Removal of endogenous ligands from a high-affinity antiserum for radioimmunoassay

Abstract

A method for removal of endogenous ligands from high-affinity antisera (stripping) is described. A thyroxine antiserum of very high affinity was used to develop the method. The antiserum was incubated at 50d`C in a glutamate buffer at pH 4.4 together with some ethanol and methyl cellulose and a large amount of activated charcoal. After incubation for up to 2 days, the stripped antibodies were separated from the ligand adsorbed to the charcoal by centri-fugation. The estimated titre increased several fold by the stripping, although the stripping method caused a loss of about 13 % of the total number of binding sites over a period of 2 days. When stripped antiserum was used instead of unstripped antiserum in an assay system, the sensitivity was up to 3 times better, and the concentration, which could be measured with the best relative precision, was 3 times lower. The stripped antiserum showed a poorer specificity than the unstripped antiserum when a short incubation period was used. However, when a long incubation period was used, the specificity was nearly the same.

Key Words:

• antiserum
• charcoal
• endogenous ligand
• radioimmunoassay
• removal
• stripping of antibodies
• stripping of antiserum
• thyroxine