Antisera and Monoclonal Antibodies Specific for Epitopes Generated during Oxidative Modification of Low Density Lipoprotein Wulf Palinski, Seppo Yla-Herttuala, Michael E. Rosenfeld, Susan W. Butler, Steve A. Socher, Sampath Parthasarathy, Linda K. Curtiss, and Joseph L Witztum

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 aldehydlc fragments of oxidized fatty acids capable of conjugating with lyslne residues of apoprotein B. By Immunizing animals with homologous malondlaldehyde-modlfled LDL (MDA-LDL), 4-hydroxynonenal-LDL (4-HNE-LDL), and Cu++-oxldlzed LDL, we developed polyvalent and monoclonal antibodies against three epitopes found In oxldatively modified LDL The present article characterizes an antiserum and monoclonal antibody (MAL-2 and MDA2, respectively) specific for MDA-lyslne, and an antiserum and monoclonal antibody (HNE-6 and NA59, respectively) specific for 4-HNE-lyslne. In addition, a monoclonal antibody (OLF4-3C10) was developed against an as yet undefined epltope generated during Cu++oxldatJon of LDL With these antibodies, we demonstrated that MDA-lyslne and 4-HNE-lyslne adducts develop on apo-llpoproteln B during copper-Induced oxidation of LDL in vitro. The application of these antibodies for immunocytochemlcal demonstration of oxidized lipoprotelns In atherosclerotic lesions of progressive severity Is described in the companion article. These antibodies should prove useful In studying the role of oxldatively modified lipoprotelns as well as other oxldatively modified proteins In atherogenesls. (Arteriosclerosis 10:325-335, May/June 1990)

H

ypercholesterolemia has long been known to be a primary risk factor for atherosclerosis. However, monocyte-derived macrophages, precursors of the vast majority of the foam cells in early atherosclerotic lesions, cannot take up native low density lipoprotein (LDL) rapidly enough in vitro to cause lipid loading.1 Oxidative modification converts LDL to a form recognized by the macrophage "scavenger" receptor 23 and possibly by other receptors as well, 45 and this oxidized LDL induces foam cell formation in vitro. An increasing number of both in vitro and in vivo observations recently reviewed8 suggests that oxidative modification of LDL actually occurs in vivo and plays an important role in atherogenesis.

Three major cell types of the arterial wall, that is, endothelial cells, smooth muscle cells, and monocytemacrophages, have been shown in vitro to be capable of oxidizing LDL. 27 - 11 Incubation of LDL in cell-free media rich in metal ions also has been shown to generate oxidized LDL that closely resembles cell-modified LDI_2.8,12 The oxidative modification of LDL is accompanied by extensive degradation of its polyunsaturated fatty acids, leading to the generation of a number of highly reactive shorter chain-length fragments, 1314 some of which could become covaJentiy attached to apoprotein (apo) B. Malondialdehyde (MDA) is a highly reactive dialdehyde generated during arachidonic acid catabolism in thrombocytes. 161617 It is also known to result from lipid peroxidation that occurs during phagocytosis by monocytes 18 and is produced during nonenzymatJc lipid peroxidation of a variety of unsaturated fatty acids. 4-Hydroxynonenal (4-HNE) is another highly reactive aldehyde that forms as a result of nonenzymatic lipid peroxidation of long-chain polyunsaturated fatty acids. 4-HNE has a relatively long half-life,19 has both a hydrophilic and a lipophilic site, and is highly reactive with proteins, including apoproteins.20 4-HNE also occurs physiologically, for example, it constitutes a major aJdehydic lipid-degradation product produced by peroxidizing liver microsomes.19 Although little is known about the generation of 4-HNE in the artery waJI, at least in concentrations sufficient to modify apoproteins, 4-HNE is known

From the Division of Endocrinology and Metabolism, Department of Medicine, University of Calitomla-San Diego, La Jolla, and the Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California. This work was supported by NHLBI Grant HL-14197 (Specialized Center of Research on Arteriosclerosis). Wulf Palinski was a Research Fellow of the Deutsche Forechungsgemelnschaft, Seppo Yla-Herttuala was supported by a Fogarty International Fellowship Award, U.S. Public Health Service Grant F5TWO4095A and a grant from the Emil Aaltonen Foundation (Finland). Steve A. Socher was supported by a Sarnoff Fellowship, and Joseph L Witztum was an Established Investigator of the American Heart Association. Address for reprints: Wulf Palinski, Division of Endocrinology and Metabolism, Department of Medicine M-013D, University of California-San Diego, La Jolla, CA 92093. Received October 3,1989; revision accepted January 11,1990.

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to be generated during the Cu ++ -induced modification of LDL in vitro.21 Apo B has approximately 360 lysine residues per molecule,22-23 and because this protein is intimately associated with lipid in the LDL particle, it is likely that the highly reactive aldehydes formed during lipid peroxidation, such as 4-HNE and MDA, will conjugate to the lysine residues. During the oxidative modification of LDL, conjugation of lipid peroxidation products to apo B occurs primarily by covalent linkage to epsilon amino groups of lysine residues. 121724 If oxidized LDL contains lysine residues conjugated to a number of such fatty acid fragments, the occurrence of oxidatively modified LDL in vivo could be demonstrated by its recognition by monospecific antibodies directed against the various lysine adducts. We have previously shown that the immunization of animals with autologous LDL modified by conjugation of lysine groups with short-chain aldehydes, such as glucose or even formaldehyde, generates monospecific antisera against the lysine adduct, such as glucitollysine or methyllysine.2526 In a similar fashion, we have now used homologous LDL conjugated with compounds generated during lipid peroxidation to produce antibodies that recognize oxidatively modified LDL, but not native LDL The present article characterizes polyvalent antisera and corresponding monoclonal antibodies directed against MDAlysine and 4-HNE-lysine, respectively. We also describe monoclonal antibodies that were generated by immunization of mice with Cu++-oxidized murine LDL Using radioimmunoassay (RIA) and Western blotting techniques, we demonstrate that MDA-lysine and 4-HNE-lysine adducts are formed with apo B during in vitro copper-mediated oxidation of LDL Using a monoclonal antibody against MDA-lysine residues, Haberland et al. 27 recently demonstrated immunocytochemically the presence of MDA-lysine residues in atherosclerotic lesions that co-localized with apo B. By immunocytochemical application of three different antibodies, we confirmed the presence of MDA-lysines in atherosclerotic lesions and demonstrated that several different oxidation-specific epitopes are found in lesioned but not in normal areas of rabbit aortas.28 Furthermore, we have specifically shown that LDL gently extracted from atherosclerotic lesions contains such epitopes 2829 and that the extracted LDL has both the physical and biological properties of in vitro oxidized LDL Our data strongly support the hypothesis that oxidatively modified LDL is indeed generated in vivo.28 In the companion article,41 all of the antibodies described in this report are used for an immunocytochemical study of the presence of oxidatively modified proteins in rabbit atherosclerotic lesions ranging from early fatty streaks to advanced fibrous plaques.

