267

Clinica Chimica Acta, 70 (1976) 267-276 0 Elsevier Scientific Publishing Company,

Amsterdam

- Printed

in The Netherlands

CCA 7815

RADIOIMMUNOASSAY

D.K. BEDFORD,

J. SHEPHERD

University Department (Received

January

FOR HUMAN PLASMA

APOLIPOPROTEIN

B

* and H.G. MORGAN

of Pathological Biochemistry,

Royal Infirmary,

Glasgow (U.K.)

29, 1976)

Summary

We have developed a simplified double antibody radioimmunoassay for human apolipoprotein B. The purified antigen, a narrow density subclass of /3 lipoprotein (d 1.030-1.050 g/ml), was isolated by a combination of gel filtration and ultracentrifugation. This material gave a single immunoprecipitin arc on crossed immunoelectrophoresis into an antibody to whole human serum. The antigen was radiolabelled using iodine monochloride at pH 10. The iodinated antigen was indistinguishable immunochemically from native material and eluted as a single radioactive peak from a Sephadex G-200 column. The lower limit of sensitivity of the assay was 15 ng protein, and the working range, 15-200 ng. The mean apolipoprotein B level (?l S.D.) in 128 healthy control subjects was 86.6 + 29.6 mg/lOO ml and is in agreement with the values published by other workers using unmodified assays.

Introduction

Normal fasting human plasma contains a number of distinct proteins specifically associated with lipids to form lipoprotein complexes which are separable into three main classes (very-low-density lipoproteins, VLDL; low-density lipoproteins, LDL; and high-density lipoproteins, HDL) according to their hydrated density [ 11, electrophoretic mobility [2], or molecular size [3,4]. Each lipoprotein class contains several kinds of lipids and one or more of the above proteins which can be regarded as belonging to one of at least four apoprotein families: apo-A, apo-B, apo-C and apo-D [5,6,7]. Recently Uterman has described, tentatively, a fifth apoprotein family, apo-E [8], which may correspond to the “arginine-rich” protein described by Shore and Shore [ 91. APO-B is predominantly although not exclusively associated with the low* To whom all correspondence Brown Building, The Methodist

should be sent. Present address: Lipid Research Clinic, Hospital. 6516 Bertner Boulevard. Houston, Texas 77025.

E‘ondrenU.S.A.

density lipoprotein class which is pathologically increased in the plasma of type II hyperlipoproteinaemic patients who characteristically demonstrate premature and severe atherosclerosis of coronary and peripheral arteries. A number of assay procedures based on immunological techniques have been devised to measure plasma levels of apolipoprotein B. The earliest of these used immunoelectrophoresis [lo] or radial immunodiffusion [ 111 in a solid agarose support. This support system permits free diffusion of LDL but limits migration of apolipoprotein B-containing VLDL through the gel and so results in an underestimation of total apolipoprotein B. More recently developed radioimmunoassay techniques, which do not suffer from this shortcoming, are now available for apolipoprotein B estimation in whole plasma from rats [12] and humans [13, 141. The authors of these double antibody radioimmunoassays have demonstrated that radioiodination of LDL with chloramine T as oxidant induced considerable lipoprotein denaturation which necessitated subsequent time-consuming gel filtration steps to isolate native labelled antigen in pure form. We have confirmed their observation [ 151 and have adopted the iodine monochloride labelling procedure of Langer et al. [16] which causes minimal antigen denaturation and gives a product in which more than 95% of the label is in the protein moiety. The radioactive material is cleared from rats monoexponentially, as determined by whole body counting, and appears as a single peak in the void volume of a Sephadex G-200 column [ 151. Therefore, the labelled antigen does not require further fractionation prior to its use in the radioimmunoassay which consequently is simplified and accelerated. This study describes the modified radioimmunoassay and defines its capabilities. A preliminary report of the method has already been published [ 17 1. Materials Bio-Gel A5m (200-400 mesh) was purchased from Bio-Rad Laboratories, Richmond, Calif. 94804. Carrier-free Na’*‘I of high specific activity for protein labelling was obtained from New England Nuclear, Boston, Mass, 02118; bovine albumin from Armour Pharmaceutical Co. Ltd., Eastbourne, England; donkey, anti-rabbit anti-serum from Wellcome Reagents Ltd., Beckenham, Kent, England; and rabbit anti whole human serum and standard P-lipoprotein from Hoechst Pharmaceuticals, Hounslow, England. Methods Preparation of low density lipoprotein Low density lipoprotein of narrow density range (d 1.030-1.050 g/ml) was purified from pooled plasma of healthy human donors which was collected into l/100 volume of 1.0 M Tris buffer, pH 7.0, containing 1.0% disodium EDTA. Earlier work by Alaupovic et al. [6] had shown that the apolipoprotein of this LDL subfraction consists almost exclusively of apo-B, with trace quantities of apo-A and apo-C in insufficient amounts to generate significant antibody levels under the experimental conditions described below. The density of the plasma was raised to 1.225 g/ml by addition of solid KBr (0.3517 g/ml) and overlayered with an equal volume of 0.01 M Tris buffer, pH

