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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:704-713.)
© 1995 American Heart Association, Inc.

Articles

Generation, Characterization, and Histochemical Application of Monoclonal Antibodies Selectively Recognizing Oxidatively Modified ApoB-Containing Serum Lipoproteins

Astrid Hammer; Gerd Kager; Gottfried Dohr; Hans Rabl; Irmgard Ghassempur; Günther Jürgens

From the Institute for Medical Biochemistry (A.H., G.K., G.J.), the Institute for Histology and Embryology (G.D., I.G.), and the Department for Surgery (H.R.), Medical Faculty, Karl-Franzens Universität Graz, Graz, Austria.

Correspondence to Dr Günther Jürgens, Institute for Medical Biochemistry, Harrachgasse 21, A-8010 Graz, Austria.

	Abstract

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Abstract To investigate either the role oxidized LDL plays in atherosclerosis or structural changes on the surface of oxidized LDL, monoclonal antibodies (mAbs) are an important tool. After immunizing mice with Cu²⁺-oxidized LDL (oxLDL) and fusion of splenocytes, hybridoma supernatants were screened and cloned. Two mAbs, OB/04 and OB/09 (IgG and IgM), were further characterized. In solid-phase fluorescence immunoassays and Western blot analysis both mAbs reacted with oxLDL, LDL oxidized by a free radical–generating azo compound, or oxVLDL but not with native LDL, acetylated LDL, oxHDL₃, azo-oxidized HDL₃, or HDL₃ modified with malondialdehyde (MDA). In competitive immunoassays with LDL modified by oxidized fatty acid–derived aldehydes, mAb OB/09 strongly reacted with MDA-LDL or MDA-VLDL and LDL modified with 4-hydroxyhexenal followed by 4-hydroxynonenal but not with 4-hydroxyoctenal or hepta-2,4-dienal. mAb OB/04 had a weak affinity for LDL after modification with these aldehydes except for MDA-LDL. LDL modified with arachidonic acid oxidation products (AAOPs) was also recognized by this mAb. However, albumin modified either by the aldehydes applied or by AAOPs did not react with either mAb. Thus, the data indicate that each of the mAbs recognizes a different epitope that is expressed only on apoB-containing lipoproteins upon oxidative modification. An immunostaining with mAb OB/04 was obtained in areas rich in macrophages and in connective tissue of a human atherosclerotic lesion.

Key Words: modified lipoproteins • apoB • lipid peroxidation • monoclonal antibodies • atherosclerosis

	Introduction

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The oxidative modification of LDL,¹ the major carrier of cholesterol in human blood, is assumed to represent a crucial point regarding the atherogenic properties of this class of lipoproteins (for review, see References 1 through 6^{1 2 3 4 5 6} ). Free radical oxidation of LDL leads to the formation of lipid hydroperoxides, derived from polyunsaturated fatty acids (PUFAs) after the intrinsic antioxidants are consumed, followed by generation of aldehydes,⁷ a cleavage and breakdown of apoB, and generation of lysolecithin.^{8 9} It is assumed that LDL entering the artery wall becomes mildly oxidized. This form is able to recruit monocytes¹⁰ that become resident macrophages.¹¹ These cells further oxidize LDL until it is recognized by the scavenger receptors.^{12 13} By accumulating lipids these cells become foam cells, a pathological hallmark of the formation of atherosclerotic lesions.¹⁴ To investigate lesions for the existence of oxidized LDL, polyclonal antisera or monoclonal antibodies (mAbs) have been raised against Cu²⁺-oxidized LDL (oxLDL) or LDL modified by lipid peroxidation–derived breakdown products such as malondialdehyde (MDA) or 4-hydroxynonenal (4-HNE).^{15 16 17 18} Using those antibodies, immunohistochemical studies of atherosclerotic lesions of Watanabe heritable hyperlipidemic (WHHL) rabbits have been performed.^{15 16 19 20} Evidence indicates the presence of oxidized LDL in atherosclerotic lesions of rabbit and humans,^{21 22} and lipid peroxidation–derived protein adducts have been demonstrated in atherosclerotic human autopsy aortas.²³ However, the antibodies generated against LDL that had been oxidized or modified by lipid peroxidation products such as MDA or 4-HNE showed a cross-reactivity with other proteins after their modification with MDA or 4-HNE.^{15 17 18 24} Thus, the lipid peroxidation–specific epitopes discovered and studied so far by immunohistochemical methods in atherosclerotic plaques do not necessarily represent oxidized LDL^{15 17 23} ; ie, they might also be located on membrane or connective tissue proteins. In the present investigation several mAbs were raised against oxLDL. The immunoreactivity to and specificity for oxidized or modified serum lipoproteins and serum albumin of two of these mAbs were investigated in detail. In addition, an immunohistochemical analysis of a human atherosclerotic lesion was performed with one of the mAbs.

