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One-step sandwich enzyme immunoassay for soluble human thrombomodulin using monoclonal antibodies
191
Clinica Chimica Acfa, 192 (1990) 291-200 Elsevier CCA 04852
One-step sandwich enzyme immunoassay for soluble human thrombomodulin using monoclonal antibodies Shuji Kodama ’
‘, Ekuko Uchijima I, Misako Nagai I, Kyoko Tatsuya Hayashi ’ and Koji Suzuki ’
Mikawatani
I,
of Bjotechn~o~, Institute, Fuji Chemical Industries, Ltd., Takaoka, Toyama and ’ Institute for Enzyme Research, University of Tokushima, Tokushima (Japan)
department
(Received 12 March 1990; revision received 16 July 1990; accepted I7 July 1990) Key wor& Sandwich enzyme immunoassay; Soluble tbrombomodulin; Systemic lupus erythematosus
Monoclonal antibodies;
Summary
Six monoclonal antibodies for human ~rombomodu~n (TM) were prepared. All of them recognized an elastase-digested fragment of TM which contains 6 epidermal growth factor (EGF)-like structural domains. We developed a one-step sandwich enzyme immunoassay for soluble TM by using 2 antibodies; one of them, which inhibited thrombin-binding to TM, was fixed to polystyrene balls, and the other, which did not inhibit the thrombin-binding, but inhibited the protein C-activating cofactor activity of TM, was used as peroxidase-labeled conjugate. The sensitivity of this assay was 1 pg/l for soluble TM. The level of soluble TM was found to be significantly increased in sera of patients with systemic lupus erythematosus in comparison to the level in sera of healthy subjects.
Thrombomodulin (TM) is a thrombin receptor on the surface of endothelial cells of arteries, veins, capillaries and lymphatics, syncytiotrophoblast cells of placenta [l, 21, and also platelets [3]. It plays a role as a cofactor for thrombin-catalyzed activation of anticoagulant protease zymogen, protein C [l]. It is a single chain glycoprotein involving 6 consecutive epidermal growth factor (EGF)-like structural
Correspondence to: Dr. S. Kodama, Institute, Fuji Chemical Industries, Ltd., 530 Chokeiji, Takaoka, Toyama 933, Japan ~9-8981/~/$03.50
0 1990 Elsevier Science Publishers B.V. (Biomedi~l
Division)
192
domains in its extracellular region [4]. Thrombin binds to the region containing the last 3 EGF-like structural domains from the NH,-terminus of TM [S]. Ishii and Majerus [6] reported the presence of soluble TM in human plasma and urine. The soluble TM consists of various molecular weight fragments which are probably proteolytically degraded from the cellular TM, or may be derived from the injured and/or inflamed endothelial cells [6]. In this study, we developed a one-step sandwich enzyme immunoassay for soluble TM by using 2 monoclonal antibodies for human TM, and found that the level of the soluble TM was significantly increased in sera of patients with systemic lupus e~hematosus (SLE) compared to that of healthy subjects. Materials and methods
Bovine serum albumin (Fraction V, BSA) and Lubrol PX were obtained from Sigma Chem. Co. (St. Louis, MO). Pepsin from porcine gastric mucosa and horseradish peroxidase (POD, Grade 1) were obtained from Worthington B&hem. Co. (Freehold, NJ), and Boehringer Mannheim GmbH (Mannheim), respectively. MonoAb-ID EIA Kit and 96-well flat-bottomed microtitration plate were obtained from Zymed Lab. Inc. (South San Francisco, CA) and Flow Lab. (McLean, VA), respectively. An Ag-Stain ‘Daiichi’ and a ~~~ellulose sheet (Tram+Blot transfer medium, 0.45 pm) were obtained from Daiichi Pure Chem. (Tokyo) and Bio-Rad Lab. (Richmond, CA), respectively. Polystyrene balls (6.5 mm in diameter) were obtained from Precision Plastic Ball Co. (Chicago, IL). N-(r-maleirnido-caproyloxy) succinimide (EMCS) and 3,3’,5,5’-tetramethylbenzidine (TMBZ) were obtained from Dojindo Lab. (Kumamoto). Ultrogel AcA44 and protein A-Cellulofine were obtained from LKB (Villeneuve la Garenne) and Seikagaku Kogyo (Tokyo), respectively. Polyethylene glycol 4000 was obtained from Merck (Darmstadt). 2aminoethanethiol - HCl and other chemicals were obtained from Wako Pure Chem. Ind., Ltd. (Osaka). PreFaratio~ of ~o~~b~ehuman TM
Human TM was isolated from placenta according to the method of Suzuki et al. 141.Purified TM was digested with elastase to obtain soluble TM by the method of Kurosawa et al. [7]. The concentration of purified soluble TM was determined by dye-binding method of Bradford 181,in which BSA was used as a standard protein. Preparation of mouse monoclonal antibodies to human TM
Monoclonal antibodies against human TM were produced according to the method of Oi and Herzenberg [9]. Two 6-wk-old female BALB/c mice were immunized intraperitoneally with 50 pg of purified human TM emulsified with an equal volume of Freund’s complete adjuvant. After 15 and 49 days, they were intrape~ton~ly administered booster injections of 50 pg of the immunogen in 20 mmol/l Tris-HCl buffer, pH 7.5, containing 0.1 mol/l NaCl and 0.5 g/l Lubrol PX. On the 3rd day after the last intravenous injection the spleen was extirpated. The
