Characterization of a monoclonal antibody that binds equally to all apolipoprotein and lipoprotein forms of human plasma apolipoprotein B. I. Specificity and binding studies

A stable mouse hybridoma cell line has been developed that produces monoclonal antibody to human plasma apolipoprotein B. This antibody was proven to be specific for apolipoprotein B immunoblotting and an enzyme immunoassay using apolipoprotein B and other apolipoproteins. The antibody bound with comparable affinities to soluble apolipoprotein B, chylomicrons, very-low-density (VLDL) and low-density lipoproteins (LDL). Coupled to agarose, this antibody allowed complete removal of apolipoprotein B-containing lipoproteins from normolipidemic, hypertriglyceridemic and hypercholesterolemic plasma. Desialyzation and deglycosylation had no effect on its binding to LDL. The described antibody had no effect on the receptor-mediated binding of radiolabeled LDL to the human hepatoma cells (HepG2) in culture. Analysis of 25 different samples of human plasma indicated identical expression of the corresponding epitope in these individuals. The described monoclonal antibody, most likely, binds to a rather stable domain of apolipoprotein B that is not altered by the interaction with lipids or polymorphism of the apolipoprotein B. We propose that this antibody be called 'Pan B' antibody.     

Immunoaffinity isolation of apolipoprotein E-containing lipoproteins

Discrete apolipoprotein E-containing lipoproteins can be identified when EDTA plasma is fractionated on columns of 4% agarose. The present study has demonstrated, by physical and metabolic criteria, that these apolipoprotein E-containing lipoprotein subclasses may be further isolated by immunoaffinity chromatography. Whole plasma was first bound to an anti-apolipoprotein E immunoadsorbent prior to gel filtration on 4% agarose. After elution from the affinity column and dialysis, the bound fraction was chromatographed on 4% agarose. Discrete subfractions of apolipoprotein E could be demonstrated within elution volumes similar to those observed in the original plasma. When whole plasma was first submitted to gel filtration and the apolipoprotein E-containing lipoproteins of either intermediate- or of high-density lipoprotein (HDL) size were subsequently bound to anti-apolipoprotein E columns, the bound eluted fractions maintained their size and physical properties as shown by electron microscopy and by rechromatography on columns of 4% agarose. The metabolic integrity of apolipoprotein E-containing very-low-density lipoproteins (VLDL) was examined by coinjection into a cynomolgus monkey of 125I-labeled apolipoprotein E-rich and 131I-labeled apolipoprotein E-deficient human VLDL which had been separated by immunoaffinity chromatography. The plasma specific activity time curves of the apolipoprotein B in VLDL, intermediate-density (IDL) and low-density (LDL) lipoproteins demonstrated rates of decay and precursor-product relationships similar to those obtained after injection of whole labeled VLDL, supporting the metabolic integrity of VLDL isolated by immunoaffinity chromatography.     

Properties of an apolipoprotein E-enriched fraction of triglyceride-rich lipoproteins isolated from human blood plasma with a monoclonal antibody to apolipoprotein B-100.

A monoclonal antibody to apolipoprotein (apo) B-100 (JI-H) with unique binding properties has been used to separate a population of triglyceride-rich lipoproteins from blood plasma of normotriglyceridemic individuals and patients with various forms of hypertriglyceridemia. This antibody fails to recognize an apoE-rich population of very low density lipoproteins (VLDL) containing apoB-100 as well as all triglyceride-rich lipoproteins containing apoB-48, but it binds other VLDL that contain apoE and almost all lipoproteins that contain apoB-100, but no apoE. The unbound triglyceride-rich lipoproteins separated by ultracentrifugation after separation from plasma by immunoaffinity chromatography contained 10-13% of the apoB of triglyceride-rich lipoproteins from three normotriglyceridemic individuals, 10-29% of that from five patients with endogenous hypertriglyceridemia, 40-48% of that from three patients with familial dysbetablipoproteinemia, and 65% of that from a patient with lipoprotein lipase deficiency. In all cases, the unbound triglyceride-rich lipoproteins contained more molecules of apoE and cholesteryl esters per particle than those that were bound to monoclonal antibody JI-H, and they were generally depleted of C apolipoproteins. These properties resemble those described for partially catabolized remnants of chylomicrons and VLDL. The affinity of the unbound lipoproteins for the low density lipoprotein (LDL) receptor varied widely, and closely resembled that of the total triglyceride-rich lipoproteins from individual subjects. Our results demonstrate that remnant-like chylomicrons and a population of remnant-like VLDL can be isolated and quantified in blood plasma obtained in the postabsorptive state from normotriglyceridemic and hypertriglyceridemic individuals alike.     