	Methods

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Lipoprotein Preparation
Lipoproteins were isolated from the plasma of normolipemic, fasting (12 to 14 hours) young male and female donors in which serum lipoprotein(a) levels were lower than 1 mg/dL. Chloramphenicol (50 mg/L; Serva Feinbiochemika GmbH & Co), kallikrein inactivator (aprotinin; 100 000 U/L; Bayer), and EDTA (1 g/L; Merck) were present during lipoprotein preparation. By differential ultracentrifugation the following fractions were obtained: LDL (d=1.020 to 1.050 g/mL), VLDL (d=0.950 to 1.006 g/mL), and HDL₃ (d=1.125 to 1.21 g/mL). HDL₃ was further purified on a heparin-coupled Sepharose CL-6B column (Pharmacia Fine Chemicals AB) to remove apoE and apoB. The fractions were sterile filtered and stored at 4°C. Protein was measured by the method of Lowry et al.²⁵

Aldehyde Modification and Oxidation of the Lipoproteins
4-HNE, 4-hydroxyhexenal (4-HHE), and 4-hydroxyoctenal (4-HOE) were synthesized,²⁶ and aqueous solutions of these aldehydes were prepared for 4-HNE²⁷ except that instead of Tris/HCL buffer, 0.1 mol/L phosphate-buffered saline (PBS), pH 7.4, containing 1 g EDTA/L and saturated with nitrogen was used. MDA was obtained by acid hydrolysis of 1,1,3,3-tetraethoxypropane,²⁸ and its content was measured.²⁹ Hepta-2,4-dienal (2,4-HDE) was supplied by Aldrich. After dialysis against the above-mentioned buffer, suitable portions of LDL, VLDL, and HDL₃ were incubated with 4 mmol/L each of 4-HNE, 4-HHE, 4-HOE, or 2,4-HDE or 30 mmol/L MDA at a final protein concentration of 1 mg/mL. Incubations were performed in the dark at 37°C for 5 hours. Excess aldehyde was removed by dialysis against 0.01 mol/L PBS, and acetylation was performed.³⁰

Prior to oxidation, the lipoproteins were dialyzed against 0.01 mol/L PBS, pH 7.4, which was carefully degassed and then saturated with nitrogen. Cu²⁺ oxidation was performed at 37°C by using 1 mg/mL lipoprotein protein with 20 or 30 µmol/L CuSO₄ at intervals between 15 minutes and 24 hours. The reaction was terminated by adding a stop solution such that the final concentration of EDTA was 2.7 mmol/L. For oxidation with the free radical–producer 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH; Polysciences Inc), 1 mg/mL lipoprotein protein and 1 to 30 mmol/L AAPH were used. Lipid hydroperoxides were evaluated.³¹ The degree of modification of the lipoproteins and apolipoproteins by oxidation or aldehyde incubation was estimated as their relative electrophoretic mobility (REM), ie, relative to the nonmodified native fraction, on agarose gels (1%) at pH 8.05 using the Lipidophor system (Immuno AG). In some samples the degree of modification of LDL was estimated by a solid-phase fluorescence immunoassay (FIA; see below).

In some experiments oxLDL was separated by gel filtration. LDL was oxidized in the presence of 30 µmol/L CuSO₄ for 24 hours. After oxidation, 1.5 mL (equivalent to 1.5 mg protein) was loaded on a column of Biogel A-5 M (Bio-Rad Laboratories) and eluted with 0.01 mol/L PBS, pH 7.4, containing 1 g EDTA/L and 50 mg chloramphenicol/L. Fractions of 1 mL were collected, assayed for lipoproteins on a nondenaturing 3.75% polyacrylamide gel by electrophoresis, and stained with Phast-Blue R (Pharmacia-LKB). The lipoproteins were sampled in three main fractions: aggregates, an intermediate fraction containing aggregated and nonaggregated LDL, and nonaggregated LDL. They were concentrated by centrifugation for 6 hours at 40 000 rpm.

Aldehyde Modification of Human Serum Albumin
The modification of essentially fatty acid–free human serum albumin (Sigma) with 4-HNE, 4-HHE, 4-HOE, 2,4-HDE, and MDA was performed as described above for the lipoproteins. The modification was confirmed by polyacrylamide gel electrophoresis on a nonreducing 10% running gel.

Generation of Thermal Auto-oxidation Products From Arachidonic Acid and Modification of LDL and Albumin
Arachidonic acid (Sigma) was subjected to auto-oxidation according to the method of Zhang et al.³² The fatty acid (25 mg) was transferred to a glass vial open to the air and heated at 37°C for 144 hours. The yellow-brown products were dissolved in 2.5 mL of 0.01 mol/L PBS, pH 7.4, and centrifuged at 5000g for 10 minutes. Aliquots of the supernatant containing the soluble residue from 1 mg oxidized fatty acid were added to LDL and human serum albumin in 0.01 mol/L PBS, pH 7.4 (final concentration, 1 mg/mL protein). After a 20-hour incubation at 20°C, the mixtures of LDL or albumin with the oxidation products were dialyzed against 0.01 mol/L PBS, pH 7.4, and 1 mg/mL EDTA.

Polyclonal Antisera Against LDL, 4-HNE–LDL, and MDA-LDL
All antisera used were from rabbits. Anti-apoB was purchased from Behring AG. Oxidation of LDL led to a decrease of reactivity with this antiserum. The generation, purification, characterization, and specificity of the antiserum against 4-HNE–LDL have been described.^{17 23 24} The antiserum against LDL incubated with 20 mmol/L MDA for 5 hours at 37°C was generated in essentially the same manner. After absorbance of the antiserum with native LDL as described for the antiserum against 4-HNE–LDL,^{17 24} the antiserum specifically reacted with MDA-protein adducts.

