Human apolipoprotein B. Evidence for its immunochemical heterogeneity using monoclonal antibodies and an immunoenzymometric assay

Clin Biochem, Vol. 21, pp. 255-261, 1988 Printed in Canada. All rights reserved.

0009-9120/88 $3.00 + .00 Copyright © 1988 The Canadian Society of Clinical Chemists.

Human Apolipoprotein B. Evidence for Its Immunochemical Heterogeneity Using Monoclonal Antibodies and an Immunoenzymometric Assay I. L U Y E Y E , 1 C. F I E V E T , 1,2 J. C. D U P O N T , 1 C. D U R I E U X J N. S L I M A N E , 1 J. F. L E C O C Q , 1 C. D E M A R Q U I L L Y , 1 and J. C. F R U C H A R T 1,2 1Service de R e c h e r c h e sur les L i p o p r o t e i n e s et I ' A t h e r o s c l 6 r o s e ( S E R L I A ) , Institut Pasteur, 1, rue du Prof. Albert Calmette, 5 9 0 1 9 Lille C e d e x , and 21nstitut National de la S a n t e et de la R e c h e r c h e Medicale ( I N S E R M ) , U 279, Institut Pasteur, 15, rue Camille Guerin, 5 9 8 0 0 Lille C e d e x , France Predefined monoclonal antibodies (Mabs) were used in an immunoenzymometric assay to study the immunochemical heterogeneity of lipoproteins and to search for potential epitopes with pathological importance. By measuring apolipoprotein B (apo B) epitopes in patients with and without angiographically documented coronary artery disease and in patients with type Ila hyperlipoproteinemia, we have found that both types of patients have a significant increase in Apo Bcontaining particles specifically recognized by one Mab (BL3). We have also observed that the effects of fenofibrate on type Ila patients vary greatly depending on the plasma concentrations of variousApo B-containing lipoproteins. The greatest effects occurred in patients with epitopes recognized by BL3. Lastly, by sequential precipitation of specific epitopes by BL3, we have obtained evidence thatthe residual epitope(s) may be related to one or more lipoprotein particles.

KEY WORDS: human apolipoprotein B; immunochemical heterogeneity; monoclonal antibodies; immunoenzymometric assay; fenofibrate. Polipoprotein B (apo B) is the major apolipoprotein in a variety of lipoproteins which vary in size from A large to small very low density lipoproteins (VLDL), to intermediate density lipoproteins (IDL) and low density lipoproteins (LDL). Each of these density classes comprises a spectrum of particles with different ratios of lipid to protein (1). It has become apparent that the antigenic determinants expressed on apo B vary greatly as a function of the hydrated density of the lipoprotein class in which it is present (2,3). It is now well established that high concentrations ofapo B-containing lipoproteins are associated with the development of atherosclerosis (4,5). Thus development of a sensitive method to quantify apo B remains a high priority. Based on the hypothesis that a particular subpopulation of such particles is atherogenic and that this lipoprotein subpopulation can be distinguished immunochemically from other nonatherogenic lipoproteins, Correspondence: Professor J. C. Fruchart SERLIA, Institut Pasteur, 1, rue du Prof. Albert Calmette, 59019 Lille Cedex, France. Manuscript received July 15, 1987; revised December 21, 1987; accepted January 15, 1988. CLINICAL BIOCHEMISTRY, VOLUME 21, AUGUST 1988

some investigators have used monoclonal antibodies (Mabs) to quantitate plasma levels of certain lipoprotein subsets in order to determine whether the presence of specific apo B epitope(s) is linked to angiographically documented coronary artery disease (CAD) or to dyslipoproteinemia (6-9). In this report, we describe the development of a noncompetitive immunoenzymometric assay (IEA) for serum apo B using three Mabs and a classic polyclonal antiserum (Pabs), and we compare the results obtained in (1) control subjects, (2) arteriographically defined CAD patients, and (3) individuals with primary type IIa hyperlipoproteinemia (phenotype associated with high risk of ischemic heart disease). We have also examined the immunochemical heterogeneity of apo Bcontaining particles in a double blind, randomized placebo-controlled pharmacological study, in which we followed the influence of fenofibrate on the plasma concentrations of the various apo B-containing lipoproteins. Lastly, by using one specific Mabs to exhaust selectively its epitopes from the apo B-containing particles we have found that some of the remaining epitopes may be expressed on different apo B-containing lipoprotein particles. All of these data are consistent with the proposal that there is considerable immunochemical heterogeneity within the spectrum of human apo B-containing particles and suggest that some Mabs may be useful in detecting individuals at risk for CAD. Materials and m e t h o d s

