Subject acyl side chain modification

If the antibody is immobilised on Sepharose , the supernatant containing the free, radioactive peptide can be separated easily and assayed in a gamma counter. With a standard curve drawn for known amounts of peptide subjected to assay under exactly the same conditions, unknown amounts of peptide can be determined by interpolation on the standard curve. There are two potential problems with this type of radioimmunoassay. First, the peptide to be assayed perhaps does not contain Tyr. If it contains His, however, this may suffice since His can be iodinated, especially by an enzymic procedure described below. Alternatively, the peptide is allowed to react with the Bolton and Hunter reagent (Bolton and Hunter, 1973), prepared by iodina-tion of the ester of 3-(4 -hydroxyphenyl)propionic acid and /V-hydroxysuccinimide. Any free amino group can be acylated by this reagent. Secondly, reaction of a peptide with Nal and chloramine-T can cause oxidation of Met, Cys and even Tyr residues, which can interfere with complexation of the iodinated peptide with antibodies raised to the un-iodinated peptide. An alternative method (Holohan et al., 1973) of iodination uses lactoperoxidase in the presence of H202. As pointed out above, this procedure is applicable to the iodination of His residues. This method avoids modification of the side-chains of Met, Cys and Tyr. 

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