Immunoassay of Pig and Human Gastric Proton Pump

GASTROENTEROLOGY 1986;90:532-9

Immunoassay of Pig and Human Gastric Proton Pump ADAM SMOLKA and WILFRED M. WEINSTEIN

Center for Ulcer Research and Education, Wadsworth Veterans Administration Medical Center, and UCLA School of Medicine, Los Angeles, California

Monoclonal antibodies against the K+ -dependent adenosine triphosphatase [ATPase) responsible for acid secretion in the pig gastric mucosa were generated by hybridoma technology. Two of these antibodies, shown to bind selectively to subunits of the ATPase and to label intracellular membranes of pig and rabbit parietal cells, were used to develop a sensitive [<1 pmol) immunoassay for the ATPase. Enzyme samples were adsorbed to the wells of polystyrene microtitration plates and then incubated sequentially with monoclonal antibody, antimouse immunoglobulin G coupled to alkaline phosphatase, and p-nitrophenyl phosphate. Standard curves relating the absorbance of the wells at 410 nm to loglD micrograms ATPase were fitted by a three-parameter logistic, with a useful assay range of 0.05-10 jLg ATPase. The immunoassay allows measurement of proton-pumping ATPase levels in human gastric biopsy specimens and may therefore be useful in studies of gastric mucosa1 function. Gastric acid secretion is mediated by a protontranslocating, K+ -dependent adenosine triphosphatase (H+ ,K+ -ATPase) in the parietal cells of the gastric mucosa (1). The enzyme is a multimeric intrinsic membrane protein with a dissociated subunit molecular weight of about 100,000 (2). Adenosine triphosphate (ATP) drives an electroneutral H+: K+ exchange via a Mg2+ -dependent phosphorylated intermediate (3,4). The number and funcReceived May 15, 1985. Accepted August 9, 1985. Address requests for reprints to: Adam Smolka, Ph.D., Wadsworth Medical Center, Building 115, Room 226A, Los Angeles, California 90073. This study was supported by National Institutes of Health grants AM 32532 and AM 17328-11 to the Center for Ulcer Research and Education and by Jet Propulsion Laboratory Contract No. 740590. The authors thank Dr. Gary van Deventer for human gastric biopsy specimens, Myriam Marin for immunocytochemistry of the human sections, and Dr. George Sachs for laboratory facilities. © 1986 by the American Gastroenterological Association 0016-5085/86/$3.50

tion of ATPase subunits remain undecided. In an earlier study, a monoclonal antibody (HK 111) was used as a marker of the H+ ,K+ -ATPase at the electron microscopic level (5). This marker revealed that with the onset of acid secretion in the rabbit, the ATPase proton pump is transferred from parietal cell cytoplasmic tubulovesicles to apical secretory canalicular membranes. Parietal cell protein and membrane transfers are not understood and may involve fusion of tubulovesicles or osmotic swelling of a preexisting canaliculus upon stimulation (6). Further study of this phenomenon would benefit from sensitive assays of ATPase distribution in subcellular compartments of the parietal cell. A specific H+,K+-ATPase assay would also have clinically significant applications, for example in the development of novel therapeutic inhibitors of acid secretion, such as omeprazole (7), and in screening of human gastric biopsy specimens. Despite their desirability for the study of ulcers, assays of the acid-secreting ATPase in gastric biopsy specimens have hitherto been impractical. The multiple A TPases present in a microsomal fraction of the gastric mucosa are not readily distinguished in a conventional phosphate-release ATPase assay. In contrast, an immunoassay with anti-H+ ,K+ -ATPase monoclonal antibodies detects only this ATPase, irrespective of contaminating ATPases. This study describes such an assay based on enzyme-linked immunosorbent assay (ELISA) (8).

Materials and Methods Monoclonal Antibodies The antibodies were prepared as described (5). BALB/CJ mice were immunized with purified vesicular pig H+ ,K+ -ATPase obtained from gastric m1.).cosal homogenates by differential and sucrose gradient centrifugation Abbreviations used in this paper: BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; PBS, phosphatebuffered saline; SDS, sodium dodecyl sulfate.

