Vox Sang. 53: 157-161 (1987)

Use of Immobilized Platelet Membrane Glycoproteins for the Detection of Platelet-Specific Alloantibodies in Solid-Phase ELISA'

Janice Collins, Richard H. Aster Blood Center of Southeastern Wisconsin and Departments of Medicine and Pathology, Medical College of Wisconsin, Milwaukee, Wisc., USA

Abstract. Platelet membrane glycoproteins were isolated from intact platelets by detergent-phase extraction, fixed to the wells of microtiter trays and used as targets for the detection of platelet-reactive alloantibodies by enzyme- linked immunospecific assay (ELISA). The final preparations contained 0.4% of total platelet protein. Antibodies reactive with antigens PIA1, PlA2, Baka, Pena and HLA-A2 were specifically detected at dilutions ranging form 1:640 to 1:1,600. Under the conditions utilized, the ELISA was more sensitive than assays involving 51Cr, radiolabeled monoclonal anti-IgG binding, and indirect immunofluorescence testing by one order of magnitude or greater. When platelets were pretreated with chloroquine to remove class I HLA antigens prior to detergent-phase extraction, reactions with HLA-specific antibodies were lost, but reactions with platelet-specific alloantibodies were retained. This approach offers a simple, sensitive and rapid method to detect and identify platelet-specific alloantibodies in sera containing HLA-reactive alloantibodies.

Introduction

Several enzyme-linked immunosorbent assays (ELISA) using intact platelets and microtiter plates for the detection of platelet-reactive antibodies have been report- ed [ 1-51. When intact platelets are used as targets, it is difficult to know whether positive reactions are the result of platelet-specific alloantibodies, HLA-specific alloanti- bodies or platelet-reactive autoantibodies. The most com- mon platelet-specific alloantibodies react with antigens of the PIA1 (Zw) 161, Bak [7] and Pen [8] systems carried on glycoproteins IIb or IIIa. Antigens of the PIE system appear to be carried on glycoprotein Ib [9]. If glycopro- tein-bearing platelet-specific antigens could be isolated from platelet membranes and fixed in a stable form to microtiter plates, it might be possible to use such prepa- rations for antibody evaluation and to assign positive reactions to target antigens carried on specific proteins. To test this possibility, we studied the reactions of a group of platelet-specific alloantibodies against a preparation of platelet membrane glycoproteins IIb and IIIa isolated

I

Blood Institute. Supported by grant HL-13629 from the National Heart, Lung and

from intact platelets by detergent extraction and immo- bilized in microtiter wells.

Materials and Methods

Platelet Membrane Extraction The principal of the isolation procedure is that amphiphilic

integral membrane proteins become incorporated into the micelles of Triton X-114 that form above the cloud point of this detergent at 37 'C. These can then be sedimented by centrifugation through sucrose, leaving the majority of platelet proteins behind in the aqueous phase [lo].

Platelets were obtained from group 0 donors of known PIA, Bak, Pen and HLA phenotypes using EDTA anticoagulant and were depleted of lymphocytes and red cells by differential centrifugation. The starting platelet preparations contained fewer than one red cell and white cell per 1,000 platelets. Platelet membranes were prepared by sonication and differential centrifugation [l I]. The membrane pellet derived from one unit of platelets (about 6 x 1Olo platelets) was solubilized in 2 ml of 146Triton X-114 (Sigma) in 10 mMTris buffer (pH 7.4) containing 150 mMNaCl and 0.4 mM phenylrnethyl-sulfo- nyl fluoride (PMSF) and 5 m M EDTA as protease inhibitors. The mixture was briefly sonicated to disperse the platelet membranes and then placed on ice for 90 min (or overnight) to achieve membrane solubilization. Detergent-phase separation was performed as de- scribed by Bordier [ 101 with modifications described by Newman et al. [ 11, 121. In brief, the Triton-solubilized membranes were centri-

