Clinical and Diagnostic Laboratory Immunology, November 1998, p. 817-822, Vol. 5, No. 6
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Preparation of a Human Standard for Determination
of the Levels of Antibodies to Oxidatively Modified Low-Density
Lipoproteins
Sinikka
Koskinen,1
Candace
Enockson,1
Maria F.
Lopes-Virella,1 and
Gabriel
Virella2,*
Division of Endocrinology, Diabetes and
Medical Genetics, Department of Medicine,1 and
Department of Microbiology and
Immunology,2 Medical University of South
Carolina, Charleston, South Carolina 29425
Received 1 June 1998/Accepted 30 July 1998
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ABSTRACT |
As part of our ongoing effort to develop a standardized competitive
enzyme immunoassay for human autoantibodies to oxidized low-density
lipoprotein (oxLDL), we have generated a reference human antibody
standard and revised some of the conditions of the assay. The
preparation of the standard involved purification of human anti-oxLDL
antibodies by affinity chromatography using immobilized oxLDL. The
total concentration of antibody in this purified human oxLDL antibody
was established by adding the concentrations of immunoglobulin G (IgG),
IgA, and IgM determined in the standard by radial immunodiffusion. The
isolated antibody was used to calibrate a whole human serum standard,
which was used to calibrate the assays to detect antibody in serum
samples. We also revisited the general conditions for performance of
our competitive assay. We determined that 1/20 was the ideal dilution
for performing the absorption step, and that 1/20 and 1/40 were optimal
dilutions to assay oxLDL antibody in unknown serum samples. We also
established that the optimal concentration of oxLDL for absorption of
free antibody in serum samples was 200 µg of oxLDL/ml; no significant decrease in the reactivity of samples with immobilized oxLDL was observed when higher concentrations of oxLDL were used for absorption. The minimum detection level of the assay is 0.65 mg/liter. Because serum samples are diluted 1/20 and 1/40 for the assay, the minimal concentration of antibody detectable in serum is 20-fold higher, i.e.,
13 mg/liter. The intraassay coefficient of variation calculated from
seven determinations of three samples containing antibody concentrations of 240, 340, and 920 mg/liter ranged from 8 to 6.1%.
The interassay coefficients of variation for sera with antibody levels
of 100 to 594 mg/liter varied from 9.2 to 7.0%, and for isolated
antibodies with concentrations of 52 to 111 mg/liter, the coefficients
varied from 5.8 to 3.9%.
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INTRODUCTION |
The role of autoantibodies against
oxidatively modified low-density lipoproteins (oxLDL) in the
pathogenesis of atherosclerosis is, presently, the object of intense
investigation. Experiments conducted in vitro have shown that LDL may
be oxidized by several types of cells, including endothelial cells,
smooth muscle cells, and macrophages (2, 11, 13, 17). oxLDL
has been found in atheromatous lesions (5, 12, 22), and LDL
extracted from atherosclerotic lesions exhibits nearly all of the
physicochemical and immunological properties of copper-oxidized LDL
(21). Antibodies against oxLDL (anti-oxLDL) have been
demonstrated in human serum (15, 19) and in atherosclerotic
lesions of rabbits and humans (6, 20, 21). Such antibodies
recognize epitopes expressed in atherosclerotic lesions of rabbits and
humans but not in normal arteries (1, 5, 12). However, the
pathogenic significance of anti-oxLDL antibodies remains uncertain due
to the discrepant results published by several groups of investigators
who found either a significant correlation between circulating
anti-oxLDL antibody levels and manifestations of atherosclerosis or no
correlation at all (4, 15, 18, 19).
The methods used for the measurement of circulating anti-oxLDL
antibodies include radioimmunoassays (15) and
enzymeimmunoassays (4, 14, 18, 19). Most of the assays are
based on a comparison of the reactivities of a sample with immobilized
oxLDL and with immobilized native LDL, and the results are expressed
either as a difference or a ratio that reflects the increased binding
to oxLDL (4, 14, 15, 18). This methodology can underestimate the antibody levels if the anti-oxLDL antibodies cross-react with native LDL (10) or may result in falsely elevated values due to the lack of correction for charge-dependent nonspecific interactions that are likely to be quite different between native LDL and oxLDL, which, as it is well known, has an increased negative charge. Furthermore, the expression of data in arbitrary units, whether they
are ratios or differences in optical density (OD), represents a
significant obstacle in the comparison of data obtained by different groups.
