(Circulation. 2000;102:1639.)
© 2000 American Heart Association, Inc.
Clinical Investigation and Reports |
Development of Antibody Against Epitope of Lipoprotein(a) Modified by Oxidation
Evaluation of New Enzyme-Linked Immunosorbent Assay for Oxidized Lipoprotein(a)
From the Central Institute (S.Y.), Shino-Test Corporation, Sagamihara, Japan; Department of Geriatric Medicine (R.M., S.N., T.O.), Osaka University Medical School, Osaka, Japan; Department of Pathology (Y.K., I. Sakurai), Nihon University School of Medicine, Tokyo, Japan; and Department of Clinical Laboratories (N.K.), Ohmiya Medical Center, Jichi Medical School, Ohmiya-shi, Japan.
Correspondence to Shingo Yamada, Shino-Test Corporation, 2-29-14 Oonodai, Sagamihara-shi, 229-0011, Japan. E-mail RD{at}shino-test.co.jp
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Abstract |
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Background—Recently, the biological effects of oxidized lipoprotein(a) [Lp(a)] have been reported to be more potent than Lp(a), the arteriosclerosis-relevant lipoprotein. Thus, investigations with oxidized Lp(a) are expected to provide viewpoints different from the conventional ones based on Lp(a).
Methods and Results—An anti-Lp(a) monoclonal antibody (161E2) was produced against synthetic peptide antigen (Arg-Asn-Pro-Asp-Val-Ala-Pro). This epitope was characterized as having various properties because its external exposure was induced as a result of oxidative modification. Using 161E2 antibody, we developed a new enzyme-linked immunosorbent assay to measure Lp(a) modified by oxidative stress. The present data demonstrated that oxidized Lp(a) that contains the epitope of 161E2 antibody was present in the serum of humans. Therefore, we used this new enzyme-linked immunosorbent assay to evaluate the role of oxidized Lp(a) in patients with hypertension, which induces oxidative stress . Interestingly, hypertensive patients with complications showed a significantly higher level of oxidized Lp(a) in serum than did normotensive subjects (P<0.01), whereas there was no significant difference in native Lp(a) between normotensive and hypertensive subjects. Importantly, positive immunostaining with 161E2 monoclonal antibody was found in the human arteriosclerotic tissue.
Conclusions—We developed a new antibody against an epitope in Lp(a) as a result of oxidation treatment but not in native Lp(a). The present data demonstrated in vivo the presence of oxidized Lp(a) in the atherosclerotic tissue and its elevation in hypertensive patients. The presence of oxidized Lp(a) may be important in understanding the role of Lp(a) in cardiovascular disease.
Key Words: lipoprotein(a) • immunoassay • monoclonal antibody • atherosclerosis • hypertension
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Introduction |
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Lipoprotein(a) [Lp(a)] is a complex of LDL in which the integral apoB-100 is linked to apo(a) via disulfide bridges. Because apo(a) has a high degree of homology with plasminogen,1 human apo(a) and plasminogen mutually compete for plasminogen receptors on endothelial cells.2 Thus, a high circulating Lp(a) level may inhibit thrombolysis and fibrin clearance. Indeed, a high serum concentration of Lp(a) is a risk factor for cardiovascular disease.3 4 5 Recently, numerous types of stress, such as oxidation, have been shown to be involved in the development of atherosclerosis. For example, oxidized LDL due to oxidative stress such as that in hypertension and diabetes, but not native LDL, has been postulated as an atherosclerosis-relevant lipoprotein. Other lipoproteins, such as Lp(a), would also be modified by oxidation. Expectedly, oxidative modification enhances the inhibitory effect of Lp(a) on plasminogen binding to U937 cell surfaces.6 These findings suggest that the oxidative form of Lp(a), rather than native Lp(a), may attenuate fibrinolytic activity through the reduction of plasminogen activation. The contribution of oxidized Lp(a) to the development of atherosclerosis is supported by several lines of evidence: (1) oxidative modification of Lp(a) enhanced Lp(a)-induced plasminogen activator inhibitor-1 (PAI-1) production in endothelial cells,7 because native Lp(a) itself increased PAI-1 production in endothelial cells8 ; (2) oxidized Lp(a) impaired endothelium-dependent vasodilatation and was more potent than oxidized LDL9 ; and (3) macrophages took up oxidized Lp(a) via scavenger receptors as well as oxidized LDL.10 However, the exact role of oxidized Lp(a) is still largely unknown due to the lack of a sensitive and specific assay system.