193
isolated spleen cells were mixed with mouse myeloma cells (P3-NSl-1) at a ratio of 5 to 1 in RPM1 1640 medium and were hybridized in 500 g/l polyethylene glycol 4000. The fused cells were cultivated in HAT media. Cell lines producing antibody against human TM were screened by ELISA, and cloned by limiting dilution. Finally, each hybridoma was injected intraperitoneally into pristane-treated BALB/c mice for ascites production, and the ascites was harvested l-2 weeks later. Immunoglobulin was purified from the ascites by ammonium sulfate fractionation, followed by a protein A-Cellulofine column chromatography. Preparation
of Fab’-POD
conjugate
Purified mouse monoclonal antibodies were digested with pepsin from porcine mucosa to obtain F(ab’), fragment as described by Hamaguchi et al. [lo]. The F(ab’)z was reduced with 2-aminoethanethiol, and the Fab’-POD conjugate was prepared by the method of Hashida et al. [ll] using EMCS as a maleimide compound and POD. Preparation
of monoclonal
antibody-coated
polystyrene
balls
Polystyrene balls were coated at 4” C for 24 h with 0.1 g/l purified mouse monoclonal antibody, MFTM4, in 0.1 mol/l Na-phosphate buffer, pH 7.5, containing 1 g/l NaN, by physical adsorption [12]. The balls were then washed 5 times with 10 mmol/l Na-phosphate buffer, pH 7.0, containing 10 g/l BSA, 0.1 mol/l NaCl and 0.01 g/l chlorhexidine, and were stored in the same buffer at 4OC. One-step sandwich EIA technique
Fifty ~1 samples of serum or aliquot of standard soluble human TM, and 300 ~1 of Fab’(MFTM-6)-POD conjugate (0.5 mg/l) in 30 mmol/l Na-phosphate buffer, pH 7.0, containing 10 g/l BSA, 0.1 mol/l NaCl, and 10 mmol/l ethylenediamine tetraacetate were incubated with a polystyrene ball which had been coated with monoclonal antibody, MFTM4, at room temperature. After 60 min incubation, the polystyrene ball was washed twice with 4 ml of 5 mmol/l Na-phosphate buffer, pH 7.0, containing 50 mmol/l NaCl, and then incubated with 300 ~1 TMBZ (0.25 g/l) and 300 ~1 hydrogen peroxide (0.075 g/l) as a substrate for 30 min at room temperature by the method of Bos et al. [13]. The reaction was stopped by adding 800 pl sulfuric acid (1.75 mol/l) and the absorbance at 450 nm was measured by a Shimazu Model UV-730 Micro-Flow Spectrophotometer (Kyoto). Serum samples
Sera of healthy subjects were obtained from the subjects without any hematological and serological abnormalities. Sera of patients with SLE were obtained from patients who were positive to diagnostic serological examinations of antinuclear antibody and anti-DNA antibody, diagnostic hematological examination of lupus erythematosus cell by Giemsa staining, and diagnostic histological examination of biopsied specimens. Serum samples were frozen at -4O’C until use and assayed within 3 mth.
194
Results Characterization
of monoclonal
antibodies
Six hybridoma cell lines producing monoclonal antibody for human TM were cloned, and purified antibodies were characterized (Table I). All antibodies belonged to IgG and recognized an elastase-digested TM which consists of 6 EGF-like structural domains. MFTM-4, -5 and -6 strongly and MFTM-1 and -3 moderately inhibited cofactor activity of TM for thrombin-catalyzed activation of protein C. MFTM-4 and -5 strongly and MFTM-1, -2 and -3 moderately inhibited thrombinbinding to TM, but MFTM-6 did not [14]. On western blotting analysis, all IgGs bound to unreduced TM, but only MFTM4 and -5 bound to reduced TM [14], suggesting that MFTM-1, -2, -3 and -6 recognize the disulfide-linked conformation of TM. Standard
curve by one-step sandwich EM for soluble human
TM
For development of one-step sandwich EIA, various combinations of 2 antibodies, in which one was coated on polystyrene balls and the other was labeled with POD, were investigated (data not shown), and it was found that the most suitable combination among antibodies was composed of MFTM4 for the solid phase and MFTM-6 (Fab’) for the conjugate coupled with POD. Figure 1 shows standard curve by one-step sandwich EIA for soluble human TM by using the above pair of the antibodies. When the sensitivity of the assay was calculated as the concentration which is 2 standard deviations (2 SD) above the zero standard, the sensitivity was 1 pg/l of sample solution. Studies on the conditions for sandwich EIA
The assay coefficient of variation (CV W) of the standards was examined at 7 different soluble human TM levels from l-64 pg per 1 of standard solution. CV values obtained were 2%6.6% (n = 8, in Fig. 1). And the values obtained by using a normal serum and two pathological sera was 3.5-6.4% (n = 8). The effect of the amount of serum added on the sandwich EIA was also studied. Three kinds of sera were used. It was confirmed that there were no differences
TABLE I Inununoglobulin
types of monoclonal
antibodies
Clone no.
Monoclonal antibody no.
