Purification of antiphospholipid antibodies using a new affinity method

THROMBOSIS RESEARCH 52; 641-648, 1988 0049-3848/88 $3.00 + .OO Printed in the USA. Copyright (c) 1988 Pergamon Press plc. All rights reserved.

BRIEF COMMUNICATION PURIFICATION OF ANTIPHOSPHOLIPID ANTIBODIES USING A NEW AFFINITY METHOD

The

H.P. McNeil, S.A. Krilis, and C.N. Chesterman University of New South Wales, School of Medicine, The St. George Hospital, Sydney, Australia

(Received 16.6.1988; accepted in revised form 20.8.1988 by Edit0r.M. Verstraete) (Received in final form by Executive Editorial Office 10.10.1988)

INTRODUCTION The development of solid phase immunoassays for the detection of circulating antibodies to phospholipids (1) has resulted in an intense interest in the study of these antibodies and of the thrombotic syndromes often associated with their presence. Whilst most work has centered on antibodies to cardiolipin (ACA), there is widespread cross-reactivity of these antibodies to the other negatively charged phospholipids, phosphatidyl-serine, phosphatidyl-inositol and phosphatidic acid (2). Hence the term antiphospholipid antibody (APLA) is more appropriate. an attempt to better characterise APLA’s, we developed an In affinity technique to extract APLA’s from patient plasma. The method involves the use of an affinity column prepared by immobilising a phosphatldyl-serine/cholesterol mixture in a polyacrylamide gel. MATERIALS Patients Plasmas from antibodies and lupus

four patients anticoagulant

AND METHODS

with activity

high levels were used.

of

anticardiolipin

Preparation of Phosphatidyl-serine/cholesterol Afflnity Column A modification of the technique described by Uchida and Filburn (3) used. Phosphatidyl-serine was (Sigma) 1Omg and cholesterol (BDH 50mg dissolved in chloroform were combined in a glass Chemicals) scintillation vial and the solvent evaporated under a stream of nitrogen. lml of ethanol was added, the vial capped, placed into boiling water, and swirled until the lipids were dissolved. The vial was quickly removed and 1Omls of a solution of 15% acrylamide, 5% BIS (Biorad) was added and of lOOU1 of mixed followed immediately by the addition vigorously mixing and an 5ul of TEMED, further ammonium persulfate, 140mg/ml additional 100~1 of ammonium persulfate. The mixture was transferred to covered with parafilm and alumlnium foil and allowed a glass test tube, to polymerize overnight at room temperature. Key Words:

phospholipid.

anticardiolipin

antibody. 641

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The rigid white gel was removed from the tube, rinsed with distilled water and minced with a razor blade. The gel was then homogenized using a hand operated loose fitting teflon pestel. The homogenized gel was washed 3 times in distilled water, allowing the gel to settle for 5 minutes and decanting the supernatant on each occasion. The settled gel particles were then assembled into a column (20mm x 70mm) and equilibrated with 5 - 10 bed volumes of O.OlM phosphate 0.15M NaCi buffer (PBS) (Dulbecco A) pH 7.3. Flow rates of up to 6OmVhour were used with minimal compaction of the relatively rigid gel particles. Phosphatidyl-choline/cholesterol A control affinity mixture was prepared phosphatidyl-serine.

affinity column utilizing in an identical

column phosphatidyl-choline/cholesterol manner to that described

above

for

Affinity Chromatography After equilibration, 8 mls of patient plasma was diluted with 32 perfused through the column at 15 mls/hour mls of PBS (1 : 5) and followed by PBS initially at the same rate until all the plasma had passed through the column, then continued at 30 mls/hour to wash unbound protein until the absorbance at 280nm of the fractions was less than 0.010 absorbance units. 35mls of eluting buffers (O.lM phosphate buffer 0.5M NaCl pH 7.3 and O.lM glycine-HCl pH 2.5) were applied sequentially to the column at 45 mls/hour. Fractions containing ACA activity were pooled and dialysed against the ion-exchange starting buffer.(see Ion-Exchange Chromatography) Radiolabelled Phosphatidyl-serine Column Ligand incorporation into the gel was determined by adding 100,000 dpm of [W]phosphatidyl-serine (Amersham) to the phosphatidyl-serine of the acrylamide. The gel cholesterol mixture prior to polymerization was then prepared and assembled into the column and chromatography performed as above. A sample of the radiolabelled gel was counted for radioactivity using NCS Solubilizer and OCS scintillation solution using the manufacturer’s recommended method (4). Following chromatography, fractions were assayed for radioactivity by counting in a scintillation counter (Packard model B4430) after addition of ASC II scintillation solution (4 mls to 200~1 of sample). Anticardiolipin antibody ELISA Anticardiolipin antibodies were measured using the enzyme linked for ACA previously described by Gharavi et. al. (2). Serial immunoassay dilutions of known positive plasma, standardised using Rayne Institute Reference sera (51, were included on each plate. The ACA level of the test samples was read from the reference sera using a log - log plot and expressed in GPL units where 1 GPL unit corresponds to the activity of 1 ug/ml of affinity purified anticardiolipin antibody (5). Lupus

anticoagulant activity The kaolin clotting time (KCT) was used as described by with a mixture of 20% test sample and 80% normal plasma 100% normal plasma. Lupus anticoagulant (LA) activity was present if the test/normal mixture clotting time was 10% longer clotting time of normal plasma. Ion-exchange Chromatography The affinity column fraction chromatography using a Pharmacia Pharmacia FI’LC (Fast Prot.ein Liquid