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followed by free-flow electrophoresis (2). When circulating anti-ATPase activity was confirmed by ELISA, the mice were killed and their lymph node cells were fused with a nonsecreting myeloma cell line (P3.X63.Ag8.653) using polyethylene glycol. Hybrids secreting anti-ATPase antibodies were cloned by limiting dilution and were expanded to mass culture in vitro or as ascites in mice primed with pristane. Antibodies were purified by precipitation with ammonium sulfate and protein A affinity chromatography (9).

Immunohistochemistry Pig and rabbit gastric mucosal samples fixed in 95% ethanol were embedded in paraffin, sectioned at 5 pm, cleared in xylene and ethanol, and incubated with monoclonal antibodies diluted at a ratio of 1: 50 in 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 30 min as described previously (5,10). The sections were then washed three times in PBS and were incubated for 30 min with 50 11-1 of a 1: 100 dilution of fluorescein isothiocyanate-conjugated goat antimouse immunoglobulin (heavy + light chain). After repeated washing in PBS, the sections were examined by fluorescence microscopy (Zeiss MP3 photomicroscope, Carl Zeiss, Inc., Thornwood, N.Y.). Endoscopic biopsy specimens obtained from 6 patients with normal fundic gland mucosa were fixed for 2-4 h in Bouin's solution, dehydrated in graded ethanols, and embedded in paraffin. Four-micron tissue sections were deparaffinized in xylene through graded alcohols to distilled water and were transferred to PBS. The sections were treated according to the method mentioned above for indirect immunofluorescence at a 1: 50 dilution of HK 7.22. Sections were also examined with the avidin-biotin-peroxidase complex method. Dilutions of the primary antibody (HK 7.22) were incubated at 1: 50 for 1 hand 1: 500 for 18 h. Biotinylated anti mouse secondary antibody was then applied for 30 min, followed by avidin-biotin-peroxidase complex for 1 h (11). Diaminobenzidine (5 mg in 10 ml of PBS with 0.2 ml of 0.01 % H 20 2) was then applied for 10 min. Between the above steps, the tissue sections were rinsed during three 10-min PBS washes. The sections were counterstained with hematoxylin and eosin.

Peptide Specificity The purified H+ ,K+ -ATPase was resolved by sodium dodecyl sulfate (SDS) polyacrylamide electrophoresis on slab gels as described previously (12). The resulting ATPase peptide patterns were transferred by electroblotting to nitrocellulose sheets (13). The sheets were incubated first for 1 h with 3% BSAIPBS to block nonspecific reactive sites, then for 2 h with 1: 100 dilutions of monoclonal antibodies in 1% BSA/PBS. The sheets were washed five times in 0.05% Nonidet P40 in PBS, incubated for 2 h with 125I-labeled protein A (10 6 cpm/ml), washed again as above, and exposed for 3 h at -70°C to Kodak X-Omat AR 5 x-ray film (Kodak Laboratory and Specialty Chemicals, Rochester, N.Y.).

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Immunoassay Polystyrene microplates with 96 wells (Flow Laboratories, Inc., McLean, Va.) were used. Antibody dilutions were made in 1% BSA/PBS, and between each reagent the wells were washed five times with PBS. Reagents and incubation times per well were as follows: 1 h with 100 11-1 of standard ATPase or unknown ATPase samples diluted in binding buffer (0.1 M carbonate buffer, pH = 9.6), 30 min with 250 11-1 of 5% BSA/PBS, 1 h with 100 11-1 of monoclonal antibody diluted 1: 5000, 1 h with 100 11-1 of a 1: 500 dilution of goat antimouse immunoglobulin covalently coupled to alkaline phosphatase, and 30 min with 100 11-1 of 1 mg/ml p-nitrophenyl phosphate in 1 M diethanolamine, pH 9.8. The color reaction was quenched by adding 50 11-1 of 2 N NaOH to each well. Absorbance of the wells was measured at 410 nm using a Titertek Multiskan microplate reader (Flow Laboratories).

