Quantification of human apolipoprotein E in plasma and lipoprotein subfractions by a non-competitive enzyme immunoassay

245

Clinica Chimica Acta, 163 (1987) 245-256 Elsevier

CCA 03709

Quantification of human apolipoprotein E in plasma and lipoprotein subfractions by a non-competitive enzyme immunoassay Monique Jean-Marie

Koffigan Bard

a, Ibrahim

a, John

Kora

Chapman

a, VQonique

Clavey

b and Jean-Charles

a,

Fruchart

a

a Se&a - Institut Pasteur and INSERM b INSERM (Received

17 February

U.279, Ldle and U.9, Pavilion B. Delassert, H;pital de la PitiC Paris C&dex (France)

1986; revision received 4 October

Key wordr: Apo E; Lipoproteins;

Enzyme-linked

1986; accepted

immunosorbent

after revision 21 October

assay ELISA;

1986)

Electroimmunoassay

Summary A non-competitive enzyme linked immunosorbent assay (ELISA) has been developed to quantitate apolipoprotein E (Apo E) concentrations in serum and in isolated lipoproteins. Microtiter plates coated with affinity-purified antibodies to Apo E were used and the Apo E bound to the plates was estimated with peroxidase-labelled antibodies to Apo E. The average concentration of Apo E in the serum from normolipidemic subjects (n = 132) was 54 f 19 mg/l. The within and between assay coefficients of variation were 4.65 and 7.088, respectively. The standard curves for Apo E in serum, in VLDL and in HDL were parallel. There was a good correlation (r = 0.81) between estimation of Apo E by our assay and that by electroimmunoassay. Assay sensitivity (1 ng of Apo E) was sufficient to enable a study of the distribution of Apo E in plasma lipoproteins separated by density gradient ultracentrifugal fractionation.

Introduction Apolipoprotein E (Apo E) is a normal constituent of all human plasma lipoprotein subfractions [l-5]. Apo E has been shown to bind with high affinity to the low-density lipoprotein (LDL) or Apo B, E receptor on cultured human skin

Correspondence and reprint requests to: Professor Professeur Calmette, 59019 Lille Ctdex, France.

0009-8981/87/$03.50

J.-C.

0 1987 Elsevier Science Publishers

Fruchart,

Serlia-Institut

B.V. (Biomedical

Division)

Pasteur,

1, rue du

246

fibroblasts in vitro, and to a distinct hepatocyte Apo E receptor [6]. Plasma lipoproteins rich in Apo E and cholesteryl esters are highly atherogenic and are characteristic of individuals with dysbetalipoproteinemia; indeed such individuals often display elevated serum Apo E concentrations [7]. Various methods have been proposed for the quantitative determination of serum Apo E concentrations. These include radioimmunoassay [1,8,9], electroimmunoassay [7,10], laser nephelometry [ll], competitive enzyme immunoassay [12,13] and radial immunodiffusion [ 141. We now describe the development of a non-competitive ‘sandwich’ enzyme-linked immunosorbance assay (ELISA) [15] for the precise determination of the concentration of Apo E in serum and lipoprotein subfractions. The advantages of this technique include its high sensitivity, requirement for small amounts of purified antiserum, elimination of radioisotopes and possible automation for use in routine clinical analyses. Methods Blood samples Samples of blood were obtained from overnight fasted donors with lower cholesterol (CT) and triglycerides (TG) values than 6.5 mmol/l and 1.7 mmol/l, respectively. For comparative purposes, sera from hypertriglyceridemic (TG > 1.7 mmol/l) or hypercholesterolemic patients (CT > 6.5 mmol/l) were also included in this study. The blood samples were allowed to clot at room temperature and were centrifuged (15 min, 4°C 2000 X g). cis-amino-caproic acid (0.9 mmol/l), After addition of EDTA (0.27 mmol/l), chloramphenicol (0.6 mmol/l), and glutathione (0.3 mmol/l), serum samples were kept at 4OC or frozen at -2OOC. Lipoprotein fractionation Lipoprotein fractions (VLDL d< 1.006 g/ml, LDL 1.019 < d < 1.063 g/ml, LpB 1.040 < d< 1.053 g/ml, and HDL 1.063 < d < 1.21 g/ml) were isolated from plasma either by sequential ultracentrifugal flotation [16], or by density gradient ultracentrifugation [17]. Isolation of Apo E VLDL from hyperlipemic donors was delipidated after lyophilisation with etherethanol [18] and solubilized in 10 mmol/l Tris (pH 8.2) containing 8 mol/l urea for 24 h at 4°C. VLDL apolipoproteins (80 mg) were separated on two Sephacryl S.200 columns (Pharmacia, Uppsala, Sweden) (100 X 2.6 cm) in series using the same urea-Tris buffer (flow rate 10 ml/h). The Apo E containing fractions were pooled and taken either for affinity column chromatography or for further purification by chromatofocusing [19]. Apo E purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [20]. The isolated Apo E displayed amino acid composition typical of purified Apo E [5] and did not cross-react with antisera against other apolipoproteins.

