Sulfonamides (SAs) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDF-E072
The Sulfonamides (SAs) ELISA Kit (RDF-E072) is an enzyme-linked immunosorbent assay for quantitative detection of SAs in Food samples with a standard range of 2-162ppb. This kit uses the Traditional ELISA format for colorimetric detection of natural SAs in samples including Muscle,Urine,Liver,Honey,Serum,Raw milk,Reconstituted milk,Finished 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 SAs in samples. For research use only.
Product Specifications
Product Name
Sulfonamides (SAs) ELISA Kit
Catalog Number
RDF-E072
Detection Range
2-162ppb
Species Reactivity
Food
Species
Food
Assay Length
45 minutes
Experimental Method
Competitive Inhibition
Detection Method
Colorimetric
Sample Type
Muscle,Urine,Liver,Honey,Serum,Raw milk,Reconstituted milk,Finished milk,Egg,Feed
Storage
2-8°C,Avoid freeze
Shelf Life
12 monthes
Product Details
Intended Use
For the quantitative detection of sulfonamides concentration in muscle, urine, liver, honey, serum, raw milk, reconstituted milk, finished milk, feed, and egg samples.
Assay Principle
This kit uses Competitive-ELISA as the method for the quantitative detection. It can detect total sulfonamides (SAs) in samples, such as muscle, feed, 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, SAs in the samples or standard competes with coupled antigen on the solid phase supporter for sites of anti-SAs 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 SAs. The concentration of SAs 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, 2 ppb, 6 ppb, 18 ppb, 54 ppb, 162 ppb) |
| HRP Conjugate | 7 mL |
| Antibody Working Solution | 10 mL |
| Substrate Reagent A | 6 mL |
| Substrate Reagent B | 6 mL |
| Stop Solution | 6 mL |
| 20xConcentrated Wash Buffer | 25 mL |
| 20xConcentated Sample Solution | 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, nitrogen evaporators, water bath, centrifuge, balance (sensibility 0.01g).
- Micropipettes: Single channel (20-200 μL, 100-1000 μL), Multichannel (30-300 μL).
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
- Urine (swine):
- Take urine sample, (if the urine sample is turbid, it should be filtered or centrifuged at 4000 r/min for 5 min until the urine sample become clear).
- Take 20 μL of the supernatant for analysis.
- Reconstituted milk (powder): Weigh 1±0.05 g of sample into a centrifuge tube. Add 8 mL of deionized water. Immediately vortex for 30 s, mix fully.
- Acidic samples such as yogurt: Weigh 1±0.05 g of homogenate sample into a centrifuge tube. Add 1 M NaOH Solution (Solution 5) (about 50 μL) for adjust PH=7.
- Take 1 mL (1 g) of sample into a 4 mL centrifuge tube, add 100 μL of ZnSO4 Solution ((Solution 2) and add 100 μL of K4Fe(CN)6 Solution (Solution 1). Immediately vortex for 30 s, mix fully.
- Add 1.8 mL of PB Solution (Solution 3). Centrifuge at 4000 rpm for 5 min at room temperature.
- Take 200 μL of the supernatant to 200 μL of deionized water, mix fully.
- Take 20 μL for analysis.
- Muscle (Method 1):
- Remove fat from sample, homogenize the sample with homogenizer.
- Weigh 1±0.05 g of homogenate sample into a 50 mL centrifuge tube. Add 9.5 mL of deionized water and add 0.5 mL of 20×Concentrated Sample Solution. Immediately vortex for 1 min, mix fully.
- Centrifuge at 4000 rpm for 10 min at room temperature.
- Take 20 μL for the supernatant analysis.
- Muscle (Method 2) (fish, shrimp, 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 0.1 mL of H3PO4 Solution (Solution 6) and add 6 mL of Acetonitrile. Immediately vortex for 2 min, mix fully.
- Centrifuge at 4000 rpm for 5 min at room temperature.
- Remove 2 mL of the supernatant to a 4 mL centrifuge tube, dry at 60-70℃ with nitrogen evaporators or water bath.(Please do it in a ventilated environment.)
- Dissolve the residue with 1 mL of N-hexane, immediately vortex for 30 s and 0.5 mL of Sample Solution (Solution 7). Vortex for 30 s
- Centrifuge at 4000 rpm for 5 min at room temperature. Remove the upper layer of N-hexane and intermediate layer impurities.
