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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:982-989.)
© 1995 American Heart Association, Inc.

Articles

Immunologic Detection and Measurement of Hypochlorite-Modified LDL With Specific Monoclonal Antibodies

Ernst Malle; Linda Hazell; Roland Stocker; Wolfgang Sattler; Hermann Esterbauer; Georg Waeg

From Karl-Franzens University, Institutes of Medical Biochemistry (E.M., W.S.) and Biochemistry (H.E., G.W.), Graz, Austria, and the Heart Research Institute, Biochemistry Group, Camperdown, Australia (L.H., R.S.).

	Abstract

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Abstract Oxidation of LDL is thought to contribute to the early stages of atherogenesis. Because myeloperoxidase is present in atherosclerotic lesions and can produce the strong oxidant hypochlorous acid (HOCl), which converts LDL into its high-uptake atherogenic form in vitro, we raised polyclonal and monoclonal antibodies (MoAbs) against HOCl-modified LDL (HOCl-LDL). Characterization of the polyclonal anti-human HOCl-LDL Abs showed that they cross-reacted strongly with 4-hydroxynonenal–, malondialdehyde-, and Cu²⁺-oxidized LDL. Similarly, polyclonal and some monoclonal Abs against aldehyde- and Cu²⁺-modified LDL cross-reacted with HOCl-LDL. In contrast to the polyclonal Abs, two selected hybridoma cell line supernatants containing MoAbs raised against HOCl-LDL (MoAb-A and MoAb-B) did not cross-react with either native LDL or aldehyde- or Cu²⁺-modified LDL. MoAb-A (clone 1B10A11, subtype IgG1) recognized an epitope that appeared to be specific for HOCl-LDL and depended on the tertiary structure of the (lipo)protein, as judged by a lack of cross-reactivity with HOCl-modified human and bovine serum albumin and a loss of reactivity associated with lipoprotein denaturation. MoAb-B (clone 2D10G9, subtype IgG2b), on the other hand, gave identical titration curves with HOCl-LDL and HOCl-modified albumins, suggesting that this antibody recognized epitopes that are commonly generated on proteins that have been oxidized with HOCl. Thus, MoAb-A and MoAb-B may be useful tools for the investigation of a possible role for HOCl-mediated damage to (lipo)proteins in atherosclerosis and other inflammatory diseases.

Key Words: myeloperoxidase • lipid peroxidation • atherosclerosis • oxidized lipoproteins

	Introduction

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Oxidation of LDL is now generally thought to contribute to the early stages of atherogenesis.^{1 2 3 4} Evidence for the occurrence of oxidized LDL (ox-LDL) in vivo includes in situ localization of material that is recognized by antibodies (Abs) directed against various types of ox-LDL.

Previous studies on the in situ identification of ox-LDL have used Abs raised against in vitro copper-oxidized LDL (Cu²⁺-ox–LDL) or LDL that has been modified by end products of lipid peroxidation, such as malondialdehyde (MDA) or 4-hydroxynonenal (HNE), as these products can convert native LDL (N-LDL) into an atherogenic form. Atherosclerotic lesions of varying severity from Watanabe heritable hyperlipidemic (WHHL) rabbits and foam cells and LDL from these lesions all contain material recognized by monoclonal antibodies (MoAbs) that recognize MDA-, HNE-, and Cu²⁺-ox–LDL.^{5 6 7} Aortas from human autopsy subjects also react positively with a polyclonal Ab raised against Cu²⁺-ox– and HNE-LDL.⁸ Epitopes that are recognized by polyclonal Abs raised against MDA-LDL have been reported to be present in the plasma of humans with established cardiovascular disease⁹ ; however, no evidence for MDA-LDL has been found in the plasma of WHHL rabbits.¹⁰ Also, autoantibodies against MDA-LDL can be demonstrated in human and rabbit sera; however, there is controversy about the relationship between these autoantibodies and atherosclerosis.^{11 12 13}

Despite much interest in this area of research, little progress has been made in the identification of the putative in vivo oxidant of LDL. Although it is generally assumed that lipid peroxidation precedes and to some extent causes oxidative modification of LDL, hypochlorous acid (HOCl) transforms LDL into its high-uptake form¹⁴ without significant lipid (per)oxidation.^{14 15} NaOCl-modified LDL is more efficient in lipid loading of macrophages than is acetylated LDL (ac-LDL).¹⁴ HOCl is a natural oxidant that is produced from H₂O₂ and Cl^- via the action of myeloperoxidase (MPO; EC 1.11.1.7), which is present in neutrophils¹⁶ and monocytes.¹⁷ The latter infiltrate the intima,¹⁸ and human lesions are known to contain large amounts of MPO.¹⁹

Oxidation of human LDL with HOCl causes cross-linking of the lipoproteins,^{15 20} and we have proposed that this occurs via formation of Schiff bases,^{14 15} similar to those that are formed when aldehydes derived from lipid peroxidation react with apo B-100.²¹ Because the reagent HOCl closely mimics the MPO/H₂O₂/Cl^- system with regard to the oxidation of different target molecules,^{14 15} we used the former rather than the latter to generate the antigens. We now report the production of polyclonal Abs and MoAbs against HOCl-LDL. We observed that polyclonal anti-human HOCl-LDL Abs cross-reacted with MDA-, Cu²⁺-ox–, and HNE-LDL and that polyclonal Abs and MoAbs raised against Cu²⁺-ox–LDL and HNE-LDL were highly cross-reactive with HOCl-LDL. We describe two monoclonal anti-human HOCl-LDL Abs that recognize HOCl-LDL and are not cross-reactive with other forms of modified LDL or N-LDL. One of these MoAbs specifically recognizes HOCl-LDL, whereas the other appears to recognize a more general epitope on HOCl-modified proteins. These Abs should provide useful tools to investigate the presence of HOCl-proteins and HOCl-LDL in atherosclerotic lesions and other diseased tissues.