269

7.0, containing 0.01% disodium EDTA and KBr to a density of 1.225 g/ml. Ultracentrifugation for 24 h (15”C, 258 000 X g) in the Ti 60 rotor of a Beckman L5 ultracentrifuge (Beckman Instruments Inc., Palo Alto, Calif. 94304) separated the buoyant orange-coloured lipoprotein fraction from the remaining plasma proteins which sedimented to the bottom of the tube. The lipoprotein fraction, which contained more than 95% of the plasma cholesterol and no non-lipoprotein proteins as detectable by crossed immunoelectrophoresis [ 181, was harvested carefully using a Pasteur pipette and concentrated in a Minicon B15 concentrating cell (Amicon Corp., Lexington, Mass. 02173). The concentrate (approximately 3.0 ml of lipoprotein from 150 ml plasma) was applied directly to a 2.6 cm X 90 cm Bio-Gel A5m agarose column (200-400 mesh) and eluted at room temperature and a flow rate of 15 ml/h with O.OlM Tris buffer, pH 7.0, containing 0.01% disodium EDTA and NaCl to 0.15 M. The AZXOnlnprofile of the eluate contained three peaks, the second of these corresponding to LDL [ 31. This material was pooled, raised to a density of 1.030 g/ ml by addition of solid KBr, and subjected to ultracentrifugation using the conditions described above. The lipoprotein in the lower third of the centrifuge tubes (d > 1.030 g/ml) was collected and respun under the same conditions and at a density of 1.050 g/ml. The buoyant LDL fraction isolated by this step (d 1.030-1.050 g/ml) was dialysed for 24 h against a 1000-fold excess of the column buffer prior to its use in antibody production, in preparation of ““I-labelled LDL, and as a reference protein in the radioimmunoassay. Production of LDL antibody 2.0 mg of LDL protein were emulsified with an equal volume of Freund’s complete adjuvant and injected subcutaneously at two weekly intervals into New Zealand White rabbits, using multiple injection sites. After the third injection, the animals were bled out by cardiac puncture and their serum collected and stored in small aliquots at -20°C after addition of NaN3 (1 mg/ml). LDL iodination Prior to iodination, the protein content of the LDL was estimated by the method of Lowry et al. [19], correction being made for differential colour development when using an albumin standard [20]. The iodination procedure was a modification of the method of Langer et al. [ 161, and has been described elsewhere [ 151. The purified radioiodinated LDL contained less than 1% free ‘*‘I as determined by paper electrophoresis, and eluted as a single peak from a Sephadex G-200 gel filtration column, even after two month’s storage. The iodinated LDL was stored at 4°C in 0.05 M barbital buffer, pH 8.6, containing bovine albumin to 3%. LDL radioimmunoassay The assay (Table I) was performed in polystyrene tubes (LP 3 tubes, Luckham Ltd., Burgess Hill, Sussex, England) and was adopted from the procedure of Schonfeld et al. [13]. All dilutions were made in 0.05 M barbital buffer pH 8.6 containing bovine albumin to 3%. 300 ~1 of appropriate standard or diluted plasma were mixed with 100 ~1 of anti LDL antibody and preincubated for 18 h. 100 ~1 of 1251-labelled LDL (approximately 20 ng protein; 5 X lo3 counts/

“70

min) were added with thorough mixing and incubation continued for a further 48 h. Finally, 50 ~1 of donkey anti rabbit antiserum were added and incubation terminated after another 24 h. The antigen-antibody complexes were precipitated by centrifugation, washed twice with barbital buffer and counted in a Packard Autogamma Scintillation Spectrometer (Packard Instrument Ltd., Caversham, Berks., England). Two blanks were included. The first contained ’ 2SI-labelled LDL and buffer only, while in the second a non-immune rabbit serum replaced the anti-LDL antibody and buffer replaced the LDL standard or diluted plasma. In both cases, the residual radioactivity of the blanks was less than 2% of the total added ‘2”I-labelled LDL.

