Gastroenterologia Japonica Copyright 9 1990 by The Japanese Society of Gastroenterology

Vol. 25, No. 3 Printed in Japan

Monoclonal antibody against human enterokinase and immunohistochemical localization of the enzyme Yasuyuki MIYOSHI 1, Toshio ONISHI 2, Toshiaki SANO 3, and Nobuhiko KOMI 1

1First Department of Surgery, 2Department of Biochemistry, and 3First Department of Pathology, University of Tokushima School of Medicine, Tokushima 770, Japan Summary: A monoclonal antibody, hek-1, was raised against enterokinase or enteropeptidase that had previously been partially purified from human duodenal fluid. Hek-1 showed staining of two glycoprotein bands of relative molecular weights of 260,000 and 240,000 on immunoblot analysis of partially purified enterokinase and of ammonium sulfate fraction of duodenal fluid. An enzyme immunoassay for human enterokinase was developed, making use of hek-1. Sensitivity to enterokinase was 20 times higher than that of the conventional assay where BAPA was used as a substrate. The immunohistochemicalstudy with hek-1 showed staining of the brush border membrane and some goblet cells of the duodenum and upper jejunum but no staining of the colon epithelium. GastroenterolJpn 1990;25:320-327 Key words:

enzyme immunoassay for enterokinase; human enterokinase; monoclonal antibody against human enterokinase

Introduction

The incidence of biliary carcinoma arising in patients with an anomalous arrangement of pancreaticobiliary ducts (APBD) is much higher than that previously assumed and increases with age 1. When the biliary ducts are congenitally dilated, biliary carcinoma incidence is high. In patietns whose biliary ducts are not dilated, gallbladder carcinoma occurs more frequently than biliary carcinoma 2. In patients with A P B D , pancreatic juice regurgitates into the biliary tract, and the juice is activated by an u n k n o w n mechanism. It has been assumed that activated pancreatic enzymes may cause pathological epithelial changes in the biliary duct, resulting in biliary carcinoma. However, the process of the activation of pancreatic zymogens or proenzymes in the biliary tract has not been elucidated. Trypsinogen, a pancreatic zymogen is nearly always present in the bile collected from patients with A P B D 3. Trypsinogen is

activated with enterokinase (EC 3.4.21.9), a glycoprotein enzyme in the small intestine. In order to detect cells producing enterokinase in the mucosal epithelium of biliary tract of patients with A P B D , it is necessary to have a monoclonal antibody against human enterokinase, however, it has not yet been obtained. Therefore, we prepared a monoclonal antibody against human enterokinase. The specificity of the antibody was immuno-histochemically tested in the small intestine before commencing studies using specimens of the biliary tract. The monoclonal antibody showed staining of the brush border membrane and goblet cells of the h u m a n d u o d e n u m and upper jejunum. The antibody was also used to develop an enzyme immunoassay for enterokinase. Materials and Methods

Duodenal fluid obtained from postoperative pa-

Redeived September 18, 1989. Accepted December 15, 1989. Address for correspondence: Yasuyuki Miyoshi, M.D., First Department of Surgery, The University of Tokushima School of Medicine, Kuramoto-cho, Tokushima 770, Japan. The authors thank Professors Shozo Yamamoto and Kazuo Hizawa, The University of Tokushima, for their valuable comments and discussions during the course of this study.

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Monoclonal antibody against human enterokinase

tients and cases with ileus was stored at -20~ and used as the source of human enterokinase. Benzoyl-DL-arginine p-nitoroanilide hydrochloride (BAPA) was supplied by the Protein Research Foundation (Osaka). Crystallized bovine trypsin, trypsinogen, crystalline porcine enterokinase, bovine serum albumin and high molecular weight standard mixture of protein for SDS-polyacrylamide gel electrophoresis were obtained from Sigma (St. Louis, MO). D E A E - S e p h a d e x A-25 and Protein A-Sepharose were purchased from Pharmacia (Uppsala). A m i c o n Far East Ltd. (Tokyo) provided the ultrafiltration membrane YM-100. Horse serum was bought from Flow Laboratories (McLean). Sea plaque agarose was from Marine Colloids (Rockland). Microbiological Associates Bioproducts (Walkersville) supplied the fetal calf serum and NCTC-109 medium. Freund's adjuvant was from Difco (Detroit, MI). Vectastain A B C Kit was obtained from Vector Laboratories (Burlingame, CA). Rabbit antimouse IgG antiserum and monoclonal typing kit were obtained from Miles Laboratories (Elkhart). Biotine conjugated affinity purified goat anti-mouse IgM was purchased from Cappel Laboratories (Turnhout). Nitrocellulose membrane filters (Type TM-2) were from Toyo Roshi (Tokyo). The Shizuoka Laboratory Animal Center (Hamamatsu) was the supplier for the BALB/c mice. The myeloma cell line (Sp2/O-Ag14) was provided by the Salk Institute (San Diego, CA) and Swiss albino 3T3 fibroblasts (CCL-92) by Dainippon Pharmaceutical Company (Osaka). Protein A-bearing Cowan I strain of Staphylococcus aureus (NCTC 8530) was a gift from Dr. W.L. Smith of Michigan State University.