Preparation of Biopsy Specimens for Immunoassay Gastric biopsy specimens were obtained with an Olympus 1T large biopsy forceps (Olympus Corp. of America, New Hyde Park, N.Y.) from fasted human subjects prepared for endoscopy under standard conditions. Written informed consent was obtained in accordance with the requirements of the Research, Development, and Human Studies Committee of the Wadsworth Veterans Administration Medical Center. Biopsy specimens were stored on dry ice. After being weighed, individual specimens were suspended in 1 ml of 20 mM Tris-Cl, 250 mM sucrose, and 1 mM phenylmethylsulfonylfluoride, pH 7.4, and were disrupted with 10 strokes at 2000 rpm in a glass-Teflon homogenizer. The homogenates were sonicated for 30 s, and centrifuged at 20,000 g for 30 min (Sorvall RC-2). Aliquots of the microsomal supernatants were applied without further processing to the microplate wells. Protein measurements were made by the SDS-Lowry procedure (14), using BSA as the standard protein.

Results Lymph node cells from mice immunized with purified microsomal membranes prepared from pig gastric mucosal homogenates were fused with the nonsecreting myeloma cell line P3.X63.Ag8.653. Primary cultures surviving 14 days in hypoxanthineaminopterin-thymidine selective medium (15) were screened by ELISA. Twelve cultures were found to secrete antibodies against the microsomal ATPase. Five cultures were cloned by limiting dilution, yielding two stable hybridoma clones (HK 4.4 and HK 7.22) which were expanded to mass culture and grown as ascites in mice. Monoclonality of the hybridomas was confirmed by subcloning by limiting dilution, with preservation of the light and heavy chain isotypes of antibodies HK 4.4 and HK 7.22 (K, y2a and K, y2b, respectively).

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Figure 1. Indirect immunofluorescent staining of parietal cells in pig (A) and rabbit (B) gastric mucosa by monoclonal antibody HK 4.4 (X400).

Figure 1 shows the cytochemical localization of antibody HK 4 .4 in the pig and rabbit gastric mucosa using a fluorescein isothiocyanate-Iabeled second antibody. In both tissues the antibody was localized in the parietal cells of the mucosa. No antibody staining was evident in the chief cells, mucus neck cells, surface epithelial cells, or enterochromaffin cells, nor was it apparent in myoepithelial cells,

plasma cells, or the interstitial supporting matrix of the mucosa. The basolateral membranes of cells lining the gastric glands were not stained. In the parietal cells, antibody HK 4.4 was concentrated in the cytoplasm, with no nuclear staining. Antibody HK 7.22 also showed high selectivity for the parietal cells of pig and rabbit gastric mucosae and strong cross-reactivity with human gastric mucosa. Figure 2

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Figure 2. Indirect avidin-biotin-peroxidase complex staining of human parietal cells by monoclonal antibody HK 7.22. A. Low power view of normal human fundic gland mucosa. There is intense staining of cells in the parietal cell zone, between the arrows. In the chief cell zone (bottom third) there are isolated positive-staining parietal cells. (Counterstained with H & E, x 120.) B. Higher power view of the same biopsy specimen. Positive-staining parietal cells (arrows) are densely packed just below the gastric pits. (x300.)

shows the localization of monoclonal antibody HK 7.22 in cells of the normal human fundic gland mucosa. Once again, all the parietal cells of the gastric glands are stained by the antibody, with no apparent staining of other cell types. To verify that the parietal cell-specific monoclonal antibodies were directed against the H+ ,K+ -ATPase responsible for gastric acid secretion, immunoblotting and immunoprecipitation of the ATPase peptides was carried out. Purified pig H+ ,K+ -ATPase was resolved by SDS electrophoresis, and the peptides were transferred to nitrocellulose sheets and probed with antibodies HK 4.4 and HK 7.22. Figure 3 shows that the purified ATPase consists predominantly of a single Coomassie Blue-stained band of molecular weight 95,000 and that both antibodies bind exclusively to this band. Confirmatory evidence that antibody HK 4.4 binds to the gastric ATPase, rather than a contaminant of the ATPase with the same molecular weight, was obtained by specific immunoprecipitation of the H+ ,K+ -A TPase. The microsomal ATPase was solubilized in Triton X-l00, incubated with antibody, and the precipitates were phosphorylated by