Methods Materials Carrier-free Na 12Siodide was purchased from Amersham (Arlington, IL). lodogen was from Pierce Chemical (Rockford, IL). Affinity-purified goat-antiguinea pig IgG and goat-antimouse IgG were from Cooper Biomedical

(Malvem, PA). Ham's F-10 medium was from GIBCO Laboratories (Grand Island, NY); butylated hydroxytoluene (BHT) was from J.T. Baker Chemical (Philippsburg, NJ); aprotinin, phenylmethylsulphonyl fluoride (PMSF), high molecular weight markers, and Freund's complete and incomplete adjuvants were from Sigma Chemical (St. Louis, MO). D-phenylalanyl-L-prolyl-L-arginine chloromettiyl ketone (PPACK) and benzamidine were from Calbiochem Behring (La Jolla, CA). Ninety-six well polyvinylchloride microttter plates were supplied by Dynatech Laboratories (Alexandria, VA). Agarose gels were purchased from Sigma Corning Diagnostics (Palo Alto, CA). Guinea pigs and mice were from Simonsen (Gilroy, CA) or Charles River Breeding Laboratories (Wilmington, MA). 4-HNE was kindly provided by Dr. Herman Esterbauer, Graz, Austria, and probucol was a gift from Merrell Dow Research Institute (Cincinnati, OH).

Procedures Lipoprotein Isolation and Modification LDL (1.020 to 1.057 g/ml) was prepared from pooled plasma of healthy human donors, male guinea pigs, and mice by sequential ultracentrifugation,30 by using 2.7 mM of ethylenediaminetetraacetate (EDTA), 2 mM of benzamidine, 1 JAM of PPACK, 0.01% of aprotinin, 50 /ig/ml of chloramphenicol, and 100 ^g/ml of gentamycin to provide antiproteolytic protection. PMSF (1 mM) was added to the plasma after separation of the blood cells. The isolated lipoprotein was extensively dialyzed against phosphate-buffered saline (PBS) (0.14 M NaCI/0.01 M phosphate buffer) containing 0.1 mM EDTA, 1 mM PMSF, and antibiotics. The protein content was then determined by the Lowry method,31 and the LDL was sterile-filtered for further use. The protein content of oxidized LDL obtained with the Lowry technique yields values 10% below the expected,28 but the values were not corrected in these studies.

Modification of Low Density Lipoprotein MDA-LDL was prepared by incubating LDL for 3 hours at 37°C with 0.5 M MDA at a constant ratio of 100 jd/mg of LDL MDA (0.5 M) was freshly generated from matonakJehyde bis dimethylacetal by acid hydrolysis: 88 /J matonaldehyde bis dimethylacetal was incubated with 12 /J 4 N HG and 400 jA H2O at 37°C for 10 minutes. The reaction was then stopped by adjusting the pH to 7.4 by the addition of 1 N NaOH, and the volume was brought to 1 ml with distilled Hj,O. After conjugation, MDA-LDL was extensively dialyzed against PBS to remove any unreacted MDA The degree of MDA modification was determined by trinitrobenzenesulfonic acid assay (TNBS)32 and averaged 77% of the lysine residues for a typical preparation. In addition, the electrcphoretic mobility of the modified lipoproteins was compared to that of native LDL by electrophoresis using 1 % agarose gels (Coming) in borate buffer (pH 8.6). Other human proteins, albumin, transferrin, and hemoglobin were conjugated with MDA by the same procedure. Polylysines, f-butoxycarbonyl (f-BOC)-lysine, and epsilon -aminocaproic acid were similarly modified by incubation with MDA but unconjugated MDA was not separated by dialysis.

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ANTIBODIES AGAINST OXIDATION SPECIFIC EPITOPES 4-HNE was conjugated to LDL under reducing conditions by a modification of the procedure described by Esterbauer et al. 14 and Jurgens et al. 20 To eliminate the solvent, an aliquot of 4-HNE in CHaCI2 was dried under nitrogen, was resolubilized in an equal volume of PBS (pH 9.0), and was exposed to vacuum for 5 minutes. LDL (2 mg) was added to and gently mixed with EDTA dissolved in PBS (pH 9.0), so that a 1 ml aliquot containing 2 mg/ml of LDL and 10 mg/ml of EDTA was obtained. Ten microliters of 2 M NaCNBH3 and 5 f*mo\ of 4-HNE were added, and the mixture was incubated at 37°C for 24 hours, followed by extensive dialysis against PBS containing 10 mg/ml EDTA to remove unconjugated 4-HNE. Conjugations of 4-HNE with other proteins were obtained by the same procedure; conjugation of 4-HNE with LDL under nonreducing conditions was performed by the same procedure, except that NaCNBH3 was omitted. Cu++-oxidized LDL was prepared by incubating 100 fig of LDL per ml with 10jiMof Cu + + in a total volume of 2 ml in Ham's F-10 medium for 18 hours at 37°C. The LDL was then dialyzed against PBS containing EDTA. More concentrated preparations of Cu++-oxidized LDL were prepared for competitive RIAs by ultracentrifugation (1.21 g/ ml). Cu++-oxidized apoprotein fragments were prepared by delipidating Cu++-oxidized LDL with methanol and chloroform and resolubilizing the resulting protein fragments in 55 mM octylglucoside as described.3

Immunization Protocol Polyvalent antisera were generated by immunizing male guinea pigs (400 to 450 g) with homologous MDALDL or homologous 4-HNE-LDL as previously described.28 The primary immunization consisted of an intradermal injection of 150 /ig of antigen (protein) in 0.5 ml PBS, suspended in 0.5 ml of Freund's complete adjuvant. Booster immunizations consisted of 100 /ig of antigen in Freund's incomplete adjuvant injected intramuscularly and subcutaneously at 14-day intervals. Antibody titers were determined in the pre-immune sera and at 5 to 7 days after the second and subsequent boosts. Monoclonal antibodies against MDA-LDL and 4-HNE-LDL were obtained by immunizing Balb/C mice with MDA- or 4-HNE-conjugated murine LDL. Fusions were performed with the P3x63Ag8.653.1 myeloma cell line by techniques previously described. 33 Primary screening of hybridoma supernatants was performed after 14 days of growth. Hybridoma supernatants were selected on the basis of their ability to bind to MDA- or 4-HNE-modified human LDL in solid-phase RIAs. Selected hybridomas were cloned by limiting dilution. Hybridoma cells were injected intraperitoneally into pristene-primed Balb/C mice to produce ascites fluid.33 Antibody titers and specificity of hybridoma supernatants and ascites were determined by solid-phase binding and competition RIA (described below). Four monoclonal antibodies against 4-HNE-LDL and two monoclonal antibodies against MDA-LDL were selected and characterized. After preliminary immunocytochemical screening studies, one monoclonal antibody against 4-HNE-LDL (NA59) and one monoclonal antibody against MDA-LDL

