A flow cytometric competition technique for measuring interaction of LDL with cellular LDL-receptors applied to patients with mutant (Arg3500-->Gln) apolipoprotein B.
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.
Heath
F
F
Department of Medicine and Cardiology, Aarhus Amtssygehus University Hospital, Denmark.
Jensen
T G
TG
Hansen
P S
PS
Jensen
L G
LG
Vissinger
H
H
Moestrup
S K
SK
Pedersen
S
S
Kølvraa
S
S
Bolund
L
L
Faergeman
O
O
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
D001055:Apolipoproteins B; D008078:Cholesterol, LDL; D011973:Receptors, LDL