Subclass/chain
21-5D2 21-7E3 21-6F7 21-4G3 21-3Hl 21-9H12
MFTM-1 MFTM-2 MFTM-3 MFTM-4 MFTM-5 MFTM-6
1gGl/~ 1gGl/~ IgGl/a IgGSa/K IgGl/K IgGl/K
195
l.O-
o “: d
2.6/ 0
0.33.2/ 0 L.0/ 0
0.1 -
003-
6.6/ o
Soluble
Human
TM
(yg/ml)
Fig. 1. Standard curve by one-step sandwich EIA for soluble human TM. The soluble human TM was determined with a combination of a mouse monoclonal antibody (MFTM-4) as a solid phase and Fab’ of a mouse monoclonal antibody (MFTM-6)-POD conjugate as described in ‘Materials and Methods’. Numbers in the figure indicate CV values (%, n = 8).
between the results from specimens under the frozen or fresh condition. Linearity was observed between soluble TM level in the tube and the amount of serum added in a range of 20 to 70 1.11of each serum.
, 30 Reaction
Time
60 (min)
, 90
Fig. 2. Time course of the immunoreaction on one-step sandwich EIA. The assay was carried out as described in Fig. 1, except that immunoreaction time was changed. Numbers in the figure indicate levels (pg/l) in the soluble human TM (). (A) and (B, C) in the figure indicate normal and pathological sera (-
-
-),
respectively.
196 TABLE II Immunoreactive
TM levels in sera at different
(A) Immunoreactive Immunoreaction
TM levels in sera at different time (min)
Normal serum Pathological sera
(B) Immunoreactive Temperature
(pg/l) (pg/l) (pg/l)
@g/l) @g/l) (pg/l)
on one-step
immunoreaction
sandwich
EIA
times on one-step
sandwich
30
60
90
3.4 16.5 33.0
3.4 17.0 32.0
3.4 17.5 32.5
TM levels in sera at different
( o C)
Normal serum Pathological sera
conditions
temperatures
on one-step
sandwich
EIA:
EIA:
15
20
25
30
37
CV (W)
3.4 16.5 32.5
3.2 16.0 33.0
3.3 17.0 32.5
3.3 17.0 33.0
3.1 16.5 32.5
3.4 2.5 0.8
Each value in (A) and (B) was mean of TM levels (n = 2) in human
serum
The time course of the immunoreaction in sandwich EIA was studied (Fig. 2). Increase of absorbance at 450 nm of the serum was parallel to that of the standard solution. As shown in Table IIA, the value obtained for the level of soluble TM in human serum was found to be constant at different incubation times of the immunoreaction. The temperature dependence on the assay was studied. Both the immunoreaction and the enzymatic reaction were carried out the same temperature. As shown in
I
1.5.
1.0.
0
z Q
0.5 -
0
’
15
_
20 25 Temperature
30 1°C 1
37
Fig. 3. The effect of temperature on one-step sandwich EIA. The assay was carried out as described in Fig. 1, except that temperature was changed. Numbers in the figure indicate levels (pg/l) in the soluble ). (A) and (B, C) in the figure indicate normal and pathological sera (- - -), human TM (respectively.
197 TABLE III Reproducibility of immunoreactive TM levels in human sera with intra-assay or inter-assay TM
A. Irma-assay
Normal serum Pathological sera
3. Inter-assay
Normal serum Patholo~~l sera
M/l
w
3.2kO.l 11.0+_0.4 22.5 + 0.5 3.2kO.2 10.7+0.4 21.710.6
(3.1) (3.6) (2.2) (6.3) (3.71 (2.81
%)
Intra- and inter-assay variations for normal and pathological samples were expressed as mean f SD and CV value.
Table IIB, the level of soluble TM measured in human serum was constant regardless of the temperature change (15-37” C). As shown in Fig. 3, the absorbance at 450 nm with both standard and sample was found to increase with increasing temperature in a range of 15-30 OC, but the absorbance at 37°C was significantly decreased. Therefore, reaction temperature was set up in a range of 15-30 o C, because ~tigen-antibody reaction seemed to be interfered with at 37 o C. The studies performed did not demonstrate any serum component which may interact with the binding of the monoclonal antibodies.
15 . t
. l
:I l
:
.
:
.
.
. .
Fig. 4. Immunor~~tive TM levels in sera of normal subjects (Nf and SLE patients. I~u~oreactive TM in human sera was determined by one-step sandwich EIA as described in Fig. 1. Vertical bars show mean f SD. Numbers in parentheses express number of samples assayed.