MONOCLONALANTIBODIES We used previously characterized Mabs specific for apo B (BL3, BL5, BL7) (10). All were produced in a mouse spleen myeloma system and purified by the Research Center Clin Midy (Montpellier). Cotitration experiments showed that all were specific for different antigenic epitopes (10). By Western blotting technique, all the three monoclonal antibodies were able to bind to B-100 and B-74. Affinity constants for apo B epitopes 255

LUYEYE, FIEVET, D U F o ~ ' r , ~1 ~L.

on intact LDL (11) were 1.48 × 101°, 3.8 × 106 and 1.54 × 10 s L/mol, respectively, for BL3, BL5 and BL7. The different epitopes expressed by all apo B-containing lipoproteins such as VLDL, IDL, LDL2 and plasma were studied in a competitive radioimmunoassay and showed that the epitopes recognized by BL3 were expressed equally by all the particles, while those specific for BL7 were significantly higher expressed in LDL2 and plasma than in VLDL or IDL. No competition was obtained in the same conditions for BL5, due probably to its very low affinity constant. CONVENTIONAL ANTIBODIES

We raised polyclonal antisera to Apo B in adult female goats and tested their specificity as reported earlier (12). From the antiserum obtained, two different fractions were prepared. First an IgG fraction was obtained by sodium sulfate precipitation followed by ionexchange chromatography. The second fraction, which contained pure immunoglobulins to LDL, was isolated by immunoaffinity chromatography. ISOLATION OF LIPOPROTEINS

VLDL, IDL and LDL2 were isolated from pooled sera of healthy human donors by sequential preparative ultracentrifugation respectively at densities d < 1.006 g/ml, 1.006 < d < 1.019 g/ml, and 1.020 < d < 1.063 g/ml (13). The protein content of these fractions was determined by a modified Lowry procedure, with bovine serum albumin as standard (14). Their immunoreactivity with Mabs BL3, BL5, and BL7 was estimated by assaying the apo B content before and after precipitation of apo B with isopropanol (15). PLASMA SAMPLES AND POPULATION SELECTION

Fasting blood samples were obtained from apparently healthy donors or from patients and centrifuged at 4°C. All biomedical studies were made on fresh sera which had been stored at 4°C for no longer than 48 h. For mapping of apo B-containing lipoproteins, the first group of patients studied consisted of 143 male subjects undergoing diagnostic coronary angiography. They were divided into two groups on the basis of the angiographic results: 1) 42 male patients had normal coronary arteries, perfectly smooth and even. 2) 101 male patients had significant vessel disease with lesions which produced >50% reduction of the lumen diameter. In a further group, 24 male patients with primary type IIa hyperlipoproteinemia, all judged from family studies to be heterozygous for familial hypercholesterolemia, were selected. All these patients were matched for age and sex with controls selected in a Center for Preventive Medicine. The pharmacological study was made on 37 patients of both sexes (22 men and 15 females) between 21 and 256

73 years old, suffering from type IIa hyperlipoproteinemia. The experiment designed to examine epitope distribution within apo B-containing particles was performed on 10 male sera obtained from apparently healthy normolipemic subjects. FENOFIBRATE STUDYDESIGN All lipid lowering drug therapy had been stopped at least two months before the 37 patients were admitted to the trial. A double blind, randomized placebo-controlled study was carried out for four months: - for 1 month, patients followed a hypocaloric (2000 cal/day) lipid-lowering diet (lipid content less than 30% of total calories with a cholesterol level < 300 mg/day and polyunsaturated fatty acid enrichment). All received a placebo. for the next 3 months, the diet was continued, but at the same time, 20 patients received 300 mg per day of fenofibrate and 17 patients received a placebo. Drug and placebo were presented and taken in absolutely identical form. Serum lipoprotein patterns were determined before (TO) and after dietary treatment (T1) and 1 and 3 months after the beginning of drug therapy (T2,T3). -

