Clinica Chimica Acta, 202 (1991) 219-226 0 1991 Elsevier Science Publishers B.V. All rights reserved 0009-8981/91/$03.50
219
CCA 05101
ELISA using monoclonal antibody to human serum arylesterase Hiroshi
Kawai ‘, Satoshi Yomoda
’ and Yoshihide
Inoue *
’ Pharmaceuticals Research Center, Kanebo, Ltd., Osaka and 2 Kanebo Hospital, Kobe (Japan) (Received 3 May 1991; revision received 16 July 1991; accepted 22 July 1991)
Key words: Serum arylesterase; Cirrhosis; Monoclonal antibody; ELISA
Summary ELBA for determining arylesterase content in human serum has been developed by the one-step sandwich method using 2 monoclonal antibodies. While the content of arylesterase in healthy adults was 81 f 25 mg/l, a decrease was observed in patients with liver cirrhosis, where the mean + SD was 37 + 7 mg/l. ELISA of human serum arylesterase correlated with the activity determined by a specific substrate assay we devised recently.
Introduction In 1958, Augustinsson [ll showed human plasma to contain two types of esterase, arylesterase (ArE; a&ester hydrolase, EC 3.1.1.2) and cholinesterase (ChE; acyl-choline acyl-hydrolase, EC 3.1.1.8). Little study, however, has been conducted to clarify the physiological functions of the former enzyme because of its wide substrate specificity [2]. Recently, we reported that serum ArE activity, determined by a new and highly specific substrate to ArE, 4-(4-fluorophenyl~thiomethyl-5-methyl-1,3-dioxol-Zone (I) [3,4], or sodium 4-[(5-methyl-2-oxo-1,3-dioxol-4-yl)methylthio]benzenesulfonate (II) [4,5], decreases remarkably in patients with liver cirrhosis [3,5].
Correspondence to: Hiroshi Kawai, Pharmaceuticals Research Center, Kanebo, Ltd., S-90, Tomobuchicho, 1-chome, Miyakojima-ku, Osaka 534, Japan.
220
The present study was conducted to develop an enzyme-linked immunosorbent assay (ELISA) that uses a highly specific monoclonal antibody directed against ArE to estimate the amount of ArE protein in human serum independently the activity of ArE. Materials and methods Chemicals
Reagent chemicals of high purity were purchased sources.
from standard
commercial
Generation of monoclonal antibodies
Monoclonal antibodies which do not bind to the active site of the enzyme antigen and do not inhibit enzyme activity [6] but specifically react to human ArE were prepared as follows. 1. Hybridoma; 9-wk-old female BALB/c mice were immunized with 50 pg of ArE preparation [3] dissolved in saline and emulsified with Freund’s complete adjuvant (Difco, USA). One ml of the mixture (1: 1) was injected i.p. After 17 days, the mice were boosted with antigen mixed with incomplete adjuvant (Difco, USA) (1: 1) and with antigen alone 15 days- later. On the third day after the last intravenous injection, the mice were killed and the spleens removed. The spleen cells (1.6 x lo*) were fused with P3X63Ag8U, myeloma cells (1.6 x 10’) in 425 g/l polyethylene glycol 1000 and hybridoma cells were established [7]. The fused cells were cultivated in HAT media. After screening, with substrate [II hydrolysis activity as an index by ELISA [Sl, 3 species of monoclonal antibody-forming hybridoma cells, 7-2C5, 9-2C3 and 12-lH8, were obtained. The resulting cells were cloned by limiting-dilution techniques. 2. Monoclonal antibody; ascites was harvested after priming male mice of BALB/c strain with 0.5 ml of Pristane (Aldrich, USA) i.p., followed 1 wk later by injection 8 x lo6 hybridoma clones in 0.8 ml of RPM1 1640 medium. After 10 days, the retained ascites was collected. Obtained ascites was fractionated by salting-out techniques with ammonium sulfate (O-40% saturation), and purified using an Affi-gel protein A MAPS-II kit (Bio-Rad, USA). Class, subclass and type of L-chains of the thus obtained monoclonal antibodies produced by hybridoma cells were IgG,/K for 7-2C5, IgG,,/K for 9-2C3, and IgGI/K for 12-lH8. Preparation of ArE standard for ELI&l
Standard preparation of ArE for ELISA was purified from human serum by one-step of immuno-affinity chromatography using the monoclonal antibody produced by 12-lH8 cells [81. The resultant preparation behaved as a single component on SDS-polyacrylamide disc gel electrophoresis, indicating high homogeneity.