We discovered a monoclonal antibody that recognizes an epitope of modified Lp(a) as a result of oxidation treatment. Because this epitope is hidden on the native Lp(a) molecule, we successfully developed a new enzyme-linked immunosorbent assay (ELISA) system with this antibody that can distinguish native and modified Lp(a), including oxidized Lp(a). Here, we demonstrate the presence of Lp(a) modified by oxidative stress in human serum and its elevation in patients with hypertension and oxidized Lp(a) in the atherosclerotic lesions of humans.
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Methods |
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Production of Anti–Modified Lp(a) Monoclonal Antibody
With a peptide synthesizer (model 430A; Applied Biosystems), the peptide was synthesized according to the t-butoxycarbonylamino acid solid-phase method (Figure 1
). The peptide synthesized was added to
maleimidobenzoyl-N-hydroxysuccinimide ester (Pierce Chemical
Co)–keyhole limpet hemocyanin (KLH; Carbiochem) or
maleimidobenzoyl-N-hydroxysuccinimide ester–BSA (Sigma
Chemical Co). The degree of peptide conjugation to BSA or KLH was 19
peptides/BSA molecule or 1289 to 3250 peptides/KLH molecule,
respectively. BALB/c mice were injected with 50 µg KLH-peptide in
Freund’s complete adjuvant. This intraperitoneal
injection was followed 2 weeks later with an injection of 15 µg
KLH-peptide in Freund’s incomplete adjuvant. Thereafter, the mice were
boosted at 2-week intervals with Lp(a) in saline for 3 months. Mice
were then administered a final intravenous injection of
Lp(a) 3 days before fusion. Fusion was carried out with
1x108 myeloma cells (NS-1) and
1x108 spleen cells.11
After fusion, cells were resuspended in S clone medium (Sanwa
Chemical) supplemented with thymidine (7.6 mg/mL), aminopterin (0.18
mg/mL), and hypoxanthine (13.6 mg/mL) and then distributed into 96-well
microtiter plates (Nunc). Two weeks later, the wells of the microtiter
plates were coated with 300 ng/100 µL BSA-peptide, and the medium was
tested by ELISA.
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Isolation of Plasminogen, LDL, and Lp(a)
Three healthy female subjects with elevated serum Lp(a) levels
(30, 45, and 21 mmol/L) served as donors for Lp(a) isolation. The
phenotypes of Lp(a) were 27, 29, and 34 kringle 4
repeats.12 As previously reported,13 the
following density fractions were isolated: (1) d<1.02, to remove VLDL
and IDL; (2) d=1.02 to 1.05, to obtain LDL; (3) d=1.05 to 1.12, to
obtain Lp(a)-rich fraction; and (4) d>1.21, to obtain
plasminogen from the plasma protein fraction.
Lp(a)-enriched fraction was subjected to lysine-Sepharose 6B affinity
chromatography (Pharmacia).14 The purity
of Lp(a) was evaluated with SDS-PAGE on 2.5% to 16%
polyacrylamide gradient gels (Tefco gel) and agarose gel. The
absolute protein mass of the isolated Lp(a) was determined by amino
acid analysis, and phospholipid, cholesterol (both
free and esterified), and triglyceride analyses
were also performed with a commercial kit (Shino-Test). The total of
protein mass plus lipid for the isolated Lp(a) was taken as the Lp(a)
concentration. Plasminogen was also prepared from the human
serum protein fraction (d>1.21).15
Evaluation of Anti–Modified Lp(a) Monoclonal Antibody (161E2)
Against Various Modified Lp(a)
The oxidation of Lp(a) was carried out with copper
ions16 and lipoxygenase17 as
oxidizing agents. Lp(a) was oxidized through incubation with
CuCl2 (0, 0.1, 0.2, 0.5, 1.0, 2.0, 4.0, 6.0, and
10.0 µg/mL; Wako Chemical) or lipoxygenase (0, 1.0,
3.0, 5.0, and 20.0 µg/mL; Sigma Chemical Co) for 12 hours at 37°C.