Protein heterogeneity of lipoprotein particles containing apolipoprotein A-I without apolipoprotein A-II and apolipoprotein A-I with apolipoprotein A-II isolated from human plasma

The protein heterogeneity of fractions isolated by immunoaffinity chromatography on anti-apolipoprotein A-I and anti-apolipoprotein A-II affinity columns was analyzed by high resolution two-dimensional gel electrophoresis. The two-dimensional gel electrophoresis profiles of the fractions were analyzed and automatically compared by the computer system MELANIE. Fractions containing apolipoproteins A-I + A-II and only A-I as the major protein components have been isolated from plasma and from high density lipoproteins prepared by ultracentrifugation. Similarities between the profiles of the fractions, as indicated by two-dimensional gel electrophoresis, suggested that those derived from plasma were equivalent to those from high density lipoproteins (HDL), which are particulate in nature. The established apolipoproteins (A-I, A-II, A-IV, C, D, and E) were visible and enriched in fractions from both plasma and HDL. However, plasma-derived fractions showed a much greater degree of protein heterogeneity due largely to enrichment in bands corresponding to six additional proteins. They were present in trace amounts in fractions isolated from HDL and certain of the proteins were visible in two-dimensional gel electrophoresis profiles of the plasma. These proteins are considered to be specifically associated with the immunoaffinity-isolated particles. They have been characterized in terms of Mr and pI. Computer-assisted measurements of protein spot-staining intensities suggest an asymmetric distribution of the proteins (as well as the established apolipoproteins), with four showing greater prominence in particles containing apolipoprotein A-I but no apolipoprotein A-II.     

A rapid micromethod for apolipoprotein E phenotyping directly in serum

A new method for the apolipoprotein E phenotyping has been developed. The method is based on isoelectric focusing of either delipidated or guanidine-HC1-treated serum or plasma in a horizontal slab gel system followed by immunoblotting using either polyclonal or monoclonal anti-apolipoprotein E antibodies as first antibody. Apolipoprotein E phenotyping with this method in 200 serum samples that had been stored at -20 degrees C for more than one year gave exactly the same results as obtained with the conventional method based on isoelectric focusing of delipidated very low density lipoproteins isolated from fresh serum followed by protein staining. Compared with the conventional method, the present method is less laborious because ultracentrifugation to isolate VLDL is not needed; it is suitable for large scale screening purposes; it needs only a few microliters of serum or plasma, and can easily be performed with samples with low concentrations of apolipoprotein E.     

Lipolytic degradation of human very low density lipoproteins by human milk lipoprotein lipase: the identification of lipoprotein B as the main lipoprotein degradation product

