The biotin capture lipid affinity assay (BCLA): a rapid method for determining lipid binding parameters for apolipoproteins

W. Sean Davidson, Amy B. Ghering, Lauren Beish, Matthew R. Tubb, David Y. Hui, and Kevin Pearson

Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45237-0507

Corresponding Author: Sean.Davidson{at}uc.edu

The ability of plasma apolipoproteins to partition between lipid-bound and lipid-free states is an important modulator of lipoprotein metabolism. To study the relationship between lipid affinity and biological function, many laboratories have begun using mutagenesis strategies to modulate apolipoprotein lipid affinity. The availability of numerous mutants has produced a need for rapid and reliable methodologies to compare apolipoprotein lipid affinities. Although numerous methods exist, most are impractical for large sample numbers while others are not designed to derive generally applicable binding parameters. Here, we describe a novel method called the biotin capture lipid affinity (BCLA) assay that measures apolipoprotein binding to a standardized preparation of small unilamellar vesicles (SUV) containing trace amounts of biotinylated and fluorescent phospholipids. After a 30 min incubation at seven different apolipoprotein concentrations, vesicle-bound protein is rapidly separated from free protein on micro-columns of immobilized streptavidin in a 96-well microplate format. Vesicle-bound protein and lipid are eluted into a microplate and are simultaneously measured in a fluorescence microplate reader for calculation of a dissociation constant (KD) and the maximum number of potential binding sites on the SUV (n). Using human apolipoprotein (apo)A-I, apoA-IV and mutants of each, we show that the assay generates binding constants that are comparable to other methods and is reproducible across time and apolipoprotein preparations. The assay is relatively easy to perform with common instrumentation and can measure triplicate binding parameters for up to 10 separate apolipoproteins in 3.5 hours consuming only 120 g of apolipoprotein in total. The benefits and potential drawbacks of the assay are discussed.

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