IMMUNOBLOT ANALYSIS OF PROTEINS ASSOCIATED WITH HEMA-MMA MICROCAPSULES - HUMAN SERUM-PROTEINS IN-VITRO AND RAT PROTEINS FOLLOWING IMPLANTATION

Human serum proteins and their fragments, associated with hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) copolymer microcapsules, were characterized using sodium dodecyl sulphate-polyacrylamide gel el ectrophoresis (SDS-PAGE) and immunoblot analysis. Capsules were incuba ted with serum for 1 week in vitro and then dissolved in ethanol to al so 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 i n the capsule eluates. These included fibronectin, plasminogen, IgG, v itronectin, Factor B, Factor H, Factor I, C3, but not beta-lipoprotein , fibrinogen, HMWK, or IgM. Complement activation fragments were detec ted in both the immunoblots of the capsule eluates and the medium cont aining serum without capsules. Thus, the adsorption of these fragments , formed independent of capsule presence, may be partially or complete ly 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 w ere also detected in immunoblots of the eluate of 'free-floating' caps ules from the rat peritoneal cavity following implantation for 1 day u sing anti-human antibodies. Detected proteins included HMWK, fibrinoge n, antithrombin III, transferrin, alpha(1)-antitrypsin, fibronectin, a lbumin, alpha(2)-macroglobulin, vitronectin, beta(2)-microglobulin, Fa ctor B and Factor I. Rat fibrinogen, IgG, and complement C3 fragments were also detected in these immunoblots, but with monoclonal antibodie s against the rat proteins. (C) 1998 Elsevier Science Ltd. All rights reserved.