Trimethoprim (TMP) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDF-E020
The Trimethoprim (TMP) ELISA Kit (RDF-E020) is an enzyme-linked immunosorbent assay for quantitative detection of TMP in Food samples with a standard range of 0.03-2.43ppb. This kit uses the Traditional ELISA format for colorimetric detection of natural TMP in samples including Muscle,Feed,Kidney,Liver,Serum,Urine,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 TMP in samples. For research use only.
Product Specifications
Product Name
Trimethoprim (TMP) ELISA Kit
Catalog Number
RDF-E020
Detection Range
0.03-2.43ppb
Species Reactivity
Food
Species
Food
Assay Length
60 minutes
Experimental Method
Competitive Inhibition
Detection Method
Colorimetric
Sample Type
Muscle,Feed,Kidney,Liver,Serum,Urine,Egg
Storage
2-8°C,Avoid freeze
Shelf Life
12 monthes
Product Details
Intended Use
For the quantitative detection of trimethoprim concentration in muscle, feed, kidney, liver, serum, urine, and egg samples.
Assay Principle
This kit uses Competitive-ELISA as the method for the quantitative detection. It can detect Trimethoprim (TMP) in samples, such as muscle, feed, etc. This kit is composed of ELISA Microtiter plate, HRP conjugate, standard liquid and other supplementary reagents. The microtiter plate in this kit has been pre-coated with antibodies. During the reaction, TMP in the samples or standard competes with Horseradish Peroxidase (HRP) conjugate for sites. Add substrate reagent is added for color development. There is a negative correlation between the OD value of samples and the concentration of TMP. The concentration of TMP 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, 0.03 ppb, 0.09 ppb, 0.27 ppb, 0.81 ppb, 2.43 ppb) |
| HRP Conjugate | 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, vortex mixer, centrifuge, graduated pipette, Balance (sensibility 0.01 g), water bath.
- Micropipettes: Single channel (20-200 μL, 100-1000 μL), Multichannel (30-300 μL).
- Reagents: methanol anhydrous, N-hexane, HCl, NaOH, acetonitrate.
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
- Feed:
- Homogenize the representative sample with a homogenizer and mix fully.
- Weigh 2±0.05 g of homogenate sample into a 50 mL centrifugal tube, add 20 mL of 0.1 M HCl Solution (Solution 2) and vortex for 15 min. Centrifuge at 3000 rpm at room temperature for 10 min.
- Take 1 mL of the supernatant into 1.5 mL centrifugal tube, adjust the pH to 6-8 with 1 M NaOH Solution (Solution 3) (The added amount of 1 M NaOH Solution is different according to the feed sample. The needed amount is generally between 70μL-100 μL), centrifuge at 3000 rpm at room temperature for 10 min.
- Take 0.5 mL of the supernatant into another 1.5 mL centrifugal tube, add 0.5 mL of Reconstitution Buffer (Solution 1), and mix thoroughly.
- Take 50 μL for analysis.
- Muscle, liver, kidney:
- Remove fat from sample. Homogenize the representative sample with a homogenizer and mix fully.
- Weigh 2±0.05 g of homogenate sample into a 50mL centrifugal tube, add 6 mL of Methanol anhydrous and 2 mL of N-hexane, vortex for 5 min.
- Centrifuge at 4000 rpm at room temperature for 10 min, remove the upper N-hexane layer, take 0.5 mL of lower liquid into clean glass test tube(do not touch the fat layer) and dry at 50-60℃ with nitrogen evaporators or water bath.
- Add 400 μL of Reconstitution Buffer (Solution 1) and 500 μL of N-hexane, vortex for 1 min. Centrifuge at 4000 rpm at room temperature for 5 min.
- Remove upper N-hexane layer, take 50 μL of lower liquid for analysis.
- Urine, serum (swine):
- Weigh 0.5 mL of sample, centrifuge at 4000 rpm at room temperature for 5 min.
- Take 50 μL of supernatant, add 200 μL of Reconstitution Buffer (Solution 1), and mix thoroughly.
- Take 50 μL for analysis.
- Egg:
- Use a homogenizer to homogenize the yolk and egg whites and mix them thoroughly.
- Weigh 2±0.05 g of homogenate sample into a 50 mL centrifugal tube, add 8 mL of Acetonitrile and vortex for 15 min. Centrifuge at 4000 rpm at room temperature for 10 min.
- Take 1 mL of supernatant into clean glass test tube (do not touch the fat layer) and dry at 50-60℃ with nitrogen evaporators or water bath.
- Dissolve the residue with 1 mL of N-hexane, and add 1 mL of Reconstitution Buffer (Solution 1), and mix thoroughly. Centrifuge at 4000 rpm at room temperature for 5 min.
- 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 2×Reconstitution Buffer with deionized water (2×Reconstitution Buffer (V): Deionized water (V) = 1:1). The Reconstitution buffer can be store at 4℃ for a month.
Solution 2: 0.1 M HCl Solution (for feed sample)
Dilute 10 mL of HCl to 1200 mL with deionized water, mix fully.
Solution 3: 1 M NaOH Solution (for feed sample)
Dissolve 4 g of NaOH to 100 mL with deionized water.
Solution 4: Wash Buffer
Dilute 20×Concentrated Wash Buffer with deionized water (20×Concentrated Wash Buffer (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 of HRP Conjugate into each well. Oscillate gently for 5 s to mix thoroughly and cover the plate with a plate sealer, then incubate at 37℃ for 45 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 4) 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 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 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℃; 45 min, 15 min
Detection limit
Feed---1.6 ppb; Muscle, Kidney, Liver---0.4 ppb; Serum, Urine ---0.4 ppb; Egg ---0.3 ppb.
Cross-reactivity
Trimethoprim---100%, Sulfapyridine, Sulfamethoxazole, Sulfisoxazole, Sulfathiazole, Sulfamerazine, Sulfadoxine ---＜0.1%
Sample recovery rate
Feed---95%±15%; Muscle, Liver, Kidney---95%±10%; Serum, Urine, Honey ---85%±10%