Enzyme assay Enterokinase activity was determined through its activating effect on trypsinogen. The assay was based on the improved methods of Erlanger et al 4 and Baratti et al s. Enterokinase solution (x ml), 10 m M Tris-acetate-50 mM NaC1, pH6.0 (0.3-x ml), 148 m M succinate buffer, pH5.6 (0.1 ml), 1 mM HCI-3 mM CaC12 (0.1 ml) and trypsinogen solution (3 mg/ml) dissolved in 1 m M HC1-3 mM CaC12 (0.1 ml) were mixed (total volume 0.6 ml)

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and the mixture was incubated at 25~ for 30 min. The activation was stopped by the addition of 1 ~1 of 2 M HC1. To the activation mixture, 0.7 ml of 1 mM B A P A (final concentration. 0.35 mM), 0.6 ml of 0.2 M Tris-HC1 containing 10 mM CaC12, pH8.0, 17 tzl of 148 m M succinate buffer, pH5.6, 33 p3 of 1 mM HC1-3 mM CaC12 and 50/~1 of 10 mM Tris-acetate containing 50 mM NaC1, pH6.0 were added. After the mixture was incubated at 25~ for 5 min, 1 ml of 30% acetic acid was added to the assay mixture and absorbance was read at 410 nm. One unit of enterokinase was defined as the amount of enzyme which was equivalent to 1/~g of porcine enterokinase the activity of which was determined at various concentrations under the same conditions as described above.

Purification of human enterokinase All procedures were performed at 4~ Two liters of duodenal fluid stored at - 2 0 ~ was brought to 40% saturation by the addition of solid (NH4)2SO 4. The mixture was stirred for 30 min and precipitate was separated by centrifugation at 10,000 x g for 10 min. The supernatant ws brought to 80% (NH4)2SO 4 saturation and the precipitate was collected as described above. The precipitate was dissolved in 20 ml of 10 mM sodium phosphate buffer, pH7.4 and dialyzed against the same buffer. The dialysate was applied to a D E A E Sephadex column (3 cm x 30 cm) equilibrated with the same buffer. After washing the column with the same phosphate buffer and the column was eluted with a linear 0-250 mM gradient of NaCI in the sodium phosphate buffer. Fractions of 3 ml were collected at a flow rate of 30 ml/h. Active fractions (75 ml) were collected and dialyzed against 10 m M sodium phosphate buffer, pH6.5. The dialyzed solution was loaded on to a phosphocellulose column (1.5 cm x 6 cm) equilibrated with 10 m M sodium phosphated buffer, pH6.5 and the column was washed with the same buffer. Enterokinase was recovered in the flow through and active fractions were applied to a D E A E - S e p h a d e x column (2.3 cm x 14 cm) equilibrated with the same phosphate buffer. After the column was washed with the equilibration buffer the enzyme was eluted with a linear 0-150

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mM gradient of NaC1 in the sodium phosphate buffer. Fractions of 3 ml were collected at a flow rate of 30 ml/h. Active fractions were pooled and concentrated with the aid of Amicon ultrafilters. The concentrated enterokinase fraction was stored at - 2 0 ~ without appreciable loss of activity for at least 12 months.

Assay of monoclonal antibody for human enterokinase A mixture of S. aureus suspension (100/zl) and rabbit anti-mouse IgG antiserum (4 tzl) was incubated at room temperature for 5 rain, followed by the addition of 1 ml of 50 mM Tris-HC1 (pH8.0). The mixture was centrifuged at 3,000 rpm for 10 min. The precipitate was suspended in 100/zl of the same buffer and 200 tzl of cultured fluids or antibody solution were added. The mixture was incubated for 1 min prior to the addition of the above mentioned buffer, followed by centrifugation at 3000 rpm for 10 min. The precipitate was suspended in 100 tzl of assay buffer (10 m M Tris-acetate, pH6.0, containing 50 mM NaC1). Enterokinase solution (10/xl) was added to the final suspension and the enterokinase activity was assyed as described above.

