Removal of endogenous ligands from a high-affinity antiserum for radioimmunoassay.
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 50 degrees 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 centrifugation. 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.
D000327:Adsorption; D000818:Animals; D000915:Antibody Affinity; D002021:Buffers; D002498:Centrifugation; D002606:Charcoal; D003429:Cross Reactions; D000431:Ethanol; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera / Q000032:analysis; D008024:Ligands; D008722:Methods; D011863:Radioimmunoassay; D013974:Thyroxine / Q000276:immunology
D002021:Buffers; D007106:Immune Sera; D008024:Ligands; D002606:Charcoal; D000431:Ethanol; D013974:Thyroxine