P1ATP in the presence and absence of K+. The precipitates were trapped and washed on nitrocellulose filters, and their 32p content was measured by scintillation counting. Retention of 32p counts on the filters was dependent on prior incubation of the enzyme with unabsorbed antibody; incubation with antibody absorbed with microsomal ATPase was ineffective. These counts were lost when K+ was included in the phosphorylation reaction, as expected of the gastric H+ ,K+ -ATPase, whose phosphorylated intermediate is sensitive to K+ (16). Neither antibody HK 4.4 nor HK 7.22 inhibited hydrolysis of A TP by purified microsomal ATPase. The H+ ,K+ -ATPase immunoassay standard curve was obtained from ELISA of purified microsomal ATPase. As a control for endogenous p-nitrophenyl phosphatase activity, ::S10 J-Lg of gastric ATPase was incubated directly with p-nitrophenyl phosphate for 30 min in 1 M diethanolamine at pH 9.B. Hydrolysis of p-nitrophenyl phosphate was not detected as measured by absorbance at 410 nm. To estimate ATPase binding to the wells, 100-J-Ll aliquots with increasing amounts of 125J-labeled ATPase were incubated in the wells for 1 h, then withdrawn and [

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SMOLKA AND WEINSTEIN

a

b

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c

d

linked antimouse immunoglobulin second antibody, and for spontaneous, nonspecific hydrolysis of the p-nitrophenyl phosphate. The curves correspond closely to simple hyperbolas, but are more exactly fitted by a three-parameter logistic of the following form:

94-

a y=----

67-

43-

30-

20.1Figure 3. Immunoblotting of pig gastric microsomal H+ ,K+ ATPase with monoclonal antibodies HK 4.4 and HK 7.22. The enzyme was resolved on 10% polyacrylamide SDS electrophoresis gels, and the peptide pattern was transferred to nitrocellulose sheets and probed with the antibodies as described in the text. Lane a, molecular weight standards; lane b, Coomassie Blue-stained peptide pattern of the ATPase; lane c, immunoblot with HK 4.4; lane d, immunoblot with HK 7.22.

their content of unbound ATPase was measured by y-counting. Figure 4 shows that at coating concentrations >0.5 j.tg/ml, ~12% of the applied enzyme remains bound to the microplate wells. For the standard curve, enzyme in binding buffer was applied to polystyrene microplate wells at coating concentrations ranging from 0.1 to 20 j.tg/ml. After processing of the wells by ELISA, A 410 values were plotted as a function of 10glO micrograms ATPase applied to the microplate wells. Figure 5 shows the standard curves obtained for pig gastric mucosal H+ ,K+ -ATPase using antibodies HK 4.4 and HK 7.22. Since the microspectrophotometer was blanked against microplate wells with no adsorbed ATPase but otherwise processed identically to the test wells, the curves are corrected for nonspecific binding of monoclonal first antibody and alkaline phosphatase-

where a is the maximum A410 when the microplate well is saturated with antigen, aO.5 is the A410 at half-maximal antigen dose, and m is the slope of the curve. The data points of Figure 5 represent the mean and standard deviation of quadruple assays at each concentration of antigen. Human gastric biopsy specimens were collected and processed, and their H+ ,K+ -ATPase content was estimated by immunoassay using the antibody HK 4.4. In the preparation of micro somes for assay, the centrifugal step partitions 70% of the H+ ,K+ -ATPase activity into the supernatant and 30% into the pellet (2). The ATPase levels in the biopsy specimens measured by the immunoassay, which samples the microsomal supernatant, must therefore be augmented by 30% to reflect the true ATPase content of the specimens. Table 1 shows the estimated gastric ATPase levels (nanograms ATPase per milligram wet weight specimen) in a series of gastric biopsy specimens taken from antral and fundic gland regions of the stomachs of 2 human subjects. Levels of H+ ,K+A TPase were immeasureably low in the antral specimens. The mean ATPase level in fundic gland biopsy specimens was 103 ng ATPase/mg wet wt specimen (SD = 20 ng/mg, n = 9). This figure is comparable to H+,K+ -ATPase levels in pig fundus, based on the yield of peptide with a molecular weight of 95,000 isolated routinely from gastric mucosal homogenates as described earlier (2).

30

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ATPase Applied to Plate (ng)

Figure 4. Binding of 125I-labeled H+ ,K+ -ATPase to polystyrene microplate wells as a function of ATPase concentration.

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Figure 5. Immunoassay standard curves using monoclonal antibodies HK 4.4 and HK 7.22. The data points depict the measured A410 (mean + SD) of quadruple assays at each of 10 coating concentrations of pig gastric microsomal ATPase.