Preparation of monoclonal antibody against human enterokinase The procedures were based on a modification by Shinjo et al 6. Two mice of BALB/c strain were immunized by intraperitoneal injection of the paritally purified human enterokinase (420 tzg of protein per mouse) emulsified with an equal volume of Freund's complete adjuvant. Second and third booster injections were performed using incomplete adjuvant at intervals of 2 weeks. Three days after the third injection the mouse with the higher titer was killed, and the spleen was removed under sterile conditions. Spleen cells and myeloma cells (SP2/O-Ag14) were fused with the aid of polyethylene glycol by the m e t h o d of Goding et al 7. Hybridoma cells were selected in H A T m e d i u m and the cells producing anti-human enterokinase were cloned in soft agar according to the m e t h o d of Kennett 8. The clone-producing antibody, referred to as hek-1, was grown in

Vol. 25. No. 3

Dulbecco's culture medium, and the cells (107 cells in 0.4 ml) were injected into the peritoneal cavity of a mouse (BALB/c) pretreated with 0.5 ml of Pristane. After about 2 weeks the mouse was killed by cervical dislocation, and the ascites fluid was collected. The antibody in the ascites fluid was precipitated with a m m o n i u m sulfate at 50% saturation. Since the antibody was proved to be of the IgM class, the IgG fraction was removed by the use of a protein A Sepharose column and the IgM fraction was further purified with the aid of an Affi-Gel Protein A MAPS TM Kit (Bio-Rad). Approximately 25mg of IgM were obtained from one mouse.

Immunoblotting of human enterokinase Electrophoretic blotting of the enzyme on a nitrocellulose m e m b r a n e was performed according to the m e t h o d of Towbin et al 9. H u m a n enterokinase solution was subjected to electrophoresis on 7.5% polyacrylamide gel (1 ram) in the presence of SDS. A Marysol gel electrophoresis apparatus model KS-8440 G M T was used for blotting proteins on a sheet of nitrocellulose membrane. 6 Electrophoresis was carried out at 35 V for 6 h at 10~ in 25 mM Tris, 192 m M glycine and 20% (V/V) methanol, pH8.3. All the following procedures described below were performed at room temperature. The nitrocellulose membrane was cut into 3 strips. One was stained with Coomassie Brilliant Blue R-250, another was stained with Schiff's reagent and the last was subjected to immunostaining. The third nitrocellulose membrane strip was washed three times for 15 min each with phosphate-buffered saline containing 0.05% Tween 20 by shaking on a rocking platform. The m e m b r a n e was immersed for 30 min in 10% goat serum in phosphate-buffered saline to prevent additional non-specific protein binding, followed by washing as described above. The nitrocellulose m e m b r a n e was placed in phosphate-buffered saline conatining 0.05% Tween 20 and hek-1 at a concentration of 3/xg/ml. After lh the membrane was washed as above, and then placed in phosphate-buffered saline containing goat biotinylated anti-mouse IgM (6.75 tzg/ml). After 1 h the m e m b r a n e was washed as above and

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treated with the Vectastain A B C Kit according to the instruction manual. After washing, the nitrocellulose m e m b r a n e was subjected to the peroxidase reaction for 70 sec in 0.6 m M 3,3'- diaminobenzidine, 3 mM hydrogen peroxide and 50 mM Tris-HC1 buffer, pH7.6.

Enzyme immunoassay for human enterokinase Enterokinase solution (100/_tl) was applied to a 96-well microplate (Nunc) and the plate was kept at 4~ for 12 h or overnight. Each well was washed 3 times with phosphate-buffered saline containing 0.05% Tween 20. The plate was blocked with 10% goat serum in phosphate-buffered saline (300/_tl) for I h at room temperature. After washing the plate, 100/zl of hek-1 (3/.tg/ml) was added to each well. After incubation for 30 rain the plate was washed as described above. Goat biotinylated anti-mouse IgM (6.75/zg/ml) was added to each well (100/zl each). After incubation for 30 min the plate was washed once more. The Vectastain A B C Kit was used according to the instruction manual. To each well, 150/zl of 50 m M phosphate citrate buffer, pH5.0, containing 4 mM ophenylenediamine and 3 mM hydrogen peroxide were added. After incubation for 10 min at 25~ the reaction was terminated by the addition of 50 /zl of 3% sodium azide. Absorbance was read with 405 nm and 492 n m filters in a Corona MTP-12 microplate photometer.

