Oxytetracycline (OTC) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDF-E112
The Oxytetracycline (OTC) ELISA Kit (RDF-E112) is an enzyme-linked immunosorbent assay for quantitative detection of OTC in Food samples with a standard range of 0.3-24.3ppb. This kit uses the Traditional ELISA format for colorimetric detection of natural OTC in samples including Muscle,Milk,Egg,Feed. 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 OTC in samples. For research use only.
Product Specifications
Product Name
Oxytetracycline (OTC) ELISA Kit
Catalog Number
RDF-E112
Detection Range
0.3-24.3ppb
Species Reactivity
Food
Species
Food
Assay Length
45 minutes
Experimental Method
Competitive Inhibition
Detection Method
Colorimetric
Sample Type
Muscle,Milk,Egg,Feed
Storage
2-8°C,Avoid freeze
Shelf Life
12 monthes
Product Details
Intended Use
For the quantitative detection of Oxytetracycline (OTC) concentration in muscle, milk, egg, and feed samples.
Assay Principle
This kit uses Competitive-ELISA as the method for the quantitative detection. It can detect Oxytetracycline (OTC) in samples, such as muscle, raw milk, feed, etc. 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, OTC in the samples or standard competes with coupled antigen on the solid phase supporter for sites of anti-OTC 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 OTC. The concentration of OTC 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, 0.3 ppb, 0.9 ppb, 2.7 ppb, 8.1 ppb, 24.3 ppb) |
| Antibody Working Solution | 7 mL |
| HRP Conjugate | 7 mL |
| 20×Concentrated Sample Diluent | 50 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, Homogenizer, Vortex mixer, Centrifuge, Balance (sensibility 0.01 g).
- Micropipettes: single-channel 20-200 µL, 100-1000 µL, and multi-channel 30-300 µL.
- Reagents: Trichloroacetic acid, Na2 Fe (CN)5 NO•2H2O, 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 1±0.05 g of homogenate muscle into the 50 mL centrifuge tube.
- Add 9 mL of Sample Diluent (Solution 4), mix fully.
- Vortex for 1 min, centrifuge at 4000 r/min for 10 min at room temperature.
- Take 50 μL of supernatant for analysis.
- Take 3 mL of homogenate fresh sample into a 5 mL centrifuge tube.
- Centrifuge for 10 min at 4000 r/min at 4-10℃. If a refrigerated centrifuge is not available, chill sample to approx 10℃ prior to centrifugation.
- Remove the upper fat layer, take 2 mL of skim milk of lower layer to another 5 mL centrifuge tube.
- Add 50 μL of 0.36 M Na2 Fe (CN)5 NO•2H2O Solution (Solution 1), vortex for 90 s. Add 50 μL of 1 M ZnSO4 Solution (Solution 2), vortex for 1 min.
- Centrifuge at 3000 r/min for 10 min at room temperature.
- Take 50 μL of the supernatant to another 2 mL centrifuge tube, add 450 μL of deionized water. Vortex for 30 s.
- Take 50 μL for analysis.
- Egg:
- Weigh 1±0.05 g of homogenate egg into the 50 mL centrifuge tube. Add 5 mL of deionized water, vortex for 2 min.
- Centrifuge at 4000 r/min for 10 min at room temperature.
- Take 1 mL of the supernatant to another centrifuge tube, and add 1 mL of Sample Diluent (Solution 4), vortex for 30 s.
- Centrifuge at 4000 r/min for 5 min at room temperature.
- Take 50 μL of supernatant for analysis.
- Feed (cattle, swine):
- Homogenize the representative sample with a homogenizer and mix fully.
- Weigh 1±0.05 g of homogenate sample into the 50 mL centrifuge tube. Add 5 mL of 1% Trichloroacetic acid Solution (Solution 3). Vortex for 5 min.
- Centrifuge at 4000 r/min for 10 min at room temperature.
- Take 40 μL of the supernatant to another centrifuge tube, and add 1560 μL of Sample Diluent (Solution 4). Vortex for 30 s.
- Take 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: 0.36 M Na2 Fe (CN)5 NO•2H2O Solution (for raw milk sample)
Weigh 0.54 g of Na2 Fe (CN)5 NO•2H2O and add 4.7 mL of deionized water, vortex until dissolved fully.
Prepare the solution fresh before use!
Solution 2: 1 M ZnSO4 Solution (for raw milk sample)
Weigh 1.44 g of ZnSO4 and add 4.32 mL of deionized water, vortex until dissolved fully.
Solution 3: 1% Trichloroacetic acid Solution (for feed sample)
Dissolve 1 g of Trichloroacetic acid to 100 mL with deionized water, mix fully.
Solution 4: Sample Diluent (for livestock, egg, feed sample)
Dilute 20×Concentrated Sample Diluent with deionized water. (20×Concentrated Sample Diluent (V): Deionized water (V) = 1:19). Mix fully.
Solution 5: Wash Buffer
Dilute 20×Concentrated Wash Buffer with deionized water. (20×Concentrated Wash Buffer (V): Deionized water (V) = 1:19). Mix fully.
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 uL HRP Conjugate and 50 μL 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 5) 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, Milk, Egg ---6 ppb; Feed---400 ppb
Cross-reactivity
Oxytetracycline ---100%; Chlortetracycline --->100%; Tetracycline--->100%; Doxycycline --- 18.5%
Sample recovery rate
90%±30%.