- Take 20 μL for analysis.
- Liver (chicken, swine):
- Remove fat from sample, homogenize the sample with homogenizer.
- Weigh 2±0.05 g of homogenate sample into a 50 mL centrifuge tube. Add 3 mL of Wash Buffer (Solution 8) and add 3 mL of Liver Extracting Solution (Solution 4). Immediately vortex for 1 min, mix fully.
- Centrifuge at 4000 rpm for 5 min at room temperature.
- Take 1 mL of the intermediate layer solution to a new centrifuge tube. Add 20 μL of 1 M NaOH Solution (Solution 5), immediately vortex for 10 s. Centrifuge at 4000 rpm for 5 min at room temperature.
- Take 20 μL of supernatant for analysis.
- Feed:
- Homogenize the representative sample with a homogenizer and mix fully.
- Weigh 1±0.05 g of homogenate sample into a 50 mL centrifuge tube. Add 10 mL of deionized water. Immediately vortex for 1 min, mix fully.
- Centrifuge at 4000 rpm for 10 min at room temperature.
- Take 20 μL of supernatant for analysis.
- Serum, egg:
- Weigh 1±0.05 g (1mL) of homogenate sample into a centrifuge tube. Add 1 mL of Methanol and 1 mL of Wash Buffer (Solution 8). Immediately vortex for 1 min, mix fully.
- Centrifuge at 4000 rpm for 5 min at room temperature.
- Take 20 μL of supernatant for analysis.
- Honey:
- Weigh 2±0.05 g of homogenate sample into a 50 mL centrifuge tube. Dissolve honey with 1 mL of deionized water, vortex for 5 min.
- Add 0.1 mL of H3PO4 Solution (Solution 6) and add 5 mL of Acetonitrile. Immediately vortex for 2 min, mix fully. Centrifuge at 4000 rpm for 5 min at room temperature.
- Remove 2 mL of the supernatant to 4 mL centrifuge tube, dry at 60-70℃ with nitrogen evaporators or water bath.
- Add 1 mL of N-hexane, immediately vortex for 30 s and 0.5 mL of Sample Solution (Solution 7). Vortex for 30 s.
- Centrifuge at 4000 rpm for 5 min at room temperature. Remove the upper layer of N-hexane and intermediate layer impurities.
- Take 20 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: K4Fe(CN)6 Solution (for raw milk, finished milk sample)
Dissolve 1.52 g of K4 Fe (CN) 6·3H2O with 10 mL of deionized water, mix fully.
Solution 2: ZnSO4 Solution (for raw milk, finished milk sample)
Dissolve 2.88 g of ZnSO4 ·7H2O with 8.64 mL of deionized water, mix fully.
Solution 3: PB Solution (for raw milk, finished milk sample)
Dissolve 6 g of Na2HPO4·12H2O and 0.5 g of NaH2PO4·2H2O with 300 mL of deionized water, mix fully.
Solution 4: Liver Extracting Solution (for chicken, swine sample)
Dissolve 1 g of C2HCl3O2 with 100 mL of deionized water, mix fully.
Solution 5: 1 M NaOH Solution (for raw milk, finished milk, chicken, swine sample)
Dissolve 4 g of NaOH with 100 mL of deionized water, mix fully.
Solution 6: H3PO4 Solution (for fish, shrimp, livestock, honey sample)
Add 2 mL of H3PO4 to 98 mL of deionized water, mix fully.
Solution 7: Sample Solution (for fish, shrimp, livestock, honey sample)
Dilute the 20×Concentrated Sample Solution with deionized water (20×Concentrated Sample Solution (V): Deionized water (V) =1:3).
Solution 8: 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 20 μL of Standard or Sample to each well, then add 50 μL of HRP Conjugate, then add 80 μL of Antibody Working Solution into each well. Oscillate gently for 10 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 260 μL of Wash Buffer (Solution 8) 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 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
Urine, Muscle (method 1, pork) ---40 ppb; Liver---10 ppb; Honey ---3 ppb; Muscle (method 1, chicken, fish, shrimp), Serum, Raw milk, Finished Milk---20 ppb; Muscle (method 2) ---2 ppb; Muscle (method 1, beef, mutton, duck), Eggs, Feed ---50 ppb.
Cross-reactivity
Sample recovery rate
90%±30%.