Enrofloxacin (ENR) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDF-E032
The Enrofloxacin (ENR) ELISA Kit (RDF-E032) is an enzyme-linked immunosorbent assay for quantitative detection of ENR in Food samples with a standard range of 0.1-8.1ppb. This kit uses the Traditional ELISA format for colorimetric detection of natural ENR in samples including Muscle,Honey,Milk,Milk 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 ENR in samples. For research use only.
Product Specifications
Product Name
Enrofloxacin (ENR) ELISA Kit
Catalog Number
RDF-E032
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,Milk,Milk powder,Egg
Storage
2-8°C,Avoid freeze
Shelf Life
12 monthes
Product Details
Intended Use
For the quantitative detection of enrofloxacin concentration in muscle, honey, milk, milk powder, and egg samples.
Assay Principle
This kit uses Competitive-ELISA as the method for the quantitative detection. It can detect Enrofloxacin (ENR) in samples, such as honey, muscle, 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, ENR in the samples or standard competes with coupled antigen on the solid phase supporter for sites of anti-ENR 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 ENR. The concentration of ENR 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 |
| 5xReconstitution 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: Acetonitrile, N-hexane, HCl, Dichloromethane
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 (livestock, fish, shrimp):
- Remove fat from sample. Homogenize the representative sample with a homogenizer and mix fully.
- Weigh 2±0.05 g of muscle homogenate into a 50 mL centrifuge tube.
- Add 8 mL of Sample Extraction Solution (Solution 2) and vortex for 5 min. Centrifuge at 4000 rpm for 10 min at room temperature.
- Remove 2 mL of the clear upper organic layer solution to a clean and dry glass tube, dry at 50-60℃ with nitrogen evaporators or water bath.
- Add 1 mL of N-hexane and vortex for 2 min. Then add 1 mL of Reconstitution Buffer (Solution 3) and vortex for 30s to mix fully. Centrifuge for 5 min at 4000 rpm at room temperature.
- Remove the N-hexane upper layer, take 50 μL of the lower water layer solution for analysis.
- Honey:
- Weigh 1±0.05 g of honey into a 50 mL centrifuge tube, add 6 mL of Sample Extraction Solution (Solution 2) and vortex for 5 min to ensure thoroughly dissolved.
- Add 3 mL of Reconstitution Buffer (Solution 3) and 11 mL of Dichloromethane, vortex for 5 min. Then centrifuge at 4000 rpm for 5 min at room temperature.
- Remove the supernatant and transfer 8 mL of the under layer organic solution to a dry tube. Dry at 50-60℃ with nitrogen evaporators or water bath.
- Dissolve the dry residue with 1 mL of Reconstitution Buffer (Solution 3). Add 1 mL of N-hexane and vortex for 30s. Centrifuge for 5 min at 3000 rpm at room temperature.
- Remove the N-hexane upper layer, take 50 μL of the lower layer solution for analysis.
- Dilute the milk with Reconstitution Buffer (Solution 3) for 20 times (e.g., add 25 μL of milk into 475 μL of Reconstitution Buffer), vortex for 1 min to dissolve it fully.
- Take 50 μL for analysis.
- Milk powder:
- Weigh 0.5±0.05 g of homogenate sample into a 10 mL centrifuge tube, add 5 mL of deionized water and vortex to dissolve it fully.
- Mix 100 μL of sample solution with 400 μL of Reconstitution Buffer (Solution 3). Vortex for 1 min.
- Take 50 μL for analysis.
- Egg:
- Weigh 1±0.05 of homogenate egg into a 10 mL centrifuge tube, add 5 mL of deionized water and vortex to dissolve it fully.
- Mix 100 μL of sample solution with 400 μL of Reconstitution Buffer (Solution 3). Vortex for 1 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 1:0.15 M HCl Solution.
Dilute 5 mL of HCl to 400 mL with deionized water, mix fully.
Solution 2: Sample Extraction Solution (for livestock, fish, shrimp, honey sample)
Add 10 mL of 0.15 M HCl Solution (Solution 1) to 90mL of Acetonitrile, mix fully.
Solution 3: Reconstitution Buffer
Dilute the 5×Reconstitution Buffer with deionized water. (5×Reconstitution Buffer (V): Deionized water (V) = 1:4). The Reconstitution buffer can be store at 4℃ for a 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℃; 45 min, 15 min
Detection limit
Muscle---0.3 ppb; Honey---0.4 ppb; Milk---3 ppb; Milk powder---6 ppb; Eggs---3 ppb.
Cross-reactivity
Enrofloxacin---100%, Oxolinic acid---28%, Levofloxacin--10%, Lomefloxacin---4%, Marbofloxacin---4%, Sarafloxacin---2%
Sample recovery rate
85%±15%.