Expression of human apolipoprotein A-I epitopes in high density lipoproteins and in serum.

Marcel, Yves L.; Jewer, D; Vézina, Camilla; Milthorp, Peter; Weech, Philip K.

doi:10.1016/s0022-2275(20)38641-7

Cited by 54 publications

(15 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…ED 50 represents the reconstituted complex concentration required to inhibit 50% of the maximal 3G10 binding on a HDL 3 -coated plate in a radioimmunometric assay. 3G10 is a monoclonal antibody directed against residues 98-121 of the human apolipoprotein A-I molecule (23). A decrease in the ED 50 value indicates an increase in the exposure of the 98-121 domain at the surface of the reconstituted Lp2A-I complex.…”

Section: Discussionmentioning

confidence: 99%

“…1-Palmitoyl 2-oleoyl phosphatidylcholine (POPC) was obtained from Avanti Polar Lipids (Birmingham, AL). The monoclonal antibody, 3G10, was produced by established protocols (23). All other reagents were analytical grade.…”

Section: Methodsmentioning

confidence: 99%

“…Immunoreactivity measurements were determined from a competitive solid phase radioimmunometric assay similar to that previously described (23). Removawells (Immulon 2, Dynatech Laboratories, MA) were coated with 100 l of HDL 3 (0.2 g in 15 m m Na 2 CO 3 , 35 m m NaHCO 3 , 0.02% NaN 3 , pH 9.6), washed with 50 m m NaPO 4 , 0.02% NaN 3 , pH 7.2 (buffer A), and saturated with 0.5% gelatin (Bio-Rad Laboratories, Hercules, CA) in buffer A. Anti-apoA-I monoclonal antibody, 3G10 (at a predetermined dilution), was mixed with serial dilutions of LpA-I or LpA-I/A-II particles in 50 m m NaPO 4 , 0.02% NaN 3 , 0.05% Tween 20, 0.1% gelatin (buffer B) and transferred to the previously coated and saturated wells for 1 h incubation at room temperature.…”

Section: Immunoreactivity Of Apoa-i On Lpa-i and Lpa-i/a-ii Complexesmentioning

confidence: 99%

See 2 more Smart Citations

Apolipoprotein A-I charge and conformation regulate the clearance of reconstituted high density lipoprotein in vivo

Braschi

Neville

Vohl

et al. 1999

Journal of Lipid Research

View full text Add to dashboard Cite

While low apolipoprotein A-I (apoA-I) levels are primarily associated with increased high density lipoprotein (HDL) fractional catabolic rate (FCR), the factors that regulate the clearance of HDL from the plasma are unclear. In this study, the effect of lipid composition of reconstituted HDL particles (LpA-I) on their rate of clearance from rabbit plasma has been investigated. Sonicated LpA-I containing 1 to 2 molecules of purified human apoA-I and 5 to 120 molecules of palmitoyl-oleoyl phosphatidylcholine (POPC) exhibit similar charge and plasma FCR to that for lipid free apoA-I, 2.8 pools/day. Inclusion of 1 molecule of apoA-II to an LpA-I complex increases the FCR to 3.5 pools/day, a value similar to that observed for exchanged-labeled HDL 3 . In contrast, addition of 40 molecules of triglyceride, diglyceride, or cholesteryl ester to a sonicated LpA-I containing 120 moles of POPC and 2 molecules of apoA-I increases the negative charge of the particle and reduces the FCR to 1.8 pools/day. Discoidal LpA-I are the most positively charged lipoprotein particles and also have the fastest clearance rates, 4.5 pools/day. Immunochemical characterization of the different LpA-I particles shows that the exposure of an epitope at residues 98 to 121 of the apoA-I molecule is associated with an increased negative particle charge and a slower clearance from the plasma. We conclude that the charge and conformation of apoA-I are sensitive to the lipid composition of LpA-I and play a central role in regulating the clearance of these lipoproteins from plasma. -Braschi, S., T. A-M. Neville, M-C. Vohl, and D. L. Sparks. Apolipoprotein A-I charge and conformation regulate the clearance of reconstituted high density lipoprotein in vivo.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Immunoreactivity Of Apoa-i On Lpa-i and Lpa-i/a-ii Complexesmentioning

confidence: 99%

See 1 more Smart Citation

Apolipoprotein A-I charge and conformation regulate the clearance of reconstituted high density lipoprotein in vivo

