A Strategy for the Identification of Differentially Expressed Proteins Secreted by Fibroblasts

Abstract

Monoclonal antibodies, which recognise markers of the modified tumour extracellular matrix, may be valuable tools for the diagnosis and therapy of cancer. The pattern of expression of some good-quality tumour-associated antigens is known to be regulated by intracellular pH. In this article, a strategy is described for the isolation of human antibodies specific for proteins which are differentially secreted by fibroblasts at different pH values. We panned a large synthetic antibody phage library against biotinylated proteins secreted by normal human dermal fibroblasts, cultured at pH 7.5, in the presence or in the absence of a molar excess of unbiotinylated proteins, secreted by fibroblasts cultured at pH 6.7. A panel of monoclonal antibodies was isolated, whose reactivity was tested by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry.