title: A flow cytometric competition technique for measuring interaction of LDL with cellular LDL-receptors applied to patients with mutant (Arg3500-->Gln) apolipoprotein B.
abstract: We report our experience with a method to evaluate binding and uptake in cells of low density lipoprotein (LDL) from heterozygous patients with familial defective apolipoprotein B-100 (FDB-LDL) and LDL from normolipidemic subjects (nonFDB-LDL). The method is based on competition for binding/uptake in Epstein-Barr Virus (EBV)-transformed lymphocytes or COS cells overexpressing an LDL-receptor transgene between fluorescently labeled LDL and the unlabeled LDL of interest, and measurements are by flow cytometry. With EBV-lymphoblasts, the ability of FDB-LDL to displace fluorescent LDL ("Dil"-LDL) from cells at 4 degrees C (binding) was reduced to approximately 1/3 of normal. Displacement of "Dil"-LDL by FDB-LDL from cells at 20 degrees C (binding/uptake) was reduced to less than 1/2 of normal. Similar results were obtained with COS cells. Freezing of serum to -80 degrees C for 24 hours did not affect results, and we could discriminate between binding/uptake of FDB-LDL and nonFDB-LDL prepared from serum that had been stored at -80 degrees C for three months.
authors.affiliation: Department of Medicine and Cardiology, Aarhus Amtssygehus University Hospital, Denmark.
authors.forename: F
authors.initials: F
authors.lastname: Heath
authors.forename: T G
authors.initials: TG
authors.lastname: Jensen
authors.forename: P S
authors.initials: PS
authors.lastname: Hansen
authors.forename: L G
authors.initials: LG
authors.lastname: Jensen
authors.forename: H
authors.initials: H
authors.lastname: Vissinger
authors.forename: S K
authors.initials: SK
authors.lastname: Moestrup
authors.forename: S
authors.initials: S
authors.lastname: Pedersen
authors.forename: S
authors.initials: S
authors.lastname: Kølvraa
authors.forename: L
authors.initials: L
authors.lastname: Bolund
authors.forename: O
authors.initials: O
authors.lastname: Faergeman
mesh_terms: D000328:Adult; D001055:Apolipoproteins B / Q000235:genetics*; D001667:Binding, Competitive / Q000235:genetics; D002461:Cell Line, Transformed; D008078:Cholesterol, LDL / Q000097:blood*; D005260:Female; D005434:Flow Cytometry; D006580:Genetic Carrier Screening; D005820:Genetic Testing; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000097:blood / Q000175:diagnosis / Q000235:genetics*; D008297:Male; D017354:Point Mutation / Q000235:genetics*; D011973:Receptors, LDL / Q000235:genetics*; D012680:Sensitivity and Specificity
chemical_list: D001055:Apolipoproteins B; D008078:Cholesterol, LDL; D011973:Receptors, LDL