Effects of polystyrene surface chemistry on the biological activity of solid phase fibronectin and vitronectin, analysed with monoclonal antibodies.
The conformation and biological activities of fibronectin (FN) and vitronectin (VN) coated on different plastic surfaces were investigated using cell adhesion and a panel of domain-specific monoclonal antibodies (mAbs). The adhesion of BHK fibroblasts was markedly better on FN coated on hydrophilic tissue culture polystyrene (TCPS) than on hydrophobic, untreated polystyrene (PS). mAbs A17 and 3E3, which inhibit the binding of BHK cells to the RGD-containing sequence within the cell binding region of FN, also bound preferentially to FN on TCPS. In contrast, two anti-FN mAbs, which have no effect on cell adhesion (A35 and A3), bound preferentially to the conformation of FN on the more hydrophobic PS. Mouse melanoma cells utilise an additional cell-binding site in the Hep II domain of FN and their preference for FN coated on TCPS was less marked than that of BHK cells. This reduced preference was again mimicked by the binding of a mAb, A32, which inhibits the binding of B16 cells to the Hep II domain of FN. In contrast, BHK cell adhesion to VN did not display a preference for TCPS over PS. The cell-binding activity of adsorbed VN was matched by the binding of a cell adhesion-inhibitory mAb, A18, which, unlike mAbs A17 and A32, displayed slightly increased binding to VN coated on PS, rather than TCPS. When the denaturating effect of coating FN and VN to PS in the presence of urea was investigated, similar correlations between BHK cell adhesion and the binding of inhibitory mAbs were observed. Urea treatment of FN significantly reduced both BHK cell adhesion and the binding of both cell adhesion-inhibitory mAbs, whereas the binding of A35 and A3 was unaffected. There was no significant effect of urea treatment of VN upon either BHK cell adhesion or mAb binding. A larger panel of anti-FN mAbs was used, together with the anti-VN mAbs, to determine whether there were differences in mAb recognition of FN and VN adsorbed on three different brands of TCPS. The mAbs segregated into four reactivity patterns, of which A17, A32, A35 and A18 respectively were representative. Significant differences were observed in mAb recognition of FN and VN adsorbed to different brands of TCPS. These may reflect differences in the ability of these surfaces to support optimal growth of different cell types. The effect of divalent cations upon adsorbed FN and VN was also investigated.(ABSTRACT TRUNCATED AT 400 WORDS)
Underwood
P A
PA
CSIRO Division of Biomolecular Engineering, Laboratory for Molecular Biology, NSW, Australia.
Steele
J G
JG
Dalton
B A
BA
D000327:Adsorption; D000818:Animals; D000911:Antibodies, Monoclonal; D001798:Blood Proteins / Q000737:chemistry* / Q000276:immunology; D002413:Cations, Divalent / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D005353:Fibronectins / Q000737:chemistry* / Q000276:immunology; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology; D011137:Polystyrenes / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012995:Solubility; D013499:Surface Properties; D014407:Tumor Cells, Cultured; D014508:Urea; D019096:Vitronectin; D014867:Water / Q000737:chemistry
D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D002413:Cations, Divalent; D005353:Fibronectins; D006023:Glycoproteins; D011137:Polystyrenes; D019096:Vitronectin; D014867:Water; D014508:Urea