A THYMOSIN-BETA-4 ELISA USING AN ANTIBODY AGAINST THE N-TERMINAL FRAGMENT THYMOSIN-BETA-4 [1-14]

A thymosin beta(4) ELISA was developed in which thymosin beta(4), absorbed on microwells, competed with thymosin beta(4) in solution for the binding sites of an anti-thymosin beta(4) antibody. The antibody molecules finally immobilized on the microwells were detected using a goat anti-rabbit immunoglobulin/horseradish peroxidase conjugate in combination with the substrate 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt, and measuring the relevant optical density values. Anti-thymosin beta(4) antibodies were raised in rabbits against intact thymosin beta(4) as well as against selected fragments of the peptide, i.e., the N terminal fragments thymosin beta(4)[1-14] and thymosin beta(4)[1-11]. The antibody against thymosin beta(4)[1-14] was used in the thymosin beta(4) ELISA, because it showed minimal cross-reactivity (0.1%) with the highly homologous peptide thymosin beta(9) as well as exhibiting the highest titre. The ELISA procedure developed, apart from showing a minimal cross-reaction with thymosin beta(9), was fast, easy to perform and exhibited good assay characteristics.