22] Immunochemical quantitation of lipoprotein lipase

Abstract

Lipoprotein lipase (LPL) has an important function in the regulation of plasma lipoprotein metabolism and is also involved in adipose tissue differentiation and in the prediction of adipocyte cell size. The enzyme hydrolyzes the triacylglycerol components of very-low-density lipoproteins (VLDL) and chylomicrons to glycerol and free fatty acids, and hence it regulates the supply of the latter to various tissues. In view of the critical role that LPL plays in normal metabolism, as well as in pathophysiological states, assays have been developed that measure LPL enzyme activity and immunoreactive mass. This chapter discusses the assessment of LPL immunoreactive material by immunological methods. Various immunoassays have previously been described here. This chapter describes two immunoassay protocols designed to quantitate human LPL (hLPL), making use of a monoclonal antibody (M40) raised against bovine milk LPL that cross-reacts with hLPL and a polyclonal anti-hLPL antibody (immunoglobulin Y, IgY) prepared in chickens. The antibody capture enzyme-linked immunosorbent assay (ELISA) is designed to detect LPL in samples with low-level contamination by other proteins, such as culture medium, whereas the two-antibody sandwich enzyme-linked immunosorbent assay (ELISA) is more sensitive and is best suited for detecting LPL in more complex samples, such as cellular extracts or post-heparin plasma. It also presents a procedure for preparing LPL from post-heparin plasma and detecting the protein by immunoblot analysis. Immunoblotting provides a semiquantitative measure of LPL immunoreactive mass as well as the apparent molecular weight of the protein.