Amantadine (Am) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDF-E085
The Amantadine (Am) ELISA Kit (RDF-E085) is an enzyme-linked immunosorbent assay for quantitative detection of Am in Food samples with a standard range of 0.5-40.5ppb. This kit uses the Traditional ELISA format for colorimetric detection of natural Am in samples including Muscle,Egg,Milk. 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 Am in samples. For research use only.
Product Specifications
Product Name
Amantadine (Am) ELISA Kit
Catalog Number
RDF-E085
Detection Range
0.5-40.5ppb
Species Reactivity
Food
Species
Food
Assay Length
45 minutes
Experimental Method
Competitive Inhibition
Detection Method
Colorimetric
Sample Type
Muscle,Egg,Milk
Storage
2-8°C,Avoid freeze
Shelf Life
12 monthes
Product Details
Intended Use
For the quantitative detection of Amantadine (AM) concentration in muscle, egg, and milk samples.
Assay Principle
This kit uses Competitive-ELISA as the method for the quantitative detection. It can detect Amantadine (Am) in samples, such as muscle, milk, etc. This kit is composed of ELISA Microtiter plate, HRP conjugate, antibody working solution, standard and other supplementary reagents. The microtiter plate provided in this kit has been pre-coated with coupled antigen. During the detection, Am in the samples or standard competes with coupled antigen on the solid phase supporter for sites of anti-Am antibody. Then Horseradish Peroxidase (HRP) conjugate is added to each well, and substrate reagent is added for color development. There is a negative correlation between the OD value of samples and the concentration of Am. The concentration of Am 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, 0.5 ppb, 1.5 ppb, 4.5 ppb, 13.5 ppb, 40.5 ppb) |
| Antibody Working Solution | 7 mL |
| HRP Conjugate | 7 mL |
| Substrate Reagent A | 6 mL |
| Substrate Reagent B | 6 mL |
| Stop Solution | 6 mL |
| 20×Concentrated Sample Solution | 10 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, Nitrogen evaporators, Water bath, Vortex mixer, Centrifuge, Balance (sensibility 0.01 g).
- Micropipettes: single-channel 20-200 µL, 100-1000 µL, and multi-channel 30-300 µL.
- Reagents: Acetonitrile, NaCl, ZnSO4·7H2O.
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 (livestock):
- Remove fat from sample, homogenize the sample with homogenizer.
- Weigh 2±0.05 g of homogenate sample into a 50 mL centrifuge tube;
- Add 0.5 g of NaCl, then add 6 mL of Acetonitrile, and fully vortex for 1min;
- Centrifuge for 5 min at 4000 r/min;
- Take 4 mL of supernatant into a new centrifuge tube, dry at 50-60℃ with nitrogen evaporators or water bath
- Add 0.5 mL of Sample Solution (Solution 3) to vortex for 30s thoroughly;
- Take 50 μL for detection.
- Egg:
- Weigh 2±0.05 g of homogenate eggs sample into a 50 mL of centrifuge tube;
- Add 6 mL of Acetonitrile, and fully vortex for 1min;
- Centrifuge for 5 min at 4000 r/min;
- Take 4 mL of supernatant into a new centrifuge tube, dry at 50-60℃ with nitrogen evaporators or water bath.
- Add 0.5 mL of Sample Solution (Solution 3), and vortex for 10s;
- Take 50 μL for detection.
- Take 0.5 mL of sample into a 4 mL centrifuge tube;
- Add 50 μL of 1 M ZnSO4 Solution (Solution 1);
- Add 2 mL of Sample Solution (Solution 3), and fully vortex for 10s;
- Centrifuge for 5 min at 4000 r/min;
- Take 50 μL for detection.
Reagent Preparation
Solution preparation:
Prepare solutions according to the number of samples. Avoid using all components in the kit at once.
Solution 1: 1 M ZnSO4 Solution (for milk sample)
Dissolve 2.88 g of ZnSO4·7H2O with 8.64 mL of deionized water, mix fully.
Note: 1 M ZnSO4 Solution is unstable, prepare fresh solution before use.
Solution 2: Wash Buffer
Dilute 20×Concentrated Wash Buffer with deionized water. (20×Concentrated Wash Buffer (V): Deionized water (V) = 1:19).
Solution 3: Sample Solution
Dilute 20×Concentrated Sample Solution with deionized water. (20×Concentrated Sample Solution (V): Deionized water (V) = 1:19).
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 50 μL HRP Conjugate and 50 uL Antibody Working Solution, and cover the plate with plate sealer. Oscillate gently for 10 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 260 μL of Wash Buffer (Solution 2) to each well and wash. Repeat wash procedure 4 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℃; 30 min, 15 min
Detection limit
Muscle, Eggs ---1 ppb; Milk---2 ppb.
Cross-reactivity
Amantadine---100%; Rimantadine---230%; Somantadine ---240%; Memantine ---＜1%
Sample recovery rate
90%±30%