The degree of oxidation was quantified through 2 methods: (1) the
increase in relative mobility on agarose gel (Helena Laboratory) or (2)
the formation of thiobarbituric acid–reactive substances (TBARS; Wako
Chemical). During this preparation, we confirmed that Lp(a) was intact
and not degraded. Glycation of purified Lp(a) was also performed
through the incubation Lp(a) (1 mg/mL) with 200 mmol/L glucose in
sterile PBS that contained 1 mmol/L Na2EDTA
(pH 7.4) at 37°C for 7 days.18 The degree of glycation
of Lp(a) was examined according to the agarose gel electrophoresis
method. The purity of Lp(a) (1 mg/mL) was also measured after 6 days of
storage at room temperature and at 37°C and after freeze/thaw cycle
repeated 10 or 30 times.
Electrophoresis and Immunoblotting
Lp(a) and modified Lp(a) were dissolved in 0.9% NaCl of a
concentration of 0.5 mg/mL and subjected to electrophoresis. The
phenotype of Lp(a) was evaluated with a high-resolution
SDS–agarose gel electrophoretic method.19 We used apo(a)
isoform standard human serum (Immuno AG) with different 5-banded
phenotypes as a molecular weight reference to control for
gel-to-gel variation.
ELISA Procedure for Modified Lp(a) and Native Lp(a)
Because this epitope exists at 
2 locations in all
apo(a),1 we used anti–modified Lp(a) monoclonal antibody
for both the solid-phase antibody and labeled antibody to improve
specificity. The monoclonal antibody was labeled with horseradish
peroxidase (Roche) as Immuno Pure Maleimide Activated
Horseradish Peroxidase Kit (Pierce Chemical). Polystyrene microtiter
plates (Nunc) were coated with anti–modified Lp(a) monoclonal antibody
(161E2; 15 µg/mL) or anti-Lp(a) polyclonal antibody (15 µg/mL)
(International Enzyme). The concentration of peptide standard ranged
from 1 to 20 ng/mL. The microtiter plates were incubated for 1 hour at
room temperature, washed, and incubated for 1 hour at room temperature
with anti–modified Lp(a) monoclonal antibody or anti-apo(a) monoclonal
antibody peroxidase conjugate. This monoclonal antibody was insensitive
to apo(a) size heterogeneity, because the detection
antibody reacted with apo(a) kringle 4 type 5-protease domain
(Shino-Test).20 After washing,
3,3'5,5'-tetra-methylbenzidine was added to each well. The enzyme
reaction was allowed to proceed for 30 minutes at room temperature. The
reaction was stopped by stop solution, and the absorbance was read at
450 nm. Lp(a) standard serum (Daiichi Chemical) was used to calibrate
the native Lp(a) ELISA. The assay calibrator was Lp(a) with 17, 27, and
34 kringle 4 repeats. Calculation of the concentration of modified
Lp(a) was based on the concentration of BSA-peptide that contributed to
16 peptides per molecule of BSA as the standard.
Measurement of Modified Lp(a) in Human Serum
One hundred thirteen normotensive control subjects (59 men and
54 women, 59±2 years old) and 223 essential hypertensive patients who
had never been treated (117 men and 106 women, 61±2 years old) were
studied. The normotensive and hypertensive groups were divided into 3
stages of hypertension according to the guidelines of the World Health
Organization (WHO)/International Society of Hypertension in
1993.21 Furthermore, untreated hypertensive patients were
divided into a group of 160 patients without complications (WHO I) and
a group of 63 patients with complications (WHO II/III). Patients with
hepatic disease, lung disease, diabetes mellitus, or
hyperlipidemia were excluded from this study.
