Mapping apolipoprotein B on the low density lipoprotein surface by immunoelectron microscopy.

Apolipoprotein B (apoB) was mapped using electron microscopy to visualize pairs of monoclonal antibodies binding to the low density lipoprotein (LDL) surface. The sites at which these monoclonals bind the apoB polypeptide sequence had already been established. The angular distances between all possible pairs of binding sites except one allowed the relative placement of six epitopes on the LDL sphere.

13C NMR evidence that substitution of glutamine for arginine 3500 in familial defective apolipoprotein B-100 disrupts the conformation of the receptor-binding domain.

Familial defective apoB-100 is a genetic mutation that is characterized by abnormal low density lipoprotein (LDL) and moderate hypercholesterolemia. Heterozygotes for this disorder possess two populations of LDL. One has normal receptor binding, and the other, which can be isolated by monoclonal antibody 19 immunoaffinity chromatography, has almost no binding activity.

THP-1 cells form foam cells in response to coculture with lipoproteins but not platelets.

The human monocytic leukemia cell line, THP-1, shares many properties with human monocyte-derived macrophages and might be a useful model for studying foam cell formation in vitro. Therefore, we examined the ability of THP-1 cells to accumulate cholesteryl esters, the hallmark feature of foam cells, in response to culture with native low density lipoprotein (LDL), modified LDL, and platelets. THP-1 cells stored more cholesteryl esters than macrophages in response to 200 micrograms/ml of LDL.

Apolipoprotein (apo) E inhibits the capacity of monosodium urate crystals to stimulate neutrophils. Characterization of intraarticular apo E and demonstration of apo E binding to urate crystals in vivo.

Factors that modulate the ability of monosodium urate crystals to stimulate leukocytes could regulate gouty inflammation. Lipoproteins that bear apo B-100 and apo E bind to urate crystals and suppress crystal-neutrophil interaction. In this study, we observed that urate crystals, coated with apo E of monocyte origin, had a diminished ability to stimulate neutrophils.

Glycosaminoglycans alter the capacity of low density lipoprotein to bind to monosodium urate crystals.

Low density lipoprotein (LDL) has previously been demonstrated to be a potent inhibitor of human inflammatory cell activation by monosodium urate (MSU) crystals in vitro. As suppression of cellular responses to crystals by LDL is known to be dependent on the binding of LDL to urate crystals we further evaluated the mechanism and regulation of LDL binding to urate crystals in vitro. Using nonlinear, least squares methodology to analyze binding, we found LDL to saturably and reversibly bind with high affinity (Kd 9.

Platelet-mediated foam cell formation in atherosclerosis.

The early fatty streak lesions of atherosclerosis are characterized by the presence of cholesteryl ester-loaded macrophages or "foam cells." Platelets are also present in the early lesions of atherosclerosis and are often found in close association with foam cells. We have investigated the hypothesis that platelets contribute to foam cell formation by inducing macrophage cholesteryl ester accumulation.

Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein.

Increasing evidence indicates that low density lipoprotein (LDL) has to be modified to induce foam cell formation. One such modification, oxidation of LDL, generates a number of highly reactive short chain-length aldehydic fragments of oxidized fatty acids capable of conjugating with lysine residues of apoprotein B. By immunizing animals with homologous malondialdehyde-modified LDL (MDA-LDL), 4-hydroxynonenal-LDL (4-HNE-LDL), and Cu+(+)-oxidized LDL, we developed polyvalent and monoclonal antibodies against three epitopes found in oxidatively modified LDL.

Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay as a candidate reference method for the measurement of apolipoprotein B-100.

A monoclonal antibody-based direct binding enzyme-linked immunosorbent assay (ELISA) for apoprotein (apo) B-100 has been developed for use as a reference method. The assay uses the two well-characterized monoclonal antibodies, MB24 and MB47. MB47, which recognizes an epitope at the low density lipoprotein (LDL) receptor-binding domain of apoB and is specific for apoB-100, is bound to the microtiter plate as the capture antibody.

A method for localizing the early products of nonenzymatic glycosylation in fixed tissue.

The process of nonenzymatic glycosylation (NEG) may play a significant role in the development of chronic complications of diabetes. Early products of NEG can be measured by various biochemical methods. A method has been developed to localize these early products of glycosylation in vivo in fixed tissue sections of normal and diabetic skin using monoclonal antibodies specific for glucitollysine, which is formed when the early products of NEG are chemically reduced in vitro.

Localization of two epitopes of apolipoprotein A-I that are exposed on human high density lipoproteins using monoclonal antibodies and synthetic peptides.

To understand the structure of apolipoprotein A-I, we have used an immunochemical approach and identified specific regions of apoA-I that may be exposed on the apoprotein as it exists on high density lipoprotein (HDL). Twelve mouse monoclonal antibodies specific for human apoA-I were generated from six fusions. Thirteen synthetic peptides of between 5 and 16 amino acid residues in length, which span the amino-terminal two-thirds of apoA-I, were tested for their ability to react with each of the 12 antibodies.

Quantitation of plasma apolipoprotein A-I using two monoclonal antibodies in an enzyme-linked immunosorbent assay.