	Methods

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Reagents
Nitrocellulose (NC) was purchased from Schleicher & Schuell; goat anti-mouse IgG (peroxidase conjugated) Abs were from Chemicon; goat anti-rabbit IgG (peroxidase conjugated), 4-chloro-1-naphthol, human serum albumin (HSA, fatty-acid free), and BSA (fatty-acid free) were from Sigma; standard-quality BSA was from Haemosan; SDS was from Serva; tetramethylbenzidine (TMB) was from Fluka; Dulbecco's modified Eagle's medium was obtained from Bio-Whittaker; polyclonal anti-human LDL Abs were from Behring AG; and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox-C) was obtained from Aldrich. All other chemicals were analytical-grade reagents obtained from Merck.

Lipoprotein Isolation
LDL (d=1.035 to 1.065 g/mL) was isolated by ultracentrifugation as described previously.²² The protein of the final LDL preparation consisted of 96% to 98% apo B-100 as measured immunochemically. Lipoprotein concentrations are expressed in milligrams or micrograms of protein per milliliter and were either determined by the bicinchoninic acid reagent kit (Pierce) using BSA as a standard or calculated from total cholesterol, as determined by the CHOD-PAP method (Boehringer Mannheim). Prior to its modification N-LDL was desalted, and the preservatives were removed by dialysis or size-exclusion chromatography on Econopac 10-DG columns (Bio-Rad).

Modification of Proteins
HOCl-LDL was prepared as described,¹⁴ and HOCl-BSA and HOCl-HSA were obtained in a similar fashion. HOCl solutions were prepared by saturation of a solution of 3 mol/L NaOH with Cl₂ until the concentration of NaOH decreased to 0.1 mol/L. The concentration of HOCl in the HOCl stock solution (about 1.4 mol/L) was determined by using the CHOD-iodine reagent (Merck) and H₂O₂ as a standard. One milligram of LDL or other protein per milliliter of PBS (pH 7.4, sometimes containing 1 mg/mL EDTA) was incubated with the HOCl solution at 4°C for as long as 2 hours at pH 7.4. For LDL incubated with 1.6 mmol/L HOCl, (final concentration), this resulted in a molar lipoprotein to oxidant ratio of 1:800. For BSA (fatty-acid free) or HSA (fatty-acid free), the corresponding molar ratios were approximately 1:100.

Preparation of HNE-LDL (1 milligram of protein per milliliter) was performed with aqueous HNE solution for 4 hours at 37°C after acidic saponification of HNE-diethylacetal as described.²³ The final HNE concentration was 2 or 5 mmol/L, corresponding to a molar LDL to HNE ratio of 1:1000 or 1:2500, respectively. Excess HNE was removed by size-exclusion chromatography on Econopac 10-DG columns.

Preparation of MDA-proteins was performed as described by Haberland and coworkers.²⁴ Briefly, LDL (1 mg protein per milliliter of PBS, pH 7.4) was incubated with increasing volumes of a freshly prepared MDA solution (0.2 mol/L in 0.1 mol/L sodium phosphate buffer, pH 6.4) for 3 hours at 37°C under N₂ to obtain increasingly modified LDL. The final MDA concentration in the reaction mixture was 10 or 20 mmol/L. The reaction was stopped by dialysis against PBS (pH 7.4).

For oxidation with copper, LDL (50 µg protein per milliliter) was incubated with 1.66 µmol/L Cu²⁺ in PBS as described.²⁵ Formation of conjugated dienes was assayed on-line by the increase in absorbance at 234 nm (within the first 3 hours, the change in absorbance at 234 nm is 0.8 to 0.9). After 24 hours, oxidation was stopped by the addition of EDTA (final concentration, 1 mg/mL).

Ac-LDL was prepared by the method of Basu and coworkers.²⁶ Briefly, 1 mL of a 0.15 mol/L NaCl solution containing 15 mg LDL protein was added to 1 mL of a saturated solution of sodium acetate with continuous stirring under N₂ at 0°C, followed by addition of multiple 2-µL aliquots of acetic anhydride to the stirred solution (final ratio of protein to acetic anhydride was 1:1.5, wt/wt). After stirring for an additional 30 minutes at 0°C the reaction solution was extensively dialyzed against PBS (pH 7.4) at 4°C.

Characterization of N-LDL and Modified LDL Preparations
The electrophoretic mobility of N-, HNE-, MDA-, HOCl- and Ac-LDL was assessed by agarose gel electrophoresis using the Lipidophor system (Immuno AG). The vitamin E contents of N- and modified LDL preparations were estimated according to Esterbauer et al.²⁷ Reactive amino groups were estimated with trinitrobenzenesulfonic acid exactly as described previously.²⁸ Cholesteryl ester content of N- and modified LDL preparations was assayed by high-performance liquid chromatography.²⁹

Preparation of Abs
Polyclonal Abs directed toward HNE-, MDA-, Cu²⁺-ox–, and HOCl-LDL were raised in rabbits. For polyclonal anti–HOCl-LDL Abs, LDL preparations treated with 200, 400, or 800 HOCl molecules per lipoprotein particle were used as antigens. MoAbs directed toward HOCl-modified LDL (800 HOCl molecules per lipoprotein particle) were raised according to standard protocols³⁰ by immunizing female BALB/c mice (Charles River). In brief, primary immunization of the mice was carried out by intraperitoneal injection of HOCl-LDL (50 µg protein in 200 µL PBS, pH 7.4) mixed with 300 µL Freund's complete adjuvant at day 0. Boosts on days 7 and 21 were given intraperitoneally with the same dose of antigen in incomplete Freund's adjuvant. On day 28, 5 to 10 µg antigen in PBS (pH 7.4) without adjuvant was injected into the tail vein. On day 32, splenocytes were collected and immortalized by polyethylene glycol fusion with Sp2/0-Ag14 myelomas (American Type Culture Collection). Clones were selected in hypoxanthine-, aminopterin-, and thymidine-containing medium³⁰ and screened by indirect enzyme-linked immunosorbent assay (ELISA) technique as outlined below. Specific clones were tested by competitive ELISA and subcloned by limiting dilution to assure monoclonality.

