Characterization of monoclonal antibodies to apolipoprotein (a) and development of a chemiluminescent assay for phenotyping apolipoprotein (a) isomorphs.
Apo(a) is linked to Lp(a) through non-covalent interactions and disulfide bond with apo B. Monoclonal antibodies were raised to reduced and carboxymethylated apo(a) in order to study apo(a) interaction with apo B and to develop a sensitive immunoassay for apo(a) and Lp(a). Nine antibodies were characterized for overlapping epitopes and for single or multiple binding sites on native Lp(a) or denatured apo(a). All monoclonal antibodies bound to Lp(a) and denatured apo(a) when these preparations were absorbed on polystyrene. In contrast, three antibodies (3D1, 4B4 and 6H9) failed to react with Lp(a) in solution, in a competitive displacement assay. This observation indicates that these epitopes are masked in native Lp(a). Cross-reactivity with plasminogen was noted for only one monoclonal antibody (4B4). An assay of competitive binding to immobilized Lp(a) or apo(a) revealed that four distinct groups of epitopes were recognized by the monoclonal antibodies: (A) 1G7, 3A5 partially overlapping with 8B6, (B) 5C4, 5B10 partially overlapping with 7C1, (C) 3D1 overlapping with 6H9, and (D) 4B4. A double antibody sandwich assay, using homologous and heterologous combinations of monoclonal antibodies, showed that monoclonal antibodies 1G7, 3A5 and 8B6 of group A, and 5C4 and 5B10 of group B recognized multiple epitopes on Lp(a) while all other antibodies (3D1, 6H9, 4B4) recognized single epitopes. Based on reports of others for the sequence of apo(a), deduced from the cDNA of the human apo(a) gene, it is proposed that monoclonal antibodies which recognize multiple epitopes are directed toward the repetitive kringle 4-like domains of apo(a) while those recognizing single epitopes are probably directed to the kringle 5 or the protease-like domain of apo(a). Monoclonal antibodies which recognized repetitive epitopes were used for the development of a highly sensitive chemiluminescent immunoblotting system for detection of apo(a) isomorphs after resolving plasma protein by polyacrylamide (4%) gel electrophoresis in the presence of sodium dodecyl sulfate. Seven relatively common isomorphs were identified and readily resolved as a mixture. The detection limit was 5-10 pg for each apo(a) isomorph. The high sensitivity allowed for the detection of isomorphs present in over 99% of plasma samples despite a wide range of ratios of apo(a) isomorphs.
Théolis
R
R
Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia, Canada.
Breckenridge
W C
WC
D000818:Animals; D000911:Antibodies, Monoclonal; D001054:Apolipoproteins A / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology*; D005260:Female; D006825:Hybridomas; D007118:Immunoassay / Q000379:methods*; D017270:Lipoprotein(a) / Q000276:immunology; D008163:Luminescent Measurements; D051379:Mice; D008807:Mice, Inbred BALB C; D010641:Phenotype
D000911:Antibodies, Monoclonal; D001054:Apolipoproteins A; D000939:Epitopes; D017270:Lipoprotein(a)