Journal of Clinical Laboratory Analysis 6:143-147 (1992)
Enzyme lmmunoassay and lmmunochemical Characterization of Pancreatic Stone Protein in Human Serum Tomoyuki Katsuzaki,' Noriyuki Tatemichi,' Chikako Takeichi,' Shinobu Hayakawa,' Tetsuo Hayakawa,' Tokimune Shibata,' Yasuyuki Nakae,* and Satoru Naruse3 'Department of Clinical Chemistry, Maruko Pharmaceutical Co., Ltd., Kasugai, Japan; 'The Second Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan; 3National Institute for Physiological Science, Okazaki, Japan Monoclonalantibodies were raised against human pancreatic stone protein (PSP) and used for one-step enzyme immunoassay (EIA). PSP-S2-5was employed as the standard in the assay. The assay's measurable range was 25-1,500 nglml and within run coefficient of variation was 3.7-6.4%.Analytical recovery of the assay was 101.5 ? Key words:
5.65% (X ? SD). The results of experiments in which serum was fractionated by Mono S (cationexchange chromatography) suggested that most of immunoreactive material in human serum is PSP-S2-5.The EIA offers simple, rapid, and specific analysis of serum PSP level for clinical diagnosis. 0 1992 Wiley-Liss,
Inc.
monoclonal antibody, pancreas, pancreatic thread protein, lithostathine, pancreatitis, pancreatic cancer, pancreatic juice
INTRODUCTION
Purification of PSP-S, and PSP-S2-5
De Car0 et al. reported the isolation of pancreatic stone protein (PSP) from human pancreatic stones (1). This protein may act as an inhibitor of spontaneous calcium carbonate precipitation in pancreatic juice (2). The four immunoreactive forms-PSP-S2-, (secretory forms)are synthesized in the acinar cell of the pancreas and secreted into the duodenum (3). PSP-S, is generated by the cleavage of the Arg-Ile bond in the N-terminal part of PSP-P2-5 (3). The fluorometric immunoassay and radioimmunoassay methods have recently been employed to the determination of immunoreactive PSP in serum and pancreatic juice (4-6). These methods used polyclonal antibodies and/or one monoclonal antibody. We prepared monoclonal antibodies to PSP and characterized them in terms of binding affinity to PSP-S, and PSP-S2_5and designed a one-step sandwich enzyme immunoassay (EIA). In this report, molecular forms of immunoreactive PSP were studied in sera from patients with pancreatic diseases.
PSP in each fraction from the column chromatography was detected by ELISA according to the method of Moriyama et al. (9). The assay procedure was described as follows: 0.1 ml of each fraction was put into the microplate. Then, the plate with 96 beads was placed onto the microplate and incubated for 18 h at 4°C. The plate was removed and placed in a trough containing 1% bovine serum albumin (BSA) in 150 mM NaCl-60 mM phosphate buffer (pH 7.2) (phosphate buffered saline, PBS). After incubation for 10 min at room temperature the plate was washed with 0.05% Tween-20 in PBS (T-PBS), and the plate was placed in the solution of monoclonal antibody (D4- 1-2 1, 1pg/ml in T-PBS) for 2 h at room temperature. Washed as above, the plate was placed in the peroxidase-conjugated rabbit anti-mouse immunoglobulin antiserum (Dako, Glostrup, Denmark) solution (0.1% in T-PBS) for 2 h at room temperature. The second antibody was washed off with T-PBS, and the plate was placed on a microplate containing 0.1 ml of 0.3% o-phenylenediamine dihydrochloride and 0.02% H202 solution in 0.1 M citratephosphate buffer (pH 6.5) and incubated for 30 min at room temperature. The reaction was stopped when the plate was removed. Absorbances were measured at 492 nm.
MATERIALS AND METHODS
Reagents Mono S (HR5/5), Q-Sepharose Fast Flow, and Sephacryl S-200HR were purchased from Pharmacia LKB Biotechnology, Ltd., Uppsala, Sweden. Specific monoclonal anti-PSP antibody D4-1-21 was obtained from Immunotech, Marseille, France (7,s). 0 1992 Wiley-Liss, Inc.
Received November 25, 1991; accepted December 30, 1991 . Address reprint requests to Dr. S . Hayakawa, Department of Clinical Chemistry, Maruko Pharmaceutical Co., Ltd., 1212 Teramae, Gejo-cho, Kasugai, 486, Japan.
144
Katsuzakiet al.