221
Enzyme-linked immunosorbent assay (ELISA) According to the one-step sandwich method using a mouse monoclonal antibody produced by 9-2C3 cells as the first antibody and a mouse monoclonal antibody produced by 7-2C5 cells labelled with peroxidase as the enzyme-labelled second antibody, the amount of ArE protein in human serum was assayed as follows. The monoclonal antibody produced by 9-2C3 ‘cells was diluted with 0.05 mol/l carbonate buffer (pH 9.6) to a concentration of 10 hg/ml and poured into each well of a 96-well microplate (Falcon 3912) at 100 ~1 per well, followed by standing at 4°C overnight. After washing each well with phosphate buffered saline (PBS, pH 7.41, 200 ~1 of PBS (pH 7.4) containing 0.5% of bovine serum albumin (BSA) were added, and the system was allowed to stand for 2 hours. The supernatant was then discharged. Next, after adding 50 ~1 of ArE standard solution dissolved in PBS containing 0.1% of BSA (pH 7.4) and 50 ~1 of the monoclonal antibody (1 pg/ml) produced by 7-2C5 cells, labelled with peroxidase according to the periodic acid cross-linking method [9], it was reacted at room temperature for 2 h. After washing each well with PBS (pH 7.41, 100 ~1 of 0.15 mol/l citrate buffer (pH 5.0) containing 0.015% of H,O, and 0.2 mg/ml of o-phenylene diamine were added. Five minutes later, 50 ~1 of 10% sulfuric acid were added to terminate the reaction, and enzyme activity was assayed using a two-wavelength (492 nm, 550 nm) microplate photometer (Corona MTP-22, Japan). Based on the results, the standard curve shown in Fig. 1 was obtained.
I 10
I
I
20
50
Aryresterase
I
loo
1
I
200
kg/ml)
Fig. 1. Standard curve by one-step sandwich ELBA for serum ArE. Serum ArE was determined by a combination of a mouse monoclonal antibody produced by 9-2C3 cells as the first antibody and a mouse monoclonal antibody produced by 7-2C5 cells, labelled with peroxidase, as the enzyme-labelled second antibody. The activity of peroxidase was assayed using a two-wavelength (492 nm, 550 nm) microplate photometer (Corona MTP-22, Japan).