To solve these problems, it seemed essential to devise assays that were
not only of satisfactory specificity and reproducibility but also
adequately standardized. Issues of specificity and reproducibility were
approached by our group several years ago, with the development of a
competitive assay, based on the measurement of binding values before
and after absorption with oxLDL, which we have routinely used in our
laboratory (19). The development of calibrator standards with known antibody concentrations, allowing expression of antibody concentration in standard mass units rather than in arbitrary units,
appears to be an important step towards standardization. Originally, we
used immunoglobulin G (IgG) isolated from a rabbit hyperimmune
anti-human LDL antiserum to standardize the assay (19), but
this approach had the important drawback that only an undetermined
proportion of this IgG reacted with oxLDL. In addition, two different
conjugates had to be used in each plate: an anti-rabbit IgG for the
calibration curve and anti-human IgG for the human samples. This report
describes the standardization of our oxLDL antibody assay for whole
human serum by using a human serum standard to calibrate the assay, as
well as other modifications in the assay conditions that appear to
improve the reproducibility of the assay.
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MATERIALS AND METHODS |
Lipoprotein isolation, modification, and characterization.
Blood for lipoprotein isolation was collected from healthy volunteers
after 12 h of fasting into a 0.4-mmol/liter concentration of EDTA.
Plasma from three to four healthy volunteers was used for separation of
LDL by preparative ultracentrifugation at 50,000 rpm for 17 h on a
Beckman L-80 ultracentrifuge after density adjustment with potassium
bromide (1.019 < density < 1.063 g/ml) using a type 70 Ti
rotor (8). Isolated LDL was washed by ultracentrifugation, dialyzed against a 0.15-mol/liter sodium chloride solution containing 300 µmol of EDTA per liter, pH 8.0, passed through an Acrodisc filter
(0.22 µm pore size) in order to sterilize and remove aggregates, and
stored under nitrogen in the dark at 4°C.
Oxidation of LDL was performed in accordance with the protocol
described by Steinbrecher (16). To remove residual KBr and EDTA, the freshly isolated LDL was passed through a PD-10 column (Pharmacia Biotech, Uppsala, Sweden). Phosphate-buffered saline (PBS),
pH 7.4, was oxygenated at 2 liters/min for 10 min, and copper chloride
was added to a final concentration of 10 µmol/liter for every 300 µg of LDL. The LDL final concentration was adjusted not to exceed 1.5 mg/ml, and the highest concentration of CuCl2 used was 40 µmol/liter when the concentration of oxLDL was between 1.2 and 1.5 mg/ml. The oxidation reaction was carried out at 37°C, and to monitor
the degree of oxidation, an aliquot (100 µg) of LDL, prepared as
described above, was diluted in 2 ml of PBS and continuously monitored
at 37°C on a luminescence spectrophotometer (SLM-Aminco Series 2;
Spectronic Instruments, Rochester, N.Y.) using a wavelength of 360 nm
for excitation and a wavelength of 430 nm to measure fluorescence
emission (3). The fluorescence profile shows a steady
increase during the first 8 to 12 h, after which a plateau is
reached, and the fluorescence values remain stable for up to 6 days
after the plateau is reached (8a). The results of oxLDL
antibody assays using oxLDL preparations obtained 5 to 48 h after
the fluorescence levels reached their peak do not differ significantly.
To avoid unnecessary degradation, we decided to stop the oxidation
reaction 4 to 6 h after the fluorescence reached its peak by
adding EDTA and butyl-hydroxytoluene (BHT) to final concentrations of
300 and 200 µmol/liter, respectively. LDL preparations obtained in
this manner have yielded reproducible results in our competitive enzyme
immunoassay. Copper and BHT were removed from the oxLDL preparations
after termination of the oxidation reaction by overnight dialysis
against 4 liters of a 0.15-mol/liter concentration of sodium chloride
containing 300 µmol of EDTA (pH 8.0) per liter. After dialysis, the
oxLDL preparations were filtered through a 0.22-µm-pore-size filter to sterilize and remove aggregates and stored at 4°C. The final protein concentration in each preparation was determined by a Lowry
assay (7).
Isolation of anti-oxLDL antibodies.
