Limitations in the use of the enzyme-linked immunosorbent assay (ELISA) for identification and quantification of thermogenin

Abstract

The use of immunological assays, ELISA and RIA, for the identification and quantification of thermogenin (the brown adipose tissue-specific, GDP-binding, 32 kDa uncoupling protein) raises doubts regarding the exclusive occurrence of thermogenin in brown adipose tissue. Weak reactions between mitochondria from rat liver, rat skeletal and heart muscle and hamster white adipose and thermogenin antibodies have been observed (Cannon et al., 1982; Lean et al., 1983; Hansen et al., 1984). In order to study whether these reactions were due to thermogenin in tissues other than brown adipose tissue (BAT) or due to non-specific binding of thermogenin antibodies, a protein from rat liver mitochondria and a protein from tubifex mitochondria were isolated by the same procedure as thermogenin. The 2 proteins had almost the same molecular weight as thermogenin and reacted with thermogenin antibodies in ELISA and dot-blotting, but did not bind GDP and had an amino acid composition different from that of thermogenin. It is concluded that the weak reactions seen between thermogenin antibodies and mitochondria from different tissues other than BAT are due to non-specific binding, and that antibody cross-reactivity alone is unsuitable for the identification of thermogenin.