This VA 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 VA in
the sample, this VA 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 VA concentration. The concentration of VA in the samples is then determined by
comparing the O.D. of the samples to the standard curve.
1.
Can’t detect the samples which contain NaN3, because NaN3 inhibits HRP activity of the horseradish
peroxidase.
2.
Extract as soon as possible after Specimen collection, Extracted according to the relevant literature.
immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw.
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 | 6.0ml | 3.0ml |
| 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 |
| Sealed bags | 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 Sample Diluent 40μl to testing sample well;
4.
Add 50μl 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 .
Calculate the mean absorbance value A450 for each set of reference standards and samples.
2.
Divide the average A450 value for each standard, control and test sample by the average A450 of
standard0 and multiply by 100 to obtain %B/B0 for each sample.
3.
Prepare a standard curve by plotting the average absorbance of each standard versus the
corresponding concentrations of the standards on linear-log graph paper or the %B/B0 value for each
standard versus the corresponding concentration of the standard on linear-log or logit-log graph paper.
logit=ln(B/ B0)/(1 － B/ B0)
4.
Any values obtained for diluted samples must be further converted by applying the appropriate
dilution factor in the calculations.
5.
The standard density is a X, the B/ B0 is a Y, sitting to mark the paper in the logit- log up draw a
standard curve. According to the B/ B0 that need to be measured the sample can from sit to mark the
density value that the paper looks up the sample up.
6.
The sensitivity by this assay is 1 μ g/L
7.
The standard curve presented here is an example of the data typically produced with this kit;
however, your results will not be identical to these.
8.
Standard curve match
Storage ：
2-8 ℃ .
Validity ： six months.
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