Acta Physiol Scand 1992, 144, 369-378
Production of monoclonal antibodies against gastric parietal cell antigens J. L. CABERO, T. SASAKI, Y.-H. SONG, R. HOL M DAHL and S. M k R D H Department of Medical and Physiological Chemistry, Biomedical Centre, Uppsala University, Sweden CABERO, J. L., SASAKI, T., SONG,Y.-H., HOLMDAHL, R. & MKRDH,S. 1992. Production of monoclonal antibodies against gastric parietal cell antigens. Acta Ph,ysiol Scund 144, 369-378. Received 19 July 1991, accepted 11 October 1991. ISSN 0001-6772. Department of Medical and Physiological Chemistry, Biomedical Centre, Uppsala University, Sweden. Two mice DBA/1 were each immunized with a single injection of one million enriched parietal cells in the hind foot pads. Monoclonal antibodies to be used as research tools in studies on regulatory mechanisms in gastric parietal cells were obtained after fusion of mouse myeloma cells (SP2) with cells from the popliteal lymph nodes of the mice. Twelve hybridomas produced antibodies reactive with structures only present in parietal cells as assessed by immunohistochemistry of oxyntic rnucosa sections. Three hybridomas were subcloned and the antibodies produced by them, designated as PC4, PC8, and PCI 17, were characterized. In an enzyme-linked immunosorbent assay, all antibodies reacted with H,K-ATPase-containing vesicles. The antibody PC8 recognized a 94 kDa protein after immunoblotting of H,K-ATPase-containing vesicles and all antibodies precipitated a 94 kDa protein from [1Z51]H,K-ATPase-containingvesicles. The antibodies PC4 and PC117 recognized extracellular structures with a polarized distribution in viable, purified parietal cells. The results suggest that the structure recognized by all three antibodies is the a-subunit of the H,K-ATPase. The antibodies produced by another hybridoma, PC43, recognized a structure present in parietal and surface epithelial cells of the oxyntic mucosa. In an enzyme-linked immunosorbent assay, they reacted with a high-activity carbonic anhydrase which had been affinitypurified from pig oxyntic mucosa and they recognized a 30 kDa protein after immunoblotting. Thus, monoclonal antibodies against both intracellular and extracellular parietal cell structures were obtained after immunization with a small number of parietal cells. Key words : carbonic anhydrase, H,K-ATPase, hybridomas
Isolated gastric parietal cells have been subjected to many studies in the last 20 years due to their production of acid and its role in peptic ulcer disease. However, various aspects of the regulation of acid secretion at the cellular level, e.g. regulation of the fusion of tubulo-vesicles and secretory channels that take place in the parietal cells during acid secretion (Forte et al. 1977, Olaisson et a/. 1990) and the regulation of some Correspondence : Sven Mirdh, Department of Medical and Physiological Chemistry, Biomedical Centre, Uppsala University, PO Box 575, S-751 23 Uppsala, Sweden.
enzymes involved in acid production, e.g. H,KATPase (Ganser & Forte 1973), are still poorly understood. Antibodies are valuable tools to characterize the cellular mechanisms and structures involved in those processes. PolyclonaI antibodies (PAB) against the H,K-ATPase were first produced by Saccomani et al. (1979) using gastric membrane fractions for immunization, while the first monoclonal antibodies (MABs) against the same antigen were produced by Smolka et al. (1983). Later, other groups have produced monoclonal antibodies against parietal cell antigens, mainly the H,K-ATPase, by using gastric microsomal vesicles (Asano et al. 1987,
-7. L. Cnbero ct al.
Benkouka et a / . 1989), or gastric mucosal cells (Mercier e l NI. 1989) for immunization. I n most cases, houeber, m u c h antigenic material a n d booster injections were required during the immunization stage which was also time-consum i n g. For many years, our group has been interested in the regulatory mechanisms of acid secretion a t the parietal cell level. I n the present study, we report the production of ZLIBs against parietal cell antigens to be used as research tools, by using a rapid and efficient procedure for immunization (Holmdahl et RI. 1985) with a single injection of a small n u m b e r of enriched parietal cells.
(Sveden). I’””I]INA was purchased from New England Nuclear (Boston, MA, USA). All other chemicals were of analytical grade and commercially available. The composition of the phosphate-buffered solution (PBS) used w a s : 137 mM NaCI, 2.7 mM KCI, 8.4 mM Na,HPO,, and 1.6 mM KH,PO,; pH 7.4.
