin G1.........................10%
Aflatoxin G2..........................5%

Sample Determination

-Grains(Rice,what,corn)

1) Weigh 2.0g homogenized sample into 50mL tube, add 10mL 50% Methanol,

shake properly with oscillator for 5min.

2) Filter with Whatman No1.paper.

3) Take 50μL supernatant + 950μL 1×Sample Diluent, mix for 30s.

4) Take 50μL for analysis.

Fold of dilution of the sample: 100

-Soy sauce, vinegar

1) Weigh 2.0g sample into 50mL tube, add 3mL Deionized water and 10mL Trichloromethane ( CHCl3), shake properly with oscillator for 5min.

2) Centrifuge the sample at 4000r/min for 10min.

3) Take 5mL lower layer to new vial, dry at 50℃ by nitrogen.

4) Dilute dry residue using 1mL 50% Methanol.

5) Take 50μL, add 950μL 1× Sample Diluent, mix for 30s.

6) Take 50μL for analysis.

Fold of dilution of the sample: 20

-Milk

1) 50μL milk+ 950μL 1×Sample Diluent, shake for 30 s.

2) Take 50μL milk for analysis.

Fold of dilution of the sample: 20

-Food Oil

1) Take 1.0g oil into 10mL tube, add 2mL n-hexane, then add 5mL 50% Methanol,

shake properly with oscillator for 5min.

2) Centrifuge at above 4000 r/min for 10min.

3) Take 50μL , add 200μL 1×Sample Diluent, mix for 30s .

4) Take 50μL for analysis.

Fold of dilution of the sample: 25

-Wine, beer, rice wine

1) Weigh 2.0g sample into 50mL tube, add 10mL Trichloromethane ( CHCl3), shake properly with oscillator for 5min.

2) Centrifuge the sample at 4000r/min for 10 min.

3) Take 5mL lower layer to new vial， into another tube, dry at 50℃ by nitrogen.

4) Dilute dry residue using 1mL 50% Methanol.

5) Take 50μL, add 950μL 1× Sample Diluent, mix for 30s.

6) Take 50μL for analysis.

Fold of dilution of the sample: 20

-Peanut

1) Weigh 1.0g of ground sample into 50mL centrifuge tube, add 5mL N-hexane, add 10mL 50% Methanol, shake properly with oscillator for 5min.

2) Centrifuge the sample at 4000r/min at room temperature for 10min.

3) Take 50μL of lower layer, mix with 450μL of 1×Sample Diluent.

4) Take 50μL for analysis.

Fold of dilution of the sample: 100

-Moon cake, peach cake, peanut butter

1) Weigh 2.0g crushed samples in 50mL centrifuge tube, add 5mL N-hexane and 10mL 50% methanol，shake properly with oscillator for 5min.

2) Centrifuge at 4000r/min at room temperature for 10min.

3) Take 5mL lower liquid into 50mL centrifuge tube; add 10mL Trichloromethane, shake for 5min , Centrifuge at 4000r/min at room temperature for 10 min.

4) Take 5mL lower layer into clean container , dry with nitrogen/air at 50℃.

5) Add 1mL of 50% methanol, shake properly.

6) Take 50μL, mix with 950μL 1×Sample Diluent, mix for 30s.

7) Take 50uL for analysis.

Fold of dilution of the sample: 40

-Wheat Powder

1) Weigh 2.0g crushed samples in 50mL centrifuge tube, add 10mL 50% methanol，shake properly with oscillator for 5min.

5) Filter with Whatman No1.paper.

2) Take 5mL liquid into 50mL centrifuge tube; add 10mL Trichloromethane, shake for 5min , Centrifuge at 4000r/min at room temperature for 10 min.

3) Take 5mL lower layer into clean container , dry with nitrogen/air at 50℃.

4) Add 1mL of 50% methanol, shake properly.

5) Take 50μL, mix with 950μL 1×Sample Diluent, mix for 30s.

5) Take 50uL for analysis.

Fold of dilution of the sample: 40

-Fresh Milk

1) Take 50µL fresh milk sample (full-cream milk or skim milk) into 1.5mL Vial.

2) Add 950µL 1×Sample Diluent, mix it well with vortex mixer..

3) Take 50µL for analysis.

Fold of dilution of the sample: 20

-Milk powder,Cream,cheese

1) Weigh 5.0g homogenized sample into 50mL centrifuge tube, add 10mL

50% Methanol, shake properly with oscillator for 5 min.

2) Centrifuge at above 4000 r/min for 10 minutes.

3) Take 2mL supernatant into another tube, dry at 50℃ by nitrogen.

4) Add 1mL N-hexane, shake properly, then add 1mL 1×Sample Diluent, mix for 30s .

5) Centrifuge at above 4000 r/min for 5min.

6) Take 50μL lower layer for analysis.

Fold of dilution of the sample: 1

-Feed

1) Weigh 1.0g homogenized sample into 50mL centrifuge tube, add 10mL 50% Methanol, shake properly with oscillator for 5min.

2) Filter with Whatman No1.paper, take supernatant.

3) Take 50μL supernatant+450μL Sample Diluent, mix for 30s .

4) Take 50μL lower layer for analysis.

Fold of dilution of the sample: 100

ELISA Procedure

1) Allow reagents, microwells, and sample extracts to reach room temperature prior to running the test.

2) Insert a sufficient number of wells into the microwell holder for all standards and samples to be tested.

3) Using a new pipette tip for each standard and sample, add 50μL of standards and prepared sample to separate wells.

4) Add 50μL of AFB HRP Enzyme conjugate into each well，add 50μL of AFB Antibody into each well, incubate for 30 min at room temperature.

5) Dump the contents of the wells. Turn the wells upside down and tap out on a paper towel until the remaining liquid has been removed.

6) Fill completely all the wells with working wash solution 250μL, mix for 30 seconds using a plate mixer, empty them by inverting the plate, repeat 4 times. Finally, remove the residual droplets by vigorous knocking on absorbent laboratory towels.

7) Add 50μL Substrate A and 50μL Substrate B to each well.

8) Incubate for 15 min at room temperature. Cover the wells with a paper towel to protect them from light sources.

9) Add 50μL of stop solution to each well.

10) After thorough mixing, the absorbance is measured using a microplate reader fitted with a 450 nm filter.

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Total Aflatoxins ELISA Kit

Description

Sample Determination

ELISA Procedure