222
Sera Serum was prepared from the blood of healthy adults (9 males and 11 females aged 28-49) and patients with cirrhosis (4 compensated and 2 decompensated, 4 males and 2 females aged 48-76). Samples were assayed within 3 days following storage at 4°C. Dete~ination
of ArE content
Serum was diluted with 0.05 mol/l carbonate buffer CpH 9.6) 500, 1,000 and 2,000 times, measured in the same manner as ELISA. ArE content was determined by conversion from the standard curve. Determination of ArE activity Serum ArE activity was measured at 37°C using substrate [III with an automatic analyzer (Hitachi Automatic Blood Chemistry Analyzer Model 705, Japan) [51. Results Standard ArE curve by one-step sandwich-ELBA The linearity of the assay for standard ArE by using a 96-well microplate was obtained from 18-144 ng/ml (0.9-72 ng/well) by combination of a monoclonal antibody of clone 9-2C3 as a solid phase and a pero~dase-labelled monoclonal antibody of clone 7-2C5 as a conjugate (Fig. 1). The detection limit, defined as the lowest concentration giving a peroxidase activity similar to that of the zero standard at 0.99 confidence, was 2 ng/ml (0.1 ng/well). When ChE (6.2 U/mg) from human plasma [31 was subjected to the above immunoassay system, there was no increase (up to 0.01 U/ml> in the peroxidase activity bound on the well (not shown), indicating there is no cross-reactivity of the present assay with human ChE. Estimation of ArE content Figure 2 shows ArE content in human serum. The values were 81 _t 25 for healthy adults and 37 + 7 mg/l for patients with cirrhosis, respectively, with significant differences (mean f SD, P < 0.001) (Table I). A marked decrease was noted in patients with cirrhosis. The 6 subjects with cirrhosis had ArE ranging from 26 to 44 mg/l. From the normal subjects, a 95% reference range of 31-131 was obtained. Co~e~at~o~ between ArE content and ArE activity Table I shows ArE specific activity in human serum. The values were 150 rt 43 for healthy adults and 167 + 36 U/mg for patients with cirrhosis, respectively, with
223
Norma
I
(!l:20>
Patients (n-6)
Fig. 2. Serum ArE content in healthy adults (01, and patients with compensated cirrhosis CD) or decompensated cirrhosis (0 ). The horizontal bars indicate mean values for each group.
non-significant differences (mean + SD, P > 0.05). A correlation between ArE in the serum as measured by ELISA (CArE, mg/l) and ArE activity in the serum measured using highly specific substrate [II] (A,,, kU/l) was found as shown in Fig. 3 and expressed as the A ArE= 5.74 log C,,
( IZ= 26, r2 = 0.43, P < 0.01).
TABLE I ArE specific activity (SA) estimate
Normal (20) Patients (6)
ArE activity (kU/I) (mean * SD)
ArE content (mg/l) (mean f SD)
ArE SA (U/mg) (mean f SD)
11.3k1.8 6.3 f 1.8 a
81k2.5 31* -I=
150 f 43 167+,36 b
a Difference from normal group statistically significant (P < 0.001). b Difference from normal group statistically non-significant (P > 0.05).
224
q
0
10
I
I
I
I
20
50
100
200
Arylesterase
by ELISA,
I
500
mg /I
Fig. 3. Correlation of serum ArE activity determined by substrate [II] (AArE, kU/O [.5] to ArE measured by ELBA CC,,,, mg/I) in healthy adults (e), and patients with compensated cirrhosis (m) or decompensated cirrhosis (0). The best fit correlation equation is: AArE = 5.74 log CArE (n = 26, r* = 0.43, P < 0.01).
A high positive correlation in the serum.
is thus shown between ArE protein content and activity
Discussion For the diagnosis of acute hepatitis, there are excellent biochemical parameters such as GOT and GPT, which can be easily measured and are widely used. However, only a few reliable biochemical parameters which indicate liver cirrhosis are presently available. ArE activity determined with phenyl acetate [lO,ll] or P-naphthyl acetate [12] as substrate, as well as ChE activity, has been reported to decrease in patients with liver disease, particularly cirrhosis. However, these substrates for the assay of ArE activity are hydrolyzed by ChE as well as ArE [2,31. Therefore these substrates are not suitable for the accurate determination of ArE activity owing to their lack of specificity. By using a new and highly specific substrate to ArE, substrate [I] [3,4], or substrate [II] [4,5], we recently showed serum ArE activity to decrease remarkably in patients with liver cirrhosis [3,5]. Using ArE activity measured by substrate [I] as an index, it was feasible to prepare antigen [3] and select hybridoma cells that form a monoclonal antibody specific to ArE [8]. We thus succeeded for the first time in developing ELISA adequate for the diagnosis of cirrhosis. In this ELISA study, we clearly showed the above decrease in ArE activity inthe serum of patients with cirrhosis, measured by substrate [II] [5], to result from reduction in ArE as an essential enzyme protein.
