HCID 25444, compound → Cholesterol

Participants

Reaction Steps

The sticks were each placed into 100 uL solution of an A-C-B reagent in wells of a 96 well microwell plate. The reagent was a conjugate of digitonin (A) linked to horseradish peroxidase (B) through a maleic anhydride-N-vinylpyrrolidone copolymer (C) and was used at a concentration of approximately 1 μg/mL. The sticks were left in the solution for 15 minutes at room temperature, after which they were removed and placed into new wells of a microwell plate containing 200 μL of wash solution. The microwell plate was agitated to effect washing and after 1 min the sticks were removed to new wells containing 200 μL of fresh wash solution and again agitated for 1 min. Washing with agitation was done a third time, after which the sticks were removed and placed in 100 uL of a substrate solution (Enhanced K-Blue reagent). The sticks were incubated with the substrate solution, in the dark, for 15 minutes at room temperature, and then removed. One hundred (100) μL of 1 N sulfuric acid were added to the wells with the substrate solution to stop further reaction and the optical density of the resulting solution was read at 450 nm on a plate reading spectrophotometer, to provide a measure of the amount of cholesterol in the skin sample.

Provenance & Evidence