ANALYTICAL
BIOCHEMISTRY
The Preparation Solid-State
65, 175-
of a Highly
Immunoassay H. JAMES
186 ( 1975)
for
Purified Porcine
Antibody Pancreatic
WEDNER, LAWRENCE N. PARKER, MICHAEL G. ROSENFELD’
and
a
a-Amylase AND
Department of Medicine. Washirlgton University School of Medicine, St. Louis. Missouri 63130. and Department oj’ Medicine, Dirxision of Endocrinology. Uni\,ersity of Calijornia. San Diego. School of Medicine, La Jolla, California 92037 Received
August
2, 1974;
accepted
November
25. 1974
Porcine pancreas and parotid cell suspensions provide model systems in which to study the mechanism of induction of a specific protein, o-amylase. by hormones acting via CAMP. A highly purified antibody against porcine pancreatic LYamylase was prepared using affinity chromatography of the total IgG fraction derived from rabbit anti-cu-amylase antiserum and was used to develop a radioimmunoassay for cr-amylase. The antigen-antibody complex was separated from unbound cu-amylase using either glutaraldehyde gelled cu-amylase or a second antibody technique: a linear standard curve was achieved over a IOO- to 1000-fold range of cY-amylase concentration. Tissue homogenates did not interfere with this assay, and assayed levels of cY-amylase in porcine pancreas were linear using IO-300 pg of homogenate. No levels or very low levels of cu-amylase were detected in control tissues.
The mechanism is unknown by which hormones acting via CAMP induce synthesis of specific proteins. On the basis of drug inhibitor studies in several target tissues, it has been suggested that CAMP can induce protein synthesis in a manner not dependent on new mRNA synthesis, presumably by acting at a posttranscriptional level (l-6). Unequivocal demonstration of posttranscriptional control by CAMP will require direct quantitation of intracellular levels of mRNA coding for an inducible protein, as has been accomplished in several mRNAs coding for proteins induced by steroid hormones (7,8). In order to study this question, induction of cu-amylase by CAMP and hormones in the porcine pancreas and parotid gland have been chosen as model systems. We here report the development and extensive purification of an antibody against porcine pancreatic a-amylase and development of a radioimmunoassay which permits quantitation of a-amylase in the pancreas. Such an antibody can also potentially be used for immunoprecipitation of polysomes bearing nascent chains of cY-amylase as has been accomplished in other tissues (9.10). I Teaching
and Research
Scholar
of the American 175
Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved
College
of Physicians
176
WEDNER,
PARKER
MATERIALS
AND
AND
ROSENFELD
METHODS
cr-Amylase from porcine pancreas was purchased from Sigma Chemical Company, Worthington Biochemical Corporation, and Calbiochem; Sepharose 4B beads were purchased from Worthington Biochemical Corporation; cyanogen bromide was obtained from Eastman Organic Chemicals; and carrier-free lz51 was obtained from New England Nuclear. Ovalbumin and hemoglobin were obtained from Sigma Chemical Company and recrystallized albumin was from Miles Laboratories, Inc. Lactoperoxidase was purchased from Calbiochem and goat anti-rabbit Ig antisera was obtained from ICN Nutritional Biochemicals Corporation. All other chemicals were reagent grade. Methods. Preparation of Specifically PuriJed Antibody. Purified CXamylase from porcine pancreas prepared by Worthington Biochemical Corporation was diluted to 2 mg/ml in 0.01 M phosphate (pH 7.39, 0.15 M NaCl (PBS) and emulsified in an equal volume of complete Freund’s aduvant (CFA). One milliliter of the cY-amylase suspension was injected into the foot pads of four New Zealand white rabbits. At 6 and 8 weeks following the initial injections, the animals received 100 pg of cY-amylase in CFA in the foot pads, and the animals were bled from an ear vein ten days following the final injection. All animals produced precipitating antibodies to cu-amylase as demonstrated by Ouchterlony double agar diffusion (1 1). Antisera from animals producing a single band on diffusion studies and Worthington Biochemical Corporation a-amylase were used for additional purification of a-amylase antibodies. IyG from anti-aamylase antisera was prepared by ammonium sulfate precipitation and DEAE-cellulose column chromatography in 0.01 M phosphate (pH 7.0), 10 mM NaCl (12). Additional purification of anti-a-amylase antibodies was prepared using a gel of a-amylase as described by Avrameas and Ternynck (13). Fifty milligrams of cY-amylase was added to 3 ml of buffer containing 25 mM sodium acetate (pH 6.0). 25 mM NaCl, and 0.15 ml of 12.5% glutaraldehyde. The mixture was agitated, allowed to sit for 60 min at room temperature, and then cooled to 4°C. An additional 2 ml of acetate buffer was added, and the partially gelled material homogenized with 10 strokes in a Dounce homogenizer. This procedure was repeated 1 h later. The a-amylase gel was then centrifuged to 500g for 1 min and washed three times with 10 ml of the acetate buffer. Following the third wash, the supernatant was discarded and the pellet resuspended in 10 c3 of 100 mM glycine (pH 6.2). The aggregated cr-amylase was repipetted in a Pasteur pipet until a uniform particle size was achieved and the “slurry” was always stored in 100 mM glycine (pH 6.2). Prior to use, the slurry was pelleted by centrifugation and washed twice in 25 mM TrisMaterials.