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fuged at 48,OOOg for 60 rnin to remove insoluble material. The supernatant was gently layered on 3 ml of 6% sucrose in Tris buffer containing 0.06% Triton X-I14 in a 12x75 ml glass test tube. The tube was then warmed above the cloud point of Triton X-114 by incubating it at 37 'C for 5 min. The tube was then centrifuged at 1,000 g for 5 min to pellet the proteindetergent mixed micelles. The top (aqueous) phase was reextracted once more with cold Triton X-114. The detergent (D-phase) was recovered as a yellowish layer on the bottom of the tube underneath the sucrose cushion. The D-phase layer was mixed with 1 ml of Tris buffer and kept frozen at -2O'C until plated for platelet antibody studies. The final preparation con- tained about 500 pg protein [ 131 per unit of platelets, representing a 0.4% recovery of total platelet protein.

In some studies, platelets were treated with 0.4 Mchloroquine to remove class I HLA antigens as described by Nordhagen et al. [ 141 before preparing the D-phase extract.

Fisation of D-Phase Proteins in Microtiter Plates The concentrated D-phase was thawed and diluted to 30 pg pro-

teinlml in ice-cold plating buffer (0.05 Msodium bicarbonate buffer, pH 9.6, containing 3.1 mM sodium azide). Fifty microliters was added to each well of a flat-bottomed, tissue culture-treated, 96-well microtiter plate (Corning Cell Wells, Elmira, N.Y., USA). The plate was covered with parafilm and stored at 4'C overnight. On the following day, the plate was inverted to remove buffer and blotted on absorbent paper. For long-term storage, plates containing the D- phase protein were kept for several hours or overnight at room temperature in a CaC12 vacuum desiccator. After covering with parafilm, they were frozen at -80 'C.

Fisation qf Intact Platelets in Microtiter Plates Platelets were prepared according to Schiffer and Young [ 11 from

blood drawn in ACD-A, washed twice in Ringers citratedextrose (RCD) solution (pH 5.8) and adjusted to 1 . 7 ~ l(r plateletdpl. Sixty microliters (lo6 platelets) were added to each well of a microtiter plate and the plate was centrifuged at 600 g for 10 min. After invert- ing the plate, the wells were washed once with buffer. For long-term storage, the plates were inverted, blotted and stored overnight at room temperature in a CaCl2 vacuum desiccator. They were then covered with parafilm and stored at 4 or -80 %.

Soiirces qf Alloantibodies Alloantibodies were obtained from 6 patients with post-transfu-

sion purpura. Serums Wha and Kro are specific for PIA1 [ 151, Kee for Baka [ 161. Lek for Leka (Bak') (courtesy of Dr. J.-L. Wautier, Paris) [ 171, Pen for a newly described, high-frequency platelet-specific marker Pena [8] and McF for PIA2 (courtesy of Dr. S h e d Slichter, Seattle) [ 181. Serum EIS, containing a monospecific antibody reac- tive with HLA-A2 [ 151, was also utilized.

ELISA Assay In preparation for the ELISA assay, the wells of freshly prepared

or thawed, frozen plates containing intact platelets or D-phase pro- tein were filled with PBSTw-gel buffer (0.0 1 M phosphate, 0.145 M NaCI, 0.05% Tween, 1 .O% gelatin; pH 7.4) and allowed to sit at room temperature for 90 rnin to block nonspecific binding of proteins. The plate was then washed twice with PBSTw buffer.