198
Recovery of standard soluble TM (8 gg/l) added to serum soluble TM (10.7 Fg/l) was 98.3 t: 2.3% (mean i SD, n = 8). Reproducibility of intra-assay and inter-assay for immunoreactive soluble TM was studied. As shown in Table III, high reproducibility was obtained by both intraand inter-assay (n = 8), and no difference of CV values was observed between intra-assay or inter-assay. Determination of soluble TM in human serum sample
The one-step sandwich EIA was applied to the determination of soluble TM in sera of healthy subjects (N) and patients with SLE. Healthy subjects consisted of both age- and sex-matched individuals, and soluble TM level in sera of healthy subjects was independent of age (20-60 yr old). I~unoreactive soluble TM in the sera of 15 SLE patients, 7.0 i 3.6 pg/l (mean + SD), was sig~ficantly higher (P < 0.001) than that of 15 healthy subjects, 3.0 rfr0.7 pg/l (Fig. 4). Discussion
A pair of monoclonal antibodies were applied to develop a one-step sandwich EIA for simple and sensitive determination of soluble human TM. This EIA was based on simultaneous imnmnoreaction of soluble TM, a monoclonal antibodycoated polystyrene ball and another antibody labeled with POD. This assay was characterized as follows: (1) Immunoreaction time and temperature do not affect the level of soluble TM in serum; (2) results are obtained less than 2 h; (3) it has high sensitivity and high reproducibility; (4) no serum component which might interfere with the interaction was found. Hayashi et al. [14] showed that MFTM-4 and MFTM-5 reacted with the 5th domain of the 6 EGF-like structural domains of human TM and MFTM-6 may react with a region involving 3rd-4th EGF-like structural domains. Therefore, our EIA probably determines the soluble TM involving at least the 1st to 5th EGF-like structural domains. Ishii et al. [15] reported that there were many proteolytically modified TM fragments with various molecular weights (28-105 kDa) in plasma, and the main fragments observed were 55, 31 and 28 kDa. In vitro digestion of cellular TM by trypsin results in 2 major fragments (54 and 27 kDa) and elastase digestion results in 2 major fragments (50 and 25 kDa) [7]. The fragments with 50-54 kDa, containing all of the EGF-like structural domains, are rather stable against proteolysis by trypsin-like enzymes and possess the protein C-activating cofactor activity, but the fragments with 25 and 27 kDa do not. Thus, our EIA could specifically determine the fragments with 50-54 kDa in plasma Ishii and Majerus [6] suggested that soluble TM is derived from injured and inflamed intravascular cell membranes. The significant increase in soluble TM levels in sera of SLE patients may confirm their suggestion. This finding also suggests that the soluble TM is useful as a molecular marker for diagnosis of intravascular cell damage. It seems that there is correlation between the soluble TM level and the anti-DNA antibody titer. Additional serum sample should be tested to establish the
199
correlation. We are at present trying to study the relations~p between level of soluble TM and other factors resulted in SLE on therapy, as well as anti-DNA antibody. Acknowledgements
We thank Dr. Kazushi Iwata, Shinichi Yoshida, .&n&o Saeki, Kyoko Matsumoto and Hidekuni Shima, for advice and discussion and Dr. Karim T.A. Malik for reading of the manuscript. References 1 Owen WG, Esmon CT. Functionat properties of an endothelia cell cofactor for thrombiu-catalog activation of protein C. J Biol Chem 1981;256:5532-5535. 2 Maruyama I, Bell C, Majerus PW. Thrombomodutin is found on endothetium of arteries, veins, capillaries, and lymphatics, and syncytiotrophoblast of human placenta. J Cell Biol 1985;101:363-371. 3 Suzuki K, Nishioka J, Hayashi T, Kosaka Y. Functionally active thrombomodutin is present in human platelets. J B&hem 1988;104:628-632. 4 Suzuki K, Kusumoto H, Deyasiki Y, et al. Structure and expression of human lhrombom~u~, a thrombin receptor on endothehum acting as a cofactor for protein C activation. EMBO J 1987;6:1891-1897. 5 Zushi M, Gomi K, Yamamoto S, et al. The last three consecutive epidermal growth factor-like structures of human thrombomoduhn comprise the minimum functional domain for protein Cactivating cofactor activity and anticoagulant activity. J Biol Chem 1989;264:10351-10353. 6 Ishii H, Majerus PW. Thrombomoduhn is present in human plasma and urine. J Chn Invest 1985;76:2178-2181. 7 Kurosawa S, Galvin JB, Esmon NL, Esmon CT. Proteolytic formation and properties of functional domains of thrombomoduiin. J BioI Chem 1987;262:2206-2212. 8 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248:254. 9 Oi VT, Herzenberg LA. ImmunogIobuIin-producing hybrid cell lines. In: M&hell BB, Shiigi SM. eds. Selected Methods in Cellular Immunology. San Francisco: W.H. Freeman and Company, 1980;351372. 10 Hamaguchi Y, Yoshitake S, Ishikawa E, Endo Y, Ohtaki S. Improved procedure for the conjugation of rabbit IgG and Fab’ antibodies with B-D-galactosidase from Escherichia cofi using N, N ‘-o-phenylenedimaleimide. J Biochem 1979;85:1289-1300. 11 Hashida S, Imagawa M, Inoue S, Ruan K-H, Ishikawa E. More useful maleimide compounds for the conjugation of Fab’ to horseradish peroxidase through thiol groups in the hinge. J Appl Biochem 1984;6:56-63. 12 Yoshida S, Bai Y, Muragaki Y, et al. A sandwich immuno~say for human prolyl4-hydroxylase using monocIonaJ antibody. CIin Cbim Acta 1986;160:3?-46. 13 Bos ES, Van der Doelen AA, Van Rooy N, Schuurs AHWM. 3,3’,5,5’-Tetramethylbenaidine as an ames test negative chromogen for horseradish peroxidase in enzyme-immunoassay. J Immunoassay 1981;2:187-204. 14 Hayashi T, Suzuki K. Monoclonal antibodies for human thrombomoduhn which recognize binding sites for tbrombin and protein C. J B&hem 1990;108:111-115. 15 Ishii H, Uchiyama H, Hiraishi S, et al. Ctinical significance of the measurement of plasma thrombomoduhn (in Japanese). Jpn J Clin Path01 1989;37:266-271.