a-AMYLASE
RADIOIMMUNOASSAY
177
HCl (pH 7.4), 25 mM KCl, 5 mM M&l, (buffer A) such that the final concentration was 0.1 mg cr-amylase per 0.2 ml. Purified IyG from 75 ml of blood was incubated with the a-amylase gel for 1 h at room temperature, and the gel was washed three times with PBS. Antibody was eluted from the gel with three washes of glycine-HCl, pH 2.8. The purified antibodies (1 mg/ml) were dialyzed against buffer containing 10 mM potassium phosphate (pH 7.2), 25 mM KCI. 5 mM MgCl,, and passed through a column containing autoclaved DEAE-cellulose (10 cm X 1 cm) layered over autoclaved carboxymethylcellulose (5 cm x 1 cm), and eluted by this same buffer. This procedure removes contaminating ribonuclease. Fractions containing peak activity were stored at -70°C. Zodination of a-amylase. Iodination was done by the solid-state lactoperoxidase method of David and Reisfeld (14), using 250-350 &i carrier-free lZJI. a-Amylase solution was diluted with water to achieve a final concentration of 0.025-2.5 mg/ml depending on final specific activity desired, 250-350 PCi of carrier-free 1251,and lo-20 ~1 of Sepharose beads were added on which lactoperoxidase has been immobilized. The reaction was initiated by the addition of H202 to 1.8 x lo-” M final concentration and continued at room temperature for 20 min: the reaction was terminated by adjusting the reaction mix to 0.025 M sodium azide and 0.05 M KI and the Sepharose beads pelleted by centrifugation. The supernatant was then dialyzed for 72 h against 4000 ml of buffer containing 25 mM Tris-HCl (pH 7.6), 25 mM KCl, and 10 mM KI with a change of buffer every 8 h. The iodinated a+amylase comigrated with an unlabeled a-amylase standard when subjected to electrophoresis on 12.5% SDS polyacrylamide gels (15) with a calculated MW of 56,000 + 500 in agreement with the MW calculated by others (16). Specific activity varied from 40,000 cpm/pg to 2,000,OOO cpm/pg. When specific activity of the a-amylase was greater than 450,000 cpmlpg, the labeled a-amylase was further processed by adsorption on immobilized antibody. Antibody was dialyzed against buffer containing 200 mM sodium citrate (pH 6.5), 25 mM NaCl, and 5 mM MgC12. Eight milligrams antibody (2 mg/ml final concentration) was added to 1 ml Sepharose 4B beads, activated with cyanogen bromide as described by Cuatrecasas (17) and allowed to react for 14 h at 4°C. The beads were then extensively washed with 100 mM glycine (pH 6.2). When used to purify [‘251]~-amylase, 0.1 ml of beads was agitated with labeled a-amylase in buffer A for 1 h at 4°C. The beads were then pelleted, washed five times with buffer, and the [ ‘ZjI]a-amylase eluted by addition of 100 mM glycine-HCl (pH 2.6) for 10 min at 4°C and dialyzed against buffer A prior to use. Solid-phase immwoassay procedure. All procedures were performed using autoclaved 10 X 75 disposable glass culture tubes. One milligram
178
WEDNER,
PARKER
AND
ROSENFELD
of albumin and 5-50 ng of [‘“jI]a-amylase were added to a volume of 0.5 ml buffer A. Samples to be assayed or known amounts of unlabeled a-amylase were added in an additional 0.5 ml of buffer A and the samples were agitated using a vortex mixer. Antibody was added at the indicated concentration (0.2-2.0 pug/ml), the samples were agitated and, following incubations at 4°C for 12 h, 0.1 M of the glutaraldehyde-fixed a-amylase slurry (0.1 mg) was added, the samples were agitated, and incubations continued for an additional 6 h. The cY-amylase slurry then was sedimented by centrifugation at 2000 rpm in an International Clinical Centrifuge and the supernatant was decanted and saved. The Pellet was