Diethylstilbestrol (DES) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDF-E001
The Diethylstilbestrol (DES) ELISA Kit (RDF-E001) is an enzyme-linked immunosorbent assay for quantitative detection of DES in Food samples with a standard range of 0.05-4.05ppb. This kit uses the Traditional ELISA format for colorimetric detection of natural DES in samples including Muscle,Liver. The kit is easy to use and comes with a pre-coated microplate and all necessary reagents and buffers. The standard curve generated in the experiment is used to calculate the concentration of DES in samples. For research use only.
Product Specifications
Product Name
Diethylstilbestrol (DES) ELISA Kit
Catalog Number
RDF-E001
Detection Range
0.05-4.05ppb
Species Reactivity
Food
Species
Food
Assay Length
75 minutes
Experimental Method
Competitive Inhibition
Detection Method
Colorimetric
Sample Type
Muscle,Liver
Storage
2-8°C,Avoid freeze
Shelf Life
12 monthes
Product Details
Intended Use
For the quantitative detection of Diethylstilbestrol (DES) concentration in muscle and liver samples.
Assay Principle
This kit uses Competitive-ELISA as the method for quantitative detection. It can detect Diethylstilbestrol (DES) in samples such as muscle, liver, etc. This kit is composed of an ELISA Microtiter plate, HRP conjugate, antibody working solution, standard and other supplementary reagents. The microtiter plate in this kit has been pre-coated with a coupled antigen. During the reaction, DES in the samples or standard competes with the coupled antigen on the solid phase supporter for sites of anti- DES antibody. Then Horseradish Peroxidase (HRP) conjugate is added to each microtiter plate well, and a substrate reagent is added for color development. There is a negative correlation between the OD values of samples and the concentration of DES. The concentration of DES in the samples can be calculated by comparing the OD of the samples to the standard curve.
Application Data
Reagents and Materials Provided
| Reagent | Quantity |
|---|---|
| ELISA Microtiter plate | 96 wells |
| Standard | 1 mL each (ppb=ng/mL=ng/g) (0 ppb, 0.05 ppb, 0.15 ppb, 0.45 ppb, 1.35 ppb, 4.05 ppb) |
| HRP Conjugate | 11 mL |
| Antibody Working Solution | 5.5 mL |
| Substrate Reagent A | 6 mL |
| Substrate Reagent B | 6 mL |
| Stop Solution | 6 mL |
| 20×Concentrated Wash Buffer | 40 mL |
| Plate Sealer | 3 |
| Sealed Bag | 1 |
| Manual | 1 |
Materials Required but not Supplied
- Equipment: Microplate reader, Printer, Homogenizer, Nitrogen evaporator, Water bath, Vortex mixer, Centrifuge, Graduated pipette, Balance (sensibility 0.01 g)).
- Micropipettes: single-channel 20-200 µL, 100-1000 µL, and multi-channel 30-300 µL.
- Reagents: Methanol, Concentrated phosphoric acid (85% H3PO4), Acetonitrile, NaOH, Acetone, Trichloromethane (CHCl3).
Storage
- Store the kit at 2-8℃. Do not freeze any kit components.
- Return any unused microwells to their original foil bag and reseal them together with the desiccant provided. Continue to store at 2-8℃.
- Shelf life is approximately 12 months from date of manufacture. Check packaging for expiry date.
Sample Preparation
- Muscle (fish, shrimp, livestock), liver:
- Remove fat from sample, homogenize the sample with homogenizer.
- Weigh 2±0.05 g of crushed homogenate into a centrifuge tube, add 6 mL of Acetonitrile-Acetone Solution (Solution 4), vortex for 2 min, centrifuge at 4000 r/min for 10 min at 15℃.
- Transfer 3 mL of the supernatant to another centrifuge tube, dry with nitrogen evaporator/water bath at 60℃. (Perform this step in a ventilated area.)
- Add 0.5 mL of Trichloromethane and vortex for 20 s, then add 2 mL of 2 M NaOH Solution (Solution 2) and vortex for 30 s, centrifuge at 4000 r/min for 5 min.
- Transfer 1 mL of the supernatant to another tube, add 200 μL of 6 M H3PO4 Solution (Solution 1) and vortex for 5 s.
- Add 3 mL of Acetonitrile for extraction, vortex for 2 min, and centrifuge at 4000 r/min for 10 min at room temperature. Then take 1.5 mL of the upper phase organic liquid and dry with nitrogen evaporator/water bath at 60℃.