Purification of PSP-SI and PSP-S2-5 was carried out using an original method developed in our laboratory. Human pancreatic juice (25 ml) was dialyzed against 50 mM Tris-HC1, pH 7.8. After centrifugation (18,000 rpm, 20 min) the supernatant was applied on Q-Sepharose Fast flow (1.6 x 40 cm) washed with same buffer (100 ml) and then eluted with a linear gradient of 0- 1 .O M NaCl (total volume 500 ml). PSP-S I fractions were dialyzed against 50 mM sodium acetate buffer, pH 4.0, containing 0.5 M NaCl and were applied to a Superose 12 prep grade column (1.6 x 50 cm). PSP-SI was eluted from Superose 12 prep grade as a symmetrical peak. The purified PSP-SI was observed as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. This preparation was confirmed to have an identical N-terminal sequence of the 10 first amino acids reported by Montalto et al. (10) and used for immunization of BALB/c mice. PSP-S2-5 was purified from human pancreatic juice. The pancreatic juice (1 ml) was diluted to 10 times with 20 mM sodium acetate buffer containing 20 mM CaC12, pH 4.0. An aliquot of 10 ml was subjected to chromatography on a Mono S column (HR 5/5) equilibrated with the buffer described above. The column was washed with the buffer for 5 min at a flow rate of 1.0 mlimin and then eluted for 35 min with a linear gradient of 0-1.0 M NaCl. The peak fraction of PSP-S2-5 was rechromatographed on the same column. SDS-PAGE of purified PSP-S2p5demonstrated motilities similar to PSP-S2-5 as reported by Montalto et al. (10). The purified PSP-S2-5 was used for the standard in the EIA.
Preparationof MonoclonalAntibodies Monoclonal antibodies were prepared according to the method fo Kohler and Milstein (1 1). Spleen cells from BALB/c mice previously immunized with human PSP-S1 were fused to myeloma cell line X63-Ag8-6.5.3. The anti-PSP-S1 antibody producing hybridoma thus obtained was injected intraperitoneally into BALB/c mice, and their ascites was collected after 2 weeks. After fractionation with ammonium sulfate, the antibodies were purified by dialysis using 40 mM potassium phosphate buffer, pH 8.0, containing 30 mM NaCl and by being passed through a DEAE-cellulose column equilibrated with the buffer described above. The subclasses of monoclonal antibodies PS-1 and PS-2 used in the present study were IgGl.
Preparationof Monoclonal Antibody Bound Polystyrene Beads Polystyrene beads (1/4 inch; Nissho Corporation, Osaka, Japan) were immersed in 10% Scat 2OX-PF (detergent: Nakarai Chemicals, Ltd., Kyoto, Japan) and washed 15 times with purified water. Then, they were immersed in the monoclonal antibody PS-I solution (0.075 mg/ml) in 0.25 M sodium phosphate buffer, pH 7.5) and kept overnight at room tempera-
ture. After the antibody solution was removed by aspiration, the beads were blocked by 10 mM sodium phosphate buffer, pH 7.0, containing 0.1% BSA and stored at 4°C until used.
Preparationof Enzyme Labeled Antibody A complex of horseradish peroxidase and monoclonal antibody PS-2 was prepared according to the two step glutaraldehyde method of Avrameas and Guilbert (12), except that the final separation was carried out using Sephacryl S-200.
Buffers for EIA 1. Standard solution: PSP-S2p5was diluted to an appropriate concentration using 10 mM sodium phosphate buffer (pH 7.0) containing 0.1% BSA and 1 mM benzamidine. 2. Diluting solution for enzyme labeled antibody: 10 mM sodium phosphate buffer (pH 7.0) containing 0.5% gelatin, O.l%BSA, 16.7% horse serum, 0.01% Triton X-405, and 0.3 M sodium chloride. 3. Washing solution: 10 mM sodium phosphate buffer (pH 7.4) containing 0.03% Triton X-405, 0.05% Tween 80, and 0.9% sodium chloride. 4. Substrate solution: 0.1 M phosphate-citrate buffer (pH 6.5) containing 0.3% o-phenylenediamine dihydrochloride and 0.02% H202.
EIA Technique for PSP For the measurement of immunoreactive PSP in the sample, one-step EIA in which two kinds of monoclonal antibodies were combined was used. The assay method is described below. Twenty microliters of a sample or standard solution was added to a test tube (inner diameter: 10 X 75 mm) and then 300 pl of labeled antibody solution (diluted 1 :100 with the diluting solution) was added. After the solution was mixed well, an antibody bound polystyrene bead was added and the mixture was incubated at 37°C for 30 min. After the solution was removed by aspiration, the bead was washed twice with the washing solution. Then, it was transferred to a new test tube and 0.5 ml of substrate solution was added. After the mixture was allowed to stand for 15 min at room temperature, the reaction was stopped by adding 2.0 ml of 1.6 N H2S04, and the absorption at 492 nm was measured. The measurement was carried out in duplicate.