washed three times with 2 ml of buffer A and the slurry was resuspended after each sedimentation. The supernatant and the pellet were then counted in a gamma counter with settings appropriate for “‘1. Immunoassay using goat anti-rabbit Zg. To further validate the solidphase immunoassay, the anti-amylase-cr-amylase complex was precipitated with goat anti-rabbit IgG. Reaction mixtures were identical to those described above except that 10h of a 1: 10 dilution of an IgG fraction of goat anti-rabbit IgG antisera (1 mglml) (previously determined to be the optimal concentration for maximal precipitation) was added: the tubes were agitated and then incubated for 26 h at 4°C. Following incubation, 2.5 ml of HZ0 was added and the tubes were spun at 3000 rpm in the IEC P-2 centrifuge for 30 min. The supernatant was decanted and the precipitate counted in a gamma counter. Under these conditions, less than 1% of the added [“51](Y-amylase was nonspecifically precipitated. Preparation of tissue for immunoassay. Fifty-pound pigs were anesthetized using pentabarbital and rapidly exsanguinated from the jugular arteries. Tissues to be assayed were immediately removed and placed in ice-cold saline. Tissues were minced and homogenized in 3 vol of buffer A per g of tissue using 20 strokes with a motor-driven Teflon pestle. The homogenates were adjusted with 0.1 M NaOH to pH 8.7 and Triton X- 100 was added to a final concentration of 0.1%. The homogenates were then sonicated at power 5 for 30 s using a Bronson sonicator and sedimented at 10,OOOg for 15 min in a Sorvall RC2. This procedure was designed to ensure quantitative release of a-amylase stored in zymogen granules or associated with nuclei and microsomes (18) in order that total cell a-amylase could be accurately determined. This procedure consistently released more than 98% of total cell a-amylase as determined by enzymatic assay of the 10,OOOg supernatant. Enzymatic assay for tissue a-amylase. a-Amylase-glycogen complexes were extracted from tissue homogenates as described by Loyter and Schramm (19), and enzymatic activity was measured by the method of Bernfeld (20). Displacement oj’[ 1251]cx-amylase by unlabeleda-amylase. Displacement
CY-AMYLASE
179
~D~O~MMUNOASSAY
0.1
1.0 IO AMYLASE ADDED
100 ingr'2.5)
IO00
FIG. 1. Fifty nanograms of [ lpjl] a-amylase (210,000 cpm/pg) was added to 1 ml of buffer A containing 1 mg albumin and varying amounts of unlabeled Lu-amylase (0.25 ng-2.5 pg). Purified anti-cr-amylase antibody (1 pg) was added and incubations continued at 4°C for 16 h. The n-amylase gel (0.3 mg protein} was then added and incubations at 4°C continued for 6 h with occasional agitation. The slurry was then pelleted and washed as described under Materials and Methods. Resuits are plotted as percentage maximal bound: free radioactivity against amount of unlabeled cu-amylase added as plotted on a log 10 scale. Similar results have been obtained in fifteen experiments of similar design.
1500 1400 1300 1200 g Ii00 8 1000 z 900 -:
800 -
t g
700 600
E 500 :: 400 300 -
'\ '-\v
200 100 IO
100
1000
1 10,000
AMYLASE ADDED
Frc. 2. Immunoassay for cY-amylase using IgG fraction of goat anti-rabbit IgG to precipitate for antigen-antibody complex. One milligram albumin, 25 ng ru-amylase (11 1,250 cpm/pg), varying concentrations of unlabeled a-amylase, IO ~1 at 1: 10 dilution of the IgG fraction of goat anti-rabbit IgG were added to 0.5 ml of buffer A. Incubations were for 36 h; the precipitate was collected and counted as described in Methods. Results are the average of duplicates differing by less than 2% and the a-amylase added is plotted on a log 10 scale.
