Development and characterization of monoclonal antibodies against canine trypsin

Veterinary Immunology and Immunopathology 80 (2001) 333±338

Short communication

Development and characterization of monoclonal antibodies against canine trypsin Takaki Waritania, Yoko Okunoa, Yoshinori Ashidaa, Ryo Tsuchiyab, Kosaku Kobayashib, Takatsugu Yamadab,* a

Animal Health Products and Chemicals Division, Animal Health Products and Chemicals Research Section, Fuji Chemical Industries Ltd., 530 Chokeiji, Takaoka-shi, Toyama 933-8511, Japan b Laboratory of Veterinary Internal Medicine, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Sagamihara-shi, Kanagawa 229-8501, Japan Received 19 September 2000; received in revised form 20 February 2001; accepted 9 April 2001

Abstract Canine cationic trypsin was puri®ed by salting-out, gel ®ltration and af®nity chromatography. Purity was con®rmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was ca. 28 kDa by SDS-PAGE. Thirty hybridomas were obtained which produced mAb to canine cationic trypsin by the cell fusion technique. Twenty-two of these recognized cationic trypsin only, while eight hybridomas recognized both cationic and anionic trypsin. Several of the anti-canine cationic trypsin mAb were puri®ed by salting-out and DEAE ion-change chromatography using ascites ¯uid of immunized BALB/c mice. The mAb proved to have very high speci®city to canine cationic trypsin as shown by immunoblotting and it may be possible to use them to develop clinical assays. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Dog; Monoclonal antibodies; Trypsin; Trypsin-like immunoreactivity

Trypsin is a digestive endopeptidase that catalyzes the hydrolysis of peptide bonds on the carboxyl side of lysine or arginine residues. The enzyme is synthesized only in the acinar cells of the pancreas as an inactive precursor, trypsinogen. It is converted to an active enzyme in the gut by the highly speci®c protease enterokinase. Once activated, trypsin in turn activates its own zymogen as well as the other pancreatic zymogens. Multiple forms of trypsin have been shown to be present in most animal species. Cationic and anionic trypsin have been isolated in dogs (Ohlsson and Tegner, 1973), humans (Guy et al., 1978), cattle Abbreviations: TLI, trypsin-like immunoreactivity; POD, peroxidase Corresponding author. Tel: ‡81-427-691636; fax: ‡81-427-691636. E-mail address: [email protected] (T. Yamada). *

0165-2427/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 1 ) 0 0 2 9 3 - 8