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(MDA2) were selected for detailed characterization and immunocytochemical studies. An Antigenex (San Francisco, CA) isotyping kit was used to type MDA2 as IgG, and NA59 as IgG^. Given the unknown nature of the epitopes and the fact that the epitopes could be distributed among multiple apo B fragments that result from Cu ++ -induced oxidation of LDL, the generation of monoclonal antibodies against oxidized apo B was particularly difficult. Several immunization series of Balb/C mice were carried out by using delipidated and octylglucoside-resolubilized apo B fragments obtained from 24-hour Cu ++ oxidized murine LDL or 4-hour Cu++-oxidized murine LDL. At days 3 and 2 before fusion, mice were boosted with apoprotein fragments obtained from 24-hour Cu++-oxidized human LDL or from a mixture of 4-hour and 24-hour Cu ++ -oxidized human LDL Primary screening of the hybridoma supernatants was performed with immunogen, that is, Cu + + oxidized murine apo B fragments, nondelipidated Cu + + oxidized human LDL, as well as native human LDL. Several hybridomas were selected and cloned, and ascites were produced. In this article, we report in detail on only one, OLF4-3C10, which has been extensively used in our immunocytochemical studies. This hybridoma was obtained from the fusion of cells from a mouse that had been immunized four times at 2-week intervals with 50 fig of 4-hour Cu++-oxidized mouse LDL and given two profusion boosts at 4 and 3 days before the fusion with 50 ^g of a mixture of 4-hour and 24-hour Cu + + oxidized human LDL fragments.33

Determination of Antibody Tlter and Specificity Solid-phase RIA techniques were used to determine the titers and specificity of antibodies. 2526 For the binding assay, 96-well polyvinylchloride microtitration plates were initially coated with 50 /J of antigen (5 jig/ml) in PBS for 2 hours at 37°C. Because preliminary experiments indicated that LDL was oxidized under these conditions, the antigen was coated in subsequent experiments in the presence of 2.7 mM EDTA and 20 nM BHT for 16 hours at 4°C. Nonadherent antigen was aspirated, and the remaining binding sites were "blocked" by incubation with 5% BSA in PBS for 45 minutes at room temperature. The wells were then aspirated, washed four times with PBS containing 0.02% NaN3, 0.05% Tween 20, 0.1% BSA, and 0.001% aprotinin by using a microtiter plate washer model 1550 (BioRad, New York, NY). Serial dilutions (50 /J) of pre- and post-immune sera were added per well and were incubated overnight at 4°C. After four washes, the amount of immunoglobulin bound was quantitated with a radiolabeled secondary antibody.25 Guinea pig IgG was detected by a goat-antiguinea pig IgG labeled with 12SI at approximately 10 000 cpm/ng with lactoperoxidase (Enzymobeads, Bio-Rad, Richmond, CA). Similarly, mouse IgG was detected with labeled goat-antimouse IgG. The plates were incubated for 4 hours at 4°C with 50 ^I/well of the second antibody, diluted to approximately 400 000 cpm/50 /J. The results are expressed as antibody binding as a function of antibody dilution. Antibody titers are defined as the reciprocal of the greatest dilution of the antiserum that

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gave a specific binding three times greater than the corresponding pre-immune serum. Competitive solid-phase RIAs were performed similarly, except that the antigen was plated at 1 /^g/ml. A fixed and limiting dilution of the primary antibody (25 /xl) was then added together with an equal volume of dilution buffer (3% BSA, 0.02% NaN3( 0.05% Tween 20, and 0.001% aprotinin in PBS) containing increasing amounts of potential competitors. The results were calculated as B/Bo, that is, the amount of antibody bound to the plated antigen in the presence of competitor (B) divided by the binding in the absence of competitor (B,,). The degree of competition in these assays is only qualitative in nature, as differences in the degree of competition may also reflect differing degrees of modification of lysine residues of the individual modified proteins.

Western Blots

1 02

1 03

10 4

105

Serum Dilution 1.0

Aliquots of 10 /ig of native or modified human LDL were electrophoresed in nonreducing 3% to 15% or 4% to 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels as previously described. 3435 The proteins were transblotted to nitrocellulose membranes and were incubated with antisera MAL-2 or HNE-6 or with monoclonal antibodies MB4736 or MB19.37 Autoradiographs were obtained after incubation with 126l-labeled goat ant-guinea pig IgG or goat anti-mouse IgG as previously described.34

0.9 0.8 0.7

0.5 0.40.30.2-

Results Polyvalent Antisera to Malondlaldehydemodttled Low Density LJpoproteln To develop antisera that would recognize epitopes generated during the oxidative modification of LDL, we conjugated MDA to LDL This adduct was chosen as a model of one of the presumed lipid products that would be generated during peroxidation of LDL polyunsaturated lipids. Under the conditions used, the conjugation yielded an average of 77% derivatization of the available lysine residues of apo B. Because human LDL is a potent immunogen, we used homologous, that is, guinea pig modified LDL to immunize guinea pigs to obtain an antibody response that was specific for MDA-lysine adducts but not for epitopes of native human LDL. Immunization of six guinea pigs with MDA-LDL resulted in pronounced antibody titers in all animals equal to or greater than 105 after a primary and two secondary immunizations. In some animals, boosting was continued biweekly, but no further increase of the titers was observed. The binding of the antisera to the respective antigen was tested by using solid-phase RIAs. Figure 1A shows the binding of two guinea pig sera (MAL-1 and MAL-2) to homologous MDA-LDL. Although the binding of the induced antibodies vastly exceeded that of the pre-immune sera, even the pre-immune sera displayed some "binding." We have previously shown that a portion of this binding to MDA-LDL in the pre-immune sera is accounted for by low titer auto-antibodies against MDALDL similar to auto-antibodies found in both rabbit and human sera. 28

0.1-

•—• o-c •—• »-* *—* v—i

human LDL human MOA-LDL human MDA— hemoglobin MDA-polyiysine human MDA-albumin MDA-t—amtnocaprolc ocid

g-o

MOA-tBOC-ly»In«

10~ l 1 0 " ' 10°

B

ng

10 1

10'