The ELISA assay was adapted from Schiffer et al. [I]. Fifty microliters of serum to be tested diluted in PBS-Tw buffer was added per well. The plate was incubated at mom temperature for 1 h

inverted and blotted in absorbent paper. Approximately 300 pl of PBSTw-Gel was added to each well to reduce nonspecific binding of Ig, and the plate was incubated at room temperature for 10 min. After inverting and washing the plate three times with PBS-Tw, 100 pl of PBSTw was added per well. Immediately, 100 p1 per well of goat anti-human immunoglobulin conjugated to alkaline-phos- phatase (Zymed) diluted 1: 400 in 0.05 MTris buffer containing 1 % bovine serum albumin and 0.1% sodium azide was added. The plate was incubated at 22-25 'C for 45 min. It was then inverted, blotted and incubated with 300 ~1 per well of PBS-Tw-Gel at room temper- ature for 10 min. After washing the plate three times with PBSTw, 100 11 of gnitrophenyl phosphate (Zymed) diluted to 3.8 mM in diethanolamine buffer (97 ml diethanolamine, 843 ml distilled water, 60 ml 1 NHCl, 100 mg MgC12/6H20,0.2 g sodium azide, pH 9.8) was added to each well. The plate was incubated at room temperature in the dark for 20-40 rnin and read at 410 nM using an automated micro-ELISA reader (Dynatech, MR 600, Alexandria, Va., USA). All test combinations were done in duplicate, and the mean optical density, corrected for absorbance at 490 nM. was used in analysis. An optical density reading of more than the mean +2 SD obtained with normal serum at the same dilution was considered positive.

Results

Optical density (OD) readings obtained with undiluted normal sera tested with D-phase protein ranged from 0.070 to 0.182 (mean 0.126, SD 0.040; 11-16). D-phase protein testedwith buffer in place of serum yielded an OD less than 0.07. When normal sera were tested with intact platelets, the OD readings ranged from 0.057 to 0.229 (mean 0.124, SD 0.046; n=28). Platelets with buffer alone regularly yielded readings less than 0.07.

Immobilized D-phase protein yielded antigen-specific reactions when used at 0.4-3.0 pg protein per well, with a background OD of less than 0.2. For routine testing, 1.5 pg per well was chosen.

A representative assay using D-phase protein isolated from a donor positive for PIA1 and HLA-A2 is shown in figure 1. Anti-PIA1 at 1 : 4 gave an OD six times greater than control serum, and anti-HLAA2 at dilution 1:64 reacted four times as strongly. Both antisera gave positive reactions at 1: 1,024. Negative reactions were obtained with D-phase proteins extracted from antigen-negative platelets. Comparable reactions were obtained with anti- PlA2, anti-Baka and anti-Penaa antibodies using appropri- ate target proteins and controls.

Table I compares the sensitivity of the ELISA for platelet antibody detection with the platelet suspension immunofluorescence test (PSIFT) [ 191, W r release [20] and an assay based on binding of 1251-labeled anti-Fc monoclonal antibody adapted [15] from the method of LoBuglio et al. [21]. The maximum dilution yielding a

Detection of Platelet-Specific Alloantibodies 159

Fig. 1. Reactions of D-phase protein prepared from platelets of a PIAI-positive, HLA-A2-positive donor with Wha antiPIA1 (m), EIS anti-HLA-A2 (A) and normal serum (0) in ELISA. Reactions of D-phase platelet protein prepared from a PIAl-negative, HLA-A2- negative donor tested with Wha, EIS and normal sera were undistin- guishable from the normal serum reactions shown (0). D-phase made from antigen-positive platelets reacted positively at serum dilutions of 1 : 1,024.

positive reaction is shown for each serum. The ELISA using D-phase or intact platelets was significantly more sensitive than the other methods. The sensitivity of D- phase protein and intact platelets for the detection of the alloantibodies in ELISA was about the same.

Figure 2 compares the reactions of D-phase protein prepared from untreated platelets and chloroquine plate- lets. D-phase protein extracted from untreated platelets reacted strongly with the anti-HLA-A2 antibody EIS (fig. 2a), but the antibody was nonreactive with D-phase protein prepared from the same platelets pretreated with chloroquine. Anti-PIA1 antibody reacted equally well with the two preparations.

