In an attempt to identify and quantitate latent and active forms of transforming growth factor β (TGFβ) without the use of cell cultures and to test for autocrine stimulation by TGFβ, rabbit antibodies were raised against native human and porcine platelet-derived TGFβ. A radio-immunoassay for TGFβ was developed using radioiodinated TGFβ, anti-TGFβ antibodies, and protein A. Inhibition in the radioimmunoassay was achieved with nanogram quantities of TGFβ, comparable to the sensitivity of radioreceptor assays. Analyses of the TGFβ levels of conditioned medium from cultured cells indicated that the latent form(s) of TGFβ is not detectable in the radioimmunoassay established using antibodies raised against native TGFβ. Immunoprecipitation analysis of radiolabeled conditioned medium revealed a specific Mr 25,000 band only after acidification. A Mr 62,000 protein was observed with and without prior acidification of the medium but could not be competed with unlabeled TGFβ in the immunoprecipitation indicating antigenic unrelatedness. The anti-TGFβ IgG inhibited the binding of [125I]TGFβ to the cell surface receptors in a radioreceptor assay. TGFβ inhibition of A549 cell growth was reversed by the antibodies, which also neutralized the growth inhibitory effects of TGFβ on AKR-2B cells in a monolayer [3H]thymidine incorporation assay as demonstrated by prevention of TGFβ inhibition of insulin and epidermal growth factor-stimulated DNA synthesis. The antibodies also effectively inhibited spontaneous soft agar growth of AKR-MCA fibroblasts, providing evidence for autocrine secretion of TGFβ as a mechanism of their anchorage-independent growth.
Supported by Grant CA 42572 awarded by the National Cancer Institute, Department of Health and Human Services, and by a contract from R & D Systems, Minneapolis, MN.