10 3

10 4 10»

competitor

Figure 1. A. Binding of guinea pig MAL-1 and MAL-2 sera to immobilized guinea pig malondlaldehyde-modifled low density lipoprotein (MDA-LDL) (coated at 5 ^fl/ml) in a solid-phase radioimmunoassay (RIA). The amount of antibody bound was detected as described in the Methods section by using 12S Habeled goat-antiguinea pig IgG (GAGPIgG). Binding was determined for pre-immune sera (closed symbols) and for sera obtained 10 days after the third booster immunization (open symbols). Each point represents the mean of triplicate determinations. B. Solid-phase competitive RIA of guinea pig antiserum MAL-2 with various potential competitors. Guinea pig MDA-LDL (1 iu8/ml) was plated as antigen, and a 1 : 10 (XX) dilution of the antiserum MAL-2 was added in the absence or presence of Increasing amounts of competitor. The results were expressed as B/Bo, where B Is the amount of MAL-2 bound In the presence, and Bo, that bound in the absence of competitor. (Figure 1B is reproduced from Paiinski et al. 28 with permission.)

The specificity of the antisera generated wtth homologous MDA-LDL was tested by solid-phase competitive RIAs; the results with one antiserum (MAL-2) are shown in Figure 1B. A partial characterization of this antiserum has previously been described. 28 Figure 1B demonstrates that MAL-2 binds the MDA-lysine epitope, which is expressed not only on human MDA-LDL but also on a variety of other MDA-conjugated proteins, including MDA-albumin and MDA-hemoglobin. Even simple MDAlysine adducts such as the polymer MDA-polylysine or the haptens MDA-epsilon-aminocaproic acid and MDAf-BOC-lysine competed for antibody binding and verified

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oxidized with Cu + + for 3 or 18 hours competed when added at high concentrations in the more sensitive RIA (Figure 3B), indicating that MDA-lysine conjugates are formed during Cu ++ -induced oxidation, that is, in the absence of exogenously added MDA. We previously published a Western blot demonstrating that Cu + + oxidized LDL contained apo B bands that were recognized by MAL-2.28

Polyvalent Antlsera to 4-Hydroxynonenalmodified Low Density Lipoprotein

10-

10° ng competitor

Figure 2. Solid-phase competitive radiolmmunoassay (RIA) of guinea pig antiserum MAL-2 with human low density lipoprotein (LDL) progressively modified with malondialdehyde (MDA). MDALDL was generated as described in the Methods section by incubation of LDL with Increasing amounts of MDA for 3 hours at 37°C. The degree of MDA modification was determined by a single trinitrobenzenesuifonic acid assay. The percentage of modification indicated for each MDA-LDL preparation was obtained by subtracting the value obtained for control LDL that had been incubated without MDA. These results assume that each LDL particle is equally modified. Extensively modified human MDA-LDL (1 /ig/ml) was plated as antigen, and a 1:5000 dilution of MAL-2 was added in the absence or presence of human MDA-LDL with varying degree of modification. The results were expressed as B/Bo (explained In the legend to Figure 1B). Each value represents the mean of duplicate determinations.

that the antiserum was specific for MDA-lysine. Most preparations of native human LDL did not compete. However, with more extensive experience, it became apparent that some fresh LDL preparations, as well as those stored for more than 2 weeks (though collected and stored in antioxidants), displayed some degree of competition when added in large excess. The extent of binding of the MAL-2 antiserum to MDA-LDL was a direct reflection of the degree of derivatization of lysine residues. Figure 2 shows that MDA-LDL, having an increasing degree of modification of its lysine residues (from 4% to 39%), competes proportionately better with plated MDA-LDL for antibody binding.

Monoclonal Antibodies to Malondialdehydemodified Low Density Lipoprotein The specificity of the monoclonal antibodies generated with mouse MDA-LDL closely resembled that of the guinea pig sera. Figure 3 shows two competitive RIAs with MDA2, the monoclonal selected for immunocytochemistry. The best competitor was MDA-LDL; MDAaibumin, MDA-transferrin, and MDA-hemoglobin also competed, whereas freshly prepared native LDL did not. The competitors shown in Panel A were human proteins, but MDA conjugated to proteins of other species were recognized to a comparable degree (data not shown). Thus, similar to the MAL-2 antiserum, the MDA2 antibody recognized the MDA-lysine epitope on LDL as well as other proteins. Compared to the MDA-modified proteins, 4-HNE-LDL did not compete significantly. However, LDL

Polyvalent antisera against homologous 4-HNE-LDL were generated in six guinea pigs in an analogous fashion. Initially, we immunized guinea pigs with LDL that had been incubated with 4-HNE in the absence of reducing conditions. Although the TNBS assays indicated that approximately 25% of lysine residues were derivatized, this nonreduced adduct proved to be a weak immunogen, and useful antisera were not obtained. The best immune responses were obtained when the immunogen was prepared under reducing conditions in the presence of NaCNBH3 (4-HNE^-LDL). Because it is unknown if similar reducing conditions are found in vivo, the recognition of non-reduced 4-HNEconjugates (4-HNEnf-LDL) or adducts reduced after conjugation with 4-HNE (post-reduced) is of essential importance. Figure 4 shows competition solid-phase RIAs that used either reduced (Panel A) or nonreduced (Pane) B) human 4-HNE-LDL for antigen coating. The antiserum, HNE-6, bound to both 4-HNEn^LDL (Panel A) and to 4-HNEnrLDL (Panel B), that is, BO=1358 and 1480 cpm, respectively. The binding of the antiserum HNE-6 to immobilized 4-HNErKrLDL was competed for by 4-HNE^LDL itself, by 4-HNE^-LDL, as well as by the free 4-HNE hapten (Panel A). Similarly, the binding of this antiserum to immobilized 4-HNEm-LDL was competed for effectively by 4-HNEn.d-LDL, as well as by 4-HNEnf-LDL (Panel B). The better competition of the reduced form probably reflects the greater degree of derivatizaton of lysine residues of the reduced form (39% vs. 13%) as well as a greater affinity of the antibody to the reduced form. Note that free 4-HNE also was an effective competitor. Cu++-oxidized LDL competed to varying degrees in both assays, demonstrating the presence of 4-HNE-lysine adducts. However, "native" human LDL did not compete against 4-HNEf8 0.2- a—B •—•

0.3

MDA-LDL MDA-transferrin MDA-albumin MDA-hemoglobin

*—* 18 hr Cu"-oxidized LDL 4—A 3 hr Cu"-oxidized LDL o-o LDL

0.1- *-* 4-HNE-LDL o-o LDL 0.0

10-' io° 101 101 103 io 4 10s io-' 10° 10" io 2 ng competitor

io 4 ^oi

0.0

Rgure 3. Two solid-phase competitive radioimmunoassays with the monoclonal antibody MDA2. Extensively modified human malondialdehyde-modified low density lipoprotein (MDA-LDL) (1 MQ/ml) was plated as antigen, and MDA2 ascites fluid was added at a dilution of 1 : 100 000 (A) or 1:5 000 000 (B) in the absence or presence of various concentrations of native and modified human proteins or lipoproteins. The amount of antibody bound was detected as described, using 126Mabeled goatantimouse IgG (GAMIgQ). The results are expressed as described In the legend for Rgure 1.

