SinoGeneClon Biotech Co.

,Ltd
INTRODUCTION

Human Adenosine A2a Receptor(ADORA2A)ELISA Kit

FOR RESEARCH USE ONLY. Not for clinical diagnosis use

Catalog No : SG-15714
For the quantitative determination of Human ADORA2A concentrations.
Reactivity: Human
Method Type: Sandwich ELISA Detection
Quantity: 96 tests
Sample type： serum, plasma, Urine,tissue homogenates, cell culture supernates
Detection range : 0.375ng/ml-10ng/ml
Sensitivity: 0.1ng/ml

Components:
Assay plate (12 × 8 coated Microwells) 1
Standard: 13.5ng/ml 1×0.5ml
Standard Diluent 1×1.5ml
HRP-Conjugate Reagent 1×6ml
Sample Diluent 1×6ml
Chromogen Solution A 1×6ml
Chromogen Solution B 1×6ml
Stop Solution 1×6ml
Wash Solution 1×20ml×30 fold
User manual 1
Adhesive Strip 2
Product Principle:
The kit is for the quantitative level of Human ADORA2A in the sample, adopt purified ADORA2A antibody to coat microtiter
plate, make solid-phase antibody, then add ADORA2A to wells, Combine ADORA2A antibody with labeled HRP to form
antibody-antigen -enzyme-antibody complex, after washing completely, add TMB substrate solution, TMB substrate becomes blue
color at HRP enzyme-catalyzed, reaction is terminated by the addition of a stop solution and the color change is measured at a
wavelength of 450 nm. The concentration of ADORA2A in the samples is then determined by comparing the O.D. of the samples to
the standard curve.

Specimen requirements:
1.Serum-coagulation at room temperature for 10-20 min，centrifuge at the speed of 2000-3000 rpm for 20-min. Remove
supernatant, if precipitation appeared, Centrifuge again. Assay immediately or aliquot and store samples at -20°C or -80°C. Avoid
repeated freeze-thaw cycles.
2.Plasma-use suited EDTA or citrate plasma as an anticoagulant, centrifuge at the speed of 2000-3000 rpm for 20-min. Remove
supernatant, if precipitation appeared, centrifuge again. Assay immediately or aliquot and store samples at -20°C or -80°C. Avoid
repeated freeze-thaw cycles. Centrifuge the sample again after thawing before the assay .
3.Urine-collect sue a sterile container, centrifuge at the speed of 2000-3000 rpm for 20-min. Remove supernatant, if precipitation
appeared, Centrifuge again. The Operation of Hydrothorax and cerebrospinal fluid reference to it. Assay immediately or aliquot
and store samples at -20°C or -80°C.
4.Cell culture supernatant-detect secretory components, Remove particulates by centrifugation for 20-min at the speed of 2000-
3000 rpm. Remove supernatant detect the composition of cells, dilute cell suspension with PBS (PH7.2-7.4), Cell concentration
reached 1 million / ml, repeated freeze-thaw cycles, damage cells and release of intracellular components, centrifugation 20-min at
the speed of 2000-3000 rpm. remove supernatant, If precipitation appeared, Centrifugal again. Assay immediately or aliquot and
store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.

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5.Tissue samples- After cutting samples, check the weight, Pipette PBS(PH7.2-7.4), Rapidly frozen with liquid nitrogen, maintain
samples at 2-8℃ after melting, Pipette PBS(PH7.4), homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-
3000 rpm. Remove supernatant. Assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw
cycles. Centrifuge the sample again after thawing before the assay.
Note:
1. Extract as soon as possible after Samples collection, and should be tested as soon as possible after the extraction. If not, samples
must be stored at -20°C (≤ 1 month) or -80°C (≤ 2 months) to avoid loss of bioactivity and contamination.
2. Can' t detect the sample which contain NaN3, because NaN3 inhibits HRP active.

