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人细胞粘附分子1试剂盒说明书（英文版）

点击次数： 421发布时间：2011/3/7

  • 上 传 者： 上海劲马生物科技有限公司
  • 上传时间： 2012/11/14 10:01:07
  • 文件大小： 12KB
  • 文件类型： jpg
  • 所属分类： 应用方法
  • 下载次数： 83次
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资料简介：

 

Goat Anti-Human intercellular adhesion molecule 1
                                                                                                   
Storage: 2 - 8 °C                                       Package size: 96 determinations  
 
PRINCIPLE OF THE METHOD
The ICAM-1 kit is a solid phase phase sandwich enzyme linked immuno sorbent assay（ELISA）. Samples , including standards of known ICAM-1 concentrations ａnd unknowns are pipetted into these wells. During the first incubation, the ICAM-1 antigen ａnd a biotinylated monoclonal antibody specific for ICAM-1 are simultaneously incubated. After washing, the enzyme（streptavidin-peroxydase）is added. After incubation ａnd washing to remove all the unbound enzyme, a substrate solution which is acting on the bound enzyme is added to induce a coloured reaction product. The intensity of this coloured product is directly proportional to the concentration of ICAM-1 present in the samples.
 
REAGENTS PROVIDED AND RECONSTTTUTION

REAGENTS（Store at 2-8℃）
1×96 WELLS
0.5×96 WELLS
RECONSTTTUTION
96/48-wells microtiter plates
1
0.5
Ready-to-use
Plastiv cover
2
1
Ready-to-use
Standard: 400ng/ml
1Vials （0.6ml）
0.5Vials （0.3ml）
See reagents preparation on page 3
Blank control
1Vials  （1.0ml）
1Vials  （0.5ml）
Ready-to-use
Standard Diluent
1Vials （4.0ml）
1Vials   （2.0ml）
Ready-to-use
Biotinylated anti-ICAM-1
1Vials （6.0ml）
1Vials   （3.0ml）
Ready-to-use
Streptavidin-HRP
1Vials （8.0ml）
1Vials   （4.0ml）
Ready-to-use
Washing Buffer
1Vials （20ml）
1Vials   （10ml）
50× concentrate
Substrate  A
1Vials （6.0ml）
1Vials   （3.0ml）
Ready-to-use
Substrate  B
1Vials （6.0ml）
1Vials   （3.0ml）
Ready-to-use
Stopping Solution
1Vials （6.0ml）
1Vials   （3.0ml）
Ready-to-use
Sample Diluent
1Vials （12ml）
1Vials   （6.0ml）
Ready-to-use

 
 
MATERIAL REQUIRED BUT NOT PROVIDED
l       Distilled water
l       Pipettes:10ul、50ul、100ul、200ul、1000ul。
l       Vortex mixer ａnd magnetic stirrer.
 
SAFETY
l       For research use only
l       Avoid any skin contact with H2SO4 ａnd TMB. In case of contact, wash thoroughly water.
l       Do not eat, drink, smoke or apply cosmetics where kit reagents are used.
l       Do not pipette by mouth.
 
PROCEDURAL NOTES/LAB.QUALITY CONTROL
l       When not in use, kit components should be stored refrigerated or frozen as indicated on vials or bottles. All reagents should be warmed to room temperature before use. Lyophilized standards should be discarded after use.
l       Once the desired number of strips has been removed, immediately reseal the bag to protect the remaining strips from edterioration.
l       Cover or cap all reagents when not in use.
l       Do not mis or interchange reagents between different lots.
l       Do not use reagents beyond the expiration date of the kit .
l       Use a clean disposable plastic pipette tip for each reagent, standard, or specimen addition in order to avoid cross-contamination, for the dispensing of H2SO4 ａnd substrate solution, avoid pipettes with metal parts.
l       Use a clean plastic container to prepare the washing solution.
l       Thoroughly mix the reagents ａnd samples before use by agitation or swirling.
l       All residual washing liquid must be drained from the wells by efficient aspiration or by decantation followed by tapping the plate forcefully on absorbent paper. Never ｉnsert absorbent paper directly into the wells.
l       The TMB solution is light sensitive. Avoid prolonged exposure to light, also, avoid contact of the TMB solution with metal to prevent colour development. Warning TMB is toxic avoid direct contact with hands. Dispose off properly. If a dark blue colour develops within a few minutes after preparation, this indicates that the TMB solution has been contaminated ａnd must be discarede. Read absorbances within 1 hour after completion of the assay.
l       When pipetting reagents, maintain a consistent order of addition from well-to-well. This will ensure equal incubation times for all wells.
l       Respect incubation times described in the assay procedure.
 