The pool of Apo S.200 was dialyzed activated Sepharose Sepharose 4 B was

E containing fractions isolated by chromato~aphy on Sephacryl against 0.1 mol/l NaHCO, (pH 8.3) and coupled to CNBr4 B (Pharmacia) [21]. Five milligrams of Apo E/ml of activated coupled.

Antiserum to ~umun Ape E Antiserum to human Apo E was prepared by injection of 0.3 mg of the chromatof~using purified Apo E into rabbits as previously described [22]. After four additional injections of 0.16 mg of purified Apo E, blood was collected and the specificity of the antiserum assessed by double immunodiffusion in agarose against purified lipoprotein B, Apo A-I, A-II, C, E and whole serum and HDL subfractions. The antiserum gave a single precipitin line against Apo E, whole serum and HDL subfractions. Antibodies to Apo E were purified by passage over the Apo E-Sepharose 4 B column (9 x 3 mm; 1 ml/h), eluted with a 200 mmol/l glycine HCl buffer at pH 2.8 and dialysed against a solution of 500 mmol/l.

In order to demonstrate that all major Apo E isoforms are recognized by the anti-APO E enzyme conjugate, the isoforms of Apo E were separated from Apo-VLDL or from purified Apo E by isoelectric focusing on polyacrylamide gel slabs con~g ampholines with pH range 4-6; these ~pho~es were synthesised in our laboratory f23f. One half of the slab was stained with Coomassie blue R-250, while proteins on the remainder of the gel slab were transferred on to nitro-cellulose paper (Millipore). The nitro-cellulose paper was then incubated with the antibodyenzyme conjugate (diluted l/250) and with substrate [24]. All the Apo E bands were stained with Coomassie blue as well as ~tib~y-else conjugate. Ape E standard Since the immunoreactivity of highly purified apolipoproteins might be less well-preserved than that of their counterparts in plasma X25],standard curves were constructed from a secondary plasma standard, which was calibrated by ELISA against purified Apo E. The protein content of this primary Apo E standard was determined by the method of Lowry et al. [26]. In order to verify calibration of our purified Apo E, its Apo E content has been determined by enzyme immunoassay using purified unsialylated Apo E3 and the purified Apo E3/3 isoform as standards. These two forms of Apo E were kindly prepared by electrofocusing by Dr. Karl H. Weisgraber at the Gladstone Laboratories, San Francisco, CA, USA. ~~e~a~at~onof the antib~-enzyme ~onj~~at~ Ten milligrams of purified antibody to Apo E were coupled to peroxidase (5 mg: Boehringer Mannheim, EC 1.11.1.7) as described by N&me and Kawaai [27]. The conjugate was mixed with an equal volume of ‘glycerol for storage in portions at - 20°C.