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(Louvard and Puigserver, 1974), pigs (Louvard and Puigserver, 1974) and rats (Brodrick et al., 1980). The proportion of anionic trypsinogen in bovine and swine pancreas does not exceed 10% of the cationic forms (Louvard and Puigserver, 1974). In humans, cationic trypsin exists at a level about twofold higher than anionic trypsin in the pancreas (Guy et al., 1978) and it is more stable than anionic trypsin (Colomb et al., 1978). Williams puri®ed canine cationic trypsin (Williams et al., 1983b) and developed a radioimmunoassay (RIA) using polyclonal antibody against canine cationic trypsin (Williams and Batt, 1983a). Polyclonal antibodies, in general, have poor in speci®city and there is much difference from batch to batch. We developed and characterized monoclonal antibodies against canine cationic trypsin, which showed high speci®city. Canine cationic trypsin was puri®ed as follow. The pancreas was removed from a humanely killed normal dog under anesthetization with pentobarbital sodium. The pancreas (27 g) was kept ice cold and minced then homogenized in a blender in twice the weight of 0.125N H2SO4. The homogenate was centrifuged at 1500 g for 30 min at 48C and the precipitate was dissolved in 0.125N H2SO4 then centrifuged at 1500 g for 30 min at 48C. The supernatant was precipitated by 20% saturated ammonium sulfate and centrifuged at 23,000 g for 30 min at 48C. This supernatant was precipitated by 80% saturated ammonium sulfate and centrifuged at 23,000 g for 30 min at 48C. Next the precipitate was dissolved in water and dialyzed against 1 mM HCl at 48C. The total amount of 400 mg powder was obtained by freeze±drying the solution. The powder was then dissolved in 10 ml of 0.2 M NaCl/0.05 M CaCl2 buffer pH 2.6 and loaded on a Sephadex G75 (Pharmacia) column (100 cm  3:2 cm). The two major peaks and one minor peak were seen. The second peak showed trypsin activity. A pool of the second peak was dialyzed against 1 mM HCl. The total amount of 200 mg powder was obtained by freeze±drying this solution. The powder from the second peak was dissolved in 5 ml of 4 mM CaCl2/100 mM Tris±HCl pH 8.0 and autoactivated for 12 h at 48C. The activated sample was loaded on a benzamidine-Sepharose 6B (Pharmacia) column (12 cm  3 cm). Chymotrypsin was eluted with 2 mM CaCl2/50 mM Tris±HCl pH 8.0 containing 0.5 M NaCl. Cationic trypsin was eluted with 2 mM CaCl2/50 mM sodium acetate buffer pH 4.25, and anionic trypsin was eluted with 2 mM CaCl2/50 mM sodium acetate buffer pH 3.25; fractions of cationic and anionic trypsin showed trypsin activity. Next each fraction was dialyzed against 1mM HCl. An amount of 20 mg powder of anionic trypsin and 17 mg of cationic trypsin were obtained by freeze±drying the solution. The cationic trypsin was used to make mAb. Ten point 3 mg of the activated sample after gel ®ltration was loaded onto Sepharose 4B (Pharmacia) column (15 cm  3 cm) conjugated with anti canine cationic trypsin mAb. The bound fraction was eluted by changing the buffer from 2 mM CaCl2/50 mM Tris±HCl (pH 8.0) to 2 mM CaCl2/200 mM glycine HCl pH 2.3. The elute was dialyzed against 1 mM HCl and freeze±dried until use. Trypsin activity was determined by the spectrophotometric procedure (Yamada and Aketa, 1982) using benzoyl-L-arginin ethyl ester as substrate. All assays were carried out at pH 8.0 in 50 mM Tris±HCl buffer containing 50 mM CaCl2 at 238C. One unit of tryptic activity was de®ned as an absorbance change of 0.001 min 1 at 253 nm. The mAbs were produced by the technique of somatic cell hybridization, as ®rst described by Kohler and Milstein (1976) and modi®ed by Oi and Herzenberg (1980). Brie¯y, 6-week-old female BALB/c mice were immunized intraperitoneally with 100 mg of canine cationic trypsin emulsi®ed with

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an equal volume of Freund's complete adjuvant. Two weeks later, the mice were boosted with an intraperitoneal injection of 100 mg of puri®ed canine cationic trypsin emulsi®ed with an equal volume of Freund's incomplete adjuvant. Four weeks later, a ®nal boost was performed intravenously using 100 mg of canine cationic trypsin dissolved in PBS pH 7.4. After 3 days, the last immunization, the mice were sacri®ced and spleens were removed and single-spleen cell suspensions were prepared. Immune spleen cells were fused with mouse myeloma cells (SP-2/0-Ag14) at a ratio of 5:1 in IMDM medium using 50% polyethylene glycol (MW 4000, Merck). Fused cells were plated onto 96 well ¯at± bottomed tissue culture plates (2:0  105 cells per well) and cultured in IMDM medium supplemented with 10% FCS containing hypoxanthine, aminopterin and tymidine in an atmosphere of humidi®ed, 7% CO2 in air at 378C. Culture supernatants from wells containing hybridomas were screened for antibody activity against canine cationic trypsin (Rennard et al., 1980). The mAb-secreting hybridomas were cloned at least twice by the limiting dilution method. Clones were expanded in a ¯ask and injected intraperitoneally into BALB/c mice (1  107 cells per mouse) primed with 2,6,10,14-tetra-methylpentadecane. The ascites ¯uid was harvested and antibodies were puri®ed by salting-out using ammonium sulfate, followed by DEAE ion-exchange chromatography (Pharmacia). The mAb protein was determined spectrophotometrically by absorption at 280 nm using an extinction coef®cient of 14.0 (Ey et al., 1978). F(ab0 )2 of mAb(A) was prepared by digesting it with pepsin. F(ab0 )2 was reduced with 2-aminoethanthiol then the Fab0 was conjugated with POD using N-(6-maleimdocaproyloxy)-succinimide (Ishikawa et al., 1983). Cationic trypsin was transferred to nitrocellulose sheets after performing 15%

Fig. 1. (A) SDS-PAGE of canine cationic trypsin. Various amounts of cationic trypsin (lanes 1±4: 1.0, 0.5, 0.25 and 0.125 mg, respectively) puri®ed from dog pancreas were analyzed by SDS-PAGE under reducing condition with 2ME on 15% gel and stained with CBB R-250. (B) Immunoblot analysis of anti-canine cationic trypsin monoclonal antibody. Various amounts of cationic trypsin (lanes 1±4: 1.0, 0.5, 0.25 and 0.125 mg, respectively) puri®ed from dog pancreas were immunostained with an anti-canine cationic trypsin mAb(A) conjugated with POD.