Polyacrylamide Gel Electrophoresis and Western Blotting Techniques
Polyacrylamide gel electrophoresis (PAGE) was performed on 3.75% (wt/vol) (for both nonreducing PAGE and SDS-PAGE) or 3.75% to 10% polyacrylamide gradient (for SDS-PAGE only) gels with electrophoresis at 150 V for 90 minutes in a Bio-Rad mini blot chamber.³¹ Samples for SDS-PAGE were treated with sample buffer (0.1 mol/L Tris/HCl, pH 6.8, 4% SDS, and 20% glycerol) at a ratio of 1:1, vol/vol. After addition of 2-mercaptoethanol (final concentration, 3%), the samples were incubated at 95°C for 5 minutes prior to application to the gels, which were later stained with Coomassie Brilliant Blue. For Western blotting experiments, proteins were electrophoretically transferred to NC membranes,³² incubated with polyclonal Abs or MoAbs, and detected with peroxidase-conjugated anti-mouse or anti-rabbit IgGs using 4-chloro-1-naphthol or TMB³³ as the substrate.

ELISA Techniques
For indirect ELISA, Maxisorp 96-well plates (Nunc) were coated, (250 µL per well) with N- or modified LDL (1 µg LDL protein per milliliter) in buffer A (PBS, pH 7.4, 1 mg/mL EDTA, and 10 µmol/L Trolox-C) for 18 hours at 4°C. Plates were washed four times with buffer B (PBS, pH 7.4, containing an additional 21.2 g/L NaCl and 0.05% [vol/vol] Tween 20) and incubated for 30 minutes at 25°C with 150 µL buffer C (buffer A containing 0.5% [wt/vol] standard-quality BSA) to block the free binding sites. After addition of polyclonal Abs or hybridoma supernatant (50 µL per well) at an appropriate dilution (1:10 to 1:100 000 in buffer C), the plates were incubated at 37°C for 2 hours. Buffer B was used to wash the plates four times, 150 µL peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG (diluted 1:20 000 in buffer C) was added to each well, and the plates were then incubated at 37°C for 45 minutes. Plates were washed four times with buffer C and then once with buffer D (citric acidxH₂O, 5.1 g/L; Na₂HPO₄x2 H₂O, 9.15 g/L; pH 5.0). The color was developed (100 µL per well) with chromogen solution (buffer D containing 0.1 mg TMB per milliliter and 1.8 mmol/L H₂O₂) for 10 to 20 minutes at 25°C and stopped by addition of 50 µL of 2 mol/L H₂SO₄. Absorbance was read at 450 nm using a Hamilton 7000 microplate reader.

For competitive ELISA, the plates were coated, washed, and blocked as described for indirect ELISA. Different concentrations of competitor (50 µL of N- as well as HNE-, MDA-, or Cu²⁺-ox–LDL) diluted in buffer C (12.5 ng to 12.5 µg protein per well) were added, followed by addition of 50 µL hybridoma supernatant at an appropriate dilution (1:100 to 1:100 000 in buffer C), and the plates were incubated for 2 hours at 37°C. Subsequent washing steps, secondary Ab reaction, and color development were performed as described above.

Subtyping of MoAbs was performed by using ultrapure rabbit anti-mouse subclass-specific antisera to mouse IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, -chain, or -chain (Bio-Rad) according to the manufacturer's protocol.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Chemical characterization of the different modified LDL preparations (ie, lysine modification, state of apo B, cholesteryl ester modification, loss of vitamin E, and relative electrophoretic mobility) for the present study is summarized in the Table. These findings are in good agreement with data previously published by us^{14 15 27 28 29} and others.^{34 35}

View this table: [in this window] [in a new window]   Table 1. Characteristics of Variously Modified LDL

We first examined the specificity of polyclonal Abs on Western blots by using the crude antisera of rabbits that had been immunized with human LDL oxidized with 800 HOCl molecules per LDL particle and equal amounts of MDA-LDL (data not shown), Cu²⁺-ox–LDL, HOCl-LDL, HNE-LDL, and N-LDL as antigens (Fig 1). With the exception of Cu²⁺-ox–LDL, which gave a diffuse smear (Fig 1, lane 4), protein bands with more or less similar intensities were seen, demonstrating that these polyclonal antisera were highly cross-reactive. A broad band at a molecular mass higher than that for apo B-100 was observed when we used LDL that had been modified with 400 to 800 molecules of HOCl per LDL. Because SDS-PAGE was carried out under reducing conditions, such high-molecular-weight bands indicate nondisulfide cross-links of apo B-100, as has been observed previously.²⁰ In contrast to auto-oxidation³⁶ or copper oxidation,³¹ fragmentation of apo B-100 was not detected in HOCl-LDL (Fig 1, lane 3). Antisera raised against LDL modified with 200 or 400 HOCl molecules per LDL gave results similar to those shown in Fig 1 (not shown).

View larger version (64K): [in this window] [in a new window]   Figure 1. Cross-reactivity of polyclonal antibodies raised against hypochlorous acid-modified LDL (HOCl-LDL) with aldehyde-modified and copper-oxidized LDL. Candidate antigens (5 µg) were tested with polyclonal anti-human HOCl-LDL antisera (1:100 dilution) after SDS–polyacrylamide gel electrophoresis of antigens on a 3.75% linear slab gel and transfer to nitrocellulose. The antigens used were native LDL (lane 1), 4-hydroxynonenal–LDL (2 mmol/L 4-hydroxynonenal, lane 2), HOCl-LDL (800 HOCl per 1 LDL, lane 3), and Cu2+-oxidized LDL (16 mol Cu2+ per mol LDL, lane 4). Arrow indicates the position of apo B-100. Antibodies were visualized with peroxidase-conjugated goat anti-rabbit IgGs.

