Immunochemical characterization of apolipoprotein A-I from normal human plasma. In vitro modification of apo A-I antigens.

Milthorp, Peter; Weech, Philip K.; Milne, Ross W.; Marcel, Yves L.

doi:10.1161/01.atv.6.3.285

Cited by 33 publications

(4 citation statements)

References 17 publications

(17 reference statements)

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“…Commercial rabbit anti-human apoB-100 and rabbit anti-human apoE polyclonal antibodies were purchased from DAKO, Denmark. Monoclonal antibody 3D4 against apoA-I was a kind gift from Professor Yves Marcel, Montreal, Canada (24). The polyclonal human apoE antibody R107 was raised in rabbits with a standard immunization protocol using purified human plasma apoE as antigen.…”

Section: Antibodiesmentioning

confidence: 99%

PLTP secreted by HepG2 cells resembles the high-activity PLTP form in human plasma

Siggins

Jauhiainen

Olkkonen

et al. 2003

Journal of Lipid Research

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Plasma phospholipid transfer protein (PLTP) is an important regulator of plasma HDL levels and HDL particle distribution. PLTP is present in plasma in two forms, one with high and the other with low phospholipid transfer activity. We have used the human hepatoma cell line, HepG2, as a model to study PLTP secreted from hepatic cells. PLTP activity was secreted by the cells into serum-free culture medium as a function of time. However, modification of a previously established ELISA assay to include a denaturing sample pretreatment with the anionic detergent sodium dodecyl sulphate was required for the detection of the secreted PLTP protein. The HepG2 PLTP could be enriched by HeparinSepharose affinity chromatography and eluted in size-exclusion chromatography at a position corresponding to the size of 160 kDa. PLTP coeluted with apolipoprotein E (apoE) but not with apoB-100 or apoA-I. A portion of PLTP was retained by an anti-apoE immunoaffinity column together with apoE, suggesting an interaction between these two proteins. Furthermore, antibodies against apoE but not those against apoB-100 or apoA-I were capable of inhibiting PLTP activity. These results show that the HepG2-derived PLTP resembles in several aspects the high-activity form of PLTP found in human plasma. -Siggins, S., M. Jauhiainen, V. M. Olkkonen, J. Tenhunen, and C. Ehnholm. PLTP secreted by HepG2 cells resembles the high-activity PLTP form in human plasma.

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Section: Antibodiesmentioning

confidence: 99%

PLTP secreted by HepG2 cells resembles the high-activity PLTP form in human plasma

Siggins

Jauhiainen

Olkkonen

et al. 2003

Journal of Lipid Research

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“…Removal of 125I-labeled apoA-II from HDL would not produce the complete binding of HDL that is observed. Complete HDL binding also suggests that the mab M-30 epitope is not "altered" as described by Milthorp et al (1986), although the different levels of reactivity with various preparations of HDL and sHDL, containing apoA-I, may be due to similar phenomena. Any possible transformation of apoA-II into an apoA-I conformational state upon mab binding was excluded.…”

Section: Discussionmentioning

confidence: 75%

Monoclonal antibodies as probes of high-density lipoprotein structure: identification and localization of a lipid-dependent epitope

et al. 1987

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Eight stable murine monoclonal antibodies (mabs) were raised against human high-density lipoproteins (HDL). Three different antibody reactivities were demonstrated by immunoblotting. A group of five antibodies were specific for apolipoprotein A-I (apoA-I) and bound to similar or overlapping epitopes. The second type of reactivity, shown by mab-32, was specific for apoA-II. In the third group, two antibodies showed high reactivity with apoA-II and slight cross-reactivity with apoA-I. The properties of two antibodies, mab M-30 specific for apoA-I and mab M-32 specific for apoAII, were characterized in detail as probes of HDL structure. The association of 125I-labeled HDL or synthetic complexes of apoA-I and phosphatidylcholine with mab M-30 was lipid dependent. Mab M-32 binding to apoA-II was independent of lipid. The lipid-dependent epitope bound by mab M-30 has been localized to an 18 amino acid synthetic apoA-I peptide. Moreover, studies with HDL2, HDL3, and immunoadsorbed HDL subfractions indicate that binding of mab M-30 to HDL is influenced by some component within the microenvironment individual HDL particles. These lines of evidence suggest that the molar ratio of apoA-I to apoA-II is the critical determinant. Binding of mab M-32 to HDL increased the reactivity of HDL to mab M-30 in a dose-dependent manner, indicating an unusual form of cooperativity between two mabs that recognize different proteins in HDL. These monoclonal antibodies will be valuable in studies of the metabolic significance of protein-protein and lipid-protein interactions in HDL.

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“…To discriminate the different apoA-I conformations on HDL particles, native monoclonal antibodies (mAbs) generated by hybridoma techniques targeting epitopes distributed along the apoA-I sequence 26–32 as well as recombinant antibody fragments isolated from phage-displayed libraries 33 have been used. A mAb that specifically recognizes an epitope of apoA-I exposed only in preβ1-HDL (or lipid-poor apoA-I) has been commercially available to measure preβ1-HDL concentration in plasma 34, 35 .…”

Section: Introductionmentioning

confidence: 99%

Immunochemical Approach for Monitoring of Structural Transition of ApoA-I upon HDL Formation Using Novel Monoclonal Antibodies

Kimura

Mikawa

Mizuguchi

et al. 2017

Sci Rep

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Apolipoprotein A-I (apoA-I) undergoes a large conformational reorganization during remodeling of high-density lipoprotein (HDL) particles. To detect structural transition of apoA-I upon HDL formation, we developed novel monoclonal antibodies (mAbs). Splenocytes from BALB/c mice immunized with a recombinant human apoA-I, with or without conjugation with keyhole limpet hemocyanin, were fused with P3/NS1/1-Ag4-1 myeloma cells. After the HAT-selection and cloning, we established nine hybridoma clones secreting anti-apoA-I mAbs in which four mAbs recognize epitopes on the N-terminal half of apoA-I while the other five mAbs recognize the central region. ELISA and bio-layer interferometry measurements demonstrated that mAbs whose epitopes are within residues 1–43 or 44–65 obviously discriminate discoidal and spherical reconstituted HDL particles despite their great reactivities to lipid-free apoA-I and plasma HDL, suggesting the possibility of these mAbs to detect structural transition of apoA-I on HDL. Importantly, a helix-disrupting mutation of W50R into residues 44–65 restored the immunoreactivity of mAbs whose epitope being within residues 44–65 against reconstituted HDL particles, indicating that these mAbs specifically recognize the epitope region in a random coil state. These results encourage us to develop mAbs targeting epitopes in the N-terminal residues of apoA-I as useful probes for monitoring formation and remodeling of HDL particles.

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Immunochemical characterization of apolipoprotein A-I from normal human plasma. In vitro modification of apo A-I antigens.

Cited by 33 publications

References 17 publications

PLTP secreted by HepG2 cells resembles the high-activity PLTP form in human plasma

PLTP secreted by HepG2 cells resembles the high-activity PLTP form in human plasma

Monoclonal antibodies as probes of high-density lipoprotein structure: identification and localization of a lipid-dependent epitope

Immunochemical Approach for Monitoring of Structural Transition of ApoA-I upon HDL Formation Using Novel Monoclonal Antibodies

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