180
WEDNER,
PARKER
I I 0.1 I PROTEIN
AND
ROSENFELD
I 1.0 100 ADDED ("g/2.5)
I 1000
FIG. 3. Specificity of competition of binding of [ ‘251]a-amylase. The assay was designed as described in Fig. 1 except that several proteins were added in the concentrations indicated on the log 10 scale of the abscissa. @---a, cY-amylase: O---O, albumin; n---n, ovalbumin; O---O, hemoglobin.
of [ 1251]a-amylase binding to antibody by unlabeled cr-amylase, as determined by a-amylase gel precipitation, was linear over a three to four log concentration of a-amylase (Fig. 1). Duplicates differed by less than 4%. The binding occurred rapidly: however, consistent duplicates were obtained with long incubations. Incubation at 4°C was as effective as incubations at room temperature. Amounts of a-amylase gel added could be varied from 0.10-0.5 mg per assay without alteration in results. Interassay variation differed by less than 10%. Comparison of solid-phase immunoassay with precipitation by second antibody. The displacement of labeled cu-amylase by known amounts of cr-amylase using precipitation by goat anti-rabbit IgG is shown in Fig. 2. The displacement curve was similar to that obtained using gelled (Yamylase. Specificity of reaction. The specificity of the assay was confirmed by failure of other proteins to compete for the binding of [‘251]a-amylase (Fig. 3). In order to document the fact that the immunoassay had appropriate specificity to allow assay of tissue without initial purification, known amounts of purified porcine a-amylase were added to homogenates of porcine kidney, liver, and muscle. The tissue homogenates had no effect on the competition of [ 1251]~-amylase binding by the added unlabeled a-amylase (Table 1). Pancreatic homogenate did compete with [ 1251]a-amylase binding and, as shown in Fig. 4, the assay was linear over a wide range of homogenate added. The specificity of the immunoassay was additionally tested by comparing the enzymatic assay and radioimmunoassay using tissues which contain little a-amylase activity or which are believed to have entirely
a!-AMYLASE
181
RADIOIMMUNOASSAY
TABLE
1
EFFECTOFTISSUEHOMOGENATESONIMMUNOASSAYOFOL-AMYLASE"
Tissue homogenate added
Unlabeled cu-amylase added
None
2.5 pg
Salivary gland Salivary gland Liver Liver Kidney Kidney
2.5 yg -
[‘251]ol-Amylase bound (cpm) 1.235 539 1,179 539 1,129 542 1.138 532
2.5 Pi2 -
2.5 pg
’ Tissue homogenates were prepared as described in Methods and, where indicated, 1Opg of each homogenate was added to 1 ml of buffer A, 250 ng [1251]ol-amylase (8200 cpm/pg), and antibody (500 @ml) was added to each reaction mix, and the bound [*251]ol-amylase was quantitated as described in Methods. Results are the average of duplicates differing by less than 3%.
12,000 I I.000 10.000 9000
G -5 is ?I g < z LL
8000 7000 6000 5000 4000
,_____
--------
30
I
60
TISSUE
90
_------z
a--__-----------I
120
I
I
I
160 190 210 240
HOMOGENATE
I
270
ASSAYED Cpg)
4. Radioimmunoassayable a-amylase as a function of the amount of tissue homogenate added to the assay. Pancreatic and liver tissue homogenates were prepared as described in Methods and radioimmunoassay was accomplished as described in Fig. 1 varying the pg of protein of homogenate added. Duplicate determinations differed by less than 4%. a-0, pancreatic homogenate; O-O, liver homogenate. FIG.