One rabbit antiserum was also assayed for specificity by indirect ELISA. For HOCl-LDL, 50% of the maximal binding occurred at a dilution of approximately 1:100 000 (Fig 2). N-LDL, Cu²⁺-ox–LDL, and HNE-LDL were somewhat less well recognized, with half-maximal binding at dilutions of 1:6000 to 1:15 000 (Fig 2). MDA-LDL as the antigen gave 50% maximal binding at a dilution of 1:7000, whereas nonspecific binding of the antiserum to uncoated wells was negligible (Fig 2).

View larger version (13K): [in this window] [in a new window]   Figure 2. Semilog plot from indirect enzyme-linked immunosorbent assay, showing cross-reactivity of anti–hypochlorous acid–modified LDL (anti–HOCl-LDL) antiserum with native (N) and forms of modified LDL. The antigens used were HOCl-LDL (800 HOCl molecules per 1 LDL particle; —), N-LDL (—), 4-hydroxynonenal–LDL (2.5 mmol/L 4-hydroxynonenal; —), malondialdehyde-LDL (5 mmol/L malondialdehyde; <——>), Cu2+-oxidized LDL (16 mol Cu2+ per mol LDL; ——), and nonspecific binding (——). Microtiter plates were coated with antigens (1 µg protein per milliliter), and the bound antiserum was detected by peroxidase-conjugated goat anti-rabbit IgGs with tetramethylbenzidine as a substrate, as described in "Methods." O.D. indicates optical density.

Indirect ELISA experiments performed with HOCl-LDL–coated plates and polyclonal antisera raised against Ac-, Cu²⁺-ox–, and HNE-LDL showed strong cross-reactivity (Fig 3). Attempts to increase the specificity of HOCl-LDL antisera by affinity chromatography with HOCl-LDL–bound Sepharose did not produce an IgG fraction that preferentially recognized HOCl-LDL (not shown). This was not surprising, on the basis of the cross-reactivity shown in Fig 2, and suggested that attempts to purify a MoAb from this antiserum would be difficult, if not impossible. We therefore decided to raise MoAbs that recognize HOCl-LDL.

View larger version (12K): [in this window] [in a new window]   Figure 3. Polyclonal antisera raised against various forms of modified LDL are cross-reactive with hypochlorous acid–modified LDL (HOCl-LDL). Microtiter plates were coated with HOCl-LDL (1 µg/mL) before addition of the antisera. The antisera (——, polyclonal anti–acetylated LDL; —, polyclonal anti–Cu2+-oxidized LDL; ——, polyclonal anti–4-hydroxynonenal LDL; and —, polyclonal anti–HOCl-LDL) were diluted 1:100 to 1:100 000. Detection and measurement were as described in "Methods." O.D. indicates optical density.

Initial screening of clones by competitive ELISA revealed 146 clones that recognized HOCl-LDL but not N-LDL. Several of these clones also showed cross-reactivity with Cu²⁺-ox–LDL (not shown) and therefore were excluded. Two clones, MoAb 1B10A11 (MoAb-A) and MoAb 2D10G9 (MoAb-B), with no cross-reactivity to Cu²⁺-ox–LDL or N-LDL, high titers to HOCl-LDL, and robust growth were selected and further characterized. Subtype determination revealed that MoAb-A belonged to subtype IgG1 and MoAb-B to IgG2b. The specificity of these two clones was initially examined by indirect ELISA experiments. Titration curves were established with serial dilutions of hybridoma cell supernatants (1:5 to 1:100 000) on HOCl-LDL–coated microtiter plates and are shown in Fig 4. MoAb-A gave half-maximal binding at a supernatant dilution of 1:200 (Fig 4a), whereas MoAb-B gave half-maximal binding at a dilution of 1:2000 (Fig 4b). Neither Cu²⁺-ox–, HNE-, MDA-, nor N-LDL bound to the Abs at these dilutions, as shown by the ELISA titration curves (Fig 4). MoAb-A and -B also showed no measurable binding to uncoated plates.

View larger version (13K): [in this window] [in a new window]   Figure 4. Supernatants containing monoclonal antibody (MoAb) A (a) and MoAb-B (b) do not cross-react with native (N), aldehyde-modified, or Cu2+-oxidized LDL (Cu2+-ox–LDL). Antigens (1 µg/mL) were coated onto microtiter plates before serial dilutions (from 1:5 to 1:78 125) of the hybridoma supernatants were added. The antigens used were Cu2+-ox–LDL (16 mol Cu2+ per mol LDL; ——); N-LDL (—); 4-hydroxynonenal LDL (5 mmol/L 4-hydroxynonenal; —); hypochlorous acid–modified LDL (800 HOCl molecules per LDL particle; —); and malondialdehyde LDL (20 mmol/L MDA; ——). Antigen binding was detected using peroxidase-conjugated rabbit anti-mouse IgGs with tetramethylbenzidine as a substrate. O.D. indicates optical density.

The specificity of MoAb-A and -B was tested further by Western blotting experiments. Protein staining of HOCl-, Ac-, Cu²⁺-ox–, HNE-, MDA-, and N-LDL separated by 3.75% PAGE under nondenaturing conditions and subsequently transferred to NC revealed a single band at a similar position, suggesting that none of the LDL modifications had an effect on the aggregation of the lipoprotein. Subsequent incubation of NC membranes with MoAb-A or MoAb-B (working dilution, 1:10) gave positive staining only with HOCl-LDL; the results obtained with MoAb-A are shown in Fig 5a.