Braschi

Neville

Vohl

et al. 1999

Journal of Lipid Research

View full text Add to dashboard Cite

show abstract

“…Antibodies. The antibodies used for these studies have been previously described and characterized (Milthorp et al, 1986;Marcel et al, 1987Marcel et al, , 1991Petit et al, 1987). The control mAb 2H2 is an antibody against rat synthetic atrial natriuretic factor (Milne et al, 1987).…”

Section: Methodsmentioning

confidence: 99%

“…Solid-Phase Radioimmunoassay of ApoA-I. Solid-phase radioimmunoassays were modified from those previously described (Marcel et al, 1987) by omission of detergent in the reaction buffer. Briefly, Immulon II Removawells were coated with 0.2 pg of apo-HDL in 100 pL of 15 mM Na3C03/ 35 mM NaHC03, pH 9.6, containing 0.02% NaN3, washed, and then saturated with 300 pL of gelatin (0.5% in PBS, pH 7.2, and 0.02% NaN3).…”

Section: Methodsmentioning

confidence: 99%

Apolipoprotein A-I conformation in discoidal particles: Evidence for alternate structures

Calabresi¹,

Meng²,

Castro³

et al. 1993

Biochemistry

View full text Add to dashboard Cite

To define the conformation of apolipoprotein A-I in discoidal particles, the immunoreactivity of a series of epitopes distributed along the apoA-I sequence has been evaluated in lipid-free apoA-I and in lipid-bound form. To this end, reconstituted discoidal lipoproteins, here called LpA-I, and defined by number of apoA-I per particle (e.g., Lp2A-I), have been prepared with palmitoyloleoylphosphatidylcholine, cholesterol, and apoA-I. Four LpA-I have been obtained and studied: two in the Lp2A-I class, 7.8 and 9.6 nm in diameter, and two in the Lp3A-I class, 10.8 and 13.4 nm. The immunoreactivity of all the epitopes tested was significantly different in LpA-I particles compared to lipid-free apoA-I, demonstrating that binding to lipids produces a drastic change in apoA-I conformation. Specific domains in the primary sequence become highly exposed while others are masked. Although the variation in immunoreactivity of the epitopes between various LpA-I was not drastic, significant differences in the calculated ED50 values were observed for a number of antibodies in small versus large particles within each class (Lp2A-I or Lp3A-I), indicating that particle size can modulate apoA-I conformation. In addition, when the competition between pairs of mAbs was analyzed in order to understand the relative position of epitopes, highly significant differences were observed as a function of particle size within each class. In particular, the competition between mAbs recognizing epitopes in the central region of apoA-I was greater in the larger particles than in their small counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)

show abstract

Enzyme‐linked immunosorbent assay to measure apolipoproteins Al and B secreted by a human hepatic carcinoma cell line (Hep G2)

Hahn

Parkes

Goldberg

1992

Clinical Laboratory Analysis

View full text Add to dashboard Cite

We describe an enzyme-linked immunosorbent assay (ELISA) to measure apolipoproteins AI and B secreted by Hep G2 cells and in cell homogenates. These assays utilize commercially available polyclonal antibodies, affinity-purified to improve their specificity, thereby achieving a dramatic increase in the sensitivity of the assay. These affinity-purified antibodies were also more sensitive than a series of monoclonal antibodies tested. We achieved a sensitivity of 0.4 ng in the apo AI assay, and a sensitivity of 5 ng in the apo B assay. By these methods, we measured secretion rates by Hep G2 cells of 358 +/- 41 ng/mg cell protein/hr for apo B and 137 +/- 8 ng/mg cell protein/hr for apo AI. These assays also allowed the measurement of intracellular apolipoproteins and thus can be used to facilitate investigations of human lipoprotein metabolism in cell culture systems.

show abstract

Expression of human apolipoprotein A-I epitopes in high density lipoproteins and in serum.

Cited by 54 publications

References 24 publications

Apolipoprotein A-I charge and conformation regulate the clearance of reconstituted high density lipoprotein in vivo

Apolipoprotein A-I charge and conformation regulate the clearance of reconstituted high density lipoprotein in vivo

Apolipoprotein A-I conformation in discoidal particles: Evidence for alternate structures

Enzyme‐linked immunosorbent assay to measure apolipoproteins Al and B secreted by a human hepatic carcinoma cell line (Hep G2)

Contact Info

Product

Resources

About