JOURNAL OF ;ER# f MilCAL ELSEVIER Journal of Immunological Methods 186 (1995) 267-274 Optimisation of CCL64-based bioassay for TGF-P Laure Garrigue-Antar, Isabelle Barbieux, Blandine Lieubeau, Olivier Boisteau, Marc Grigoire * zyxwvutsrqponmlkjihgfedcbaZYXWV INSERM U 419, Insfitrct de Biologic, 9 Quai Moncousu, 44035 Nantes Ceder, France Received 19 January 1995; revised 30 March 1995; accepted 2 June 1995 Abstract Transforming growth factor /3 (TGF-P) IS released by a variety of cells and known to be involved in many different processes including the immune response, wound healing and carcinogenesis. As most experimental investigations have been based on quantitative analysis of TGF-/3 production using a bioassay. it scemcd important to test the validity and limitations of this method. This paper analyses several parameters that may impair TGF$ quantification by bioassay. Recommendations are made concerning the influence of technical parameters and the prcsencc of other cytokines (EGF and bFGF) commonly released by cultured cells to which the MvlLu mink lung epithelial ccl1 line (CCL641 is sensitive Keywords: Transforming growth factor /?; Bioassay zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 1. introduction TGF-P, is a multifunctional cytokine secreted in vivo in an inactive (latent) form which includes a glycosylated precursor of 75 kDa (Purchio et al., 1988). The precursor is linked to a large glycopro- tein, the latent TGF-P-binding protein (Miyazono Abbreviations: bFGF, basic fibroblast growth factor; ICso, demi-inhibitory concentration; ELISA, enzyme-linked im- munosorbcnt assay; EGF, epidermal growth factor; EDTA, ethylenediaminetetraacetic acid: NRK, normal rat kidney; PDGF, platelet-derived growth factor; SELJSA, sandwich en- zyme-linked immunosorbent assay; TGF-P, transforming growth factor /3; TNF-, tumor necrosis factor cu; TNF-P, tumor necrosis factor fi. ’ Corresponding author. Tel.: (33140-08-41-50; Fax: (33)40- OS-‘U-82. et al., 1988; Wakefield et al., 1988; Kanzaki et al., 19901, which is functionally undefined (Flaumenhaft et al., 1993). In vitro, most of the cells secreting TGF-P in variable amounts have receptors for this factor. The different approaches developed to quantify soluble TGF-/3 released by cells include biologi- cal methods (Meager, 19911, radioreceptor assays (Danielpour et al., 1989) and immunological methods such as ELISA (Dash et al., 1989) or SELISA (Danielpour, 1993). Since each of these methods have advantages and drawbacks, the ex- perimental protocol must be carefully controlled. Consequently, we have analysed the existing CCL64-based bioassay in order to optimise each parameters. Previous papers have used different experimental methods to quantify TGF-P pro- duction (Meager, 1991; Kelley et al., 1992; Ran- llO22- 1759/95/$09.50 10 1995 Elsevier Science 13.V. All rights reserved S.S/~I 11022.1759~05~001s1-4