Human phosphatidicacid (PA)ELISA Kit
FOR RESEARCH USE ONLY. Not for clinical diagnosis use
CATALOG #:11256
INTRODUCTION
? This kit allows for the determination of PA concentrations in Human serum
? Detection of species: Human
? Detection medium: cell culture supernates and other biological fluids
? Assay range：6.0ng/L -160 ng/L
PRINCIPLE OF TEST
The kit assay Human PA level in the sample, use Purified Human PA antibody to
coat microtiter plate wells, make solid-phase antibody, then add PA to wells, Combined
PA 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 PA in the samples is then
determined by comparing the O.D. of the samples to the standard curve.
COMPOSITION OF THE KIT
1 wash solution 20ml×1bottle 7 Stop Solution 6ml×1 bottle
2
HRP-Conjugate
reagent
6ml×1 bottle 8
Standard
（320ng/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
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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
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. Step 1: Dilute and add sample:Dilute Original density Standard as follow table:
160ng/L 5 Standard 150μl Original density Standard+150μl Standard diluent
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80ng/L 4 Standard 150μl 5 Standard+150μl Standard diluent
40ng/L 3 Standard 150μl 4 Standard+150μl Standard diluent
20ng/L 2 Standard 150μl 3 Standard +150μl Standard diluent
10ng/L 1 Standard 150μl 2 Standard +150μl Standard diluent
Step 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.
Step 3: Incubate: Cover with the adhesive strip provided, incubate for 30 min at 37℃.
Step 4: Configurate liquid: Dilute wash solution 30-fold (or 20-fold) with distilled
water.
Step 5: Washing: Uncover the adhesive strip, discard liquid, Pipette washing buffer to
every well, still for 30s then drain, repeat 5 times.
Step 6: Add enzyme: Pipette HRP-Conjugate reagent 50μl to each well, except blank
well.
Step 7: Incubate: Operation with 3.
Step 8: Washing: Operation with 5.
Step 9: Color: Pipette Chromogen Solution A 50ul and Chromogen Solution B to each
well, avoid the light preservation for 15 min at 37℃
Step 10: Stop the reaction: Pipette Stop Solution 50μl to each well, Stop the reaction
(the blue change to yellow).
Step 11: Calculate: take blank well as zero, Read absorbance at 450nm after Pipetteing
Stop Solution within 15min.
CALCULATION OF RESULT
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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].
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.