Immunohistochemistry Some portions of the human d u o d e n u m , upper jejunum and colon which had been resected from cases of gastric or colon cancer were fixed overnight in Periodate-Lysine-Paraformaldehyde (PLP) solution. After the tissues were successively washed for 6 h each by immersing in 10%, 15% and 20% sucrose dissolved in phosphatebuffered saline, they were placed in a Cryo-MBed, frozen in liquid nitrogen and stored at -80~ The frozen specimens were cut into 6/a,m thick sections with a Cryostat, followed by staining with the HE or with the A B C m e t h o d ~~ using hek-1 as the first antibody and diaminobenzidine as a substrate. The HE staining of the preparations showed normal histologies.

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Table 1 Purification process of human enterokinase from duodenal fluid Specific Activity Protein activity (units) (rag) (units/mg yield (%) protein) Duodenal fluid 1398 Ammonium sulfate fraction 1 1 7 2 DEAE-SephadexA-25 489 (pH7.4) Phosphocellulose 376 DEAE-SephadexA-25 293 (pH6.5)

8000 1409 68.9 35.2 11,7

0.175 0.832 7.09 10.7 25.0

100 83.8 35.0 26.9 21.0

Results

Purification of enterokinase H u m a n enterokinase was partially purified from duodenal fluid stored at -20~ by ammonium sulfate fractionation, chromatography on D E A E - S e p h a d e x A-25, pH7.4, on phosphocellulose, pH6.5 and on D E A E - S e p h a d e x A-25, pH6.5. The purity of enterokinase in the various steps is showsn in Table 1. Enterokinase was purified about 140-fold from the duodenal fluid with respect to specific activity. SDS-polyacrylamide gel electrophoresis showed two major bands after staining with Coomassie Brilliant Blue R-250 as shown in Figure la. Their molecular weights are about 260,000 and 240,000, respectively. Since enterokinase is a glycoprotein enzyme 12, we treated an electrophoretogram with Schiff's reagent (PAS staining). The bands were faint but two corresponding bands with molecular weights of 260,000 and 240,000 were noted (data not shown).

Preparation of monoclonal antibody against human enterokinase Hybrids were obtained in 68 wells and three of them were positive in the screening asay. One line was further cloned and named hek-1. The chain class of the monoclonal antibody hek-1 was examined using the Ouchterlony technque. The antibody formed a single precipitin line with antimouse IgM (data not shown).

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OD 2.0.

T.5"

1.0-

Fig. 1 SDS-polyacrylamide gel electrophoresis of human enterokinase. a; Partially purified enterokinase (lane 2). Protein bands were stained with Coomassie Brilliant Blue R-250. Lane 1, a molecular weight standard mixture containing myosine from rabbit muscle (molecular weight 205,000) 8-galactosidase (116,000), phosphorylase b from rabbit muscle (97,400), bovine serum albumin (68,000) and egg albumin (44,000). b; Immunostaining of enterokinase with hek-l. Ammonium sulfate fraction of duodenal fluid was subjected to electrophoresis and Western blot anaysis with hek-l.

0.5-

10'

i0 3

(0 2

T0

rig/m,

Concentration of enterokinase

Fig. 2 Calibraiton curve of enzyme immunoassay for human enterokinase. The enzyme immunoassay was performed in the presence of various amounts of partially purified human enterokinase. The abscissa shows amounts of enterokinase which are equivalent to authentic porcine enterokinase.

Binding of hek-1 to enterokinase T h e binding of hek-1 to enterokinase was examined by Western blot analysis. The a m m o n i u m sulfate fraction of duodenal fluid was separated by SDS-polyacrylamide gel electrophoresis and the bands were transferred to a sheet of nitrocellulose m e m b r a n e by electrophoresis. A strip of the nitrocellulose membrane was stained with the avidine-biotin complex m e t h o d (ABC method) in the presence and absence of hek-113. Two distinctive bands with molecular weights of 260,000 and 240.000, respectively, were stained as shown in Figure lb. Similar results were obtained with partially purified enterokinase.