182
WEDNER,
PARKER
TABLE COMPARISON
OF ENZYMATIC
AND
ROSENFELD
2
AND RADIOIMMUNOASSAYABLE
(Y-AMYLASE~
Tissue homogenate assayed
o-Amylase enzymatic activity (Uimg protein) % maximal activity
RIAo-amylase (ng/lO~g total protein)
Liver Salivary gland Kidney Muscle Parotid gland Pancreas
1.5 3.4 8.3 35.5 100.0 68.2
90%, are obtained when binding to gelled CYamylase is used as the criterion for purity. The latter estimate is probably more accurate since the antibodies with highest affinity for txamylase may not be eluted from the gel, resulting in an underestimation of the purity of the antibody. DISCUSSION It is well established that CAMP serves as a second messenger and mediates many of the intracellular effects produced by polypeptide hormones and biogenic amines (19,20). The mechanism by which hormones acting via CAMP rapidly induce synthesis of specific proteins in their evidence target tissues remains unresolved. There is inconclusive suggesting that new RNA synthesis may not be required for induction of proteins in several model systems (l-6). In order to study this question, model systems have been selected in which CAMP rapidly induces synthesis of a specific protein. In pancreatic minces, CAMP, PGEI, and pancreozymin induce a rapid increase in the synthesis of cu-amylase
a-AMYLASE
RADIOIMMUNOASSAY
185
(M.G.R., unpublished data). In isolated cell suspensions from parotid gland, epinephrine or CAMP stimulate synthesis of cY-amylase (5,6). These studies require a highly purified antibody to porcine a-amylase and a sensitive radioimmunoassay to permit its quantitation. Rabbit anti-a-amylase antibody was prepared by standard techniques and purified by affinity chromatography of the IgG fraction of the rabbit antiserum with at least an 18- to 20-fold increase in specific activity. The antibody preparation was further purified by DEAE-cellulose, CMcellulose chromatography to remove traces of contaminating ribonuclease in order to permit use of the antibody for additional studies involving immuno-precipitation of the polyribosomes bearing nascent cY-amylase chains. The immunoassay which was devised permits reproducible quantitation of a-amylase in the range of 10 ng- 10 pg of ac-amylase. The immunoassay demonstrates a linear a-amylase content over a broad range of concentrations of tissue homogenates assayed and has been successfully utilized to quantitate porcine pancreatic a-amylase levels. The sensitivity of the assay is entirely adequate to permit quantitation of QIamylase in minces or suspension cultures of pancreatic acinar cells. In addition, it appears that the antibody to pancreatic cu-amylase is not applicable to studies of the porcine parotid gland. Studies in other species have been interpreted to suggest that pancreatic and parotid CYamylase are not immunologically identical, although a weak cross reactivity was seen with anti-pancreatic a-amylase serum and parotid tissue homogenates (16). Other studies have suggested pancreatic and salivary a-amylases were immunologically very similar (21). The apparently low estimates of a-amylase content of porcine parotid gland using competition with [‘““I] pancreatic a-amylase as the assay could mean (i) that only a small percentage of the total parotid a-amylase is identical to that in the pancreas, or (il’) that there is limited immunological cross reactivity between the parotid and pancreatic forms with the apparently low levels of parotid a-amylase reflecting competition with pancreatic CYamylase which was used to generate the standard curve. Resolution of this question will be achieved when an antibody to porcine parotid (Yamylase is available for study. The assay described appears to be specific for a-amylase since it detects a-amylase only in tissues which are known to contain the enzyme and the protein precipitated by the immunoassay technique comigrates with a porcine a-amylase standard when analyzed by SDS-polyacrylamide gel electrophoresis. The use of glutaraldehyde gelled cY-amylase to precipitate antigen-antibody complexes in the immunoassay was chosen because of the desirability of this method in preparation of the polysomes bearing a-amylase chains. A double antibody precipitation method for immunoassay of cy-amylase is an effective alternative.
186
WEDNER,
PARKER
AND
ROSENFELD
The preparation of a highly purified ribonuclease-free antibody to porcine pancreatic cY-amylase and its use in a highly sensitive and specific radioimmunoassay permits documentation of the CAMP stimulation of a-amylase synthesis in the porcine pancreas and parotid gland. Exploration of the mechanism of this induction will be reported elsewhere. ACKNOWLEDGMENTS The authors gratefully acknowledge the aid of Dr. Gary David for iodination techniques and Dr. Charles Parker for advice and review of the manuscript. This investigation was supported by PHS Research Grant No. AM13 149 from the National Institute of Arthritis, Metabolism & Digestive Diseases and by Grant No. BC169 from the American Cancer Society.