1.2

B

1.1

o m

•1.2 •1.1

1.0-

•1.0

0.9-

•0.9

0.8

•0.8

0.7

•0.7

0.6

•0.6

0.5-

0.5

0.4-

•0.4

0.3-

•0.3

0.20.1

1-4 •-• «-» »-•

•-HNE 18 hr Cu**-o»ld. LDL (h) LDL (h)

10-M0-' 10° 10' 101 101 104 10»

,^-LDL (h) 4-HNE 4-»C n r -LD«. (h) 18 hr Cu~oxld. LDL (h) LDL(h)

o m

•0.2 0.1

10-' 10-' 10° 10' 1CV 101 104 10»

ng competitor Rgure 4. Solid-phase competitive radioimmunoassays (RIAs) with guinea pig antiserum HNE-6. Reduced human 4-HNE-LDL (4-HNErKrLDL) was plated as antigen (1 Mg/ml) for the RIA shown in A, and nonreduced human 4-HNE-LDL (4-HNEn,-LDL) was plated as antigen (5 nglm\) for the RIA in B. HNE-6 was added in a dilution of 1 : 20 000 (A) or 1:2000 (B), together with increasing amounts of competitors. Antibodies bound were detected with 123l-labeled goat antiguinea pig IgG (GAGPIgG). The results are expressed as described in the legend for Figure 1. Bo=1358 cpm (A) and 1480cpm(B).

molecular weight and 4-HNEred-f-BOC-lysine. LDL conjugated under reducing conditions with other aldehydes, such as hexanaUd-LDL and butyraldehydered-LDL, did not compete at all, whereas nonenaJdehydeIBd-LDL competed slightly. 4-HNE alone competed when added at high con-

centrations. However, in this assay, native human LDL did not compete. As previously mentioned, Cu++-oxidation of LDL (in the absence of any reducing agent) generated epitopes recognized by these antibodies, and LDL oxidized for 24 hours with Cu + + ions competed with 4-HNE^d-LDL

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1.21.11.00.90.8O 00 \ CD

0.70.60.50.40.30.20.1-

• - • 4-HNEped-LDL (9P) •DL(h) • - • 4-HNEred-poddlzed LDL (h) • - • 4-HNE • - - LDL(h) I-LDL (h)

...

00

m-u Acetyi-LDL (h) » - • MDA-LDL (h)

10"3 10"2 10- 1 10°

101

102

103

104 105

ng competitor Figure 5. Solid-phase competitive radioimmunoassay of guinea pig antiserum HNE-6 with various competitors. Guinea-pig 4-HNE^j-LDL (1 fig/mi) was plated as antigen, and a 1 : 7500 dilution of HNE-6 was added in the absence or presence of competitors. Binding of antibodies was determined by using 1S5l-GAGPIgG. The results are expressed as described in the legend for Figure 1B.

Monoclonal Antibodies to 4-Hydroxynonenalmodlfied Low Density Upoproteln Based on initial immunocytochemicaJ studies, we selected one of the four 4-HNE-rysine specific monoclonal antibodies for future immunocytochemical application (NA59). As shown in Figure 6, 4-HNEnrf-LDL competed best. Other proteins conjugated with 4-HNE under reducing conditions also were recognized to varying degrees by the antibody, confirming specificity for the 4-HNE-lysine epitope. Human 4-HNE-LDL prepared under nonreducing conditions also was bound by the antibody. Similar to our results with the polyvalent HNE-6 antiserum, free 4-HNE, not conjugated to protein, and 18-hour Cu++-oxidized LDL also competed for binding, whereas MDA-LDL did not (data not shown). As noted above, the competitive behavior of "native" LDL depended very much on the individual LDL preparation and on its storage time. The human preparation shown in Figure 6 competed slightly at high concentrations. Such moderate recognition of native LDL by the antibody can probably be attributed to partial oxidative modification of the individual preparation, which most likely occurred after isolation. In fact, most LDL preparations did not compete for binding with this antibody. Monoclonal Antibodies to Cu++-oxldized Density Upoprotein

Low

The oxidative modification of LDL by endothelial cells or metal ions such as Cu + + is accompanied by complex changes in its chemical and physical properties (reviewed in reference 6), including extensive degrada-

10'

10»

nq competjtor

Figure 6. Solid-phase competitive radioimmunoassay with the monoclonal antibody NA59. Human 4-HNEnxrLDL (1 /ig/ml) was plated as antigen, and NA59 ascttes fluid was added at a dilution of 1 : 10 000 in the absence or presence of various concentrations of human proteins or lipoproteins. The amount of antibody bound was detected as described by using 126l-labeled goat antimouse IgG (GAMIgG). The results are expressed as described in the legend for Figure 1.

tjon of apo B-100. Figure 7 shows a Western blot with a mixture of two monoclonal antibodies: MB47, specific for the receptor-binding domain of native apo B,36 and MB19, specific for an amino-terminal eprtope of apo B.34 Human LDL oxidized for 2, 4, 6, 8, 20, and 24 hours showed progressive degradation of apo B, with predominantly intermediate size bands in 2-hour to 8-hour Cu + + oxidized LDL, which were then further degraded into

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0

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ilwa Figure 7. Western blot of "native" human low density llpoprotein (LDL) (0) and LDL oxidized in vitro by incubation with Cu + + Ions for 2, 4, 6, 8, 20, and 24 hours as described in the Methods section. Apolipoproteins (apo) (20 /tg/lane) were separated by electrophoresis in a nonreducing 3% to 15% sodium dodecyl sutfate-polyacrylamide gel electrophoresis gel and were transferred to a nitrocellulose membrane as described. Western blotting was performed with a mixture of two monoclonal antibodies: one specific for the receptor-binding domain of apo B, MB47 (dilution 1:2500), and one specific for an amlno-terminal epttope of apo B, MB19 (dilution 1:3000). 12S!-labeled goat-antimouse IgG was used as secondary antibody, as described in the Methods section. Note the fragmentation of native LDL; this preparation was isolated without addition of phenylmethylsutfonyi fluoride.