Production of mAbs
Primary immunization of BALB/c mice by using 60 µg oxLDL for 4 hours (REM, 1.45) in 0.3 mL PBS (0.2 mL IP and 0.1 mL IM) was performed followed by a second immunization with oxLDL for 4 hours (REM, 1.5) from the same donor 2 weeks later. Four and 6 weeks later and 4 days before the fusion, mice were boosted with 60 µg oxLDL for 24 hours (REM, 4.0, 3.9, and 3.6, respectively). Fusions were performed with the NS1 myeloma cell line^{33 34} followed by culturing in hypoxanthine, aminopterin, and thymidine selection medium. Primary screening of hybridoma supernatants was performed after 14 days of growth. The supernatants were screened by FIA using oxLDL, MDA-LDL, 4-HNE–LDL, and native LDL as the coated antigen. Selected hybridomas were cloned by limiting dilution. To identify the immunoglobulin class of the mAbs, a kit for mouse immunoglobulins from GIBCO BRL (Life Technologies GmbH) was used.

Solid-Phase FIA
The use of an FIA for lipoprotein estimation and epitope characterization of lipoproteins has been described.^{24 35} Briefly, for the binding assay, polyvinyl chloride microtitration plates (Nunc Maxisorb) were coated with 200 µL of the respective antigen (1 µg/mL) in 50 mmol/L sodium carbonate buffer, pH 9.6, containing 1 mg/mL EDTA at 4°C overnight. After washing once with washing buffer (50 mmol/L Tris/HCl, pH 7.7, 9 g/L NaCl, and 0.2 g/L Tween 20), 250 µL blocking buffer (50 mmol/L NaH₂PO₄ · H₂O, 5 g/L bovine serum albumin, and 60 g/L sorbitol) was added to the wells to block the remaining binding sites. The wells were then washed twice, and 200 µL hybridoma supernatants diluted 1:10 with assay buffer (50 mmol/L Tris/HCl, pH 7.7, 5 g/L bovine serum albumin, 0.5 g/L bovine globulin, 8 mg/L diethylenetriaminepentaacetic acid, 9 g/L NaCl, and 100 µg/L Tween 20) was added and incubated for 90 minutes at room temperature. After three washes, the amount of mouse immunoglobulin bound was detected by adding 50 ng sheep anti-mouse IgG or sheep anti-mouse IgM (Sigma) labeled with europium (DELFIA Eu-labeling kit, No. 1244-302, Wallac Oy) per well according to the manufacturer's instructions. The labeling yield was 16 Eu³⁺/IgG (mol/mol). After incubation for 1 hour and six washes, bound Eu³⁺ was released in the presence of 200 µL of the enhancement solution (Wallac Oy). The fluorescence was measured with a 1234 DELFIA research fluorometer (Wallac Oy). The degree of modification of oxLDL or modified LDL in some samples was estimated as the reactivity with an antiserum to apoB before and after oxidation and modification, respectively. Microtitration plates were coated with 200 µL of the respective antigen (1 µg/mL) at 4°C overnight. After blocking and washing, essentially as described above, the amount of apoB was measured with 150 ng Eu³⁺-labeled anti-apoB.

Competitive solid-phase FIAs were performed similarly, except that 100 µL of a fixed dilution of primary antibody was added together with an equal volume of assay buffer containing increasing amounts of potential competitors. The results were expressed as B/B₀, where B is the amount of antibody bound to the coated antigen in the presence of competitor and B₀ that in the absence of competitor.²⁴

Electrophoresis and Western Blot Analysis
Electrophoresis was performed on 3.5% nonreducing and nondenaturing polyacrylamide gels or 5% sodium dodecyl sulfate (SDS) polyacrylamide gels under reducing conditions. Aliquots of 15 to 20 µg native or modified human LDL protein dissolved in sample buffer (0.075 mol/L Tris, pH 8.8, containing 20% glycerol and 0.01% Orange G [Aldrich]) were applied per lane. Electrophoresis was performed in a Mini Protean II electrophoresis chamber (60 minutes at 50 mA and 150 V; Bio-Rad). Transfer to nitrocellulose membranes (0.1-µm pore size; Hoefer Scientific Instruments) was done with an LKB NovaBlot electrophoretic transfer kit (Pharmacia-LKB) for 90 minutes at 50 mA and 17 V. The transfer solution contained 48 mmol/L Tris, 39 mmol/L glycine, 0.037% SDS, and 20% methanol (vol/vol). Nonspecific binding sites were blocked with 3% skim milk in 20 mmol/L Tris, 90 mmol/L NaCl, 1 mmol/L NaN₃, and 0.05% Tween 20, pH 7.4, for 3 hours at 20°C. Afterward the hybridoma culture medium (1:10 diluted in blocking solution) was incubated at 4°C overnight. After three washes with TTBS, alkaline phosphatase–conjugated anti-mouse IgG or anti-mouse IgM (both from Sigma) was added, followed by incubation at 20°C for 3 hours. After washing, bound antibody was visualized with 0.5 mg/mL 5-bromo-4-chloro-3-indolylphosphate in 1 mol/L 2-amino-2-methyl-1-propane, pH 10.3.

Immunohistochemistry
Cryosections were made from an atherosclerotic artery section obtained from the femoral artery after amputation of the leg from a patient suffering from open gangrene due to an advanced arterial occlusive disease. Immediately after its removal the tissue was carefully rinsed with 0.01 mol/L PBS, pH 7.4, containing 1 mg/mL EDTA and 100 mg/L Trolox (Hofmann LaRoche) to prevent tissue oxidation. The material was kept in this buffer for about 30 minutes until it was embedded in Tissue-Tek (Miles Inc.) and frozen to -80°C in a cryocut instrument. Immunohistochemical analysis was performed by means of the alkaline phosphatase–anti–alkaline phosphatase (APAAP) complex technique. Frozen arterial sections (5 µm thick) were incubated with the cell culture supernatant of mAb OB/04 as the primary antibody (30 minutes in a humid chamber at 20°C). Anti-mouse immunoglobulin (Dako Corp) was used as second antibody (diluted 1:25) and incubated for 30 minutes. Before addition of APAAP (Dako) the sections were washed three times in 0.05 mol/L Tris-buffered saline, pH 7.6. APAAP (diluted 1:25) was applied for 45 minutes, and the color reaction was performed with the New Fuchsin Substrate System from Dako. Microscopy was done on Axiophot (Zeiss).

