Pre-beta high-density lipoprotein determined by immunoblotting with chemiluminescent detection.
We describe a novel assay of pre-beta high-density lipoprotein (HDL), a unique apolipoprotein A-I (apo A-I)-containing lipoprotein particle. The pre-beta and alpha lipoproteins are separated by electrophoresis in agarose and transferred onto a membrane by capillary blotting. The membrane blot is sequentially incubated with sheep anti-human apo A-I antiserum and then with a conjugate of rabbit anti-sheep immunoglobulin and horseradish peroxidase. Chemiluminescence formed by the peroxidase-catalyzed oxidation of luminol in the presence of an enhancer is captured on photographic film, and the pre-beta HDL band is quantified by transmission densitometry. The assay is calibrated with standards prepared from a reference serum diluted in 9 mol/L urea. Within-batch precision (CV) at pre-beta HDL concentrations of 22.1 and 44.3 mg/L was 7% and 4.9% respectively. Pre-beta HDL contained 1.6% (0.65-2.6%, mean and range) of total serum apo A-I in 30 normolipidemic subjects.
O'Kane
M J
MJ
Department of Clinical Biochemistry, Royal Victoria Hospital, Belfast, UK.
Wisdom
G B
GB
McEneny
J
J
McFerran
N V
NV
Trimble
E R
ER
D000328:Adult; D016632:Apolipoprotein A-I / Q000032:analysis*; D004355:Drug Stability; D004587:Electrophoresis, Agar Gel; D005260:Female; D053442:High-Density Lipoproteins, Pre-beta; D006801:Humans; D015151:Immunoblotting / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D008075:Lipoproteins, HDL / Q000097:blood*; D008163:Luminescent Measurements; D008297:Male; D012016:Reference Values
D016632:Apolipoprotein A-I; D053442:High-Density Lipoproteins, Pre-beta; D008075:Lipoproteins, HDL