Tetracyclines (TCs) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDF-E046
The Tetracyclines (TCs) ELISA Kit (RDF-E046) is an enzyme-linked immunosorbent assay for quantitative detection of TCs in Food samples with a standard range of 0.05-4.05ppb. This kit uses the Traditional ELISA format for colorimetric detection of natural TCs in samples including Muscle,Liver,Egg,Honey,Urine,Milk,Milk powder. 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 TCs in samples. For research use only.
Product Specifications
Product Name
Tetracyclines (TCs) ELISA Kit
Catalog Number
RDF-E046
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,Egg,Honey,Urine,Milk,Milk powder
Storage
2-8°C,Avoid freeze
Shelf Life
12 monthes
Product Details
Intended Use
For the quantitative detection of tetracyclines concentration in muscle, liver, honey, milk, egg, urine, and milk powder samples.
Assay Principle
This kit uses Competitive-ELISA as the method for the quantitative detection. It can detect Tetracyclines (TCS) in samples, such as muscle, honey, 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, TCS in the samples or standard competes with coupled antigen on the solid phase supporter for sites of anti-TCS 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 TCS. The concentration of TCS 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, 0.05 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 |
| 20xConcentrated Wash Buffer | 40 mL |
| 5xReconstitution Buffer | 50 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, printer, homogenizer, vortex mixer, centrifuge, graduated pipette, Balance (sensibility 0.01 g).
- Micropipettes: Single channel (20-200 μL, 100-1000 μL), Multichannel (30-300 μL).
- Reagents: N, N-Dimethylformamide (DMF)
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, egg:
- Homogenize muscle, liver and egg samples with homogenizer.
- Weigh 2±0.05 g of homogenate sample into the a 50 mL centrifuge tube, add 4 mL of DMF. Vortex for 5 min, the sample is fully mixed with the liquid. Centrifuge at 4000 rpm for 10 min at room temperature.
- Take 250 μL supernatant to another centrifuge tube, add 750 μL of Reconstitution Buffer (Solution 1) and mix fully.
- Take 50 μL for analysis.
- Honey:
- Weigh 1±0.05 g of honey into the centrifuge tube, add 2 mL of DMF. Vortex for 2 min, centrifuge at 4000 rpm for 10 min at room temperature.
- Take 100 μL of the supernatant to another centrifuge tube, add 1900 μL of Reconstitution Buffer (Solution 1) for diluting, mix fully for 30 s.
- Take 50 μL for analysis.
- Urine (swine):
- Dilute the urine sample with Reconstitution Buffer (Solution 1) for 10 times (if the urine sample is turbid, it should be filtered or centrifuged at 4000 rpm for 10 min until the urine sample become clear).
- 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: Reconstitution Buffer
Dilute the 5×Reconstitution Buffer with deionized water (5×Reconstitution Buffer (V): deionized water (V) =1:4), mix fully. The Reconstitution buffer can be store at 4℃ for a month.
Solution 2: Wash Buffer
Dilute the 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℃.
- Standard Preparation:
- Prepare the Standard Liquid. Standard Liquid of low concentration is unstable, prepare fresh solution before use.
- Take 3 mL of Reconstitution Buffer (Solution 1) into 0 ppb bottle. Take 2 mL of Reconstitution Buffer (Solution 1) into 0.05 ppb bottle, 0.15 ppb bottle, 0.45 ppb bottle, 1.35 ppb bottle respectively. Take 3mL of Reconstitution Buffer (Solution 1) into 4.05 ppb bottle.
- Standard Liquid 6: Take 12 μL of High Standard Liquid (1.0 ppm) into 4.05 ppb bottle, then mix fully. The concentration of Standard Liquid 6 is 4.05 ppb.
- Standard Liquid 5: Take 1 mL of Standard Liquid 6 into 1.35 ppb bottle, then mix fully. The concentration of Standard Liquid 5 is 1.35 ppb.
- Standard Liquid 4: Take 1 mL of Standard Liquid 5 into 0.45 ppb bottle, then mix fully. The concentration of Standard Liquid 5 is 0.45 ppb.
- Standard Liquid 3: Take 1 mL of Standard Liquid 4 into 0.15 ppb bottle, then mix fully. The concentration of Standard Liquid 5 is 0.15 ppb.
- Standard Liquid 2: Take 1 mL of Standard Liquid 3 into 0.05 ppb bottle, then mix fully. The concentration of Standard Liquid 5 is 0.05 ppb.
- Standard Liquid 1: Use Reconstitution Buffer directly. The concentration of Standard Liquid 1 is 0 ppb.
- Assay Procedure:
- 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 37℃ 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 2) 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 37℃ 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 37℃ 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 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)
37℃, 30 min, 30 min, 15 min
Detection limit
Muscle, Liver, Eggs---0.4 ppb; Honey---2 ppb; Urine---0.5 ppb.
Cross-reactivity
Tetracycline ---100%; Oxytetracycline ---100%; Chlortetracycline ---16.7%; Deoxytetracycline ---4.2%.
Sample recovery rate
Muscle, Liver, Eggs---85%±20%, Honey---75%±20%, Urine---80%±20%.