Quantitation of cell membrane glycoproteins in pathological conditions using a lectin-bound enzyme-linked immunosorbent assay (ELISA)

Journal of Immunological Methods, 110 (1988) 217-223

217

Elsevier JIM04776

Quantitation of cell membrane glycoproteins in pathological conditions using a lectin-bound enzyme-linked immunosorbent assay (ELISA) Application to human platelets in the Bernard-Soulier syndrome * Jeanne Drouin, Carlos A. Izaguirre and Pierre Patenaude Department of Medicine, Universityof Ottawa, Ottawa GeneralHospital, Ottawa, Ontario, Canada

(Received 26 October 1987, revised received 21 December 1987, accepted 18 January 1988)

Several techniques are available to study cell m e m b r a n e glycoproteins in pathological conditions but these either lack sensitivity or require radiolabelled material or expensive apparatus. We have, therefore, developed a sandwich-type enzyme-linked immunosorbent assay (ELISA) to study patients with the Bernard-Soulier syndrome (BSS), a hereditary platelet disorder characterized primarily, at the molecular level, by glycoprotein Ib (Gplb) deficiency. We have used the lectin wheat germ agglutinin to capture G p l b in the microtiter wells. Following incubation with monoclonal antibody AN51 which recognizes an epitope on G p l b , the immune complex was detected using the streptavidin-peroxidase-biotin complex. Platelet samples from 24 normal controls gave a mean value of 106% whereas in the six BSS patients the mean value was 14% and in the eight obligatory heterozygotes it was 78%. The technique is simple, inexpensive and sensitive and does not require the use of radioactive material. The assay method could be applied to quantitate other cellular glycoproteins where specific lectins and monoclonal antibodies are available. Key words: ELISA; Lectin; Cellular glycoprotein; Bernard-Soulier syndrome

Correspondence to: J. Drouin, Department of Medicine,

University of Ottawa, Ottawa General Hospital, Ottawa, Ontario K1H 8L6, Canada. * This research was supported by the Ottawa General Hospital Research Fund, the University of Ottawa Medical Research Fund and a donation from the Friends of the Ottawa General Hospital. Dr. Izaguirre is a Basic Science Associate of the Arthritis Society (C). Abbreviations: BSA, bovine serum albumin; BSS, Bernard-Soulier syndrome; EGTA, ethyleneglycol-bis-(/3aminoethyl ether)-N,N,N',N'-tetraacetic acid; ELISA, enzyme-linkedimmunosorbent assay; PAS, periodate-Schiff; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Tris, tris(hydroxymethyl)methylamine; WGA, wheat germ agglutinin.

Introduction Several techniques are available to study cell m e m b r a n e glycoproteins in pathological conditions and these have served to elucidate the nature of m e m b r a n e disorders in diseases such as Glanzmann's thrombasthenia (Nurden and Caen, 1974), the Bernard-Soulier syndrome (Nurden and Caen, 1975), hematological neoplasms (Gahmberg et al., 1982) and the Wiskott-Aldrich syndrome (Remold-O'Donnell et al., 1987). Techniques used b y Nurden and Caen (1974, 1975) to establish a molecular basis for the hemo-

