A rapid, simple high capacity cholesterol crystal growth assay.

Abstract

Cholesterol crystal “nucleation time”, more recently referred to as cholesterol crystal observation time, is defined as the first appearance of cholesterol crystals from isotropic crystal-free biles on light microscopy. This test is used to assess the potency of nucleating agents. Crystal appearance has conventionally been determined by polarizing light microscopy and crystal growth by counting the number of crystals. In this study we adapted a spectrophotometric method to a microtiter plate reader to generate cholesterol crystal growth curves. Model biles were prepared with a cholesterol saturation of 1.2 to 1.3 and total lipid concentration of 10.7 g/dl (taurocholate, 125 mM; cholesterol, 16.8-18.4 mM; phospholipid, 43 mM). Pronucleating IgM samples were used to establish and validate the assay. Cholesterol crystal growth curves were generated by reading absorption at 630 nm daily on a Dynatech microplate reader. Results were correlated to cholesterol crystal counts as determined by polarizing light microscopy. Standard curves generated from absorbencies of known masses of cholesterol crystals were used to quantify the mass of cholesterol crystals formed over the observation period. The assay was applied to known pronucleating biliary immunoglobulins. Results obtained were similar to our previous report that biliary IgM is more potent than biliary IgG. We conclude that using microplates and a microtiter plate reader provides a rapid high capacity method for detecting cholesterol crystal growth to assess potential nucleating agents in nucleation assays.