Jo,lrnal of Immunological Methods, 153 (1992) 41-48

41

© 1992 ElsevierScience Publishers B.V. All rights reserved (}022-1759/92/$05.00

JIM06383

A monoclonal antibody-based enzyme immunoassay for human GMP-140/P-selectin M a s a h i k o K a t a y a m a a M a k o t o H a n d a b H i r o n o b u A m b o b, Y o h k o Araki h Sayuri Hirai '' Ikunoshin K a t o a, Y o h k o Kawai b, Kiyoaki W a t a n a b e b and Y a s u o Ikeda h ,aBiotechnology Research Laboratories, Takara Shuzo Co., Ltd, Otsu, Shiga 520-21, Japan, and h BImMCenter, Departments of Internal Medicine and Laboratory Medicine, Keio Unicersity Hospital, Shinjuku, Tokyo 160, Japan

(Received 19 December 1991. revised received18 February Iq92, accepted 30 March 1992)

Two hybridoma cell lines producing monocional antibodies WGA-I and PL7-6, reactive only with thrombin-stimulated human platclet have been established. Both these antibodies were investigated for their specific reactivity against GMP-140, based on the amino acid composition analysis of immunopurifled antigen and N terminal amino acid sequencing of its prote, ase fragments. A two-site enzyme immunoassay for quantification of human GMP-140 was developed using WGA-I monoclonal antibody immobilized on 96-well microplates and horseradish peroxidase-labeled PL7-6 monoclonal antibody as detector. The assay was able to measure GMP-140 in serum and plasma with a sensitivity of about 5 n g / m l and a precision better than 10%. This assay will be useful for the detection of GMP-140 derived from platelcts or endothelium in biological fluids and tissue extracts. Key words: Granule membrane protein-140; Monoclonalantibody; Enzyme immunoassay;Plasma; CD62; P-Selectin

Introduction Granule membrane protein-t40 (GMP-140, PADGEM, CD62 molecule, and designated as P-seleetin by Bevilacqua et al., 1991), a member of the selectin family with an apparent molecular weight of 140 kDa, is rapidly redistributed to the

Correspondence to: M. Katayama, Cell Technology Reagent

Section, BiotechnologyResearch Laboratories, Takara Shuzo Co., Ltd, Seta 3-4-1, Otsu, Shiga520-21, Japan. Tel.: 81-77543-7216; Fax: 81-775-43-2494). Abbreciations: GMP-140, granule membrane protein-140; WP, washed platelets; PG, platelet glycoprotein; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; BSA, bovine serum albumin.

plasma membrane of stimulated platelcts or endothelial cells, and interacts with stimulated neutrophils through a Ca2+-dependent Icctin-likc mechanism (McEver and Martin, 1984; Moore et al., 1991). GMP-140 is a useful marker exposed on the stimulated cell surface which indicates the activation of platelets and cndothclium in experimental inflammation (Hamburger and McEver, 1990; Larsen et al., 1989), and has potential clinical applications (Palabrica et al., 1989; Savage et al., 1989; Fijnheer et al., 1990). The complementary DNA-derived amino acid sequence indicates that it contains a lectin-like domain, an EGF-like domain, nine consensus repeats similar to those in complement-regulatory proteins, a transmembrahe domain and a short cytoplasmic tail (Johnston et al., 1989a). The other known selectin

molecules structurally related to GMP-140 are endothelial leukocyte adhesion molecule-1 and peripheral lymph node lymphocyte homing receptor, or ELAM-1 and Mel 14, respectively (Bevilacqua et al., 1989; Lasky et al., 1989). Complementary DNA also predicts variant forms of GMP-140, including a putative soluble form lacking the transmembrane domain that appears to arise from alternative splicing of messenger RNA (Johnston et al., 1989a). Potential soluble GMP140 seems to be present in plasma, serum, and other bod~' fluids. Quantitative measurements of GMP-140 concentrations in biological fluids or tissue extracts have not been reported. In the present study, we developed a sandwich enzyme immunoassay for GMP-140 using two different monoclonal antibodies and determined that immunoreactive GMP-140 is present in normal human plasma and serum. This assay may be useful for monitoring levels of body fluid GMP-140 in diseases involving degranulation of platelets and/or endothelial cells.