High Performance Liquid Chromatography (HPLC) Technique Subjects. Eighteen patients admitted to the Nagoya University Hospital have been studied. In all cases the diagnosis of pancreatic diseases was made on clinical grounds (biochemical, pancreozymin secretin test, endoscopic retrograde cholangiopancreatography). Serum PSP levels were determined by the proposed method. The normal range of serum PSP was 75.7 ? 56.4 nglml (mean ? 2 SD, n = lo). The
Enzyme lmmunoassay for PSP
mean values of serum PSP levels in pancreatic cancer, acute pancreatitis, and chronic pancreatitis were 8 17.2 ng/ml (range: 127-2,340 ng/ml, n = 6), 1,443.1 ng/ml (range: 304-3,250 ng/ml, n = 6), and 280.3 ng/ml (range: 84-731 nglml, n = 6), respectively. In this study, sera containing high level of PSP were used for the analysis on HPLC. HPLC system. The HPLC system included a 2249 LC gradient pump, injector valve V7, and a 2510 Uvicord SD detector (detection wavelength: 280 nm). All were purchased from Pharmacia LKB Biotechnology Co., Uppsala, Sweden. 1. Pancreatic juice: Human pancreatic juice was diluted 1:lO with 20 mM sodium acetate buffer (pH 4.0) containing 20 mM CaC12. An aliquot of 2.0 ml was applied to a Mono S (HR5/5)column equilibrated with the buffer described above. Then the column was washed with the buffer for 5 min at a flow rate of 1.0 ml/min, and then eluted for 35 min with a linear gradient of 0- 1.O M NaCl. 2. Serum: Human serum was diluted 1:20 with 20 mM sodium succinate buffer (pH 3.8). An aliquot of 1 .O ml was applied to a Mono S (HR5/5) column equilibrated with the buffer described above. Then the column was washed with the buffer for 5 min at a flow rate of 1.0 mumin, and then eluted for 30 min with a linear gradient of 0-1 .O M NaCl.
RESULTS Reactivity of EIA System The clones were selected according to the affinities of the monoclonal antibodies. The antibodies PS-1 and PS-2 had high affinity to both PSP-S, and PSP-2-5 molecules. The standard curves for purified PSP-SI and PSP-2-5 are shown in Figure 1. The EIA system showed similar reactivities toward PSP-S1 and PSP-S2-5. In this study, PSP-S2-5 was used as a standard. The detection limit of the assay, taken as 3 SD above the mean absorbance of the zero standard, was 25 nglml. High-
145
TABLE 1. Analytical Recovery of Standard Added to Ten Different Serum Samples, as Measured by the Proposed Method Standard Sample no. 1
2 3 4 5 6 1
8 9 10
Endogenous (ngW
Added (ngW
Recovery"
43.4 46.8 118.8 191.8 306.0 51.1 82.9 86.2 94.3 111.0
80 80 80 80 80 600 600 600 600 600
91.4 100.0 108.6 100.0 102.I 100.6 98.6 99.4 112.3 101.7
aAveragerecovery%(&SD)
=
(%)
101.5 2 5.65.
dose hook effect was not observed in the range tested up to 100 pg/ml of PSP.
Precisionand Analytical Recovery Assay precision was studied at three concentrations of serum for every assay. Within-run CVs (n = 10) were between 3.7% and 6.4% for sera containing PSP at 92.2, 134.6, and 466.9 ng/ml. CVs of the between-run assay (n = 10)ranged between 3.7% and 6.6% for PSP (81.5, 115.0, 185.2, and 268.1 ng/ml) assay. PSP was added to normal human serum and quantified. Analytical recovery of added PSP was essentially quantitative (Table I ) .
Linearity The relationship between PSP concentration and sample volume is shown in Figure 2. The four specimens studied were from patients with chronic pancreatitis and acute pancreatitis. We diluted a serum sample with the zero standard of EIA. Good linearity was observed.
300
100
0
PSP (ng/ml)
Fig. 1. Standard curve for immunoreactive PSP with proposed method. Open circles and broken line indicate standard curve for PSP-S Closed circles and solid line indicate standard curve for PSP-Sz_s.
,.
0
115
215
3/5
415
Dilution
Fig. 2. Dilution study with four different sera.
5/5
Katsuzaki et at.
146
Analysis of PSP in Human Serum by Mono S
I
500
a
2
-
300
.-9
2p 2
-iE
200
2.0
I
E 0 a , 01
100
4-4
m
0
0
0
0
10
20
30
40
0.0
50
Fraction No. (1.O rnl/tube)
Fig. 3. Cation exchange (Mono S) chromatography of human pancreatic juice. Closed circles and broken line indicate amounts of immunoreactive PSP. Solid line indicates OD at 280 nm.
Analysis of PSP in Human Pancreatic Juice by Mono S
DISCUSSION
When human pancreatic juice was diluted and subjected to Mono S cation-exchangechromatography,PSP of each fraction was measured by the EIA. A single peak of PSP-S2-5 was observed (Fig. 3). Similar results were obtained with two other human pancreatic juices.
Fraction No. (0.5 rnl/tube)
n .O Fraction
Cation-exchange chromatography of patient sera on Mono S (HR 5/5), followed by the EIA of column fractions, was performed to characterize the molecular form of PSP. An elution pattern for PSP of serum from a pancreatic cancer patient (serum PSP: 2,340 ng/ml) is shown in Figure 4A. Two peaks of immunoreactive PSP were observed. The major peak of immunoreactive PSP was eluted in a position corresponding to that of PSP-S2_5.The minor peak of immunoreactive material was eluted at a position consistent with that of PSP-SI. An elution pattern for PSP of serum from an acute pancreatitis patient (serum PSP: 1,731 ngiml) is shown in Figure 4B. A single peak of PSP- S2-5 was observed. Similar results were obtained in serum from one patient each with chronic pancreatitis (serum PSP: 73 1 ng/ml), pancreatic cancer (serum PSP: 1,836 ng/ml), and acute pancreatitis (serum PSP: 3,250 ng/ml).