248

Peroxidase

substrate solution

O-Phenylenediamine dihydrochloride (Sigma, St. Louis, MO, USA) was dissolved at a concentration of 16 mmol/l in a 100 mmol/l phosphate 100 mmol/l citrate buffer at pH 5.5, containing hydrogen peroxide 3.5 mmol/l and used within 30 min of its preparation. Immunoassay

Coating, washing, addition of conjugate, substrate, HCl and finally spectrophotometer reading were performed by use of an automatic ELISA Processor (Behring Institute, Marburg, FRG). In order to minimise non-specific binding to ~crotiter wells, the assay buffer (dilution of antigen or conjugate) contained 10 g/l BSA. Samples were usually diluted with 100 mmol/l phosphate-buffered saline at pH 7.4 containing BSA 1 OOO- and 2000-fold with an electronic dilutor (Dilutrend, Boehringer Mannheim) and the plasma standard diluted from 1,000 to l/6400. Polystyrene microtiter plates (Flat Bottom 96-well EIA plates, catalog no. 3590, Costar, Cambridge, MA, USA) were washed with phosphate-buffered saline before they were coated with 100 ~1 of antibodies to Apo E in each well (150 mg/l in phosphate-buffered saline at pH 7.4 containing 3 mmol/l NaN,) by incubation overnight at room temperature or for 3 h at 37°C and subsequently stored at 4°C until use (not tested for a longer than one month). After washing with phosphate-buffered saline four times, 100 ~1 of each dilution of the samples and of the standards was added to the wells and incubated for 2 h at 37“C. The plate was washed and incubated again for 2 h at 37°C with 100 yl of peroxidase-antibody conjugate (diluted 4000-fold in phosphate-buffered saline containing BSA) per well. After washing, 100 ~1 of a fresh substrate solution was added to each well. The enzyme reaction was performed for 30 min at room temperature in the dark. On completion, the reaction was terminated by addition of 100 ~1 of 1 mol/l HCl per well, and the absorbance read at 492 nm. The absorbance was plotted against Apo E concentration to generate the standard curve from which the Apo E concentration in serum or lipoprotein subfractions could be determined. The concentration of Apo E in normolipe~c and h~er~pemic plasma samples was also measured by EIA [12] in order to compare the precision and reproducibility of both methods. Delipidation

of serum

In order to test the influence of delipidation 30 sera and plasma standard were delipidated with hydroxypolyethoxydodecane (’ thesit’, Behringwerke AG, Marburg, FRG) and with n-butanol/diisopropyl ether [283. The TG levels of these sera were between 0.60 and 8.07 mmol/l and CT levels between 2.37 and 7.89 mmol/l.

Optimal assay ~anditia~s

Optimal coating of microtiter plates with our purified antiserum to Apo E was

249

evaluated by coating at different immunoglobulin concentrations (0 to 20 mg/dl) followed by incubation with a fixed dilution (1 OOO-fold) of the working standard. The remainder of the experiment was carried out at several conjugate dilutions (500 to 16000-fold). A concentration of 15 mg/l of antibodies for plate coating (1.5 pg/well), and a 1 : 4000 dilution of the conjugate were optimal for high sensitivity and low zero-dose. Optimal conditions for the period of incubation of antigen with anti-APO E coated plates and of conjugate binding under different periods of incubation were determined. Serial dilutions of the plasma standard (l/10-1/105) were added to the wells and incubated for 30, 60, 90, 120 and 180 min at 37OC. After washing, the conjugate (1 : 4000) was added to each well and incubated as indicated above. The remainder of the assay procedure was carried out as described above. A standard curve, giving both a good working range (l-40 ng) and sensitivity (the minimum detectable content was 1 rig/assay)) for the assay, was obtained by use of a 2-h incubation at 37OC for each step. Precision of the assay The mean within-assay coefficient of variation (CV), calculated from measurement of the Apo E content of three samples (with Apo E concentrations of 58, 70 and 107 mg/l, respectively), repeated 60 times, was 4.65 f 0.14%. To determine the CV for between-assay variability, duplicate measurements were performed on 20 serum samples (with Apo E levels ranging from 47-118 mg/l) in 20 consecutive assays on 3 consecutive working days. The mean between-assay CV was 7.08 k 0.43%. Accuracy of the assay To determine whether our method measures Apo E accurately in purified lipoprotein fractions, we prepared standard curves for Apo E present in plasma, in VLDL, in LDL and in HDL fractions. The parallelism of the curves (Fig. 1) were tested after linearisation and the slopes of the corresponding lines are respectively 1.11; 1.31; 1.14 and 1.23 for VLDL, HDL, LDL and plasma suggesting that our method permits estimation of Apo E content in all lipoprotein density classes, and that Apo E in these fractions reacts in an immunologically similar fashion. We also evaluated the accuracy of the assay by mixing various quantities of chylomicrons or VLDL to the infranatant at d = 1.006 g/ml of the corresponding hypertriglyceridemic plasma; or inversely by mixing various quantities of delipidated apo VLDL to a serum. Each fraction was quantified for Apo E before and after mixing (Fig. 2). The origin ordinates represent the value of residual Apo E before adding. All the regression coefficients are good (r = 0.98) and the slopes are near 1. The Apo E contents of 30 sera have been quantified before and after treatment. Detergent or delipidation does not modify the response. The linear regression between non-delipidated samples (x) and treated samples (y) are with thesit and with y = 0.16 + 0.82x, r = 0.934, X = 78.3 + 43 mg/l, 7 = 80.4 f 37 mg/l; n-butanol diisopropyl ether, y = 0.12 + 0.995x, r = 0.979, X = 78.3 -t 43 mg/l, j = 79.2 k 43 mg/l.