View larger version (44K): [in this window] [in a new window]   Figure 5. Lack of cross-reactivity of monoclonal antibody (MoAb) A and B with native (N), aldehyde-modified, or Cu2+-oxidized LDL (Cu2+-ox–LDL) as analyzed by Western blotting. Antigens (5 µg protein) were separated by nondenaturing polyacrylamide gel electrophoresis (PAGE, a) or SDS-PAGE (b) on 3.75% linear polyacrylamide gels, transferred to nitrocellulose, detected with MoAb-A (a) or MoAb-B (b), and visualized with peroxidase-conjugated goat anti-mouse IgGs with tetramethylbenzidine (TMB) as a substrate. Visualization of native apo B-100 was performed by incubation of nitrocellulose with polyclonal rabbit anti-human LDL antibodies and peroxidase-conjugated goat anti-rabbit IgGs with TMB as a substrate. The antigens used were acetylated LDL (lane 1), Cu2+-ox–LDL (lane 2), 4-hydroxynonenal-LDL (lane 3), hypochlorous acid–modified LDL (HOCl-LDL, lane 4), malondialdehyde-LDL (lane 5), and N-LDL (lane 6). Bold arrow indicates the position of apo B-100. Smaller arrow (marked with * in b) indicates the presence of HOCl-LDL in the stacking gel.

To examine whether denaturation of HOCl-LDL affected the immunoreactivity of the two MoAbs, different concentrations of HOCl-LDL (0.05 to 5 µg LDL protein) in the absence or presence of SDS were dotted onto NC, reacted with MoAb-A or MoAb-B, and visualized with peroxidase-conjugated goat anti-mouse IgGs. Immunoreactivity of MoAb-A toward SDS-treated HOCl-LDL was significantly reduced compared with untreated HOCl-LDL samples. In contrast, MoAb-B recognized HOCl-LDL whether SDS was present or not (not shown). Western blotting of HOCl-LDL run on a 3.75% SDS-PAGE and detected with MoAb-B (Fig 5b) gave a broad band with an apparent molecular mass slightly higher than that of native apo B-100, together with a band present in the stacking gel. The latter was probably due to cross-linked HOCl-LDL.¹⁸ Consistent with the aforementioned results, MoAb-A did not detect HOCl-LDL after SDS-PAGE (not shown).

To test whether the MoAbs were specific for HOCl-LDL, BSA (fatty-acid free) or HSA (fatty-acid free) was modified at 4°C with 50 or 100 mol HOCl per mol protein. This results in the same ratio of HOCl per amino acid (mol/mol) as in LDL modified with 400 or 800 mol HOCl per mol LDL. Whereas MoAb-B recognized both HOCl-BSA (fatty-acid free; Fig 6b) and HOCl-HSA (fatty-acid free; data not shown) by indirect ELISA to an extent similar to HOCl-LDL, MoAb-A recognized neither HOCl-BSA (fatty-acid free; Fig 6a) nor HOCl-HSA (fatty-acid free; data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 6. Lack of cross-reactivity of monoclonal antibody (MoAb) A (a) and MoAb-B (b) supernatants with variably oxidized hypochlorous acid (HOCl)–BSA (fatty-acid free) and HOCl-LDL, as judged by indirect ELISA. Antigens (1 µg/mL) were coated onto microtiter plates, serial dilutions (from 1:5 to 1:78 125) of hybridoma supernatants added, the plates incubated, and the bound antibodies detected as described in "Methods." The antigens used were HOCl-LDL (800 HOCl molecules per LDL particle; —), HOCl-LDL (400 HOCl molecules per LDL particle; —), "old" HOCl-LDL (800 HOCl molecules per LDL particle; ——), HOCl-BSA (100 HOCl molecules per BSA molecule; —), and HOCl-BSA (50 HOCl molecules per BSA molecule; ——). O.D. indicates optical density.

The immunoreactivity of MoAb-A toward HOCl-LDL depended on both the degree and duration of HOCl modification. The highest response was observed when LDL was treated with 800 HOCl molecules per lipoprotein particle and subsequently stored for 7 days at 4°C (Fig 6a). Decreased recognition was observed with freshly prepared HOCl-LDL (800 HOCl molecules per LDL particle), whereas freshly prepared HOCl-LDL (400 HOCl molecules per LDL particle) showed less immunoreactivity in indirect ELISA. In contrast, MoAb-B recognized HOCl-LDL to the same extent whether it was oxidized with 400 or 800 mol HOCl per mol LDL, and no effect of storage of HOCl-LDL was observed under our assay conditions (Fig 6b).

To further study to what extent the formation of secondary products from HOCl might alter epitope recognition, freshly prepared HOCl-LDL (400 and 800 HOCl molecules per LDL particle) at 4°C was incubated at 37°C for different periods of time. For MoAb-A, a substantial increase in immunoreactivity was achieved by incubation of freshly prepared HOCl-LDL (800 HOCl molecules per LDL particle) for as long as 6 hours (Fig 7a). LDL that had been modified with 400 HOCl molecules per LDL particle similarly increased its immunoreactivity with MoAb-A upon incubation. On the other hand, the incubation time at 37°C showed only negligible influence of immunoreactivity of MoAb-B toward LDL that had been modified with 800 HOCl molecules per LDL particle (Fig 7b) or 400 HOCl molecules per LDL particle.

View larger version (17K): [in this window] [in a new window]   Figure 7. Time-dependent development of epitopes recognized by monoclonal antibody (MoAb) A (a) and MoAb-B (b) supernatants on hypochlorous acid (HOCl)–modified LDL. LDL was oxidized at 4°C with 800 HOCl molecules per LDL particle and then incubated at 37°C for up to 6 hours. Samples were taken after 0 (—), 0.5 (——), 1 (—), 2 (——), 4 (—), and 6 (——) hours of incubation and coated onto microtiter plates (1 µg/mL); serial dilutions (from 1:5 to 1:78 125) of hybridoma supernatants were added, the plates incubated, and the bound antibodies detected as described in "Methods."