Anti-oxLDL antibodies
were isolated by using an affinity chromatography protocol previously
described by Mironova et al. (10). In summary, freshly
isolated LDL was dialyzed against a coupling buffer containing 0.1 mol
of NaHCO3 per liter and 0.5 mol of NaCl (pH 8.3) per liter
and incubated overnight on a rocker at 4°C with CNBr-activated
Sepharose 4B (Pharmacia Biotech) prepared in accordance with the
manufacturer's instructions and equilibrated with the same coupling
buffer. At the end of this incubation, free binding sites were blocked
with a solution containing 0.2 mol of glycine (pH 8.0) per liter, and
after extensive washing and degassing, the gel suspension was
transferred to a chromatography column and washed thoroughly with PBS,
pH 7.4. The column was then equilibrated with PBS containing 10 µmol
of CuCl2 per liter, and oxidation was allowed to proceed
for 18 h at 37°C. The protein concentration is estimated to
correspond to about 300 to 400 µg/ml of gel, based on the coupling
efficiency data published by Pharmacia Fine Chemicals. Therefore, the
conditions of immobilized LDL oxidation were similar to those used for
LDL in solution. The oxidation reaction was stopped by washing the
column extensively with PBS containing 300 µmol of EDTA per liter and
200 µmol of BHT per liter followed by washes with NaHCO3
buffer and acetate buffer. Finally, the column was equilibrated with
0.01 mol of NaHCO3 buffer (pH 8.3) per liter.
To isolate anti-oxLDL antibodies from serum samples, 1 ml of serum was
diluted with 4 ml of the bicarbonate buffer used to equilibrate the
affinity chromatography column, and it was allowed to diffuse into the
column. After the column loaded with diluted serum was incubated
overnight at 4°C, unbound proteins were washed off with the
equilibrating buffer. The bound antibodies were eluted with a
0.1-mol/liter concentration of NaHCO3 (pH 8.3) buffer
containing 0.5 mol of NaCl per liter, and after the OD returned to
baseline, the column was eluted a second time with 0.2 mol of
glycine-HCl buffer (pH 2.3) per liter. All of the bound antibody was
eluted with 0.1 mol of NaHCO3 (pH 8.3) per liter. As shown
in Table 1, the antibody purified in this
manner was predominantly of the IgG isotype, but IgA and IgM antibodies
were also isolated. No contaminating proteins were detected in the
purified antibody preparations by immunoelectrophoresis. The purified
antibodies were aliquoted and stored at 
20°C without addition of
preservatives until use. Under these conditions, the purified antibody
remained stable for at least 6 months, as demonstrated by its
reproducible reactivity with immobilized oxLDL.
Standards and quality control sera.
Purified human oxLDL
antibody, prepared as described above, was used to calibrate a whole
human serum sample, which was subsequently used as the standard for all
assays measuring serum oxLDL antibody levels. Since the assay, as
described in this manuscript, will detect antibodies of all isotypes,
and since the oxLDL antibodies purified by affinity chromatography can
be distributed among the three major immunoglobulin isotypes (Table 1),
we used the total antibody concentration in the purified human antibody
preparation, obtained by adding the concentrations of IgG, IgA, and IgM
determined by radial immunodiffusion (medium-level
radioimmunodiffusion kits; The Binding Site), as our reference value.
As quality controls for the assay, we used three serum samples with
different levels of oxLDL antibodies (Table 1). The isotype distribution and the dissociation constant values
(Kd) for our human antibody standard and for the
oxLDL antibodies isolated from three of the quality control serum
samples, determined as previously described (10), are also
shown in Table 1. Table 1 also includes, for reference purposes, the
Kd value for a rabbit anti-human LDL antibody
isolated by affinity chromatography using the same column and general
procedure outlined for isolation of human oxLDL antibodies. Standards
and quality controls were aliquoted and stored at 20°C without
additives and have remained stable for at least 6 months (their
reactivity in the oxLDL antibody enzyme immunoassay has remained
basically unchanged).
Enzyme immunoassay.