250

oL_._. 0

___-L-_

_-_b_

_I_

10"

‘0 Protein

cow37trotion

103 #,7q

.34.

/m j

Fig. 1. Calibration curves for Apo E quantitation in lipoprotein fractions isolated by sequential ultracentrifugation: VLDL (d i 1.006 g/ml) (A), LDL (1.019 <: d < 1.063 g/ml) (B), total HDL (1.063 < d < 1.21 g/ml) (C) and in the plasma standard (D). The mean of duplicate determinations at each concentration is shown. Absorbance is plotted on the ordinate against protein concentration (ng/ml) on the abscissa, on a log scale.

10 Added

opoE

20

I

(mgfdl)

Fig. 2. Plotting of measured Apo E vs. added Apo E. When Apo E is added as VLDL to the infranatant at d = 1.006 g/l (x); as chylomicrons to the infranatant at d = 1.006 g/I (0); as dehpidated Apo VLDL to serum (+) the linear regression are: X, y = 1.69 + 1.105x, r = 0.997; 0, y = 2.22 •t 0.903x, r = 0.993; + , y = 6.21+ 1.035~. r = 0.987.

251

o-

O-

OApoproteln

concentrot,on

(ng/ml)

Fig. 3. Calibration curves for Apo E quantitation obtained with purified apolipoproteins A-I (o), A-II (v),C-III (m) and E (X), respectively, with lipoprotein B (0). Absorbance is plotted on the ordinate against Apo E concentration (mg/ml) on the abscissa, on a log scale.

We measured Apo E in 40 normo- and hyperlipidemic samples (with Apo E concentrations ranging from 42-140 mg/l) by our enzyme immunoassay (x) and by electroimmunoassay (y). There was a good correlation between these two methods r = 0.81, y = 0.78x + 17.74, x = 83.9 f 22.4 mg/l, y = 83.6 k 21.6 mg/l. Specificity of the assay The specificity of our assay for Apo E was demonstrated by the construction of calibration curves with lipoprotein B and Apo E, A-I, A-II and C-III, respectively. The curves obtained demonstrated that our method did not quantify apoproteins other than Apo E (Fig. 3). Reactivity of isoforms of Apo E Immunoblotting demonstrated that all Apo E isoforms were recognized by our anti-APO E enzyme conjugate. Apo E3 and nonsialylated Apo E3/3 isoforms gave standard curves which were superimposable on that of a standard curve constructed from Apo E isolated from a pool of plasma, indicating that the immunological reactivity of these Apo E isoforms was identical to that of total Apo E.

I

limits (g/ml)

sera serum

h

in human

3.2 2.5 84.0

c: 1.019

1

Gradient

0.7 1.5 1.2

a

isolated

2.0 7.6 0.1

1.0231.043

3

number

fractions

1.0191.023

2

fraction

serum lipoprotein

2.5 8.5 0.2

1.0431.058

4

3.2 11 0.3

1.0581.069

5

over the entire density

6.0 17 0.5

1.0691.091

6

gradient

8.5 8 0.6

1.0911.121

7

14.5 3 0.7

1,1211.148

8

19.0 4 3.0

1.1481.186

9

18.4 8 1.9

1.1861.211

10

21.8 26 8.4

> 1.211

11

--

a All fractions were of 1 ml volume with the exception of fractions 3 and 5 which were removed in volumes of 1.8 and 0.5 ml, respectively. ’ Density limits are taken from density profile obtained in ‘control’ NaCI-KBr gradient after ultracentrifugation for 48 h. as a function of volume [17]. ’ Values are average data from gradients of three different normolipidemic, of two type IIA homozygous hypercholesterolemic and of one type hypertriglyceridemic serum. Each fraction was analyzed in duplicate.

