The detection of "purified" incubation damage by several radioimmunoassay separation methods

Damage to labeled peptides has been recognized as an almost unavoidable consequence of exposure to plasma during radioimmunoassay incubat ions, which can cause serious errors in estimating plasma hormone concentrations. Surprisingly little is known about the nature and behavior of damaged labeled peptides in various systems for separating antibody-bound from free labeled hormones. This is due in part to lack of a generally applicable definition of "incubation damage" as an altered molecule rather than a phenomenon observed in the separation system. We have proposed the definition, "nonimmunoreactive radioactive peptide(s) produced during incubation of radioactively labeled peptide antigens." With 125I-hbetaMSH used as a model labeled peptide antigen, incubation damage was produced under defined conditions and purified by gel filtration. It appeared smaller than 125I-hbetaMSH, suggesting that it resulted from enzymic proteolysis. The behavior of purified incubation damage in five representative separation systems was not consistently predictable. In the paper chromatoelectrophoresis and QUSO-plasma systems, it behaved like a large molecule, partitioning in the "bound" phase. In these two systems, incubation damage must be detected in appropriate control tubes containing the specimen without antibody. In the dextran-charcoal, polyethylene glycol, and second-antibody systems, damage behaved like a small nonimmunoreactive peptide and partitioned in the "free" phase. In these systems, since incubation damage cannot be separated from free 125I-labeled peptide, damage control tubes must have excess antibody added after incubation. The apparent lack of incubation damage in some radioimmunoassays may be due to inappropriate controls for its detection, leading to erroneous estimation of plasma hormone concentrations.