	Results

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Establishment of Hybridomas Producing mAbs Against oxLDL
Female 8-week-old BALB/c mice whose spleens were used for cell fusion were immunized with oxLDL. To select hybridomas that produce antibodies against oxLDL, an FIA was performed for screening of the hybridoma culture media. Ten hybridoma clones producing antibodies highly reactive to oxLDL but negative to native LDL were obtained from 240 wells on the first screening. Four clones were subjected to limiting dilution. Two stable and rapidly growing hybridomas OB/04, an IgG₁, and OB/09, an IgM, were obtained.

Immunoreactivity of mAbs OB/04 and OB/09 During the Time Course of Oxidation With LDL and VLDL but Not HDL
The three main classes of lipoproteins, LDL, VLDL, and HDL₃, were oxidized in the presence of 30 µmol/L CuSO₄. At different times an aliquot of 0.2 mL was withdrawn from the incubation mixture, and the oxidation was stopped by adding EDTA and cooling to 4°C. The progress of oxidation was recorded by measuring the formation of lipid hydroperoxides³¹ (Fig 1A) and estimating the REM at certain times. The samples were also examined for the development of epitopes recognized by the mAbs. Microtiter plates were coated with the oxidized lipoproteins (1 µg/mL protein), and the reactivity of the culture supernatants from mAbs OB/04 (diluted 1:150) and OB/09 (diluted 1:75) was measured by means of an FIA. mAb OB/04 reacted strongly with oxLDL and moderately with oxVLDL but did not react with oxHDL₃ (Fig 1B). Similar results were obtained with mAb OB/09 except that the recognition was not as high (Fig 1C). The extent of binding of both mAbs to oxLDL and oxVLDL increased with the duration of the oxidation. However, none of the samples taken from oxHDL₃ showed any reaction with the mAbs, even though the protein part was also clearly modified by oxidation. REM after 6 hours of oxidation was 1.25 and increased further with the progress of oxidation. Thus, the increase in the reactivity of the mAbs with oxidizing lipoproteins is a direct reflection of the degree of oxidative modification of LDL and VLDL.

View larger version (10K): [in this window] [in a new window]   Figure 1. Line graphs showing immunoreactivity of monoclonal antibodies (mAbs) OB/04 and OB/09 during the time course of Cu2+-mediated oxidation of human serum lipoproteins. LDL, VLDL, and HDL3 (1 mg/mL protein) were oxidized at 37°C in the presence of 30 µmol/L CuSO4. The degree of oxidation wasrecorded by measuring the formation of lipid hydroperoxides in Cu2+-oxidized LDL (ox-LDL), Cu2+-oxidized VLDL (ox-VLDL), and Cu2+-oxidized HDL3 (ox-HDL) after their reaction with a color reagent. The amount of lipid hydroperoxide is equivalent to the amount of free iodine formed. Its absorbance is monitored at 365 nm (A). For the binding assay, microtitration plates were coated with the oxidized lipoproteins (1 µg/mL protein). The amount of antibody bound was detected as described under "Methods" by using Eu3+-labeled goat anti-mouse IgG for mAb OB/04 (B) and goat anti-mouse IgM for mAb OB/09 (C). The results are given in fluorescence counts. Each point represents the mean of duplicate determinations.

Characterization of the Specificity of mAbs OB/04 and OB/09 in Competitive Immunoassays With Oxidized or Modified Lipoproteins
For further determination of the specificity of both mAbs, we tested native and oxidized lipoproteins for their ability to compete with oxLDL for binding to these mAbs in an FIA. oxLDL was a strong competitor (nearly 100%), while oxVLDL reacted more weakly with OB/04, probably because apoB represents only 30% of its apoprotein (Fig 2A); however, the native forms did not compete. Native or oxHDL₃ did not compete at all. Application of mAb OB/09 in this assay revealed generally similar results, but oxLDL and especially oxVLDL were rather weak competitors under the assay conditions used (Fig 2B). However, when the wells were coated with MDA-LDL, MDA-LDL and MDA-VLDL but not MDA-HDL₃ strongly competed for the binding of mAb OB/09 to the wells. oxLDL and oxVLDL were weaker competitors, and oxHDL₃ did not compete at all (Fig 2C). Thus, we conclude that MDA linked to apoB but not to apolipoproteins of HDL₃ generates a distinct epitope recognized by mAb OB/09.

