Characteristics of human lipoproteins isolated by selected-affinity immunosorption of apolipoprotein A-I.
We have isolated high density lipoproteins (HDL) from human serum by a new strategy, selected-affinity immunosorption, avoiding perturbation from ultracentrifugation or polyanion precipitation. The principle of this strategy is to utilize a subpopulation of monospecific antibodies directed against apolipoprotein A-I (apo A-I), which was preselected for dissociation under mild conditions of elution. Particles containing more than 90% of the apo A-I contained in serum were isolated uncontaminated by any other serum proteins. The immunoaffinity columns have been cycled over 300 times without apparent diminution in capacity. The apo A-I-containing particles sequestered from serum by immunosorption were more polydisperse in diameter and contained more protein than in ultracentrifugally isolated HDL. They also contained minor apolipoproteins that were not observed in ultracentrifuged HDL. Furthermore, the apo A-I-containing particles had different electrophoretic mobilities from those of ultracentrifugally isolated HDL when run under nondenaturing conditions in polyacrylamide gels. The apo A-I-containing particles isolated by our procedure separate into discrete bands similar to the subspecies visualized when whole serum is subjected to electrophoresis, whereas ultracentrifuged HDL migrate as two broad featureless zones. This suggests that selected affinity immunosorption does not subject the lipoproteins to structural disruption like that which occurs during ultracentrifugation. The principle of selected-affinity immunosorption should be of widespread utility in the isolation of fragile biological complexes.
D000936:Antigen-Antibody Complex; D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D001053:Apolipoproteins / Q000097:blood / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007106:Immune Sera; D007525:Isoelectric Focusing; D008075:Lipoproteins, HDL / Q000097:blood / Q000302:isolation & purification*
D000936:Antigen-Antibody Complex; D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D001053:Apolipoproteins; D007106:Immune Sera; D008075:Lipoproteins, HDL