0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

218 static defects observed in Glanzmann's thrombasthenia and the Bernard-Soulier syndrome (BSS) consisted of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of platelet extracts followed by periodate-Schiff staining and densitometric scanning of gels. Sensitive methods were later developed, making use of radiolabelled platelet membrane preparations such as two-dimensional SDSPAGE and autoradiography (Nurden et al., 1981), or isoelectrofocusing followed by one-dimensional SDS-PAGE and fluorography (Clemetson et al., 1982). Quantitative assays for platelet glycoproteins have been developed using crossed immunoelectrophoresis (Kunicki et al., 1981) or electroimmunoassay on washed platelets (Kristopeit et al., 1984) but such assays also require the use of radioactive reagents that are not readily available. Similarly, glycoprotein deficiencies have been quantitated using monoclonal antibodies in a flow cytofluorimeter (Johnston et al., 1984) but such facilities are limited to larger research centers. In this paper, we report a micro-enzyme-linked immuosorbent assay (micro-ELISA) which makes use of lectin binding to glycoproteins and their recognition by monoclonal antibodies. The assay is sensitive, reproducible and can be carried out using simple laboratory materials and equipment. We have used this assay in order to quantitate glycoprotein Ib (Gplb), a platelet membrane constituent which is deficient in BSS (George et al., 1984). In this case, we have used the lectin wheat germ agglutinin (WGA) which has a high affinity for Gplb (Moroi et al., 1984) and monoclonal antibody AN51 which recognizes this glycoprotein (Ruan et al., 1981). The assay differentiated clearly between subjects affected by BSS, heterozygotes and healthy controls. This method could be used widely to measure glycoprotein levels in other cell types whenever a specific lectin and monoclonal antibody are available.

Methods

Patients and normal subjects Following approval of the Human Experimentation Committee and after obtaining informed consent, blood was drawn from the following: (1) 24 age matched healthy controls; (2) six siblings

who fulfilled the characteristics of BSS and who have been the subjects of several previous studies (Drouin et al., 1979; Rock et al., 1980; Adams et al., 1983; McGill et al., 1984); (3) one parent and seven children of patients with BSS.

Reagents All the reagents for the SDS-PAGE and Western blotting were purchased from Bio-Rad, Richmond, CA or LKB, Bromna, Sweden. WGA, o-phenylenediamine and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO). The monoclonal antibodies with GpIb (AN51) and G p I I b / I I I a 015) specificity were a gift from A. McMichael, Oxford, England. The biotinylated sheep anti-mouse and the streptavidin biotin peroxidase complex were from Amersham (Oakville, Ontario) or Zymed (South San Francisco, CA). Immulon II ELISA plates were from Dynatech (Alexandria, Virginia).

Preparation of platelets Nine parts of whole blood, obtained using a standard venipuncture technique, were added to one part of 3.8% sodium citrate. Citrated blood v~as centrifuged at 140 x g for 10 min at room temperature (RT) to obtain platelet-rich plasma (PRP). PRP was pipetted carefully to avoid contamination with the leukocyte layer. Platelets were centrifuged at 800 x g for 15 rain and resuspended in 5 ml of Phillips buffer (NaC1 5.63 g/l, glucose 15.44 g/l, EGTA 3.81 g/l, Tris 1.039 g/l) with 0.35% BSA. This process was repeated twice. Platelets were resuspended in Phillips buffer only and contaminating erythrocytes were removed by centrifuging at 140 x g for 5 min while saving the supernatant. This process was repeated three times so as to ensure a homogeneous platelet preparation. The platelet containing supernatants were centrifuged at 800 x g for 15 min and resuspended in TEG buffer (0.05 M Tris, 0.01 M EGTA, pH 7.6) and counted on a Coulter instrument. They were then sonicated (3 x 15 s using a microtip Fisher dismembrator at 33% relative output) centrifuged at 7000 X g for 5 min, aliquotted in polystyrene tubes and frozen at - 8 0 o C. Protein concentrations were determined using a Bio-Rad protein determination kit.

219

SDS-PAGE, electroblotting and staining with WGA Platelet extracts of normal subjects and BSS patients were analyzed by SDS-PAGE at a protein concentration of 20/~g/lane in a 7.5% acrylamide gel prepared according to the method of Laemmli (1970) in a vertical slab apparatus. The gels were stained with silver nitrate using the method of Morrissey (1981) following oxidation of proteins with 0.7% sodium periodate (Tsai et al., 1982). Proteins were also transferred electrophoretically from gels to nitrocellulose paper using a transfer apparatus (Trans-Blot system, Bio-Rad). The nitrocellulose paper was then stained with WGAcoupled peroxidase according to the method of Moroi and Jung (1984).