View larger version (10K): [in this window] [in a new window]   Figure 2. Line graphs showing results of solid-phase competitive fluorescence immunoassays of monoclonal antibodies (mAbs) OB/04 and OB/09 with native and oxidatively modified lipoproteins. Cu2+-oxidized LDL (ox-LDL; relative electrophoretic mobility [REM], 3.2; modified apoB, 40%) (A and B) or malondialdehyde (MDA)-LDL (REM, 1.9; modified apoB, 37%) (C) was plated as antigen (1 µg/mL) on microtiter plates, and the cell culture supernatant of mAb OB/04 (dilution, 1:200) (A) or OB/09 (dilution, 1:100) (B and C) was added in the absence or presence of increasing amounts of native LDL, oxLDL, MDA-LDL, Cu2+-oxidized VLDL (ox-VLDL; modified apoB, 55%) or MDA-VLDL (modified apoB, 40%) and Cu2+-oxidized HDL3 (ox-HDL) as competitors. Each lipoprotein (2 mg lipoprotein protein) was oxidized in 2 mL 0.01 mol/L phosphate-buffered saline, pH 7.4, in the presence of 30 µmol/L CuSO4 for 24 hours. The amount of antibody bound was detected as described under "Methods" by using Eu3+-labeled goat anti-mouse IgG for mAb OB/04 (A) and goat anti-mouse IgM for mAb OB/09 (B and C). The results are expressed as B/B0, where B is the amount of antibody bound in the presence and B0 in the absence of the competitor. Each point represents the mean of triplicate determinations.

Acetylation of LDL transforms this lipoprotein to a substrate recognized by scavenger receptors.³⁶ We studied a possible cross-reactivity of the mAbs with acetylated LDL (REM, 3.3), comparing it with oxLDL (REM, 3.2; modified apoB, 40%) in preventing the binding of mAbs OB/04 and OB/09 to oxLDL in the wells at competitor concentrations from 0.01 µg/mL to 1 mg/mL. Acetylated LDL competed up to 8%, but oxLDL competed close to 100%.

A variety of aldehydes are generated during peroxidation of PUFAs transported by lipoproteins^{2 7 37} and involved in the formation of new epitopes on their surface.^{17 24 27} LDL modified with one of the aldehydes, ie, MDA, 4-HNE, 4-HHE, 4-HOE, or 2,4-HDE, and oxLDL were tested in an FIA for their ability to compete with oxLDL on the wells for binding mAbs OB/04 and OB/09. With mAb OB/04, oxLDL itself was the only strong competitor. MDA-LDL did not compete at all, and the LDL samples modified with the other aldehydes were weak competitors (Table).

View this table: [in this window] [in a new window]   Table 1. Solid-Phase Competitive Fluorescence Immunoassay of mAbs OB/04 and OB/09 With LDL Modified by Various Aldehydes as Competitor

For the competition assay with mAb OB/09, MDA-LDL was chosen for coating the wells since a higher reactivity was obtained with MDA-LDL than with oxLDL (Fig 2B and 2C). MDA-LDL and 4-HOE–LDL were almost equivalent in their competing ability, followed closely by 4-HNE–LDL, whereas 4-HHE– and 2,4-HDE–LDL did not compete at all.

Characterization of the Specificity of mAbs OB/04 and OB/09 in Competitive Immunoassays Using Modified Albumin or LDL
As far as the polyclonal antisera and the mAbs generated hitherto against oxLDL or aldehyde-modified LDL were characterized, they showed a strong cross-reactivity with other proteins modified by aldehydes such as MDA or 4-HNE and with fragments from oxHDL.^{15 17 18 24} We tested the potency of albumin modified with one aldehyde (MDA, 4-HNE, 4-HOE, 4-HHE, or 2,4-HDE) to prevent binding of the mAbs to oxLDL (REM, 3.2; modified apoB, 40%) or MDA-LDL (REM, 1.9; modified apoB, 37%). oxLDL (1 mg/mL protein) almost completely prevented (>95%) binding of mAb OB/04 to oxLDL on the wells, whereas aldehyde-modified albumin competed very weakly. MDA-, 2,4-HDE–, 4-HHE–, 4-HNE–, or 4-HOE–modified albumin (1 mg/mL) was able to compete with mAb OB/04 by only 8% to 14%. mAb OB/09 was in no case prevented from binding to MDA-LDL by aldehyde-modified albumin, whereas MDA-LDL itself competed by about 90%. Thus, modification of albumin with certain aldehydes stemming from lipid peroxidation was not able to create epitopes that were recognized by the two mAbs.

However, during oxidation of LDL a variety of compounds, different from the aldehydes detected so far, are formed that might also react with apoB³⁷ and take part in the formation of new epitopes against which our mAbs were reactive. Thus, thermal arachidonic acid auto-oxidation products (AAOPs) were used for the modification of human serum albumin and LDL.³² One of the substances presumably present in such a reaction mixture is 4-HNE.^{2 7 37} We used an antiserum against 4-HNE–LDL^{17 23 24} to prove that albumin and LDL were in fact modified and expressed an epitope composed of this aldehyde. AAOP-albumin and AAOP-LDL were equally effective in preventing binding of this antiserum to 4-HNE–LDL in the wells (Fig 3A). However, AAOP-albumin did not prevent mAb OB/04 from binding to oxLDL in the wells, whereas AAOP-LDL did (Fig 3B). This further strengthens the assumption that this mAb exclusively recognized LDL modified by AAOP. Neither AAOP-albumin nor AAOP-LDL was able to prevent binding of mAb OB/09 to MDA-LDL in the wells. This could be explained by the lack of MDA-derived epitopes on either AAOP-albumin or AAOP-LDL (Fig 3D). An antiserum against MDA-modified LDL also failed to recognize MDA-specific epitopes on AAOP-modified albumin and LDL (Fig 3C).

