Stoichiometric binding of apolipoprotein B-specific monoclonal antibodies to low density lipoproteins.
The structure of apolipoprotein B and its stoichiometry on plasma lipoproteins has been a major issue and one refractory to a variety of analyses. Immunochemical analyses represent an independent approach. Examinations of apolipoprotein B (apo-B) epitopes on human plasma low density lipoproteins (LDL) using monoclonal antibodies have consistently revealed the existence of extensive apo-B heterogeneity. In the present study, we have addressed the solution of the stoichiometry problem using quantitative analysis of the maximum number of identical antibodies that can be bound per LDL particle in which we take into account this ligand heterogeneity. We have estimated the molecular weight of apo-B by quantifying the number of times a given apo-B epitope is expressed on the surface of LDL. The quantitative binding of eight previously characterized monoclonal antibodies was measured in a fluid phase radioimmunoassay. The results were analyzed by Scatchard analysis and expressed on the basis of independent measurements of the maximum amount of LDL that could be bound by each antibody. Affinity constants for each of the eight antibodies varied between 8.5 X 10(7) and 80 X 10(7) M-1. For these same antibodies, the concentration of maximally bound antibody at a normalized LDL concentration of 1000 ng/ml was estimated to be 0.9-1.8 nM with a mean of 1.23 nM. Adopting a molecular mass from physicochemical analysis for LDL apo-B of 550,000 daltons, the molar ratio between bound antibody and LDL varied between 0.5 and 1.2 (mean 0.75 +/- 0.15). The results supported the hypothesis that apo-B is present as a single large molecular weight polypeptide in LDL.
O
O
Wiklund
C A
CA
Dyer
B P
BP
Tsao
L K
LK
Curtiss
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001055:Apolipoproteins B / Q000097:blood* / Q000276:immunology; D000939:Epitopes / Q000032:analysis; D006801:Humans; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000097:blood* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001055:Apolipoproteins B; D000939:Epitopes; D008077:Lipoproteins, LDL