Discussion The preparation of and some properties of monoclonal antibodies directed against the gastric mucosal H+ .K+ -ATPase. which is directly responsible for acid secretion into the stomach. were recently described (5). In the present study, anti-H+,K+Table 1. H+, K+ -ATPase Levels in Human Gastric Mucosal Biopsy Specimens Obtained at Endoscopy Nanograms H+, K+-ATPase/mg wet wt specimen Biopsy specimens Gastric body Gastric body Gastric body Gastric body Gastric body Prepyloric antrum Prepyloric antrum Prepyloric , antrum a

Patient 1a 100

b

71 86

129 <14 <14

Patient 2 100 86

100 129 129 <14 <14 <14

For each patient, multiple specimens were taken at a single endoscopic session. b Values are ATPase levels derived from the standard curve shown in Figure 5, plus the 30% correction allowing for partitioning of microsomes into the nuclear-mitochondrial pellet during preparation of gastric biopsy specimens for assay.

ATPase monoclonal antibodies form the basis of an immunoassay of the gastric proton pump. Enzymelinked immunosorbent assay using polyclonal antisera has previously been applied to the sarcoplasmic reticulum ATPase of rabbit fast muscle (17). The sensitivity of ELISA, allied with the unique specificity of monoclonal antibodies. yields an assay capable of detecting 10 ng, or 100 fmol, of the acid-secreting ATPase. The assay is shown to detect pig and human H+ ,K+ -ATPase and has also been used in the isolation of pepsinogen granules from rabbit gastric mucosa (18) and of H+ ,K+ -ATPase from both rat and rabbit gastric mucosa (unpublished observations). Screening of hybridoma supernatants by ELISA using microsomal ATPase as the bound antigen obviously identifies antibodies against membranebound non-ATPase antigens that happen to copurify with the ATPase. The fact that both HK 4.4 and HK 7.22 bind to an SDS electrophoresis band (of molecular weight 95,000) that constitutes 90% of the protein in the gastric microsomal ATPase preparation and that is specifically phosphorylated from ATP in a K+-sensitive manner (16) is strong presumptive evidence that these monoclonal antibodies are directed against the H+ ,K+ -ATPase. The immunoprecipitation results further suggest that antibody

538 SMOLKA AND WEINSTEIN

HK 4.4 binds to a K+ -sensitive phosphorylatable peptide of the ATPase, most probably a catalytic subunit of the enzyme. In addition, the immunoblot and immunoprecipitation experiments argue that the antigens recognized by HK 4.4 and HK 7.22 are sequence determinants rather than conformation determinants, as antigenicity was preserved after treatment with SDS and Triton X-l00. The immunohistochemical and ELISA crossreactivity of the monoclonq.l antibodies with ATPa.ses of different species is most striking, and suggests that the K+ -dependent. proton-translocating ATPases are highly conserved. Also noteworthy is the absence of immunohistpchemical crossreactivity with Na+ ,K+ -ATPase and mitochondrial Fi/Fo ATPase (no antibody staining in cells other than parietal cells, and no staining of mitochondria). In addition, neither HK 4.4 nor HK 7.22 bind to pig kidney Na+,K+-ATPase and rabbit kidney Na+,K+A TPilse as measured by ELISA using purified preparations of these enzymes as bound alltigens (data not shown). Total homogenates of the human gastric mucosal biopsy specimens could not be used for immunoassay for the following reasons. The immunpassay is based on nonspecific adsorption of membralles to the microplate wells. Microsomal membranes constitute only a fraction of the membranes present in total homogenates. When unfractionated homogenates were plated into the wells, the adsorption of irrelevant membralles limited th!'J amount of microsomal membranes that could be bound. Under these conditions insufficient ATPase was bound to allow detection by the immunoassay, and a preliminary step for concentrating the microsomal membranes became necessary. The inevitable loss of micro somes into the centrifugal pellet requires, therefore, that a correction be made to ATPase levels measured by this immunoassay. The incubation times specified in the immunoassay protocol were selected to allow completion of the assay within 5 h. Th!'J sensitivity of the assay can be improved simply by lengthening exposure of the microplates to the ATPase and by longer incubations with antibodies. Thus, overnight binding of antigen gave 20% more A4i0 than binding for 1 h, while a 15% increase in A4i0 resulted from 2-h rather than l-h incubations with first and second antibodies. A potentially important application of the immunoassay described here is measurement of H+ ,K+ -ATPase levels in human gastric biopsy specimens. Total H+ ,K+ -ATPase content of the biopsy specimen is measured, whether or not the ATPase is hydrolytically active. The immunoassay therefore complements phosphate-release ATPase assays of gastric biopsy specimens, which measure only the

