Structure-function studies on human C4b-binding protein using monoclonal antibodies.
Human C4b-binding protein (C4BP) is a multimeric regulatory complement component interacting with vitamin K-dependent protein S and complement C4b. Using hybridoma technology, a panel of monoclonal antibodies (mAb) specific for intact human C4BP and its 160-kDa chymotryptic central core fragment were prepared to study the structure-function relationships of C4BP. By Western blot analysis and competition experiments, four distinct groups of mAb were identified and mapped on the C4BP molecule. By rotary shadowing, spider-like images of C4BP-antibody complexes were obtained and immunoelectron microscopy provided some information on the stoichiometry of the antibody-C4BP interaction. Certain antibodies interacted with C4BP molecules only at a ratio of 1:1. Others formed complexes of two or more antibodies bound to homologous sites on the C4BP molecule. Using an enzyme-linked immunosorbent sandwich assay for the measurement of the complex formation between protein S and C4BP, mAb against the central core and the disulfide-linked beta chain of C4BP were identified that inhibited the binding of protein S to C4BP. In a binding assay using 125I-labeled C4BP and solid-phase C4b, the inhibitory effect of one group of anti-C4BP mAb on the binding of C4BP to C4b was demonstrated.
Hessing
M
M
Department of Haematology, University Hospital Utrecht, The Netherlands.
Kanters
D
D
Heijnen
H F
HF
Hackeng
T M
TM
Sixma
J J
JJ
Bouma
B N
BN
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D015153:Blotting, Western; D002352:Carrier Proteins / Q000276:immunology*; D002874:Chromosome Mapping; D015935:Complement C4b / Q000378:metabolism; D003169:Complement Inactivator Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D016253:Microscopy, Immunoelectron; D017293:Protein S; D011951:Receptors, Complement / Q000276:immunology*; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D000939:Epitopes; D006023:Glycoproteins; D017293:Protein S; D011951:Receptors, Complement; D015935:Complement C4b