Instructions for Zearalenone (ZEN) ELISA Kit

Instructions for Zearalenone (ZEN) ELISA Kit

This kit is for research use only

1 Purpose: This kit is used for residues of zearalenone (ZEN) in feed, fish, shrimp and meat tissues (such as chicken, beef and pork), eggs, honey, milk, serum and urine samples Quantitative testing.

2 Experimental principle

This kit uses a competitive ELISA method, coated with zearalenone (Zearalenone, ZEN) conjugated antigen in a microplate, added zearalenone (ZEN) standard or sample, free zearalene Zone (Zearalenone, ZEN) and pre-coated zearalenone (ZEN) conjugated antigen on microwell strips compete with each other for anti-zearalenone (ZEN) abzyme label, using TMB substrate Color development, the color changes from blue to yellow after adding the stop solution, and the microplate reader is used to detect at 450nm wavelength. The absorbance value is inversely proportional to the content of zearalenone (ZEN) in the sample, and the sample is calculated by the standard curve The content of zearalenone (Zearalenone, ZEN).

3 Kit composition

3.1 Pre-coated zearalenone (Zearalenone, ZEN) conjugated antigen detachable enzyme labeling plate: 1 piece (12 wells × 8 strips).
3.2 Standard zearalenone (Zearalenone, ZEN): 6 bottles (1ml / bottle), the contents are: 0 ng / ml, 0.5 ng / ml, 2 ng / ml, 20 ng / ml, 200 ng / ml , 400 ng / ml.
3.3 Anti-zearalenone (Zearalenone, ZEN) antibody conjugate: 1 bottle (6ml).
3.4 Color Rendering Solution A: 1 bottle (6ml).
3.5 Color Rendering Solution B: 1 bottle (6ml).
3.6 Stop solution: 1 bottle (6ml), 2M sulfuric acid.
3.7 Sample diluent: 1 bottle (10 ×, 6ml), used for sample dilution.
3.8 Concentrated washing solution: 1 bottle (20 ×, 20ml), used for washing plates.
3.9 A manual.

4 Materials needed but not provided
4.1 Equipment
4.1.1 Wavelength 450nm microplate reader.
4.1.2 Crusher.
4.1.3 Measuring cylinder.
4.1.4 Oscillator.
4.1.5 Funnel.
4.1.6 Whatman No 1 or equivalent filter paper.
4.1.7 Micro pipette.
4.2 Reagent
4.2.1 Deionized water or distilled water.
4.2.2 Methanol.
5 Storage
5.1 The kit is stored at 2 ~ 8 ℃, do not freeze
5.2 Unused microplates should be sealed and dried
6 Notes
6.1 Please read the instructions carefully before using the kit.
6.2 Do not use expired kits.
6.3 Before using the kit, return the reagent to room temperature (25 ± 2 ℃). It is recommended to return to temperature for at least 2 hours.
6.4 The standard product contains zearalenone (Zearalenone, ZEN). Special care should be taken when using, and gloves should be worn during operation.
6.5 The stop solution contains sulfuric acid, which prevents burns to the skin and corrosion of clothing when used.
6.6 The tips used for different standards and samples can not be mixed, otherwise it will affect the test results.
6.7 The reagents in different batch kits should not be mixed; the tips used for different standards and samples should not be mixed, otherwise it will affect the experimental results.
6.8 When diluting the sample, the sample diluent in this kit must be used, otherwise it will affect the experimental results
6.9 Avoid foaming when mixing reagents.
7 Working fluid preparation
7.1 Zearalenone (ZEN) standard solution: 0 ng / ml, 0.5 ng / ml, 2 ng / ml, 20ng / ml, 200 ng / ml, 400 ng / ml
7.2 Concentrated washing solution: dilute with distilled water at 1:20 (1 + 19) for later use

