Sulfamethoxazole (SMZ) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDF-E021
The Sulfamethoxazole (SMZ) ELISA Kit (RDF-E021) is an enzyme-linked immunosorbent assay for quantitative detection of SMZ in Food samples with a standard range of 0.1-8.1ppb. This kit uses the Traditional ELISA format for colorimetric detection of natural SMZ in samples including Muscle,Honey,Serum,Urine,Egg,Milk,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 SMZ in samples. For research use only.
Product Specifications
Product Name
Sulfamethoxazole (SMZ) ELISA Kit
Catalog Number
RDF-E021
Detection Range
0.1-8.1ppb
Species Reactivity
Food
Species
Food
Assay Length
60 minutes
Experimental Method
Competitive Inhibition
Detection Method
Colorimetric
Sample Type
Muscle,Honey,Serum,Urine,Egg,Milk,Feed
Storage
2-8°C,Avoid freeze
Shelf Life
12 monthes
Product Details
Intended Use
For the quantitative detection of sulfamethoxale concentration in muscle, honey, serum, urine, egg, milk, and feed samples.
Assay Principle
This kit uses Competitive-ELISA as the method for the quantitative detection. It can detect Sulfamethoxazole (SMZ) in samples, such as muscle, milk, honey, 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, SMZ in the samples or standard competes with coupled antigen on the solid phase supporter for sites of anti-SMZ 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 SMZ. The concentration of SMZ 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, 0.1 ppb, 0.3 ppb, 0.9 ppb, 2.7 ppb, 8.1 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, 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: Ethyl acetate, concentrated HCl, N-hexane, acetonitrile, Na2HPO4·12H2O, NaOH, NaH2PO4·2H2O.
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.
- Weigh 2±0.05 g of homogenate muscle into 50 mL centrifuge tube. Add 1 mL of 0.1 M PBS Buffer (Solution 1), vortex for 10 min. Add 7 mL of Acetonitrile-ethyl acetate Solution (Solution 2), Vortex for 4 min, centrifuge at 4000 r/min for 5 min at room temperature.
- Take 4 mL of the clean upper organic solution to a another centrifuge tube, dry at 50-60℃ of nitrogen evaporators or water bath.
- Redissolve the dry residual sediment with 1 mL of N-hexane. Add 1 mL of Reconstitution Buffer (Solution 5) and mix for 30s. Centrifuge at 4000 rpm for 5 min at room temperature.
- Remove the upper layer, and take 50 μL of the lower layer for analysis.
- Muscle (Method 2):
- Remove fat from sample, homogenize the sample with homogenizer.
- Weigh 1±0.05 g of homogenate muscle into a 50 mL centrifuge tube, add 9 mL of 0.1 M PBS Buffer (Solution 1) and vortex for 5 min, centrifuge at 4000 rpm for 5 min at room temperature..
- Take 50 μL of the supernatant for analysis.
- Serum (swine):
- Stand the serum for 30 min at room temperature. Centrifuge at 4000 rpm for 10 min at room temperature.
- Take 1 mL of supernatant. Add 3 mL of 0.1 M PBS Buffer (Solution 1) and vortex fully for 30s.
- Take 50 μL for analysis.
- Honey:
- For laboratory samples without crystallization, stir well. For the sample with crystallization phenomenon, place it in a closed water bath not exceeding 60℃, heat it, shake, stir after the sample is all melted, and cool to room temperature.
- Weigh 1±0.05 g of honey sample into a 50 mL centrifuge tube. Add 1 mL of 0.5 M HCl Solution (Solution 3). Incubate at 37℃ for 30 min.
- Add 2.5 mL of 0.2 M NaOH Solution (Solution 4) (adjust PH≈5), then add 4 mL of Ethyl acetate. Vortex for 5 min, centrifuge at 4000 rpm for 5 min at room temperature.
- Take 2 mL of the supernatant to a another centrifuge tube, dry at 50-60℃ of nitrogen evaporators or water bath.
- Redissolve the dry residual sediment with 0.5 mL of Reconstitution Buffer (Solution 5). Mix for 30s.
- Take 50 μL for analysis.
- Urine (swine):
- Add 3 mL of 0.1 M PBS Buffer (Solution 1) into 1 mL of centrifuged clear urine sample, vortex for 30s.
- Take 50 μL for detection and analysis.
- Dilute 100 μL of milk with 0.1 M PBS Buffer (Solution 1) (100μL of milk +1.9 mL of 0.1M PBS Buffer). Mix for 30s.
- Take 50 μL for analysis.
- Feed:
- Homogenize the representative sample with a homogenizer and mix fully.
- Weigh 2±0.05 g of feed sample into 50 mL centrifuge tube, add 8 mL of Acetonitrile, vortex 5 min, centrifuge at a 4000 rpm for 5 min at room temperature.
- Take 1 mL of the upper organic layer to 10 mL clean dry glass tube, dry at 50-60℃ of nitrogen evaporators or water bath.
- Redissolve the dry residual sediment with 1 mL of N-hexane, Vortex for 30s, then add 1 mL of 0.1 M PBS Buffer (Solution 1), Vortex sample for 30s. Centrifuge at 4000 rpm for 5 min at room temperature.
- Remove the upper organic layer, take 100 μL of the lower water layer to 2 mL centrifuge tube, add 900 μL of 0.1 M PBS Buffer (Solution 1), vortex sample for 1 min, mix well;
- Take 50 μL sample 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 2 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.
- Remove the upper layer, and take 50 μL of the lower layer 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: 0.1 M PBS Buffer (for muscle, serum, urine, milk, feed sample)
Dissolve 25.8 g of Na2HPO4·12H2O and 4.4 g of NaH2PO4·2H2O to 1000 mL with deionized water, mix fully.
Solution 2: Acetonitrile-ethyl acetate Solution (for muscle sample)
Add 50 mL of Acetonitrile and 50 mL of Ethyl acetate to 100 mL glass bottle, mix fully.
Solution 3: 0.5 M HCl Solution (for honey sample)
Add 4.3 mL of Concentrated HCl to 100mL with deionized water, mix fully.
Solution 4: 0.2 M NaOH Solution (for honey sample)
Dissolve 0.8 g of NaOH to 100 mL with deionized water, mix fully.
Solution 5: Reconstitution Buffer (for muscle, honey sample)
Dilute the 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 6: 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, 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 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 6) 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℃; 45 min, 15 min
Detection limit
Muscle (method 1) ---0.1 ppb; Muscle (method 2) ---1 ppb, Honey---0.1 ppb, Serum, Urine ---0.4 ppb; Milk---2 ppb; Feed---4 ppb; Egg ---0.2 ppb.
Cross-reactivity
Sulfamethoxazole---100%
Sample recovery rate
Muscle, Honey ---85%±25%, Serum, Urine, Milk, Feed---80%±25%