0
No. (0.5 ml/tube)
Fig. 4. Cation exchange (Mono S) chromatography of human serum. A: Pancreatic cancer (PSP value: 2,340 ngiml). B: Acute pancreatitis (PSP value: 1,731 ng/ml). Closed circles and broken line indicate amounts of imrnunoreactive PSP. Solid line indicates OD at 280 nm.
We developed the EIA for serum PSP and the EIA system showed the same reactivities toward PSP-S1 and PSP-S2-5. The assay can be completed within 1 h by mixing the sample, peroxidase-IgG conjugate, and IgG coated bead together in a one-step incubation. The EIA provided a detection limit sufficient for serum determination and excellent analytical recovery and precision. The high-dose hook effect did not occur at the high concentration below 100 pgiml of PSP and this gave no problem in the assays for PSP in serum. When non-activated human pancreatic juice was analyzed by Mono S cation-exchange chromatography, only one peak of immunoreactive PSP was detected. This peak was eluted at the position corresponding to PSP-S2-5. Therefore, the EIA has high specificity for PSP and offers simple, rapid, and specific analysis of serum PSP level for clinical diagnosis. In sera from a chronic pancreatitis patient and an acute pancreatitis patient, the immunoreactivity consists of only PSP-S2-5, but in serum from a patient with pancreatic cancer, an immunoreactive fraction appears at the position corresponding to that of PSP-SI in addition to the PSP-S2-5 fraction. The PSP-SI may represent that PSP-S2-5 was cleaved by proteinase. Therefore, the finding of PSP-SI in serum reflects the activation of proteinase in the pancreatic juice or pancreas. The mechanism of production of PSP-SI is not clear at present, and requires further study. Differential measurement of PSP-S2-5 and PSP-S ] may offer pathophysiological information; that is, elevation of PSP-S1 in serum suggests activation of proproteinases in the pancreaticjuice or pancreas.
AC KNOWLEDGMENTS This study was supported by the grant of the Intractable Pancreatic Disease Research Committee from the Health and Welfare Ministry of Japan.
Enzyme lmmunoassay for PSP
REFERENCES 1 . De Caro A, Lohse J , Sarles H: Characterization of a protein isolated
from pancreatic calculi of men suffering from chronic calcifying pancreatitis. Biochem Biophys Res Commun 87:1 176- 1 182, 1979. 2. Multigner L, De Caro A, Lombard0 D, Campese D, Sarles H: Pancreatic stone protein, a phosphoprotein which inhibits calcium carbonate precipitation from human pancreatic juice. Biochem Biophys Res Commun 110:69-74,1983. 3. De Caro A, Multigner L, Dagorn JC, Sarles H: The human pancreatic stone protein. Biochimie 70:1209-1214, 1988. 4. Schmiegel WH, Burchert M, Kalthoff H, Thiele HG, Biitzow HG, Klose G, Greten H: Pancreatic stone protein in serum of patients with pancreatitis. Lancer 2:686-687, 1986. 5. Provansal-Cheylan M, Mariani A, Bernard JP, Sarles H, Dupuy P: Pancreatic stone protein: Quantification in pancreaticjuice by enzyme-linked immunosorbent assay and comparison with other methods. Pancreas 4:680-689, 1989. 6. Schmiegel W,Burchert M, Kalthoff H, Roeder C, Butzow G, Grimm H, Kremer B, Soehendra N, Schreiber HW, Thiele HG. Greten H: Immunochemical characterization and quantitative distribution of pancreatic
147
stone protein in sera and pancreatic secretions in pancreatic disorders. Gastroenterology 99:1421-1430,1990. 7. Montalto G, Luscher M, De Caro A, Multigner L, Sarles H , Delaage M: Analyse au moyen d’un anticorps monoclonal des composants du suc pancriatique humain apparente i la protiine des calculs pancreatiques. C RAcadSci 300:189-202,1985. 8. Provansal-Cheylan M, Lusher M, De Car0 A, Multigner L, Montalto G, Sarles H, Delaage M: Monoclonal antibodies to pancreatic stone protein. Radioimmunoassay and immunological comparison with trypsin 1. Biochimie68:1109-lll3,1986. 9. Moriyama T,Yamadera K, Takabe T, Makino I , Kato H , Nobuoka M, Makino M: Purification of the pancreatic stone protein by highperformance liquidchromatography. JChromarogr 493:164- 169,1989. 10. Montalto G , Bonicel J , Multigner L, Rovery M, Sarles H, De Caro A: Partial amino acid sequence of human pancreatic stone protein, a novel pancreatic secretory protein. Biochem J 238:227-232, 1986. 1 I . Kohler G, Milstein C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495-497,1975. 12. Avrameas S, Guilbert B: Enzyme-immunoassay for the measurement of antigens using peroxidase conjugates. Biochimie 54:837-842, 1972.