7.3 Sample diluent: 1:10 (1 + 9) diluted with distilled water for use
7.3 Developer: reserved, avoid direct light
7.4 Reaction stop solution: reserved
8 Sample processing: (Sample should be operated strictly in accordance with the instructions during the extraction process, and it should be accurately diluted during the extraction process, otherwise the results will be inaccurate, and the sample should be stored in a cool and dark place and refrigerated)

General sample handling

8.1 Take 10g crushed sample and add 20ml 70% methanol solution
8.2 Vibrate vigorously for 3 minutes
8.3 Filter with Whatman No 1 filter paper
8.4 Take 100µl of processed sample and add 400µl of sample diluent
8.5 Take 100μl of diluent for analysis

Animal tissue pretreatment

8.6 Accurately weigh 1 ± 0.05 g of homogenized tissue sample into a 50 ml polystyrene centrifuge tube; add 3 ml of acetonitrile-acetone extraction solution, shake vigorously at 2000 rpm for 20 s, and centrifuge at 4000 r / min for 5 min

8.7 Take 0.8ml of supernatant and blow dry at 50 ℃ under nitrogen flow

8.8 Add 3.2mL sample diluent, vortex at 750rpm for 20s

8.9 Take 100ml for analysis

Feed pretreatment method

8.10 Accurately weigh 1 ± 0.05 g of crushed feed sample into a 50 ml polystyrene centrifuge tube; add 4 ml of acetonitrile, 1 ml of acetone, shake vigorously at 2000 rpm for 20 s, and centrifuge at 4000 r / min for 3 min

8.11 Take 0.7ml of supernatant and blow dry under nitrogen flow at 50 ℃

8.12 Add 2.8mL sample diluent, vortex for 20s, mix and take 100ml for analysis

Milk pretreatment method

8.13 Take 1 ml milk sample into 5 ml polystyrene centrifuge tube; add 4 ml sample diluent, mix and take 100 ml for analysis

Milk powder pretreatment method

8.14 Accurately weigh 0.3g milk powder sample into a 7ml polystyrene centrifuge tube; add 2ml PBS solution, 2ml n-hexane, mix by shaking

8. Centrifuge above 154000r / min for 5min, remove the organic layer and the middle layer, take the lower layer solution from 100ml to 400ml of sample dilution

