Identification of immunoreactive forms of thymosin alpha 1 in serum and supernatants by combining HPLC and RIA.

Thymosin alpha 1 (T alpha 1) is a biologically active peptide, originally isolated from the thymus and currently undergoing clinical trials as an immunomodulator in cancer patients, in individuals with chronic active hepatitis, and as an immunoenhancer of vaccines in immunocompromised individuals. Absorption of rabbit antibody to thymosin alpha 1 with a synthetic C-14 fragment of T alpha 1 results in an antiserum with increased affinity for the amino terminal region of T alpha 1 and the precursor protein prothymosin alpha (ProT alpha). Using HPLC methodologies, the predominant form of immunoreactivity in serum and thymus was T alpha 1 not the precursor. Using this assay we detected a decline in mouse serum T alpha 1 following irradiation but not thymectomy, an observation consistent with the existence of an important radiation sensitive lymphoid source of serum T alpha 1. The secretion of authentic T alpha 1 but not the precursor into culture medium by thymic epithelial cells as well as in mitogen-stimulated peripheral blood lymphocytes was also demonstrated by HPLC/RIA. HPLC analysis by molecular weight sizing columns demonstrated that unlike thymic epithelial cells or peripheral blood lymphocytes, the immunoreactive T alpha 1 (IRT alpha 1) form in the supernatants from tumor cells such as MCF-7 breast carcinoma was of a lower molecular weight than authentic T alpha 1. These studies suggest that the authentic form of T alpha 1 is the major immunoreactive form in normal serum and that it is secreted by the medullary thymic epithelial cells as well as by peripheral blood lymphocytes. An additional immunoreactive form, secreted by tumor cells has also been identified and is the subject of future studies.