The concentration of oxLDL antibody was
determined in all samples (whole serum samples and isolated antibodies)
by the competitive enzyme immunoassay previously described by us
(19). The assay was conducted on flat-bottom Immulon type 1 enzyme immunoassay plates purchased from Dynatech (Chantilly, Va.). The
plates were coated immediately prior to use by overnight incubation at
4°C with 100 µl of oxLDL (7.5 mg/liter in a 1-mol/liter
concentration of carbonate-bicarbonate buffer [pH 9.6]). Serum
calibrators, controls, and unknown samples were diluted 1/10 in PBS
containing 1% bovine serum albumin (PBS-BSA) and were then separated
into two 200-µl aliquots. One of the aliquots was absorbed with an equal volume of oxLDL (400 µg/ml in PBS-BSA), with the same batch of
oxLDL used to coat the plates in the absorption step. The other aliquot
(unabsorbed) was mixed with an equal volume of PBS-BSA. The final
dilution of these aliquots was 1/20. All samples (absorbed and
unabsorbed) were incubated overnight at 4°C on a rocker. After incubation, the plates were washed with 0.05% Tween 20 (Sigma Chemical
Co.) in PBS, pH 7.8, to remove unbound oxLDL, blocked with 5% BSA in
PBS (1 h at room temperature), and washed four more times. Neither the
coating buffer nor any of the remaining buffers used to perform the
assay contained antioxidants because a comparison of assays performed
with and without addition of antioxidants (EDTA at 0.27 mmol/liter and
BHT at 20 µmol/liter) to the buffers showed no difference in the
final results. The absorbed and unabsorbed aliquots were spun at
9,000 × g in an Eppendorf centrifuge (model 5413) for
30 min. The supernatants of the centrifuged samples, unabsorbed and
absorbed, were divided into two portions, one to be tested as it was
absorbed (1:20 dilution) and the other one to be tested after being
diluted 1:2 (final dilution, 1:40). One-hundred-microliter volumes of
both dilutions were transferred to the wells of the oxLDL-coated
plates. After overnight incubation at 4°C and extensive washing,
peroxidase-conjugated rabbit anti-human IgG (IgG fraction), reacting
with both IgG heavy chains and light chains (Cappel Organon Teknika,
Durham, N.C.), was added to the wells. After incubation for 1 h at
4°C the unbound antibody was washed off and the plates were incubated
for 10 min in the dark with the substrate (0.5 mmol of
2,2'-azino-di-[3-ethylbenzthiazoline-6-sulfonate] [ABTS; Sigma
Chemical Co., St. Louis, Mo.] per liter and 3% [vol/vol] hydrogen
peroxide in a 45-mmol/liter concentration of citric acid buffer [pH
4.0]). The reaction was stopped with 0.1 mol of citric acid (pH 2.1)
per liter and the absorbance was measured at 414 nm in a VMax
enzyme-linked immunosorbent assay reader (Molecular Devices, Sunnyvale,
Calif.).
The OD values used to calculate antibody concentrations in both
standards and unknown samples were obtained by subtracting the OD
values of the oxLDL-absorbed samples from those of the unabsorbed
samples. Five serial dilutions of the whole human serum standard were
used to calibrate the assay. The best fit for the calibration curve was
obtained with a third-degree polynomial function. The concentration of
the antibody in unknown samples represents the average of two
determinations (1/20 and 1/40 dilutions) after correction by the
dilution factor.
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RESULTS |
Characterization of the calibration standard.
The
dose-response curves obtained with purified human oxLDL antibody and
the calibrated whole human serum standard are reproduced in Fig.
1. Each point represents the mean of
results obtained in four (human antibody standard) to six (purified
anti-oxLDL) different assays run in duplicate.
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FIG. 1.
Composite concentration versus OD interpolation plots
for oxLDL standards used for calibration of our oxLDL antibody assay.
The two plots correspond to data generated with isolated human
antibodies and whole human serum (plot of the means of six successive
determinations of isolated human anti-oxLDL antibodies [ ] and plot
of the means of four successive determinations of the whole human serum
standard [ ]. The concentrations of antibody in each standard were
determined as detailed in the text. Each value corresponds to the mean
of six assays ± 1 standard deviation for the isolated antibody
and of four assays ± 1 standard deviation for the serum standard.
Each point in the figure corresponds to the difference in
OD414 readings between unadsorbed and oxLDL-adsorbed
samples.
