9. 在450nm波长处测定各孔的OD值。
Assay procedure
1.Dilute and add sample:Dilute Original density Standard as follow table:
20μg/L5 Standard150μl Original density Standard+150μl Standard diluent
10μg/L4 Standard150μl 5 Standard+150μl Standard diluent
5μg/L3 Standard150μl 4 Standard+150μl Standard diluent
2.5μg/L2 Standard150μl 3 Standard +150μl Standard diluent
1.25μg/L1 Standard150μl 2 Standard +150μl Standard diluent
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 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).
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