Clinica Chimica Acfa, 192 (1990) 291-200 Elsevier CCA 04852
One-step sandwich enzyme immunoassay for soluble human thrombomodulin using monoclonal antibodies Shuji Kodama ’
‘, Ekuko Uchijima I, Misako Nagai I, Kyoko Tatsuya Hayashi ’ and Koji Suzuki ’
Mikawatani
I,
of Bjotechn~o~, Institute, Fuji Chemical Industries, Ltd., Takaoka, Toyama and ’ Institute for Enzyme Research, University of Tokushima, Tokushima (Japan)
department
(Received 12 March 1990; revision received 16 July 1990; accepted I7 July 1990) Key wor& Sandwich enzyme immunoassay; Soluble tbrombomodulin; Systemic lupus erythematosus
Monoclonal antibodies;
Summary
Six monoclonal antibodies for human ~rombomodu~n (TM) were prepared. All of them recognized an elastase-digested fragment of TM which contains 6 epidermal growth factor (EGF)-like structural domains. We developed a one-step sandwich enzyme immunoassay for soluble TM by using 2 antibodies; one of them, which inhibited thrombin-binding to TM, was fixed to polystyrene balls, and the other, which did not inhibit the thrombin-binding, but inhibited the protein C-activating cofactor activity of TM, was used as peroxidase-labeled conjugate. The sensitivity of this assay was 1 pg/l for soluble TM. The level of soluble TM was found to be significantly increased in sera of patients with systemic lupus erythematosus in comparison to the level in sera of healthy subjects.
Thrombomodulin (TM) is a thrombin receptor on the surface of endothelial cells of arteries, veins, capillaries and lymphatics, syncytiotrophoblast cells of placenta [l, 21, and also platelets [3]. It plays a role as a cofactor for thrombin-catalyzed activation of anticoagulant protease zymogen, protein C [l]. It is a single chain glycoprotein involving 6 consecutive epidermal growth factor (EGF)-like structural
Correspondence to: Dr. S. Kodama, Institute, Fuji Chemical Industries, Ltd., 530 Chokeiji, Takaoka, Toyama 933, Japan ~9-8981/~/$03.50
0 1990 Elsevier Science Publishers B.V. (Biomedi~l
Division)
192
domains in its extracellular region [4]. Thrombin binds to the region containing the last 3 EGF-like structural domains from the NH,-terminus of TM [S]. Ishii and Majerus [6] reported the presence of soluble TM in human plasma and urine. The soluble TM consists of various molecular weight fragments which are probably proteolytically degraded from the cellular TM, or may be derived from the injured and/or inflamed endothelial cells [6]. In this study, we developed a one-step sandwich enzyme immunoassay for soluble TM by using 2 monoclonal antibodies for human TM, and found that the level of the soluble TM was significantly increased in sera of patients with systemic lupus e~hematosus (SLE) compared to that of healthy subjects. Materials and methods
Bovine serum albumin (Fraction V, BSA) and Lubrol PX were obtained from Sigma Chem. Co. (St. Louis, MO). Pepsin from porcine gastric mucosa and horseradish peroxidase (POD, Grade 1) were obtained from Worthington B&hem. Co. (Freehold, NJ), and Boehringer Mannheim GmbH (Mannheim), respectively. MonoAb-ID EIA Kit and 96-well flat-bottomed microtitration plate were obtained from Zymed Lab. Inc. (South San Francisco, CA) and Flow Lab. (McLean, VA), respectively. An Ag-Stain ‘Daiichi’ and a ~~~ellulose sheet (Tram+Blot transfer medium, 0.45 pm) were obtained from Daiichi Pure Chem. (Tokyo) and Bio-Rad Lab. (Richmond, CA), respectively. Polystyrene balls (6.5 mm in diameter) were obtained from Precision Plastic Ball Co. (Chicago, IL). N-(r-maleirnido-caproyloxy) succinimide (EMCS) and 3,3’,5,5’-tetramethylbenzidine (TMBZ) were obtained from Dojindo Lab. (Kumamoto). Ultrogel AcA44 and protein A-Cellulofine were obtained from LKB (Villeneuve la Garenne) and Seikagaku Kogyo (Tokyo), respectively. Polyethylene glycol 4000 was obtained from Merck (Darmstadt). 2aminoethanethiol - HCl and other chemicals were obtained from Wako Pure Chem. Ind., Ltd. (Osaka). PreFaratio~ of ~o~~b~ehuman TM
Human TM was isolated from placenta according to the method of Suzuki et al. 141.Purified TM was digested with elastase to obtain soluble TM by the method of Kurosawa et al. [7]. The concentration of purified soluble TM was determined by dye-binding method of Bradford 181,in which BSA was used as a standard protein. Preparation of mouse monoclonal antibodies to human TM
Monoclonal antibodies against human TM were produced according to the method of Oi and Herzenberg [9]. Two 6-wk-old female BALB/c mice were immunized intraperitoneally with 50 pg of purified human TM emulsified with an equal volume of Freund’s complete adjuvant. After 15 and 49 days, they were intrape~ton~ly administered booster injections of 50 pg of the immunogen in 20 mmol/l Tris-HCl buffer, pH 7.5, containing 0.1 mol/l NaCl and 0.5 g/l Lubrol PX. On the 3rd day after the last intravenous injection the spleen was extirpated. The
