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 TGF alpha was established utilizing monoclonal and polyclonal anti-synthetic human TGF alpha antibodies with defined epitopes. A polyclonal antibody which was raised in a rabbit and affinity purified by C terminal peptide (hTGF alpha (34-50)) recognized both intact and denatured human TGF alpha. Murine monoclonal antibodies isolated bound only to the denatured form of TGF alpha at the second loop (hTGF alpha (16-33)). However, the rabbit antibody was found to induce a conformation change of intact TGF alpha and the resultant immunocomplex was recognized by monoclonal antibodies. By virtue of this property, the ELISA could detect both native and denatured TGF alpha with the same efficiency with a detection limit of 0.1 ng/ml. Human EGF did not interfere with the ELISA. Production of TGF alpha in several transformed human cell lines was quantitatively examined. Some cell lines were found to secrete TGF alpha, but the production rate was very low, except one melanoma, suggesting that TGF alpha may function only locally in a very limited area in vivo.
Inagaki
H
H
Laboratory for Biochemistry, Hoechst Japan Limited, Saitama, Japan.
Katoh
M
M
Kurosawa-Ohsawa
K
K
Tanaka
S
S
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000032:analysis; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011487:Protein Conformation; D011817:Rabbits; D015291:Transforming Growth Factors / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology
D000911:Antibodies, Monoclonal; D015291:Transforming Growth Factors; D066246:ErbB Receptors