人白蛋白(ALB)ELISA试剂盒详细说明书

人白蛋白(ALB)ELISA试剂盒详细说明书Human albumin（ALB）FOR RESEARCH USE ONLY
Drug Names
Generic Name：Human albumin(ALB) ELISA Kit.
Purpose
This kit allows for the determination of ALB concentrations in Human serum, blood
plasma, and other biological fluids.
Principle of the assay
The kit assay Human ALB level in the sample，use Purified Human ALB antibody to coat
microtiter plate wells, make solid-phase antibody, then add ALB to wells, Combined ALB
antibody which With HRP labeled,become 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 sulphuric acid solution and the
color change is measured spectrophotometrically at a wavelength of 450 nm. The
concentration of ALB in the samples is then determined by comparing the O.D. of the samples
to the standard curve.5
Materials provided with the kit
Materials provided with
the kit
48determinations 96 determinations Storage
User manual 1 1
Closure plate membrane 2 2
Sealed bags 1 1
Microelisa stripplate 1 1 2-8℃
Standard：90ng/ml 0.5ml×1 bottle 0.5ml×1 bottle 2-8℃
Standard diluent 1.5ml×1 bottle 1.5ml×1 bottle 2-8℃
HRP-Conjugate reagent 3ml×1 bottle 6ml×1 bottle 2-8℃
Sample diluent 3ml×1 bottle 6ml×1 bottle 2-8℃
Chromogen Solution A 3ml×1 bottle 6ml×1 bottle 2-8℃
Chromogen Solution B 3ml×1 bottle 6ml×1 bottle 2-8℃
Stop Solution 3ml×1 bottle 6ml×1 bottle 2-8℃
wash solution
（20ml×20 fold）
×1bottle
（20ml×30 fold）
×1bottle
2-8℃
人白蛋白(ALB)ELISA试剂盒详细说明书Specimen requirements
1. serum- coagulation at room temperature 10-20 mins， centrifugation 20-min at the speed of
2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.
2. plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20
mins ,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If
precipitation appeared, Centrifugal again.
3. Urine-collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m.
remove supernatant, If precipitation appeared, Centrifugal again. The Operation of
Hydrothorax and cerebrospinal fluid Reference to it.
4. cell culture supernatant-detect secretory components, collect sue a sterile container,
centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant,detect the
composition of cells, Dilut 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 r.p.m. remove
supernatant, If precipitation appeared, Centrifugal again.6
5. Tissue samples- After cutting samples, check the weight,add PBS（PH7.2-7.4）, Rapidly
frozen with liquid nitrogen, maintain samples at 2-8℃ after melting,add PBS（PH7.4）,
Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 r.p.m.
remove supernatant.
6. 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.
7. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.
Assay procedure
1.Dilute and add sample to Standard: set 10 Standard wells on the ELISA plates coated, add
Standard 100µl to the first and the second well, then add Standard dilution 50µl to the first and
the second well, mix; take out 100µl form the first and the second well then add it to the third
and the forth well separately. then add Standard dilution 50µl to the third and the forth
well ,mix ; then take out 50µl from the third and the forth well discard, add 50µl to the fifth and
the sixth well ,then add Standard dilution 50µl to the fifth and the sixth well, mix ; take out 50µl
from the fifth and the sixth well and add to the seventh and the eighth well, then add Standard
dilution 50µl to the seventh and the eighth well ,mix ; take out 50µl from the seventh and the
eighth well and add to the ninth and the tenth well, add Standard dilution 50µl to the ninth and
the tenth well, mix , take out 50µl from the ninth and the tenth well discard(add Sample 50µl to
each well after Diluting ,(density: 60ng/ml，40 ng/ml，20 ng/ml，10 ng/ml，5 ng/ml)
2.add sample：Set blank wells separately (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.
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.7
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 to each well, evade the
light preservation for 15 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.
Important notes
1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes in
the room temperature, ELISA plates coated if has not use up after opened, the plate should
be stored in Sealed bag.
2. washing buffer will Crystallization separation, it can be heated the water helps dissolve
when dilute . Washing does not affect the result.
3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the
experimental error. add sample within 5 mins, if the number of sample is much ,
recommend to use Volley .
4. 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）.
5. Closure plate membrane only limits the disposable use, to avoid cross-contamination.
6. The substrate evade the light preservation.
7. Please according to use instruction strictly, The test result determination must take the
microtiter plate reader as a standard.
8. All samples, washing buffer and each kind of reject should according to infective material
process.
9. Do not mix reagents with those from other lots.8
Calculate
人白蛋白(ALB)ELISA试剂盒详细说明书Assay range
2ng/ml -80 ng/ml
Storage and validity
1．Storage： 2-8℃.
2．validity： six months.
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.
This chartis for reference only