Fig. ads to

1. Immunochemical was whole

subjected human

purity

to crossed serum.

of

isolated

LDL.

immunorlectr[lph~,resis

Low-density using

lipoprotein, a 7 .O% agarose

purified support

as described and

3% rabbit

in Methantibwlv

Results Immunochemical

purity

and stability

of ‘251-labclled

LLII,

The immunochemical purity of the ‘2”1-lahelled LDL antigen (d = 1.0301.050 g/ml) was checked by crossed immunoelectrophoresis [ 171 into anti-total human antiserum (Fig. 1). The single immunoprecipitin arc confirmed that the LDL was immunochemically pure: and Ouchterlony double diffusion analysis 1201 of native and radioiodinated LDL demonstrated that the labelling procedure had not altered the immunochemical reactivity of the molecule (Fig. 2). The precipitin arcs of native and labelled LDL showed reactions of identity and the immunoprecipitate of the latter coincided with the autoradiographic precipitin line [ 151. Freshly prepared ‘*“I-labelled LDL eluted as a single peak from a Sephadex G-200 column (Fig. 3). The same result was obtained after two month’s storage at 4°C in the presence of 3% bovine albumin to minimise self irradiation damage, this finding obviating the need for repeated purification of the labelled antigen prior to its use in the assay.

Fig. 2. Effect of radioiodination on immunochemical reactivity of LDL. Native and ‘25 I-labt~llrdI,DL were compared by double immunodiffusion in a 1% agarose support. Alternate wriyheral wrlls from A contained native LDL and ‘251-labelled LDL was inserted into alternate wells from B. The wntrc well contained a purified anti-LDL antibody.

272

15

I

r-=ay

10

\ I

20

-------v---*--=1 30

40

Fraction Fig. 3. Scphadrx G-200 chrr)matography of ’ * ’ I-lab control plasma (1); *, control

Displacemant curves of I251-labelled LDL by plasma are shown. The antiserum dilution was plasma

(2);

J. abetalipoproteinemic

Pfasma.

275

The assay specificity was investigated as described in the legend of Fig. 7, in which percentage of label bound is plotted against the reciprocal of the antibody dilution. There is distinct parallelism between the antibody reactivity against the standard LDL preparation and against diluted plasma specimens from two control subjects. Moreover, the plasma from a patient with abetalipoproteinemia, in which there is absolute deficiency of apoprotein B, caused no ‘2sI-labelled LDL displacement in the assay, even at plasma dilutions of only l/1000. Apolipoprotein B levels were measured in the plasma of 128 control subjects (60 male, 68 female; mean age 48 years, range 16-66 years; mean cholesterol (2 1 SD.) 240 + 35 mg/lOO ml). The frequency distribution was Gaussian, with a mean value (+l S.D.) of 86.6 + 29.6 mg/lOO ml. There was no statistically significant difference between male and female values. Finally, a comparison was made between the Apo B levels in 100 subjects as measured by radial immunodiffusion and by the radioassay. A correlation coefficient of 0.66 was obtained. Discussion Plasma lipoproteins are, in general, measured indirectly by quantitating (/3 quantification) their lipid content by a procedure [22] which requires expensive equipment and which is not amenable to automation. Radioimmunoassay of the apoprotein components of the lipoproteins is a viable alternative or adjunct to ,!? quantification, and a number of specific and sensitive double antibody radioimmunoassays for apolipoprotein B have been described in the literature [12,13,14]. In this study we have modified the LDL isolation and labelling technique. These modifications have permitted the preparation of a labelled antigen which, on storage at 4°C in the presence of 3% bovine albumin, is stable for long periods (at least two months) and does not require repurification prior to its use in the assay. Therefore, the gel filtration steps required to purify the antigen in previous assays [12,13,14] may be omitted from the method. This benefit has not been achieved at the expense of assay sensitivity or specificity. The assay sensitivity (15 rig/assay)) is in excess of that required to measure apolipoprotein B levels in plasma and has potential value in in vitro studies of LDL metabolism. The specificity of the assay was checked in several ways. The standard LDL displaced, competitively, more than 87% of the ‘251-labelled LDL from the antibody; the plasma dilution curves of standard and test plasmas showed distinct parallelism; and abetalipoproteinaemic plasma, even at high concentration, failed to compete with the ‘251-labelled LDL for antibody combining sites. The mean apolipoprotein B level (+l S.D.) in the plasma of 128 control subjects was 86.6 f 29.6 mg/lOO ml, which correlates with radial immunodiffusion data (r = 0.66). This result is in agreement with the value obtained by other workers using an unmodified assay [ 13,141. Our experience of this simplified radioimmunoassay for apolipoprotein B has confirmed our prediction of its usefulness as an addition to or replacement for p quantification. Future ability to assay the other apolipoproteins in this way

276

may eventually provide a diagnostic test for dyslipoproteinaemias superior to the qualitative techniques currently employed.

which

is

Acknowledgement This work was supported Research Trust.

by a grant frdm the Scottish

Hospital

Endowments

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