Materials and methods

Preparation of bnmunogen Washed platelets (WP) were prepared from fresh citrated blood drawn from healthy human subjects by albumin gradient and gel filtration techniques (Timmons and Hawiger, 1989). 2 × l09 washed platelets were subjected to ultrasonication and ultracentrifuged at 100,000 × g for 30 min, at 4°C. The pellet was extracted in a solution containing 1% deoxycholate (Sigma Chemical Co.) and ultracentrifuged again as indicated above. The supernatant was separated from sedimented material. Platelet glycoproteins (PG) bound to a wheat germ agglutinin column (Pharmacia) were isolated from the supernatant as described previously (Handa et al., 1986). WP and PG were used as immunogens, for the construction of distinct hybridomas.

fled in complete Freund's adjuvant; the second and third immunizations consisted of 2 × 10s WP per mouse in incomplete adjuvant administered at 5 and 10 weeks, respectively, after the first immunization. All injections were given intraperitoneally. Spleen cells were obtained 3 days after the last immunization. Cell fusion with P3X63 AgSU1 BALB/c myeloma cells were performed using 50% polyethylene glycol 1540 (Wako Pure Chemical) and 15% dimethyl suifoxide (Dojin Chemical). The cell suspension in HAT medium was seeded onto the microtiter plates. A second fusion experiment was performed in a similar fashion, using 200 p,g of PG as immunogen instead of 2 × l0 s WP. Supernatants were screened for antibodies specific to platelet 14 days after fusion by an ELISA method using 0.025% glutaraidehyde-fixed WP on the microtiter plates. Cells from positive cultures were cloned by limiting dilution.

Flow cytometric analysis 1 × 107 platelets were stimulated by thrombin (Sigma Chemical Co.) for 5 rain. Stimulated and non-stimulated platelets were fixed for 2 min in phosphate-buffered saline (PBS) containing 0.05% glutaraldehyde. After fixation, platelets were washed with PBS, suspended in hybridoma culture supernatants, and incubated for 30 min at room temperature. FITC-labeled anti-mouse immunoglobulin (Capped was used in flow cytometric analysis (Cytoron, Ortho Diagnostic Systems) for the detection of monoclonal antibodies highly reactive with thrombin-stimulated platelets, and less reactive with non-stimulated platelets.

Purification of monoclonal antibodies Monocional antibodies were purified from mouse ascites by ammonium sulfate precipitation and an IgG purification kit with immobilized protein A (Pierce Chemical Co.). Purified monoclonal antibodies were coupled to CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer's instruction.

Construction of hybridomas

Purification of immunoreactire antigen

Female BALB/c mice were immunized with washed platelets as follows: the first immunization comprised 2 x l0 ~ of WP per mouse emulsi-

The antigen was immunopurified using immobilized WGA-I on Scpharose from the PG fraction. Purified antigen was then electrophoresed

on a 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), and transferred to an Immobilon-P membrane (Millipore) for Western blot analysis. The antigen for standardization of the enzyme immunoassay was first isolated as described above and further purified by application to a 3.9 x 300 mm /~Bondapak C4 reverse-phase high-performance liquid chromatography (HPLC) column (Waters Associates). The eluent compositions were set for continuous gradient elution using 0.1% trifluoroacetic acid in water as starting clution and 100% aeetonitrile as final elution. The amino acid composition of highly purified antigen was analyzed using the L-8500 analyser (Hitachi) to determine the amount of standard antigen. The tryptie or V8-protease fragments of the purified antigen were prepared according to a previously published method (Handa et al., 1986), and

analysis of the N terminal amino acid residue was performed using Edman degradation in the PSO-! automated protein sequencer system (Shimadzu Corporation) as described previously (Matsudaira, 1987).

Enzyme immunoassayprocedure First, 96-well plates coated with WGA-I were blocked with bovine serum albumin (BSA). PL7-6 was labeled with horseradish peroxidase (Boehringer Mannheim) according to a previous method (Wilson et al., 1978). To each well, 100 g.I of standard GMP-140 (0, 10, 20, 40, 8(I, 160, 32(I, 640 ng/ml) or samples were added. GMP-140 standard was diluted in PBS containing I% BSA to various noted concentrations. The plate was incubated for I h at 37°C and washed with PBS. 100 /.d of peroxidase-labeled monoelonal antibody solution were then added to each well and

THRONBN

UNSTIMULATED

0.| U/m~ ;=-st

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t0;

P L 7-6

|

C"

| U/m~ ;u-S¢

to~

to6

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. . .

•

FU-SC

1

z

,

a,A

~u-s¢

tO~

1=4 ,~ .- :L .,..:; .

WGA- ]

l'°~

,..

.'.i~.~ ~

s~

L06

:..

zo •

Fig. 1. Flow cytometric analysis o f monoclonal antibodies to thrombin-stimulated platelets. Platelets were incubated with thromhin at concentrations o f 0, 0 . 6 and I U / m l . T h e reactivities o f the monoclonal antibodies were detccted using FITC-conjugated anti-mouse I g G and flow cytometry.