EXHAUSTION OF EPITOPES BL3 IN SERA We have optimized a procedure which accurately reproduces the conditions of the immunoenzymometric assay (IEA) described below. Disposable polystyrene tubes were first coated with BL3 in the following way: 2 mL of an antibody solution containing 16 ~g of antibody protein per mL in phosphate-buffered isotonic saline (PBS; phosphate 10 NaC1 mmol/L(pH 7.2), 150 mmol/L) were added to each tube. The tubes were incubated at room ~emperature overnight and then emptied by suction. Immediately before assay, the tubes were washed thrice with PBS and aspirated dry. Two ml aliquots of serum samples (n = 10) and ultracentrifugally isolated lipoprotein fractions (VLDL, IDL, LDL2) from which it was desired to eliminate the BL3 epitopes were diluted with PBS containing 10 g of bovine albumin (BSA) per liter (BSA-PBS) and then incubated in one of the above tubes at 37°C for 2h. The sample was then transferred to another coated tube, under the same conditions. This operation was repeated five times. At the end, it was possible to quantitate in the liquid phase BL3, BL5, BL7.

Noncompetitive immunoenzymometric assay a) Solid-phase antibody. Flexible PVC plates with 96 wells of 270 ~L capacity (Costar, Cambridge, MA 02139) were used. Each well was coated with 0.1 mL (2 ~g protein) of a diluted IgG polyclonal or monoclonal antibody solution against apo B. The diluting buffer was 0.1 M phosphate buffer, pH 7.2. The plates were incubated overnight at room temperature and then washed three times with PBS before aspirating dry. b) Antibody-enzyme conjugate. The labelled second CLINICAL BIOCHEMISTRY, VOLUME 21, AUGUST 1988

IMMUNOCHEMICAL HETEROGENEITY OF APOLIPOPROTEIN B antibody (goat polyclonal affinity-purified antibodies, peroxidase conjugated) was prepared as described previously (12). c) Peroxidase substrate solution. O-phenylenediamine dihydrochloride (Sigma Chemical Co., St. Louis, MO), 3 g/L, was dissolved in a 0.1 mol/L phosphate-citrate buffer (pH 5.5) containing hydrogen peroxide (3.5 mmol/L). d) Standards. The apo B content of LDL used as primary standard was estimated by protein assay before and after precipitation of apo B with isopropanol (14,15). For the assays, we used a secondary standard prepared by pooling frozen normal sera and adding antibiotics, protease inhibitors, and antioxidants. These additives (and their final concentrations) were as follows: EDTA (0.27 mmol/L), cis-amino-caproic acid (0.9 mmol/L), chloramphenicol (0.6 mmol/L), and glutathione (0.3 mmol/L). e) Quantitation ofapo B. Dilutions of all samples and standards were prepared in BSA-PBS; 0.1 mL of each was added to the wells, and the reaction was allowed

to proceed for 2h at 37°C (a blank, consisting of 0.1 mL of BSA-PBS only, was included in each series). The wells were then washed three times with PBS and aspirated dry. 0.1 mL of conjugate (diluted 15000-fold in BSA-PBS) was added to the wells, and the plates were incubated at 37°C for exactly 2h. The plates were washed as previously and thereafter incubated with 0.1 mL per well of freshly prepared substrate solution for 30 min at room temperature. The reaction was stopped by addition of 0.1 mL of 0.1 mol/L HCL to each well, and the developed color was read at 492 nm. All of the above additions and washes were made automatically by an Elisa ProcessorR (Behring Institute, Marburg). LIPID ANALYSES Total cholesterol and triglycerides were determined by enzymatic methods (16,17) adapted to centrifugal analysis.

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257

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The s t a n d a r d curves obtained with the serum calibrator for Pabs and Mabs are shown in Figure 1. The effective working r a n g e of the assay is from 100 to 1000 n g / m L of apo B using Pabs, corresponding to from 104to 5 × 102-fold dilutions of the s e r u m calibrator when Mabs are used. 3000- and 6000-fold dilutions of sera are m a d e routinely for clinical study, regardless of the n a t u r e of the antibodies. The within-run assay precisions, estimated by 32 replicate analyses in a pooled h u m a n sera are respectively 5.7, 3.8, 3.9 and 5.3% for Pabs and Mabs BL3, BL5, BL7. The between-run assay precisions, assessed by measuring 10 aliquots of a pooled s e r u m each day for 4 consecutive days are, respectively, 10.8, 5.7, 7.1 and 8.9% for Pabs and Mabs BL3, BL5, BL7.

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Figure 2--Apo B-containing lipoproteins in controls (n = 104) and angiographically defined patients without (n = 42) or with (n = 101) ~ coronary artery disease. Bars indicate -+ 2 SD.

