Aflatoxin M1 Elisa Kit for Milk

• Environmental Protection: Yes
• Certification: CE/ISO
• Color: White
• Classification: Food Diagnostic
• Function: Food Testing
• Appearance: Liquid
• Model NO.: KA07201H
• Transport Package: Foam Box with Ice Bag
• Specification: 96T/Kit
• Trademark: kwinbon
• Origin: China
• HS Code: 38229000
• Production Capacity: 50000PCS/Year

Product Description

Competitive Enzyme Immunoassay for Quantitative Analysis of Aflatoxin M1

 

1. Background

Aflatoxin M1 is a species of aflatoxin. The rate of appear is very high in food and feed in hot and humid area. The physicochemical property of it is steady and it is not easy destroyed by pasteurization. After mammal intake the feed or food polluted by aflatoxin B1, it will be turned into aflatoxin M1 with the mean of hydroxylation. The main dangers of Aflatoxin M1 are carcinogenicity and mutagenicity. It can destroy the animal liver and result in liver cancer and even die.
This kit is a new generation of drug residue test with enzyme immunoassay technology. It has characteristics of speediness, convenient, accuracy and high sensitivity. The operation time only need 60 minutes and it can furthest reduce the operation error and workload.
2. Test Principle
This ELISA kit is designed to detect aflatoxin M1 based on "indirect-competitive" enzyme immunoassay. The microtiter wells are coated with BSA-linked aflatoxin M1 antigen. Aflatoxin M1 in the sample competes with the precoated antigen for binding to the limited number of antibody. After TMB substrate, the signal is measured with an ELISA photometer. The absorption is inversely proportional to the aflatoxin M1 concentration in the sample, compared with the standard curve. Then multiply by corresponding dilution ratio.  And you can calculate the residue of aflatoxin M1 in the sample.

3. Applications
This kit can be used for qualitative and quantitative rapid test of aflatoxin M1 in milk powder, raw milk, cheese, Yoghourt, Finished milk (peanut milk, walnut milk, breakfast milk, high calcium milk).

4. Cross reactions

Aflatoxin M1	100%
Aflatoxin B1	164%
Aflatoxin B2	14%
Aflatoxin G1	85%
Aflatoxin G2	<1%

5. Equipment needed but not provided
5.1 Equipments
----ELISA reader (450nm/630nm)
----Shaker
----Vortex mixer
----Analytical balance (inductance: 0.01g)
----Graduated pipette: 10ml
----Rubber pipette bulb
----Polystyrene centrifuge tube: 50ml, 2ml 
----Micropipettes: 20μl-200μl, 100μl-1000μl
50-300μl-multipipette
5.2 Reagents
---- Acetonitrile (AR)
---- Trichloromethane (AR)
---- N-hexane (AR)
----Deionized water
6. Kit Components

1. Microtiter plate with 96 wells coated with antigen
2. Standard solutions(6 bottles×1ml/bottle)

0ppb, 0.03ppb, 0.09ppb, 0.27ppb, 0.81ppb, 2.43ppb

1. Enzyme conjugate 12ml.........….….red cap
2. Antibody solution  7ml .........….....green cap
3. Substrate solution A  7ml ..........…white cap
4. Substrate solution B  7ml....…..…........red cap
5. Stop solution  7ml ...........….…yellow cap
6. 20×Concentrated wash solution 40ml
7. ........................….transparent cap
8. 2×Concentrated extraction solution  50ml.....…blue cap