Although the direct conversion of very low density lipoproteins (VLDL) into low density (LDL) and high density (HDL) lipoproteins only requires lipoprotein lipase (LPL) as a catalyst and albumin as the fatty acid acceptor, the in vitro-formed LDL and HDL differ chemically from their native counterparts. To investigate the reason(s) for these differences, VLDL were treated with human milk LPL in the presence of albumin, and the LPL-generated LDL1-, LDL2-, and HDL-like particles were characterized by lipid and apolipoprotein composition. Results showed that the removal of apolipoproteins B, C, and E from VLDL was proportional to the degree of triglyceride hydrolysis with LDL2 particles as the major and LDL1 and HDL + VHDL particles as the minor products of a complete in vitro lipolysis of VLDL. In comparison with native counterparts, the in vitro-formed LDL2 and HDL + VHDL were characterized by lower levels of triglyceride and cholesterol ester and higher levels of free cholesterol and lipid phosphorus. The characterization of lipoprotein particles present in the in vitro-produced LDL2 showed that, as in plasma LDL2, lipoprotein B (LP-B) was the major apolipoprotein B-containing lipoprotein accounting for over 90% of the total apolipoprotein B. Other, minor species of apolipoprotein B-containing lipoproteins included LP-B:C-I:E and LP-B:C-I:C-II:C-III. The lipid composition of in vitro-formed LP-B closely resembled that of plasma LP-B. The major parts of apolipoproteins C and E present in VLDL were released to HDL + VHDL as simple, cholesterol/phospholipid-rich lipoproteins including LP-C-I, LP-C-II, LP-C-III, and LP-E. However, some of these same simple lipoprotein particles were present after ultracentrifugation in the LDL2 density segment because of their hydrated density and/or because they formed, in the absence of naturally occurring acceptors (LP-A-I:A-II), weak associations with LP-B. Thus, the presence of varying amounts of these cholesterol/phospholipid-rich lipoproteins in the in vitro-formed LDL2 appears to be the main reason for their compositional difference from native LDL2. These results demonstrate that the formation of LP-B as the major apolipoprotein B-containing product of VLDL lipolysis only requires LPL as a catalyst and albumin as the fatty acid acceptor. However, under physiological circumstances, other modulating agents are necessary to prevent the accumulation and interaction of phospholipid/cholesterol-rich apolipoprotein C- and E-containing particles.     

Clusterin (complement lysis inhibitor) forms a high density lipoprotein complex with apolipoprotein A-I in human plasma

Clusterin/human complement lysis inhibitor (CLI) is incorporated stoichiometrically into the soluble terminal complement complex and inhibits the cytolytic reaction of purified complement components C5b-9 in vitro. Using an anti-clusterin affinity column, we found that an additional protein component with a molecular mass of 28-kDa co-purifies with clusterin from human plasma. We show by immunoblotting and amino acid sequencing that this component is apolipoprotein A-I (apoA-I). By using physiological salt buffers containing 0.5% Triton X-100, apoA-I is completely dissociated from clusterin bound to the antibody column. Free clusterin immobilized on the antibody-Sepharose selectively retains apoA-I from total human plasma. Delipidated apoA-I and to a lesser extent ultracentrifugation-purified high density lipoproteins (HDL) adsorbed to nitrocellulose also have a binding affinity for purified clusterin devoid of apoA-I. The isolated apoA-I-clusterin complex contains approximately 22% (w/w) lipids which are composed of 54% (mole/mol) total cholesterol (molar ratio of unesterified/esterified cholesterol, 0.58), 42% phospholipids, and 4% triglycerides. In agreement with the low lipid content, apoA-I-clusterin complexes are detected only in trace amounts in HDL fractions prepared by density ultracentrifugation. In free flow isotachophoresis, the purified apoA-I-clusterin complex has the same mobility as the native clusterin complex in human plasma and is found in the slow-migrating HDL fraction of fasting plasma. Our data indicate that clusterin circulates in plasma as a HDL complex, which may serve not only as an inhibitor of the lytic terminal complement cascade, but also as a regulator of lipid transport and local lipid redistribution.     

Characterization of monoclonal anti-rabbit apolipoprotein E antibodies and chemical composition of lipoproteins separated by anti-apolipoprotein E immuno-affinity chromatography