8.16 After mixing, take 100ml for analysis


9 Enzyme-free analysis steps
9.1 Experimental notes
9.1.1 Before starting the experiment, please restore all reagents to room temperature (25 ± 2 ℃) outside the box for about 2 hours. After warming to room temperature (25 ± 2 ℃), take out the microporous strips again. Re-seal the microporous strips and dry immediately at 2 ~ 8 ℃. Note: Make sure that the temperature is sufficient, otherwise the accuracy and accuracy of the test will be affected.
9.1.2 Please put the reagent back to 2 ~ 8 ℃ immediately after use
9.1.3 Please do not change the analysis program
9.1.4 Please use an accurate micropipette
9.1.5 Once the operation starts, please do not interrupt any program
9.1.6 The reproducibility of ELISA results greatly depends on the operating procedures, please strictly follow the requirements
9.1.7 To avoid cross-contamination, each standard and sample should be loaded with different tips
9.1.8 Do not let the tip touch the solution or inner surface of the microwell when loading
9.2 Analysis steps
9.2.1 Number in advance, mark the position of B0, standard and sample, double hole detection is recommended
9.2.2 Take the required number of micropores (micropore strips are removable), reseal the excess strips and immediately put them back at 2 ~ 8 ℃ to store
9.2.3 Dilute the sample and concentrated washing solution (20 ×) into working solution (diluted with distilled or deionized water)
9.2.4 Add 50µl of 0.0 ng / ml standard solution to well B0
9.2.5 Add 50 μl of standard solution to each standard well
9.2.6 Add 50µl of sample solution to each sample well
9.2.7 Add 50µl of anti-zearalenone (ZEN) abzyme conjugate to all wells
9.2.8 Gently shake the reaction plate for a few seconds.
9.3 Warm bath at 37 ° C for 30min (Tap the reaction plate from time to time during the warm bath to reduce double-hole errors)
9.3.1 Shake off the liquid in the well and wash the microplate 5 times with lotion. The last time should be tapped on absorbent paper to completely remove the liquid in the well.
9.4 Response
9.4.1 After the washing procedure is completed, immediately add 50µl of chromogenic solution A and then 50µl of chromogenic solution B to each microwell with a micropipette; shake the reaction plate slightly to mix thoroughly
9.4.2 37 ℃ warm bath for 10min
9.4.3 Add 50µl of stop solution to each well and mix well
9.4.4 Detect the absorbance at 450nm and read the result within 5min.
10 Result calculation
10.1 Quantitative analysis
10.1.1 The average value (B) of the absorbance value of each concentration standard solution and sample obtained is divided by the absorbance value (B0) of the first standard (0 standard) and multiplied by 100%, which is the percent absorbance value.
B—average absorbance value of standard solution or sample solution
Average absorbance value of B0—0 ng / ml standard solution
10.1.2 With the logarithmic value of zearalenone (ZEN) concentration as the X axis and the percent absorbance value as the Y axis, draw a standard curve. According to the percent absorbance of the sample, the abscissa of the corresponding point can be obtained from the curve, which is the logarithmic value of the concentration of zearalenone (ZEN). Zearalenone, ZEN) Concentration C (ng / ml)
10.1.3 Because the sample has been pre-diluted, the sample concentration obtained from the standard curve must be multiplied by its dilution factor.
10.2 Semi-quantitative determination
10.1.1 Visual semi-quantitative determination: first select an appropriate standard solution to run with the sample, and judge whether the concentration value of the sample is less than or greater than the standard value based on the comparison of the absorbance value of the sample and the standard.
10.1.2 Instrument semi-quantitative determination: first select an appropriate standard solution to run with the sample, and determine whether the concentration value of the sample is less than or greater than the standard value according to the color depth comparison of the sample and the standard product.

11 Specific substances Cross-reactive substances Cross-reactive

Zearalenone ……………………………… 100%
Zearalenone …………………………………… 13%
Zearalenol …………………………………… <1%

12 Kit parameters The lower detection limit of this kit is 0.5 ng / ml
The best value of B0 absorbance should be greater than 1.0
The error in the absorbance plate of the kit is less than 8%, and the error between the plates is less than 15%.
The recovery rate of the tissue sample extraction method provided in this specification is greater than 80%.
13 Standard curve mode (for reference only)
The standard curve range provided by the kit is 0.5 ng / ml ~ 400 ng / ml.

14 Limitations of analysis The samples tested positive by this kit should be confirmed by another method such as HPLC or GC / MS.

Shanghai Yanjin Biological Technology Co., Ltd. specializes in selling ELISA kits of various brands and price levels. Serving universities and immunology research units. Quality assurance and perfect after-sales service. And can be free to detect, better service for you.

This women Straight Umbrella bulk sale offers an unbeatable deal on one of our most popular women`s umbrella items. Our Ladies Straight Umbrella features a water and wind resistant wide canopy that can keep you dry even in stormy rain showers. With special Ladies Straight Umbrella bulk pricing, you can get a discount on this top rated feminine item when you purchase in bulk. Place a Straight Umbrella for women bulk order today to stay dry with this elegant style find.

Ferrule For Womens Straight Umbrella
Hyades Umbrella is a one-stop company that provides Support, Knowledge and Advice for your umbrellas. It is important to work with a company that has in depth knowledge of umbrella manufacturing. To ensure all technical elements are communicated accurately, we can provide precise specification sheets including materials, color swatches, samples as well as pre-production samples so the bulk production meets your design.


You can be assured of the best outcome and smoothest development process to market with the help of our team of experienced and knowledgeable Umbrella Masters.






Women's Straight Umbrella

Women's Straight Umbrella, Women's Straight Umbrella, Women's Stick Umbrella, Ladies Straight Umbrella

Hyades Umbrella Co., Ltd. , https://www.hyadesumbrella.com