Determination of Cu-Containing Metallothionein: Comparison of Ag Saturation Assay, Thiomolybdate Assay, and Enzyme-Linked Immunosorbent Assay

Abstract Three methods for the quantification of Cu-containing metallothionein (MT) (Ag saturation assay, thiomolybdate assay, and enzyme-linked immunosorbent assay (ELISA)) were compared for their ability to recover in vitro prepared standard Cu-MT both in absence and presence of rat liver cytosol. Uniform molar calibration of the assays was achieved using the nitrogen content of the standard Cu-MT measured by the Kjeldahl procedure. With all three methods Cu-MT reliably could be quantified. The Ag saturation assay and the thiomolybdate assay, dependent on the amount of Cu-MT and the presence of hepatic cytosol, showed a tendency to over- or underestimate the theoretical expectation. The ELISA generally performed best and moreover was three orders of magnitude more sensitive than the other two assays. With all three methods corresponding MT levels were found in the Cu-rich liver of a Long-Evans Cinnamon rat.