A new sandwich enzyme immunoassay for measurement of plasma pre-β1-HDL levels

Abstract

Pre-β1-HDL, a putative discoid-shaped high density lipoprotein (HDL) of approximately 67-kDa mass that migrates with pre-β mobility in agarose gel electrophoresis, contains apolipoprotein A-I (apoA-I), phospholipids, and unesterified cholesterol. It participates in the retrieval of cholesterol from peripheral tissues. In this study we established a new sandwich enzyme immunoassay (EIA) for measuring plasma pre-β1-HDL using mouse anti-human pre-β1-HDL monoclonal antibody (MAb 55201) and goat anti-human apoA-I polyclonal antibody. MAb 55201 reacted with apoA-I in lipoprotein [A-I] with molecular mass less than 67 kDa, and with pre-β1-HDL separated by nondenaturing two-dimensional electrophoresis, whereas it did not react with apoA-I in α-HDL. Pre-β1-HDL levels measured by this method declined when incubated at 37°C for 2 h, whereas this decrease was not observed in the presence of 2 mM lecithin:cholesterol acyltransferase inhibitor 5,5′-dithiobis (2-nitrobenzoic acid). To clarify the clinical significance of measuring pre-β1-HDL by this method, 47 hyperlipidemic subjects [male/female 22/25; age 55 ± 14 years; body mass index 25 ± 4.5 kg/m2; total cholesterol (TC) 245 ± 64 mg/dl; triglyceride (TG) 232 ± 280 mg/dl; HDL cholesterol (HDL-C) 51 ± 23 mg/dl] and 25 volunteers (male/female 15/10; age 36 ± 9.3 years; body mass index 23 ± 3.5 kg/m2; TC 183 ± 28 mg/dl; TG 80 ± 34 mg/dl; HDL-C 62 ± 15 mg/dl) were involved. Plasma pre-β1-HDL levels were significantly higher in hyperlipidemic subjects than in volunteers (39.3 ± 10.1 vs. 22.5 ± 7.5 mg/ml, P < 0.001) whereas plasma apoA-I levels did not differ (144.2 ± 28.4 vs. 145.3 ± 16.3 mg/dl). These results indicate that this sandwich EIA method specifically recognizes apoA-I associated with pre-β1-HDL.—Miyazaki, O., J. Kobayashi, I. Fukamachi, T. Miida, H. Bujo, and Y. Saito. A new sandwich enzyme immunoassay for measurement of plasma pre-β1-HDL levels. J. Lipid Res. 2000. 41: 2083–2088.

Keywords

• BALB/c mice
• hypoxanthine-aminopterin-thymidine medium
• SDS-PAGE
• anti-apoA-II polyclonal antibody
• horseradish peroxidase