225
Acknowledgements The authors thank A. Sugita, A. Kono and T. Yamamoto for assistance. References 1 Augustinsson K-B. Electrophoretic separation and classification of blood plasma esterases. Nature 1958;181:1786-1789. 2 Augustinsson K-B, Ekedahl G. On the specificity of arylesterases. Acta Chem Stand 1962;16:240241. 3 Tanaka H, Kitamura M, Inoue Y. A simple assay for serum arylesterase using a specific substrate. Clin Chim Acta 1987;170:105-110. 4 Kawai H, Sakamoto F, Taguchi M, Kitamura M, Sotomura M, Tsukamoto G. 2-Oxo-1,3-dioxoles as specific substrates for measurement of arylesterase activity. Chem Pharm Bull 1991;39:1422-1425. 5 Kawai H, Sakamoto F, Inoue Y. Improved specific assay for serum arylesterase using a water-soluble substrate. Clin Chim Acta 1990;188:177-182. 6 Rosalki SB. Monoclonal antibodies to enzymes. Clin Chim Acta 1989;183:45-58. 7 Yelton DE, Diamond BA, Kwan S-P, Scharff MD. Fusion of mouse myeloma and spleen cells. Curr Top Microb Immunol 1978;81:1-7. 8 Yomoda S, Kawai H, Iwamoto S, Sotomura M. Monoclonal antibody. Jpn Kokai Tokkyo Koho JP 63,267,799 1988. 9 Nakane PK, Kawaoi A. Peroxidase-labeled antibody. A new method of conjugation. J Histochem Cytochem 1974;22:1084-1091. 10 Takahashi Y, Aoyama I, Ito F, Yamamura Y. Evaluation of phenyl acetate esterase activity as an index of liver damage. Clin Chim Acta 1967;18:21-32. 11 Lorentz K, Flatter B, Augustin E. Arylesterase in serum: elaboration and clinical application of a fixed-incubation method. Clin Chem 1979;25:1714-1720. 12 Burlina A, Michielin E, Galzigna L. Characteristics and behavior of arylesterase in human serum and liver. Europ J Clin Invest 1977;7:17-20.
219
CCA 05101
ELISA using monoclonal antibody to human serum arylesterase Hiroshi
Kawai ‘, Satoshi Yomoda
’ and Yoshihide
Inoue *
’ Pharmaceuticals Research Center, Kanebo, Ltd., Osaka and 2 Kanebo Hospital, Kobe (Japan) (Received 3 May 1991; revision received 16 July 1991; accepted 22 July 1991)
Key words: Serum arylesterase; Cirrhosis; Monoclonal antibody; ELISA
Summary ELBA for determining arylesterase content in human serum has been developed by the one-step sandwich method using 2 monoclonal antibodies. While the content of arylesterase in healthy adults was 81 f 25 mg/l, a decrease was observed in patients with liver cirrhosis, where the mean + SD was 37 + 7 mg/l. ELISA of human serum arylesterase correlated with the activity determined by a specific substrate assay we devised recently.
Introduction In 1958, Augustinsson [ll showed human plasma to contain two types of esterase, arylesterase (ArE; a&ester hydrolase, EC 3.1.1.2) and cholinesterase (ChE; acyl-choline acyl-hydrolase, EC 3.1.1.8). Little study, however, has been conducted to clarify the physiological functions of the former enzyme because of its wide substrate specificity [2]. Recently, we reported that serum ArE activity, determined by a new and highly specific substrate to ArE, 4-(4-fluorophenyl~thiomethyl-5-methyl-1,3-dioxol-Zone (I) [3,4], or sodium 4-[(5-methyl-2-oxo-1,3-dioxol-4-yl)methylthio]benzenesulfonate (II) [4,5], decreases remarkably in patients with liver cirrhosis [3,5].
Correspondence to: Hiroshi Kawai, Pharmaceuticals Research Center, Kanebo, Ltd., S-90, Tomobuchicho, 1-chome, Miyakojima-ku, Osaka 534, Japan.