Antecubital venous blood was taken during the morning after an
overnight fast. Blood pressure (BP) was measured with a standard
sphygmomanometer in a standardized setting in the clinic in the morning
with the patient supine. BP measurement was repeated 3 times in a
blinded fashion.
Immunohistochemical Staining by Anti–Modified Lp(a) Monoclonal
Antibody (161E2)
The blood vessels were removed 2 hours after death at the
autopsy. Immediately after removal, tissue rings from the aortic arch
were fixed in buffered formalin and paraffin embedded, according to
standard procedures. Subsequently, immunohistochemical staining was
performed with anti-apoB polyclonal antibody (Biostride), anti-Lp(a)
polyclonal antibody (International Enzyme), anti–modified Lp(a)
monoclonal antibody (161E2), anti-rabbit Ig HRP-labeled polyclonal
antibody, anti-goat Ig HRP-labeled polyclonal antibody, or anti-mouse
Ig HRP-labeled polyclonal antibody (DAKO).
Statistical Analysis
All values are expressed as mean±SEM. ANOVA with subsequent
Bonferroni’s test was used to determine the significance of
differences in multiple comparisons. Multiple regression
analyses were used to assess the relation between BP and other
parameters. Values of P<0.05 were considered
statistically significant.
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Results |
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Characterization of Anti–Modified Lp(a) Monoclonal Antibody (161E2)
We selected a peptide that consisted of 9 residues from kringle 4 type 2 of apo(a) (Arg-Asn-Pro-Asp-Ala-Val-Ala-Ala-Pro) by using prediction methods for the position of antigenic sites in proteins (Figure 1
). Hybrids obtained from the fusion between the
splenocytes of BALB/c mice immunized with KLH-peptide and the mouse
hybridoma cell line P3U1 were screened by solid-phase ELISA against
BSA-peptide, plasminogen, and LDL and subcloned several
times. To evaluate whether native Lp(a), Lp(a) oxidized with
CuCl2, plasminogen, and LDL reacted
with the monoclonal antibodies, we performed Western blot
analysis. The 2 monoclonal antibodies reacted with oxidized
Lp(a) but did not react with native Lp(a), plasminogen, or
LDL (Figure 2). Moreover, we tested the
effects of various oxidants on the signal detected by anti–modified
Lp(a) monoclonal antibody (161E2). Electrophoretic mobility of native
and oxidized Lp(a) on agarose gel as a parameter of the
different concentrations of CuCl2 or
lipoxygenase is shown in Figures 3 and 4.
After the treatment of Lp(a) with either CuCl2 or
lipoxygenase, a change in mobility of Lp(a) could be
detected in agarose gel electrophoresis. In addition, the treatment
with CuCl2 at concentrations of >0.5 µg/mL
induced a change in mobility detected with agarose gel electrophoresis,
accompanied by an increase in TBARS (lipid peroxide) level (Figure 3).
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Development of Oxidized Lp(a) ELISA Method
Differences in the signals of the modified Lp(a) ELISA method
between native Lp(a) and modified Lp(a) were investigated after the
subjection of purified Lp(a) to various treatments for modification,
including glycation, freeze/thaw cycles, and 6-day storage at 37°C or
room temperature. Glycation of Lp(a) was confirmed in the experiments,
on the basis of a change of mobility in agarose gel electrophoresis. No
differences in the parameters were observed between native
Lp(a) and Lp(a) subjected to various treatments for modification. Only
oxidized Lp(a) could be detected with the 161E2 monoclonal antibody
(Figure 5).