193
isolated spleen cells were mixed with mouse myeloma cells (P3-NSl-1) at a ratio of 5 to 1 in RPM1 1640 medium and were hybridized in 500 g/l polyethylene glycol 4000. The fused cells were cultivated in HAT media. Cell lines producing antibody against human TM were screened by ELISA, and cloned by limiting dilution. Finally, each hybridoma was injected intraperitoneally into pristane-treated BALB/c mice for ascites production, and the ascites was harvested l-2 weeks later. Immunoglobulin was purified from the ascites by ammonium sulfate fractionation, followed by a protein A-Cellulofine column chromatography. Preparation
of Fab’-POD
conjugate
Purified mouse monoclonal antibodies were digested with pepsin from porcine mucosa to obtain F(ab’), fragment as described by Hamaguchi et al. [lo]. The F(ab’)z was reduced with 2-aminoethanethiol, and the Fab’-POD conjugate was prepared by the method of Hashida et al. [ll] using EMCS as a maleimide compound and POD. Preparation
of monoclonal
antibody-coated
polystyrene
balls
Polystyrene balls were coated at 4” C for 24 h with 0.1 g/l purified mouse monoclonal antibody, MFTM4, in 0.1 mol/l Na-phosphate buffer, pH 7.5, containing 1 g/l NaN, by physical adsorption [12]. The balls were then washed 5 times with 10 mmol/l Na-phosphate buffer, pH 7.0, containing 10 g/l BSA, 0.1 mol/l NaCl and 0.01 g/l chlorhexidine, and were stored in the same buffer at 4OC. One-step sandwich EIA technique
Fifty ~1 samples of serum or aliquot of standard soluble human TM, and 300 ~1 of Fab’(MFTM-6)-POD conjugate (0.5 mg/l) in 30 mmol/l Na-phosphate buffer, pH 7.0, containing 10 g/l BSA, 0.1 mol/l NaCl, and 10 mmol/l ethylenediamine tetraacetate were incubated with a polystyrene ball which had been coated with monoclonal antibody, MFTM4, at room temperature. After 60 min incubation, the polystyrene ball was washed twice with 4 ml of 5 mmol/l Na-phosphate buffer, pH 7.0, containing 50 mmol/l NaCl, and then incubated with 300 ~1 TMBZ (0.25 g/l) and 300 ~1 hydrogen peroxide (0.075 g/l) as a substrate for 30 min at room temperature by the method of Bos et al. [13]. The reaction was stopped by adding 800 pl sulfuric acid (1.75 mol/l) and the absorbance at 450 nm was measured by a Shimazu Model UV-730 Micro-Flow Spectrophotometer (Kyoto). Serum samples
Sera of healthy subjects were obtained from the subjects without any hematological and serological abnormalities. Sera of patients with SLE were obtained from patients who were positive to diagnostic serological examinations of antinuclear antibody and anti-DNA antibody, diagnostic hematological examination of lupus erythematosus cell by Giemsa staining, and diagnostic histological examination of biopsied specimens. Serum samples were frozen at -4O’C until use and assayed within 3 mth.
194
Results Characterization
of monoclonal
antibodies
Six hybridoma cell lines producing monoclonal antibody for human TM were cloned, and purified antibodies were characterized (Table I). All antibodies belonged to IgG and recognized an elastase-digested TM which consists of 6 EGF-like structural domains. MFTM-4, -5 and -6 strongly and MFTM-1 and -3 moderately inhibited cofactor activity of TM for thrombin-catalyzed activation of protein C. MFTM-4 and -5 strongly and MFTM-1, -2 and -3 moderately inhibited thrombinbinding to TM, but MFTM-6 did not [14]. On western blotting analysis, all IgGs bound to unreduced TM, but only MFTM4 and -5 bound to reduced TM [14], suggesting that MFTM-1, -2, -3 and -6 recognize the disulfide-linked conformation of TM. Standard
curve by one-step sandwich EM for soluble human
TM
For development of one-step sandwich EIA, various combinations of 2 antibodies, in which one was coated on polystyrene balls and the other was labeled with POD, were investigated (data not shown), and it was found that the most suitable combination among antibodies was composed of MFTM4 for the solid phase and MFTM-6 (Fab’) for the conjugate coupled with POD. Figure 1 shows standard curve by one-step sandwich EIA for soluble human TM by using the above pair of the antibodies. When the sensitivity of the assay was calculated as the concentration which is 2 standard deviations (2 SD) above the zero standard, the sensitivity was 1 pg/l of sample solution. Studies on the conditions for sandwich EIA
The assay coefficient of variation (CV W) of the standards was examined at 7 different soluble human TM levels from l-64 pg per 1 of standard solution. CV values obtained were 2%6.6% (n = 8, in Fig. 1). And the values obtained by using a normal serum and two pathological sera was 3.5-6.4% (n = 8). The effect of the amount of serum added on the sandwich EIA was also studied. Three kinds of sera were used. It was confirmed that there were no differences
TABLE I Inununoglobulin
types of monoclonal
antibodies
Clone no.
Monoclonal antibody no.