REFERENCES 1. Ferguson, J. J. Jr., (1963) J. Biok Chem. 238, 2754-2759. 2. Garren, L. D., Ney, R. L., and Davis, W. W. (1965) Proc. Nat. Acad. Sci. [/,%I 53, 1443-1450. 3. Grand, R. J., and Gross, R. P. (1970) Proc. Nat. Acad. Sci. USA 65, 1081-1088. 4. Wicks, W. D. (1971) J. Biol. Chem. 246, 2 17-223. 5. Labrie. F., Beraud, G.. Gauthier, M., and Lemay, A. (1971) J. Biol. Chem. 246, 1902-1908. 6. Grand, R. J., and Gross, P. R. (1969)J. Biol. Chem. 244, 5608-5615. 7. Comstock. J. P., Rosenfeld, G. C., O’Malley. B. W., and Means, A. R. (1972) Proc. Nat. Acad. Sci. USA 69, 2377-2380. 8. O’Malley, B. W., Rosenfeld, G. C., Comstock. J. P., and Means, A. R. (1972) Nature New Biol. 240, 45-47. 9. Palacios, R., and Schimke, R. T. (1973) J. Biol. Chem. 248, 1424-1430. 10. Palmiter, R. D.. Palacios, R., and Schimke, R. T. (1972) J. Biol. Chem. 247, 3296-3304. 11. Ouchterlony. 0. (1948) Acta Pathol. Microbial. Scnnd. 25, 186- 19 1. 12. Fahey. J. L. (1971) in Methods in Immunology and Immunochemistry (Williams, C. A.. and Chase, M. W., eds), Vol. I, p. 321, Academic Press, New York. 13. Avrameas, S., and Ternynck, T. (1969) Immunochemistry 6, 53-66. 14. David, G. S., and Reisfeld, R. A. (1974) Biochemistry 13, 1014-1021. 15. Neville, D. M.. Jr. (1971) J. Biol. Chem. 246, 6328-6334. 16. Sanders. T. G.. and Rutter, W. J. (1972) Biochemistry 11, 130-136. 17. Cuatrecasas, P. (1970) /. Biol. Chem. 245, 3059-3065. 18. Schramm. M., and Danon, D. (1961) Biochim. Biophys. Acta 50, 102-I 12. 19. Loyter. A.. and Schramm, M. (1962) Biochim. Biophys. Acta 65, 200-206. 20. Bernfeld, P. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds). Vol. 1, p. 149, Academic Press, New York. 21. McGeachin. R. L., Pavord. M. W., Widmer, D. N.. and Prell, P. A. (1966) Comp. Biochem. Physiol. 18, 767-772.
BIOCHEMISTRY
The Preparation Solid-State
65, 175-
of a Highly
Immunoassay H. JAMES
186 ( 1975)
for
Purified Porcine
Antibody Pancreatic
WEDNER, LAWRENCE N. PARKER, MICHAEL G. ROSENFELD’
and
a
a-Amylase AND
Department of Medicine. Washirlgton University School of Medicine, St. Louis. Missouri 63130. and Department oj’ Medicine, Dirxision of Endocrinology. Uni\,ersity of Calijornia. San Diego. School of Medicine, La Jolla, California 92037 Received
August
2, 1974;
accepted
November
25. 1974
Porcine pancreas and parotid cell suspensions provide model systems in which to study the mechanism of induction of a specific protein, o-amylase. by hormones acting via CAMP. A highly purified antibody against porcine pancreatic LYamylase was prepared using affinity chromatography of the total IgG fraction derived from rabbit anti-cu-amylase antiserum and was used to develop a radioimmunoassay for cr-amylase. The antigen-antibody complex was separated from unbound cu-amylase using either glutaraldehyde gelled cu-amylase or a second antibody technique: a linear standard curve was achieved over a IOO- to 1000-fold range of cY-amylase concentration. Tissue homogenates did not interfere with this assay, and assayed levels of cY-amylase in porcine pancreas were linear using IO-300 pg of homogenate. No levels or very low levels of cu-amylase were detected in control tissues.