D-phase protein stored concentrated in Triton X-114 buffer for 6 months before being thawed and plated reacted as well as freshly prepared D-phase protein with the antibodies tested (fig. 3a). D-phase proteins desic- cated and frozen in microtiter trays and stored for 3 months also reacted as well as the fresh preparation.

In confirmation with Schiffer and Young [ 11, we found that desiccated intact platelets could also be stored in microtiter trays at 4 'C for 9 months with only minor loss of sensitivity for the detection of anti-PIA1 (fig. 3b).

Fig. 2. Reactions of anti-PlAl (m), EIS anti-HLA-A2 (A) and nor- mal serum (0) with D-phase protein from untreated platelets (a) and chloroquine-treated platelets (b) from a PIA1-positive, HLA-A2-pos- itive donor. Chloroquine treatment abolished the reaction of anti- HLA-A2 with subsequently prepared D-phase protein, but had only a minimal effect on reactions of antiPlA1.

Table I. Reactions of different alloantisera against immobilized D-phase proteins and intact platelets

Se- Specif- Reciprocal serum titer in: rum icity

ELISA ELISA PSIFT SICr mono- with with release clonal D-phase platelets binding

Wha P P 1,280 1,280 10 50 300 Kro P P 1,280 1,280 50 50 100 McF PIQ 640 640 5 NT 5 EIS HLA-A2 1,280 1,280 10 10 10 Lek L,ek4Baka) 1,600 800 100 NT 200 Kee Baka 640 640 16 128 25 Tru Pena 640 1,280 16 10 20

NT = Not tested.

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Fig.& Reactions of anti-PIA1 antibody Kro against D-phase protein stored concentrated for 6 months at -20 'C (0) or in microtiter plates for 3 months (m) at -80 'C. The stored D-phase preparations were as sensitive as freshly prepared D-phase protein (A) for antibody detection. Reactions of the same preparations with normal serum (a) were superimposable. b Reactions of anti-PIA1 against intact PIA1- positive platelets stored for 1 month (D) or 9 months (0) at -80 'C in microtiter trays or freshly prepared (A). Normal serum reactions with the same preparations were superimposable (a).

Discussion

Our findings confirm those of several other groups [ 1-51 concerning the utility of the microtiter ELISA assay for the detection of platelet-reactive antibodies. In our hands, ELISA was up to 100 times more sensitive for antibody detection than PSIFT, W r release and anti-Fc monoclonal binding assays for the detection of alloanti- bodies reacting with platelet-specific and HJA antigens (table I). The ELISA is rapid and inexpensive, requiring about 4.5 h using preloaded microtiter plates. With sem- iautomated reagent dispensers and plate washers, many plates can be run concurrently. No loss of sensitivity for the detection of anti-PIA1 was seen after storage of trays

preloaded with D-phase protein for 3 months or concen- trated D-phase for 6 months (fig. 3). Studies to determine the maximum permissible storage time are in progress.

Our data indicate that integral platelet membrane pro- teins isolated in the D phase with Triton X-114 and representing only about 0.4% of the total protein of intact platelets are as sensitive as intact platelets for the detec- tion of the alloantibodies studied, which were specific for PIA1, PlA2, Baka, Pena and HLA-A2. Because nearly all platelet IgG is released after platelet sonication, we had hoped that D-phase proteins might give lower back- ground reactions with anti-IgG reagents than intact plate- lets, but this was not the case. Our average background reaction of an OD of 0.170 achieved with both intact platelets and D-phase proteins using undiluted serum is lower than that described in most other reports. This may reflect modifications of the ELISA procedure developed empirically (see 'Methods') We also hoped that the use of a detergent-extracted integral membrane preparation would eliminate class I HLA antigens, thought by some workers to be passively acquired by platelets from plasma, which might be expected to concentrate in the aqueous phase in the separation procedure. However, HLA-A2 as reflected by the reactions of antibody EIS was recovered in the D phase in amounts sufficient to yield reactions as strong as those obtained with intact platelets (fig. 2a). These reactions were eliminated when platelets pre- treated with chloroquine were used for the D-phase prep- aration (fig. 2b). Their recovery in the D-phase prepara- tion suggests that a significant fraction of the class I HLA antigens of platelets are carried on integral membrane proteins [ 10, 111.

