This Melanin ELISA kit is intended Laboratory for Research use only and is not for use in diagnostic or
therapeutic procedures.The Stop Solution changes the color from blue to yellow and the intensity of the
color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of Melanin
in the sample, this Melanin ELISA Kit includes a set of calibration standards. The calibration standards
are assayed at the same time as the samples and allow the operator to produce a standard curve of Optical
Density versus Melanin concentration. The concentration of Melanin in the samples is then determined by
comparing the O.D. of the samples to the standard curve.
10 minutes at approximately 3000×g. Remove serum and assay immediately or aliquot and store samples
at -20 ℃ or -80 ℃ .Avoid repeated freeze-thaw cycles.
at 3000×g at 2-8 ℃ within 30 minutes of collection. Store samples at -20 ℃ or -80 ℃ . Avoid repeated
freeze-thaw cycles.
assay immediately or aliquot and store samples at -20 ℃ or -80 ℃ . Avoid repeated freeze-thaw cycles.
The samples shoule be centrifugated dequately and no hemolysis or granule was
allowed.
1.
Standard microplate reader(450nm).
2.
Precision pipettes and Disposable pipette tips.
3.
37 ℃ incubator.
1.
Do not substitute reagents from one kit to another. Standard, conjugate and microplates are
matched for optimal performance. Use only the reagents supplied by manufacturer.
2.
Do not remove microplate from the storage bag until needed. Unused strips should be stored at
2-8°C in their pouch with the desiccant provided.
3.
Mix all reagents before using.
Remove all kit reagents from refrigerator and allow them to reach room temperature ( 20-25°C).
| Name | 96 determinations | 48 determinations |
| Microelisa stripplate | 8*12strips | 8*6strips |
| Standard | 0.3ml*6tubes | 0.3ml*6tubes |
| Sample Diluent | 6.0ml | 3.0ml |
| HRP-Conjugate reagent | 10.0ml | 5.0ml |
| 20X Wash solution | 25ml | 15ml |
| Chromogen Solution A | 6.0ml | 3.0ml |
| Chromogen Solution B | 6.0ml | 3.0ml |
| Stop Solution | 6.0ml | 3.0ml |
| Closure plate membrane | 2 | 2 |
| User manual | 1 | 1 |
20×wash solution:Dilute with Distilled or deionized water 1:20.
1.
Prepare all reagents before starting assay procedure. It is recommended that all Standards and
Samples be added in duplicate to the Microelisa Stripplate.
2.
Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.
3.
Add Sample: Add testing sample 10μl then add 40μl of Sample Diluent to testing sample wel.
4.
Add 100μl of HRP-conjugate reagent to each well, c over with an adhesive strip and incubate for 60
minutes at 37°C.
5.
Aspirate each well and wash, repeating the process four times for a total of five washes. Wash by
filling each well with Wash Solution (400 μl ) using a squirt bottle, manifold dispenser or autowasher.
Complete removal of liquid at each step is essential to good performance. After the last wash, remove
any remaining Wash Solution by aspirating or decanting. Invert the plate and blot it against clean paper
towels.
6.
Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix and
incubate for 15 minutes at 37°C. Protect from light.
7.
Add 50 μl Stop Solution to each well. The color in the wells should change from blue to yellow. If the
color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure
thorough mixing.
8
Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.
1. This standard curve is used to determine the amount in an unknown sample. The standard curve is
generated by plotting the average O.D. (450 nm) obtained for each of the six standard concentrations on
the vertical (Y) axis versus the corresponding concentration on the horizontal (X) axis.
2. First, calculate the mean O.D. value for each standard and sample. All O.D. values, are subtracted by
the mean value of the zero standard before result interpretation. Construct the standard curve using graph
paper or statistical software.
3. To determine the amount in each sample, first locate the O.D. value on the Y-axis and extend a
horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and
read the corresponding concentration.
4. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age
can cause variation in result. Each user should obtain their own standard curve.
5. The sensitivity by this assay is 1 mg/mL.
6. Standard curve.
Storage ：
2-8 ℃ .
Validity ： six months.
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