220
The present study was conducted to develop an enzyme-linked immunosorbent assay (ELISA) that uses a highly specific monoclonal antibody directed against ArE to estimate the amount of ArE protein in human serum independently the activity of ArE. Materials and methods Chemicals
Reagent chemicals of high purity were purchased sources.
from standard
commercial
Generation of monoclonal antibodies
Monoclonal antibodies which do not bind to the active site of the enzyme antigen and do not inhibit enzyme activity [6] but specifically react to human ArE were prepared as follows. 1. Hybridoma; 9-wk-old female BALB/c mice were immunized with 50 pg of ArE preparation [3] dissolved in saline and emulsified with Freund’s complete adjuvant (Difco, USA). One ml of the mixture (1: 1) was injected i.p. After 17 days, the mice were boosted with antigen mixed with incomplete adjuvant (Difco, USA) (1: 1) and with antigen alone 15 days- later. On the third day after the last intravenous injection, the mice were killed and the spleens removed. The spleen cells (1.6 x lo*) were fused with P3X63Ag8U, myeloma cells (1.6 x 10’) in 425 g/l polyethylene glycol 1000 and hybridoma cells were established [7]. The fused cells were cultivated in HAT media. After screening, with substrate [II hydrolysis activity as an index by ELISA [Sl, 3 species of monoclonal antibody-forming hybridoma cells, 7-2C5, 9-2C3 and 12-lH8, were obtained. The resulting cells were cloned by limiting-dilution techniques. 2. Monoclonal antibody; ascites was harvested after priming male mice of BALB/c strain with 0.5 ml of Pristane (Aldrich, USA) i.p., followed 1 wk later by injection 8 x lo6 hybridoma clones in 0.8 ml of RPM1 1640 medium. After 10 days, the retained ascites was collected. Obtained ascites was fractionated by salting-out techniques with ammonium sulfate (O-40% saturation), and purified using an Affi-gel protein A MAPS-II kit (Bio-Rad, USA). Class, subclass and type of L-chains of the thus obtained monoclonal antibodies produced by hybridoma cells were IgG,/K for 7-2C5, IgG,,/K for 9-2C3, and IgGI/K for 12-lH8. Preparation of ArE standard for ELI&l
Standard preparation of ArE for ELISA was purified from human serum by one-step of immuno-affinity chromatography using the monoclonal antibody produced by 12-lH8 cells [81. The resultant preparation behaved as a single component on SDS-polyacrylamide disc gel electrophoresis, indicating high homogeneity.
221
Enzyme-linked immunosorbent assay (ELISA) According to the one-step sandwich method using a mouse monoclonal antibody produced by 9-2C3 cells as the first antibody and a mouse monoclonal antibody produced by 7-2C5 cells labelled with peroxidase as the enzyme-labelled second antibody, the amount of ArE protein in human serum was assayed as follows. The monoclonal antibody produced by 9-2C3 ‘cells was diluted with 0.05 mol/l carbonate buffer (pH 9.6) to a concentration of 10 hg/ml and poured into each well of a 96-well microplate (Falcon 3912) at 100 ~1 per well, followed by standing at 4°C overnight. After washing each well with phosphate buffered saline (PBS, pH 7.41, 200 ~1 of PBS (pH 7.4) containing 0.5% of bovine serum albumin (BSA) were added, and the system was allowed to stand for 2 hours. The supernatant was then discharged. Next, after adding 50 ~1 of ArE standard solution dissolved in PBS containing 0.1% of BSA (pH 7.4) and 50 ~1 of the monoclonal antibody (1 pg/ml) produced by 7-2C5 cells, labelled with peroxidase according to the periodic acid cross-linking method [9], it was reacted at room temperature for 2 h. After washing each well with PBS (pH 7.41, 100 ~1 of 0.15 mol/l citrate buffer (pH 5.0) containing 0.015% of H,O, and 0.2 mg/ml of o-phenylene diamine were added. Five minutes later, 50 ~1 of 10% sulfuric acid were added to terminate the reaction, and enzyme activity was assayed using a two-wavelength (492 nm, 550 nm) microplate photometer (Corona MTP-22, Japan). Based on the results, the standard curve shown in Fig. 1 was obtained.