Exner (6) compared to considered than the

was subjected to cation exchnnge Mono S HR 5/5 column, operating with Chromatography) system.

a

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643

The sample was applied to the column in a starting buffer of 0.05M acetate 0.05M NaCl pH 4.8 with a flow rate of O.Sml/min. A linear gradient of eluting buffer 0.05M acetate 0.65M NaCl pH 5.2 from 0% to 100% over 30 minutes was applied. 1 ml fractions were collected and the absorbance at 280nm of the fractions was monitored. Fractions were assayed for ACA using the ELISA technique described above and for LA activity. ACA positive fractions were pooled and concentrated in an Amicon concentrator using a PM 30 membrane and 200 kPa pressure of nitrogen. Sodium-dodecyl-sulfate Polyacrylamide Gel Electrophoresis 50~1 samples for SDS-PAGE were added to equal volumes of 0.125M Tris pH 6.8, 20% glcerol, 4% SDS, 0.004% Bromphenol blue and heated in boiling water for 2 minutes, then run on a 5 - 20% exponent.ial gradient polyacrylamide using the Laemmli technique (7) with a 3% gel polyacrylamide stacking gel. After electrophoresis the gel was stained with Coomassie Brilliant Blue. Protein

Assay and Immunoglobulin Concentration Protein content of samples was assayed using a modification of the method of Lowry and immunoglobulin determination by (81, immunodiffusion (Mancini technique) using anti-human IgG antisera (Kallestad Laboratories).

RESULTS AND DISCUSSION Incorporation of phosphatidyl-serine into the polyacrylamide gel as assessed [14C]phosphatidyl-serine was greater than 95%. During by homogenizing and washing of the gel prior to column assembly, 5% of the added radioactivity was recovered in the supernatants. Following affinity in the fractions was within chromatography the radioactivity detected background limits thus confirming that the ligand remained immobilised even during elution with either 0.5M NaCl or glycine-HCl pH 2.5. This affinity method thus represents a significant advance over liposome techniques for antibody purification as these result in the contamination of the eluted protein with lipid ligand (9) thus requiring organic solvent extraction (10). The phosphatidyl-choline/cholesterol control column did not bind APLA with the plasma fall-through containing identical amounts of ACA and LA activity as the applied plasma (Table 1). No protein was eluted either 0.5M NaCl or O.lM glycine-HCl, from this column with confirming that phosphatidyl-serine is the true ligand. The patients plasma contained IgC-ACA with a level of 260 GPL units (260 ug/ml of antibody) or 3.1 GPL units/mg protein. A total of applied to the affinity column. The 2000 ug IgG-ACA (8mls plasma) was fall through contained IgG-ACA activity but reduced in amount to t.he applied plasma (Figure 1). In addition, LA activity was absorbed by the being complete in the early fall-through fractions but only partial column with 0.5M NaCl in O.lM in the later fractions (Table 1). Upon elution phosphate buffer, ACA activity was found in the eluted protein, but LA The total phospholipid specific activity was or absent. weak eluant was 720 ug immunoglobulin in this affinity column activity antibody. The average specific representing a 36% recovery of applied activity of l.his eluant was 306 GPL units/mg protein thus representing a one hundred fold purification on this column. Following elution with 0.5M NaCl, no further protein was eluted with O.lM glycine-HCl.

ANTIPHOSPHOLIPID ANTIBODIES

644

TABLE 1 of various fractions during purification and control phosphatidyl-choline columns

and LA activity on phoshatidyl-serine

ACA

LA ACTIVITY dKCT KCTr/KCTc (seconds)

SAUPLE

Plasma (K.R. 1 Phosphatidylcholine Column Plasma/PBS (1:5) Fall-through - # 7 - I 10 Phosphatidylserine Plasma/PBS (1: 5) Fall-through - # - I Eluted # (Pooled Ion-exchange

193

260

359 / 214 359 / 214 360 I 214

145 145 146

40 40 43

324 197 235 235

194 194 194 210

130 3 41 20

52 13 10 200

210 / 215

0

70

Column 9 14 & concentrated)

eluted

I

/ / / I

t 80% normal plasma + 80% normal plasma

J PBS

1 PLASMA Z17T

IgG-ACA ACTIVITY (GPL units)

341 / 154

KCTT = KCT of 20% test plasma or fractions KCTc = KCT of 20% control buffer / plasma dKCT = KCTr - KCTc (see methods)

./O.SMNaCl

I

0

10

20 FRACTION

Figure

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1: Affinity

30

40

50

NUMBER

chromatography of plasma from patient K.R. using phosphatidyl-serine column. Approximately 60% of the applied ACA activity (broken line) passed through the column and the remaining 40% of the applied activity eluted with 0.5M NaCI.

