Tilmicosin (TMC) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDF-E063
The Tilmicosin (TMC) ELISA Kit (RDF-E063) is an enzyme-linked immunosorbent assay for quantitative detection of TMC in Food samples with a standard range of 0.5-40.5ppb. This kit uses the Traditional ELISA format for colorimetric detection of natural TMC in samples including Muscle,Liver,Honey,Milk,Egg. 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 TMC in samples. For research use only.
Product Specifications
Product Name
Tilmicosin (TMC) ELISA Kit
Catalog Number
RDF-E063
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,Liver,Honey,Milk,Egg
Storage
2-8°C,Avoid freeze
Shelf Life
12 monthes
Product Details
Intended Use
For the quantitative detection of tilmicosin concentration in muscle, liver, honey, milk, and egg samples.
Assay Principle
This kit uses Competitive-ELISA as the method for the quantitative detection. It can detect Tilmicosin (TMC) in samples, such as muscle, liver, 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, TMC in the samples or standard competes with coupled antigen on the solid phase supporter for sites of anti- TMC 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 TMC. The concentration of TMC 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) |
| HRP Conjugate | 5.5 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 |
| 2xReconstitution 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, nitrogen evaporators, water bath, vortex mixer, centrifuge, graduated pipette, Balance (sensibility 0.01 g).
- Micropipettes: Single channel (20-200 μL, 100-1000 μL), Multichannel (30-300 μL).
- Reagents: Methanol, Ethyl acetate, Sodium Bicarbonate (NaHCO₃), Sodium carbonate (Na2CO3),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
- Muscle (Method 1):
- Remove fat from sample, homogenize the sample with homogenizer.
- Add 2±0.05 g of homogeneous sample to centrifuge tube, then add 2 mL of 0.1M (PH=10.6) CB Buffer (Solution 1), vortex and mix thoroughly. Add 8 ml of Ethyl acetate, vortex for 3 min, and centrifuge at 3000 r/min for 5 min.
- Take 4 mL of upper organic liquid into glass tube, dry at 50-60℃ with nitrogen evaporators or water bath.
- Dissolve the residual with 1 mL of N-hexane, add 1 mL of Reconstitution Buffer (Solution 3) and vortex for 1 min. Centrifuge at 3000 r/min for 5 min.
- Discard the upper liquid, take 50 μL of lower liquid for analysis.
- Muscle (Method 2):
- Remove fat from sample, homogenize the sample with homogenizer.
- Add 1.0±0.05 g of homogeneous sample to centrifuge tube, then add 4 mL of Wash Buffer (Solution 5), vortex for 3 min, centrifuge at 3000 r/min for 5 min in room .
- Take 200 µL of supernatant to new centrifuge tube, add 600 µL of Reconstitution Buffer (Solution 3), and mix for 30s.
- Take 50 µL of liquid for analysis.
- Liver:
- Add 1.0±0.05 g of homogeneous sample to centrifuge tube, then add 2 mL of 0.5 M (PH=10.6) CB Buffer (Solution 2) and add 8 ml of Ethyl acetate, vortex for 3 min, centrifuge at 3000 r/min for 5 min.
- Take 2 mL of upper organic liquid into glass tube, dry at 50-60℃ with nitrogen evaporators or water bath.
- Dissolve the residual with 1 mL of N-hexane, add 1 mL of Reconstitution Buffer (Solution 3) and vortex for 1 min. Centrifuge at 3000 r/min for 5 min.
- Discard the upper liquid. Take 100 µL of lower liquid, add 400 µL of Reconstitution Buffer (Solution 3), and mix for 30s.
- Take 50 μL of liquid for analysis.
- Weigh 2±0.05 g of honey sample into 50 mL centrifuge tube, add 2 mL of 0.1M (PH=10.6) CB Buffer (Solution 1) and add 8 ml of Ethyl acetate, vortex for 3 min, centrifuge at 3000 r/min for 5 min..
- Take 2 mL of upper organic liquid into glass tube, dry at 50-60℃ with nitrogen evaporators or water bath. Add 1 mL of Reconstitution Buffer (Solution 3) and mix for 30s.
- Take 50 µL of liquid for analysis.
- Egg:
- Mix 100µL of homogeneous egg sample and 900µL of Sample Diluent (Solution 4) for 30s.
- Take 50 µL of liquid to analysis.
- Dilute 100 µL fresh milk sample with 900µL of Sample Diluent (Solution 4), mix for 30s.
- Take 50 µL of liquid to analysis.
Reagent Preparation
Solution preparation:
Prepare solutions according to the number of samples. Avoid using all components in the kit at once.
Solution 1: 0.1M (PH=10.6) CB Buffer (for muscle, honey sample)
Dissolve 0.932 g of Na2CO3 and 0.1 g of NaHCO₃ to 100 mL with deionized water.
Solution 2: 0.5 M (PH=10.6) CB Buffer (for liver sample)
Dissolve 4.66 g of Na2CO3 and 0.5 g of NaHCO3 to 100 mL with deionized water.
Solution 3: Reconstitution Buffer (for muscle, liver, honey sample)
Dilute the 2×Reconstitution Buffer with deionized water. (2×Reconstitution Buffer (V): Deionized water (V) =1:1).
Solution 4: Sample Diluent (for egg, milk sample)
Dilute Reconstitution Buffer with Methanol. (Reconstitution Buffer (V): methanol (V) = 19:1).
Solution 5: Wash Buffer
Dilute 20×Concentrated Wash Buffer with deionized water. (20×Concentrated Wash Buffer (V): Deionized water (V) = 1:19). The Wash Buffer can be store at 4℃ for a month.
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 of HRP Conjugate, then add 50 μL of Antibody Working Solution into each well. Oscillate gently for 5 s to mix thoroughly and cover the plate with a plate sealer, 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).
- 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, 15 min
Detection limit
Muscle (method 1) ---0.5 ppb; Muscle (method 2) ---10 ppb; Liver ---10 ppb; Honey---1 ppb; Milk, eggs ---5 ppb.
Cross-reactivity
Tilmicosin ---100%; Tylosin---1%
Sample recovery rate
Muscle ---90%±20%, Liver---75%±15%; Honey, Milk, Eggs---95%±25%.