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In situ identification of ox-LDL by the use of Abs directed against various types of the oxidized lipoprotein^{5 6 7 8 37} has provided important support for the oxidative theory of atherosclerosis.² The usefulness of these immunohistochemical studies for identification of the putative in vivo oxidant responsible for LDL oxidation is, however, uncertain, owing to the complex nature of the tissue samples examined and the necessarily limited extent of epitopes tested for cross-reactivity. The MoAbs that were selected and characterized in this study showed high selectivity for HOCl-LDL, as their cross-reactivity with N-LDL and other forms of ox-LDL (ie, HNE-, MDA-, and Cu²⁺-ox–LDL) was negligible (Figs 4 and 5). In particular, MoAb-A appeared to be specific for HOCl-LDL, as it did not recognize HOCl-modified HSA (fatty-acid free) or BSA (fatty-acid free). Also, the extent of MoAb-A reactivity increased with an increasing degree of LDL oxidation (Fig 6a) and incubation time with HOCl (Fig 7a) and was lost when the lipoprotein particle disintegrated. Because HOCl is consumed within 5 minutes or less after its addition to LDL, these findings indicate that the epitope recognized by MoAb-A was derived, at least in part, from secondary reaction(s) of the oxidant with LDL and was thus dependent on the tertiary structure of the lipoprotein. Therefore, MoAb-A is likely to be specific for HOCl-LDL. MoAb-B, on the other hand, gave identical titration curves with HOCl-LDL, HOCl-BSA, and HOCl-HSA when used at the same protein concentration per well (Fig 6b). Furthermore, the epitopes recognized by MoAb-B were formed rapidly and fully at relatively low concentrations of the oxidant (Figs 6b and 7b), suggesting that this Ab recognized those epitopes that are commonly generated on proteins that have been oxidized with HOCl. Together, our data suggest that MoAb-A and -B, specific for HOCl-LDL and HOCl-protein, respectively, may be useful tools for studying the occurrence and hence, potential relevance of HOCl-mediated (lipo)protein damage in various inflammatory disorders.

A striking feature of our studies was the strong cross-reactivity of polyclonal and even some of the monoclonal anti-HOCl Abs with HNE-, MDA-, and Cu²⁺-ox–LDL and vice versa. This apparent high degree of cross-reactivity of polyclonal anti–HOCl-LDL Abs with HNE-, MDA-, and Cu²⁺-ox–LDL may be rationalized on the basis of the similar chemical modifications that are induced by these oxidants. We have shown that although treatment of LDL with HOCl does not lead to substantial lipid oxidation,^{14 15} large amounts of chloramines are formed initially.¹⁵ Some of these chloramines are converted to intermediate imines, which themselves can produce aldehydes. We propose that these protein aldehydes give rise to intramolecular and intermolecular cross-links via reversible formation of Schiff bases with an (unoxidized) lysine or histidine residue, if this is followed by irreversible reduction.¹⁵ The resulting structure may show some similarity with that produced from interaction of lipid aldehydes, such as MDA and HNE, or those produced during copper-mediated oxidation of LDL with apo B-100.²⁷

A number of previous studies employed polyclonal Abs and MoAbs in attempts to localize MDA-, HNE-, and Cu²⁺-ox–LDL in biological samples, such as atherosclerotic lesions from WHHL rabbits^{5 6 7 10 38} and humans^{8 38} and WHHL rabbit and human plasma^{10 38} and from synovial tissue from subjects suffering from rheumatoid arthritis.³⁹ Although the Abs used were tested for cross-reactivity with at least some additional antigens,¹³ none of the studies mentioned above included HOCl-modified (lipo)protein in the panel of antigens tested. Because our studies demonstrated a large degree of cross-reactivity of anti–HNE- and Cu²⁺-ox–LDL Abs with HOCl-LDL and because HOCl may be formed at inflammatory sites where aldehyde- or radical-mediated damage has been suspected, HOCl-modified (lipo)proteins may have been responsible for at least some of the epitopes recognized by the anti–aldehyde- and Cu²⁺-ox–LDL antibodies used.

Hypochlorite reacts with a wide range of biological molecules.⁴⁰ Exposure of mouse peritoneal macrophages to HOCl-ox–LDL resulted in increased intracellular concentrations of cholesterol and cholesteryl esters¹⁴ ; cholesterol engorged "foam cells," the hallmark of "fatty streaks," are the first histologically recognizable stage of atherogenesis.^{2 3} Whether HOCl-LDL is relevant to the process of atherosclerosis in humans remains to be seen. While it is generally believed that neutrophils are not present in significant numbers, a recent report showed that MPO, the enzyme that produces HOCl from H₂O₂ and Cl^-, is present in large amounts in human atherosclerotic lesions.¹⁹ MPO may be derived from freshly recruited monocytes.³⁹ That LDL modification may be independent of formation of lipid hydroperoxides is also supported by the finding that early atherosclerosis is accompanied by a decreased rather than an increased accumulation of fatty acid hydroxy derivatives.⁴¹

In contrast to the situation in atherosclerosis, neutrophils have been implicated in causing part of the symptoms or tissue damage in rheumatoid arthritis, myocardial infarction, ischemia/reperfusion, inflammatory bowel disease, and asthma (for a review, see Reference 42⁴² ). These cells contain MPO as a major constituent and, like monocytes, can release this protein upon appropriate activation. For example, MPO and/or HOCl have been implicated specifically in causing damage to the sarcoplasmic reticulum after ischemia/reperfusion⁴³ ; inactivating the 1-protease inhibitor, thereby allowing other neutrophil enzymes to inflict endothelial cell damage⁴⁴ ; crosslinking immune complexes⁴⁵ ; and modulating the inflammatory response by oxidation of complement.⁴⁶ Assessment of the involvement of oxidants in tissue damage in vivo has been difficult, and traditional methods have relied on indirect studies using oxidant scavengers or inhibitors of oxidant production. The HOCl-specific Abs presented in this article may be useful tools for the investigation of the role of HOCl-mediated damage in these and other diseases.