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The mean concentration of the purified human antibody, calculated by
adding the concentrations of IgG, IgA, and IgM in the purified
reference antibody preparation determined in triplicate by radial
immunodiffusion, was 117 mg/liter. The antibody concentration on the
lowest dilution (1/20) of the whole human serum standard used for
calibration purposes, determined against the calibration curve
constructed with purified human oxLDL antibody, was 40.8 ± 5.38 mg/liter (mean ± standard deviation of three different assays,
each one of which included three dilutions of the serum standard run in
duplicate). By running seven series of dilutions of the serum standard,
we found that the lowest concentration of antioxLDL antibodies
that we could determine with satisfactory accuracy was 0.65 mg/liter,
which translates to a minimal concentration of 13 mg/liter on serum
samples (which are diluted 1/20 and 1/40 for the assay) and 1.3 mg/liter on purified antibody preparations (which only need to be
diluted 1/2 for the absorption step).
The slopes of the concentration versus OD plots of purified antibody
and serum antibody are slightly different, probably reflecting the
selection of antibodies of relatively higher affinity in the purification process. Thus, the preparation of a whole human serum standard has the advantage not only of making frequent antibody purification unnecessary but also of providing a more accurate calibration curve for the assay of circulating antibodies.
Precision parameters.
The intraassay coefficient of variation
(CV) calculated from seven determinations of three samples containing
antibody concentrations of 240, 340, and 920 mg/liter ranged from 8 to
6.1%. The interassay CVs for serum samples with antibody levels of 100 to 594 mg/liter varied from 9.2 to 7.0%, and for isolated antibodies
with concentrations of 52 to 111 mg/liter, the CVs varied from 5.8 to
3.9%. Three to five runs were performed to calculate the interassay
CVs (Table 2).
Determination of the optimal conditions for the assay of oxLDL
antibodies.
In a competitive immunoassay such as the one that we
developed, three major variables need to be considered: the degree of LDL oxidation and its consistency from batch to batch, the
concentration of antigen used in the absorption step, and the dilution
of the unknown samples in order to fall in the linear part of the
calibration curve.
The degree of LDL oxidation can be monitored by several different
parameters, such as formation of thiobarbituric acid reactive substances (7), formation of conjugated dienes
(7), and generation of fluorescent compounds during
oxidation (3). We have used the last parameter to ensure
that the degree of oxidation was similar in the different batches of
oxLDL used in our experiments, thus ensuring optimal and reproducible
reactivity with oxLDL antibodies. Such optimal reactivity of anti-oxLDL
antibodies was observed with LDL preparations that had been stopped
from further oxidation 4 to 6 h after fluorescence levels reached
their maximum level.
To determine the concentration of oxLDL that led to maximal absorption
of oxLDL antibody, we absorbed both our reference whole serum (at a
final dilution of 1/20) and our reference isolated antibody (at a final
dilution of 1/2) with various concentrations of oxLDL (12.5 to 800 µg
of oxLDL per ml). As shown in Fig. 2, we
observed a direct correlation between the concentration of oxLDL used
for absorption and the difference in OD values between absorbed and
unabsorbed samples. Figure 2 also shows that the degree of absorption
starts to reach a plateau at an oxLDL concentration of 100 µg/ml, and
there is no evidence for significant increases in absorption with
concentrations higher than 200 µg/ml. The percentage reduction of OD
after absorption with 200 µg of oxLDL for serum samples containing a
high titer of antioxLDL antibodies was similar for dilutions
ranging from 1/10 to 1/80 (results not shown). In accordance with these
results, we have chosen 200 µg/ml as the concentration of oxLDL used
for absorption.
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FIG. 2.
Concentration of oxLDL required for maximal absorption
of free anti-oxLDL antibodies. Each point in the figure corresponds to
the difference in OD readings at 414 nm between unabsorbed and absorbed
aliquots of our reference purified antibody and serum D (see Table 1).
Identical dilutions of the antibody preparations (1/2 for isolated
antibody, 1/20 for serum) were absorbed with increasing concentrations
of oxLDL, as shown in the figure. In the unabsorbed aliquots, oxLDL was
replaced by PBS. Each value corresponds to the mean duplicate
values ± 1 standard deviation.
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To determine the optimal sample dilution to adequately measure oxLDL
antibody concentrations in whole serum, we prepared several dilutions
of our reference serum, ranging from 1/5 to 1/800, to determine which
dilutions would fall in the linear part of the calibration curve (Fig.