Normoli~mic sera H~erchoIesterolemic Hypertriglyceridemic

Total Apo E (W) From gradient ’ of:

Density

Apo E distribution

TABLE

IV

253

Apo E concentration in normolipidemic plasma The mean Apo E concentration found in our normal 54 5 19 mg/l.

subjects

(n = 132) was

Distribution of Apo E among lipoprotein subfractions The distribution of Apo E among lipoprotein fractions was investigated in healthy subjects and in hyperlipidemic patients either by a density gradient ultracentrifugation procedure [17] or by preparative ultracentrifugation [16]. Apo E contents were measured in density gradient fractions from 3 normolipidemic, 2 hypercholesterolemic and 1 hypertriglyceridemic plasma sample (Table I), in which mean Apo E concentrations were 65,108 and 229 mg/l, respectively. In healthy subjects and in the hypercholesterolemic patients, Apo E was distributed over a distinct density range, i.e. 1.069 < d < 1.21 g/ml, and corresponding to that of HDL, and HDL, [17]. The hypercholesterolemic patients possessed less Apo E in the HDL, density range as compared to that seen in the equivalent density range in normal plasmas, but in contrast, rather more lipoprotein-associated Apo E was detected in these subjects over the density range corresponding to LDL, HDL, and HDL, (1.020 < d< 1.100 g/ml). In the hypertriglyceridemic patient (type IV), > 80% of Apo E was associated with the triglyceride-rich lipoproteins (chylomicrons and VLDL), thereby contrasting with the distribution typical of healthy and of hypercholesterolemic subjects. The large amount of Apo E in the infranatant of d > 1.22 g/ml (21%) is presumably an artefact produced by ultracentrifugation. In these density gradient ultracentrifugation studies, the mean recovery of Apo E was 84 + 11.3%. Discussion We have developed a highly specific, non-competitive enzyme-linked immunoassay for the quantification of human serum apolipoprotein E. The advantages of this method include its rapidity, simplicity, high precision and a sensitivity similar to that of radioimmunoassay [8] (the minimum detectable contents were 1 ng and 0.8 ng, respectively). The coated microtiter plates are stable for a month at least and the labelled second antibody is stable for at least one year at -2OOC. The method avoids the use of radioisotopes. Repeated freezing and thawing of plasma samples (up to five times) did not alter their behaviour in the enzyme immunoassay. Moreover, the conditions of storage (frozen for up to 6 mth at - 35” C or refrigerated for up to 7 days at + 4OC) did not appear to affect the immunoassayable Apo E. The precision of our assay is comparable to that typical of other methods [8,15]. The specificity of our ELISA was established by the failure of purified human Apo A-I, A-II, and C-III and lipoprotein B to generate a measurable response. The accuracy of our method was demonstrated by the identical forms of standard curves obtained with three different Apo E preparations, a good correlation in levels of Apo E determined by the ELISA and EIA, and a highly satisfactory recovery of

