Generic Name：Mouse α-galactoyl,Gal ELISA Kit
This kit allows for the determination of α-galactoyl concentrations in Human serum, blood plasma, and other biological fluids.
Principle of the assay
The kit assay Mouse α-galactoyl level in the sample，use Purified Mouse α-galactoyl antibody to coat microtiter plate wells, make solid-phase antibody, then add α-galactoyl to wells, Combined α-galactoyl 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 α-galactoyl in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Materials provided with the kit
|Materials provided with the kit||48determinations||96 determinations||Storage|
|Closure plate membrane||2||2|
|Standard：540pg/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）
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: 360pg/mL，180 pg/mL ，90 pg/mL，45 pg/mL，22.5pg/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.
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.