I 10
I
I
20
50
Aryresterase
I
loo
1
I
200
kg/ml)
Fig. 1. Standard curve by one-step sandwich ELBA for serum ArE. Serum ArE was determined by a combination of a mouse monoclonal antibody produced by 9-2C3 cells as the first antibody and a mouse monoclonal antibody produced by 7-2C5 cells, labelled with peroxidase, as the enzyme-labelled second antibody. The activity of peroxidase was assayed using a two-wavelength (492 nm, 550 nm) microplate photometer (Corona MTP-22, Japan).
222
Sera Serum was prepared from the blood of healthy adults (9 males and 11 females aged 28-49) and patients with cirrhosis (4 compensated and 2 decompensated, 4 males and 2 females aged 48-76). Samples were assayed within 3 days following storage at 4°C. Dete~ination
of ArE content
Serum was diluted with 0.05 mol/l carbonate buffer CpH 9.6) 500, 1,000 and 2,000 times, measured in the same manner as ELISA. ArE content was determined by conversion from the standard curve. Determination of ArE activity Serum ArE activity was measured at 37°C using substrate [III with an automatic analyzer (Hitachi Automatic Blood Chemistry Analyzer Model 705, Japan) [51. Results Standard ArE curve by one-step sandwich-ELBA The linearity of the assay for standard ArE by using a 96-well microplate was obtained from 18-144 ng/ml (0.9-72 ng/well) by combination of a monoclonal antibody of clone 9-2C3 as a solid phase and a pero~dase-labelled monoclonal antibody of clone 7-2C5 as a conjugate (Fig. 1). The detection limit, defined as the lowest concentration giving a peroxidase activity similar to that of the zero standard at 0.99 confidence, was 2 ng/ml (0.1 ng/well). When ChE (6.2 U/mg) from human plasma [31 was subjected to the above immunoassay system, there was no increase (up to 0.01 U/ml> in the peroxidase activity bound on the well (not shown), indicating there is no cross-reactivity of the present assay with human ChE. Estimation of ArE content Figure 2 shows ArE content in human serum. The values were 81 _t 25 for healthy adults and 37 + 7 mg/l for patients with cirrhosis, respectively, with significant differences (mean f SD, P < 0.001) (Table I). A marked decrease was noted in patients with cirrhosis. The 6 subjects with cirrhosis had ArE ranging from 26 to 44 mg/l. From the normal subjects, a 95% reference range of 31-131 was obtained. Co~e~at~o~ between ArE content and ArE activity Table I shows ArE specific activity in human serum. The values were 150 rt 43 for healthy adults and 167 + 36 U/mg for patients with cirrhosis, respectively, with
223
Norma
I
(!l:20>
Patients (n-6)
Fig. 2. Serum ArE content in healthy adults (01, and patients with compensated cirrhosis CD) or decompensated cirrhosis (0 ). The horizontal bars indicate mean values for each group.
non-significant differences (mean + SD, P > 0.05). A correlation between ArE in the serum as measured by ELISA (CArE, mg/l) and ArE activity in the serum measured using highly specific substrate [II] (A,,, kU/l) was found as shown in Fig. 3 and expressed as the A ArE= 5.74 log C,,
( IZ= 26, r2 = 0.43, P < 0.01).
TABLE I ArE specific activity (SA) estimate
Normal (20) Patients (6)
ArE activity (kU/I) (mean * SD)
ArE content (mg/l) (mean f SD)
ArE SA (U/mg) (mean f SD)
11.3k1.8 6.3 f 1.8 a
81k2.5 31* -I=
150 f 43 167+,36 b
a Difference from normal group statistically significant (P < 0.001). b Difference from normal group statistically non-significant (P > 0.05).