MEASUREMENT OF APO B CONCENTRATION IN SERA In Figures 2 and 3 we present apo B values obtained with the three Mabs and the P a b s in sera from the patients examined, and show for all apo B parameters

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STATISTICAL ANALYSES The results were expressed as m e a n -+ SD. Statistical evaluation was performed by Student's t-test or M a n n and W h i t n e y ' s non p a r a m e t r i c U-test (18). Probability (p) values were considered significant when less t h a n 0.05.

100

Results ABILITY OF THE ANTIBODIESTO BIND ALL FORMS OF THE ANTIGEN APO B A.U

To test the ability of the Pabs- and Mabs-based assays to detect apo B in all its forms, we constructed standard curves with isolated lipoproteins (VLDL, IDL, LDL2) and the s e r u m calibrator, and compared statistically the slopes of these different curves. No significant difference was obtained w h e n testing Pabs on VLDL, IDL, LDL2 or calibrator, thus these antibodies showed equal i m m u n o r e a c t i v i t y to all these apo B-containing particles. W h e n using the Mabs, significant differences were calculated for the slopes obtained with the different antigens, corresponding to variable reactivities of each Mab for the fractions tested. Consequently, we have expressed the final results ofapo B values in sera, measured with Mabs, in a r b i t a r y units (AU), an a r b i t r a r y value 100 being given to the control group and the patients receiving drug at time TO. 258

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Figure 3--Apo B-containing lipoproteins in controls (n = 24) and patients (n = 24) with familial hypercholesterolemia (type IIa) ~ . Bars indicate -+ 2 SD. CLINICAL BIOCHEMISTRY, VOLUME 21, AUGUST 1988

IMMUNOCHEMICAL HETEROGENEITY OF APOLIPOPROTEIN B

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Figure 4---Effect of fenofibrate on Apo B-containing lipoproteins recognized by polyclonal and 3 different monoclonal antibodies in patients with familial hypercholesterolemia (n = 37). Bars indicate -- 2 SD. - - fenofibrate; - - - placebo; *p < 0.05; **p < 0.01; ***p < 0.001 significant differences between controls and arteriographically defined CAD patients, and between controls and type IIa patients. Mab BL3 appears to be the best discriminator in this regard. Between controls and subjects with arteriographically normal coronary arteries, no significant differences were detected. Only the Pabs and Mab BL3 were able to discriminate between subjects with normal and occluded coronary arteries. EFFECT OF FENOFIBRATE ON APO B-CONTAINING LIPOPROTEINPARTICLES Figure 4 shows the changes observed in apparent epitope expression at each stage of the fenofibrate

study. It is of particular interest t h a t fenofibrate induced various effects on lipoprotein particles recognized by three Mabs and t h a t after 1 month of t r e a t m e n t (T2), the serum concentration of the epitopes detected by BL3 and BL7 were significantly lower in the treated t h a n in the placebo group. The observed differences were, respectively, 33.2 and 28.5%. After 3 months of treatment (T3), these differences became 32.4 and 49.7%. BL5 was lowered by 24.3 and 12.6% in T2 and T3, respectively, but these differences were not significant. There was a tendency for the differences to decrease at CLINICAL BIOCHEMISTRY, VOLUME 21, AUGUST 1988

the T3 interval, this being probably due to incomplete following of the diet by the patients.

EPITOPE BL3 EXPRESSION IN APO B-CONTAINING PARTICLES RECOGNIZED BY BL5 AND BL7 Figure 5 represents the m e a n efficacy of BL3 exhaustion in 10 sera from normolipemic subjects and in lipoprotein fractions (VLDL, IDL, LDL2), and the changes which occurred in expression of BL5 and BL7 epitopes. The results were expressed as per cent of initial measured value. Under the conditions used, about 10% of the epitopes of BL3 were not removed from the sera and the isolated fractions. In sera and LDL this resulted in a reduction in the expression of BL7 but not of BL5 epitopes, whereas in VLDL and IDL, the expression of BL7 and BL5 epitopes was unchanged. Discussion

Several investigators have developed panels of murine Mabs against h u m a n apo B (2,6,19,20). These antibodies have made significant contributions (reviewed in Ref. 21) to the elucidation of the molecular weight 259

LUYEYE, FIEVET, DUPONT, ET AL.