254

amounts of Apo E added to a sample by addition of chylo~cron or VLDL or Apo-VLDL fractions. A good correlation is also obtained between Apo E of intact and delipidated sera. We have found serum Apo E concentrations in normolipemic subjects similar to those of Castro and Fielding [14] who used radial immunodiffusion and of Have1 et al [l] who used radioimmunoassay. By contrast, others have reported lower, using a radioimmunoassay [8], or higher values, using an electroimmunoassay [7] and a laser immunonephelometric assay [ll], respectively. It seems likely that the different Apo E concentrations are relatives to the calibration procedure used. Measurement of the Apo E concentration in plasma and of its distribution in lipoproteins and in lipoprotein subfractions in relation to genetic variation in its isoforms was a major reason for development of our assay. Our results showed that the major isoforms react with the antibodies which we have raised against total Apo E. This finding is consisting with others [1,9,29]. In normolipidemic and hypercholesterolemic subjects, Apo E was primarily associated with high density lipoprotein fractions (d > 1.060 g/ml). By contrast, the density distribution of Apo E in the plasma of the hypertriglyceridemic patient was distinguished by its almost exclusive association with triglyceride-rich lipoproteins. These results differ from those of Kushwaha et al [30] who found that Apo E in hypertriglyceridemic plasma was not concentrated in VLDL and chylomicron fractions. However, in agreement with our results, Blum et al. f8], using agarose column chromatography, showed that nearly all Apo E was associated with the triglyceride-rich lipoproteins in h~ert~glyce~de~c, and that 64% and 74% of Apo E was associated with HDL in normals, and in h~ercholesterole~cs subjects, respectively. The remainder of APO-E was mainly associated with VLDL in these subjects. In contrast, Utermann 1311 found Apo E to occur predominantly in the VLDL of both normal and hyperlipoproteinemic subjects but also detected Apo E in HDL. Curry et al [7] showed that apolipoprotein E in the serum of normolipidemic subjects was equally distributed among three major lipoprotein density classes. Thus, they showed that lipoprotein E occurred mainly in association with lipoprotein B and C in the VLDL fraction, but that Apo E constituted the protein moiety of a distinct lipoprotein family (LpE). Have1 et al (11 observed that the dissociation of Apo E from human lipoproteins during ultracent~fug~ isolation is more pronounced for VLDL than HDL, that Apo E was mainly associated with VLDL, IDL and LDL,, and that its recovery in HDL and LDL fractions could result from ultracentrifugal dissociation from VLDL. Our ultracentrifugal studies have shown that isolated LDL and HDL contains significant proportions of Apo E, a finding in good agreement with the earlier observations of other authors [7,8]. We have also detected large amounts of Apo E in the d > 1.22 g/ml fraction, which presumably results from the dissociation of Apo E from lipoprotein particles during ultracentrifugation. Apo E distribution in lipoprotein subfractions does appear to vary from one normolipidemic (or hypercholesterolemic) individual to another, and further studies

255

on the apoprotein composition of human serum lipoprotein fractions are therefore in progress in our laboratory in order to evaluate the clinical significance of the present results. Acknowledgement

