Nitrofurantoin (AHD) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDF-E004
The Nitrofurantoin (AHD) ELISA Kit (RDF-E004) is an enzyme-linked immunosorbent assay for quantitative detection of AHD in Food samples with a standard range of 0.02-1.62ppb. This kit uses the Traditional ELISA format for colorimetric detection of natural AHD in samples including Muscle,Liver,Honey,Milk,Milk powder,Feed,Egg powder,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 AHD in samples. For research use only.
Product Specifications
Product Name
Nitrofurantoin (AHD) ELISA Kit
Catalog Number
RDF-E004
Detection Range
0.02-1.62ppb
Species Reactivity
Food
Species
Food
Assay Length
60 minutes
Experimental Method
Competitive Inhibition
Detection Method
Colorimetric
Sample Type
Muscle,Liver,Honey,Milk,Milk powder,Feed,Egg powder,Egg
Storage
2-8°C,Avoid freeze
Shelf Life
12 monthes
Product Details
Intended Use
For the quantitative detection of Nitrofurantoin (AHD) concentration in muscle, liver, honey, milk, milk powder, egg, egg powder, and feed samples.
Assay Principle
This kit uses Competitive-ELISA as the method for the quantitative detection. It can detect Nitrofurantoin (AHD) in samples, such as muscle, honey, milk, 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, AHD in the samples or standard competes with coupled antigen on the solid phase supporter for sites of anti-AHD 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 AHD. The concentration of AHD 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, 0.02 ppb, 0.06 ppb, 0.18 ppb, 0.54 ppb, 1.62 ppb) |
| 10 mL | |
| 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 |
| 20×Concentrated Wash Buffer | 40 mL |
| 2×Reconstitution Buffer | 50 mL |
| Plate Sealer | 3 |
| Sealed Bag | 1 |
| Manual | 1 |
Materials Required but not Supplied
- Equipment: Microplate reader, Printer, Homogenizer, Nitrogen evaporator, Water bath, Vortex mixer, Centrifuge, Graduated pipette, Balance (sensibility 0.01 g)).
- Micropipettes: single-channel 20-200 µL, 100-1000 µL, and multi-channel 30-300 µL.
- Reagents: Ethyl acetate, N-hexane, NaOH, Concentrated HCl, K2HPO4•3H2O, ZnSO4•7H2O, Na2Fe (CN)5 NO •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
Milk:
- Take 5 mL of milk into centrifuge tube, add 250 μL of 0.36 M Na2Fe (CN)5 NO Solution (Solution 1) and vortex for 30 s, then add 250 μL of 1.04 M ZnSO4 Solution (Solution 2) and vortex for 30 s, centrifuge at 4000 r/min for 10min at 15℃. (If a refrigerated centrifuge is not available, chill sample to approx 15℃ prior to centrifugation.)
- Take 1.1 mL of supernatant, add 4 mL of deionized water, 0.5 mL of 1 M HCl Solution (Solution 4) and 100 μL of Derivatization Reagent, vortex for 5 min.
- Incubate overnight at 37℃(about 16 hours) or incubate in water bath at 50℃ for 3 hours (the effect of stratification will be affect when more than 50℃).
- Add 5 mL of 0.1 M K2HPO4 Solution (Solution 3), 0.4 mL of 1 M NaOH Solution (Solution 5) and 5 mL of Ethyl acetate, vortex for 5 min.
- Centrifuge at 4000 r/min at room temperature for 10 min.
- Take 2.5 mL of upper liquid to another tube, dry at 50-60℃ with nitrogen evaporators or water bath. (Please do it in a ventilated environment.)
- Dissolve the residual with 1 mL N-hexane, add 1 mL of Reconstitution Buffer (Solution 6) and vortex for 30 s. Centrifuge at 4000 r/min at room temperature for 10 min.
- Discard the upper n-hexane, take 50 μL lower liquid for analysis.
- Weigh 1±0.05 g of sample into centrifuge tube, add 4 mL of deionized water, 0.5 mL of 1 M HCl Solution (Solution 4) and 100 μL of Derivatization Reagent, vortex for 5 min.
