Characterization of monoclonal antibodies against human apolipoprotein E.

Milne, Ross W.; Douste-Blazy, P.; Marcel, Yves L.; Retegui, Lilia A.

doi:10.1172/jci110225

Cited by 81 publications

(24 citation statements)

References 21 publications

(9 reference statements)

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“…11 The specificity of these Mabs and the identification of their epitopes have been described in several reports. 111213 The properties of the Mabs against apo E 25 and against apo D 26 have been reported earlier. Mab 3D7 was obtained from mice immunized with chylomicrons.…”

Section: Antibodies Against Human Apolipoproteinsmentioning

confidence: 77%

Expression of apolipoprotein B epitopes in lipoproteins. Relationship to conformation and function.

Marcel¹,

Hogue²,

Weech³

et al. 1988

Arteriosclerosis

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The immunochemical properties of apolipoprotein (apo) B have been studied in very low density lipoprotein (VLDL), (Sf 100 to 400), VLDL 2 (Sf 60 to 100), VLDL 3 (Sf20 to 60), different intermediate density lipoprotein (IDL), and low density lipoprotein (LDL) subfractions isolated from patients with type IV hypertriglyceridemia. In these lipoproteins, we characterized the association of apo B with other apolipoproteins and the expression and immunoreactivity of several apo B epitopes close to the apo B receptor binding sites (3F5, 4G3, 3A8, and 5E11) and of other epitopes located on the apo B100-B48 common region (1D1 and 2D8). Immunoprecipitation showed that the proportion of lipoprotein particles expressing each apo B epitope increased from VLDL, to LDL*; this was more apparent with 3A8 and SE11 than with 3F5. The VLDL that were negative for apo E epitopes (60% or more of the total) were enriched in apo C. The lipoprotein particles containing apo E and/or apo C-lll decreased progressively from VLDL, (30% and 85%, respectively) to LDL 2 (10% and 25%, respectively). Similar observations were made for apo C-l and apo D, demonstrating that apolipoprotein heterogeneity is greatest in the lightest lipoproteins. By competitive radioimmunoassay, the epitope for 4G3 was equally immunoreactive in each lipoprotein subclass, and the affinity constant (Ka) of 4G3 for different lipoproteins showed little variation. In contrast, both immunoreactivity and Ka of 3A8 and 5E11 increased progressively and significantly with the increasing density of the lipoprotein subclasses. This phenomenon is correlated with the increasing binding affinity of apo B in these lipoprotein subclasses to the LDL receptor of fibroblasts. We conclude that, as the apo B-containing lipoproteins become smaller, the conformation of specific regions of apo B is modified: in the receptor binding domain, the conformation of epitope 4G3, which is mapped between residues 2980 and 3080, remains constant, while that of 3A8 and 5E11 (residues 3441 to 3568) changes progressively. We propose the theory that the change in conformation in the domain spanning residues 3441 and 3568 allows the maximum expression of epitopes 3A8 and 5E11 and of the receptor binding site. 23 The size and density heterogeneity of the apo B-containing lipoproteins appear to be introduced not only at the time of secretion of nascent lipoproteins 4 but also during catabolism, 5 the end-point of which is the cellular uptake via receptors that recognize either apo B-100 or apo E or both. 6 In the course of the intravascular transformation of VLDL, the ligand responsible for the binding of the lipo- Received March 31, 1988; revision accepted June 24, 1988. protein to the apo B/E receptor changes progressively from apo E to apo B as the particle becomes smaller, 78 and this transformation is most pronounced at the stage of VLDL 3 (Sf 20 to 60). The studies of Gianturco et al. 7 and Bradley et al. 9 have shown that the apo E-mediated affinity of the VLDL for the apo B/E receptor is depen...

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Section: Antibodies Against Human Apolipoproteinsmentioning

confidence: 77%

Expression of apolipoprotein B epitopes in lipoproteins. Relationship to conformation and function.

Marcel¹,

Hogue²,

Weech³

et al. 1988

Arteriosclerosis

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“…Apo A-I, 12 apo E, 13 and apo B 14 in study 1 were measured by radioimmunoassay as described previously. In study 2, apo A-I and apo B were measured immunoturbidimetrically with the use of kits from Orion Diagnostica, London, UK.…”

Section: Immunoassaysmentioning

confidence: 99%

Plasma concentrations of cholesteryl ester transfer protein in hyperlipoproteinemia. Relation to cholesteryl ester transfer protein activity and other lipoprotein variables.