View larger version (22K): [in this window] [in a new window]   Figure 3. Line graphs showing results of solid-phase competitive fluorescence immunoassays of monoclonal antibodies (mAbs) OB/04 and OB/09 and the polyclonal antibodies to 4-hydroxynonenal (4-HNE)–LDL and malondialdehyde (MDA)-LDL with LDL and human serum albumin (Alb.) modified by thermal arachidonic acid auto-oxidation products (AAOP) as competitors. Microtiter plates were coated with 4-HNE–LDL (A), Cu2+-oxidized LDL (ox-LDL) (B), or MDA-LDL (C and D) as antigens (1 µg/mL). The rabbit antibodies to 4-HNE–LDL (dilution, 1:3000) (A) or MDA-LDL (dilution, 1:1500) (C) and the cell culture supernatant of mAb OB/04 (dilution, 1:200) (B) or mAb OB/09 (dilution, 1:100) (D) were added in the absence or presence of increasing amounts of AAOP-LDL or AAOP-albumin as competitors. The amount of antibody bound was detected as described under "Methods" by using Eu3+-labeled goat anti-rabbit IgG (A and C), goat anti-mouse IgG for mAb OB/04 (B), and goat anti-mouse IgM for mAb OB/09 (D). The results are expressed as B/B0 (explained in the legend to Fig 2). Each point represents the mean of triplicate determinations.

Recognition of Aggregated and Nonaggregated oxLDL by mAb OB/04 or mAb OB/09 in Western Blot Analysis
Modification of LDL by aldehydes derived from lipid peroxidation or by oxidation leads to aggregation of apoB or the lipoprotein particle.^{27 38 39} To determine whether the recognized epitopes on oxLDL are located on aggregated or on nonaggregated lipoprotein particles, oxLDL (REM, 3.55) was separated by gel filtration. LDL, oxLDL, and the three fractions of the column fractionation were subjected to electrophoresis on 3.75% nondenaturing polyacrylamide gels followed by Western blot analysis with mAbs OB/04 and OB/09 and anti-apoB antibody. mAb OB/04 recognized aggregated and nonaggregated oxLDL but not native LDL (Fig 4).

View larger version (78K): [in this window] [in a new window]   Figure 4. Western blot analysis of native LDL and Cu2+-oxidized LDL (oxLDL) with monoclonal antibody (MAB) OB/04. Native LDL (lane 1), oxLDL (lane 2), and its three main fractions obtained by gel filtration on Biogel A-5 M (aggregated LDL [lane 3], aggregated and nonaggregated LDL [lane 4], and nonaggregated LDL [lane 5]) were subjected to nondenaturing polyacrylamide gel electrophoresis (3.75% running gel) and transferred to nitrocellulose membranes. Western blotting was performed with the cell culture supernatant of mAb OB/04 (dilution, 1:10) with rabbit anti-human apoB (dilution, 1:750) as a control. Alkaline phosphatase goat anti-mouse IgG (dilution, 1:400) and goat anti-rabbit IgG (dilution, 1:1200) were used as secondary antibodies.

mAb OB/09 showed good staining with aggregated oxLDL (fractions 1 and 2) but only a very faint staining with the nonaggregated fraction 3 of oxLDL (Fig 5A). Comparison of native LDL, oxLDL, acetylated LDL, or MDA-LDL showed a strong reaction of mAb OB/09 with the broad band of MDA-LDL but only with the aggregated forms of oxLDL (Fig 5B). Native and acetylated LDL did not react. ApoB from MDA-LDL was separated on a 5% reducing SDS-polyacrylamide gel by electrophoresis followed by Western blot analysis with anti-apoB antibody or mAb OB/09. A reaction with mAb OB/09 was obtained only with a higher molecular cross-linked form of apoB or its fragments from MDA-LDL (data not shown).

View larger version (28K): [in this window] [in a new window]   Figure 5. Western blot analysis of native LDL, Cu2+-oxidized LDL (oxLDL), malondialdehyde (MDA)-LDL, and acetylated LDL with monoclonal antibody (MAB) OB/09. A, oxLDL (lane 1), native LDL (lane 2), and its three main fractions obtained by gel filtration on Biogel A-5 M: aggregated LDL (lane 3), aggregated and nonaggregated LDL (lane 4), and nonaggregated LDL (lane 5). B, Native LDL (lane 1), MDA-LDL (lane 2), oxLDL (lane 3), and acetylated LDL (lane 4). Lipids were subjected to nondenaturing polyacrylamide gel electrophoresis (3.75% running gel) and transferred to nitrocellulose membranes. Western blotting was performed with the cell culture supernatant of mAb OB/09 (dilution, 1:5) (A and B) with rabbit anti-human apoB (dilution, 1:750) as a control (B). Alkaline phosphatase goat anti-mouse IgM (dilution, 1:400) and goat anti-rabbit IgG (dilution 1:1200) were used as secondary antibodies.

Recognition of AAPH-Oxidized Lipoproteins
The oxidation of lipoproteins used in the experiments so far was performed by adding copper ions in micromolar concentrations. It is assumed that the copper ions bind to the surface of LDL at certain sites where they develop a lipid peroxidation catalyzing activity.⁴⁰ To prove that the epitopes recognized by the two mAbs are not only expressed on oxLDL, oxidation of LDL was also performed by means of a water-soluble free radical–generating compound, AAPH. LDL was oxidized by either 30 µmol/L CuSO₄ or 10 mmol/L AAPH for 24 hours. Competitive FIAs performed with either oxLDL or AAPH-oxidized LDL and using either oxLDL or AAPH-oxidized LDL as the coating agent showed a slightly stronger competition with oxLDL than with AAPH-oxidized LDL for mAb OB/04 no matter which species of oxidized LDL was used for coating. Since oxLDL (REM, 3.45) was more strongly modified than AAPH-oxidized LDL (REM, 2.8), the degree of oxidation and not the mode seems to be the only parameter for recognition by this mAb. The results obtained with the FIA were confirmed by Western blot analysis of oxLDL or AAPH-oxidized LDL with mAb OB/04 (Fig 6).