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acrylamide SDS-PAGE (Laemmil and Farve, 1973). Next the immobilized proteins were characterized by immunodetection using POD conjugated Fab' fraction of mAb(A), followed by detection with diaminobenzidine in the presence of 0.003% hydrogen peroxide (Tanabe, 1983). Three point 8 mg of cationic trypsin was obtained from a canine pancreas. Puri®ed canine cationic trypsin appeared as a single band at 28 kDa by electrophoresis under reducing conditions (Fig. 1A). Thirty hybridomas, secreting mAbs against canine cationic trypsin, were selected by ELISA after the cell fusion. Twenty-two hybridoma recognized only cationic trypsin, but eight hybridoma recognized both cationic and anionic trypsins. According to an isotype assay of the monoclonal antibodies using a MonoAb-ID EIA kit (Boehringer Mannheim), the light chain of all mAbs were k type but the heavy chains were variable as shown in Table 1. One mAb (clone 009-303) reacted speci®cally with canine cationic trypsin at 28 kDa as shown by immunoblotting (Fig. 1B). Table 1 The evaluation of mAb activities against canine cationic and anionic trypsin were shown by absorbance of ELISAa Cell names

Subtype

Reaction to cationic trypsin

Reaction to anionic trypsin

004-301 004-312 004-203 004-214 005-201 005-202 005-203 005-204 005-205 005-206 005-208 007-202 007-203 007-205 007-206 007-207 007-209 008-202 008-204 008-205 008-206 008-207 009-201 009-202 009-303 009-204 009-205 009-206 009-207 009-209

IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgM, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG1, k chain IgG2a, k chain IgG1, k chain IgG1, k chain

4‡ 4‡ 4‡ 4‡ 4‡ 4‡ 4‡ 3‡ 4‡ 3‡ 4‡ 3‡ 3‡ 3‡ 3‡ 3‡ 3‡ 3‡ 3‡ 3‡ 3‡ 3‡ 4‡ 4‡ 5‡ 4‡ 3‡ 3‡ 3‡ 3‡

3‡ 3‡

a

 ‡  ‡

3‡



Here: ( ) 0±0.1; () 0.1±0.2; (‡) 0.2±0.4; (2‡) 0.4±0.6; (3‡) 0.6±0.8; (4‡) 0.8±1.0; (5‡) >1.0.