ELISA method The method used to quantitate Gplb in our platelet preparations followed the general principles for the double antibody sandwich-ELISA described by Voller et al. (1980). Optimal experimental conditions were determined (see results section) and the following protocol was found to give the best results. Immulon II plates (96 well fiat bottomed) were coated with 0.2 ml of WGA at a concentration of 10 #g/ml in carbonate buffer pH 9.6 at 4 ° C overnight. Wells were then washed three times with 0.2 ml of phosphate-buffered saline (PBS) 0.1% (v/v) Tween 20 at pH 7.4. Remaining free protein binding sites were then blocked using 0.3 ml/well of 3% (w/v) BSA in PBS pH 7.4 for 2 h at RT. The platelet extract (0.2 ml) was added to the plates at protein concentrations of 100, 50, 10, 1 and 0.1 /~g/ml and incubated at 37°C in a saturating humidity for 2 h. Plates were washed twice in PBS-Tween 20. Monoclonal antibody AN51 was added (0.2 ml at a 1 in 800 dilution in PBS 1% BSA) to each well and incubated at 37 °C for 2 h. Wells were again washed twice with PBS/Tween 20 and incubated with 0.2 ml of a 1/200 dilution of a biotinylated sheep anti-mouse IgG (Amersham) antibody in PBS 1% (v/w) BSA at 37°C for 1 h. Wells were washed twice with PBS-Tween 20 buffer. The wells were overlayed with a 1/1000 dilution of streptavidin-biotin horseradish peroxidase complex in PBS, pH 7.4, for 1 h at 37 ° C. Prior to incubation with substrate, plates were washed five times with PBS-

Tween 20 to ensure complete removal of unbound complex. 0.3 ml of freshly prepared enzyme substrate solution (4 mg/10 ml o-phenylenediamine and 0.001% H202 in 0.1 M citrate buffer pH 5.0 was added to each well. After 5 min the reaction was stopped by adding 0.05 ml of 4 N H2SO4 to the wells. The color change in each well (which is proportional to the amount of captured antigen) was read spectrophotometrically at a wavelength 490 nm using a Dynatech ELISA reader (Minireader II). Results were plotted as optical density (OD) against increasing protein concentration on semilog paper.

Results

Assessment of the affinity of WGA for Gplb Staining of nitrocellulose paper with peroxidase-conjugated WGA following electrophoretic transfer of platelet proteins from polyacrylamide gels gave a clear-cut band for Gplb in normals but no staining in BSS as indicated in Fig. 1. At the platelet protein concentration used (20 #g per lane) there was only faint staining with other GP in the nitrocellulose paper indicating the high degree of specificity of this lectin for Gplb.

Characterization of the ELISA assay for Gplb The effect of using different concentrations of WGA (0.1-10 t~g/ml) and of platelet protein (0.1-500 ~tg/ml) on the OD at 490 nm is shown in Table I. The optimal WGA concentration was found to be 10 #g/ml. A platelet protein concentration of 50/~g/ml was chosen as standard because it was within the linear portion of the slope (range: > 10-100 /~g/ml). Platelet protein concentrations higher than 100 # g / m l gave variable results, usually well below the maximum value obtained at 100/~g/ml of platelet protein. This effect is probably due to competition for WGA binding sites by other platelet glycoproteins. These experiments were carried out at RT for 2 h; this probably accounts for the differences in OD observed in Tables I and II. Optimal dilutions of antibody were determined using a checkerboard titration. They were found to be 1/800 for AN51 and

220 TABLE I ELISA F O R G p l b ON PLATELETS Effect of using differing concentrations of wheat germ agglutinin (WGA) and of platelet protein on optical density at 490 nm. Monoclonal antibody AN51 was used at a concentration of 1/800. Incubations were done for 2 h at 22 ° C. WGA

Platelet protein ( # g / m l )

(~,g/m])