Enzyme immunoassay for human enterokinase A n enzyme immunoassay was developed for human enterokinase using hek-1. Figure 2 shows a standard curve for partially purified human enterokinase which is detectable in the range of

0.1 p,g to 7/zg/ml (equivalent to concentrtion of authentic porcine enterokinase). The sensitivity is approximately 20 times higher than that of conventional enzyme assay where B A P A is used as a substrate. Similar results were obtained when bile was used as an enterokinase source. Four groups of 50 wells containing 0, 0.13, 0.4 and 1.2/zg/ml of human enterokinase were subjected to the standard enzyme immunoassay, giving an intraassay coefficient of variation of 4.1, 3.8, 3.6 and 3.6%, respectively. Three groups of 5 wells containing 0.13, 0.4 and 1.2/~g/ml of human enterokinase, were examined by enzyme immunoassay 5 times for the period of 6 weeks, and the interassay coefficient of variation was 1.7, 1.4 and 3.1%, respectively. These results indicate that the enzyme imunoassay for human enterokinase is reliable.

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Fig. 3 Immunohistochemical localization of human enterokinase (• a, Brush border membrane of the duodenum; b, demonstration of goblet cells reactive with hek-1 in the duodenum; c, brush border membrane of the upper jejunum; d, demonstration of hek-l-reactive goblet cells in the upper jejunum; e, mucosa of the colon. Arrows indicate staining with hek-l.

Immunohistochemical localization o f enterokinase in the human intestine Since it has been reported that h u m a n enterokinase is present in the brush border membrane of the d u o d e n u m and upper portion of the jejunum 14-18, we tested whether or not hek-1 could specifically demonstrate the localization of enterokinase in the mucosa of intestines resected

during surgery. The brush border membrane and some goblet cells of the d u o d e n u m and upper jej u n u m were stained as shown in Figure 3a-d, but no staining was observed in the mucosa of the colon (Fig. 3e) where the presence of enterokinase has not been detected 19. Our results are similar to those observed by the immunofluorescent technique developed by Hermon-Taylor

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et a118, except that they did not observe staining of goblet cells. In Figure 3 some interstitial cells are also stained. We believe them to be mast cells which contain high peroxidase activity. Sectioned tissues were treated with 0.3% H 2 0 2 in methanol to inhibit endogenous peroxidase but some remaining peroxidase might be responsible for the interstinal staining. Only these cells were stained in the absence of hek-1. Discussion

Human enterokinase was partially purified from duodenal fluid and the monoclonal antibody, hek1, was prepared. The antibody showed staining of two bands of approximate molecular weights of 260,000 and 240,000 on Western blot analyses of ammonium sulfate-precipitated duodenal fluid and of partially purified enterokinase. Similar bands were noted on staining with Schiff's reagent as well as with Coomassie Brilliant Blue R-250. Grant and Hermon-Taylor identified two isoenzymes by isoelectric focusing of highly purified human enterokinase 12'2~ They also observed bands of approximate molecular weights of 235,000 -255,000,210,000 (less-distinct band)and 150,000 in four runs on SDS-polyacrylamide gel electrophoresis of enterokinase after reduction with/3-mercaptoethanol. In two other runs bands corresponding to molecular weights of 200,000220,000, 150,000 and 110,000 were noted. Enterokinase is a glycoprotein containing 57% carbohydrate a2'2~. It is considered that the high sugar content is probably the reason for the variable behaviour of human enterokinase on SDS-polyacrylamide gel electrophoresis 12. Enzyme immunoassay was developed using hek-1 and the sensitivity of the assay was 20 times higher than that of the conventional assay where B A P A was hydrolyzed and absorbance was read at 410nm. Although the sensitivity was not increased very much, the method is useful from a clinical point of view. Duodenal fluid and bile contain bilirubin which interferes with colorimetry. In order to avoid the interference of bilirubin, duodenal fluid must be diluted, resulting in occasional low and unreliable activity of entero-