Six mouse monoclonal antibodies against rabbit apolipoprotein E (apo E) have been developed. Of these monoclonal antibodies, clone 5 revealed a high affinity for purified apo E, very low density lipoprotein (VLDL) and beta-VLDL. This monoclonal antibody was used to prepare an immunoaffinity column. Coupled to Sepharose 4B, this antibody allowed complete removal of lipoproteins containing apo E from plasma of New Zealand white (NZW) rabbits; 62, 46, 14, and 3% of VLDL-, IDL-, LDL-, and HDL-protein, respectively, were bound to the anti-apo E affinity column. The bound VLDL was significantly rich in free cholesterol (FC) and cholesteryl esters (CE) relative to the unbound VLDL, whereas bound IDL, LDL and HDL were significantly rich in FC only. All of the bound fractions were characterized by significantly increased ratios of FC/phospholipids (PL). These results indicate that the two lipoprotein populations with and without apo E have different lipid compositions. The relatively high content of cholesterol in lipoproteins containing apo E suggests a contribution of apo E to plasma cholesterol transport.     

Isolation of high-density lipoproteins by immunoaffinity column chromatography from hog plasma

High density lipoprotein (HDL) was isolated from hog plasma by a simple immunoaffinity column chromatography procedure using immobilized anti-apolipoprotein AI. The composition of HDL isolated by immunoaffinity chromatography was nearly identical to that of a control sample that was isolated by an alternate method utilizing ultracentrifugation and gel chromatography. The HDL isolated by immunoaffinity chromatography had a larger number of polypeptide components that the control as indicated by acrylamide gel electrophoresis in the presence of urea. When the HDL isolated by immunoaffinity chromatography was applied to a heparin-agarose column the amount of protein retained was approximately twice that of the control. These findings indicate that the ultracentrifugation procedure probably induced the loss of apolipoprotein E containing components from the HDL complex.     

Studies on the isolation and partial characterization of apolipoprotein D and lipoprotein D of human plasma.

This report describes further studies on the characterization of apolipoprotein D (ApoD), a recently recognized human plasma apolipoprotein, and presents results on the isolation and distribution of its lipoprotein form, lipoprotein D (LP-D). ApoD, isolated by a procedure combining hydroxylapatite and Sephadex G-100 column chromatography, migrated on 7% polyacrylamide gel as a single band with a mobility intermediate between those of A-II and C-II polypeptides. On double diffusion and immunoelectrophoresis, ApoD reacted only with antiserum to ApoD. It was characterized by the presence of all common amino acids including half-cystine. The amino terminal acid was blocked. Carbohydrate analysis demonstrated that ApoD is a glycoprotein with glucose, mannose, galactose, glucosamine, and sialic acid accounting for 18% of the dry weight of ApoD. The estimated molecular weight of ApoD IS 22 100. ApoD occurs in the serum as a lipoprotein which was isolated from high density lipoproteins3 by two different chromatographic procedures. In the first procedure, high density lipoproteins3 were treated with neuraminidase and chromatographed on concanavlin A. The retained fraction containing LP-D was purified by hydroxylapatite column chromatography. Alternatively, LP-D was isolated by a procedure combining chromatography of high density lipoproteins3 or whole serum on an immunosorber containing antibodies to ApoD, and hydroxylapatite column chromatography. LP-D displayed a single, symmetrical boundary in the analytical ultracentrifuge and a single band on 7% polyacrylamide gel electrophoresis. When injected into rabbits it produced antisera that reacted only with ApoD. On immunoelectrophoresis LP-D had a mobility different from that of lipoprotein A (LP-A). A direct immunological comparison of LP-D and LP-A showed a reaction of nonidentity. LP-D consists of 65-75% protein and 25-35% lipid. The lipid moiety contains cholesterol, cholesterol ester, triglyceride, and phospholipid. The phospholipid. composition is characterized by a relative high content of lysolecithin and sphingomyelin and a relatively low content of lecithin. We have concluded from these studies that ApoD is a unique apolipoprotein that exists in the form of a distinct lipoprotein family with a macromolecular distribution extending from very low density lipoproteins into very high density lipoproteins, but with a maximum concentration in high density lipoproteins3 and a minimum concentration in high density lipoproteins.     

