Total Aflatoxins ELISA Test Kit for Feed Corn Peanut Tissue 96 Wells/Kit

1. Mycotoxin Testing Kits Principle

This kit is based on the indirect competitive enzyme immunoassay for the detection of total aflatoxins. Conjugated antigens are pre-coated on microwell strips. Aflatoxin in the sample competes with the conjugated antigen pre-coated on the microwell strip for anti-aflatoxin antibodies. After the enzyme conjugate is added, TMB substrate is added for staining. The optical density (OD) value of a sample is negatively correlated with the aflatoxin in the sample. This value is compared to a standard curve, and the residual aflatoxin level is then obtained.

2. Technical Specifications

Sensitivity: 0.02 ppb
Incubator temperature: 25℃
Culture time: 30min～15min
Detection limit:
Feed, rice, corn, peanut, tissue, edible oil 2ppb
Cross Reaction Rate:
Aflatoxin B1 100%
Aflatoxin M1 91.2%
Aflatoxin B2 68.4%
Aflatoxin G1 4.7%
Aflatoxin G2 2.7%
Recovery rate:
Feed, rice, corn, peanut, tissue, edible oil 95±35%

3. Mycotoxin Testing Kits Components

4. Sample Preparation

Instructions for use (the following points must be dealt with before preprocessing)
1) The experiment can only use disposable tips, and the tips must be replaced when aspirating different reagents;
2) Before the experiment, each experimental utensil must be cleaned and re-cleaned if necessary to avoid contamination interfering with the experimental results.

Solution preparation before sample pretreatment:
1) Sample dissolution solution
Use 1 part 20X concentrated redissolving solution and dissolve with 19 parts deionized water to obtain a ready-to-use sample redissolving solution.
2) Sample extract
Use 7 parts methanol and dissolve in 3 parts deionized water for a ready-to-use sample extract.

Sample Preparation
4.1 Preparation of tissue, feed, rice and maize samples
1) Put 1.0±0.05g of the ground sample into a 50ml centrifuge tube, add 5ml of sample extract, shake for 3min, centrifuge at 20°C for 10min above 4000r/min;
2) Take 100ul supernatant, add 700ul sample redissolving solution, shake well;
3) Take 50μl for testing
Dilution factor: 40

4.2 Preparation of edible oil samples
1) Take 1.0±0.05g edible oil sample into a 50ml centrifuge tube; add 5ml of sample extract, then add 4ml of n-hexane, shake for 3min, centrifuge at 4000r/min at 20℃ for 10min;
2) Discard the supernatant, take 100ul of the middle layer solution, add 700ul of sample redissolving solution, and shake well;
3) Take 50μl for testing
Dilution factor: 40

4.3 Preparation of peanut samples
1) Put 1.0±0.05g peanut crushed sample into a 50ml centrifuge tube; add 5ml of sample extract, then add 4ml of n-hexane, shake for 3min, centrifuge at 4000r/min above 20℃ for 10min;
2) Discard the supernatant, take 100ul of the middle layer solution, add 400ul of sample redissolving solution, and shake well;
3) Take 50μl for testing
Dilution factor: 25

5. ELISA procedures

5.1 Instructions for use
1) Bring ELISA reagents to room temperature (20 - 25 °C) before use.
2) Put the ELISA reagent back to 2-8°C immediately after use.
3) The reproducibility of the ELISA analysis process largely depends on the consistency of the plate washing, and the correct plate washing operation is the focus of determining the ELISA procedure.
4) During all processes of constant temperature incubation, avoid light, and seal the microplate with a cover film.

5.2 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, andincubate 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).

6. Result judgment

There are two methods to judge the results; the first one is the rough judgment, while the second is the quantitative determination. Note that the OD value of the sample has a negative correlation with the Total Aflatoxin in the sample.

6.1 Qualitative determination
The concentration range (ppb) can be obtained by compared the average absorbance value with standards. Suppose absorbance value of Sample One is 0.3, Sample Two is 1.0, and the standards are: 0ppb of 2.243; 0.02ppb of 1.816; 0.06ppb of 1.415; 0.18ppb of 0.74; 0.54ppb of 0.313; 1.62ppb of 0.155. Then the concentration of the sample one is in the range of 0.54ppb ~ 1.62ppb; Sample Two is 0.06ppb ~ 0.18ppb. The concentration range of total aflatoxin in the samples can be obtained by multiplied by the corresponding dilution of the sample.

6. 2 Quantitative Analysis
In order to calculate the concentration of samples, a standard curve should be made. Before standard curve is made, the concept of % absorbance should be know.
Calculation of % absorbance:

B—the average OD value of the sample or the standard solution.
B0—the average OD value of the 0 ng/mL standard solution.
The zero standard is thus made equal to 100 % and the absorbance values are quoted in percentages. The values calculated for the standards are entered in a system of coordinates on semilogarithmic graph paper against the the total aflatoxin concentration [ng/L]. The total aflatoxin concentration in ng/L (ppb) corresponding to the absorbance of each sample can be read from the calibration curve.
A special software for result analysis of ELISA will facilitate double or multiple determinations. If you need, please call to request.

7. Precautions

1) The room temperature below 25 ℃ or the temperature of the reagents and the samples being not returned to the room temperature (20-25 ℃) will lead to a lower standard OD value.
2) Dryness of the microplate in the washing process will be accompanied by the situations including the non-linear standard curves and the undesirable reproducibility; So continue to next step immediately after washing.
3) Mix evenly before adding any reagents.
4) The stop solution is the 2 M sulfuric acid solution, avoid contacting with the skin.
5) Do not use the kit exceeding its expiry date. The use of diluted or adulterated reagents from the kits will lead to the changes in the sensitivity and the detecting OD values. Do not exchange the reagents from the kits of different lot numbers to use.
6) Storage: store at 2-8 ℃, not frozen. Put the unused microplate into an auto-sealing bag to re-seal it. The standard substance and the colourless color former is light sensitive, and thus they cannot be directly exposed to the light.
7) Discard the colouration solution with any color that indicates the degeneration of this solution. The detecting value(450/630nm) of the 0 standard solution (0 ppb) of less than 0.5((A450nm<0.5)) indicates its degeneration.
8) The optimum reaction temperature is 25 ℃, and too high or too low temperatures will result in the changes in the detecting sensitivity and OD values.

8. Storage and expiry date

Storage: store at 2-8 ℃, not frozen.
Expiry date: 12 months; date of production is on box.
Note: If the Vacuum package of microplate has leakage, it is still valid to use, do not affect the test result, be relax to use.

Q1: When will it be shipped?
A1: We will ship the goods for you as soon as possible within 7 working days after receiving the payment. (In case of external factors such as the epidemic, the delivery may be delayed)

Q2: Does it support OEM/ODM?
A2: It can be supported, but the specific quantity needs to be more than 100,000 pieces, which is convenient for customized products.

Q3: How is your factory doing in terms of quality control?
A3: We have nationally certified ISO9001 and ISO13485. Our production process conforms to standard procedures to ensure optimum product quality.

Q4: How to provide after-sales service?
A4: We provide professional online technical after-sales service. We can provide you with one-on-one guidance via video, telephone, etc.

Q5: What is the payment method?
A5: We receive payment by T/T.

Q6: How to ship?
A6: Choose the best shipping method for you by getting quotes from our many cooperative carriers, and also ship according to your requirements.