Doramectin (DRM) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDF-E131
The Doramectin (DRM) ELISA Kit (RDF-E131) is an enzyme-linked immunosorbent assay for quantitative detection of DRM in Food samples with a standard range of 0.2-16.2ppb. This kit uses the Traditional ELISA format for colorimetric detection of natural DRM 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 DRM in samples. For research use only.
Product Specifications
Product Name
Doramectin (DRM) ELISA Kit
Catalog Number
RDF-E131
Detection Range
0.2-16.2ppb
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 Doramectin (DRM) concentration in muscle and liver samples.
Assay Principle
This kit uses Competitive-ELISA as the method for the quantitative detection. It can detect Doramectin (DRM) in samples, such as raw milk, muscle. This kit is composed of ELISA Microtiter plate, HRP conjugate, antibody working solution, standard and other supplementary reagents. The microtiter plate in this kit has been pre-coated with coupled antigen. During the reaction, DRM in the samples or standard competes with coupled antigen on the solid phase supporter for sites of anti-DRM antibody. Then Horseradish Peroxidase (HRP) conjugate is added to each microtiter plate well, and substrate reagent is added for color development. There is a negative correlation between the OD value of samples and the concentration of DRM. The concentration of DRM 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 Liquid | 1 mL each (ppb=ng/mL=ng/g) (0 ppb, 0.2 ppb, 0.6 ppb, 1.8 ppb, 5.4 ppb, 16.2 ppb) |
| 11×Concentrated HRP Conjugate | 1 mL |
| HRP Conjugate Diluent | 10 mL |
| 10×Concentrated Sample Diluent | 25 mL |
| Substrate Reagent A | 6 mL |
| Substrate Reagent B | 6 mL |
| Stop Solution | 6 mL |
| 20×Concentrated Wash Buffer | 25 mL |
| Plate Sealer | 3 |
| Sealed Bag | 1 |
| Manual | 1 |
All reagent bottle caps should be tightly closed to prevent evaporation and microbial contamination.
Materials Required but not Supplied
- Equipment: Microplate reader, Vortex mixer, Centrifuge.
- Micropipettes: single-channel 20-200 µL, 100-1000 µL, and multi-channel 30-300 µL.
- Reagents: Methanol, N-hexane.
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
Raw milk:
- Take 1 mL of fresh sample into a 10 mL centrifuge tube, then add 3 mL of Methanol to fully vortex for 1 min.
- Centrifuge for 5 min at 4000 r/min.
- Take 1 mL of the supernatant into a new centrifuge tube. Dry at 60℃ with nitrogen evaporators or water bath.
- Dissolve the dry residue with 0.5 mL of Sample Diluent (Solution 2).
- Take 50 μL for detection and analysis.
- Muscle and liver:
- Take 2±0.05 g of homogenate sample into 50 mL centrifuge tube, and add 3 mL of N-hexane and 6 mL of Acetonitrile to fully vortex for 1 min.
- Centrifuge for 5 min at 4000 r/min. Remove the upper N-hexane layer,
- Take 1 mL of the middle layer into a new centrifuge tube; Dry at 60℃ with nitrogen evaporators or water bath.
- Following different redissolve methods according to below different sample types.
- Muscle: add 250 μL of Methanol, vortex for 1 min. Then add 750 μL of Sample Diluent (Solution 2).
- Liver: add 150 μL of Methanol, vortex for 1 min. Then add 850 μL of Sample Diluent (Solution 2). Vortex for 1 min.
- Take 50 μL for detection and analysis.
Reagent Preparation
Solution preparation:
Prepare solutions according to the number of samples. Avoid using all components in the kit at once.
Solution 1: Wash Buffer
Dilute 20×Concentrated Wash Buffer with deionized water. (20×Concentrated Wash Buffer (V): Deionized water (V) = 1:19).
Solution 2: Sample Diluent
Dilute 10×Concentrated Sample Diluent with deionized water. (20×Concentrated Wash Buffer (V): Deionized water (V) = 1:9).
Solution 3: HRP Conjugate Diluent
Dilute 11×Concentrated HRP Conjugate with HRP Conjugate Diluent. (11×Concentrated HRP Conjugate (V): Deionized water (V) = 1:10).
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 (25℃) 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 80 uL HRP Conjugate (Solution 3) into each well and cover the plate with plate sealer. Oscillate gently for 10 s to mix thoroughly, then incubate at 25℃ for 40 min in the dark.
- Wash: Carefully remove the plate sealer, then remove the liquid from each well. Immediately add 260 μL of Wash Buffer (Solution 1) 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).
- Color Development: add 50 μL of Substrate Reagent A to each well, and then add 50 μL of Substrate Reagent B. Gently oscillate for 10 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 for 10 s 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 5 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℃; 40 min, 15 min
Detection limit
Muscle, raw milk ---4 ppb
Cross-reactivity
Sample recovery rate
90%±30%