From table I, it is apparent that the platelet-specific antigens PIA1, P P , Baka, (Leks) and Pena are isolated intact in the D phase with the glycoprotein IIb/IIIa com- plex with which they are associated. The specificity of D-phase preparations as targets for alloantibody has been demonstrated by concordant reactions of intact platelets and D-phase proteins from 20 donors when tested with antibodies specific for each of these alloantigens (data not shown).

The observations suggest that further studies of the utility of isolated immobilized platelet membrane glyco- proteins for the detection of platelet-reactive alloantibod- ies by ELISA are warranted. The method is rapid and sensitive, and it seems possible that the target antigens will remain stable in the frozen state indefinitely either solubilized in detergent or after plating. In several days, a single technologist can prepare trays containing D-phase- derived platelet glycoproteins from a panel of as many as

Detection of Platelet-Specific Alloantibodies 161

10 donors of varying platelet alloantigen phenotypes. These preparations can be used to detect and identify platelet-specific alloantibodies in more than 500 sera from patients with post-transfusion purpura, neonatal alloimmune thrombocytopenia and, perhaps, refractori- ness to platelet transfusions resulting from alloimmuni- zation. As noted, interference from HLA-specific anti- bodies can be eliminated using D-phase proteins prepared from chloroquine-treated platelets.

An alternative approach to the detection of platelet- specific alloantibodies in a solid-phase assay is to use the glycoprotein IIb/IIIa complex or other platelet membrane glycoproteins purified by immunoaffinity chromatogra- phy, Furihata et al. [22] and Kickler et al. [23] have employed monoclonal antibodies specific for glycopro- teins IIb, IIIa and the glycoprotein IIbflIIa complex fmed in microtiter wells and treated with platelets solubilized in Triton X-100 for alloantibody detection. In this way, specific glycoproteins or glycoprotein complexes can be immobilized in individual wells. The stability of these preparations after storage for various periods of time is now being evaluated.

References

1 Schiffer, C. A.; Young, V.: Detection ofplatelet antibodies using a micro-enzyme-linked immunosorbent assay (ELISA). Blood 61.

2 Howe, S.E.; Lynch, D.M.; Lynch, J.M.: An enzyme-linked immunosorbent assay for the quantitation of serum platelet- bindable IgG. Transfusion 24: 348-352 (1984).

3 Taaning, E.: Microplate enzyme immunoassay for detection of platelet antibodies. Tissue Antigens 25: 19-27 (1985).

4 Fabris, F. ; Casonato, A; Crociani, E.; Zanchetta, R.; Busolo, F.; Girolami, A.: A new ELISA method for the detection of serum bindable anti-platelet antibodies (SPBIG). Clinica chim. Acta

5 Tamerius, J. D.; Curd, J. G. ; Tani, P.; McMillan, R.: An enzyme- linked immunosorbent assay for platelet compatibility testing.

6 Kunicki, T.; Aster, R. H.: Isolation and immunologic characteri- zation of the human platelet alloantigen, PIA1. Molec. Immunol.

7 Borne, A. E. G. Kr. von dem; Riesz, E. von; Verheugt, F. W.A.; Ten Cate, J. W.; Koppe, J. G. ; Engelfriet, C. P. ; Nijenhuis, L. E.: Baka, a new platelet-specific antigen involved in neonatal alloim- mune thrombocytopenia. Vox Sang. 39: 113-120 (1980).

8 Friedman, J. M.; Aster, R. H.: Neonatal alloimmune thrombo- cytopenic purpura and congenital porencephaly in two siblings associated with a ‘new’ maternal antiplatelet antibody. Blood 65:

9 Furihata, K.; Hunter, J.; Aster, R.H.; Coewing, G.; Shulman, N.R.; Kunicki, T.J.: The association of the human platelet

311-317 (1983).