A purification method for apolipoprotein A-I and A-II

Apolipoproteins A-I and A-II were isolated from precipitates obtained by cold ethanol fractionation of human plasma. The starting material used in this report was precipitate B of the Kistler and Nitschmann method which corresponds approximately to fraction III of the Cohn and Oncley procedure. Through the use of urea, chloroform, and ethanol in appropriate concentrations, apolipoproteins A-I and A-II were isolated by a simple extraction technique avoiding time-consuming ultracentrifugation. Starting from 10 g of centrifuged precipitate B, approximately 100 mg of apolipoprotein A-I and 10 mg of apolipoprotein A-II were obtained. When incubated with normal human or rabbit plasma, both apolipoproteins were readily incorporated into high-density lipoproteins. Apolipoprotein A-I obtained by the cold ethanol method activated lecithin-cholesterol acyltransferase to the same extent as apolipoprotein A-I prepared by the classical flotation method. Apolipoprotein A-II had no such properties by itself, but was capable of potentiating lecithin-cholesterol acyltransferase activity of apolipoprotein A-I.     

Selective isolation of human plasma low-density lipoprotein particles containing apolipoproteins B and E by use of a monoclonal antibody to apolipoprotein B

A monoclonal antibody to human plasma apolipoprotein B was used in a single-step immunoaffinity chromatography procedure to isolate a subpopulation of low-density lipoprotein particles from normolipidemic human plasma. The isolated particles were homogeneous in terms of size (20 nm), flotation coefficient (Sf = 9.5), and electrophoretic mobility (beta band). Their protein moiety consisted of apolipoproteins B and E in a molar ratio close to 2. The lipid moiety consisted of 47.3% cholesterol, 4.7% triglycerides, and 48.0% phospholipids. To indicate its characteristic apolipoprotein composition and hydrated density properties, this family of particles was named LP-B:EL2. In most normolipidemic subjects, LP-B:EL2 particles accounted for less than 10% of the total plasma apolipoprotein B content. The LP-B:EL2 particles bound to the membranes of the human hepatoma HepG2 cells in a specific and saturable manner indicative of receptor-mediated binding. Their binding was significantly higher than that of low-density lipoprotein particles containing only apolipoprotein B.     

Metabolism of apolipoprotein E-containing human plasma lipoproteins by rat and human cells in culture.

Cultured preadipocytes from rat epididymal fat pads were able to bind, internalize, and degrade human plasma very-low-density lipoproteins (VLDL) more efficiently than low-density lipoproteins (LDL). VLDL, but not LDL, activated acyl-CoA: cholesterol acyltransferase (ACAT) and increased cholesterol accumulation in these cells. However, trypsin-treated VLDL (T-VLDL) lost the capacity to bind, activate ACAT, and increase cholesterol accumulation. After the treatment of VLDL with trypsin, SDS/polyacrylamide-gel electrophoresis and immunoblotting showed that apolipoprotein E (apo E) was completely degraded, whereas apolipoprotein CII (apo C-II) was preserved. ApoE complexed with dimyristoyl phosphatidylcholine (DMPC) was able to complete with VLDL for binding to the cells. Although T-VLDL did not bind to the preadipocytes, these cells accumulate triacylglycerols from T-VLDL, presumably after lipolysis, as efficiently as from native VLDL. Rat smooth muscle cells and skin fibroblasts also bind and metabolize human VLDL better than LDL. However, human skin fibroblasts and omental preadipocytes metabolized LDL better than VLDL. These studies indicate that rat tissues can recognize and metabolize apoE-containing human plasma VLDL although they cannot recognize human LDL.     

Characterization of disulfide-linked heterodimers containing apolipoprotein D in human plasma lipoproteins.

