Introduction

The test kit is detecting for the quantitative analysis of Aflatoxin M1 in milk, milk powder.

Principle of Test

The Aflatoxin M1 (AFM1) ELISA Test Kit is based on a competitive colorimetric ELISA assay.

The toxin of interest has been coated in the plate wells. During the analysis, sample is added along with the primary antibody specific for the target toxin and enzyme-conjugate. If the target is present in the sample, it will compete for the antibody, thereby preventing the antibody from binding to the toxin attached to the well. The enzyme-conjugate, will bined with the primary antibody that is complexed to the toxin coated on the plate wells, last, take coloration with TMB substrate. The samples absorbance value is an inverse relationship with AFM1 content. Lastly, compared with the standard curve can be concluded the AFM1 residues in sample.

Technique Data

• Kit sensitivity: 0.02ppb (ng/ml)
• reactive mode: 25℃, 30min～15min
• Detection Limits:

• Cross-reaction rate:

Aflatoxin M1 ………………………….100%

• Sample Recovery rate:

Milk …………………………………..……. 85±15%

Milk powder …………………………………….. 80±15%

Materials required but not supplied

• Equipments: microplate reader, printer, mixer or stomacher, nitrogen-drying device, oscillator, centrifuge, measuring pipets, and balance with a reciprocal sensibility of 0.01g;
• Micropipettors: single-channel 20 to 200µl and 100 to1000µl, and multi-channel 300µl;
• Reagents: acetonitrile, deionized water.

Sample pre-treatment

• Instructions

Labware must be clean and the use of disposable pipette tips to avoid contamination of interference results

• Solution preparation before sample pre-treatment:

Liquor 1:  Sample extracts solution:

84% Acetonitrile-water solution, V (Acetonitrile): V (Deionized water) = V21: V4

Liquor 2:  redissolving solution:

2 times dilute the2× concentrated redissolving solution with deionized water to be used for sample redissolving, it can be stored at 4 ℃ environment up to a month.

• Sample pretreatment step:

1. Milk sample

• Take 1ml milk samples into 50ml centribuge tube, pipette 4ml Acetonitrile, mix for 5min and centrifuge at 4000r/min at room temperature for
• Take 2.5ml supernatant and blow dry at 50 to 60 ℃ with nitrogen or dry with water bath; add 1ml redissolving solution (Liquor 2), oscillate fully.
• Take out 50µl for test.

dilution times of the sample:2     detection Limits: 0.04 ppb

1. Milk powder sample

• Weigh 5.0g milk powder samples into 50ml centribuge tube, add 20ml Sample extract solution (Liquor 1 ), oscillate for 5min, filtrate or centrifuge at 4000r/min at room temperature for 10min.
• Take 1ml filterable liquid or clear liquid and blow dry at 50 to 60 ℃ with nitrogen or dry with water bath; add 750µl redissolving solution (Liquor 2 ), oscillate fully.
• Take out 50µl for test.

dilution times of the sample:3     detection Limits: 0.06 ppb

Enzyme-linked immune test steps

• Take out the microtiter plate and the reagents required from 4 ℃ cold storage environment , put it in a place with room temperature for over 30 min , The wash buffer would crystallize in refrigeration, in order to make the Wash Buffer dissolved thoroughly, it needs indoor temperature. Shake the reagent bottle before usage. Take out required quantity of microwell plates and frames. The remaining strips are stored in the foil bag and zip-locked closed. Store the remaining kit in the refrigerator (2-8°C).
• Before experiment : dilute 40 ml of the concentrated washing buffer (20×concentrated) with the distilled or deionized water to 800ml (or just to the required volume) for using;

Step 1: Number: determine the number of well (samples and standards) to be used and store unused wells in 4 ℃. Every sample and standard must be parallel well (2well), and record their location

Step 2: Addtion reaction: Add 50μl standard or sample into marked well, then add 50μl HRP-conjugate solution into each well, next, add 50μl antibody working solution into each well

Step 3: Incubate: Cover with the adhesive Membrane, oscillate gently for 5s and incubate for 30 min at 25℃.

Step 4: Washing: Uncover the adhesive Membrane, discard liquid, pipette 250μl washing buffer to every well, still for 30s then drain, repeat 5 times, Pat dry with a blotting paper

Step 5: Color: Pipette 50ul Substrate A Solution, then pipette 50ul Substrate B Solution to each well, oscillate gently for 5s, avoid the light preservation for 15 min at 25℃

Step 6: Stop the reaction: Pipette Stop Solution 50μl to each well, oscillate gently, stop the reaction (the blue change to yellow).

Step 7: Calculate: Read absorbance at 450nm with microplate reader (Recommend reading the OD value at the dual-wavelength 450/630nm).finish this step within 10min.

Interpretation of result

• Calculate the percentage of absorbance value

A—the average (double wells) OD value of the sample or the standard solution;

A0—the average OD value of the 0 ppb standard solution.

• Draw the standard curve and calculate
• Take absorbance percentage of standards as Y-axis, the corresponding log of standards concentration (ppb) as X-axis.
• Draw the standard semilog curves with X-axis and Y-axis.
• Take absorbance percentage of samples substitute into standard curve, then can get the corresponding concentration from standard curve; last, Multiplied by the corresponding dilution times is the actual concentration of AFM1 of samples
• It is more convenient for a large amount of samples to use professional analyzing software to calculate, this will be accurate and rapid. (Welcome to contact us for this software)
• Attention
• Before test, the reagents and samples should be balanced to room temperature (25℃). If below 25℃, it will lead to all the standard OD value is low
• In washing process, the dry micro plate will lead to the non-linear standard curves and undesirable reproducibility, so continue to next step immediately after washing.
• The reproducibility is largely determined by consistency of washing step, so please mix uniformly and wash thoroughly.
• On Incubate step, cover micro plates with adhesive Membrane to avoid light.
• Do not mix reagents with those from other lots
• Substrate A/B solution is colourless, if not, please discard.
• If absorbance value of 0ppb is below5, it means that the reagent may be metamorphic.