ABSTRACT
A new, specific and sensitive radioimmunoassay, using a polyclonal antiserum raised in rabbits, is described for quantitating plasma thymulin. As little as 300 fg thymulin can be measured in one assay tube. The method has been used to measure thymulin in human blood (umbilical vessel blood, 2191 +/- 123 fg/ml; children and adults up to the age of 20 years, 1499 +/- 119 fg/ml; and adults between 21-65 years, 371 +/- 18 fg/ml). There is a highly significant decrease within these three groups (P less than 0.001 by one way analysis of variance). Also plasma thymulin levels were determined in rats (601 +/- 127 fg/ml) and in pooled plasma samples from mice (638 +/- 56 fg/ml). No thymulin was detected in plasma obtained from nude rats, nude mice and thymectomised mice. These results show that the radioimmunoassay described here is a useful quantitative tool for measuring plasma thymulin that will have applications in basic, applied and clinical research.
Subject(s)
Thymic Factor, Circulating/analysis , Thymus Hormones/analysis , Animals , Chromatography, High Pressure Liquid , Female , Humans , Male , Mice , Mice, Inbred C57BL , Rabbits , Radioimmunoassay , Rats , Rats, Inbred Strains , Thymic Factor, Circulating/immunology , Thymic Factor, Circulating/isolation & purificationABSTRACT
Thymulin, a metallononapeptide with the following aminoacid sequence: pyroGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-AsnOH is a thymic hormone involved in T cell differentiation requiring zinc to express biological activity as measured by the rosette assay. We established an enzyme immunoassay (EIA) for synthetic zinc-free thymulin with a thymulin-acetylcholinesterase conjugate as tracer and specific polyclonal rabbit antithymulin antibodies. The assay is performed as a classical competition assay in microtiter plates previously coated with mouse monoclonal IgG to rabbit IgG. A quantitative thymulin assay more sensitive than radioimmunoassays (RIAs) previously described was obtained with a sensitivity (IC50) of 32.5 +/- 5 pg/ml and a detection limit of 5 pg/ml. Analysis in the EIA of synthetic thymulin analogs showed that the minimal peptidic structure necessary for enzymatic tracer competition is the C-terminal part Lys3 to Asn9. It was also shown that the biologically active form of thymulin (zinc-bound) has the same immunoreactivity as zinc-free thymulin and that other thymic hormones, thymosin alpha 1 and thymopoietin II (or TP5) and unrelated short peptides do not cross-react with thymulin. These data demonstrate the specificity of this EIA for thymulin and show its suitability for application in biological fluids.