McPherson

Mann

Tall

et al. 1991

Arterioscler Thromb

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198

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Cholesteryl ester transfer protein (CETP) mediates an important pathway for reverse cholesterol transport. Concentrations of CETP in fasting plasma were measured by radioimmunoassay in two different groups of hyperlipoproteinemic subjects. Plasma CETP concentrations measured by radioimmunoassay correlated closely with cholesterol ester transfer activity in normal plasma (r=0.86). In the first group of 58 patients, plasma CETP concentrations were significantly increased, as compared with those in 79 normal subjects and in hypercholesterolemic (+26%) and combined hyperlipoproteinemic (+25%) subjects but were not altered in moderately hypertriglyceridemic subjects. Marked elevations in plasma CETP levels were documented in patients with dysbetalipoproteinemia (+68%) and severe chylomicronemia (+85%). Similar results were obtained in a second population of 50 hyperlipoproteinemic subjects. Significant correlations were found between plasma CETP levels and total cholesterol (r=0.52), very low density lipoprotein (VLDL) cholesterol (r=0.63), and apolipoprotein E concentration (r=0.40). Correction of the lipoprotein phenotype by dietary means resulted in significant reductions in plasma CETP concentrations in patients with chylomicronemia and dysbetalipoproteinemia. In these subjects, plasma high density lipoprotein cholesterol concentrations increased as CETP decreased. These studies indicate that CETP levels increase in association with enhanced peripheral cholesterol transport via low density lipoprotein, /3-VLDL, or chylomicron remnants. (Arteriosclerosis and Thrombosis 1991;ll:797-804) E arly studies by Rehnborg and Nichols 1 and Nichols and Smith 2 demonstrated that on incubation of normolipemic plasma, cholesteryl ester (CE) increased in very low density lipoprotein (VLDL), whereas triglyceride (TG) concentration decreased in VLDL but increased in high density lipoprotein (HDL) and low density lipoprotein (LDL). These experiments were the first to document CE transfer between lipoproteins, a phenomenon that was further clarified by the demonstra- tion by Zilversmit et al 3 that the d>1.21 g/ml fraction contained a protein that catalyzed CE transfer between lipoproteins, a protein that is now identified as cholesteryl ester transfer protein (CETP). More recently, two research groups have purified and characterized CETP, which is a very hydrophobic protein with a molecular size of M r 74,000, 45 in agreement with the data derived from the cloning and sequencing of the cDNA encoding for CETP. 6 While CETP mediates only the exchange of CEs between HDL and LDL, 7 it does promote net transfer of CEs from HDL to VLDL and chylomicrons. 8 This specificity of the transfer reaction makes it a major pathway for reverse cholesterol transport in many mammalian species. However, the significance of plasma levels of CETP in atherosclerosis must be considered in terms of the two facets of the CETPmediated system -factors facilitating the accumulation of HDL CE (secretion of nascent HDL, its interaction with peripheral cells...

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“…10 Following fusion, the cells, suspended in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 15 mM Hepes, 40 fiM 2-mercaptoethanol, penicillin-streptomycin, 30% fetal bovine serum (FBS), 0.11 mM hypoxanthine, 0.38 fiM aminopterine and 16 fiM thymidine were distributed in 576 microculture wells (Costar, Cambridge, Massachusetts). When sufficient growth had occurred, normally at 10 to 15 days, the supernatants were screened by a solid phase radioimmunoassay (RIA) 10 for the presence of anti-LDL antibodies using Removawells (Dynatech Laboratories Incorporated, Dynatech Corporation, Alexandria, Virginia) which had been coated overnight at room temperature with 200 fi\ of a solution of LDL (50 fig LDL protein/ml in 5 mM glycine pH 9.2). The cells in the positive wells were recloned by limiting dilutions in 96 well microculture plates at a cell density that produced growth in approximately 10% of the wells.…”

Section: Production Of Monoclonal Antibodiesmentioning

confidence: 99%

“…The RIA for apo B was essentially the same as that for apo E. 10 We distributed 200 fi\ of the IgG fraction diluted in 5 mM glycine in Removawells and left it overnight. The concentration of IgG chosen on the basis of preliminary experiments ranged from 5 to 20 /xg/ml depending upon the individual clone.…”

Section: Radioimmunoassay Of Apo Bmentioning

confidence: 99%

Characterization of monoclonal antibodies against human low density lipoprotein.

Milne¹,

Théolis²,

Verdery³

et al. 1983

Arteriosclerosis

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139

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Apolipoprotein B (apo B) is the major protein species of LDL, but the elucidation of its structure has proven to be a most difficult task mainly because of its total insolubility in a delipidated form which may be related to high oxidation sensitivity.1 While widely varying molecular weights for apo B have been reported in recent years, it is now thought to have a molecular weight in excess of 200.000.2 ' 3 The LDL receptor present on the surface of mammalian cells binds certain plasma lipoproteins by recognizing sites which are present on apo B as well as on apo E. 4 ' 5 The binding of LDL to the LDL receptor initiates a series of events which has been called the LDL pathway.6 ' 7 The LDL is taken into the cell where it is hydrolyzed in lysosomes. The released cholesterol inhibits endogenous cholesterol synthesis at the level of the rate-limiting enzyme, hydroxymethyl glu- taryl coenzyme A reductase (HMG CoA reductase), stimulates cholesterol esterification, and inhibits synthesis of the LDL receptor itself.Monoclonal antibodies have proven useful in the study of protein structure and in the identification of functional domain of proteins. With the aid of monoclonal antibodies against human LDL, we are trying to identify and eventually purify and characterize the regions of apo B recognized by the LDL receptor. Here we describe seven monoclonal antibodies against LDL which are differentiated on the basis of their cross-reactivities with chemically modified LDL and their abilities to interfere with the LDL pathway. In addition, we describe a novel adaptation of the cotitration method 8 which allows a tentative mapping of the antigenic determinants. Methods Preparation of LDLBlood from normal subjects was collected into EDTA and the red blood cells were removed by centrifugation. Lipoprotein subfractions were prepared in a Beckman L5-65 ultracentrifuge with a 50.2 Ti rotor (Beckman Instruments Incorporated, Spinco Division, Palo Alto, California). LDL were isolated by successive ultracentrifugations 9 at 4° between densities of 1.030-1.050 g/ml, dialyzed exhaustively against phosphate-buffered saline (PBS) containing 1 mM EDTA, sterilized by ultrafiltration and stored at 4°C.

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Characterization of monoclonal antibodies against human apolipoprotein E.

Cited by 81 publications

References 21 publications

Expression of apolipoprotein B epitopes in lipoproteins. Relationship to conformation and function.

Expression of apolipoprotein B epitopes in lipoproteins. Relationship to conformation and function.

Plasma concentrations of cholesteryl ester transfer protein in hyperlipoproteinemia. Relation to cholesteryl ester transfer protein activity and other lipoprotein variables.

Characterization of monoclonal antibodies against human low density lipoprotein.

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