- Incubate overnight at 37℃(about 16 hours) or incubate with water bath at 50℃ for 3 hours (the effect of stratification will be affect when more than 50℃).
- Add 250 μL of 0.36 M Na2Fe (CN)5 NO Solution (Solution 1), vortex for 30 s, then add 250 μL of 1.04 M ZnSO4 Solution (Solution 2), vortex for 30 s, centrifuge at 4000 r/min at 15℃ for 10 min.
- Take all the supernatant to another tube, add 5 mL of 0.1 M K2HPO4 Solution (Solution 3), 0.4 mL of 1 M NaOH Solution (Solution 5) and 5 mL of Ethyl acetate, vortex for 5 min.
- Centrifuge at 4000 r/min at room temperature for 10 min.
- Take 2.5 mL of upper liquid to another tube, dry at 50-60℃ with nitrogen evaporators or water bath.
- Dissolve the residual with 1 mL N-hexane, add 1 mL of Reconstitution Buffer (Solution 6) and vortex for 30 s. Centrifuge at 4000 r/min at room temperature for 10 min.
- Discard the upper n-hexane, take 50 μL lower liquid for analysis.
- Honey, muscle (livestock, fish, shrimp), liver, feed, egg:
- Weigh 1±0.05 g of homogenate sample into centrifuge tube, add 4 mL of deionized water, 0.5 mL of 1 M HCl Solution (Solution 4) and 100 μL of Derivatization Reagent, vortex for 5min.
- Incubate overnight at 37℃(about 16 hours) or incubate in water bath at 50℃ for 3 hours (the effect of stratification will be affect when more than 50℃).
- Add 5 mL of 0.1 M K2HPO4 Solution (Solution 3), 0.4 mL of 1 M NaOH Solution (Solution 5) and 5 mL of Ethyl acetate, vortex for 5 min.
- Centrifuge at 4000 r/min at room temperature for 10 min.
- Take 2.5 mL of upper liquid to another tube, dry at 50-60℃ with nitrogen evaporators or water bath.
- Dissolve the residual with 1 mL N-hexane, add 1 mL of Reconstitution Buffer (Solution 6) and oscillate for 30 s. Centrifuge at 4000 r/min at room temperature for 10 min.
- Discard the upper n-hexane, take 50 μL lower liquid for analysis.
- Detection limit of fish, shrimp: 0.06 ppb
Reagent Preparation
Solution preparation:
- Prepare solutions according to the number of samples. Avoid using all components in the kit at once.
- Solution 1: 0.36 M Na2Fe (CN)5 NO •2H2O Solution (for milk and milk powder samples).
- Dissolve 10.7 g of Na2Fe (CN)5 NO •2H2O to 100 mL with deionized water.
- Solution 2: 1.04 M ZnSO4 Solution (for milk and milk powder, egg powder samples)
- Dissolve 29.8 g of ZnSO4•7H2O to 100 mL with deionized water, mix fully.
- Solution 3: 0.1 M K2HPO4 Solution
- Dissolve 11.4 g of K2HPO4•3H2O to 500 mL with deionized water, mix fully.
- Solution 4: 1 M HCl Solution
- Dilute 8.6 mL of HCl to 100 mL with deionized water, mix fully.
- Solution 5: 1 M NaOH Solution
- Dissolve 4 g of NaOH to 100 mL with deionized water.
- Solution 6: Reconstitution Buffer
- 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 7: 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 (25oC) 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 per well, then add 50 μL of HRP Conjugate to each well, then add 50 μL of Antibody Working Solution, cover the plate with plate sealer, oscillate for 5 s and mix thoroughly, 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 7) 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 (livestock), Liver, Honey, Milk ---0.04 ppb; Milk powder, Feed, Egg powder, Egg---0.04 ppb; Muscle (fish, shrimp) ---0.06 ppb
Cross-reactivity
AHD---100%; AOZ, SEM, AMOZ---< 0.1%.
Sample recovery rate
Muscle, Liver---85%±25%; Honey, Milk ---80%±20%; Milk powder, Feed, Egg powder, Egg---85%±25%.