lower molecular weight fragments. Despite extensive antioxidative and antjproteolytic protection during routine blood collection, preparation of LDL, and immunoblotting, even native LDL frequently showed several such high molecular weight degradation fragments. Which fragments might contain epitopes specific to the oxidath/e modification and what was the nature of such epitopes was unknown a priori. We previously published a Western blot of Cu ++ -oxidized LDL stained with our polyvalent antisera to MDA-lysine (MAL-2), which demonstrated that several apo B bands contained MDA-lysine residues.28 Figure 8 shows that 4-HNE-lysine epitopes present in Cu ++ -oxidized LDL also can be identified with Western blotting techniques. The figure displays control LDL and LDL oxidized with Cu + + for 3 hours and 18 hours and stained with the apo B specific monoclonals, MB47 and MB-19, and with the 4-HNE-lysine specific antiserum, HNE-6. Whereas the two monoclonaJs detected different spectra of fragments, HNE-6 recognized epitopes of Cu ++ -oxidized LDL and, of course, multiple bands of the 4-HNE-LDL standard. To increase the probability of generating an immune response against oxidation-specific epitopes present only on the apoprotein fragments (and possibly uncover

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epitopes masked by lipid), we used apoprotein fragments from 4- or 24-hour Cu++-oxidized mouse LDL for immunizations. At days 2 and 3 before fusion, mice were boosted with apoprotein fragments obtained from a mixture of 4-hour and 24-hour Cu++-oxidized human LDL The use of human LDL in such pre-fusion boosts just before sacrifice of the mice does not generate antibodies against epitopes of the heterologous protein but is used because it may stimulate expression of B-cell clones containing the epitopes most likely to react with human modified LDL, as previously described.33 The hybridoma culture supernatants were screened against the respective immunogen, and secondary selection of the clones was based on binding to the immunogen, 4- and 24-hour Cu++-oxidized human LDL, 24-hour Cu++-oxidized apoprotein fragments, and native LDL. Initially, we observed significant binding of these monoclonal antibodies to native human LDL, despite the fact that the mice had not been exposed to human LDL long enough to generate an antibody response to human apo B. Subsequent experiments revealed that during the coating of native LDL to wells of the microtiter plate, considerable oxidation occurred. The addition of EDTA (1 mM) and BHT (20 fiM) or of probucol (60 ng/ml dissolved in ethanol), or high density lipoprotein (HDL) at three times the amount of LDL prevented oxidation during the 2-hour plating at 37°C. Plating under a nitrogen atmosphere was equally effective but did not further enhance the protective effect of the other additives. To prevent the generation of oxidative artifacts, further antigen coating of LDL was performed for 16 hours at 4°C in the presence of BHT (20 /AM) and EDTA (2.7 mM). Despite the precautions noted above, there was still slight recognition of native LDL by the monoclonal antibodies. Figure 9 shows the binding of OLF4-3C10 to 18-hour Cu++-oxidized human LDL, 4-hour Cu++-oxidized LDL, and native human LDL It appears that native LDL is binding at about 45% of the binding to oxidized LDL However, when the binding to nonspecific proteins (e.g., BSA used as post-coat) is also plotted, the specific binding of the antibodies to native LDL (obtained by subtracting the nonspecific binding) is less than 10% of the binding to oxidized LDL This slight binding to native LDL could represent either incomplete antioxidative protection during the isolation and plating of LDL or could conceivably represent oxidative epitopes that are present on LDL in vivo. Again, based on preliminary immunostaining results, one monoclonal antibody (OLF4-3C10) was selected and characterized in further detail. Figure 10 shows two representative competition RIAs. OLF4-3C10 recognized apo B fragments of LDL oxidized to various degrees (8 hours, 18 hours, 24 hours). Nondelipidated LDL oxidized for short time periods was poorly recognized (data not shown), whereas after 48 hours of oxidation, Cu ++ oxidized LDL competed effectively (Panel A). The poor recognition of nondelipidated LDL oxidized for short time periods, as compared to apoprotein fragments, is likely to be due to masking of epitopes by the lipid component. In this assay, native LDL did not compete. Another RIA (Panel B) showed that octylglucoside, the agent used to resolubilize the delipidated apoprotein fragments, did not "com-

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Figure 8. Sodium dodecyl sulfate-polyacryiamide gel electrophoresis (SDS-PAGE) and Western blot analysis of human native low density lipoprotein (LDL) (Lanes 1), 3-hour Cu++-oxidized LDL(Lanes2), 18-hour Cu++-oxidized LDL (Lanes 3), and 4-HNE-LDL (Lane 4). Apolipoproteins were separated in nonreducing 4% to 12% SDS-PAGE gels, were transferred to a nitrocellulose membrane, and were Western blotted with either MB47 (dilution 1 MOOO), MB19 (dilution 1:1000), or HNE-6, the polyvalent antiserum against 4-HNE-lysines (dilution 1 : 100). 126l-labeled goat antimouse IgG was used as secondary antibody for MB47 and MB19, and 12sl-labeled goat antiguinea pig IgG was used for HNE-6.

oxidized HDL apoproteins also competed. Although to date we have not determined the identity of the epitope recognized by OLF4-3C10, the recognition of Cu++-modified HDL suggests that it most likely is a lipid-protein adduct similar to the others described in this report.

Discussion

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DILUTION 1st ANTIBODY Figure 9. Antibody binding curves of OLF4-3C10 ascites fluid to different antigens in a solid-phase radtoimmunoassay (RIA). Human 18-hour Cu++-oxldized low density lipoprotein (LDL) (O), human 3-hour Cu++-oxidized LDL (•), and native human LDL (A) were each plated at 5 Aig/ml. Bovine serum albumin (BSA) (A) was plated at 5%; i.e., at the standard concentration used for post-coating of plates. After incubation wtth increasing dilutions of ascites fluid, the amount of antibody bound to each antigen was detected by using 125 l-labeled goat-antimouse IgG (GAMIgG), as described in the Methods section. In this assay, the background binding (binding to BSA-post-coated wells) was not subtracted.

pete" at the concentrations used. Other models of oxidatively modified LDL for example, MDA-LDL and acetylLDL also displayed slight competition, although at much higher concentrations. Most likely, the epitopes recognized were generated during the preparation of MDA-LDL and acetyl-LDL 4-HNE-LDL did not compete (not shown). It was initially hoped that this antibody would be specific for oxidized apo B; however, fragments of 24-hour Cu ++ -

