Clin Biochem, Vol. 23, pp. 149-153, 1990 Printed in Canada. All rights reserved.
Copyright
¢
0009-9120/90 $3.00 + .00 1990 The Canadian Society of Clinical Chemists.
Enzyme Immunoassay for Human Pancreatic Amylase TAKASHI FUJISAWA, TAKAHIKO NAKAMURA, MASATOSHI FUJII, SATOSHI TANI, YOSHINORI OKABAYASHI, and MAKOTO OTSUKI Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan An enzyme immunoassay (EIA) for human pancreatic amylase has been developed for the detection of human serum amylase content. Our monoclonal antibody is highly specific for human pancreatic amylase; it cross reacted negligibly with the salivary isoenzyme. We developed a solid phase enzyme immunoassay for determination of pancreatic amylase in human serum with this antibody. The assay required 20 p,L of serum and the standard curve was linear to at least 1000 ng/mL of pancreatic amylase. Inter- and intra-assay CVs were less than 10%. The results obtained by the EIA correlated well with those determined by the conventional electrophoretic method. In normal subjects, the mean concentration of serum pancreatic amylase determined by the EIA was found to be 92.3 -- 26,1 ng/mL (mean __. SD). The EIA we describe is useful for directly determining pancreatic amylase in human serum. Specifically distinguishing pancreatic from salivary amylase may have considerable clinical value.
ificity. Recently, the specific recognition of the salivary amylase isoenzyme by monoclonal antibodies has been reported (9-14). Measurement of amylase activity in the supernatant fluids from sera treated with these antibodies provides an assay of pancreatic amylase. In contrast to these indirect methods, here we describe a direct enzyme immunoassay (EIA) for human P-amylase using a monoclonal antibody that specifically binds to the P-amylase without any detectable cross reaction against the S-amylase.
KEY WORDS: amylase isoenzyme; pancreatic amylase; salivary amylase; enzyme immunoassay; monoclonal antibody; acute pancreatitis.
PURIFICATION OF HUMANP-AMYLASE AND PREPARATION OF THE MONOCLONAL ANTIBODY
Introduction
a-amylase (EC 3.2.1.1.) in human serum and urine can be separated into two isoenzymes; the salivary (S) and the pancreatic amylase (P). Several methods for differential assay of amylases have been developed based on electrophoresis (1,2) and amylase inhibitor from wheat germ (3). Electrophoresis is a sensitive method but is time consuming and technically complex. The amylase inhibitor method is easy, but the inhibitor does not totally differentiate the isoenzymes, S- and P-amylase being inhibited by approximately 80-85% and 10-15%, respectively (4). Consequently, a specific, rapid and simple method is required for precise quantitative measurement of P-amylase in serum and urine. Several immunochemical methods for separation of amylase isoenzymes involving polyclonal antisera have been reported (5-8). However, the antibodies for P-amylase used in these previous studies crossreacted with S-amylase, and thus they lacked spec-
Correspondence: Dr. Makoto Otsuki, Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan. Manuscript received January 5, 1989; revised April 10, 1989; accepted April 15, 1989. CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990
Materials a n d M e t h o d s
Human P-amylase was purified from human pancreatic juice by the method of Matsuura et al. (15). The P-amylase preparation was free of deamination products, because no anodically migrating bands were visible by poly-acrylamide gel electrophoresis. The monoclonal antibody for human P-amylase was prepared as follows. Female Balb/C mice were immunized with 100 Ixg of human P-amylase in complete Freund's adjuvant. These immunized mice were killed and their spleen cells removed for fusion with the myeloma cell line P3-Ag8-U1 according to the methods described by Kohler and Milstein (16), and Hales (17). Primary cultures were screened for antibody production and cross reactivity by an enzyme linked immunoassay method. Cell clones that produced antibodies showing a selective binding to human P-amylase were further cultured. The monoclonal antibody was purified by passing through a protein A column (18). ENZYME
I M M U N O A S S A Y FOR H U M A N
P-AMYLASE
Using the aforementioned monoclonal antibody, a solid-phase EIA for human P-amylase was developed based on the two-site assay principle. Wells of microtiter plates were coated with anti-human Pamylase monoclonal antibody. 20 ~L of P-amylase standards (0-1000 ~g/mL) or samples and 200 ~L of phosphate buffer were placed in the wells and mixed 149
FUJISAWA, NAKAMURA, FUJII, ET AL.
2,0
--
~.
= Standard curve
;~
Serum~Dilution curve ?.: U r i n e ~
6.