SPECIMEN COLLECTION\\ PROCESSING AND STORAGE
l       Serum－－-Avoid any inintentional stimulation of the cells by the procedure. Use pyrogen\\endotoxin free collecting tubes. Serum should be removed rapidly ａnd carefully from the red cells after clothing. For that, after clothing, centrifuge at approximately 1000×g for 10 min ａnd remove serum.
l       Plasma－－-EDTA\\ citrate ａnd heparin plasma can be assayed. Spin samples at 1000×g for 30 min remove particulates. Harvest plasma.
l       Cell culture supernatants－－-Remove particulates ａnd aggregates by spinning at approximately 1000×g for 10 min.
l       Storage－－-If not analyzed shortly after collection, samples should be aliquoted（250-500ul） to avoid freeze-thaw cycles ａnd stored frozen at -70℃. Avoid multiple freeze-thaw cycles of frozen specimens. When possible, avoid use of badly hemolyzed or lipemic sera. If large amounts of particles are present, this should be removed prior to assay by centrifugation or filtration.
l       Recommendation－－-Do not thaw by heating at 37℃ or 56℃. Thaw at room temperature ａnd make sure that sample is completely thawed ａnd homogenous before assaying.
 
PREPARATION OF REAGENTS
l       Standards: Standard have to be reconstituled with the volume of standard buffer diluent indicated on the vial. This reconstitution produces a stock solution of 400ng/ml ICAM-1. Allow standard to stand for 5
l       minutes with gentle swirling prior to making dilutions. Serial dilutions of standard must be made before each assys ａnd cannot be stored.
 

400 ng/ml
（6 Standard）
Original density 50ul。
200 ng/ml
（5 Standard）
100ul 6 Standard +100ul diludent
100 ng/ml
（4 Standard）
100ul 5 Standard +100ul diludent
50 ng/ml
（3 Standard）
100ul 4 Standard +100ul diludent
25 ng/ml
（2 Standard）
100ul 3 Standard +100ul diludent
12.5 ng/ml
（1 Standard）
100ul 2 Standard +100ul diludent
0 ng/ml
Blank Control
50ul。

 
l       Washing buffer 50×concentrate: Dilute 50 times in distilled water.
 
ASSAY METHOD
l       Before use, mix all reagents thoroughly without making foam.
l       Determine the number of microwell strips required to test the desired number of samples,plus appropriate number of wells needed for running blanks standards. Each sample, standard ａnd blank should be assayed in duplicate. Remove sufficient microwell strips from the pouch.
l       Add 50ul of standard diluent to standard wells B1,B2, C1,C2, D1,D2, E1, E2, F1,F2. Reconstitute standard vial with the appropriate volume as described in the chapter reagents preparation. Preparation. Pipet 100ul of standard into wells A1 ａnd A2 （see plate scheme below）. Transfer 50ul from A1 ａnd A2 to B1 ａnd B2 wells. Mix the contents by repeated aspirations ａnd ejections. Take care not to scratch the inner surface of microwells. Repat this procedure from the wells B1,B2 to wells C1,C2 ａnd from wells C1,C2 to D1,D2 ａnd so on creating two parallel rows of ICAM-1 standard dilutions ranging，Add 50ul of standard diluent to the bland wells.
l       Dilute samples 1:1 distribing 50ul of sample into 50ul of dilluent ,Add 50ul of diluted sample to wells..
l       Add 50ul of diluted biotinylated anti-ICAM-1 to all wells.
l       Cover with a plate vover ａnd incubate for 1 hour at 37℃.
l       Remove the cover ａnd wash the plate as follows: ⑴　aspirate the liquid from each well, ⑵　dispensse 0.3ml of washing solution into each well. ⑶　Aspirate again the contet of each well after 0.5 minute. ⑷　Repeat steps ⑵ ａnd ⑶ three times.
l       Distribute 60ul of streptavidin-HRP solution to all wells, including blank wells.
l       Cover ａnd incubate 30 min at 37℃.
l       Remove the cover ａnd empty wells, Wash microwell strips according to step, Proceed immediately to the next step.
l       Add 50ul Substrate A ａnd Substrate B to each well。Incubate for 10 min at 37℃。
l       The enzyme-substrate reaction is stopped by quickly pipetting 50ul of H2SO4. stop reagent into each well, including the blank wells, to completely ａnd uniformly inactivate the enzyme. Results must be red immediately after the addition of H2SO4.
l       Read absorbance of each well on a spectrophotometer using 450nm as the primary wavelength ａnd optionally 620nm （610nm to 650nm is acceptable ） as the reference wavelength.
 
 
SUGGESTED PLATE SCHEME

 
Standard
concentrations（ng/ml）
 
A
400
400
sample
sample
sample
sample
sample
sample
sample
sample
sample
sample
B
200
200
sample
sample
sample
sample
sample
sample
sample
sample
sample
sample
C
100
100
sample
sample
sample
sample
sample
sample
sample
sample
sample
sample
D
50
50
sample
sample
sample
sample
sample
sample
sample
sample
sample
sample
E
25
25
sample
sample
sample
sample
sample
sample
sample
sample
sample
sample
F
12.5
12.5
sample
sample
sample
sample
sample
sample
sample
sample
sample
sample
G
0
0
sample
sample
sample
sample
sample
sample
sample
sample
sample
sample
H
sample
sample
sample
sample
sample
sample
sample
sample
sample
sample
sample
sample

 
LIMITATIONS OF THE PROCEDURE
Do not extrapolate the standard curve beyond the max  standard curve point. The dose-response is non-linear in this region ａnd good accruacy is difficult to obtain.
 
CALCULATION OF RESULTS
The minimum detectable concentration in this assay is estimated to be 1.0ng/ml
 

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