146: 223-228 (1985).

Blood 62: 744-749 (1983).

16: 353-360 (1979).

1412-1415 (1985).

alloantigen PIE1 with the glycoprotein Ib complex. Blood 66: suppl. 1, p. 304a (1 985).

10 Bordier, C.: Phase separation of integral membrane proteins in Triton X-114 solution. J. biol. Chem. 256: 1604-1607 (1981).

1 Newman, P. J.; Knipp, M. A.; Kahn, R.A.: Extraction and iden- tification of human platelet integral membrane proteins using Triton X-114. Thromb. Res. 27: 221-224 (1982).

2 Newman, P. J.; Kahn, R. A.; Hines, A.: Detection and character- ization of monoclonal antibodies to platelet membrane proteins. J. Cell Biol. 90: 249-253 (198 1).

3 Markwell, M. A. K.; Haas, S. M.; Bieber, L. L.; Tolbert, N. E.: A modification of the Lowry procedure to simplify protein deter- mination in membrane lipoprotein samples. Analyt. Biochem.

14 Nordhagen, R.; Flaathen, S.T.: Chloroquine removal of HLA antigens from platelets for the platelet immunofluorescence test. Vox Sang. 48: 156-159 (1985).

15 Janson, M.; McFarland, J.; Aster, R. H.: Quantitative determina- tion of platelet surface alloantigens using a monoclonal probe. Hum. Immunol. 15: 251-262 (1986).

16 Keimowitz, R.M.; Collins, J.; Davis, K.; Aster, R.H.: Post- transfusion purpura associated with alloimmunization against the platelet-specific antigen, Bar . Am. J. Hematol. 21: 79-88 (1 986).

17 Boizard, B.; Wautier, J. L.: h k a , a new platelet antigen absent in Glanzmann’s thrombasthenia. Vox Sang. 46: 47 (1 984).

18 Slichter, S.J.; Ballem, P.; Teramura, G.: Characteristics of PIAi antibodies produced in neonatal alloimmune thrombocytopenia (NAT) versus post-transfusion purpura (PTP). Blood 62: suppl. 1, pp. 894 (1983).

19 Borne, A. E. G. Kr. von dem; Verheugt, F. W. A.; Oosterhof, F., von Riesz, E. van, Brute1 de la Riviere, A.; Engelfriet, C.P.: A simple immunofluorescence test for the detection of platelet antibodies. Br. J. Haemat. 39: 195 (1978).

20 Aster, R. H.; Enright, S. E.: A platelet and granulocyte membrane defect in paroxysmal nocturnal hemoglobinuria: usefulness for the detection of platelet antibodies. J. clin. Invest. 48: 1199 (1 969).

21 LoBuglio, A. F.; Court, W. S.; Vincour, L.; Maglott, G.; Shaw, G. M.: Immune thrombocytopenic purpura. Use of a 12sI-labeled anti-human IgG monoclonal antibody to quantify platelet-bound IgG. New Engl. J. Med. 309: 459 (1 983).

22 Furihata, K.; Nugent, D. J.; Aster, R. H.; Kunicki, T. J.: Anti-Pena binds specifically to an epitope on platelet glycoprotein IIIa. Blood 68: suppl. 1, p. 308 (1986).

23 Kickler, T. S.; Furihata, K.; Kunicki, T. J.; Herman, J. H.: Aster, R. H.: A new platelet alloantigen allelic to Bak. and its association with post-transfusion purpura. Blood 68: suppl. 1, p. I 1 la (1986).

87: 206-207 (1978).

Received January 5, 1987 Revised manuscript received: March 25, 1987 Accepted March 25, 1987

Janice Collins Blood Center of Southeastern Wisconsin 1701 West Wisconsin Avenue Milwaukee, WI 53233 (USA)