Determination of the LDL receptor binding capacity of human lymphocytes by immunocytofluorimetric assay.
The determination of the LDL receptor binding capacity of human blood lymphocytes was assessed by indirect immunocytofluorimetric assay. To produce the maximal synthesis of the LDL receptor, the cholesterol efflux was enhanced by incubation of lymphocytes with HDL3 subfractions. The binding capacity of the LDL receptor was measured by incubation at 4 degrees C either with LDL and rabbit anti-LDL immunoglobulins or with peptide receptor antibody (ARP-Ig) raised against the NH2-terminal sequence of the LDL receptor. Thereafter complexes were incubated with fluorescein-labelled anti-rabbit immunoglobulin (FITC-Ig). Fluorescence flow cytometry was used to quantify the number of fluorescent lymphocytes and results were expressed as the percentage of lymphocytes with a fluorescent intensity above the threshold. Using preimmune rabbit immunoglobulin and then FITC-Ig, only 5-10% of cells were fluorescent. Neither LDL nor ARP-Ig could bind to homozygous familial hypercholesterolemia (FH) lymphocytes. Normal lymphocytes preincubated with HDL3 could bind LDL or ARP-Ig, the number of fluorescent cells being 59 and 39.2% respectively. Subjects with confirmed or suspected heterozygous FH demonstrated cell fluorescence at about half the normal level.
Benhamamouch
S
S
Institut Pasteur, Lille, France.
Kuznierz
J P
JP
Agnani
G
G
Marzin
D
D
Lecerf
J M
JM
Fruchart
J C
JC
Clavey
V
V
D005260:Female; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000097:blood; D007118:Immunoassay; D008077:Lipoproteins, LDL / Q000097:blood; D008214:Lymphocytes / Q000378:metabolism*; D008297:Male; D010835:Phytohemagglutinins / Q000494:pharmacology; D011973:Receptors, LDL / Q000378:metabolism*; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D008077:Lipoproteins, LDL; D010835:Phytohemagglutinins; D011973:Receptors, LDL; D013861:Thiocyanates; D016650:Fluorescein-5-isothiocyanate