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Therefore, we developed a new ELISA to detect oxidized Lp(a) by using
antioxidized Lp(a) monoclonal antibody (161E2) as the capture antibody
and the detection antibody. To increase the precision and the
comparability of the oxidized Lp(a) ELISA method, we designed it for
use with a relatively low dilution of plasma (1:100) and calibrator
(1:100). A standard curve obtained with BSA-peptide is shown in Figure 6. The working range of the assay was
between 0 and 0.345 nmol/L BSA-peptide, and the assay was linear within
this range. The intra-assay and interassay coefficients of variation
for mean values of triplicate determinations (n=10) of high-level
oxidized Lp(a) serum (0.115 nmol/L BSA-peptide) were 1.2% and 5.0%,
respectively; those for low-level oxidized Lp(a) serum (0.035 nmol/L
BSA-peptide) were 4.5% and 7.2%, respectively. Figure 7 shows the results of our investigation
of whether the ELISA serves as a quantitative assay for purified
oxidized Lp(a) with the different phenotypes. Using serial
dilutions of oxidized Lp(a) with the phenotypes of 27, 29, and
34 kringle 4 repeats, we found the linearity of the response curve
constructed with the oxidized Lp(a) ELISA method. Levels of each
oxidized Lp(a) (BSA-peptide) were 0.23, 0.2, and 0.18 nmol/L. These
data suggest that the assay with the 161E2 antibody is quantitative for
oxidized Lp(a) independent of the phenotypes.
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Serum Native Lp(a) and Oxidized Lp(a) Concentrations in
Normotensive Subjects and Untreated Hypertensive Patients
Using the new ELISA for oxidized Lp(a), we measured native and
oxidized Lp(a) concentrations in normotensive subjects and untreated
hypertensive patients, because hypertension is well known to stimulate
oxidative stress.22 23 Oxidized Lp(a) concentration in
untreated hypertensive patients was significantly associated with
systolic, but not diastolic, BP
(P<0.05, r=0.13). In contrast, there was no
significant association between BP and serum Lp(a) concentration. To
further analyze the relationship between serum oxidized Lp(a)
concentration and BP, we evaluated the effect of hypertensive
complications on serum native Lp(a) and oxidized Lp(a) concentrations
in hypertensive subjects, according to WHO guidelines. There was no
apparent difference in serum native Lp(a) concentration among the
normotensives, hypertensives without complications, and hypertensives
with complications. Moreover, the oxidized Lp(a) concentration in
hypertensive subjects with complications (WHO II/III) was significantly
higher than that in those without complications (WHO I) (Figure 8, P<0.01), whereas serum
oxidized Lp(a) concentrations in hypertensive patients without
complications showed a tendency to be higher than that in normotensive
subjects. In normotensive subjects, oxidized Lp(a) level did not
correlate with serum concentration of native Lp(a).
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Immunocytochemical Findings
Finally, we examined the presence of oxidized Lp(a) in the vessel
wall, because Lp(a) has been reported to easily penetrate the vessel
wall and to accumulate within the atherosclerotic
lesion.24 25 Similar to previous
reports,24 25 apoB and native Lp(a) showed a similar
distribution in the cap region and around the necrotic core of the
elevated intimal lesion. On the other hand, oxidized Lp(a) was highly
localized in the cap region, which was superimposed by native Lp(a) and
apoB accumulation (Figure 9).