0.1

1.0

10

50

100

500

0.1 1 10

0.05 0.02 0.04

0.06 0.01 0.04

0.03 0.1 0.07

0.04 0.04 0.24

0.07 0.03 0.36

0.07 0.06 0.027

1/200 for the biotinylated sheep anti-mouse IgG. Using a platelet protein concentration of 50 /~g/ml and AN51 dilution of 1/800, incubation times of 1, 2, 3 and 24 h were tested at RT and at 37 ° C. Optimal binding occurred with incubation for 2 h at 37 °C (Table II). Using various concentrations of platelet protein in the assay system (0.001-100 /xg/ml) mono-

T A B L E II

a

b

ELISA F O R G p l b ON PLATELETS Effect of temperature and length of incubation on AN51 binding (optical density at 490 nm). Monoclonal antibody AN51 was diluted at 1/800. Platelet protein concentration was

50/Lg/ml. Temperature

OD49o

( ° C)

Length of incubation (h) 1

2

3

24

22 37

0.21 0.68

0.55 1.06

0.54 1.09

0.30 0.68

0.70

AN51 NORMAL

0.60

Gplb,

0.50

-

o~ .,d"

>- 0 . 4 0

--

Z 0.30

--

._1

.< ¢0 ~_ 0 . 2 0

AN51 BSS

Q.

o 0.10 w

0.001

0.01

NORMAL .L

~

2_

.IL

I

I

I

I

0.1

1

10

100

P R O T E I N C O N C E N T R A T I O N (,ug/ml)

Fig. 1. Affinity of W G A for Gplb. Peroxidase-conjugated W G A staining of nitrocellulose paper was carried out following Western blotting of SDS-PAGE gels. This gave a discrete band corresponding to G p l b in a normal control (lane a) but no counterpart in material from a patient with BSS (lane b). There was only faint staining of other glycoproteins. The platelet protein concentration was 20 # g / l a n e .

Fig. 2. Sensitivity and specificity of the ELISA assay at differing concentrations of platelet protein. The concentration of W G A was kept constant at 10 /~g/rnl. Monoclonal antibodies J15 (with G p l I b / I I I a specificity) and AN51 (with G p l b specificity) were used at a dilution of 1/800. Incubation time was 3 h at 3 7 ° C . AN51 b o u n d to W G A - b o t m d platelet glycoproteins (normal (e) and BSS (11)) whereas J15 (A) was unreactive with W G A - b o u n d platelet glycoproteins.

221

clonal antibodies AN51 (specific for platelet membrane Gplb) and J15 specific for platelet membrane G p l I b / I I I a (Vainchenker et al., 1982) were compared. Fig. 2 demonstrates clearly that J15 was unreactive with WGA-bound platelet glycoproteins. Thus, using WGA as the lectin in this system, the assay was very specific for Gplb.

Quantitation of Gplb in BSS and normals A standard curve was constructed using pooled platelet extract prepared from 12 normal donors and stored at - 7 0 ° C. The optical density of the standard curve was arbitrarily given a value of 100%. The optical density of platelet extracts from individual normal donors and patients at a given platelet protein concentration (50/zg/ml) was expressed as a percentage of the standard.

Using W G A at a concentration of 10 /zg/ml (0.1 ml/well) and 50 t~g/ml of platelet extract < 0.1 ml/well), a clear separation of GpIb concentrations was obtained in normal subjects versus the BSS patients and the eight relatives of the BSS patients (Fig. 3). The mean value of platelet extracts obtained from normal subjects was 106% while that for BSS was 14%. This difference was highly significant (P < 0.001). The values obtained in eight relatives of the BSS patients (one parent and seven children) were located either in the low normal range (five cases) or between the normal and the BSS values (three cases); the mean value was 78%. This difference is highly significant, both relative to BSS patients and to normal subjects (P < 0.001). Thus, the assay can be used to study heterozygosity in this disease.

160

Discussion

Gplb 140

120

>, lOO ._1

LL/ I-< "J Q,..

