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Aflatoxin M1

This test kit is based on the indirect competitive enzyme immunoassay for the detection of Aflatoxins M1. The coupling antigen is pre-coated on the micro-well stripes. The Aflatoxins M1 in the sample and the coupling antigens pre-coated on the micro-well stripes compete for the anti- Aflatoxins M1 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 M1 in the sample. This value is compared to the standard curve and the Aflatoxins M1 residues is subsequently obtained.

2.  Technical specifications

Sensitivity 0.03 ppb

Incubator temperature:

Incubator time:  30min~15min

Detection limit:

Milk …………………………………………………………...0.1ppb

Milk powder, Yogurt ………………………………………...0.3ppb

Cross-reaction rate:

Aflatoxins M1… ….100%

Aflatoxin B1… ….12.9%

Aflatoxin B2… ….1.4%

Aflatoxin G1… ….1.9%

Aflatoxin G2… ….0.3%

Recovery rate:

Milk……………………………………………………………………….… 90±25%

Milk powder, Yogurt……………………………………………………… 100±30%

 



3.  Components

1

Micro-well strips

12 strips with 8   removable wells each

2

6× standard solution   (1 mL each)

0ppb

0.03ppb

0.09ppb

0.27ppb

0.81ppb

2.43ppb

3

Enzyme conjugate

7ml

red cap

4

Antibody working   solution

7ml

blue cap

5

Substrate A

7ml

white cap

6

Substrate B

7ml

black cap

7

Stop solution

7ml

yellow cap

8

20× concentrated   washing buffer

40ml

white cap

9

10× concentrated redissolving solution

50ml

transparent cap



4.  Materials required but not provided

1) Equipment: ELISA Reader (450 nm/630nm), homogenizer, shaker, centrifuge, balance: 0.01g quantity sensitive, nitrogen-drying device, incubator, graduated pipettes, printer

2) Micropipettes: single-channel 20ml ~ 200ml, 100ml ~ 1000ml, multi-channel 30~300 μl

5. Sample pre-treatment

Instructions (The following points must be dealt with before the pre-treatment)

1) Only the disposable tips can be used for the experiments and the tips must be changed when used for absorbing different reagents;

2)  Before the experiment, each experimental utensil must be clean and should be re-cleaned if necessary, in order to avoid the contamination that interferes with the experimental results.

Solution preparation before sample pre-treatment:

1)  Sample redissolving solution

Use 1 part of 10x concentrated redissolving solution and dissolve with 9 parts of deionized water to obtain the ready to use sample redissolving solution.

Samples Preparation

5.1  Preparation of raw milk and finished milk samples

1) Take raw milk and finished milk samples, put them at room temperature, test them directly when samples return back to room temperature.

2) Take 50 μl to test

Dilution factor: 1

5.2 Preparation of milk powder sample

1) Take 1.0±0.05g milk powder sample into a 50ml centrifuge tube; add 10ml Sample redissolving solution, shake thoroughly for 3min, centrifuge at above 4000r/min at 20℃ for 10 min;

2) Take 50µl supernatant to test.

Dilution factor: 10

5.3  Preparation of Yogurt sample

1) Take 1ml yogurt sample, dilute with Sample redissolving solution at 1:9 (100ul yogurt + 900ul Sample redissolving solution), mix for 30s;

      2)  Take 50 μl to test

Dilution factor: 10

6.  ELISA procedures

6.1 Instructions

1.  Bring ELISA reagents to room temperature (20 - 25 °C) before use.

2.  Put ELISA reagents back to 2-8 ℃ immediately after use

3.  The ELISA reproducibility in the analysis process is largely depends on the consistency of the washing plate, the correct washing plate operation is the point of determination ELISA program

4.  In all process of constant temperature incubation, avoid light exposure, seal the microplate with the cover membrane 

6. 2  Operation procedures

1.  Bring test kit to the room temperature (20-25 ℃) for at least 30 min, note that each reagent must be shaken evenly before use;

2.  Put the required micro-well strips into plate frames. Re-sealed the unused microplate, stored at 2-8 ℃, not frozen.

3.  Solution preparation: take the 40ml 20× concentrated washing buffer, dissolve with deionized water at 1:19 (1 part 20× concentrated washing buffer + 19 parts deionized water), or prepare as quantity needed.

4.  Numbering: number the micro-wells according to samples and standard solution; each sample and standard solution should be performed in duplicate; record their positions.

5.  Add standard/sample: Add 50 µL of the sample or the standard solution into separate duplicate wells, then add enzyme conjugate, 50 µL/well; then antibody working solution, 50 µL/well. Mix gently by shaking the plate manually, seal the microplate with the cover membrane, incubate at 25 °C for 30 min in the dark.

6.  Wash microplate: Carefully open the cover membrance,pour liquid out of microwell; add 250 µL/well of washing buffer, wash fully for 4-5 times, 15-30 s each time, then take out and flap to dry with absorbent paper.(Use unused spear to pierce bubble after dry)

7.  Coloration: add 50 µL of substrate A solution then 50 µL B solution into each well. Mix gently by shaking the plate manually, and incubate at 25 °C for 15min in the dark for coloration.

8.  Determination: add 50 µL of the stop solution into each well. Mix gently by shaking the plate manually. Set the wavelength of the microplate reader at 450 nm to determine the OD value of every well. (Recommend to read the OD value at the dual-wavelength 450/630 nm within 5 min).