Human plasma apolipoprotein (apo) D is a glycoprotein with an apparent molecular weight of 29,000 M(r). It is present, mainly, in high density lipoproteins (HDL) and very high density lipoproteins (VHDL). Western blot analysis of HDL and VHDL using rabbit antibodies to human apoD revealed major immunoreactive bands at 29,000 and 38,000 M(r), with minor bands ranging from 50,000 to and 80,000 M(r). Only the 29,000 M(r) band corresponding to apoD remained when the electrophoresis was conducted under reducing conditions, demonstrating that apoD is cross-linked to other proteins via disulfide bonds. The broad pattern of immunoreactivity was also observed under nonreducing conditions when the blood was collected into a solution of sulfhydryl-trapping reagents, or when these reagents were added to the isolated lipoproteins. These results indicated that the disulfide bonds were not the result of disulfide exchange during the experimental procedures. On the basis of amino acid sequencing and reactions to antibodies, the 38,000 M(r) band was identified as an apoD-apoA-II heterodimer. The apoD-apoA-II was also demonstrated in plasma. In both HDL and plasma, the apoD-apoA-II heterodimer constituted the major form of apoD. Disulfide-linked heterodimers of apoD and apoB-100 were also found in low and very low density lipoproteins, and in whole plasma. It is concluded that a fraction of human apoD, like other cysteine-containing apolipoproteins, exists as a disulfide-linked heterodimer with other apolipoproteins in all major human lipoprotein fractions.     

Human apolipoprotein C-I accounts for the ability of plasma high density lipoproteins to inhibit the cholesteryl ester transfer protein activity

The aim of the present study was to identify the protein that accounts for the cholesteryl ester transfer protein (CETP)-inhibitory activity that is specifically associated with human plasma high density lipoproteins (HDL). To this end, human HDL apolipoproteins were fractionated by preparative polyacrylamide gradient gel electrophoresis, and 30 distinct protein fractions with molecular masses ranging from 80 down to 2 kDa were tested for their ability to inhibit CETP activity. One single apolipoprotein fraction was able to completely inhibit CETP activity. The N-terminal sequence of the 6-kDa protein inhibitor matched the N-terminal sequence of human apoC-I, the inhibition was completely blocked by specific anti-apolipoprotein C-I antibodies, and mass spectrometry analysis confirmed the identity of the isolated inhibitor with full-length human apoC-I. Pure apoC-I was able to abolish CETP activity in a concentration-dependent manner and with a high efficiency (IC(50) = 100 nmol/liter). The inhibitory potency of total delipidated HDL apolipoproteins completely disappeared after a treatment with anti-apolipoprotein C-I antibodies, and the apoC-I deprivation of native plasma HDL by immunoaffinity chromatography produced a mean 43% rise in cholesteryl ester transfer rates. The main localization of apoC-I in HDL and not in low density lipoprotein in normolipidemic plasma provides further support for the specific property of HDL in inhibiting CETP activity.     

Characteristic binding of human plasma apolipoprotein B to gangliotetraosylceramide and gangliotriaosylceramide.

The binding of human plasma low-density lipoproteins (LDL), freshly prepared by discontinuous ultracentrifugation, to several neutral and acidic glycosphingolipids was examined by TLC immunostaining with the anti [apolipoprotein B (apoB)] antibody. ApoB was found to bind characteristically to the asialogangliosides, gangliotetraosylceramide (Gg4Cer) and gangliotriaosylceramide (Gg3Cer), the former being a more potent receptor than the latter, indicating that the sequences Gal beta 1-3GalNAc beta 1-4Gal and GalNAc beta 1-4Gal are involved in the binding of apoB. A weak positive reaction with fucosylgangliotetraosylceramide (IV2Fuc-Gg4Cer), which has the same internal recognition sequences, was also observed (the binding ability was only 1/7 of that in the case of Gg4Cer). No binding to other neutral glycosphingolipids, or glycosphingolipid sulfates (I3-SO3-GalCer) and gangliosides, was detected, and therefore substitution of the receptor glycolipid with sialic acid was thought to inhibit the binding. The results indicate that, along with the binding of apoB to the LDL-binding domain of the receptor glycoprotein, interaction with some carbohydrate chains in the receptor, or with glycolipids coexisting on the plasma membrane, may be important for the binding of apoB to cells.     

