Description

1. Principle

AflatoxinM1(AFM1) ELISA Kit is a competitive enzyme immunoassay for the quantitative analysis of AflatoxinM1 in milk, grain and feed, etc.

The test is a competitive direct ELISA that provides exact concentrations in parts per billion (ppb). Free toxin in the sample and controls competes with enzyme-labeled toxin (conjugate) for the antibody binding sites. After a wash step, substrate reacts with the bound enzyme conjugate to produce blue color. A stop solution is then add which changes the color from blue to yellow. The intensity of the color is inversely proportional to the concentration of AflatoxinM1 in the samples and standards.

2. Technical specifications

Sensitivity : 0.025ng/mL (0.025ppb)

Detect time: Approx. 75 min

Detection limit:
Fresh Milk............................................0.25ppb
Yogurt,Milk powder,Cheese..........0.025ppb

Recovery rate:
85 ±10%

Cross-reaction rate:
Aflatoxin M1.....................................100 %
Aflatoxin B1............................approx.1 %
Aflatoxin G1 ..........................approx.1 %
Aflatoxin G2 ...........................approx.1%

Sample Determination

-Fresh Milk
1) Take 50µL fresh milk sample (full-cream milk or skim milk) into 1.5mL Vial.
2) Add 450µL 1×Sample Diluent, mix it well with vortex mixer..
3) Take 50µL for analysis.
Fold of dilution of the sample: 10

-Yogurt ,Milk powder,Cheese
1) Weigh 5.0g of yogurt in 50mL centrifuge, add 10mL 50% Methanol, mix it well with vortex mixer for 5min.
2) Centrifuge the sample at 4000r/min for 10min.
3) Carefully take 2mL supernatant for analysis and transfer it into a 1.5mL Vial.
4) Add 1mL N-hexane and 1.0mL 1×Sample Diluent, mix it well with vortex mixer.
5) Centrifuge the sample at 4000r/min for 5min.
6) Take 50µL lower layer for analysis.
Fold of dilution of the sample: 1

-Feed
1) Weigh 2.0g of ground sample in an 50mL centrifuge tube, add 10mL 50% Methanol, mix it well with vortex mixer for 5min.
2) Filter entire extract by using Whatman No.1.
3) Take 50μL of supernatant , mix with 950μL of 1×Sample Diluent for 30s.
4) Take 50μL 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 HRP Enzyme conjugate into each well, add 50μL AFM1 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 450nm filter.

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Aflatoxin M1 ELISA Kit

Description

Sample Determination

ELISA Procedure