Melamine (MEL) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDF-E010
The Melamine (MEL) ELISA Kit (RDF-E010) is an enzyme-linked immunosorbent assay for quantitative detection of MEL in Food samples with a standard range of 2-162ppb. This kit uses the Traditional ELISA format for colorimetric detection of natural MEL in samples including Milk powder,Milk,Muscle,Liver,Feed,Egg,Serum. 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 MEL in samples. For research use only.
Product Specifications
Product Name
Melamine (MEL) ELISA Kit
Catalog Number
RDF-E010
Detection Range
2-162ppb
Species Reactivity
Food
Species
Food
Assay Length
45 minutes
Experimental Method
Competitive Inhibition
Detection Method
Colorimetric
Sample Type
Milk powder,Milk,Muscle,Liver,Feed,Egg,Serum
Storage
2-8°C,Avoid freeze
Shelf Life
12 monthes
Product Details
Intended Use
For the quantitative detection of melamine concentration in milk powder, milk, muscle, liver, feed, egg, and serum samples.
Assay Principle
This kit uses Competitive-ELISA as the method for the quantitative detection. It can detect Melamine (MEL) in samples, such as milk, feed, 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, MEL in the samples or standard competes with coupled antigen on the solid phase supporter for sites of anti-MEL 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 MEL. The concentration of MEL 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, 2 ppb, 6 ppb, 18 ppb, 54 ppb, 162 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 |
| 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 evaporators, Water bath, Oscillators, Centrifuge, Graduated pipette, Balance (sensibility 0.01 g).
- Micropipettes: single channel (20-200 μL, 100-1000 μL), Multichannel (30-300 μL).
- Reagents: N-hexane, Acetonitrile, NaOH, Concentrated HCl, Methanol.
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 600 μL of milk sample into 2 mL centrifuge tube and add 1 mL Acetonitrile, vortex until it mixed fully. Centrifuge at 4000 rpm for 5 min.
- Take 100 μL of supernatant and add 900 μL of Reconstitution Buffer (Solution 5), mix fully.
- Take 50 μL for analysis.
- Milk powder:
- Weigh 2±0.05 g of milk powder sample into 50 mL centrifuge tube, add 4 mL of Methanol, vortex until it mixed fully. Centrifuge at 4000 rpm for 10 min.
- Take 100 μL of supernatant and add 900 μL of Reconstitution Buffer (Solution 5). Mix fully.
- Take 50 μL for analysis.
- Milk, milk powder (Method 2):
- Take 2 mL of milk sample or 2 g of milk powder sample into a centrifuge tube.
- Add 8 mL of Acetonitrile-0.1 M NaOH Solution (Solution 3) and vortex fully for 2 min. Centrifuge at 4000 rpm for 10 min. Take 4 mL of the supernatant liquid and dry at 50-60℃ with nitrogen evaporators or water bath (Please do it in a ventilated environment).
- Add 1 mL of N-hexane to dissolve the remaining dry material, then add 1 mL of Reconstitution Buffer (Solution 5). Vortex for 30 s and centrifuge to remove the upper layer n-hexane phase.
- Take 50 μL of the lower layer liquid for analysis.
- Muscle (livestock, fish, shrimp), liver:
- Remove fat from sample. Homogenize the representative sample with a homogenizer and mix fully.
- Weigh 2±0.05 g of homogenate muscle sample into a 50 mL centrifuge tube.
- Add 8 mL of Acetonitrile-0.1 M NaOH Solution (Solution 3) and vortex fully for 2 min. Centrifuge at 4000 rpm for 10 min. Take 2 mL of the upper layer liquid and dry at 50-60℃ with nitrogen evaporators or water bath (Please do it in a ventilated environment).
- Add 1 mL of N-hexane to dissolve the remaining dry material, then add 1 mL of Reconstitution Buffer (Solution 5). Vortex for 30 s and centrifuge to remove the upper layer n-hexane phase.
- Take 50 μL of the lower layer liquid for analysis.
- Feed:
- Homogenize the representative sample with a homogenizer and mix fully.