Subclass/chain
21-5D2 21-7E3 21-6F7 21-4G3 21-3Hl 21-9H12
MFTM-1 MFTM-2 MFTM-3 MFTM-4 MFTM-5 MFTM-6
1gGl/~ 1gGl/~ IgGl/a IgGSa/K IgGl/K IgGl/K
195
l.O-
o “: d
2.6/ 0
0.33.2/ 0 L.0/ 0
0.1 -
003-
6.6/ o
Soluble
Human
TM
(yg/ml)
Fig. 1. Standard curve by one-step sandwich EIA for soluble human TM. The soluble human TM was determined with a combination of a mouse monoclonal antibody (MFTM-4) as a solid phase and Fab’ of a mouse monoclonal antibody (MFTM-6)-POD conjugate as described in ‘Materials and Methods’. Numbers in the figure indicate CV values (%, n = 8).
between the results from specimens under the frozen or fresh condition. Linearity was observed between soluble TM level in the tube and the amount of serum added in a range of 20 to 70 1.11of each serum.
, 30 Reaction
Time
60 (min)
, 90
Fig. 2. Time course of the immunoreaction on one-step sandwich EIA. The assay was carried out as described in Fig. 1, except that immunoreaction time was changed. Numbers in the figure indicate levels (pg/l) in the soluble human TM (). (A) and (B, C) in the figure indicate normal and pathological sera (-
-
-),
respectively.
196 TABLE II Immunoreactive
TM levels in sera at different
(A) Immunoreactive Immunoreaction
TM levels in sera at different time (min)
Normal serum Pathological sera
(B) Immunoreactive Temperature
(pg/l) (pg/l) (pg/l)
@g/l) @g/l) (pg/l)
on one-step
immunoreaction
sandwich
EIA
times on one-step
sandwich
30
60
90
3.4 16.5 33.0
3.4 17.0 32.0
3.4 17.5 32.5
TM levels in sera at different
( o C)
Normal serum Pathological sera
conditions
temperatures
on one-step
sandwich
EIA:
EIA:
15
20
25
30
37
CV (W)
3.4 16.5 32.5
3.2 16.0 33.0
3.3 17.0 32.5
3.3 17.0 33.0
3.1 16.5 32.5
3.4 2.5 0.8
Each value in (A) and (B) was mean of TM levels (n = 2) in human
serum
The time course of the immunoreaction in sandwich EIA was studied (Fig. 2). Increase of absorbance at 450 nm of the serum was parallel to that of the standard solution. As shown in Table IIA, the value obtained for the level of soluble TM in human serum was found to be constant at different incubation times of the immunoreaction. The temperature dependence on the assay was studied. Both the immunoreaction and the enzymatic reaction were carried out the same temperature. As shown in
I
1.5.
1.0.
0
z Q
0.5 -
0
’
15
_
20 25 Temperature
30 1°C 1
37
Fig. 3. The effect of temperature on one-step sandwich EIA. The assay was carried out as described in Fig. 1, except that temperature was changed. Numbers in the figure indicate levels (pg/l) in the soluble ). (A) and (B, C) in the figure indicate normal and pathological sera (- - -), human TM (respectively.
197 TABLE III Reproducibility of immunoreactive TM levels in human sera with intra-assay or inter-assay TM
A. Irma-assay
Normal serum Pathological sera
3. Inter-assay
Normal serum Patholo~~l sera
M/l
w
3.2kO.l 11.0+_0.4 22.5 + 0.5 3.2kO.2 10.7+0.4 21.710.6
(3.1) (3.6) (2.2) (6.3) (3.71 (2.81
%)
Intra- and inter-assay variations for normal and pathological samples were expressed as mean f SD and CV value.
Table IIB, the level of soluble TM measured in human serum was constant regardless of the temperature change (15-37” C). As shown in Fig. 3, the absorbance at 450 nm with both standard and sample was found to increase with increasing temperature in a range of 15-30 OC, but the absorbance at 37°C was significantly decreased. Therefore, reaction temperature was set up in a range of 15-30 o C, because ~tigen-antibody reaction seemed to be interfered with at 37 o C. The studies performed did not demonstrate any serum component which may interact with the binding of the monoclonal antibodies.
15 . t
. l
:I l
:
.
:
.
.
. .
Fig. 4. Immunor~~tive TM levels in sera of normal subjects (Nf and SLE patients. I~u~oreactive TM in human sera was determined by one-step sandwich EIA as described in Fig. 1. Vertical bars show mean f SD. Numbers in parentheses express number of samples assayed.