Binding of human apolipoprotein A-IV to human hepatocellular plasma membranes

We have investigated the binding of human apolipoprotein A-IV (apo A-IV) to human hepatocellular plasma membranes. Addition of increasing concentrations of radiolabeled apo A-IV to hepatic plasma membranes, in the presence and absence of a 25-fold excess of unlabeled apo A-IV, revealed saturation binding to the membranes with a KD of 154 nM and a binding maximum of 1.6 ng/microgram of membrane protein. The binding was temperature-insensitive, partially calcium-dependent, abolished when apo A-IV was denatured by guanidine hydrochloride or when the membranes were treated with Pronase and decreased when apo A-IV was incorporated into phospholipid/cholesterol proteoliposomes. In displacement studies using purified apolipoproteins and isolated lipoproteins, only unlabeled apo A-IV, apo A-I and high-density lipoproteins effectively competed with radiolabeled apo A-IV for membrane binding sites. We conclude that human apo A-IV exhibits high-affinity binding to isolated human hepatocellular plasma membranes which is saturable, reversible and specific.     

Bacterial expression and in vitro refolding of a single-chain fv antibody specific for human plasma apolipoprotein B-100

From the cloned heavy and light chains of a murine monoclonal antibody (mAbB23) which is specific for human apolipoprotein (apo) B-100 of plasma low-density lipoproteins, a vector was designed for expression of a single-chain antibody (scFv) of mAbB23 in Escherichia coli. The expression vector was constructed so that the scFv gene (V(L)-linker-V(H)) was expressed under the control of the T7 promoter. The inclusion body of scFv was isolated from E. coli lysate and solubilized in 6 M guanidine-hydrochloride without reducing agents, followed by refolding through slow dilution into refolding buffer. After complete removal of the remaining denaturant by dialysis, the soluble scFv was purified through an apo B-100-coupled affinity column, and an active fraction, which had an antigen-binding activity comparable with that of native Fab, was easily obtained. The expression and in vitro refolding of scFv resulted in production of an active molecule in a yield of 15-20 mg per 1-liter flask cultivation.     

Enzyme-linked immunosorbent assay for human plasma apolipoprotein B

A noncompetitive enzyme-linked immunosorbent assay (ELISA) has been developed for measuring total plasma apolipoprotein (apo) B using affinity purified polyclonal and monoclonal antibodies. Microtiter plates from different manufacturers were tested with regard to their IgG binding characteristics; only one plate yielded consistent coefficients of variation of less than 5%. The optimal plasma dilution in this assay was 1:3000. IgG anti-apoB antisera conjugated to alkaline phosphatase was used as a second antibody. p-Nitrophenyl phosphate was utilized as substrate for color development, and the absorbance (410 nm) was read utilizing an ELISA reader interfaced with a microcomputer for data processing. Plasma apoB levels in plasma have been determined in 1115 male and female participants in the Framingham Offspring Study. Mean (+/- SD) plasma concentrations were 89 +/- 28 mg/dl. Significant age and sex related differences in apoB levels were noted.     

Identification and partial characterization of discrete apolipoprotein A-containing lipoprotein particles secreted by human hepatoma cell line HepG2

The purpose of this study was to identify the apolipoprotein A-containing lipoprotein particles produced by HepG2 cells. The apolipoprotein A-containing lipoproteins separated from apolipoprotein B-containing lipoproteins by affinity chromatography of culture medium on concanavalin A were fractionated on an immunosorber with monoclonal antibodies to apolipoprotein A-II. The retained fraction contained apolipoproteins A-I, A-II and E, while the unretained fraction contained apolipoproteins A-I and E. Both fractions were characterized by free cholesterol as the major and triglycerides and cholesterol esters as the minor neutral lipids. Further chromatography of both fractions on an immunosorber with monoclonal antibodies to apolipoprotein A-I showed that 1) apolipoprotein A-II only occurs in association with apolipoprotein A-I, 2) apolipoprotein A-IV is only present as part of a separate lipoprotein family (lipoprotein A-IV), and 3) apolipoprotein E-enriched lipoprotein A-I:A-II and lipoprotein A-I are the main apolipoprotein A-containing lipoproteins secreted by HepG2 cells.