A new sandwich enzyme immunoassay for measurement of plasma pre-beta 1-HDLlevels

Pre-beta1-HDL, a putative discoid-shaped high density lipoprotein (HDL) of approximately 67-kDa mass that migrates with pre-beta mobility in agarose g el electrophoresis, contains apolipoprotein A-I (apoA-I), phospholipids, an d unesterified cholesterol. It participates in the retrieval of cholesterol from peripheral tissues. In this: study we established a new sandwich enzy me immunoassay (EIA) for measuring plasma pre-beta1-HDL using mouse anti-hu man pre-beta1-HDL monoclonal antibody (MAb 55201) and goat anti-human apoA- I polyclonal antibody. MAb 55201 reacted with apoA-I in lipoprotein [A-I] w ith molecular mass less than 67 kDa, and with pre-beta1-HDL separated by no ndenaturing two-dimensional electrophoresis, whereas it did not react with apoA-I in alpha -HDL. Pre-beta1-HDL levels measured by this method declined when incubated at 37 degreesC for 2 h, whereas this decrease was not obser ved in the presence of 2 mM lecithin:cholesterol acyltransferase inhibitor 5,5'-dithiobis (2-nitrobenzoic acid). To clarify the clinical significance of measuring pre-beta1-HDL by this method, 47 hyperlipidemic subjects [male /female 22/25; age 55 +/- 14 years; body mass index 25 +/- 4.5 kg/m(2); tot al 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/m(2); TC 183 +/- 2 8 mg/dl; TG 80 +/- 34 mg/dl; HDL-C 62 +/- 15 mg/ dl) were involved. Plasma pre-beta1-HDL levels were significantly higher in hyperlipidemic subjects t han 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 reco gnizes apoA-I associated with pre-<beta>1-HDL.