0.1ppb Zearalenone Mycotoxin Testing Kits For Feed Corn 96 Wells/Kit

Zearalenone ELISA Test Kit for Feed Corn 96 Wells/Kit Sensitivity 0.1 ppb

1. Principle

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

2. Technical specifications

Sensitivity: 0.1 ppb
Incubator temperature: 25℃
Incubator time: 30min～15min

Detection limit:
Feed 10ppb
Rice, Corn, Peanut 5ppb

Cross-reaction rate:
Zearalenone 100%

Recovery rate:
Feed, rice, Peanut, corn 100±30%

3. Components

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 20l ~ 200l, 100l ~ 1000l, multi-channel 30～300 μl
3) Reagents: Methanol, n-hexane.

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:
Solution no. 1: Sample redissolving
The 2×concentrated redissolving solution is diluted with deionized water at 1:1(1part concentrated redissolving solution + 1part deionized water), used for sample redissolving.
Solution no. 2: Sample extract solution
7 parts of Methanol + 3 parts of deionized water to obtain the ready to use sample extract solution.

Samples Preparation
5.1 Preparation of Feed sample
1) Take 1.0±0.05g grinded feed sample into 50ml centrifuge tube, add 5ml sample extract solution;
2) Shake completely for 3min(or shake by hand for above 5min), centrifuge at above 4000r/min at 20℃ for 10 min;
3) Take 50ul supernatant(up-layer), add 950ul sample redissolving, shake to evenly;
4) Take 50μl to test
Dilution factor: 100
Note: if the drug residue concentration in the sample is out of curve range, the sample can be further diluted for test (for example: take 50ul supernatant(up-layer) into a new centrifuge tube, add 1450ul deionized water, the dilution factor is 150 ).

5.2Preparation of corn, rice sample
1) Take 1.0±0.05g grinded sample into 50ml centrifuge tube; add 5ml sample extract solution;
2) Shake completely for 3min(or shake by hand for above 5min), centrifuge at above 4000r/min at 20℃ for 10 min;
3) Take 100ul supernatant(up-layer), add 900ul sample redissolving, shake to evenly;
4) Take 50μl to test
Dilution factor: 50
Note: If the drug residue concentration of the sample is out of curve range, the sample can be further diluted for test (for example: take 50ul supernatant(up-layer) into a new centrifuge tube, add 950 deionized water, the dilution factor is 100).

5.3Preparation of Peanut sample
1) Take 1.0±0.05g grinded peanut sample into 50ml centrifuge tube; add 5ml sample extract solution, then add 4ml n-hexane;
2) Shake completely for 3min (or shake by hand for above 5min), centrifuge at above 4000r/min at 20℃ for 10 min;
3) Discard up-layer clear liquid, take 100ul middle-layer liquid, add 900ul sample redissolving, shake to evenly;
4) Take 50μl to test
Dilution factor: 50
Note: If the drug residue concentration of the sample is out of curve range, the sample can be further diluted for test (for example: take 50ul middle-layer liquid into a new centrifuge tube, add 950 deionized water, the dilution factor is 100).

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. Put the kit at room temperature (20-25°C) for at least 30 minutes, note that each reagent must be shaken well before use;
2. Place the desired microwell strips in the plate frame. Unused microplates should be resealed and stored at 2-8°C, not frozen.
3. Solution preparation: Take 40ml of 20× concentrated washing buffer and dissolve it in deionized water at a ratio of 1:19 (1 part of 20× concentrated washing buffer + 19 parts of deionized water), or prepare as needed.
4. Numbering: Number the microwells according to the samples and standard solutions; each sample and standard solution should be done in duplicate; record their positions.
5. Add standard/sample: Add 50 µL of sample or standard solution to separate double wells, then add enzyme conjugate, 50 µL/well; then antibody working solution, 50 µL/well. Mix gently by shaking the plate by hand, seal the microplate with a cover film, and incubate at 25 °C for 30 min in the dark.
6. Wash the microplate: Carefully open the cover film and pour out the liquid in the microwell; add 250 µL/well of washing buffer, wash thoroughly 4-5 times for 15-30 s each time, then remove it with absorbent paper Pat dry. (Prick the air bubbles with an unused spear after drying)
7. Color development: Add 50 µL of Substrate A solution to each well, followed by 50 µL of Solution B. Mix gently by shaking the plate by hand and incubate for 15 min at 25 °C in the dark for staining.
8. Assay: Add 50 µL of Stop Solution to each well. Mix gently by shaking the plate by hand. Set the wavelength of the microplate reader to 450 nm to determine the OD value of each well. (It is recommended to read OD values ​​at dual wavelengths 450/630 nm within 5 minutes).

7. 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 Zearalenone in the sample
7.1 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; 0.1ppb of 1.816; 0.3ppb of 1.415; 0.9ppb of 0.74; 2.7ppb of 0.313; 8.1ppb of 0.155. Then the concentration of the sample one is in the range of 2.7ppb ~ 8.1ppb; Sample Two is 0.3ppb ~ 0.9ppb. The concentration range of Zearalenone in the samples can be obtained by multiplied by the corresponding dilution of the sample.
7. 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 Zearalenone concentration [ng/mL]. The Zearalenone concentration in ng/ml 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.

8. 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.

9. 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?
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