Olaquindox Metabolites (MQCA) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDF-E008
The Olaquindox Metabolites (MQCA) ELISA Kit (RDF-E008) is an enzyme-linked immunosorbent assay for quantitative detection of MQCA in Food samples with a standard range of 0.3-24.3ppb. This kit uses the Traditional ELISA format for colorimetric detection of natural MQCA in samples including Muscle,Liver. 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 MQCA in samples. For research use only.
Product Specifications
Product Name
Olaquindox Metabolites (MQCA) ELISA Kit
Catalog Number
RDF-E008
Detection Range
0.3-24.3ppb
Species Reactivity
Food
Species
Food
Assay Length
75 minutes
Experimental Method
Competitive Inhibition
Detection Method
Colorimetric
Sample Type
Muscle,Liver
Storage
2-8°C,Avoid freeze
Shelf Life
12 monthes
Product Details
Intended Use
For the quantitative detection of 3-methyl quinoxaline-2-carboxylic acid concentration in muscle and liver samples.
Assay Principle
This kit uses Competitive-ELISA as the method for the quantitative detection. It can detect 3-methyl quinoxaline-2-carboxylic acid (MQCA) in samples, such as tissue, liver, 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, MQCA in the samples or standard competes with coupled antigen on the solid phase supporter for sites of anti-MQCA 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 MQCA. The concentration of MQCA 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, 0.3 ppb, 0.9 ppb, 2.7 ppb, 8.1 ppb, 24.3 ppb) |
| HRP Conjugate | 11 mL |
| Antibody Working Solution | 6 mL |
| Substrate Reagent A | 6 mL |
| Substrate Reagent B | 6 mL |
| Stop Solution | 6 mL |
| 20×Concentrated 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 evaporator, Water bath, Oscillators, Centrifuge, Graduated pipette, Balance (sensibility 0.01 g).
- Micropipettes: single-channel 20-200 µL, 100-1000 µL, and multi-channel 300 µL.
- Reagents: Ethyl acetate, Concentration H2SO4, N-hexane.
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
- Tissue (livestock, fish, shrimp):
- Weigh 1±0.05 g of homogenate sample without fat, add 8 mL of Ethyl acetate, add 1 mL of 2 M H2SO4 Solution (Solution 1), oscillate for 30s, Centrifuge at 4000 r/min at room temperature for 5 min.
- Take 4 mL of upper liquid (Ethyl acetate layer ) to a clean glass tube, dry at 50-60℃ nitrogen evaporators or water bath.
- Dissolve the dried material with 1 mL of n-hexane, oscillate 30s and add 1mL of Reconstitution Buffer (Solution 2), mix fully, centrifuge at 4000 r/min at room temperature for 5min.
- Remove the upper organic phase, take 50 μL of lower liquid for analysis.
- Liver:
- Weigh 1±0.05 g of homogenate sample without fat, add 8 mL of Ethyl acetate, then add 1mL of 2 M H2SO4 Solution (Solution 1), oscillate for 30s, centrifuge at 4000 r/min at room temperature for 5min.
- Take 2 mL of upper liquid (Ethyl acetate layer ) to a clean glass tube, dry at 50-60℃ nitrogen evaporators or water bath.
- Dissolve the dried material with 2 mL of N-hexane, oscillate 30s and add 1 mL of Reconstitution Buffer (Solution 2), mix fully, centrifuge at 4000 r/min at room temperature for 5 min.
- Remove the upper organic phase, 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: 2 M H2SO4 Solution
- Take 15 mL Concentrated H2SO4, slowly add to 120 mL deionized water, mix well, store at room temperature
- Solution 2: Reconstitution Buffer
- Dilute the 2×Reconstitution Buffer with deionized water. It can be stored at 4℃ for one month. (2×Reconstitution Buffer (V): Deionized water (V) = 1:1)
- Solution 3: 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 Antibody Working Solution, and cover the plate with plate sealer. Oscillate gently for 5 s to mix thoroughly, 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 3) 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).
- HRP Conjugate: add 100 μL of HRP Conjugate to each well, incubate at 25℃ for 30 min in the dark.
- Wash: repeat step 3.
- 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 for 10 s 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)
37℃; 30 min, 30 min, 15 min
Detection limit
Muscle---1.5 ppb; Feed---150 ppb
Cross-reactivity
Olaquindox---100%, Carbadox---< 0.1%
Sample recovery rate
Muscle ---80%±15%, Feed---85%±15%.