Reagent preparation
1：Wash Buffer (1x) - If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals
have completely dissolved. Dilute 20 ml of Wash Buffer Concentrate (30 x) into deionized or distilled water to prepare 600 ml of
Wash Buffer (1 x).
2：Standard
Dilute the standard：Pipette 50μl standard diluent in each tube. Pipette 100μl standard (13.5ng/ml) in the fifth tube. And take out
100μl from the fifth five tube into the fourth. Pipette 50μl from the fourth tube to the third tube and produce dilution series as
below. The undiluted Standard serves as the high standard (13.5ng/ml ). Sample Diluent serves as the zero standard（blank well）
(0ng/ml).

Tube 6 5 4 3 2 1 0
ng/ml 13.5 9 6 3 1.5 0.75 0

Assay procedure:
Step 1: Prepare all reagents, working standards, Blank and samples as directed in the previous sections.
Step 2: Refer to the Assay Layout Sheet to determine the number of wells to be used and put any remaining wells and the
kkkkkdesiccant back into the pouch and seal the ziploc, store unused wells at 4°C
Step 3：Pipette standard 50μl to testing standard well，Pipette Sample diluent 40μl to testing sample well, then add kk kk
k______testing sample 10μl (sample final dilution is 5-fold), Pipette sample to wells, don’t touch the well wall as far as
kkkkkkpossible, and mix gently.
Step 4: Incubate: Cover with the adhesive strip provided, incubate for 30 min at 37℃.
Step 5: Configurate liquid: Dilute wash solution 30-fold with distilled water.
Step 6: Washing: Uncover the adhesive strip, discard liquid, pipette washing buffer to every well, still for 30s then drain,
kkkkkrepeat 5 times.
Step 7: Add enzyme: Pipette HRP-Conjugate reagent 50μl to each well, except blank well.
Step 8: Incubate: Operation with 4
Step 9: Washing: Operation with 6.
Step10: Color: Pipette Chromogen Solution A 50ul and Chromogen Solution B 50ul to each well, avoid the light
kkkkkpreservation for 15 min at 37℃.
Step 11: Stop the reaction: Pipette Stop Solution 50μl to each well, stop the reaction (the blue change to yellow).
Step 12: Calculate: take blank well as zero. Read absorbance at 450nm after pipette Stop Solution within 15min.

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Calculation of result:
Take the standard concentration as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find
out the corresponding concentration 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 concentration and the OD value, with the
sample OD value in the equation, calculate the sample concentration, multiplied by the dilution factor, the result is the sample
actual concentration.
Graphical Representation as following:

Expiration:
Twelve months [see label on the outer box for the specific date]
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 ℃

Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Human ADORA2A were tested 20
times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Human ADORA2A were tested on 3
different plates, 8 replicates in each plate.
CV(%) = SD/mean X 100
Intra-Assay: CV<8%
Inter-Assay: CV<10%

Attention:
1: The kit takes out from the refrigeration should be balanced 15-30 minutes in the room temperature, if the coated ELISA
kkplates have not been used up after opening, the plate should be stored in sealed bag.
2: Washing buffer will Crystallization separation, it can be heated in water to dissolve.
3: Pipette sample with pipettors each step, and proofread its accuracy frequently to avoid the experimental error. Pipette kksample
within 5 min, if the number of sample is big, recommend using multichannel pipettor.
4: If the testing material concentration is excessively high (The sample OD is higher than the first standard well)，please dilute the
sample (n-fold).
5: Adhesive Strip only limits the disposable use to avoid cross-contamination.
6: The substrate should evade the light to be preserved.
7: Please refer to the user instruction strictly, the test result determination must take the microtiter plate reader as a standard.
8: The preparation of samples and all the reagents should refer to infective material process.
9: Do not mix reagents with those from other lots.

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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 .

If You Have Problems

Technical Service Contact information
Email: tech@sinogeneclon.com
Web: www.sinogeneclon.com
In order to obtain higher efficiency service, please ready to supply the lot number of the kit to us (found on the
outside of the box).

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