A rapid sandwich enzyme-linked immunosorbent assay (ELISA) for the quantitation of human apolipoprotein (apo) A-I was developed. The assay uses a pair of noncompeting purified monoclonal antibodies to detect apoA-I in plasma. The antibodies used in this assay were selected because they bind greater than 90% of radioiodinated high density lipoprotein (HDL), they identify "fresh" nondeamidated epitopes on apoA-I, and they have comparable binding affinities for isolated HDL and HDL in plasma.

Apoprotein E-rich high density lipoproteins inhibit ovarian androgen synthesis.

The influence of high density lipoproteins (HDL) on luteinizing hormone-stimulated rat ovarian theca/interstitial cell steroidogenesis was studied. Without HDL the cells produced primarily androgens from progestin precursors. In the presence of rat or human HDL steroid output increased 3-5-fold, but the type of steroid produced was dependent on the source of the HDL.

Platelet-enhanced apolipoprotein E production by human macrophages: a possible role in atherosclerosis.

Cholesterol-loaded human monocyte-derived macrophages increase their production of apolipoprotein E (apoE). Although cholesterol loading is often achieved with modified plasma lipoproteins, macrophages can be loaded also by coculture with platelets. Therefore, the relationship between platelet-mediated cholesteryl ester accumulation and apoE secretion was examined.

Lipoprotein B37, a naturally occurring lipoprotein containing the amino-terminal portion of apolipoprotein B100, does not bind to the apolipoprotein B,E (low density lipoprotein) receptor.

In 1979, Steinberg and colleagues described a unique kindred with familial hypobetalipoproteinemia (Steinberg, D., Grundy, S. M.

New mechanism for foam cell generation in atherosclerotic lesions.

Because of a close association between platelets and macrophages in early fatty streak lesions, the hypothesis was tested that platelets contribute to lesion progression by directly enhancing macrophage cholesteryl ester (CE) accumulation. Both the rate of cholesterol esterification and the accumulation of CE were increased within 24 h of the co-culture of adherent macrophages with platelets. Maximum increases in esterification and CE accumulation were observed within 3 to 4 d of culture and were greater than 10-fold over controls.

Characterization of an abnormal species of apolipoprotein B, apolipoprotein B-37, associated with familial hypobetalipoproteinemia.

Steinberg and colleagues have previously described a unique kindred with normotriglyceridemic hypobetalipoproteinemia (1979. J. Clin.

Genetic analysis of a kindred with familial hypobetalipoproteinemia. Evidence for two separate gene defects: one associated with an abnormal apolipoprotein B species, apolipoprotein B-37; and a second associated with low plasma concentrations of apolipoprotein B-100.

In 1979 Steinberg and colleagues recognized a unique kindred with normotriglyceridemic hypobetalipoproteinemia (1979. J. Clin.

Detection of two apolipoprotein B species (apoBc and apoBg) by a monoclonal antibody.

A monoclonal antibody (MB-19) was used to investigate the polymorphism of apolipoprotein B in a large East Finnish family and in unrelated subjects. Apolipoprotein B was shown to exhibit high, intermediate or low affinity binding to this antibody. Thus, MB-19 bound strongly to the Ag(c) epitope, an Ag antigenic domain previously characterized by human antisera, while it bound only weakly to the allelic epitope Ag(g).

Kinetics of C1 activation by a monoclonal anti-C1q antibody and its (Fab)2 fragments.

Monoclonal antibody 1H11, which binds to the "head" portion of C1q, has been shown to be a strong, stoichiometric activator of C1, the first component of human complement, maximal activation being achieved at a ratio of one antibody-combining site per one C1q head; moreover, this activation occurs even in the presence of C1-inhibitor, as reported previously. In the present paper, the kinetics of activation are shown to be biphasic; that is, a portion of the C1 is activated very rapidly, and the remainder slowly. These two processes can be separated by the order of mixing of preincubated components; thus, only the rapid activation rate is observed if C1q and the monoclonal antibody are preincubated together and are added subsequently to a mixture of C1r2C1S2 and C1-inhibitor.

Apolipoprotein B allotypes MB19(1) and MB19(2) in subjects with coronary artery disease and hypercholesterolemia.

We recently characterized a common form of genetic polymorphism in human apolipoprotein (apo) B, using the specific monoclonal antibody MB19 (Young SG, et al. Proc Natl Acad Sci USA 1986; 83:1101-1105). Antibody MB19 binds apo B with one of three distinct patterns of immunoreactivity (strong, intermediate, or weak).

Apolipoprotein B mediates the capacity of low density lipoprotein to suppress neutrophil stimulation by particulates.

Low density lipoprotein (LDL) inhibits phagocytosis of certain negatively charged particulates and also inhibits subsequent cellular secretory and oxidative responses to these particulates. In the present work, we have defined the structural features of LDL involved in this activity. Starch-heptane extraction depleted greater than 95% of neutral lipids but had little effect on the capacity of LDL to inhibit monosodium urate crystal- or polystyrene latex bead-induced neutrophil chemiluminescence (CL).

Two new monoclonal antibody-based enzyme-linked assays of apolipoprotein B.

We describe two new monoclonal antibody-based, solid-phase immunoenzymometric assays for the quantification of apolipoprotein (apo) B in plasma: a competitive assay and a direct assay. For both, we utilize 96-well microtiter plates and native low-density lipoprotein (LDL) for preparing the standard curve. A single monoclonal antibody, MB24, is used in the competitive assay.