View larger version (40K): [in this window] [in a new window]   Figure 6. Western blot analysis of native LDL, Cu2+-oxidized LDL, and 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH)–oxidized LDL with monoclonal antibody (MAB) OB/04. oxLDL (30 µmol/L CuSO4; lane 1), 10 mmol/L AAPH-oxidized LDL (lane 2), 1 mmol/L AAPH-oxidized LDL (lane 3), and native LDL (lane 4) were subjected to nondenaturing polyacrylamide gel electrophoresis (3.75% running gel) and transferred to nitrocellulose membranes. Western blotting was performed with the cell culture supernatant of mAb OB/04 (dilution, 1:10). Alkaline phosphatase goat anti-mouse IgG (dilution, 1:400) was used as the secondary antibody.

mAb OB/09 revealed somewhat different results. Experiments performed by competitive FIAs using oxLDL for coating the wells revealed oxLDL or MDA-LDL as strong competitors, while AAPH-oxidized LDL had a weaker activity in preventing binding of this mAb to oxLDL. Although MDA-LDL had a weaker increase in its REM than AAPH-oxidized LDL (2.3 versus 2.8), it was by far the strongest competitor. Furthermore, in Western blot analysis only the aggregates of oxLDL and AAPH-oxidized LDL reacted with this mAb (data not shown). The staining of AAPH-oxidized LDL was rather weak and corresponded well with the result from the FIA. Since both mAbs recognized AAPH-oxidized LDL, they were able, in principle, to detect epitopes on oxLDL independent of the mode of oxidation.

HDL₃ was also oxidized with 20 mmol/L AAPH for 24 hours, and a high degree of modification was obtained (REM>5.5). In the competition assay AAPH-HDL₃ prevented binding of the polyclonal aldehyde-specific antibody (against 4-HNE–LDL) to 4-HNE–LDL at 1 mg/mL concentration of the competitor by about 80%. However, this AAPH-HDL₃ was not able to compete in any manner for binding of mAb OB/04 to oxLDL or mAb OB/09 to MDA-LDL used for coating the wells.

Immunohistochemical Staining of a Human Atherosclerotic Plaque With mAb OB/04
Fig 7A shows the staining pattern of a cryosection of human atherosclerotic tissue from the femoral artery. Atherosclerotic areas reacted with mAb OB/04 and also with a mAb against macrophage antigen (data not shown). A detail from the lesion is shown in Fig 7B. Since the frozen tissue sections were not treated with lipid-extracting solvents, multiple small lipid droplets could be detected due to lipid accumulation in macrophages. A sharp intracellular staining was obtained with mAb OB/04 in these macrophages that were transforming into foam cells. The tissue surrounding the smooth muscle cells revealed a predominantly extracellular, more diffuse staining along laminar structures. Controls applying nonimmune mouse serum were negative.

View larger version (4K): [in this window] [in a new window]   Figure 7. Alkaline phosphatase–anti–alkaline phosphatase immunostaining of a cryosection from human atherosclerotic tissue. The primary antibody was monoclonal antibody OB/04. B shows a section from A (arrow) at a higher magnification. L indicates lumen (original magnification x40 [A] and x200 [B]).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The polyclonal antisera or mAbs used so far to detect oxLDL were generated either against oxLDL itself^{16 18 19} or against LDL having been modified by distinct aldehydes^{16 17 18 24 41} or other undefined products derived from lipid peroxidation of PUFAs.³² In one report the float-up fraction of the atherosclerotic arterial homogenate from WHHL rabbits was used to modify LDL for immunization.⁴² However, the antibodies characterized thus far not only reacted with oxLDL and/or modified LDL but also showed a cross-reactivity with proteins modified with 4-HNE, MDA,^{18 24} or other products derived from lipid peroxidation of PUFAs,³² or with oxHDL and 4-HNE–modified HDL.^{18 24} In contrast to a previous study¹⁸ in which apoprotein fragments of oxLDL resolubilized in octylglucoside were used for immunization, we used entire oxLDL particles for injection in mice. By this strategy we hoped to obtain an immune response specifically directed against certain surface structures on LDL or apoB that are not formed on other proteins modified with lipid peroxidation–derived products, such as aldehyde-lysine adducts.¹⁸

Neither mAb reacted with oxHDL₃ or AAPH-oxidized or MDA-modified HDL₃ since these modified lipoproteins were not able to compete in any of the performed assays for binding of the antibodies to oxLDL (mAb OB/04) or MDA-modified LDL (mAb OB/09). From these data we deduce that both mAbs are specifically directed against distinct epitopes formed only upon oxidation of apoB-containing lipoproteins like LDL or VLDL. Acetylation of the lipoproteins had little influence.

The experiments performed to characterize compounds involved in the creation of the epitope recognized and bound by mAb OB/04 showed that alkenals like 4-HNE and 4-HOE followed by 4-HHE and 2,4-HDE played a certain role when these aldehydes were used to modify LDL, but the divalent aldehyde MDA could not form an epitope detected by this mAb. Furthermore, modification of human serum albumin with these aldehydes did not lead to the creation of a corresponding epitope.