To verify the occurrence of oxidized LDL in vivo, to study its prevalence in arterial vessel walls, and to assess the effect of therapeutic intervention on the accumulation of oxidized lipoproteins will require a method of detecting oxidation-specific epitopes of LDL without inducing oxidative artifacts. In this article, we describe the development of polyvalent antisera and monoclonal antibodies against several epitopes generated during the oxidative modification of LDL Lipid peroxidation generates a number of highly reactive aldehyde products, which in turn can be covalently linked to lysine residues of proteins, such as apo B. 1 2 1 7 2 4 The demonstration of the simultaneous presence of a variety of such adducts on LDL would provide convincing evidence for the occurrence of oxidative modification of LDL MDA and 4-HNE are two lipid peroxidation products known to occur in vivo and to be formed during the oxidative modification of LDL in vitro. 2021 Our current studies, together with our previous reports,28'29 clearly demonstrate that MDA-lysine and 4-HNE-tysine adducts with apo B are generated during the in vitro and in vivo oxidative modification of LDL This was shown both in RIA format and by Western blots of oxidized LDL protein. Similarly, the epitope recognized by OLF43C10 is generated during oxidation of LDL as demonstrated by RIAs. However, to date we have not been able to

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ng competitor Figure 10. Two solid-phase competitive radioimmunoassays with the monoclonal antibody 0LF43C10. Human 24-hour Cu ++ -oxidized low density lipoprotein (LDL) was plated as antigen at 1.5 /ig/ml (A) or 1 /ig/ml (B), and OLF4-3C10 ascites fluid was added in a 1 : 750 dilution in the absence or presence of the indicated competitors. The amount of antibody bound was detected as described by using 126l-labeled goat antimouse IgG (GAMIgG). The results were expressed as B/Bo as explained In the legend to Figure 1.

obtain staining of in vitro Cu++-oxidized LDL with this monoclonal antibody by Western blotting, although 0LF43C10 has stained some LDL preparations extracted from the artery wall (Yla-Herttuala, unpublished observation). Recently Mown et al. 38 described a monoclonal antibody raised against homogenized atherosclerotic plaque that, in turn, bound to in vitro oxidized LDL, but they have not characterized the epitope recognized. Haberland et al. 27 found that their monoclonal antibody against MDAlysine did not recognize oxidized LDL, possibly as a result of the addition of insufficient amounts of oxidized LDL in the competition assay. Salmon et al. 39 also generated an MDA-lysine specific antjserum by immunizing rabbits with MDA-modified rabbit LDL They too demonstrated the presence of MDA-lysine residues in apo B of in vitro Cu ++ -oxidized LDL with this antiserum. Finally, Boyd et al. 40 recently described a monoclonal antibody generated against in vitro Cu++-oxidized LDL that immunostained atherosclerotic lesions in Watanabe heritable hyperlipidemic rabbits.

The authors thank Daniel Steinberg for his support and valuable advice, Hermann Esterbauer for his generous gift of 4-HNE, and David Wadleigh for excellent technical help.

The immunocytochemical application of five different antisera and monoclonal antibodies on serial sections of atherosclerotic lesions provides a reliable method to localize oxidized proteins and oxidized LDL Using the polyvalent antisera against 4-HNE-lysines and MDA-lysines, as well as OLF4-3G10, we have demonstrated the occurrence of oxidized proteins in atherosclerotic lesions but not in normal areas of arterial walls.28 We also have shown that the staining material is at least in part oxidized LDL by using the above antibodies for Western blots of LDL gently extracted from the same arterial walls.28-29 In the companion article**1, we report that the full spectrum of our antibodies was used to study the distri-

1. Goldstein JL, Ho YK, Basu SK, Brown MS. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc NaH Acad Sci USA 1979;76:333-377 2. Stelnbrecher UP, Parthasarathy S, Leake DS, WHztum JL, Steinberg D. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids. Proc Nat) Acad Sci USA 1984;81:3883-3887 3. Parthasarathy S, Fong LG, Otero D, Steinberg D. Recognition of resolubilized apoproteins from delipidated, oxidatJvely modified low density lipoprotein (LDL) by the acetyi-LDL receptor. Proc Natl Acad Sci USA 1987:84:537-540

bution of oxidized proteins in rabbit atherosclerotic lesions ranging from very early fatty streaks to advanced fibrous lesions. Furthermore, this article shows the similarity in staining patterns between our induced antibodies and the auto-antibodies recognizing MDA-LDL which we discovered in the serum of several species.28 The antibodies reported here have thus enabled us to establish the occurrence in vivo of oxidized LDL and should provide a tool to establish its potential pathogenetic role for atherosclerosis. In addition, because lipid peroxidation and subsequent modification of associated proteins is a widespread biological process, these immunological reagents should be useful in studies of a number of biological systems.

Acknowledgments

References

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ANTIBODIES AGAINST OXIDATION SPECIFIC EPITOPES 4. Sparrow CP, Parthasarathy S, Steinberg D. A macrophage receptor that recognizes oxidized low density lipoprotein but not acetytated low density lipoprotein. J Biol Chem 1989;264:2599-2604 5. Aral HT, Klta M, Yokode S, Narumlya S, Kawal C. Multiple receptors for modified low density lipoproteins in mouse peritoneal macrophages: different uptake mechanisms for acetylated and oxidized low density lipoproteins. Biochem Biophys Res Commun 1989; 159:1375-1382 6. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Wrtztum Jl_ Beyond cholesterol. Modifications of low density lipoprotein that increase its atherogenicity. N Engl J Med 1989;320:915-924 7. Henrlksen T, Mahoney EM, Steinberg D. Enhanced macrophage degradation of biologically modified low density lipoprotein. Arteriosclerosis 1983:3:149-159 8. Heinecke JW, Rosen H, Chart A. Iron and copper promote modification of low density lipoprotein by human arterial smooth muscle cells in culture. J Clin Invest 1984; 74:1890-1894 9. Morel DW, DICorleto PE, Chlsolm GM. Endothelial and smooth muscle cells alter low density lipoprotein in vitro by free radical oxidation. Arteriosclerosis 1984;4:357-364 10. Cathcart MK, Morel DW, Chlsolm GM III. Monocytes and neutrophils oxidize low density lipoprotein making it cytotoxic. J Leucocyte Biol 1985;38:341-350 11. Parthasarathy S, Printz DJ, Boyd D, Joy L, Steinberg D. Macrophage oxidation of low density lipoprotein generates a modified form recognized by the scavenger receptor. Arteriosclerosis 1986:6:505-510 12. Stelnbrecher UP, Wltztum JL, Parthasarathy S, Steinberg D. Decrease in reactive amino groups during oxidation or endothelial cell modification of LDL. Correlation with changes in receptor-mediated catabolism. Arteriosclerosis 1987;7:135-143 13. Esterbauer H, Cheeseman KH, Dlanzanl MU, Poll G, Slather TF. Separation and characterization of the aldehydic products of lipid peroxidation stimulated by ADP-Fe2* in rat liver microsomes. Biochem J 1982;208:129-140 14. Esterbauer H, Jurgens G, Quehenberger O, Keller E. Autooxidation of human low density lipoprotein: Loss of polyunsaturated fatty acids and vitamin E and generation of aldehyde. J Lipid Res 1987;28:495-509 15. Hamberg M, Svensson J, Wakabayashl T, Samuelson B. Prostaglandin endoperoxides. A new concept concerning the mode of action and release of prostaglandins. Proc Nati Acad Sci USA 1974;71:345-349 16. Smith JB, Interman CM, Silver MJ. Malondialdehyde formation as an indicator of prostaglandin production by human platelets. J Lab Clin Med 1976;88:167-172 17. Fogelman AM, Schechter JS, Saeger J, Hokom M, Child JS, Edwards PA. Malondialdehyde alteration of low density lipoprotein leads to cholesterol accumulation in human monocyte-macrophages. Proc Natl Acad Sci USA 1980;77: 2214-2218 18. Stossel TP, Mason RJ, Smith Al_ Lipid peroxidation by human blood phagocytes. J Clin Invest 1974;54:638-645 19. BenedettJ A, Comport! M, Esterbauer H. Identification of 4-hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal lipids. Biochim Biophys Acta 1980;620:281-296 20. Jurgens G, Lang J, Esterbauer H. Modification of human low-density lipoprotein by the lipid peroxidation product 4-hydroxynonenal. Biochim Biophys Acta 1986:875:103-114 21. Esterbauer H, Jurgens G, Quehenberger O, Koller E. Autooxidation of human low density lipoproteins: loss of potyunsaturated fatty acids and vitamin E and generation of aldehydes. J Lipid Res 1987:28:495-509 22. Law SW, Grant SM, Hlguchi K, et al. Human liver apolipoprotein B-100 cDNA: complete nucleic acid and derived amino acid sequence. Proc Nati Acad Sci USA 1988;83:8142-8146