	Acknowledgments

 
This investigation received support from the Austrian Fonds zur Förderung der Wissenschaftlichen Forschung P 8433 MED and P 11276 MED (to Dr Malle), P 10145 MED (to Dr Sattler), and SO 7102 MED (to Drs Esterbauer and Waeg); the National Health and Medical Research Council of Australia (grants 910284 and 940915 to Dr Stocker); and the Community Health and Anti-Tuberculosis Society (to Dr Hazell). We also thank the Faculty of Medicine, University of Sydney, for travel funding. The authors are indebted to Edith Pursch for invaluable help in setting up MoAb techniques and the Franz Lanyar Foundation, KFU Graz.

	Footnotes

 
Reprint requests to Ernst Malle, Karl-Franzens University, Institute of Medical Biochemistry, Harrachgasse 21, A-8010 Graz, Austria.

Received November 14, 1994; accepted March 22, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Munro JM, Cotran S. The pathogenesis of atherosclerosis: atherogenesis and inflammation. Lab Invest. 1988;58:249-261. [Medline] [Order article via Infotrieve]

2. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modification of low-density lipoprotein that increase its atherogenicity. N Engl J Med. 1989;320:915-924. [Medline] [Order article via Infotrieve]

3. Steinbrecher UP, Zhang H, Lougheed M. Role of oxidatively modified LDL in atherosclerosis. Free Radic Biol Med. 1990;9:155-168. [Medline] [Order article via Infotrieve]

4. Luc G, Fruchart JC. Oxidation of lipoproteins and atherosclerosis. Am J Clin Nutr. 1991;53:206S-209S. [Abstract/Free Full Text]

5. Boyd HC, Gown AM, Wolfbauer G, Chait 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. [Abstract]

6. Rosenfeld ME, Palinski W, Ylä-Herttuala S, Butler S, Witztum JL. Distribution of oxidation specific lipid-protein adducts and apolipoprotein B in atherosclerotic lesions of varying severity from WHHL rabbits. Arteriosclerosis. 1990;10:336-349. [Abstract/Free Full Text]

7. Rosenfeld ME, Khoo JC, Miller E, Parthasarathy S, Palinski W, Witztum JL. Macrophage-derived foam cells freshly isolated from rabbit atherosclerotic lesions degrade modified lipoproteins, promote oxidation of low-density lipoproteins, and contain oxidation-specific lipid-protein adducts. J Clin Invest. 1991;87:90-99.

8. Jürgens G, Chen Q, Esterbauer H, Mair S, Ledinski G, Dinges HP. Immunostaining of human autopsy aortas with antibodies to modified apolipoprotein B and apoprotein(a). Arterioscler Thromb. 1993;13:1689-1699. [Abstract/Free Full Text]

9. Salmon S, Maziere C, Theron L, Beucler I, Ayrault-Jarrier M, Goldstein S, Polonovski J. Immunological detection of low-density lipoproteins modified by malondialdehyde in vitro or in vivo. Biochim Biophys Acta. 1987;920:215-220. [Medline] [Order article via Infotrieve]

10. Haberland ME, Fong D, Cheng L. Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits. Science. 1988;241:215-218. [Abstract/Free Full Text]

11. Palinski W, Rosenfeld ME, Ylä-Herttuala S, Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witztum JL. Low density lipoprotein undergoes oxidative modification in vivo. Proc Natl Acad Sci U S A. 1989;86:1372-1376. [Abstract/Free Full Text]

12. Salonen JT, Ylä-Herttuala S, Yamamoto R, Butler S, Korpela H, Salonen R, Nyyssönen K, Palinski W, Witztum JL. Autoantibodies against oxidised LDL and progression of carotid atherosclerosis. Lancet. 1992;339:883-887. [Medline] [Order article via Infotrieve]

13. Vaarala O, Alfthan G, Jauhiainen M, Leirisalo-Repo M, Aho K, Palosuo T. Crossreaction between antibodies to oxidised low-density lipoprotein and to cardiolipin in systemic lupus erythematosus. Lancet. 1993;341:923-925. [Medline] [Order article via Infotrieve]

14. Hazell LJ, Stocker R. Oxidation of low-density lipoprotein with hypochlorite causes transformation of the lipoprotein into a high-uptake form for macrophages. Biochem J. 1993;290:165-172.

15. Hazell LJ, van den Berg JJM, Stocker R. Oxidation of low-density lipoprotein by hypochlorite causes aggregation that is mediated by modification of lysine residues rather than lipid oxidation. Biochem J. 1994;302:297-304.

16. Lampert MB, Weiss SJ. The chlorinating potential of the human monocyte. Blood. 1983;62:645-651. [Abstract/Free Full Text]

17. Thomas EL, Grisham MB, Jefferson MM. Myeloperoxidase-dependent effects of amines on functions of isolated neutrophils. J Clin Invest. 1983;72:441-445.

18. Gerrity RG. The role of monocytes in atherogenesis, I: transition of blood-borne monocytes into foam cells in fatty lesions. Am J Pathol. 1981;103:181-190. [Abstract]

19. Daugherty A, Dunn JL, Rateri DL, Heinecke JW. Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions. J Clin Invest. 1994;94:437-444.