We also tried to perform Western blot analysis from reducing SDS-polyacrylamide gels. However, mAb OB/04 consistently failed to react with apoB from oxLDL regardless of whether it was present in an aggregated or fragmented form. Based on these results, we believe that the domain detected by this mAb is either destroyed under reducing, delipidating, or denaturing conditions or masked by SDS. As a third possibility, certain surface lipids of LDL and VLDL could be considered candidates for expressing the specific epitope after lipid peroxidation. 4-HNE reacts with phosphatidylethanolamine and phosphatidylserine from rat liver microsomes or mitochondria, leading to fluorescent chromolipids.⁴³ However, why should those epitopes not be formed upon oxidation of HDL₃?

In studying the creation of new structures on the surface of LDL formed during oxidative modification, not only aldehydes but also lipid hydroperoxides have been used for modification of LDL.^{32 44} In the present study AAOPs were used to modify human serum albumin and LDL. That both of them were modified showed their almost equal ability to prevent binding of the antibody against 4-HNE–LDL to 4-HNE–LDL in the wells. However, mAb OB/04 reacted with AAOP-modified LDL but not with AAOP-modified albumin, supporting the strong specificity of this mAb for only modified LDL.

The epitope detected by mAb OB/09 was formed by endogenous generation of MDA during oxidation of LDL and VLDL or by modification of these two lipoproteins by exogenously added MDA. However, oxidation or exogenous modification of HDL₃ was not able to create such an epitope. Moreover, MDA-modified human serum albumin did not bind to this mAb. This suggests that this particular epitope is formed by a change of the structural conformation of apoB induced by its interaction with MDA rather than composed of MDA itself or its adduct with a free amino group of the protein. Further support for this assumption comes from the competition studies of LDL exogenously modified by 4-HOE and 4-HNE. 4-HOE–LDL was almost as potent in inhibiting binding of mAb OB/09 to MDA-LDL in the wells as MDA-LDL itself in solution, although the modification of 4-HOE–LDL in terms of charge (REM, 1.45) was weaker than for MDA-LDL (REM, 1.9). 4-HNE–LDL was also a strong competitor in this assay, whereas albumin modified with 4-HOE or 4-HNE was not.²⁴ The fact that structurally unrelated aldehydes, such as these alkenals and the divalent aldehyde MDA, lead to similar potent competitors when used for modification of LDL underline our assumption that the epitope recognized by mAb OB/09 is created during oxidation of LDL by conformational changes of apoB. This idea was further strengthened by the results obtained by Western blot analysis from nondenaturing or SDS–polyacrylamide gel electrophoresis of MDA-LDL. In each case only the high-molecular-weight aggregates of oxLDL or the larger cross-linked form of apoB (or its fragments) from MDA-LDL reacted with mAb OB/09, suggesting that the epitope recognized by this antibody is formed and preserved on cross-linked apoB. Such high-molecular-weight bands of apoB were found in early studies of LDL treated with MDA and HNE.^{45 27} Due to its bifunctionality, MDA can cause intramolecular as well as intermolecular cross-links reacting with free amino groups of proteins⁴⁶ that might lead to structural changes of the protein chain. Only 16% of the lysine residues of apoB have to be modified by MDA to make LDL a substrate for the scavenger receptor, whereas in the case of acetylation or succinylation, a modification of more than 60% of the lysines is necessary.⁴⁷ This example shows the potency of MDA to create specific epitopes on apoB.

The thermal auto-oxidation products obviously did not contain reactive MDA (probably due to a self-condensation reaction), since neither AAOP-modified albumin nor AAOP-modified LDL could prevent binding of a polyclonal antibody specific for MDA-LDL to MDA-LDL. mAb OB/09 also did not recognize AAOP-modified albumin or LDL. Since 4-HNE–LDL reacted with mAb OB/09, it is surprising that the antibody did not recognize AAOP-modified LDL, although an epitope derived from 4-HNE was found on the latter. Obviously 4-HNE is able to form more than one kind of epitope on LDL, the expression of which might differ between 4-HNE– and AAOP-modified LDL. Although we do not know more details of epitopes for which mAb OB/09 is specific, we showed that certain aldehydes from lipid peroxidation of PUFAs, when conjugated with apoB, are involved in the formation of this epitope.

The investigation of a human atherosclerotic lesion with mAb OB/04 revealed that this antibody also reacts with physiologically oxidized apoB-containing lipoproteins. The intracellular staining of macrophages in a fatty streak was in accordance with studies performed in WHHL rabbits.²⁰ A large portion of the staining was also extracellular and associated with matrix elements surrounding smooth muscle cells, which agrees with former investigations of lesions from WHHL rabbits^{15 20} or from human autopsy material.²³ From the characterization of the specificity of mAb OB/04 we deduce that the staining is due to an extracellular deposition and attachment of oxidized apoB-containing lipoproteins to matrix components. Oxidized apoB stemming from digestion of LDL by macrophages might be released after cell death and bind to connective tissue such as collagen fibers.⁴⁸

The mAbs reported on here will bring about further insights into the distribution of oxidized apoB-containing lipoproteins in atherosclerotic plaques. They also might be used to establish immunoassays for the investigation of a possible presence of modified or oxidized apoB-containing lipoproteins in circulating human blood. Furthermore, the characterization of the epitopes recognized by the two mAbs presented could contribute new details to the knowledge of the structural changes caused by oxidative modification of human serum lipoproteins.

	Acknowledgments

 
This work was supported by a grant from the Austrian Research Council (project No. P 8271-Med) to Dr Jürgens. We wish to thank Astrid Blaschitz and Gerhard Ledinski for their expert technical assistance.

Received November 8, 1994; accepted February 14, 1995.

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