PL 7- 6

Results

WGA-1

Characterization of monoclonal antibodies

kDa

200=..

•'q1135 116~

....

:

93

Two monoclonai antibodies, PL7-6 and WGA1, specific to stimulated platelets were obtained from hybridomas in the first and second fusions, respectively. In flow cytometric analysis, these two immunoglobulin GI antibodies were found to react strongly with thrombin-stimulated platelets in a dose-dependent manner (Fig. 1). In preliminary experiments, Scatchard analysis showed about 8000 binding sites for IZ~l-labeled WGA-I or PL7-6 on each thrombin-stimulated platelet surface (data not shown). We observed that WGA-I did not compete with PL7-6 in binding to the stimulated platelet surface (data not shown).

661~

Nonreduced Reduced

Nonreduced Reduced

Fig. 2. Western blot analysis of monoclonal antibodies. The reduced and nonreduced antigen was separated on SDSPAGE, transferred to Immobilon-Pmembrane,and immunostained with PL7-6and WGA-I. the plate incubated for l h at 37°C. After PBS washing 100 p,I of 5.5 mM o-phenylenediamine • 2HCI (Sigma Chemical Co.) solution were added as substrate and the mixture left for 10 min at room temperature. The enzyme reaction was stopped by the addition of 100 #l of l N HCI and absorbance at 492 nm was measured in a Titertek Muitiscan (Flow Laboratories).

0.2

0.1

0

0

"~ ' J " 10

I 20 3JO Elution Volume (ml)

410

5'0

1.5E g,

Preparation of blood samples Fresh whole blood was obtained from ten healthy individuals in our laboratory using a 21 gauge needle and a disposable plastic syringe. Serum as well as citrated plasma were obtained from all individuals. Whole blood for plasma preparation was immediately anticoagulated with a 1/10 volume of 3.14% sodium citrate and centrifuged for l0 min at 3000 x g at 4°C. Further whole blood for serum preparation was incubated for l h at 37°C, then incubated again for 12 h at 4°(2. Plasma and serum were stored frozen at -20°C. Untimed urine samples were also collected from these individuals.

.........

1.0o~ .o

0.5-

0

10

20 30 Elution Volume (ml)

40

50

Fig..3, HPLC purification of immunoreactive antigen, Tvp: HPLC elution profile of the antigen purified by immobilized

WGA- 1; bottom:detection of immunoreactiveantigen in the fraction eluted from HPLC by enzyme immunoassay.Arrows indicate the fractioncontainingimmunoreactiveantigen.

TABLE I AMINO ACID COMPOSITION OF PURIFIED ANTIGEN Amino acid Asp Thr Ser Glu Gly Ala Cys Val Met lie Leu Tyr Phe Lys Pro His Arg Total

A 8.68 6.99 8.81 9.97 8.29 6.87 8.42 4.27 1.55 3.11 7.90 3.63 3.89 3.89 7.51 2.85 3.37

B 8.88 6.88 7.25 I 1.0(I 8.25 7.111} 6.25 5.38 1.511 8.511 2.88 3.88 4.38 7.38 3.25 3.88

C 8.96 6.96 9.27 I 1.68 111.48 6.78 6.38 4.94 2.111 3.98 7.58 3.112 3.66 3.7 I 7.2 I 3.27 3.32

100.00

1011.114

I(X).(12

3.511

Key: A, amino acid composition of GMP-141I deduced from the reported sequence of human endothelial cell derived DNA (Johnston et al., 1989a); B, reported amino acid composition of human platelet GMP-140 (Johnston et al., 1989b); C, purified antigen by HPLC.

Tryptic

fragment

GMP-140

(239-246)

Tryptic

fragment

GMP-!40

(320-326)

V~-protease fragment GMP-140

(117-1271

v~-p~otease fragment GMP-140

1291-2961

I:

W e s t e r n blot analysis s u g g e s t e d that both W G A - ! a n d PL7-6 are reactive with a n t i g e n with an app a r e n t m o l e c u l a r weight o f 135,000 u n d e r non-red u c i n g conditions on S D S - P A G E , b u t are not reactive with r e d u c e d a n t i g e n (Fig. 2). T h e purified antigen, e l u t e d in a single peak from H P L C , was also highly i m m u n o r e a c t i v e with the two m o n o c l o n a l antibodies constituting a sandwich i m m u n o a s s a y format (Fig. 3). T h u s , PL7-6 was able to react with the protein b o u n d o n a W G A 1-immobilized agarose c o l u m n , s u g g e s t i n g that PL7-6 a n d W G A - I recognize t h e s a m e molecule in platelets. A m i n o acid compositional analysis o f protein purified by H P L C was p e r f o r m e d (Table 1), a n d t h e r e s u l t a n t c o m p o s i t i o n was close to that of G M P - 1 4 0 ( J o h n s t o n et al., 1989a,b). in N terminal a m i n o acid s e q u e n c i n g , no s e q u e n c e could be o b t a i n e d from intact purified protein, b u t N terminal a m i n o acid s e q u e n c i n g of two tryptic f r a g m e n t s a n d two V8 p r o t e a s e f r a g m e n t s g e n e r a t e d from purified a n t i g e n was p e r f o r m e d (Fig. 4). T h e s e q u e n c e s o b t a i n e d were all identical with s e q u e n c e s ~,f G M P - 1 4 0 predicted from t h e c o m p l e m e n t a r y D N A s e q u e n c e ( J o h n s t o n et

Ala-Phe-Gln-His-Gln-Ser-Xaa

: 2:

Lys-Ala-Phe-Gln-His-Gln-Ser-SerGly-Leu-Asp-Met-Leu-Xaa

:

i: :

2: :

Arg-Gly-Leu-Asp-Met-L~u-Cys-

His-Xaa-Leu-Lys-Lys-Lys-Xaa-Ala-Leu-Xaa Glu-His-Cys-Leu-Lys-Lys-Lys-His-Ala-Leu-Cys-

Gly-Thr-Met-Asp-Xaa Glu-Gly-Thr-Met-Asp~Cys-

Fig. 4. Partial amino acid sequences of purified antigen. Tryptic and V8 protease digestion of ~urified antigen were performed as described in the materials and methods section. Xaa indicates indeterminant residues, lluman umbilical vein endothelial cell GMP-14{; sequences are taken from the previous report by Johnston et al. {1989a).

¢q

TABLE II PRECISION OF EMZYME IMMUNOASSAY Three standard samples were assayed ten times on a single plate as described in Materials and methods (intra-assay)or in duplicate on ten different days (interassay)

1.5

Mean v a l u e

(ng/ml)

1.0 lntra-assay

0

~

i

I

i

Coefficient variation (%)

52

8.9

(N= 10)

126 367

6.3 4.8

Interassay (N= 10)

61 136 345

9.2 6.9 5.4

I

GMP-140 (ng/ml) Fig. 5. Representative standard curve for GMP-140 assay. GMP-140 standard was diluted in PBS containing I% BSA to yield various concentrations and then assayed. al., 1989a). Highly purified antigen was also found to be reactive with murine monoclonal antiGMP-140 antibody S12 (McEver and Martin, 1984).

Precision of enzyme immunoassay A two-site enzyme immunoassay using immobilized WGA- 1 and peroxidase-labeled PL7-6 made it possible to assay the concentration of GMP-140. The representative calibration curve of the assay permitted quantification of GMP-140 within the concentration range 10-640 n g / m i (Fig. 5). The minimal detectable level, defined as the analyte concentration that results in a signal corresponding to the signal of the assay blank plus two standard deviations (SDs), was determined to be 4.8 n g / m i . The intra-assay coefficients of variation obtained from ten repeat analyses of standard GMP-140 with an antigen concentration of 52-367 n g / m l ranged from 4.8% to 8.9%, and the interassay coefficients of variation obtained from ten different assays of standard GMP-140 with an antigen concentration of 61-345 n g / m i ranged from 5.4% to 9.2% (Table I1).

Detection of GMP-140 in human plasma and serum We assayed GMP-140 levels in citrated plasma and serum samples obtained from ten healthy donors. Plasma GMP-140 levels in ten healthy

subjects ranged from 15 to 190 (mean + SD, 98 + 49) n g / m l . Serum GMP-140 levels ranged from 65 to 460 (mean + SD, 262 + 85) n g / m l . There was no detectable GMP-140 in untimed urine samples from these healthy individuals.