80

UJ Z < 1:13 tr' O t,,O <

60

40

20

99

NORMAL n=24

BSS

RELATIVES

n=6

OF BSS n=8

Fig. 3. Percent absorbance of platelet lysates at 490 n m using monoclonal antibody AN51 directed against O p l b in an ELISA system. G p l b mean values of the BSS patients were m u c h lower than those of normal controls ( P < 0.001). Heterozygotes gave intermediate values with a m e a n that was significantly different from normal controls ( P < 0.001).

The assay that we report takes advantage of the affinity of lectins for certain glycoproteins and their capture on the plastic microtiter wells of a microELISA plate. The lectin-glycoprotein complex is then reacted with a monoclonal antibody which recognizes an epitope on the glycoprotein. The reaction is amplified using the streptavidinperoxidase-biotin complex which leads to a strong signal without increasing the background. We have used this assay to study a kindred with the BSS, a condition characterized primarily by deficiency of the platelet membrane protein GpIb (George et al., 1984). Other methods have been developed for the study of platelet membrane abnormalities in BSS. However, the original assay consisting of one-dimensional SDS-PAGE of platelet extracts followed by staining of the gels with periodic acid-Schiff (Nurden and Caen, 1975) required a large amount of protein and was not sensitive enough to quantitate glycoprotein defects. More sensitive techniques, such as two-dimensional SDS-PAGE (Jenkins et al., 1976; Nurden et al., 1981), crossed-immunoelectrophoresis (Kunicki et al., 1981) or electroimmunoassay (Kristopeit et al., 1984), all required radioactive material which is not readily available. Finally, platelet glycoprotein abnormalities have also been quantitated using a monoclonal antibody and

222

non-radioactive labels in a cell sorter (Johnston et al., 1984) but such equipment is costly and of limited availability. In this assay, we have used WGA because of its affinity for glycoprotein Ib (Moroi et al., 1984) and monoclonal antibody AN51 which recognizes an epitope on GpIb (Ruan et al., 1981) and were able to separate homozygotes and obligatory heterozygotes from normals. We obtained similar results when using this lectin with monoclonal antibody AP1 which also recognizes an epitope on GpIb (Kristopeit et al., 1984) (results not shown). When using monoclonal antibody J15 which is directed against another platelet membrane glycoprotein, G p I I b / I I I a , (Vainchenker et al., 1982), very low OD readings were obtained with the WGA lectin, indicating the specificity of the assay for GpIb (Fig. 2). However, when we used lentil lectin with affinity for G p I I b / I I I a , (McGregor et al., 1980) together with J15, comparable levels of G p I I b / I I I a were obtained in BSS patients, heterozygotes and normal controls (results not shown). This indicates that the platelet samples were of comparable quality in both patient groups and normals and suggests that the assay system is versatile. The advantages of the present assay method are its low cost, safety, sensitivity and relative simplicity. The technique is superior to that reported by De Marco et al. (1986) where platelets were simply adsorbed to the plastic surface through centrifugation. Here, platelet glycoproteins are covalently bound to the solid-phase adsorbed lectin, thus minimizing greatly the risks of elution with washes. The fact that radioactive material is not required is a particular advantage over radioimmunoassay methods to which it is comparable in sensitivity: at 5 /~g protein/well the microELISA method compares favorably with electroimmunoassay. In conclusion, we have found the microELISA method particularly useful as a quantitative assay for GpIb in patients with BSS. The assay principle could be applied to measure other cellular glycoproteins where specific lectins and monoclonal antibodies are available. This may be worthwhile in disease conditions where cell surface glycoproteins are known to be altered such as in Glanzmann's thrombasthenia (Nurden and Caen, 1974), hematological malignancies (Gahmberg et al.,

1982), congenital dyserythropoietic anemia (Baines et al., 1982) and in the Wiskott-Aldrich syndrome (Remold-O'Donnell et al., 1987).

Acknowledgements We wish to thank Dr. A. McMichael and Dr. T. Kunicki for the gift of the monoclonal antibodies and Dr. P. Clezardin and Ms. S. Parmentier for their helpful comments.

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