Journal of Immunological Methods, 119 (1989) 65-73 65 Elsevier JIM05135 Local tissue injury induced by glucose oxidase conjugated with anti-collagen antibody Vladimir R. Muzykantov, Mikhail M. Peclo and Olga Yu. Printseva Institute of Experimental Cardiology of the U.S.S.R. CardiologyResearch Center (IEC UCRC), Academy of Medical Sciences, Moscow, U.S.S.R. (Received17 May 1988,revisedreceived 3 October1988, accepted28 November1988) The conjugation of glucose oxidase with anti-collagen antibody using periodate oxidation of the enzyme carbohydrate moiety is described. After conjugation, the antibody retained its antigen-binding capacity and the enzyme retained hydrogen peroxide-generating activity. Intradermal administration of the immune conjugate into rats induced local tissue injury at doses 10-100/~g. Pronounced damage (local hyperemia and edema) occurred 24 h after injection and necrosis developed 1 week later. The enzyme was tightly bound to the fibrillar components of the extracellular matrix and retained its activity in vivo for a prolonged period of time. In contrast, non-immune IgG-conjugated glucose oxidase was removed rapidly from the site of injection and did not induce tissue damage. Pure native anti-collagen antibody was retained at the site of injection for 8 days, but caused no tissue injury. These results suggest that active glucose oxidase conjugated with antibodies to tissue antigen can be accumulated and retained in the tissues. At the site of accumulation local 'proinflammatory' damage develops even in the absence of the halide-peroxidase system. Similar conjugates could be potential agents for local modulation of inflamma- tion. Key words: Hydrogen peroxide; Drug targeting;Inflammation; Tissuedamage; Immunotoxin Introduction Hydrogen peroxide released by activated phagocytes produces a direct cytotoxic effect (Nathan et al., 1979; Badwey and Karnovsky, 1980), degrades extracellular matrix (Weiss et al., Correspondence to: V.R. Muzykantov, U.S.S.R. Cardiology Research Center, 3rd Cherepkovskaya Str. 15A, Moscow 121552, U.S.S.R. Abbreviations: PBS,phosphate-buffered saline;BSA, bovine serum albumin; GARGG, goat antibody against rabbit im- munoglobulins; HRPO, horseradish peroxidase; DAB, di- aminobenzidine; HAP, hydroxylapatite; PEG, polyethylene glycol; BAM,biologicalactivitymodifier; SMCC, succinimi- dyl-4-( N-maleimidomethyl)cyclohexane-l-carboxylate; SPDP, succinimidyl-3-(pyridyl-dithio)propionate. 1985, 1986), modulates plasma proteases and in- duces chemoattractant activity (Shingu and Nobunaga, 1984). In vivo and in vitro studies have demonstrated both protective 'anti-inflammatory' properties of the HEOE-destroying enzyme catalase (Nathan et al., 1979; Fliegel et al., 1984) and damaging 'proinflammatory' properties of the H2OE-generating enzymes glucose oxidase and xanthine oxidase (Johnson et al., 1981). It has been suggested that catalase and its PEG or lipo- somal derivatives protect host cells at inflamma- tory or ischemic sites against injury induced by hydrogen peroxide (Fliegel et al., 1984; Freeman et al., 1985). To achieve a prolonged local effect the targeting of protective substances has been proposed (Muzykantov et al., 1987; Sakharov et al., 1987). It is obvious, however, that in certain 0022-1759/89/$03.50 © 1989 ElsevierSciencePublishersB.V.(Biomedical Division)