Monoclonal antibodies as probes to investigate the molecular changes of C5 associated with the different stability of the molecule on sheep erythrocytes and Escherichia coli 0111:B4.
The fifth C component (C5) exhibits a different stability when bound to sheep E or Escherichia coli 0111:B4, being fairly stable on the bacterial intermediate sensitized E. coli 0111:B4 coated with C components up to C5 (BAC1-5) and extremely labile on the RBC intermediate sensitized sheep E coated with C components up to C5 (EAC1-5). We examined the possibility that molecular changes of membrane-bound C5 might be responsible for the different functional behavior of the two intermediates using mAb to C5 and sensitive immunoassays to detect bound C5. The decay of EAC1-5 over 30 min of incubation at 37 degrees C was associated with a significant drop in the reactivity of bound C5 with three of four mAb used. These results contrasted with those obtained with BAC1-5, which showed unchanged reactivity with all mAb tested over the same period of incubation. The effect of mAb on the activity of C5 was then investigated in an attempt to relate the change of the reactivity pattern of EAC1-5 with the functional modification of bound C5. MAb 1.5 and 1.6 were the only antibodies that interfered with the functional activity of C5, although through a different mechanism. In particular, mAb 1.5 was active both on fluid-phase and on membrane-bound C5 and is therefore likely to interact with the binding site for the late components on C5. Conversely, mAb 1.6 was only effective on fluid-phase C5 and acted by promoting a decay of BAC1-5 similar to the spontaneous decay of EAC1-5. We suggest that the bacterial outer membrane may protect C5 from functional decay and that mAb 1.6 interferes with the stabilizing effect of the bacteria in an as yet unclear manner.
Rottini
G
G
Istituto di Patologia Generale, Università di Trieste, Italy.
Roncelli
L
L
Narchi
G
G
Patriarca
P
P
Protani
G
G
Perissutti
S
S
Tedesco
F
F
D000818:Animals; D000911:Antibodies, Monoclonal; D003182:Complement C5 / Q000378:metabolism*; D004912:Erythrocytes / Q000276:immunology*; D004926:Escherichia coli / Q000276:immunology*; D006801:Humans; D011487:Protein Conformation; D012756:Sheep; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D003182:Complement C5