Human NP-S ELISA Kit
FOR RESEARCH USE ONLY. Not for clinical diagnosis use
CATALOG #:13698
INTRODUCTION
? This kit allows for the determination of NPS concentrations in Human serum
? Detection of species: Human
? Detection medium: serum, cell culture supernates.
PRINCIPLE OF TEST
The kit assay Human NP-S level in the sample, use Purified Human NP-S antibody
to coat microtiter plate wells, make solid-phase antibody, then add NP-S to wells,
Combined NP-S antibody which With HRP labeled, become antibody - antigen -
enzyme-antibody complex, after washing Compley, Add TMB substrate solution,TMB
substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the
addition of a sulphuric acid solution and the color change is measured
spectrophotometrically at a wavelength of 450 nm. The concentration of Human NP-S
in the samples is then determined by comparing the O.D. of the samples to the
standard curve.
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COMPOSITION OF THE KIT
1 wash solution 20ml×1bottle 7 Stop Solution 6ml×1 bottle
2
HRP-Conjugate
reagent
6ml×1 bottle 8
Standard
（160ng/L）
0.5ml×1 bottle
3 Microelisa stripplate 12well×8strips 9 Standard diluent 1.5ml×1bottle
4 Sample diluent 6ml×1 bottle 10 Instruction 1
5 Chromogen Solution A 6ml×1 bottle 11
Closure plate
membrane
2
6 Chromogen Solution B 6ml×1 bottle 12 Sealed bags 1
STORAGE CONDITIONS
? The unopened kit shall be stored at [2-8 ℃] .
? For opened kit can be stored at [2-8 ℃] for up to 1 month. If not be used recently,
the standard should be kept in -20 ℃.
WASHING METHOD
? Manually washing method: shake away the remained liquid in the enzyme
plates; place some bibulous papers on the test-bed, and flap the plates on the
upside down strongly. Inject at least 0.35ml after-dilution washing solution into the
well, and marinate 1~2 minutes. Repeat this process according to your
requirements.
? Automatic washing method: if there is automatic washing machine, it should
only be used in the test when you are quite familiar with its function and
performance.
SAMPLE PREPARATION
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1. extract as soon as possible after Specimen collection,and according to the relevant
literature, and should be experiment as soon as possible after the extraction. If it
can’t, specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw
cycles.
2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.
ASSAY PROCEDURE
1. Dilute and add sample:Dilute Original density Standard as follow table:
80ng/L 5 Standard 150μl Original density Standard+150μl Standard diluent
40 ng/L 4 Standard 150μl 5 Standard+150μl Standard diluent
20 ng/L 3 Standard 150μl 4 Standard+150μl Standard diluent
10 ng/L 2 Standard 150μl 3 Standard +150μl Standard diluent
5.0 ng/L 1 Standard 150μl 2 Standard +150μl Standard diluent
2. Add sample: Set blank wells separay (blank comparison wells don’t add sample
and HRP-Conjugate reagent, other each step operation is same). testing sample well.
add Sample dilution 40μl to testing sample well, then add testing sample 10μl (sample
final dilution is 5-fold), add sample to wells , don’t touch the well wall as far as
possible, and Gently mix.
3. Incubate: After closing plate with Closure plate membrane , incubate for 30 min at
37℃.
4. Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with
distilled water and reserve.
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5. Washing: Uncover Closure plate membrane, discard Liquid, dry by swing, add
washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.
6. Add enzyme: Add HRP-Conjugate reagent 50μl to each well, except blank well.
7. Incubate: Operation with 3.
8. Washing: Operation with 5.
9. Color: Add Chromogen Solution A 50ul and Chromogen Solution B 50ul to each well,
evade the light preservation for 10 min at 37℃
10. Stop the reaction: Add Stop Solution50μl to each well, Stop the reaction(the blue
color change to yellow color).
11. Assay: take blank well as zero , Read absorbance at 450nm after Adding Stop
Solution and within 15min.
CALCULATION OF RESULT
Take the standard density as the horizontal, the OD value for the vertical ,draw
the standard curve on graph paper, Find out the corresponding density according to the
sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate
the straight line regression equation of the standard curve with the standard density
and the OD value ,with the sample OD value in the equation, calculate the sample
density, multiplied by the dilution factor, the result is the sample actual density.
EXPIRATION
Six months [see label on the outer box for the specific date].
eBioscience, Inc.
Headquarters 10255 Science Center Drive San Diego, CA 92121 USA
Customer Service:888.999.1371 858.642.2058 info@eBioscience.com
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ATTENTION
? The kit takes out from the refrigeration should be balanced 15-30 minutes in the
room temperature, if the coated ELISA plates have not been used up after opening,
the plate should be stored in sealed bag.
? washing buffer will Crystallization separation, it can be heated the water helps dissolve
when dilute . Washing does not affect the result.
? Pipette sample with pipettors each step, and proofread its accuracy frequently to
avoid the experimental error. Pipette sample within 5 min, if the number of sample
is big, recommend using multichannel pipettor.
? if the testing material content is excessively higher (The sample OD is bigger than
the first standard well ),please dilute Sample (n-fold), Please diluente and
multiplied by the dilution factor.（×n×5）
? Adhesive Strip only limits the disposable use to avoid cross-contamination.
? The substrate should evade the light to be preserved.
? Please refer to the user instruction strictly, the test result determination must take
the microtiter plate reader as a standard.
? The preparation of samples and all the reagents should refer to infective material
process.
? Do not mix reagents with those from other lots