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kinase. Now we can effectively assay enterokinase in the bile from APBD patients. We have collected bile specimens from A P B D patients at operation, stored them at -20~ and performed enzyme immunoassay. The presence of enterokinase could suggest a mechanism of the activation of regurgitated pancreatic enzymes in the biliary tract with A P B D patients. The monoclonal antibody, hek-1, stained the brush border membrane and some goblet cells of the duodenum and upper jejunum in our immunohistochemical study. Neither epithelium nor goblet cells of the colon was stained with hek-1. Enterokinase appears to be localized in the brush border membrane of the human upper intestine 15'18'22'23. Localization or production of enterokinase in goblet cells of the intestine, however, is controversial. Hermon-Taylor et al did not observe fluorescence in goblet cells using antisera against human enterokinase purified to apparent homogeneity 18. Takano, Suzuki and Yasuda have proposed that enterokinase may be produced by goblet cells 19. Our preparation of the monoclonal antibody, hek-1, stained some goblet cells of the duodenum and upper jejunum but did not stain any goblet cells or mucous epithelium of the colon where enterokinase has not been detected. We regard hek-1 as a specific monoclonal antibody against enterokinase. References 1. Komi N, Tamura T, Tsuge S, et al: Relation of patient age to premalignant alterations in choledochal cyst epithelium: Histochemical and immunohistochemical studies. J Pediatr Surg 1986;21:430-433 2. Komi N, Tamura T, Miyoshi Y, et al: Histochemical and immunohistochemical studies on development of biliary carcinoma in forty-seven patients with choledochal cyst -Special reference to intestinal metaplasia in the biliary duct-. Jpn J Surg 1985;15: 273-278 3. Ohkawa H, Sawaguchi S, Yamazaki Y, et al: Research on animal models of the anomalous pancreatico-biliary ductal union. Study on the activation of pancreatic proteolytic enzymes refluxing into the biliary tract. J Pediatr Surg 1982;18:185-191 4. Erlanger BF, Kokowsky N, Cohen W: The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 1961;95:271-278 5. Baratti J, Maroux S, Louvard D: Effect of ionic strength and calcium ions on the activation of trypsinogen by enterokinase. Biochim Biophys Acta 1973;321:632-638

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6. Shinjo F, Yoshimoto T, Yokoyama C, et al: Studies on porcine arachidonate 12-1ipoxygenasc using its monoclonal antibodies. J Biol Chem 1986;261:3377-3381 7. Goding JW: Antibody production by hybridomas. J Immunol Methods 1980;39:285-308 8. Kennett RH, McKean TJ: Monoclonal Antibodies. Plenum Press, New York. 1980;372-373 9. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 1979;76:4350-4354 10. Bergroth V: Comparison of various immunohistochemical methods. Histochemistry 1983;77:177-184 11. Wagcner C, Yang YHJ, Crawford FG: Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: a systemic approach for the determination of epitope specificities of monoclonal antibodies. J Immunol 1983;130: 2308-2315 12. Grant DAW, Hermon-Taylor J: The purification of human enterokinase by affinity chromatography and immunoadsorption. Biochem J 1976;147:243-254 13. Yolken RH, Leister FJ, Whitcomb LS: Enzyme immunoassays for the detection of bacterial antigens utilizing biotin-labeled antibody and peroxidase biotin-avidin complex. J Immunol Methods 1983;56:319-327 14. Hadorn B, Steiner N, Sumida C: Intestinal enterokinase. Mech-

15. 16. 17.

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anisms of its "secretion" into the lumen of the small intestine. Lancet 1971;23:165-166 Nordstrom C, Dahlqvist A: Localization of human enterokinase. Lancet 1972;28:933-934 Rutgeerts L, Tytgat G, Eggermont E: Localization of enterokinase in man. Gastroenterology 1972;63:382-383 Nordstrom C, Dahlqvist A: Quantitative distribution of some enzymes along the villi and crypts of human small intestine. Scand J Gasgroenterol 1973;8:407-416 Hermon-Taylor J, Perrin J, Grant DAW: Immunofluorescnet localisation of enterokinase in human small intestine. Gut 1977;18:259-265 Takano K, Suzuki T, Yasuda K: Immunohistochemical localization of enterokinase in the porcine intestine. Okajimas Fol Anat Jap 1971;48:15-28 Grant DAW, Hermon-Taylor J: Purification of porcine enterokinase by affinity chromatography. Biochem J 1975;147:363-366 Grant DAW, Magee AI~ Hermon-Taylor J: Optimisation of condition for the affinity chromatography of human enterokinase on immobilised p-aminobenzamidine. Eur J Biochem 1978;88:183-189 Woodley JF, Keane R: Enterokinase in normal intestinal biopsies and those from patients with untreated coeliac disease. Gut 1972;13:900-902 Lobley GW, Moss S, Holmes R: Brush-border localization of human enterokinase. Br Soc Gastroenterol 1973;14:816-831

Monoclonal antibody against human enterokinase and immunohistochemical localization of the enzyme.

A monoclonal antibody, hek-1, was raised against enterokinase or enteropeptidase that had previously been partially purified from human duodenal fluid...
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