20. O'Connell AM, Gieseg SP, Stanley KK. Hypochlorite oxidation causes cross-linking of Lp(a). Biochim Biophys Acta. 1994;1225:180-186. [Medline] [Order article via Infotrieve]

21. Kikugawa K, Kato T, Hayasaka A. Formation of dityrosine and other fluorescent amino acids by reaction of amino acids with lipid hydroperoxides. Lipids. 1991;26:922-929. [Medline] [Order article via Infotrieve]

22. Malle E, Ibovnik A, Steinmetz A, Kostner GM, Sattler W. Identification of glycoprotein IIb as the lipoprotein(a)-binding protein on platelets: lipoprotein(a) binding is independent of an arginyl-glycyl-aspartate tripeptide located in apolipoprotein(a). Arterioscler Thromb. 1994;14:345-352. [Abstract/Free Full Text]

23. Jürgens G, Lang H, Esterbauer H. Modification of human low-density lipoprotein by the lipid peroxidation product 4-hydroxynonenal. Biochim Biophys Acta. 1986;875:103-114. [Medline] [Order article via Infotrieve]

24. Haberland ME, Oleh CL, Fogelman AM. Role of lysines in mediating interaction of modified low density lipoproteins with the scavenger receptor of human monocyte macrophages. J Biol Chem. 1984;259:11305-11311. [Abstract/Free Full Text]

25. Puhl H, Waeg G, Esterbauer H. Methods to determine oxidation of low-density lipoproteins. Methods Enzymol. 1994;233:425-441. [Medline] [Order article via Infotrieve]

26. Basu SK, Goldstein JL, Anderson RGW, Brown MS. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc Natl Acad Sci U S A. 1976;73:3179-3182.

27. Esterbauer H, Gebicki J, Puhl H, Jürgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic Biol Med. 1992;13:341-390. [Medline] [Order article via Infotrieve]

28. Malle E, Ibovnik A, Leis HJ, Kostner GM, Verhallen PFJ, Sattler W. Lysine modification of low density lipoproteins or lipoprotein(a) by 4-hydroxynonenal or malondialdehyde decreases platelet serotonin secretion without affecting platelet aggregability and eicosanoid formation. Arterioscler Thromb Vasc Biol. 1995;15:377-384. [Abstract/Free Full Text]

29. Sattler W, Mohr D, Stocker R. Rapid isolation of lipoproteins and assessment of their peroxidation by high-performance liquid chromatography postcolumn chemiluminescence. Methods Enzymol. 1994;233:469-489. [Medline] [Order article via Infotrieve]

30. Ostberg L, Pursch E. Humanx(mousexhuman) hybridomas stably producing human antibodies. Hybridoma. 1983;2:361-367. [Medline] [Order article via Infotrieve]

31. Sattler W, Kostner GM, Waeg G, Esterbauer H. Oxidation of lipoprotein Lp(a). A comparison with low-density lipoproteins. Biochim Biophys Acta. 1991;1081:65-74.[Medline] [Order article via Infotrieve]

32. Malle E, Heß H, Münscher G, Knipping G, Steinmetz A. Purification of serum amyloid A and its isoforms from human plasma by hydrophobic interaction chromatography and preparative isoelectric focusing. Electrophoresis. 1992;13:422-428. [Medline] [Order article via Infotrieve]

33. McKimm-Breschkin JL. The use of tetramethylbenzidine for solid phase immunoassays. J Immunol Methods. 1990;135:277-280.[Medline] [Order article via Infotrieve]

34. Vanderyse L, Devreese AM, Baert J, Vanloo B, Lins L, Ruyaschaert JM, Rosseneu M. Structural and functional properties of apolipoprotein B in chemically modified low density lipoproteins. Atherosclerosis. 1992;97:187-199. [Medline] [Order article via Infotrieve]

35. Frei B, Gaziano JM. Content of antioxidants, preformed lipid hydroperoxides, and cholesterol as predictors of the susceptibility of human LDL to metal ion-dependent and -independent oxidation. J Lipid Res. 1993;34:2135-2145.[Abstract]

36. Schuh J, Fairclough GF Jr, Haschmeyer RH. Oxygen-mediated heterogeneity of apo-low-density lipoprotein. Proc Natl Acad Sci U S A. 1978;75:3173-3177. [Abstract/Free Full Text]

37. Itabe H, Takeshima E, Iwasaki H, Kimura J, Yoshida Y, Imanaka T, Takano T. A monoclonal antibody against oxidized lipoprotein recognizes foam cells in atherosclerotic lesions: complex formation of oxidized phosphatidylcholines and polypeptides. J Biol Chem. 1994;269:15274-15279. [Abstract/Free Full Text]

38. Ylä-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989;84:1086-1095.

39. Winyard PG, Tatzber F, Esterbauer H, Kus ML, Blake DR, Morris CJ. Presence of foam cells containing oxidised low density lipoprotein in the synovial membrane from patients with rheumatoid arthritis. Ann Rheum Dis. 1993;52:677-680. [Abstract/Free Full Text]

40. Winterbourn CC. Comparative reactivities of various biological compounds with myeloperoxidase-hydrogen peroxide-chloride, and similarity of the oxidant to hypochlorite. Biochim Biophys Acta. 1985;840:204-210. [Medline] [Order article via Infotrieve]

41. De Meyer GR, Bult H, Herman AG. Early atherosclerosis is accompanied by a decreased rather than an increased accumulation of fatty acid hydroxyderivatives. Biochem Pharmacol. 1991;42:279-283. [Medline] [Order article via Infotrieve]

42. Malech HL, Gallin JI. Current concepts: immunology—neutrophils in human diseases. N Engl J Med. 1987;317:687-694. [Medline] [Order article via Infotrieve]

43. Kukreja RC, Weaver AB, Heiss ML. Stimulated human neutrophils damage cardiac sarcoplasmic reticulum function by generation of oxidants. Biochim Biophys Acta. 1989;990:198-205. [Medline] [Order article via Infotrieve]

44. Stroncek DF, Vercellotti GM, Huh PW, Jacob HS. Neutrophil oxidants inactivate alpha-1-protease inhibitor and promote PMN-mediated detachment of cultured endothelium: protection by free methionine. Arteriosclerosis. 1986;6:332-340. [Abstract/Free Full Text]

45. Jasin HE. Oxidative cross-linking of immune complexes by human polymorphonuclear leukocytes. J Clin Invest. 1988;81:6-15.

46. Clark RA. Modulation of the inflammatory response by the neutrophil myeloperoxidase system. In: Rossi F, Patriarca P, eds. Biochemistry and Function of Phagocytes. Adv Exp Med Biol. New York, NY: Plenum Press; 1982;141:207-216.

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