| Research Abstract |
Apolipoproteins (apo) are protein components of lipoproteins which play an important role in regulation of lipoprotein metabolism. A regulatory role of apolipoproteins in lipoprotein metabolism was studied using monoclonal antibodies against apolipoproteins. Firstly, plasma levels of apo B isoproteins such as apo B-100 and apo B-48 were evaluated by SDS-PAGE, protein bands of which were stained with Coomassie brilliant blue R or Western blotting using polyclonal antibody against pan-apo B. Secondly, apolipoproteins which were used for immunization to obtain monoclonal antibodies were isolated by fast protein liquid chromatography system and chromatofocusing etc. Thirdly, mouse monoclonal antibodies against pan-apo B, apo B isoproteins and apo C_s were produced in two fusions with spleen cells of Balb/c mouse and SP 2-0/Ag 14 mouse syeloma cells. Several monoclonal antibodies against pan-apo B and apo B isoproteins were obtained. However, production of monoclonal antibody which react sp
… More
ecifically with each of apo B-100 and apo B-48 was not successful. Finally, a competitive solid-phase radioimmunoassay (RIA) was developed employing one of the anti-apo C-I antibodies. VLDL was adsorbed to plastic microtiter wells, and a limiting amount of the antibody was reacted with the adsorbed VLDL. The amount of monoclonal antibody that bound to the immobilized VLDL-apo C-I was determined with a ^<125>I-labeled goat anti-mouse IgG antibody. The addition of competitor apo C-I complexed with lipids resulted in reduced binding of the anti-apo C-I antibody to the immobilized VLDL-apo C-I. The 40:1 (w/w) complex of Intralipid phospholipid/apo C-I was used as the primary assay standard because its displacement curve paralleled the curves obtained with whole plasma or isolated lipoproteins. Mean plasma concentration of apo C-I of 48 normolipidemic subjects was 25.0<plus-minus>11.7 (SD) mg/l. Plasma apo C-I contents correlated significantly with plasma triglyceride concentrations (n=93, r=+0.833, p < 0.001). Further studies will be needed to obtain more specific monoclonal antibodies against apo B and apo E isoproteins. Less
|