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Discussion |
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Recently, oxidized Lp(a) has been reported to have additional specific biological properties compared with native Lp(a). Because the biological effects of oxidized Lp(a) are more potent compared with native Lp(a),6 7 8 9 10 it is noteworthy to investigate the role of oxidized Lp(a) in the pathogenesis of atherosclerosis. However, the lack of a suitable antibody that reacts with oxidized Lp(a) limited the investigation of oxidized Lp(a). Fortunately, we obtained a new monoclonal antibody to react with oxidized Lp(a) but not native Lp(a). Here, we report a newly developed ELISA to detect modified Lp(a), especially oxidized Lp(a). The profile of this monoclonal antibody (161E2) against modified Lp(a) was characterized by the following features: (1) The epitope detected by the 161E2 monoclonal antibody recognized a part of the apo(a) molecule. Consequently, the 161E2 monoclonal antibody can react with Lp(a) modified by oxidative stress [oxidized Lp(a)] but not native Lp(a). (2) It is possible to quantify oxidized Lp(a) with the peptide used as the standard, because the recognized epitope is well defined due to the antibody against the known synthetic peptide. It allows extremely good stability and large-scale preparation for ELISA. Because 161E2 antibody reacted with an epitope on kringle IV type 2, it seems to lead to a difficult problem in distinguishing between the number of modified kringle 4 repeats versus the number of Lp(a) particles. Although the former possibility is unlikely due to the linearity with dilution of Lp(a) containing different single isoforms, further studies must be necessary to define the effects of kringle size. Serial dilutions of each of these serums containing a single isoform gave a linear range of presumed concentrations. In addition, the kringle 4 repeats did not provide a large difference in the value of serum Lp(a) concentration measured by antibody against kringle 4. For example, even if their kringle repeats increase from 19 to 38, the value of Lp(a) would not be increased to 19-fold or even to 2-fold.20 26 Antibody would probably not be able to bind to all of epitopes in the kringle 4 due to the 3-dimensional structure or the size of protein.
With this newly developed ELISA, we evaluated the presence of oxidized Lp(a) in the serum of normotensive subjects and hypertensive patients, because hypertension is a well-known oxidative stress.22 23 Interestingly, the present data demonstrated the presence of oxidized Lp(a) in human serum. More importantly, oxidized Lp(a) was significantly increased in hypertensive patients, especially in subjects with hypertensive complications. This finding is extremely important, because vasodilatation is inhibited by oxidized Lp(a).9 Elevation of oxidized Lp(a) may explain the endothelial dysfunction observed in hypertensive patients.27 Moreover, because oxidized Lp(a) enhanced Lp(a)-induced PAI-1 production in vascular endothelial cells,7 the elevation of oxidized Lp(a) might be related to the pathogenesis of hypertensive complications such as stroke.
During the development of atherosclerotic lesions,24 bypass vein grafts,28 and aortic aneurysms,29 Lp(a) accumulates within fibrin clots attached to blood vessel walls. Because Lp(a) accumulates within the fibrin clot with time, fatty streaks are formed that develop into occlusive atherosclerotic plaques. The accumulation of Lp(a) within the blood vessel is believed to promote an antifibrinolytic environment, foam cell formation, the generation of a fatty streak, and an increase in smooth muscle cells.24 Importantly, immunohistochemical staining with 161E2 antibody could detect a positive signal in the human atherosclerotic vessel. Because oxidized Lp(a) is a potent stimulus of monocyte adhesion to endothelial cells,30 the presence of oxidized Lp(a) might promote atherogenic changes in the human blood vessel. The present results failed to demonstrate which cells can be stained with this antibody. Because previous reports revealed that oxidative modification converted Lp(a) particles to a form readily recognized by macrophage scavenger receptors,10 the positive cells might be macrophages that had taken up oxidized Lp(a) via scavenger receptors. In addition, oxidized Lp(a) caused more pronounced stimulation of super oxide production, whereas native Lp(a) itself caused a moderate, dose-dependent stimulation of superoxide production.31 Accumulation of native Lp(a) may enhance the stimulation of oxidized Lp(a), a more potent atherogenic lipoprotein, in the vessel wall.
We developed a new monoclonal antibody (161E2) against the epitope in Lp(a) that was revealed externally as a result of oxidative treatment but not native Lp(a). The present data demonstrated the in vivo presence of oxidized Lp(a) in the arteriosclerotic human blood vessel and its elevation in hypertensive patients compared with normotensive subjects. The presence of oxidized Lp(a) may be important in understanding the role of Lp(a) in cardiovascular disease.
Received February 2, 2000; revision received April 28, 2000; accepted May 11, 2000.
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