198
Recovery of standard soluble TM (8 gg/l) added to serum soluble TM (10.7 Fg/l) was 98.3 t: 2.3% (mean i SD, n = 8). Reproducibility of intra-assay and inter-assay for immunoreactive soluble TM was studied. As shown in Table III, high reproducibility was obtained by both intraand inter-assay (n = 8), and no difference of CV values was observed between intra-assay or inter-assay. Determination of soluble TM in human serum sample
The one-step sandwich EIA was applied to the determination of soluble TM in sera of healthy subjects (N) and patients with SLE. Healthy subjects consisted of both age- and sex-matched individuals, and soluble TM level in sera of healthy subjects was independent of age (20-60 yr old). I~unoreactive soluble TM in the sera of 15 SLE patients, 7.0 i 3.6 pg/l (mean + SD), was sig~ficantly higher (P < 0.001) than that of 15 healthy subjects, 3.0 rfr0.7 pg/l (Fig. 4). Discussion
A pair of monoclonal antibodies were applied to develop a one-step sandwich EIA for simple and sensitive determination of soluble human TM. This EIA was based on simultaneous imnmnoreaction of soluble TM, a monoclonal antibodycoated polystyrene ball and another antibody labeled with POD. This assay was characterized as follows: (1) Immunoreaction time and temperature do not affect the level of soluble TM in serum; (2) results are obtained less than 2 h; (3) it has high sensitivity and high reproducibility; (4) no serum component which might interfere with the interaction was found. Hayashi et al. [14] showed that MFTM-4 and MFTM-5 reacted with the 5th domain of the 6 EGF-like structural domains of human TM and MFTM-6 may react with a region involving 3rd-4th EGF-like structural domains. Therefore, our EIA probably determines the soluble TM involving at least the 1st to 5th EGF-like structural domains. Ishii et al. [15] reported that there were many proteolytically modified TM fragments with various molecular weights (28-105 kDa) in plasma, and the main fragments observed were 55, 31 and 28 kDa. In vitro digestion of cellular TM by trypsin results in 2 major fragments (54 and 27 kDa) and elastase digestion results in 2 major fragments (50 and 25 kDa) [7]. The fragments with 50-54 kDa, containing all of the EGF-like structural domains, are rather stable against proteolysis by trypsin-like enzymes and possess the protein C-activating cofactor activity, but the fragments with 25 and 27 kDa do not. Thus, our EIA could specifically determine the fragments with 50-54 kDa in plasma Ishii and Majerus [6] suggested that soluble TM is derived from injured and inflamed intravascular cell membranes. The significant increase in soluble TM levels in sera of SLE patients may confirm their suggestion. This finding also suggests that the soluble TM is useful as a molecular marker for diagnosis of intravascular cell damage. It seems that there is correlation between the soluble TM level and the anti-DNA antibody titer. Additional serum sample should be tested to establish the
199
correlation. We are at present trying to study the relations~p between level of soluble TM and other factors resulted in SLE on therapy, as well as anti-DNA antibody. Acknowledgements
We thank Dr. Kazushi Iwata, Shinichi Yoshida, .&n&o Saeki, Kyoko Matsumoto and Hidekuni Shima, for advice and discussion and Dr. Karim T.A. Malik for reading of the manuscript. References 1 Owen WG, Esmon CT. Functionat properties of an endothelia cell cofactor for thrombiu-catalog activation of protein C. J Biol Chem 1981;256:5532-5535. 2 Maruyama I, Bell C, Majerus PW. Thrombomodutin is found on endothetium of arteries, veins, capillaries, and lymphatics, and syncytiotrophoblast of human placenta. J Cell Biol 1985;101:363-371. 3 Suzuki K, Nishioka J, Hayashi T, Kosaka Y. Functionally active thrombomodutin is present in human platelets. J B&hem 1988;104:628-632. 4 Suzuki K, Kusumoto H, Deyasiki Y, et al. Structure and expression of human lhrombom~u~, a thrombin receptor on endothehum acting as a cofactor for protein C activation. EMBO J 1987;6:1891-1897. 5 Zushi M, Gomi K, Yamamoto S, et al. The last three consecutive epidermal growth factor-like structures of human thrombomoduhn comprise the minimum functional domain for protein Cactivating cofactor activity and anticoagulant activity. J Biol Chem 1989;264:10351-10353. 6 Ishii H, Majerus PW. Thrombomoduhn is present in human plasma and urine. J Chn Invest 1985;76:2178-2181. 7 Kurosawa S, Galvin JB, Esmon NL, Esmon CT. Proteolytic formation and properties of functional domains of thrombomoduiin. J BioI Chem 1987;262:2206-2212. 8 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248:254. 9 Oi VT, Herzenberg LA. ImmunogIobuIin-producing hybrid cell lines. In: M&hell BB, Shiigi SM. eds. Selected Methods in Cellular Immunology. San Francisco: W.H. Freeman and Company, 1980;351372. 10 Hamaguchi Y, Yoshitake S, Ishikawa E, Endo Y, Ohtaki S. Improved procedure for the conjugation of rabbit IgG and Fab’ antibodies with B-D-galactosidase from Escherichia cofi using N, N ‘-o-phenylenedimaleimide. J Biochem 1979;85:1289-1300. 11 Hashida S, Imagawa M, Inoue S, Ruan K-H, Ishikawa E. More useful maleimide compounds for the conjugation of Fab’ to horseradish peroxidase through thiol groups in the hinge. J Appl Biochem 1984;6:56-63. 12 Yoshida S, Bai Y, Muragaki Y, et al. A sandwich immuno~say for human prolyl4-hydroxylase using monocIonaJ antibody. CIin Cbim Acta 1986;160:3?-46. 13 Bos ES, Van der Doelen AA, Van Rooy N, Schuurs AHWM. 3,3’,5,5’-Tetramethylbenaidine as an ames test negative chromogen for horseradish peroxidase in enzyme-immunoassay. J Immunoassay 1981;2:187-204. 14 Hayashi T, Suzuki K. Monoclonal antibodies for human thrombomoduhn which recognize binding sites for tbrombin and protein C. J B&hem 1990;108:111-115. 15 Ishii H, Uchiyama H, Hiraishi S, et al. Ctinical significance of the measurement of plasma thrombomoduhn (in Japanese). Jpn J Clin Path01 1989;37:266-271.