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Canine trypsin-like immunoreactivity (TLI) which includes trypsin and trypsinogen, is a highly speci®c enzyme in pancreas. The nucleotide and amino acid sequence for both cationic and anionic trypsinogen has been determined (Pinsky et al., 1985). The amino acid sequences of cationic and anionic trypsins resemble each other very closely. There are many reports on the puri®cation of trypsin by several different methods (Ohlsson and Tegner, 1973; Guy et al., 1978; Louvard and Puigserver, 1974; Brodrick et al., 1980). We used the method of Williams et al. because it was simple. Williams et al has also developed radioimmunoassay (RIA) for canine TLI using polyclonal antibody against canine cationic trypsin. But no one has developed mAb to canine trypsin, though mAbs against human trypsin have been developed (Crotte et al., 1992). We developed 30 mAbs against canine cationic trypsin and prepared peroxidase (POD) labeled Fab0. Twenty-two of the 30 mAbs, recognized only cationic trypsin while eight antibodies recognized both anionic and cationic trypsin. These antibodies may react with the same epitope of the amino acid sequence because the amino acid sequence is very close between anionic and cationic trypsins. We will soon be able to determine the 3D structure of canine trypsins and trypsinogens to discover epitopes these mAbs recognized. Our experiments proved that mAbs 008-207 and 009-303 recognized the peptide sequence of canine cationic trypsin, and that mAb 004-203, 004-214 and 005-201 recognized its 3D structure (data not shown). It is not yet clear which epitopes were recognizing by the other monoclonal antibodies. The mAb 009-303 had high speci®city to canine cationic trypsin and did not cross-reacting with bovine trypsin, swine trypsin or canine anionic trypsin (data not shown). For measurement of canine TLI, only the RIA kit (double antibody canine TLI, DPC) is commercially available. TLI values are commonly used in veterinary medicine to diagnose exocrine pancreas insuf®ciency. However, it is dif®cult to carry out the RIA procedure in most veterinary clinics and general laboratories. Thus, we plan to develop simpler ELISA and immunochromatographic assays using these mAbs for clinical application of canine TLI. Acknowledgements We thank Onda Masaaki, Yasuhiko Inagaki and Mitsuru Furuichi for technical assistance. References Brodrick, J.W., Largman, C., Geokas, M.C., O'Rourke, M., Ray, S.B., 1980. Clearance of circulating anionic and cationic pancreatic trypsinogens in the rat. Am. J. Physiol. 239, G511±G515. Colomb, E., Guy, O., Deprez, P., Michel, R., Figarella, C., 1978. The two human trypsinogens: catalytic properties of the corresponding trypsins. Biochim. Biophys. Acta 525, 186±193. Crotte, O.G., Jamska, B.M., Brayle, A., Lafont, P., Figarella, C., 1992. Monoclonal antibodies to human pancreatic trypsin1 inhibit the activation of human trypsinogens 1 and 2. Eur. J. Biochem. 204, 133±136. Ey, P.L., Prowse, S.J., Jenkin, C.R., 1978. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-Sepharose. Immunochemistry 15, 429±436. Guy, O., Lombardo, D., Bartlett, D.C., Anie, J., Figurella, C., 1978. Two human trypsinogens. Puri®cation, molecular properties, and N-terminal sequences. Biochemistry 17, 1669±1675.

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Ishikawa, E., Imagawa, M., Hashida, S., Yoshitake, S., Hamaguchi, Y., Ueno, T., 1983. Enzyme-labeling of antibodies and their fragments for enzyme immunoassay and immunohistchemical staining. J. Immunoassay 4, 209±237. Kohler, G., Milstein, C., 1976. Derivation of speci®c antibody-producing tissue culture and tumor lines by cell fusion. Eur. J. Immunol. 6 (7), 511±519. Laemmil, U.K., Farve, M., 1973. Maturation of the head of bacteriophage T4.1. DNA packing events. J. Mol. Biol. 80, 597±601. Louvard, M.N., Puigserver, A., 1974. On bovine and porcine anionic trypsinogens. Biochim. Biophys. Acta 371, 177±185. Ohlsson, K., Tegner, H., 1973. Anionic and cationic dog trypsin. Isolation and partial characterization. Biochim. Biophys. Acta 317, 328±337. Oi, V.T., Herzenberg, L.A., 1980. Immunoglobulin-producing hybrid cell lines. In: Mishell, B.B., Shigi, S.M. (Eds.), Selected Methods in Cellular Immunology. Freeman, San Francisco, pp. 351±371. Pinsky, S.D., Laforge, K.S., Scheele, G., 1985. Differential regulation of trypsinogen mRNA translation: fulllength mRNA sequences encoding two oppositely charged trypsinogen isoenzymes in the dog pancreas. Mol. Cell. Biol. 5, 2669±2676. Rennard, S.I., Berg, R., Martin, G.R., Foidart, J.M., Robey, P.G., 1980. Enzyme-linked immunoassay (ELISA) for connective tissue components. Anal. Biochem. 104, 205±214. Tanabe, K., 1983. Western blotting. Cell Technol. 1/2, 1061±1063. Williams, D.A., Batt, R.M., 1983a. Diagnosis of canine exocrine pancreatic insuf®ciency by the assay of serum trypsin-like immunoreactivity. J. Small Anim. Pract. 24, 583±588. Williams, D.A., Batt, R.M., Beynon, R.J., 1983b. A simple procedure for the puri®cation of the zymogens and active forms of canine anionictrypsin, cationic trypsin and chymotrypsin. Biochem. Soc. Trans. 11 (4), 351. Yamada, Y., Aketa, K., 1982. Puri®cation and characterization of trypsin-like enzyme of sperm of the sea urchin, hemicentrotus pulcherrimus. Dev. Growth Differ. 24, 125±134.