Enzyme lmmunoassay and lmmunochemical Characterization of Pancreatic Stone Protein in Human Serum Tomoyuki Katsuzaki,' Noriyuki Tatemichi,' Chikako Takeichi,' Shinobu Hayakawa,' Tetsuo Hayakawa,' Tokimune Shibata,' Yasuyuki Nakae,* and Satoru Naruse3 'Department of Clinical Chemistry, Maruko Pharmaceutical Co., Ltd., Kasugai, Japan; 'The Second Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan; 3National Institute for Physiological Science, Okazaki, Japan Monoclonalantibodies were raised against human pancreatic stone protein (PSP) and used for one-step enzyme immunoassay (EIA). PSP-S2-5was employed as the standard in the assay. The assay's measurable range was 25-1,500 nglml and within run coefficient of variation was 3.7-6.4%.Analytical recovery of the assay was 101.5 ? Key words:
5.65% (X ? SD). The results of experiments in which serum was fractionated by Mono S (cationexchange chromatography) suggested that most of immunoreactive material in human serum is PSP-S2-5.The EIA offers simple, rapid, and specific analysis of serum PSP level for clinical diagnosis. 0 1992 Wiley-Liss,
Inc.
monoclonal antibody, pancreas, pancreatic thread protein, lithostathine, pancreatitis, pancreatic cancer, pancreatic juice
INTRODUCTION
Purification of PSP-S, and PSP-S2-5
De Car0 et al. reported the isolation of pancreatic stone protein (PSP) from human pancreatic stones (1). This protein may act as an inhibitor of spontaneous calcium carbonate precipitation in pancreatic juice (2). The four immunoreactive forms-PSP-S2-, (secretory forms)are synthesized in the acinar cell of the pancreas and secreted into the duodenum (3). PSP-S, is generated by the cleavage of the Arg-Ile bond in the N-terminal part of PSP-P2-5 (3). The fluorometric immunoassay and radioimmunoassay methods have recently been employed to the determination of immunoreactive PSP in serum and pancreatic juice (4-6). These methods used polyclonal antibodies and/or one monoclonal antibody. We prepared monoclonal antibodies to PSP and characterized them in terms of binding affinity to PSP-S, and PSP-S2_5and designed a one-step sandwich enzyme immunoassay (EIA). In this report, molecular forms of immunoreactive PSP were studied in sera from patients with pancreatic diseases.
PSP in each fraction from the column chromatography was detected by ELISA according to the method of Moriyama et al. (9). The assay procedure was described as follows: 0.1 ml of each fraction was put into the microplate. Then, the plate with 96 beads was placed onto the microplate and incubated for 18 h at 4°C. The plate was removed and placed in a trough containing 1% bovine serum albumin (BSA) in 150 mM NaCl-60 mM phosphate buffer (pH 7.2) (phosphate buffered saline, PBS). After incubation for 10 min at room temperature the plate was washed with 0.05% Tween-20 in PBS (T-PBS), and the plate was placed in the solution of monoclonal antibody (D4- 1-2 1, 1pg/ml in T-PBS) for 2 h at room temperature. Washed as above, the plate was placed in the peroxidase-conjugated rabbit anti-mouse immunoglobulin antiserum (Dako, Glostrup, Denmark) solution (0.1% in T-PBS) for 2 h at room temperature. The second antibody was washed off with T-PBS, and the plate was placed on a microplate containing 0.1 ml of 0.3% o-phenylenediamine dihydrochloride and 0.02% H202 solution in 0.1 M citratephosphate buffer (pH 6.5) and incubated for 30 min at room temperature. The reaction was stopped when the plate was removed. Absorbances were measured at 492 nm.
MATERIALS AND METHODS
Reagents Mono S (HR5/5), Q-Sepharose Fast Flow, and Sephacryl S-200HR were purchased from Pharmacia LKB Biotechnology, Ltd., Uppsala, Sweden. Specific monoclonal anti-PSP antibody D4-1-21 was obtained from Immunotech, Marseille, France (7,s). 0 1992 Wiley-Liss, Inc.
Received November 25, 1991; accepted December 30, 1991 . Address reprint requests to Dr. S . Hayakawa, Department of Clinical Chemistry, Maruko Pharmaceutical Co., Ltd., 1212 Teramae, Gejo-cho, Kasugai, 486, Japan.
144
Katsuzakiet al.