7. Reagents Preparation
Solution 1: Extraction solution
Dilute the 2×Concentrated extraction solution with deionized water in the volume ratio of 1:1(1ml of 2×concentrated extraction solution + 1ml of deionized water), which will be used to dilute the sample.
Solution 2: Washing solution
Dilute the 20×concentrated wash solution with deionized water in the volume ratio of 1:19(e.g. 5ml of 20×concentrated wash solution + 95ml of deionized water), which will be used to rinse the plates. The diluted wash solution can be conserved for a month at 4ºC.
8. Sample Preparations
8.1 Notice and precautions before operation:
(a) Please use one-off tips in the process of experiment, and change the tips when absorbing different reagent.
(b) Make sure that all experimental instruments are clean.
(c)Untreated milk sample should be stored in frozen environment.
(d) Treated samples can't be stored more than 4 hours.
8.2 Milk (raw milk, finished milk)
Take 50μl of milk sample to directly assay.
8.3 Yoghourt
----Mix the caked yoghourt sample completely.
------Take 200μl yoghourt sample after mix into a 2ml polystyrene centrifuge tube.
-----Add 800μl wash solution (see solution 2), shake to mix it completely;
------Take 50μl for assay.
8.4 Milk powder
----Weight 1.0±0.05g of milk powder sample into a 50ml polystyrene centrifuge tube.
----Add 8ml deionized solution and shake to mix it completely.
----Take 50μl for assay.
8.5 Cheese
----Mash the cheese sample before detection.
----Weight 1.0±0.05g cheese sample into 50ml polystyrene centrifuge tube. Add 3ml deionized water, add 3ml acetonitrile, vortex to mix it completely.
---- Centrifuge for separation: 3000g / 20-25ºC/ 5min. Take 3ml of the supernatant into a 50ml clean dry tube. Add 6ml trichloromethane and vortex for 15s.
----- Centrifuge for separation: 3000g / 20-25ºC/ 5min. Remove the supernatant and take 3ml of the lower organic phase into a 10ml clean glass tube. Dry with 50-60ºC(122-140ºF)water bath under nitrogen flow.
----Add 1ml n-hexane and vortex for 1min. Add 1ml extraction solution (solution 1) and vortex for 20s. Centrifuge for separation: 3000g / 20-25ºC/ 5min.
----Remove the supernatant organic phase and take 50μl of the lower solution for assy.
9. Assay process
9.1 Notice before assay
9.1.1 Make sure all reagents and microwells are all at room temperature (20-25ºC).
9.1.2 Return all the rest reagents to 2-8ºC immediately after used.
9.1.3 Washing the microwells correctly is an important step in the process of assay; it is the vital factor to the reproducibility of the ELISA analysis.
9.1.4 Please avoid direct sunlight during the incubation, which means the plate should be covered with the plate cover provided in the kit.
9.2 Assay Steps
9.2.1 Take all reagents out at room temperature (20-25ºC) for more than 30min. Shake gently before use. 
9.2.2 Get the microwells needed out and return the rest into the zip-lock bag at 2-8ºC immediately.
9.2.3 The concentrated wash solution and concentrated extraction solution should be brought to room temperature before use.
9.2.4 Number: Number every microwell position and all standards and samples should be run in duplicate. Record the standards and samples positions.
9.2.5 Add standard /sample and antibody solution: Add 50µl of standard solution (kit component) or prepared sample to corresponding wells. Add 50µl of antibody solution (kit component). Mix gently by shaking the plate manually and incubate for 30min at 25ºC with cover (or in dark place).
9.2.6 Wash: Remove the cover gently and pour the liquid out of the wells and rinse the microwells with 250µl of diluted wash solution (solution 2) at interval of 10s for 4-5 times. Absorb the residual water with absorbent paper (the rest air bubble can be eliminated with unused tip).
9.2.7 Add the enzyme conjugate: Add 100ul of enzyme conjugate to each well and shake gently. Incubate for 30min at 25ºC with cover (or in dark place). And then repeat the step 9.2.6.
9.2.8 Coloration: Add 50µl of solution A (kit component) and 50µl of solution B (kit component) to each well. Mix gently by shaking the plate manually and incubate for 15 min at 25ºC with cover(see 12.8).
9.2.9 Measure: Add 50µl of stop solution (kit component) to each well. Mix gently by shaking the plate manually and measure the absorbance at 450nm (It's suggested measure with the dual-wavelength of 450/630nm. Read the result within 5min after addition of stop solution). (If in the absence of microplate reader, you can judge it by visual method with no stop solution.).

10. Results
10.1 Percentage absorbance
The mean values of the absorbance values obtained from the standards and the samples are divided by the absorbance value of the first standard (zero standard) and multiplied by 100%.
   =
B --the mean absorbance value of each standards or each samples
B₀ --absorbance value of zero standard
(2)Standard Curve
---To draw a standard curve, the absorbance value of standards as y-axis, semilogarithmic of the concentration of the standards (ppb) as x-axis.
---The aflatoxin M1 concentration of each sample (ppb), which can be read from the calibration curve, is multiplied by the corresponding dilution factor of each sample followed, and the actual concentration of sample is obtained.
Please notice:
Special software has been developed for all data reduction, which can be provided on request.
Sample dilution ratio:
Milk sample (raw milk, finished milk): ...…1
Yoghourt, Cheese: ...............….5
Milk powder: ..................…..8
11. Sensitivity, accuracy and precision
Test Sensitivity: 0.03ppb
Detection limit
Milk..................…......…0.03ppb
Cheese,Yoghourt..................…0.15ppb
Milk powder........................0.25ppb
Accuracy
Milk ...........................…85%±15%
Yoghourt.........................85%±15%
Milk powder .....................….85%±15%
Cheese ........................…85%±15%
Precision
C.V. of the ELISA kit is less than 10%.
12. Notice
12.1 The mean values of the absorbance values obtained for the standards and the samples will be reduced if the reagents and samples have not been regulated to room temperature (20-25ºC). 
12.2 Do not allow microwells to dry between steps to avoid unsuccessful reproducibility and operate the next step immediately after tap the microwells holder.
12.3. Shake each reagent gently before use.
12.4. Keep your skin away from the stop solution for it is the high concentration of H₂SO₄ solution.
12.5 Don't use the kits out of date. Don't exchange the reagents of different batches, or else it will drop the sensitivity.
12.6 Keep the ELISA kits at 2-8ºC, do not freeze. Seal rest microwell plates, Avoid straight sunlight during all incubations. Covering the microtiter plates is recommended.
12.7 Substrate solution should be abandoned if it turns colors. The reagents may be turn bad if the absorbance value (450/630nm) of the zero standard is less than 0.5(A450nm<0.5).
12.8 The coloration reaction need 15min after the addition of solution A and solution B, but you can prolong the incubation time ranges to 20min or more if the color is too light to be determined, never exceed 25min, on the contrary, shorten the incubation time properly.
12.9 The best reaction of temperature will be 25ºC. Please make sure the temperature is correct during all steps. Higher or lower temperature will lead to experiment failure. 
13. Storage
Storage condition: 2-8ºC.
Storage period: 12months