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User Manual

ELK05J0194, Leucine

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B/B0)
LOGIT- LOG
standards concentration (X)
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
leucine ELISA Kit instruction
Intended use
This leucine 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
leucine in the sample, this leucine 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 leucine concentration. The concentration of leucine 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

   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
   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.

   8. 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.
   9. 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.
   10. Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.
       Calculation of results
   11. Calculate the mean absorbance value A450 for each set of reference standards and samples.
   12. 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.
   13. 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)
   14. Any values obtained for diluted samples must be further converted by applying the appropriate
       dilution factor in the calculations.
   15. 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.
   16. The sensitivity by this assay is 1ng/mL
   17. 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.
   18. Standard curve match
       LOGIT- LOG
       B/()
       standards concentration (X)
       Storage: 2-8℃. Validity:six months.
       FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!