\ I 4 ‘ T E R I 1 1 - S AND M E T H O D S
P r e p r a t r on
Isolution mid enrrrhment qf purieta f cells
Cells from the osyntic area of pig gastric mucosa were isolated by sequential incubations with pronase, EGTA, and collagenase and parietal cells were enriched by means of centrifugation on a densitygradient of Percoll followed by centrifugal elutriation as previously described (Cabero et al. 1989). of
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H, K-.I TPase. Vesicular membranes containing H,K-ATPase from pig and human gastric niucosa (resection specimens) were prepared as previously Boi ine serum albumin (BSA), phenylmethylsuldescribed (Ljungstriim et a/. 1984). phonhlfluoride (PZISF), pepstatin, 4-(2-hydrosy.Vu,K-.qTPase. Membranes containing Na,Keth!l)-I-piperazine-ethanesulphonic acid (HEPES), and pol!ethylene gl!col 1500 (PEG) were from ATPase were obtained from the outer medulla of pig kidney and subsequentll- treated with SDS and Iloehringer (XIannheim, Germany). CM-Sephadeu” has from Pharniacia Fine Chemicals (Lppsala, purified as described by Jsrgensen (1974). C ~ r b o i zanh,ydrasr,fiom i~ the oxyntir ~ I L I ‘ O S U .Soluble Sweden). Goat anti-mouse IgG-peroxidase conjugate, goat anti-mouse IgG, rabbit anti-mouse IgG-FITC carbonic anhydrase from fresh pig gastric mucosa was conjugate. goat anti-mouse IgG coupled to agarose purified by affinity chromatography. T h e affinity beads, mouse peroxidase-anti-perosidase complex column was prepared by coupling p-(aminomethyl) (P.4P). o-phenylenediamine dihydrochloride (OPT)), benzenesulphonaniide to Chl-Sephadex with freshly p - ( amino m e t h y 1)- benzenes u 1p h o n a m i d e , hypo- dissolved EDCDI as described (Osborne & Tashian 19i5). The osyntic region of a pig stomach was dine, ethylene-gl! colxantine, aminopterin, t his(/i-aniinoethyl ether) ,.l“,.V‘-tetraacetic acid dissected and the mucosa was scraped off from the ( E G T l ) , Tris (hydroxymeth!-I) amino-methane muscular layer. The scrapings were homogenized in (TKIS), l-ethyl-3-(3-dimethj-lamino-propyl) carbo- 50 m%rHepes-Tris buffer, pII 8.3, containing 100 mM diimide €fCI (EDCDI), carbonic anh!-drase I1 from sucrose. After filtration through glass wool, the hoi ine er! throcytes, and ovalbumin were obtained homogenate was centrifuged at 70000 g for 60 min at from Sigma (St Louis, LIO, USA). Complete Freund’s 4 “C. About 100 ml of the obtained supernatant was adjuiant was from Difco Laboratories (Detroit, 111, mixed with an equal volume of 5 0 mM Hepes-Tris buffer, pI-1 8.3. T o this mixture was added 30 ml of L-S.4) Kormal rabbit serum was from \.ector 1 .ahoratories (Burlingame, C.4, LS.4). Sormal goat the sulphonamide-C~I-Sephadexgel, which had been serum %as from GIRCO (Grand Island, NY.,LSA4). equilibrated in the Hepes-Tris buffer. The mixture q-eth! I-3-aniinocarbazol (rIEC) isas from .\Idrich Has stirred overnight at 4 “C. The gel was packed in a Chemie (Steinheim, Germany). Isotype-specific goat column and eluted stepwise with 50 mM Tris-II,So, antibodies conjugated to peroxidase were from Nordic buffer, p H 6.5, which contained the following salts: 1.abs (Tilhurg, The Netherlands). TweenR 20, 0 . 4 ~Na,So, (100 ml); 2 M NaCl (150ml); and Triton ’ N 100, and ethylenedinitrilo tetraacetic acid finally 0.4 M NaN, (I50 ml). The protein-containing ( E n T 1) were from Zlerck (Darmstadt, Germany). fractions obtained with the last elution buffer were Plastic 96-wells Immulon‘ flat-bottom plates were pooled, concentrated in a collodion bag, and espurchased from Dj-natech Laboratories, Inc. (Sussex, tensively dialysed against 10 mM Na-phosphate buffer, UK). l’itrocellulose paper was from Schleicher and pI I 7.2. This fraction was stored frozen until used. It Schuell (Dassel, German!-). IODO BE.4DS were contained a high carbonic anhydrase activity when from Pierce (Rockford, IL, CSA). Carbonic anh!-drase assayed as described by hIcIntosh (1968) and it I l l purified from bovine skeletal muscle cells was a eshibited a major protein band of 30 kDa after generous gift from Dr -4. Elisabeth Eriksson, De- polyacr!-lamide gel electrophoresis in sodium dodecylpartment of .\lolecular Biology, Uppsala University sulphate. o’h~flll~~li/s
