A NEW SANDWICH ENZYME-LINKED-IMMUNOSORBENT-ASSAY (ELISA) FOR TRANSFORMING GROWTH FACTOR-ALPHA (TGF-ALPHA) BASED UPON CONFORMATIONAL MODIFICATION BY ANTIBODY-BINDING

A sandwich-type enzyme-linked immunosorbent assay (ELISA) for human TGFα was established utilizing monoclonal and polyclonal anti-synthetic human TGFα antibodies with defined epitopes. A polyclonal antibody which was raised in a rabbit and affinity purified by C terminal peptide (hTGFα(34-50)) recognized both intact and denatured human TGFα. Murine monoclonal antibodies isolated bound only to the denatured form of TGFα at the second loop (hTGFα(16-33)). However, the rabbit antibody was found to induce a conformation change of intact TGFα and the resultant immunocomplex was recognized by monoclonal antibodies. By virtue of this property, the ELISA could detect both native and denatured TGFα with the same efficiency with a detection limit of 0.1 ng/ml. Human EGF did not interfere with the ELISA. Production of TGFα in several transformed human cell lines was quantitatively examined. Some cell lines were found to secrete TGFα, but the production rate was very low, except one melanoma, suggesting that TGFα may function only locally in a very limited area in vivo. © 1990.