Purification of PSP-SI and PSP-S2-5 was carried out using an original method developed in our laboratory. Human pancreatic juice (25 ml) was dialyzed against 50 mM Tris-HC1, pH 7.8. After centrifugation (18,000 rpm, 20 min) the supernatant was applied on Q-Sepharose Fast flow (1.6 x 40 cm) washed with same buffer (100 ml) and then eluted with a linear gradient of 0- 1 .O M NaCl (total volume 500 ml). PSP-S I fractions were dialyzed against 50 mM sodium acetate buffer, pH 4.0, containing 0.5 M NaCl and were applied to a Superose 12 prep grade column (1.6 x 50 cm). PSP-SI was eluted from Superose 12 prep grade as a symmetrical peak. The purified PSP-SI was observed as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. This preparation was confirmed to have an identical N-terminal sequence of the 10 first amino acids reported by Montalto et al. (10) and used for immunization of BALB/c mice. PSP-S2-5 was purified from human pancreatic juice. The pancreatic juice (1 ml) was diluted to 10 times with 20 mM sodium acetate buffer containing 20 mM CaC12, pH 4.0. An aliquot of 10 ml was subjected to chromatography on a Mono S column (HR 5/5) equilibrated with the buffer described above. The column was washed with the buffer for 5 min at a flow rate of 1.0 mlimin and then eluted for 35 min with a linear gradient of 0-1.0 M NaCl. The peak fraction of PSP-S2-5 was rechromatographed on the same column. SDS-PAGE of purified PSP-S2p5demonstrated motilities similar to PSP-S2-5 as reported by Montalto et al. (10). The purified PSP-S2-5 was used for the standard in the EIA.
Preparationof MonoclonalAntibodies Monoclonal antibodies were prepared according to the method fo Kohler and Milstein (1 1). Spleen cells from BALB/c mice previously immunized with human PSP-S1 were fused to myeloma cell line X63-Ag8-6.5.3. The anti-PSP-S1 antibody producing hybridoma thus obtained was injected intraperitoneally into BALB/c mice, and their ascites was collected after 2 weeks. After fractionation with ammonium sulfate, the antibodies were purified by dialysis using 40 mM potassium phosphate buffer, pH 8.0, containing 30 mM NaCl and by being passed through a DEAE-cellulose column equilibrated with the buffer described above. The subclasses of monoclonal antibodies PS-1 and PS-2 used in the present study were IgGl.
Preparationof Monoclonal Antibody Bound Polystyrene Beads Polystyrene beads (1/4 inch; Nissho Corporation, Osaka, Japan) were immersed in 10% Scat 2OX-PF (detergent: Nakarai Chemicals, Ltd., Kyoto, Japan) and washed 15 times with purified water. Then, they were immersed in the monoclonal antibody PS-I solution (0.075 mg/ml) in 0.25 M sodium phosphate buffer, pH 7.5) and kept overnight at room tempera-
ture. After the antibody solution was removed by aspiration, the beads were blocked by 10 mM sodium phosphate buffer, pH 7.0, containing 0.1% BSA and stored at 4°C until used.
Preparationof Enzyme Labeled Antibody A complex of horseradish peroxidase and monoclonal antibody PS-2 was prepared according to the two step glutaraldehyde method of Avrameas and Guilbert (12), except that the final separation was carried out using Sephacryl S-200.
Buffers for EIA 1. Standard solution: PSP-S2p5was diluted to an appropriate concentration using 10 mM sodium phosphate buffer (pH 7.0) containing 0.1% BSA and 1 mM benzamidine. 2. Diluting solution for enzyme labeled antibody: 10 mM sodium phosphate buffer (pH 7.0) containing 0.5% gelatin, O.l%BSA, 16.7% horse serum, 0.01% Triton X-405, and 0.3 M sodium chloride. 3. Washing solution: 10 mM sodium phosphate buffer (pH 7.4) containing 0.03% Triton X-405, 0.05% Tween 80, and 0.9% sodium chloride. 4. Substrate solution: 0.1 M phosphate-citrate buffer (pH 6.5) containing 0.3% o-phenylenediamine dihydrochloride and 0.02% H202.
EIA Technique for PSP For the measurement of immunoreactive PSP in the sample, one-step EIA in which two kinds of monoclonal antibodies were combined was used. The assay method is described below. Twenty microliters of a sample or standard solution was added to a test tube (inner diameter: 10 X 75 mm) and then 300 pl of labeled antibody solution (diluted 1 :100 with the diluting solution) was added. After the solution was mixed well, an antibody bound polystyrene bead was added and the mixture was incubated at 37°C for 30 min. After the solution was removed by aspiration, the bead was washed twice with the washing solution. Then, it was transferred to a new test tube and 0.5 ml of substrate solution was added. After the mixture was allowed to stand for 15 min at room temperature, the reaction was stopped by adding 2.0 ml of 1.6 N H2S04, and the absorption at 492 nm was measured. The measurement was carried out in duplicate.