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SDS-PAGE analysis of the affinity column eluant (Figure 2) revealed a major protein band corresponding to IgG (Molecular weight 15OkDa) with some minor contaminating protein of lower molecular weight. When the specific immunoglobulin fraction from the affinity column was subjected to cation exchange chromatography, 3 protein peaks were demonstrated (Figure 3). ACA activity was found exclusively in the second peak and SDS-PAGE analysis (Figure 2) showed a single broad band of IgG. Protein assay and immunoglobulin determination of this ion-exchange fraction revealed a protein content of 7Oug/ml and IgG concentration of 68ug/ml thus confirming the high 095%) purity of the antibody.

Figure

2: SDS-polyacrylamide gel electrophoresis on a 5-20% exponential gradient gel of the affinity column eluted fraction (lane 2) and the ion-exchange eluted fraction (lane 1) stained with Coomassie Blue.

Activity of this fraction in the ACA-ELISA was calculated to be 70 GPL units or 70ug/ml of specific antibody. This shows a parallel dose response curve when compared with the ACA activity of the native plasma (Figure 4). This fraction contains 1000 GPL units/mg protein representing a greater than 320 fold purification. The purified IgG is a specific antiphospholipid antibody with activity in the ACA-ELISA identical to native plasma. In addition there is excellent agreement between Rayne Institute reference values (based on affinity purified antibodies using cardiolipin liposome techniques) (6) and the affinity purified IgG using this technique.

645

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100

80 E

c

z N

W

Y a

0

5

10

15

FRACTION

20

0

25

NUMBER

Figure 3: Ion-exchange chromatography of the affinity column eluted fraction. ACA activity (broken line) eluted in the middle protein peak (solid line) at 30% eluting buffer (0.2M NaCll (see text for details of buffers)

-PATIENT c_rPURIFIED

PLASMA IgG

.0

.6

. 4

.2

0

2800

1400

ANTIBODY

700

350

175

CONCENTRATION

88

44

22

[ng/mll

Figure 4: Dilution curve in the ACA-ELISA of the highly purified IgG-APLA compared to that of the native plasma at dilutions of 1:lOO to 1:12,800.

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ANTIPHOSPHOLIPID ANTIBODIES

TABLE 2 ACA and LA activity of native plasma and affinity column purified fractions from 4 patients showing purification of ACA but not LA activity. Native Patient

Purified

Plasma

IgG-ACA (GPL units)

LA

IgG-ACA (GPL units)

KR

260

tt

200

FM

90

tt

36

cs

26

t

19

TB

25

t

30

Antibody LA

t_

The purified antibody did not possess LA activity at concentrations equivalent to those present in native plasma where LA activity was strong. phosphatidyl-serine affinity column However, as seen in Table 1, the absorbed LA activity, yet this activity was not recoverable in the eluted immunoglobulin. It is possible that antibodies responsible for LA activity bind to the column with higher affinity than ACA’s and are not We are currently investigating this possibility eluted with 0.5M NaCl. We have purified a total of 4 patient plasmas on the affinity further. column and isolated immunoglobulin with strong ACA activity but LA activity has been weak or absent (Table 2). Harris et. al. (9) have said to possess LA activity, but the purified ACA’s which are effect been observed at high antibody anticoagulant has only al. (10) have affinity purified LA antibodies concentrations. Pengo et. which demonstrate LA activity at concentrations as low as 3.3ug/ml and in solid phase immunoassays, although the bind to anionic phospholipids This disproportionate is not stated. amount of ACA activity present purification of one activity (ACA) over the other (LA), is consistent with our data and suggests that the antibody responsible for LA activity may be a different APLA than those binding in solid phase immunoassays. It is also possible that there are a range of antibodies with similar specificities but varying affinities for different phospholipids (11). described here provides a In summary, the purification procedure of obtaining highly purified antiphospholipid simple and rapid means over liposome techniques. In antibody with significant advantages particular, the complete incorporation and immobilisation of lipid ligand an immobile support, together with the ability to monitor the ACA into and LA activity during separation represent a significant, advance. the demonstration that antibodies purified in this Additionally, whilst possessing ACA activity, do not, show LA activity, SUggeStS manner, responsible for each activity are different. Further that the antibodies antibodies from a number of patients characterization of these purified regarding the nature of phospholipid may provide additional information into possible mechanisms of insight binding antibodies and thus some thrombosis.

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Acknowledgements This work was supported by the National Health and Medical Research Council of Australia and the National Heart Foundation of Australia. REFERENCES 1.

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