GASTROENTEROLOGY Vol. 90, No.3

activated H+ ,K+ -ATPase, and measurements of acid secretion in gastric biopsy specimens by the [14C]aminopyrine accumulation technique (19). The assay may also be used to identify H+ ,K+ -ATPase synthesis and assembly pathways in isolated subcellular fractions of the parietal cell, before the enzyme has achieved its functional multimeric structure. Similarly, after loss of the enzyme from the apical membrane by the normal processes of protein turnover, with presumed concomitant loss of hydrolytic activity, the immunoassay may be useful in localizing the ATPase to fractionated lysosomes or other intracellular recycling compartments.

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a model for gastric secretion. Rev Physiol Biochem Pharmacol 1977;79:133-6~.

2. Saccomani G, Stewart HB, Shaw D, Lewin M, Sachs G. Characterization of gastric mucosal membranes. IX. Fractionation and purification of potassium ATPase-contajning vesicles by zonal centrifugation and fr!3e flow electrophoresis technique. Biochim Biophys Acta 1977;465:311-30. 3. Sachs G, Chang HH, Rabon E, Schackman R, Lewin M, Saccomani G. A non-electrogenic H+ pump in plasma membranes of hog stomach. J BioI Chern 1976;251:7690-8. 4. Saccomani G, Dailey DW, Sachs G. The action ?f trYPsin on the gastric (H++K+) ATPase. J BioI Chern 1979;254;2821-7. 5. Smolka A, Helander HF, Sachs G. Monoclonal antibodi!3s against gastric proton potassium ATPase. Am J Physiol 1983;245:G589-96. 6. DiBona DR, Ito S, Berglindh T, Sachs G. Cellular site of gastric acid secretion. froc Nat! Acad Sci USA 1979;76:6689-93. 7. Fellenius E, Berglindh T, Sachs G, et al. Substituted benzimidazoles inhibit gastric acid secretion by blocking hydrogen potassium ATPase. Nature 1981;290:159-61. 8. Engvall E, Perlmann P. Enzyme-linked immunosorbent assay, ELISA. III. Quantitation of specific antibodies by enzymelabelled anti-immunoglobulin in antigen-coated tubes. J Immunol 1972;109:129-35. 9. Seppala I, Sarvas H, Peterfy F, Mabila O. The four subclasses of immunoglobulin G can be isolated from mouse S!3rum by using protein A Sepharose. Scand JImmunoI1981;14:335-42. 10. Sainte-Marie G. A paraffin embedding technique for studies employing immunofluorescence. J Histochem Cytoche~ 1962;10:250-6. 11. Gallyas F, Gorc T, Merchenthaler I. High grade intensification of the end product of the diamine benzidine reaction for peroxidase histochemistry. J Histochem Cytochem 1982; 30:183-4. 12. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680-4. 13. Burnette WN. Western blotting. Electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 1981;112:195-203.

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14. Peterson GL. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 1977;83:346-56. 15. Littlefield JW. Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 1964; 145:709-10. 16. Wallmark B, Stewart HB, Rabon E, Saccomani G, Sachs G,

The catalytic cycle of gastric hydrogen potassium ATPase. J BioI Chern 1980;255:5313-9. 17. Betto R, Damiani E, Biral D, Mussini 1. Enzyme linked

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immunoassay for study of sarcoplasmic reticulum ATPase. J Immunol Methods 1981;46:289-98. 18. Peerce B, Smolka A, Sachs G. Isolation of pepsinogen granules from rabbit gastric mucosa. J BioI Chern 1984;259: 9255-62. 19. Fellenius E, Elander B, Wallmark B, Haglund U, Helander HF,

Olbe L. A micro-method for the study of acid secretory function in isolated oxyntic glands from gastroscopic biopsies. Clin Sci 1983;64:423-31.