High Performance Liquid Chromatography (HPLC) Technique Subjects. Eighteen patients admitted to the Nagoya University Hospital have been studied. In all cases the diagnosis of pancreatic diseases was made on clinical grounds (biochemical, pancreozymin secretin test, endoscopic retrograde cholangiopancreatography). Serum PSP levels were determined by the proposed method. The normal range of serum PSP was 75.7 ? 56.4 nglml (mean ? 2 SD, n = lo). The
Enzyme lmmunoassay for PSP
mean values of serum PSP levels in pancreatic cancer, acute pancreatitis, and chronic pancreatitis were 8 17.2 ng/ml (range: 127-2,340 ng/ml, n = 6), 1,443.1 ng/ml (range: 304-3,250 ng/ml, n = 6), and 280.3 ng/ml (range: 84-731 nglml, n = 6), respectively. In this study, sera containing high level of PSP were used for the analysis on HPLC. HPLC system. The HPLC system included a 2249 LC gradient pump, injector valve V7, and a 2510 Uvicord SD detector (detection wavelength: 280 nm). All were purchased from Pharmacia LKB Biotechnology Co., Uppsala, Sweden. 1. Pancreatic juice: Human pancreatic juice was diluted 1:lO with 20 mM sodium acetate buffer (pH 4.0) containing 20 mM CaC12. An aliquot of 2.0 ml was applied to a Mono S (HR5/5)column equilibrated with the buffer described above. Then the column was washed with the buffer for 5 min at a flow rate of 1.0 ml/min, and then eluted for 35 min with a linear gradient of 0- 1.O M NaCl. 2. Serum: Human serum was diluted 1:20 with 20 mM sodium succinate buffer (pH 3.8). An aliquot of 1 .O ml was applied to a Mono S (HR5/5) column equilibrated with the buffer described above. Then the column was washed with the buffer for 5 min at a flow rate of 1.0 mumin, and then eluted for 30 min with a linear gradient of 0-1 .O M NaCl.
RESULTS Reactivity of EIA System The clones were selected according to the affinities of the monoclonal antibodies. The antibodies PS-1 and PS-2 had high affinity to both PSP-S, and PSP-2-5 molecules. The standard curves for purified PSP-SI and PSP-2-5 are shown in Figure 1. The EIA system showed similar reactivities toward PSP-S1 and PSP-S2-5. In this study, PSP-S2-5 was used as a standard. The detection limit of the assay, taken as 3 SD above the mean absorbance of the zero standard, was 25 nglml. High-
145
TABLE 1. Analytical Recovery of Standard Added to Ten Different Serum Samples, as Measured by the Proposed Method Standard Sample no. 1
2 3 4 5 6 1
8 9 10
Endogenous (ngW
Added (ngW
Recovery"
43.4 46.8 118.8 191.8 306.0 51.1 82.9 86.2 94.3 111.0
80 80 80 80 80 600 600 600 600 600
91.4 100.0 108.6 100.0 102.I 100.6 98.6 99.4 112.3 101.7
aAveragerecovery%(&SD)
=
(%)
101.5 2 5.65.
dose hook effect was not observed in the range tested up to 100 pg/ml of PSP.
Precisionand Analytical Recovery Assay precision was studied at three concentrations of serum for every assay. Within-run CVs (n = 10) were between 3.7% and 6.4% for sera containing PSP at 92.2, 134.6, and 466.9 ng/ml. CVs of the between-run assay (n = 10)ranged between 3.7% and 6.6% for PSP (81.5, 115.0, 185.2, and 268.1 ng/ml) assay. PSP was added to normal human serum and quantified. Analytical recovery of added PSP was essentially quantitative (Table I ) .
Linearity The relationship between PSP concentration and sample volume is shown in Figure 2. The four specimens studied were from patients with chronic pancreatitis and acute pancreatitis. We diluted a serum sample with the zero standard of EIA. Good linearity was observed.
300
100
0
PSP (ng/ml)
Fig. 1. Standard curve for immunoreactive PSP with proposed method. Open circles and broken line indicate standard curve for PSP-S Closed circles and solid line indicate standard curve for PSP-Sz_s.
,.
0
115
215
3/5
415
Dilution
Fig. 2. Dilution study with four different sera.
5/5
Katsuzaki et at.
146
Analysis of PSP in Human Serum by Mono S
I
500
a
2
-
300
.-9
2p 2
-iE
200
2.0
I
E 0 a , 01
100
4-4
m
0
0
0
0
10
20
30
40
0.0
50
Fraction No. (1.O rnl/tube)
Fig. 3. Cation exchange (Mono S) chromatography of human pancreatic juice. Closed circles and broken line indicate amounts of immunoreactive PSP. Solid line indicates OD at 280 nm.
Analysis of PSP in Human Pancreatic Juice by Mono S
DISCUSSION
When human pancreatic juice was diluted and subjected to Mono S cation-exchangechromatography,PSP of each fraction was measured by the EIA. A single peak of PSP-S2-5 was observed (Fig. 3). Similar results were obtained with two other human pancreatic juices.
Fraction No. (0.5 rnl/tube)
n .O Fraction
Cation-exchange chromatography of patient sera on Mono S (HR 5/5), followed by the EIA of column fractions, was performed to characterize the molecular form of PSP. An elution pattern for PSP of serum from a pancreatic cancer patient (serum PSP: 2,340 ng/ml) is shown in Figure 4A. Two peaks of immunoreactive PSP were observed. The major peak of immunoreactive PSP was eluted in a position corresponding to that of PSP-S2_5.The minor peak of immunoreactive material was eluted at a position consistent with that of PSP-SI. An elution pattern for PSP of serum from an acute pancreatitis patient (serum PSP: 1,731 ngiml) is shown in Figure 4B. A single peak of PSP- S2-5 was observed. Similar results were obtained in serum from one patient each with chronic pancreatitis (serum PSP: 73 1 ng/ml), pancreatic cancer (serum PSP: 1,836 ng/ml), and acute pancreatitis (serum PSP: 3,250 ng/ml).