- Weigh 2±0.05 g of crushed feed sample into a centrifuge tube. Add 2 mL of 1 M HCl Solution (Solution 1) and 16 mL of deionized water, then homogenate the sample. Vortex for 5 min to mix fully.
- Centrifuge at 4000 rpm for 15 min. Take 10 mL of the supernatant and adjust the pH to 6-8 with 1 M NaOH Solution (Solution 4). (The added amount of 1 M NaOH Solution is different according to the feed sample. The needed amount is generally between 0.5 mL-1 mL).
- Centrifuge at 4000 rpm for 15 min. Take the supernatant to another centrifuge tube (It is recommended to increase the centrifuge speed or filter the supernatant with filter paper if the supernatant is muddy).
- Dilute the supernatant for 10 times with the Reconstitution Buffer (Solution 5) (Take 100 μL of supernatant and add 900 μL of Reconstitution Buffer, mix fully).
- Take 50 μL for analysis.
- Eggs:
- Homogenate the egg sample with homogenizer to mix fully.
- Weigh 2±0.05 g of homogenate egg sample into centrifuge tube. Add 8 mL of Acetonitrile- 0.1 M NaOH Solution (Solution 3) and vortex fully for 2 min. (After adding the Acetonitrile-0.1M NaOH Solution to vortex, it may be jelly, which is normal).
- Centrifuge at 4000 rpm for 10 min at room temperature. Take 1 mL of the upper layer liquid and dry at 50-60℃ with nitrogen evaporators or water bath (Please do it in a ventilated environment).
- Add 1 mL of N-hexane to dissolve the remaining dry material, and then add 1 mL of Reconstitution Buffer (Solution 5). Vortex fully for 30 s and centrifuge to remove the upper layer n-hexane phase.
- Take 50 μL of the lower layer liquid to another centrifuge tube and add 150 μL of Reconstitution Buffer (Solution 5), mix fully for 30 s.
- Take 50 μL for analysis.
- Serum (swine):
- Take 0.5 mL of sample into a 50 mL centrifuge tube.
- Add 2 mL of Acetonitrile- 0.1 M NaOH Solution (Solution 3) and vortex for 2 min. Centrifuge at 4000 rpm for 10 min. Take 1 mL of the upper layer liquid to another centrifuge tube and dry at 50-60℃ with nitrogen evaporators or water bath.
- Add 1 mL of N-hexane to dissolve the remaining dry material, then add 1 mL of Reconstitution Buffer (Solution 5). Vortex for 30 s and centrifuge to remove the upper layer n-hexane phase.
- Take 50 μL of the lower layer 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: 1 M HCl Solution (for feed sample)
- Dilute 8.6 mL of Concentrated HCl to 100 mL with deionized water.
- Solution 2: 0.1 M NaOH Solution
- Dissolve 0.4 g of NaOH to 100 mL with deionized water.
- Solution 3: Acetonitrile-0.1 M NaOH Solution (for milk, milk powder, livestock, fish, shrimp, liver, eggs, serum sample)
- Mix 84 mL of Acetonitrile and 16 mL of 0.1M NaOH Solution (Solution 2) fully.
- Solution 4: 1 M NaOH Solution (for feed sample)
- Dissolve 4 g of NaOH to 100 mL with deionized water.
- Solution 5: Reconstitution Buffer
- Dilute the 2×Reconstitution Buffer with deionized water. (2×Reconstitution Buffer (V): Deionized water (V) = 1:1). The Reconstitution Buffer can be stable for 1 month at 4℃.
- Solution 6: 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 6) 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, 30 min, 15 min
Detection limit
Milk powder---40 ppb; Milk---54 ppb; Milk powder (method 2)---2 ppb; Muscle, Liver---4 ppb; Feed---200 ppb; Eggs---40 ppb; Serum---8 ppb.
Cross-reactivity
Melamine---100%; Cyanuric Acid---60%; s-Triazine---< 1%.
Sample recovery rate
Milk, Milk powder---90±20%; Muscle, Feed---85±20%; Eggs---80±20%.