Structural modifications of monoclonal antibodies following direct versus indirect labelling with 99Tcm: does fragmentation really occur?
In this study, the influence of direct and indirect 99Tcm-labelling on the molecular structural integrity of monoclonal antibodies and other immunoglobulin preparations was investigated. Molecular composition of antibody preparations [two IgG monoclonal antibodies, one F(ab')2 fragment (all directly labelled), one indirectly labelled polyclonal human immunoglobulin preparation] and of serum samples after antibody injection were studied using polyacrylamide gel electrophoresis (PAGE; non-reducing and reducing conditions) and gel filtration chromatography. With PAGE, depending on the conditions used, a variety of lower molecular weight products could be detected. When analysing the same antibody preparations by gel filtration chromatography, all complete antibody preparations appeared as homogenous proteins of IgG molecular weight (150 kD). In F(ab')2 fragments, some further fragmentation to Fab' was noticed. Neither in vitro nor in vivo (serum) evidence of smaller fragments could be detected by gel filtration, despite their presence in PAGE. We therefore conclude that through the reductive step of direct 99Tcm-labelling, interchain disulphide linkages are broken but the polypeptide chains of complete IgG remain associated by non-covalent linkages, whereas (F(ab')2 is fragmented further to form essentially Fab'. The protein-denaturating conditions of PAGE (even if performed non-reducingly) seem to produce artifacts, not representing the real in vivo condition. PAGE results should therefore be interpreted only with great care.
Behr
T
T
Department of Nuclear Medicine, Friedrich-Alexander University of Erlangen-Nuremberg, Germany.
Becker
W
W
Hannappel
E
E
Wolf
F
F
D000911:Antibodies, Monoclonal / Q000302:isolation & purification / Q000528:radiation effects*; D002272:Carcinoembryonic Antigen / Q000276:immunology; D002850:Chromatography, Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification / Q000528:radiation effects; D007074:Immunoglobulin G / Q000097:blood / Q000302:isolation & purification / Q000528:radiation effects*; D007553:Isotope Labeling / Q000379:methods; D013667:Technetium
D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D013667:Technetium