Parietal cell MABs Production of monoclonal antibodies Immunization. Two million cells (85 yn parietal cells) were suspended in 100 pl PBS and emulsified with an equal volume of Freund's complete adjuvant. The emulsion was then injected subcutaneously in the hind foot pads of two male DBA/1 mice (originally obtained from Jackson Laboratory, Barharbor, ME, USA). Fusion. Hybridomas were produced essentially as described (Holmdahl et al. 1985). In brief, 9 days after immunization, the popliteal lymph nodes were removed from the mice and their cells were fused with mouse myeloma cells (SP2 line) using 50% (w/w) PEG 1500. After the fusion, the hybridomas were distributed in 480 microwells and incubated in D.MEM culture medium containing 5 % foetal calf serum, 50 ,ug ml-' gentamycin, and hypoxantinel aminopterin/thymidine (Kohler & Milstein 1975). Growing hybridomas were detected by microscopical examination. Aliquots of 100 pl of the culture medium were taken from each well for screening when an area of 1/4-1/2 of the bottom of the wells was covered with cells. Cloning and isotyping. The selected hybridomas were cloned several times by limiting dilution (0.5 cell well -') until all the wells containing cells were positive against the specific antigen. Isotypes were determined using an ELISA with peroxidase-conjugated, isotype specific goat antibodies and alkaline phosphataseconjugated rat monoclonal antibody no. 187.1.10, specific for mouse K chains (Ware et al. 1984). Purzjcataon of the antibodies. The antibodies were purified from the culture medium by using the thiophilic adsorbent (T gel) described by Belew et al. (1987). Characterzzation of the monoclonal antibodies Enzyme-linked immunosorbent assay (ELISA).Plastic 96-well flat-bottomed plates were coated for 2-3 h at 37 "C with 0.05 M NaC0,-/NaHCO, buffer, p H 9.5, containing 5-10 p g ml-' of the indicated antigen. Free binding sites were blocked overnight at 4 "C with 194 (w/v) ovalhumin in PBS, p H 7.4. The medium containing the primary antibody was then added to each well and incubated for 1 h at 37 "C. Goat antimouse IgG-peroxidase conjugate was used as a secondary antibody. Binding of mouse IgG was detected after adding to each well 0.04% (w/v) OPD and 14 mM hydrogen peroxide in 0.1 M citric acid/ 0.2 M NaHPo,, p H 5 . After stopping the colour development with 2 M H,So,, the plates were read at 492 nm in a Titertek multiscan spectrophotometer. All the washing steps were performed with PBS containing 0.05% (v/v) Tweenn 20. Negative controls were performed either by omission of the primary antibody or by using normal mouse serum.
Immunohzstochemtstry. For most experiments, 6 pm-thick sections were cut from frozen, non-fixed, tissue blocks and placed on gelatin/chromealumcoated glass slides. They were air-dried and kept at -70 "C until used. Each working day, the tissue sections were fixed with ice-cold 50% acetone for 30 s followed by ice-cold 100% acetone for 10 min and air-dried. After rehydration in PBS, the sections were treated with normal rabbit or goat serum (diluted 1 :4 in PBS) for 15 min to block non-specific binding sites followed by incubation with the monoclonal antibodies for 60 min. During the initial screening the immunoperoxidase method was used to detect binding of the primary antibodies; the sections were incubated with goat anti-mouse IgG for 30min followed by incubation with mouse PAP complex for 30 min. AEC was used as a chromogen and haematoxylin for counter-staining. Antibody binding sites were recognized by the presence of a red precipitate. After the initial screening, immunofluorescence was used for detection of MAB binding; the sections were incubated with rabbit anti-mouse IgG FITC-conjugate for 60min and specific labelling was detected in a microscope equipped with epifluorescence. Negative controls were performed either by omitting the primary antibody or by using an unrelated monoclonal antibody against complement C9. When sections of oxyntic mucosa were used, positive controls were obtained by using a monoclonal antibody raised against porcine pepsin. T o detect the binding of the antibody against carbonic anhydrase (see Results) it was necessary to fix the tissues before freezing and sectioning. In preliminary experiments, various fixation procedures were used to find optimal conditions. Fixation of the tissues with Bouin's fluid for 6 h did not improve the binding of the MAB. Fixation at room temperature with 0.25% (v/v) glutaraldehyde in 0.1 M phosphate buffer, p H 7.4, for 3 h improved the binding but it was not optimal. The best staining was obtained by using 4% (w/v) EDCDI in 0.1 M phosphate buffer, p H 7.4, for 3 h at room temperature for fixation and by omitting the treatment with acetone before starting the staining procedure. To detect the antibodies bound to extracellular epitopes, about 1 x los freshly isolated pig parietal cells (8&90°/, purity) were incubated in suspension at room temperature for 60 min in a solution of Hanks' balanced salts, also containing 25 mM Hepes, 11 mM glucose, 0.2% (w/v) BSA, and the primary antibodies. The cells were then washed three times with the incubation buffer without antibodies, incubated with rabbit anti-mouse IgG FITC-conjugate (diluted in incubation buffer) for another 60 min, and finally washed three times with incubation buffer. Specific labelling was detected in a microscope equipped with epifluorescence. Immunoblottzng. Polyacrylamide gel electrophoresis
J. L. Cnbero et al.
Table 1. Reactivitv pattern of the monoclonal antibodies selected after the initial screening using different immunological techniques EI