We thank Doctor P. Alaupovic for his help in EIA method. References 1 Havel RJ, Kotite L, Vigne JL, et al Radioimmunoassay of human arginine-rich apoiipoprotein, apoprotein E. Concentration in blood plasma and lipoproteins as affected by apoprotein E-3 deficiency. J. Clin Invest 1980;66:1351-1362. 2 Zannis VI, Breslow JL. Apolipoportein E. Molec Cell Biochem 1982;42:3-20. 3 Shore B, Shore V. An apolipoprotein preferentially enriched in cholesteryl ester-rich very low density lipoproteins. Biochem Biophys Res Commun 1974;58:1-7. 4 Mahley RW, Weisgraber KH, Rail JC, Innerarity TL. Structure and function of apolipoprotein E. In: Lippel K, ed. Proceedings of the Workshop on Apo~poprotein Qu~tification. Lipid metabolism. Bethesda, MD: Atherogenesis Branch, National Heart, Lung and Blood Institute, 1984: 123-135. 5 Rali Jr SC, Weisgraber KH, Mahley RW. Human apolipoprotein E. The complete amino acid sequence. J Biol Chem 1982;257:4171-4178. 6 Mahley RW, Hui DY, Innerarity TL, Weisgraber KH. Two independent lipoprotein receptors on hepatic membranes of dog, swine, and man. J Clin Invest 1981;68:1197-1206. 7 Curry MD, McConathy WJ, Afaupovic P, Ledford JH, Popovic M. Determination of human apolipoprotein E by electroimmunoassay. B&hem Biophys Acta 1976;439:413-425. 8 Blum CB, Aron L, Sciacca R. Radioimmuno~say studies of human apolipoprotein E. J Clin Invest 1980; 66: 1240-1250. 9 Milne RW, Douste-Blazy P, Retegui L, Marcel YL. Characterization of monoclonal antibodies against human apolipoprotein E. J Clin Invest 1981;68:111-117. 10 Bittolo Bon G, Cazzolato G, Saccardi M, Kostner GM, Avogaro P. Total plasma apo E and high density lipoprotein apo E in survivors of myocardial infarction. Atherosclerosis 1984;53:69-75. 11 Weisweiler P, Schwandt P. Determination of human apol~poproteins A-I, B, and E by laser nephelometry. J Clin Chem Clin 1984;22:113-118. 12 Holmquist L. Quantitation of human serum very low density apo C-I, C-II, C-III and E by enzyme immunoassay. J Immunol Methods 1980;34:243-251, 13 Wright DA, Beck DL, Garcia RE, Karim R, Holten D. Quantitation of apolipoprotein E in rabbit sera with a competitive enzyme-linked immunosorbent assay. J Immunol Methods 1983:58:143-153. 14 Castro GR, Fielding CJ. Evidence for the distribution of apoiipoprotein E between lipoprotein classes in human no~~hoiesterolemic plasma and for the origin of unassociated apolipoproteins E (Lp-E). J Lipid Res 1984;25:58-67. 15 EngvaB E, Perlmann P. Enzyme linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulins G. Immunochemistry 1971;8:874-879. 16 Fruchart JC, Desreumaux C, Dewailly P, et al. Enzyme immunoassay for human apolipoprotein B, the major protein moiety in low-density and very-low density lipoprotein. Clin Chem 1978;24:455-460. 17 Chapman MJ, Goldstein S, Lagrange D. Laplaud PM. A density gradient ultracentrifugal procedure for the isolation of the major lipoprotein classes from human serum. J Lipid Res 1981;22:339-358. 18 Huff MW, Fidge NH, Nestel PJ, Billington T, Watson B. Metabolism of C apolipoproteins: kinetics of C-II, C-III, and C-III,, and VLDL apolipoprotein B in normal and hyperlipoproteinemic subjects. J Lipid Res 1981;22:1235-1246. 19 W&w&r P, Schwandt P. Isolation of human apolipoprotein E by chromatofocusing Clin Chim Acta 1982:124:45-50.

256 20 Kane JP. A rapid electrophoretic technique for identification of subunit species of apoproteins in serum lipoproteins. Anal Biochem 1973;53:350-364. 21 Axen R, Porath J, Ernback S. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature 1967;214:1302-1304. 22 Fruchart JC, Kora I, Cachera C, et al. Simultaneous measurement of plasma apolipoproteins A-I and B by electroimmunoassay. Clin Chem 1982;28:59-62. 23 Bugugnani MJ, Koffigan M, Kora I, Ouvry D, Clavey V, Fruchart JC. Rapid assessment of the distribution of apolipoproteins C-II and C-III subspecies in triglyceride-rich lipoproteins by isoclectric focusing. Clin Chem 1984;30:349-351, 24 Kittler JM. A general immunochemical method for detecting proteins on blots. Analyt Biochem 1984;137:210-212. 25 Albers JJ, Cheung MC. Radial immunodiffusion assay of lipoproteins and apoproteins: application to high density lipoproteins. In: Lippel K, ed. Report of the High Density Lipoprotein Methodology Workshop. U.S. Dept. of Health, Education, and Welfare, Public Health Service, National Institutes of Health Publication no. (NISI) 79-1616, 1979: 251-264. 26 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-275. 27 Nakane P, Kawaai J. Peroxidase laballed antibody, a new method of conjugation. J Histochem Cytochem 1974;22:1084-1091, 28 Cham BE, Knowles BR. A solvent system for delipidation of plasma or serum without protein precipitation. J Lipid Res 1976;17:176-181. 29 Weisweiler P, Schwandt P. Immunonephelometric quantitation of apohpoprotein E in human serum. J Clin Chem CIin Biochem 1983;21:227-230. 30 Kushwaha RS, Hazzard WR, Wahl PW, Hoover JJ. Type III hyperlipoproteinemia: diagnosis in whole plasma by apolipoprotein E immunoassay. Ann Intern Med 1977;86:509-516. 31 Utermarm G. Isolation and partial characterization of our arginine-rich apohpoprotein from human plasma very low density lipoproteins: apolipoprotein E. Hoppe-Seyler’s Z Physiol Chem 1975;356:1113-1121.