3). Dilutions near the middle range of the calibration curve are preferred to avoid errors due to either antigen excess or excessively low antigen concentrations. Therefore, 1/20 and 1/40 appeared to be optimal. We adopted 1/20 as the dilution to be absorbed, and each sample is assayed at 1/20 and 1/40 dilutions. The correlation between the values obtained with these two dilutions after correction by the dilution factor is excellent (Fig.
4). Linear regression analysis showed a
highly significant (P < 0.0001) correlation between
the two sets of values (r = 0.984).
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FIG. 3.
Effect of dilution on the measurement of OD differences
between unabsorbed and oxLDL-absorbed samples of serum sample D (see
Table 1). The initial absorbed and unabsorbed aliquots were prepared at
a 1/5 dilution, and a series of dilutions was prepared from those
aliquots. Each point in the graph corresponds to the difference in OD
between identical dilutions of the unabsorbed and absorbed aliquots run
in duplicate.
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FIG. 4.
Degree of agreement between oxLDL antibody
concentrations calculated from 1/20 and 1/40 dilutions of the unknown
samples. The plotted data correspond to a single run in which all
samples were tested at both dilutions. Values entered in the plot were
corrected by multiplication by the respective dilution factors.
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DISCUSSION |
There is a recognized need to establish standardized assays for
anti-oxLDL antibodies that would allow different groups to generate
comparable data. Several issues need to be addressed in this effort,
including choice of the assay procedure, choice of a suitable antigen,
and development of calibrators that would allow the expression of data
in standard units of antibody concentration.
We report in this publication the results of our efforts leading to the
establishment of calibration standards for oxLDL antibody assays,
which, to our knowledge, had not been previously established. This was
possible because of the development of a reliable affinity chromatography method for the isolation of oxLDL antibodies
(10) that allowed us to isolate antibodies from human sera.
The isolated oxLDL antibodies always include molecules of the three
major isotypes (IgG, IgA, and IgM), and since our assay is designed to
measure total antibody concentrations, the total antibody concentration in the isolated antibody preparation was established by adding the
concentrations of IgG, IgA, and IgM, determined by radial immunodiffusion, and this concentration was used as the starting point
to calibrate a whole serum sample.
The advantages of establishing a whole serum standard are manifold. On
the one hand, it is easier to calibrate a serum sample from which a
large number of aliquots can be prepared and properly stored, avoiding
the problems inherent to repeated and frequent antibody isolations that
would be a major drawback if the standard was prepared with purified
antibody. In addition, Fig. 1 shows that the concentration versus OD
curves obtained with the antibody standard and the serum standard have
different slopes. The most likely cause for this difference in the
calibration curves is the selection of an antibody population of higher
affinity during purification by affinity chromatography. Those
antibodies with lower affinity are likely to have been washed off the
column, while the population with a relatively higher affinity would
have remained bound to the column. For practical purposes, using a purified antibody standard would add an element of imprecision to the assay.
To further increase the precision of the assay, it is important to
avoid confounding factors and to standardize as many of the assay steps
as possible. Our decision to develop a competitive assay which allows
the calculation of specific binding, i.e., the difference in binding
between unabsorbed and oxLDL-absorbed aliquots, rather than a
comparative assay based on the different reactivities of sera with
oxidized and native LDL, was based on the desire to avoid the
interference of two sources of nonspecific binding ("charged" oxLDL
and native LDL). Most of the oxLDL antibody immunoassays compare the
reactivity of a given sample with oxidized LDL and that of a sample
with native LDL. The values for oxLDL antibodies obtained in these
assays are expressed in arbitrary units, either as the ratios of the OD
value recorded with oxLDL to that with native LDL or as the differences
between these two OD values (4, 14, 15, 18). The problem
with this approach is that oxidized LDL has a much stronger negative
charge than native LDL. Therefore, the measurement of the reactivity
with native LDL as an index of nonspecific binding ignores the fact that the nonspecific binding levels of IgG molecules (which at pH 7.4 have a charge between neutral and weakly positive) to oxidized and
native LDL are significantly different and constitutes an uncontrolled
variable factor which interferes with the final calculation of antibody concentrations.