- Perform the following redissolving steps according to your sample type(s):
- 7a. Fish, shrimp:
- Dissolve the residue with 1 mL of 40% Methanol Solution (Solution 3), vortex for 30 s and use 50 μL for analysis.
- 7b. Muscle (livestock), liver:
- Dissolve the residue with 2.5 mL of 40% Methanol Solution (Solution 3), vortex for 30 s and use 50 μL for analysis.
Reagent Preparation
Solution preparation:
- Prepare solutions according to the number of samples. Avoid using all components in the kit at once.
- Solution 1: 6 M H3PO4 Solution (for fish, shrimp, livestock, liver samples)
- Dilute 100 mL of 85% H3PO4 to 150 mL with deionized water, mix thoroughly.
- Solution 2: 2 M NaOH Solution (for fish, shrimp, livestock, liver samples)
- Dissolve 8 g of NaOH to 100 mL with deionized water.
- Solution 3: 40% Methanol Solution (for fish, shrimp, livestock, liver samples)
- Dilute Methanol with deionized water (Methanol (V): Deionized water (V) =2:3), mix thoroughly.
- Solution 4: Acetonitrile-Acetone Solution (for fish, shrimp, livestock, liver samples)
- Dilute Acetonitrile with Acetone (Acetonitrile (V): Acetone (V) =4:1),
- mix thoroughly.
- Solution 5: Wash Buffer
- Dilute 20×Concentrated Wash Buffer with deionized water (20×Concentrated Wash Buffer (V): Deionized water (V) = 1:19), mix thoroughly. Notes:
- Bring all reagents and samples to room temperature before use.
- Turn on the microplate reader 30 min in advance to preheat the instrument and set the testing parameters.
- All experimental apparatus should be clean, and disposable pipette should be used and changed frequently to avoid cross-contamination during the experiment.
Assay Procedure
Bring all reagents and samples to room temperature (25oC) before use. All reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Any unused Microtiter plate wells should be resealed as soon as possible and stored at 2-8℃.
- Number: number the samples and standards in order (multiple wells), and keep a record of the plate layout. Standards and Samples must be tested in duplicate.
- Add Sample: add 50 μL of Standard or Sample to each well, then add 50 μL Antibody Working Solution, and cover the plate with plate sealer. Oscillate gently for 5 s to mix thoroughly, then incubate at 25℃ for 30 min in the dark.
- Wash: Carefully remove the plate sealer, then remove the liquid from each well. Immediately add 300 μL of Wash Buffer (Solution 5) to each well and wash. Repeat wash procedure 5 times, 30 s intervals each time. Invert the plate and pat it against a thick sheet of absorbent paper (If there are bubbles in the wells, clean pipette tips can be used to pop them).
- HRP Conjugate: add 100 μL of HRP Conjugate to each well, incubate at 25℃ for 30 min in the dark.
- Wash: repeat step 3.
- Color Development: add 50 μL of Substrate Reagent A to each well, and then add 50 μL of Substrate Reagent B. Gently oscillate for 5 s to mix thoroughly. Incubate at 25℃ for 15 min in the dark (The reaction time can be extended according to the amount of color change).
- Stop Reaction: add 50 μL of Stop Solution to each well. Gently oscillate to mix thoroughly.
- OD Measurement: determine the optical density (OD value) of each well at 450 nm (reference wavelength 630 nm) with a microplate reader. This step should be performed no later than 10 min after the reaction has been stopped.
Calculation of Results
Absorbance (%)=A/A₀×100%
- A: Average absorbance of standard or sample
- A₀: Average absorbance of 0 ppb Standard
- Drawing and calculation of standard curve
- Create a standard curve by plotting the absorbance percentage of each standard on the y-axis against the log concentration on the x-axis to draw a semi-logarithmic plot. Add the average absorbance value of each sample to the standard curve to calculate the corresponding concentration. If samples have been diluted, the concentration calculated from the standard curve must be multiplied by the dilution factor.
- We recommend using professional software for fast and accurate analysis of large numbers of samples.
Product Data
Reaction conditions (Incubation time and temperature)
25℃; 30 min, 30 min, 15 min
Detection limit
Muscle (fish, shrimp) ---0.2 ppb; Muscle (livestock), Liver ---0.5 ppb
Cross-reactivity
Diethylstilbestrol---100%, Dienestrol---38.5%, Hexestrol---8.5%, Ethinyl estradiol--＜0.1%, Estriol---＜0.1%
Sample recovery rate
85%±10%