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User Manual

ELK05J0246, Sialic acid (SA)

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
SA ELISA Kit instruction
Intended use
This SA 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 SA in
the sample, this SA 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 SA concentration. The concentration of SA in the samples is then determined by
comparing the O.D. of the samples to the standard curve.
Sample collection and storages

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.
   Cell culture supernates and plant exact fluids - Remove particulates by centrifugation and assay
   immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw.
   Materials required but not supplied
3. Standard microplate reader(450nm)
4. Precision pipettes and Disposable pipette tips.
5. 37 ℃incubator
   Precautions
6. 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.
7. 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.
8. Mix all reagents before using.
   Remove all kit reagents from refrigerator and allow them to reach room temperature ( 20-25°C)
   Materials supplied

   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
   Note: Standard (S0 ￫S5) concentration was followed by:0､0.75､1.5､3､6､12 ng/mL
   Reagent preparation
   20×wash solution:Dilute with Distilled or deionized water 1:20.
   Assay procedure

   9. Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate.
   10. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.
   11. Add Sample: Add testing sample 10μl then add Sample Diluent 40μl to testing sample well;
   12. Add 50μl of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37°C.
   13. Aspirate each well and wash, repeating the process four times for a total of five washes. Wash by filling each well with Wash Solution (400μl) 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.
   14. 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.
   15. Add 50μl 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.
   16. Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.
       Calculation of results
   17. Calculate the mean absorbance value A450 for each set of reference standards and samples.
   18. 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.
   19. 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)
   20. Any values obtained for diluted samples must be further converted by applying the appropriate dilution factor in the calculations.
   21. 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.
   22. The sensitivity by this assay is 0.1ng/mL
   23. 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.
   24. Standard curve match
       Storage: 2-8℃. Validity:six months.
       FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!