There is general agreement about the use of copper-oxidized LDL as an
antigen in oxLDL antibody assays because, according to observations of
Ylä-Herttuala et al., copper-oxidized LDL carries nearly the same
properties as the oxLDL found in human atheromatous lesions
(21). It must be noted, however, that we and others
(15) have experienced a considerable degree of variability when testing for anti-oxLDL antibodies using different batches of
oxLDL, even when they are prepared under the same conditions and from
the same donor pool. This seems to be related to differences in
oxidation susceptibility between different batches of LDL, which
results in difficulties when attempts are made to reproduce the same
degree of oxidation at different times under the same apparent
experimental conditions. Thus, it is likely that the epitopes exposed
by different batches of oxLDL may be quite variable, unless the degree
of oxidation is properly controlled.
In our laboratory, copper-oxidized LDL has allowed us to obtain data
with the highest reproducibility, providing that the degree of
oxidation of LDL is properly standardized to ensure that different
batches have similar degrees of reactivity with the corresponding
antibodies. This reproducibility has been achieved with oxLDL
preparations collected 3 to 5 h after the formation of fluorescent
LDL protein compounds reaches its peak. Under those conditions,
regardless of the time required for the fluorescence values to reach
their peak, each batch of LDL used in our experiments is at the same
stage of oxidation and the reactivity with reference antibodies
remained reproducible.
In addition, we decided it was also important to establish the optimal
concentrations of oxLDL required for the absorption step as well as the
optimal sample dilution for performance of the assay. The parameters
now recommended are slightly different from those included in the
original description of the assay, increasing the concentration of
oxLDL used for absorption to 200 µg/ml and increasing the serum
dilution used in the absorption step from 1/10 to 1/20. These changes
were adopted to ensure a more complete blocking of free antibody sites
and to ensure that the OD values obtained with most unknown samples
fall in the optimal range of the calibration curve. The antibody
concentrations calculated from the two dilutions correlated extremely
well (Fig. 4). Testing two different dilutions has an advantage over
duplicates of a single dilution because in the occasional serum sample
in which the antibody concentrations may be excessive, the two
dilutions will give dissimilar results (the concentration calculated
from the 1/40 dilution exceeding the concentration calculated from the
1/20 dilution), and the lack of concordance between the two dilutions
will alert us to the problem. Such samples are then tested at higher
dilutions, seeking those that give concordant results.
The final assessment of the performance of our assay was to determine
the intra- and interassay CVs both for the measurement of isolated
anti-oxLDL antibodies and serum samples. The CVs corresponding to the
intraassay variability varied from 8 to 6.1%, and the CVs corresponding to the interassay variability ranged from 5.8 to 3.9%
with purified antibodies and from 9.2 to 7.0% with serum samples
(Table 2). Comparing the CVs for purified antibodies and serum samples
at similar antibody concentrations, it is obvious that the
reproducibility is better with purified antibodies, which is usually
the case in all enzyme immunoassays. Still, an interassay CV of 9.2%
at the lower level of detection is an excellent index of the stability
and precision of the method, particularly when we take into
consideration that the analyte is an antibody of moderate to low affinity.
In conclusion, we have developed a human antibody standard for the
assay of oxLDL antibodies and have optimized the competitive enzyme
immunoassay for oxLDL antibodies that was previously described (19). These new developments will allow a more precise and
reproducible determination of the levels of circulating anti-oxLDL
antibodies. As the assays for oxLDL antibodies become more accurate and
reproducible, we expect that the discrepancies found in the literature
on the correlation between circulating anti-oxLDL antibody levels and manifestations of atherosclerosis (4, 15, 18, 19) will be
finally resolved.
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ACKNOWLEDGMENTS |
This work was supported by National Institutes of Health grant
HL-55782, by the Research Service of the Department of Veterans Affairs, and by grants from the Academy of Finland, Pohjois-Karjala Cultural Foundation, Aarne Koskelo Foundation, and Jalmari and Rauha
Ahokas Foundation. Sinikka Koskinen is a postdoctoral fellow of the
Juvenile Diabetes Foundation International.
We thank Charlyne Chassereau for her skilled technical assistance and
Alva Mullins for editorial assistance.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Phone: (803) 792-4339. Fax: (803)
792-2464. E-mail: virellag{at}musc.edu.
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Clinical and Diagnostic Laboratory Immunology, November 1998, p. 817-822, Vol. 5, No. 6
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