224
q
0
10
I
I
I
I
20
50
100
200
Arylesterase
by ELISA,
I
500
mg /I
Fig. 3. Correlation of serum ArE activity determined by substrate [II] (AArE, kU/O [.5] to ArE measured by ELBA CC,,,, mg/I) in healthy adults (e), and patients with compensated cirrhosis (m) or decompensated cirrhosis (0). The best fit correlation equation is: AArE = 5.74 log CArE (n = 26, r* = 0.43, P < 0.01).
A high positive correlation in the serum.
is thus shown between ArE protein content and activity
Discussion For the diagnosis of acute hepatitis, there are excellent biochemical parameters such as GOT and GPT, which can be easily measured and are widely used. However, only a few reliable biochemical parameters which indicate liver cirrhosis are presently available. ArE activity determined with phenyl acetate [lO,ll] or P-naphthyl acetate [12] as substrate, as well as ChE activity, has been reported to decrease in patients with liver disease, particularly cirrhosis. However, these substrates for the assay of ArE activity are hydrolyzed by ChE as well as ArE [2,31. Therefore these substrates are not suitable for the accurate determination of ArE activity owing to their lack of specificity. By using a new and highly specific substrate to ArE, substrate [I] [3,4], or substrate [II] [4,5], we recently showed serum ArE activity to decrease remarkably in patients with liver cirrhosis [3,5]. Using ArE activity measured by substrate [I] as an index, it was feasible to prepare antigen [3] and select hybridoma cells that form a monoclonal antibody specific to ArE [8]. We thus succeeded for the first time in developing ELISA adequate for the diagnosis of cirrhosis. In this ELISA study, we clearly showed the above decrease in ArE activity inthe serum of patients with cirrhosis, measured by substrate [II] [5], to result from reduction in ArE as an essential enzyme protein.
225
Acknowledgements The authors thank A. Sugita, A. Kono and T. Yamamoto for assistance. References 1 Augustinsson K-B. Electrophoretic separation and classification of blood plasma esterases. Nature 1958;181:1786-1789. 2 Augustinsson K-B, Ekedahl G. On the specificity of arylesterases. Acta Chem Stand 1962;16:240241. 3 Tanaka H, Kitamura M, Inoue Y. A simple assay for serum arylesterase using a specific substrate. Clin Chim Acta 1987;170:105-110. 4 Kawai H, Sakamoto F, Taguchi M, Kitamura M, Sotomura M, Tsukamoto G. 2-Oxo-1,3-dioxoles as specific substrates for measurement of arylesterase activity. Chem Pharm Bull 1991;39:1422-1425. 5 Kawai H, Sakamoto F, Inoue Y. Improved specific assay for serum arylesterase using a water-soluble substrate. Clin Chim Acta 1990;188:177-182. 6 Rosalki SB. Monoclonal antibodies to enzymes. Clin Chim Acta 1989;183:45-58. 7 Yelton DE, Diamond BA, Kwan S-P, Scharff MD. Fusion of mouse myeloma and spleen cells. Curr Top Microb Immunol 1978;81:1-7. 8 Yomoda S, Kawai H, Iwamoto S, Sotomura M. Monoclonal antibody. Jpn Kokai Tokkyo Koho JP 63,267,799 1988. 9 Nakane PK, Kawaoi A. Peroxidase-labeled antibody. A new method of conjugation. J Histochem Cytochem 1974;22:1084-1091. 10 Takahashi Y, Aoyama I, Ito F, Yamamura Y. Evaluation of phenyl acetate esterase activity as an index of liver damage. Clin Chim Acta 1967;18:21-32. 11 Lorentz K, Flatter B, Augustin E. Arylesterase in serum: elaboration and clinical application of a fixed-incubation method. Clin Chem 1979;25:1714-1720. 12 Burlina A, Michielin E, Galzigna L. Characteristics and behavior of arylesterase in human serum and liver. Europ J Clin Invest 1977;7:17-20.
