Florfenicol (FF) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDF-E062
The Florfenicol (FF) ELISA Kit (RDF-E062) is an enzyme-linked immunosorbent assay for quantitative detection of FF in Food samples with a standard range of 0.15-12.15ppb. This kit uses the Traditional ELISA format for colorimetric detection of natural FF in samples including Muscle,Liver,Honey,Milk,Milk powder,Feed,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 FF in samples. For research use only.
Product Specifications
Product Name
Florfenicol (FF) ELISA Kit
Catalog Number
RDF-E062
Detection Range
0.15-12.15ppb
Species Reactivity
Food
Species
Food
Assay Length
45 minutes
Experimental Method
Competitive Inhibition
Detection Method
Colorimetric
Sample Type
Muscle,Liver,Honey,Milk,Milk powder,Feed,Egg
Storage
2-8°C,Avoid freeze
Shelf Life
12 monthes
Product Details
Intended Use
For the quantitative detection of florfenicol concentration in muscle, liver, honey, milk, milk powder, feed and egg samples.
Assay Principle
This kit uses Competitive-ELISA as the method for the quantitative detection. It can detect Florfenicol (FF) in samples, such as feed, muscle, milk and honey, 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, FF in the samples or standard competes with coupled antigen on the solid phase supporter for sites of anti-FF 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 FF. The concentration of FF 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, 0.15 ppb, 0.45 ppb, 1.35 ppb, 4.05 ppb, 12.15 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: Ethyl acetate, N-hexane, Na2Fe(CN)5(NO)▪2H2O, ZnSO4▪7H2O.
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 (fish, shrimp, livestock), liver:
- Homogenize muscle, liver samples with homogenizer.
- Weigh 3±0.05 g of homogenate sample into the a 50 mL centrifuge tube, add 6 mL of Ethyl acetate. Vortex for 5 min, centrifuge at 4000 r/min for 10 min at room temperature.
- Take 4 mL of supernatant to new centrifuge tube, dry at 50-60℃ with nitrogen evaporators or water bath. (Please do it in a ventilated environment.)
- Dissolve the residual with 1 mL of N-hexane, add 1 mL of Reconstitution Buffer (Solution 3) and vortex for 1 min. Centrifuge at 4000 r/min at room temperature for 10 min.
- Discard the upper n-hexane, take 50 μL lower liquid for analysis.
- Honey:
- Weigh 2±0.05 g of honey sample into the centrifuge tube, add 4 mL of Ethyl acetate and 4 mL of deionized water. Vortex for 2 min, centrifuge at 4000 r/min for 10 min at room temperature.
- Take 2 mL of supernatant to new centrifuge tube, dry at 50-60℃ with nitrogen evaporators or water bath.
- Dissolve the residual with 0.5 mL of Reconstitution Buffer (Solution 3), mix fully.
- Take 50 μL for analysis.
- Egg:
- Homogenize egg sample with homogenizer.
- Weigh 2±0.05 g of homogenate egg into the centrifuge tube, add 8 mL of Ethyl acetate. Vortex for 2 min, centrifuge at 4000 r/min for 10 min at room temperature.
- Take 4 mL of supernatant to new centrifuge tube, dry at 50-60℃ with nitrogen evaporators or water bath.
- Dissolve the residual with 2 mL of N-hexane, add 0.5 mL of Reconstitution Buffer (Solution 3) and vortex for 4 min. Centrifuge at 4000 r/min at room temperature for 5 min.
- Discard the upper n-hexane, take 50 μL lower liquid for analysis.
- Centrifuge the milk at 4000 r/min for 10 min at 15℃, discard upper fat layer. Take 5 mL of fat free milk sample into 50 mL centrifuge tube, add 250 μL of 0.36M Na2Fe(CN)5(NO)▪2H2O Solution (Solution 1) and vortex for 30 s, then add 250 μL of 1.04M ZnSO4▪7H2O Solution (Solution 2) and vortex for 30 s, centrifuge at 4000 r/min for 10 min at 15℃.
- Take 2.2 mL of the supernatant to another centrifuge tube, add 4 mL of Ethyl acetate and vortex for 2 min, centrifuge at 4000 r/min for 10 min at room temperature.
- Take 2 mL of supernatant to another centrifuge tube, dry at 50-60℃ with nitrogen evaporators or water bath.
- Dissolved the residue with 0.5 mL of Reconstitution Buffer (Solution 3), mix fully.
- Take 50 μL for analysis.
- Milk powder:
- Weigh 2±0.05 g milk powder into 50 mL centrifuge tube, dissolved with 10 mL deionized water, add 1 mL of 0.36M Na2Fe(CN)5(NO)▪2H2O Solution (Solution 1) and 1mL of 1.04M ZnSO4▪7H2O Solution (Solution 2). Vortex for 2 min and centrifuge at 4000 r/min for 10 min at 15℃ (If a refrigerated centrifuge is not available, chill sample to approx 15℃ prior to centrifugation ).
- Take 3.6 mL of the supernatant to another centrifuge tube, add 6 mL of Ethyl acetate and vortex for 5 min, centrifuge at 4000 r/min for 10 min at room temperature.
- Take 4 mL of supernatant to another centrifuge tube, dry at 50-60℃ with nitrogen evaporators or water bath
- Dissolve the residue with 0.4 mL of Reconstitution Buffer (Solution 3), mix fully.
- Take 50 μL for analysis.
- Feed:
- Homogenize the representative sample with a homogenizer and mix fully.
- Weigh 2±0.05 g of homogenate feed into 50 mL centrifuge tube, add 8 mL of Ethyl acetate. Vortex for 5 min, centrifuge at 4000 r/min for 10 min at room temperature.
- Take 3 mL of supernatant to new centrifuge 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), mix fully. Centrifuge at 4000 r/min at room temperature for 5 min.
- Discard the upper n-hexane, take 50 μL lower liquid 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: 0.36M Na2Fe(CN)5(NO)▪2H2O Solution (for milk, milk powder sample)
Dissolve 10.7 g of Na2Fe(CN)5(NO)▪2H2O to 100 mL with deionized water.
Solution 2: 1.04M ZnSO4▪7H2O Solution (for milk, milk powder sample)
Dissolve 29.8 g of ZnSO4▪7H2O to 100 mL with deionized water.
Solution 3: 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 1 month.
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, 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 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 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, Liver, Honey, Milk, Egg ---0.075 ppb; Feed, Milk powder ---0.15 ppb.
Cross-reactivity
Florfenicol ---100%; Thiamphenicol --- < 0.1%
Sample recovery rate
Muscle, Liver ---85%±20%; Honey ---85%±25%; Feed, Milk, Egg ---75%±25%.