Norfloxacin (NOR) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDF-E127
The Norfloxacin (NOR) ELISA Kit (RDF-E127) is an enzyme-linked immunosorbent assay for quantitative detection of NOR in Food samples with a standard range of 0.1-8.1ppb. This kit uses the Traditional ELISA format for colorimetric detection of natural NOR in samples including Muscle,Honey,Milk,Milk powder,Egg,Serum,Urine. 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 NOR in samples. For research use only.
Product Specifications
Product Name
Norfloxacin (NOR) ELISA Kit
Catalog Number
RDF-E127
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,Serum,Urine
Storage
2-8°C,Avoid freeze
Shelf Life
12 monthes
Product Details
Intended Use
For the quantitative detection of Norfloxacin (NOR) concentration in muscle, honey, milk, milk powder, egg, serum, and urine samples.
Assay Principle
This kit uses Competitive-ELISA as the method for the quantitative detection. It can detect Norfloxacin (NOR) in samples, such as honey, muscle, milk, milk powder, egg, etc. This kit is composed of ELISA Microtiter plate, HRP conjugate, antibody working solution, standard liquid and other supplementary reagents. The microtiter plate in this kit has been pre-coated with coupled antigen. During the reaction, NOR in the samples or standard competes with coupled antigen on the solid phase supporter for sites of anti-NOR 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 NOR. The concentration of NOR 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, 0.3 ppb, 0.6 ppb, 1.2 ppb, 2.4 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 |
| 5×Reconstitution Buffer | 50 mL |
| 20×Concentrated Wash Buffer | 40 mL |
| Plate Sealer | 3 |
| Sealed Bag | 1 |
| Manual | 1 |
| HRP Conjugate | 5.5 mL |
All reagent bottle caps should be tightly closed to prevent evaporation and microbial contamination.
Materials Required but not Supplied
- Equipment: Microtiter plate reader, Printer, Homogenizer, Nitrogen Evaporators, Water bath, Vortex mixer, Oscillators, Centrifuge, Graduated pipette, Balance (sensibility 0.01 g).
- Micropipettes: single-channel 20-200 µL, 100-1000 µL, and multi-channel 30-300 µL.
- Reagents: N-hexane, Anhydrous acetonitrile, Concentrated 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 (chicken, pork, fish, shrimp):
- Homogenize the representative sample with a homogenizer and mix fully.
- Weigh 2±0.05 g of homogenized sample into 50 mL centrifuge tube, add 8 mL of Sample Extraction Solution (Solution 2), oscillate for 5 min, centrifuge at 4000 rpm for 10 min at room temperature.
- Take 2 mL of supernatant to another 10 mL centrifuge tube, dry with nitrogen evaporators/water bath at 50-60℃ (Please do it in the fume hood).
- Add 1 mL of N-hexane, oscillate for 2 min. Add 1 mL of Reconstitution Buffer (Solution 3), oscillate for 30 s. Centrifuge at 4000 rpm for 5 min at room temperature.
- Discard the upper N-hexane, take 50 μL lower liquid for analyze.
- Honey:
- Weigh 1±0.05 g of honey sample into 50 mL centrifuge tube, and add 6 mL of Sample Extraction Solution (Solution 2), oscillate for 5 min and mix fully.
- Add 3 mL of Reconstitution Buffer (Solution 3) and 11 mL of Dichloromethane, oscillate for 5 min. Centrifuge at 4000 rpm for 5 min at room temperature.
- Remove the upper liquid, take 8 mL lower organic phase into another centrifuge tube, dry with nitrogen evaporators/water bath at 50-60℃(Please do it in the fume hood).
- Dissolve the residual with 1 mL of Reconstitution Buffer (Solution 3), and add 1 mL of N-hexane, oscillate for 30 s. Centrifuge at 4000 rpm for 5 min at room temperature.
- Discard the upper liquid, take 50 μL lower liquid for analyze.
- Take 25 μL of fresh milk sample into centrifuge tube. Add 475 μL of Reconstitution Buffer (Solution 3), oscillate for 1 min and mix fully.
- Take 50 μL for detection and analysis.
- Milk powder:
- Homogenize the sample with a homogenizer and mix fully.
- Weigh 0.5±0.05 g of homogenized milk into 10 mL centrifuge tube, and add 5 mL of deionized water, oscillate for 1 min and mix fully.
- Take 100 μL of sample liquid into another centrifuge tube, and add 400 μL of Reconstitution Buffer (Solution 3), oscillate for 1 min.
- Take 50 μL for detection and analysis.
- Egg:
- Homogenize the representative sample with a homogenizer and mix fully.
- Weigh 1±0.05 g of homogenized sample into 10 mL centrifuge tube, and add 5 mL of deionized water, oscillate for 1 min and mix fully.
- Take 100 μL of sample liquid into another centrifuge tube, and add 400 μL of Reconstitution Buffer (Solution 3), oscillate for 1 min.
- Take 50 μL for detection and analysis.
- Urine (animal):
- Take 4 mL of Reconstitution Buffer (Solution 3) into centrifuge tube, and add 1 mL of centrifuged clear urine sample, oscillate for 30 s.
- Take 50 μL for detection and analysis.
Reagent Preparation
Solution preparation:
Prepare solutions according to the number of samples. Avoid using all components in the kit at once.
Solution 1: 0.15M HCl Solution
Dissolve 5 mL of Concentrated HCl to 400 mL with deionized water, mix fully.
Solution 2: Sample Extraction Solution (for muscle, honey sample)
Take 10 ml of 0.15M HCl Solution (solution 1) and 90 ml of anhydrous acetonitrile, mix fully.
Solution 3: Reconstitution Buffer
Dilute 5×Reconstitution Buffer with deionized water. (5×Reconstitution Buffer (V): Deionized water (V) = 1:4), it could be stored at 4℃ for one 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 uL HRP Conjugate, then add 50 μL Antibody Working Solution, and cover the plate with plate sealer. Oscillate gently for 5 s to mix thoroughly, 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 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, Honey---0.3 ppb; Milk---3 ppb; Milk powder---7.5 ppb; Egg---4.5 ppb; Urine ---0.75 ppb
Cross-reactivity
Norfloxacin---100%
Sample recovery rate
90%±10%