and antigenic structure of apo B, the apolipoproteins that specify cellular binding, the apolipoprotein polymorphisms, and, finally, the role of the lipoproteins in atherosclerosis and coronary heart disease. Lipoproteins represent a class of extremely heterogeneous immunogens, consisting of a mixture of particles differing in their apolipoprotein composition (1). RESIDUAL EPITOPES

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Figure 5--Exhaustion of BL3 epitopes with expression of epitopes BL5 and BL7. Sera were absorbed 6 times with BL3 coated to polystyrene tubes as described in Methods. Results are expressed as mean percentage of the initially measured value (n = 10). Bars indicate --- 2 SD. 260

To explore such heterogeneity and to elucidate its potential clinical implications, we have developed new methodologies (22) designed to recognize only a limited kind of particle within the spectrum. The development of specific Mabs have made such an approach possible. Several immunological assays have been developed for the quantitation of apo B. These techniques usually employ Pabs (23), but Mabs have also been used (24). In contrast to the Pabs, which may recognize several different epitopes on the antigen molecule, Mabs are directed toward a single epitope. Such antibodies have the advantage that they are highly specific and reproducible. However, when measuring total apo B, they may have the drawback of recognizing only a part of the entire apo B in plasma. This selectivity may be useful in providing the possibility of an association between specific apo B epitopes and different pathological cases. This report describes our experience with an immunoenzymometric assay for apo B in which three Mabs are used. These Mabs are directed toward three different epitopes of LDL and do not bind to all LDL particles (10). The assay we describe offers distinct advantages over previously described apo B immunoassays: no centrifugation steps are required, radioisotopes are not used, the antigen to be measured is not chemically modified, manipulation can be entirely automated, and monoclonal antibodies are available in large quantities and easily purified. The coefficients of variation obtained with the three Mabs are lower than those obtained under the same conditions with Pabs. This may derive from the extreme specificity of Mabs. The particularly good precision of Mabs BL3 is perhaps related to the fact that it alone recognizes an epitope sequence (this antibody reacts with the C-terminus of the apo B molecule and more precisely with residues 4082-4525; results not published) that is apparently not expressed to the same extent in all individuals. Different studies support the view that increased apo B is a potential marker for patients with CAD (25). To search for a correlation between lipoprotein subpopulations and predisposition to CAD, we determined apo B values in patients with and without angiographically documented CAD and in patients with type IIa hyperlipoproteinemia, who have a greater incidence of CAD than normal. Each patient group was compared with age- and sex-matched control subjects. Our results suggest that atherosclerotic and type IIa patients have a significant increase in apo B-containing particles specifically recognized by BL3 antibody. This raises two questions: first, does this antibody really recognize a specific epitope on atherogenic particles, or, alternatively, is it due in part to better precision obtained in the assay using BL3? With regard to the effect of fenofibrate on type IIa patients, it is interesting that the drug did not affect all the apo B-containing particles to the same extent. No significant change was seen in concentrations of particles identified by BL5. These results confirm that there is a lipoprotein immunoheterogeneity and indicate that fenofibrate reduces significantly the circulating mass of atherogenic particles. CLINICAL BIOCHEMISTRY, VOLUME 21, AUGUST 1988

IMMUNOCHEMICAL HETEROGENEITY OF APOLIPOPROTEIN B A Mab recognizes one epitope related to one or more lipoprotein particles. To try to investigate this relationship, we exhausted most of epitopes specific for BL3 in 10 sera and in lipoprotein fractions (VLDL, IDL, LDL2) and assessed the resulting changes in epitopes BL5 and BL7. After exhaustion of BL3 epitopes in sera and in all fractions, no change was detectable in BL5 expression, suggesting t h a t epitopes BL3 and BL5 are not both expressed on the surface of the same apo Bcontaining lipoproteins. Of BL7 expression in the same conditions, 30 and 40% was still m e a s u r a b l e in sera and LDL2, respectively, while no change was obtained in VLDL and IDL. This fact suggests t h a t BL3 and BL7 epitopes m a y be associated on some of lipoprotein particles particularly a b u n d a n t in LDL2 class density. Our results m a y be related to the different specificities of Mabs which appear to recognize different subpopulations of apo B-containing particles, alternatively and variably to the same particles whose lipids m i g h t participate in m a s k i n g the accessibility of the epitopes.

9.

10.

11. 12.

13.

14.

Acknowledgements We would like to thank Dr. J. Shepherd for helpful assistance in the preparation of this manuscript.

15.

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