Radioimmunoassay of the phagocytosis‐stimulating peptide tuftsin in normal and splenectomized subjects

A radioimmunoassay was developed for the phagocytosis‐stimulating peptide tuftsin, H‐Thr‐Lys‐Pro‐Arg‐OH, and preliminary results of its use in evaluating tuftsin levels in human blood are presented. Synthetic tuftsin was rendered antigenic through coupling of p‐diazonium phenylacetyl‐tuftsin to bovine serum albumin (BSA). Using the BSA‐tuftsin conjugate to immunize rabbits, antisera were obtained that bound ¹²⁵I‐labeled p‐aminophenylacetyl‐tuftsin at dilutions up to 1:1500 (maximal binding 25–3 0 %). Binding of the radiolabeled peptide was inhibited by tuftsin and some of its synthetic N‐terminal analogs but was not affected by various unrelated natural and synthetic peptides. The amino acid sequence Lys‐Pro‐Arg‐OH appears to be the antigenic determinant recognized by the rabbit antibodies. Using the radioimmunoassay, quantitative determination of material immuno‐chemically related to tuftsin was performed in the sera of intact and splenectomized subjects after treatment of the sera with trypsin. Sera from normal patients (21 cases) were found to contain an average ± standard error of 278.47 ± 13.49 ng/ml, whereas those of patients who underwent traumatic (5 cases) and elective (6 cases) splenectomy contained 239.0 ± 47.68 ng/ml and 94.71 ± 22.5 ng/ml, respectively. No correlation was found between levels of IgG in serum and that of tuftsin‐like material. Analysis of purified IgG fragments, Fab from man and from rabbit and Fc from rabbit, clearly demonstrated that tuftsin is located or bound to the latter polypeptide. It was, of course, also found to be present in the intact purified IgG from humans.