Discussion

Some selectin molecules function as leukocyte receptors, mediating leukocyte emigration in inflammatory responses to tissue injury and infection by binding a sialylated carbohydrate structure found on neutrophils and monocytes (Poiley et al., 1991). A recent report showed that fluidphase GMP-140 isolated from platelets was able to strongly inhibit the adhesion of activated neutrophils to endothelium (Gamble et al., 1990), suggesting that GMP-140 in tissue and body fluids may also serve an anti-adhesive function in intravascular inflammation. In this study, we directly detected GMP-140 in some body fluids by a simple enzyme immunoassay employing two specific monoclonal antibodies. Surprisingly, no sequence could be obtained from intact purified protein, suggesting the presence of a blocked amino terminus. This contrasts with previously reported results (Johnston et al., 1989a). We found that amino acid sequences derived from protease-generated peptides of purified antigen were identical to sequences predicted from GMP-140 complementary D N A of endothelial cells, and that the amino acid compo-

sition of the antigen was similar to that of GMP140 reported previously (Johnston et al., !989a). This finding suggests that protein immunoreactive with PL7-6 and WGA- 1 is identical to GMP140. Although several groups have developed m o w oclonal antibodies to GMP-140 by immunization with activated human platelets (McEver and Martin, 1984; Palabrica et al., 1989; Gamble et al., 1990), these monoclonal antibodies have not been used in two-site immunoassays. The present sandwich-type immunoassay is recognized to be advantageous because its use of two different monoclonal antibodies permits rapid, precise, and specific measurement of GMP-140 in a convenient two-step procedure. The enzyme immunoassay requires only equipment and skills which are normally available in both research and clinical laboratories. Normal plasma and serum contains immunoreactive GMP-140. A n increase in the amount of GMP-140 in serum samples may result from in vitro proteolytic destruction of platelet membrane or platelet granules. It is unlikely, however, that the citrated plasma GMP-140 identified in our study derived from residual platelet microparticles. Some measurements of GMP-140 were performed with in vitro release and proteolysis minimized by drawing the blood directly into a container including prostaglandin E) and agents known to inhibit proteolysis. However, this did not reduce the plasma GMP-140 concentrations (Katayama et al., manuscript in preparation). 'These results suggest that GMP-140 levels in citrated plasma, if prepared exactly as described in the materials and methods section, accurately represent the levels of GMP-140 circulating in blood. Immunoreactive GMP-140 is present in normal plasma at a median concentration of 100 n g / m l . A fluid-phase GMP-140 has been shown to selectively limit the adhesion of activated neutrophils to endothelium in vitro (Gamble et al., 1990). It is tempting to speculate that released GMP-140 may, under appropriate conditions, regulate some inflammatory reactions in vivo which are largely mediated by neutrophils. The mechanism of GMP-140 release from platelets and endothelial cells is not understood. To inter-

pret the functions of plasma GMP-140 fully, it will be necessaB, to understand the mechanism of GMP-140 expression, its source, and its intravascular survival. We suspect that cells secrete GMP-140 directly, although it remains possible that a soluble form is derived from a membraneassociated form fragmented by proteolysis on the activated cell surface. Further investigations are necessary to resolve these mechanisms in detail. This assay for GMP-140 is sensitive, precise, rapid, and relatively simple for laboratory testing, The assay may also represent a potential clinical marker which will be informative in assessing the pathophysiology of intravascular inflammatory disorders, as well as in predicting the patient's prognosis and response to therapy.

Acknowledgements

We thank Dr. R.P. McEver (University of Oklahoma) for providing S12 monoclonal antibody.

References

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Johnston, G.I., Cook, R.G. and McEver, R.P. (1989a) Cloning of GMP-140, a granule membrane protein of platelets and endothelium. Cell 56, 1033-1044. Johnston, G.I,, Kurosk3,, A, and McEvor, R.P. (1989b) Structural and biosynthetic studies of the granule membrane protein, GMP-140, from human platelets and endothelial cells, J. Biol. Chem. 264. 1816-1823, Larsen, E., Cell A.~ Gilbert, G.E., Furie, B.C., Erhan, J.K., Bonfanti, R., Wagner, D.D. and Furie, B. (1989) PADGEM protein: a receptor that mediates the interaction of activated platclets with neutrophils and monoeytes. Cell 59, 305-312. Lasky, L.A., Singer, M.S., Yednock, T.A., Dowhenko, D., Fennie, C., Rodrigucz, H., Nguyen, T., Stachel, S. and Rosen, S.D. (1989) Cloning of a lymphocyte homing receptor reveals a lectin domain. Cell 56, 1045-1055. Matsudaira, P. (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262. 10035-10038. McEver, R.P. and Martin, M.N. (1984) A monoclonal antibody to a membrane glycoprotein binds only to activated p'~latelets. J. Biol. Chem. 259, 9799-9804. b'~oore, K.L., Varki, A. and McEver, R.P. (1991) GMP-140 binds to a Glycoprotein receptor on human neutrophils: evidence for a lectin-like interaction. J. Cell Biol. 112, 491-499.

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