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23. Knott J, Pease RJ, Powell LM, et al. Complete protein sequence and identification of structural domains of human apolipoprotein B. Nature 1986;323:734-738 24. Stelnbrecher UP. Oxidation of human low density lipoprotein results in derivatization of lyslne residues of apolipoprotein B by lipid peroxide decomposition products. J Biol Chem 1987:262:3603-3608 25. Wltztum JL, Stelnbrecher UP, Fisher M, Kesanleml A. Nonenzymatic glucosyiation of homologous LDL and albumin render them immunogenic in the guinea-pig. Proc Nati Acad Sci USA 1983:80:2757-2761 26. Stelnbrecher UP, Fisher M, Wltztum JL, Curtiss LK. Immunogenictty of homologous low density lipoprotein after methyiation, ethylation, acetyiarjon or carbamyiation: Generation of antibodies specific for derivatyzed tysine. J Lipid Res 1984;25:1109-1116 27. Haberland ME, Fong D, Cheng L Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperiipidemlc rabbits. Science 1988;241:215-241 28. Palinski W, Rosenfeld ME, Yla-Herttuala S, et al. Low density lipoprotein undergoes oxidatlve modification In vivo. Proc Natl Acad Sci USA 1989;86:1372-1376 29. Yla-Herttuala S, Palinski W, Rosenfeld ME, et al. Evidence for the presence of oxidatively modified low density lipoprotein In atherosclerotic lesions of rabbit and man. J Clin Invest 1989;84:1086-1095 30. Havel RJ, Eder HA, Bragdon JH. Distribution and chemical composition of ultracentrifugally separated lipoproteins In human serum. J Clin Invest 1955:34:1345-1353 31. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951:193:1831-1841 32. Habeeb ASSA. Chemical evaluation of conformational differences in native and chemically modified proteins by trinitrcbenzenesulfonic acid. Biochim Biophys Acta 1966; 115:440-454. 33. Curtiss LK, Wltztum JL A novel method for generating region specific monoclonal antibodies to modified proteins: Application to the identification of human glucosylated low density lipoproteins. J Clin Invest 1983:72:1427-1438 34. Young SG, Bertlcs SJ, Scott TM, Dubols BW, Curtiss LK, Wltztum J L Parallel expression of the MB19 genetic polymorphism in apoprotein B-100 and apoprotein B-48: Evidence that both apoprotelns are products of the same gene. J Biol Chem 1986:261:2995-2998 35. Parthasarathy S, Stelnbrecher UP, Barnett J, Wltztum J, Steinberg D. Essential role of phospholipase A? activity in endothelial cell-induced modification of low density lipoprotein. Proc Natl Acad Sci USA 1985;82:3000-3004 36. Young SG, Wltztum J L Casal DC, Curtiss LK, Bernstein S. Conservation of the low density lipoprotein receptor-binding domain of apoprotein B: Demonstration by a new monoclonal antibody, MB47. Arteriosclerosis 1986;6:178-188 37. Young SG, Bertics SJ, Curtiss LK, Casal DC, Wltztum J L Monoclonal antibody MB19 detects genetic polymorphism in human apolipoprotein B. Proc Nati Acad Sci USA 1986; 83:1101-1105 38. Mowrl H, Ohkuma S, Takano T. Monoclonal DLRI./104G antibody recognizing peroxidized lipoproteins in atherosclerotic lesions. Biochim Biophys Acta 1988;963:208-214 39. Salmon S, Mazlere C, Theron L et al. Immunological detection of low density lipoproteins modified by malondialdehyde in vitro or in vivo. Biochim Biophys Acta 1987;920:215-220 40. Boyd HC, Gown AM, Wolfbauer G, Chalt A. Direct evidence for a protein recognized by a monoclonal antibody against oxidatively modified LDL in atherosclerotic lesions from a Watanabe Heritable Hyperlipidemic rabbit. Am J Pathol 1989:135:815-825 41. Rosenfeld ME, Palinski W, Yla-Hertuala S, Butler S, Wltztum J L Distribution of oxidation specific lipid-protein adducts and apoliprotein B in atherosclerotic lesions of varying severity from WHHL rabbit Arteriosclerosis 1990;10:336-349

oxidized low density lipoproteins •

monoclonal antibodies • atherosclerosis

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Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein. W Palinski, S Ylä-Herttuala, M E Rosenfeld, S W Butler, S A Socher, S Parthasarathy, L K Curtiss and J L Witztum Arterioscler Thromb Vasc Biol. 1990;10:325-335 doi: 10.1161/01.ATV.10.3.325 Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1990 American Heart Association, Inc. All rights reserved. Print ISSN: 1079-5642. Online ISSN: 1524-4636

The online version of this article, along with updated information and services, is located on the World Wide Web at: http://atvb.ahajournals.org/content/10/3/325

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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...
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