0
No. (0.5 ml/tube)
Fig. 4. Cation exchange (Mono S) chromatography of human serum. A: Pancreatic cancer (PSP value: 2,340 ngiml). B: Acute pancreatitis (PSP value: 1,731 ng/ml). Closed circles and broken line indicate amounts of imrnunoreactive PSP. Solid line indicates OD at 280 nm.
We developed the EIA for serum PSP and the EIA system showed the same reactivities toward PSP-S1 and PSP-S2-5. The assay can be completed within 1 h by mixing the sample, peroxidase-IgG conjugate, and IgG coated bead together in a one-step incubation. The EIA provided a detection limit sufficient for serum determination and excellent analytical recovery and precision. The high-dose hook effect did not occur at the high concentration below 100 pgiml of PSP and this gave no problem in the assays for PSP in serum. When non-activated human pancreatic juice was analyzed by Mono S cation-exchange chromatography, only one peak of immunoreactive PSP was detected. This peak was eluted at the position corresponding to PSP-S2-5. Therefore, the EIA has high specificity for PSP and offers simple, rapid, and specific analysis of serum PSP level for clinical diagnosis. In sera from a chronic pancreatitis patient and an acute pancreatitis patient, the immunoreactivity consists of only PSP-S2-5, but in serum from a patient with pancreatic cancer, an immunoreactive fraction appears at the position corresponding to that of PSP-SI in addition to the PSP-S2-5 fraction. The PSP-SI may represent that PSP-S2-5 was cleaved by proteinase. Therefore, the finding of PSP-SI in serum reflects the activation of proteinase in the pancreatic juice or pancreas. The mechanism of production of PSP-SI is not clear at present, and requires further study. Differential measurement of PSP-S2-5 and PSP-S ] may offer pathophysiological information; that is, elevation of PSP-S1 in serum suggests activation of proproteinases in the pancreaticjuice or pancreas.
AC KNOWLEDGMENTS This study was supported by the grant of the Intractable Pancreatic Disease Research Committee from the Health and Welfare Ministry of Japan.
Enzyme lmmunoassay for PSP
REFERENCES 1 . De Caro A, Lohse J , Sarles H: Characterization of a protein isolated
from pancreatic calculi of men suffering from chronic calcifying pancreatitis. Biochem Biophys Res Commun 87:1 176- 1 182, 1979. 2. Multigner L, De Caro A, Lombard0 D, Campese D, Sarles H: Pancreatic stone protein, a phosphoprotein which inhibits calcium carbonate precipitation from human pancreatic juice. Biochem Biophys Res Commun 110:69-74,1983. 3. De Caro A, Multigner L, Dagorn JC, Sarles H: The human pancreatic stone protein. Biochimie 70:1209-1214, 1988. 4. Schmiegel WH, Burchert M, Kalthoff H, Thiele HG, Biitzow HG, Klose G, Greten H: Pancreatic stone protein in serum of patients with pancreatitis. Lancer 2:686-687, 1986. 5. Provansal-Cheylan M, Mariani A, Bernard JP, Sarles H, Dupuy P: Pancreatic stone protein: Quantification in pancreaticjuice by enzyme-linked immunosorbent assay and comparison with other methods. Pancreas 4:680-689, 1989. 6. Schmiegel W,Burchert M, Kalthoff H, Roeder C, Butzow G, Grimm H, Kremer B, Soehendra N, Schreiber HW, Thiele HG. Greten H: Immunochemical characterization and quantitative distribution of pancreatic
147
stone protein in sera and pancreatic secretions in pancreatic disorders. Gastroenterology 99:1421-1430,1990. 7. Montalto G, Luscher M, De Caro A, Multigner L, Sarles H , Delaage M: Analyse au moyen d’un anticorps monoclonal des composants du suc pancriatique humain apparente i la protiine des calculs pancreatiques. C RAcadSci 300:189-202,1985. 8. Provansal-Cheylan M, Lusher M, De Car0 A, Multigner L, Montalto G, Sarles H, Delaage M: Monoclonal antibodies to pancreatic stone protein. Radioimmunoassay and immunological comparison with trypsin 1. Biochimie68:1109-lll3,1986. 9. Moriyama T,Yamadera K, Takabe T, Makino I , Kato H , Nobuoka M, Makino M: Purification of the pancreatic stone protein by highperformance liquidchromatography. JChromarogr 493:164- 169,1989. 10. Montalto G , Bonicel J , Multigner L, Rovery M, Sarles H, De Caro A: Partial amino acid sequence of human pancreatic stone protein, a novel pancreatic secretory protein. Biochem J 238:227-232, 1986. 1 I . Kohler G, Milstein C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495-497,1975. 12. Avrameas S, Guilbert B: Enzyme-immunoassay for the measurement of antigens using peroxidase conjugates. Biochimie 54:837-842, 1972.