~ Salivary amylase
E 1.5t-
O .0
10~
tO -Q
Copyright
¢
0009-9120/90 $3.00 + .00 1990 The Canadian Society of Clinical Chemists.
Enzyme Immunoassay for Human Pancreatic Amylase TAKASHI FUJISAWA, TAKAHIKO NAKAMURA, MASATOSHI FUJII, SATOSHI TANI, YOSHINORI OKABAYASHI, and MAKOTO OTSUKI Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan An enzyme immunoassay (EIA) for human pancreatic amylase has been developed for the detection of human serum amylase content. Our monoclonal antibody is highly specific for human pancreatic amylase; it cross reacted negligibly with the salivary isoenzyme. We developed a solid phase enzyme immunoassay for determination of pancreatic amylase in human serum with this antibody. The assay required 20 p,L of serum and the standard curve was linear to at least 1000 ng/mL of pancreatic amylase. Inter- and intra-assay CVs were less than 10%. The results obtained by the EIA correlated well with those determined by the conventional electrophoretic method. In normal subjects, the mean concentration of serum pancreatic amylase determined by the EIA was found to be 92.3 -- 26,1 ng/mL (mean __. SD). The EIA we describe is useful for directly determining pancreatic amylase in human serum. Specifically distinguishing pancreatic from salivary amylase may have considerable clinical value.
ificity. Recently, the specific recognition of the salivary amylase isoenzyme by monoclonal antibodies has been reported (9-14). Measurement of amylase activity in the supernatant fluids from sera treated with these antibodies provides an assay of pancreatic amylase. In contrast to these indirect methods, here we describe a direct enzyme immunoassay (EIA) for human P-amylase using a monoclonal antibody that specifically binds to the P-amylase without any detectable cross reaction against the S-amylase.
KEY WORDS: amylase isoenzyme; pancreatic amylase; salivary amylase; enzyme immunoassay; monoclonal antibody; acute pancreatitis.
PURIFICATION OF HUMANP-AMYLASE AND PREPARATION OF THE MONOCLONAL ANTIBODY
Introduction
a-amylase (EC 3.2.1.1.) in human serum and urine can be separated into two isoenzymes; the salivary (S) and the pancreatic amylase (P). Several methods for differential assay of amylases have been developed based on electrophoresis (1,2) and amylase inhibitor from wheat germ (3). Electrophoresis is a sensitive method but is time consuming and technically complex. The amylase inhibitor method is easy, but the inhibitor does not totally differentiate the isoenzymes, S- and P-amylase being inhibited by approximately 80-85% and 10-15%, respectively (4). Consequently, a specific, rapid and simple method is required for precise quantitative measurement of P-amylase in serum and urine. Several immunochemical methods for separation of amylase isoenzymes involving polyclonal antisera have been reported (5-8). However, the antibodies for P-amylase used in these previous studies crossreacted with S-amylase, and thus they lacked spec-
Correspondence: Dr. Makoto Otsuki, Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650, Japan. Manuscript received January 5, 1989; revised April 10, 1989; accepted April 15, 1989. CLINICAL BIOCHEMISTRY, VOLUME 23, APRIL 1990
Materials a n d M e t h o d s
Human P-amylase was purified from human pancreatic juice by the method of Matsuura et al. (15). The P-amylase preparation was free of deamination products, because no anodically migrating bands were visible by poly-acrylamide gel electrophoresis. The monoclonal antibody for human P-amylase was prepared as follows. Female Balb/C mice were immunized with 100 Ixg of human P-amylase in complete Freund's adjuvant. These immunized mice were killed and their spleen cells removed for fusion with the myeloma cell line P3-Ag8-U1 according to the methods described by Kohler and Milstein (16), and Hales (17). Primary cultures were screened for antibody production and cross reactivity by an enzyme linked immunoassay method. Cell clones that produced antibodies showing a selective binding to human P-amylase were further cultured. The monoclonal antibody was purified by passing through a protein A column (18). ENZYME
I M M U N O A S S A Y FOR H U M A N
P-AMYLASE
Using the aforementioned monoclonal antibody, a solid-phase EIA for human P-amylase was developed based on the two-site assay principle. Wells of microtiter plates were coated with anti-human Pamylase monoclonal antibody. 20 ~L of P-amylase standards (0-1000 ~g/mL) or samples and 200 ~L of phosphate buffer were placed in the wells and mixed 149
FUJISAWA, NAKAMURA, FUJII, ET AL.
2,0
--
~.
= Standard curve
;~
Serum~Dilution curve ?.: U r i n e ~
6.
~ Salivary amylase
E 1.5t-
O .0
10~
tO -Q
