STRUCTURAL MODIFICATIONS OF MONOCLONAL-ANTIBODIES FOLLOWING DIRECT VERSUS INDIRECT LABELING WITH TC-99M - DOES FRAGMENTATION REALLY OCCUR

In this study, the influence of direct and indirect 99Tc(m)-labelling on the molecular structural integrity of monoclonal antibodies and oth er 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 h uman immunoglobulin preparation] and of serum samples after antibody i njection were studied using polyacrylamide gel electrophoresis (PAGE; non-reducing and reducing conditions) and gel filtration chromatograph y. With PAGE, depending on the conditions used, a variety of lower mol ecular weight products could be detected. When analysing the same anti body preparations by gel filtration chromatography, all complete antib ody preparations appeared as homogenous proteins of IgG molecular weig ht (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 dire ct 99Tc(m)-labelling, interchain disulphide linkages are broken but th e 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 n on-reducingly) seem to produce artifacts, not representing the real in vivo condition. PAGE results should therefore be interpreted only wit h great care.