title: Immunoblot analysis of proteins associated with HEMA-MMA microcapsules: human serum proteins in vitro and rat proteins following implantation.
abstract: Human serum proteins and their fragments, associated with hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) copolymer microcapsules, were characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. Capsules were incubated with serum for 1 week in vitro and then dissolved in ethanol to also precipitate the adsorbed protein. The precipitate was dissolved in 2% (w/v) SDS (the 'capsule eluate') to be assayed by electrophoresis. The majority of proteins probed for in the immunoblots were detected in the capsule eluates. These included fibronectin, plasminogen, IgG, vitronectin, Factor B, Factor H, Factor I, C3, but not beta-lipoprotein, fibrinogen, HMWK, or IgM. Complement activation fragments were detected in both the immunoblots of the capsule eluates and the medium containing serum without capsules. Thus, the adsorption of these fragments, formed independent of capsule presence, may be partially or completely responsible for the complement fragments associated with capsules. The prevention of complement activation by the addition of 5.8 mM EDTA, at the beginning of the week-long incubation, resulted in fewer low-molecular-weight C3 fragments associated with capsules. Rat proteins were also detected in immunoblots of the eluate of 'free-floating' capsules from the rat peritoneal cavity following implantation for 1 day using anti-human antibodies. Detected proteins included HMWK, fibrinogen, antithrombin III, transferrin, alpha1-antitrypsin, fibronectin, albumin, alpha2-macroglobulin, vitronectin, beta2-microglobulin, Factor B and Factor I. Rat fibrinogen, IgG, and complement C3 fragments were also detected in these immunoblots, but with monoclonal antibodies against the rat proteins.
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, Centre for Biomaterials, University of Toronto, Ont., Canada.
authors.forename: J E
authors.initials: JE
authors.lastname: Babensee
authors.forename: R M
authors.initials: RM
authors.lastname: Cornelius
authors.forename: J L
authors.initials: JL
authors.lastname: Brash
authors.forename: M V
authors.initials: MV
authors.lastname: Sefton
mesh_terms: D000818:Animals; D001672:Biocompatible Materials; D001798:Blood Proteins / Q000032:analysis* / Q000187:drug effects; D002118:Calcium / Q000097:blood; D002214:Capsules; D003169:Complement Inactivator Proteins / Q000187:drug effects; D003165:Complement System Proteins / Q000032:analysis / Q000187:drug effects; D003470:Culture Media; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006801:Humans; D015151:Immunoblotting; D008297:Male; D008768:Methylmethacrylates; D011102:Polyhydroxyethyl Methacrylate; D019736:Prostheses and Implants; D051381:Rats; D017208:Rats, Wistar
chemical_list: D001672:Biocompatible Materials; D001798:Blood Proteins; D002214:Capsules; D003169:Complement Inactivator Proteins; D003470:Culture Media; D008768:Methylmethacrylates; D011102:Polyhydroxyethyl Methacrylate; C040206:HTR composite; D003165:Complement System Proteins; D004492:Edetic Acid; D002118:Calcium