The Application of a Radioimmunoassay for Sensitive Detection of Metallothionein (Thionein) in Physiologic Fluid of Humans and Rats

Abstract

Recently a radioimmunoassay (RIA) has been developed in my laboratory for the sensitive detection of metallothionein (Mt) or its precursor, thionein (Th). These metal-binding proteins are of considerable interest to toxicologists (see this volume, Chapters 25 and 26). The assay (VanderMallie and Garvey, 1979; Chang et al., 1980a) is a competitive, double antibody (Ab) type of RIA, set up in tubes and involving three stages of reaction at 4° C. For standardization, the first stage is incubation of primary Ab (specific Ab vs Mt) with known amounts of unlabeled Mt; the second stage is a continued incubation after addition of [¹²⁵I]-Mt; and the third stage is addition of secondary Ab and continued incubation. Cen-trifugation is then used to separate the bound [¹²⁵I]-Mt from the free [¹²⁵I]-Mt and the radioactivity in the precipitate is determined by gamma counting. Along with the assay tubes with varying amounts of known unlabeled Mt, there is an assay for “maximum bound” (B₀), i.e., a tube that lacks any nonlabeled competing Mt, and also an assay for “nonspecific bound”(NSB), i.e., a tube that lacks the primary Ab. Using the precipitated counts (cpm) in the relationship (cpm - NSB)/(B₀ - NSB), a plot is prepared relating % bound (F) to log Mt protein. This customarily sigmoid curve is called a standard curve. In the case of the RIA for Mt it characteristically has an essentially linear region which can be well-represented by a linear-log regression relating Y and log Mt. This regression is then used to quantitate samples containing unknown amounts of Mt.