The mechanism is unknown by which hormones acting via CAMP induce synthesis of specific proteins. On the basis of drug inhibitor studies in several target tissues, it has been suggested that CAMP can induce protein synthesis in a manner not dependent on new mRNA synthesis, presumably by acting at a posttranscriptional level (l-6). Unequivocal demonstration of posttranscriptional control by CAMP will require direct quantitation of intracellular levels of mRNA coding for an inducible protein, as has been accomplished in several mRNAs coding for proteins induced by steroid hormones (7,8). In order to study this question, induction of cu-amylase by CAMP and hormones in the porcine pancreas and parotid gland have been chosen as model systems. We here report the development and extensive purification of an antibody against porcine pancreatic a-amylase and development of a radioimmunoassay which permits quantitation of a-amylase in the pancreas. Such an antibody can also potentially be used for immunoprecipitation of polysomes bearing nascent chains of cY-amylase as has been accomplished in other tissues (9.10). I Teaching
and Research
Scholar
of the American 175
Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved
College
of Physicians
176
WEDNER,
PARKER
MATERIALS
AND
AND
ROSENFELD
METHODS
cr-Amylase from porcine pancreas was purchased from Sigma Chemical Company, Worthington Biochemical Corporation, and Calbiochem; Sepharose 4B beads were purchased from Worthington Biochemical Corporation; cyanogen bromide was obtained from Eastman Organic Chemicals; and carrier-free lz51 was obtained from New England Nuclear. Ovalbumin and hemoglobin were obtained from Sigma Chemical Company and recrystallized albumin was from Miles Laboratories, Inc. Lactoperoxidase was purchased from Calbiochem and goat anti-rabbit Ig antisera was obtained from ICN Nutritional Biochemicals Corporation. All other chemicals were reagent grade. Methods. Preparation of Specifically PuriJed Antibody. Purified CXamylase from porcine pancreas prepared by Worthington Biochemical Corporation was diluted to 2 mg/ml in 0.01 M phosphate (pH 7.39, 0.15 M NaCl (PBS) and emulsified in an equal volume of complete Freund’s aduvant (CFA). One milliliter of the cY-amylase suspension was injected into the foot pads of four New Zealand white rabbits. At 6 and 8 weeks following the initial injections, the animals received 100 pg of cY-amylase in CFA in the foot pads, and the animals were bled from an ear vein ten days following the final injection. All animals produced precipitating antibodies to cu-amylase as demonstrated by Ouchterlony double agar diffusion (1 1). Antisera from animals producing a single band on diffusion studies and Worthington Biochemical Corporation a-amylase were used for additional purification of a-amylase antibodies. IyG from anti-aamylase antisera was prepared by ammonium sulfate precipitation and DEAE-cellulose column chromatography in 0.01 M phosphate (pH 7.0), 10 mM NaCl (12). Additional purification of anti-a-amylase antibodies was prepared using a gel of a-amylase as described by Avrameas and Ternynck (13). Fifty milligrams of cY-amylase was added to 3 ml of buffer containing 25 mM sodium acetate (pH 6.0). 25 mM NaCl, and 0.15 ml of 12.5% glutaraldehyde. The mixture was agitated, allowed to sit for 60 min at room temperature, and then cooled to 4°C. An additional 2 ml of acetate buffer was added, and the partially gelled material homogenized with 10 strokes in a Dounce homogenizer. This procedure was repeated 1 h later. The a-amylase gel was then centrifuged to 500g for 1 min and washed three times with 10 ml of the acetate buffer. Following the third wash, the supernatant was discarded and the pellet resuspended in 10 c3 of 100 mM glycine (pH 6.2). The aggregated cr-amylase was repipetted in a Pasteur pipet until a uniform particle size was achieved and the “slurry” was always stored in 100 mM glycine (pH 6.2). Prior to use, the slurry was pelleted by centrifugation and washed twice in 25 mM TrisMaterials.
a-AMYLASE
RADIOIMMUNOASSAY
177
HCl (pH 7.4), 25 mM KCl, 5 mM M&l, (buffer A) such that the final concentration was 0.1 mg cr-amylase per 0.2 ml. Purified IyG from 75 ml of blood was incubated with the a-amylase gel for 1 h at room temperature, and the gel was washed three times with PBS. Antibody was eluted from the gel with three washes of glycine-HCl, pH 2.8. The purified antibodies (1 mg/ml) were dialyzed against buffer containing 10 mM potassium phosphate (pH 7.2), 25 mM KCI. 5 mM MgCl,, and passed through a column containing autoclaved DEAE-cellulose (10 cm X 1 cm) layered over autoclaved carboxymethylcellulose (5 cm x 1 cm), and eluted by this same buffer. This procedure removes contaminating ribonuclease. Fractions containing peak activity were stored at -70°C. Zodination of a-amylase. Iodination was done by the solid-state lactoperoxidase method of David and Reisfeld (14), using 250-350 &i carrier-free lZJI. a-Amylase solution was diluted with water to achieve a final concentration of 0.025-2.5 mg/ml depending on final specific activity desired, 250-350 PCi of carrier-free 1251,and lo-20 ~1 of Sepharose beads were added on which lactoperoxidase has been immobilized. The reaction was initiated by the addition of H202 to 1.8 x lo-” M final concentration and continued at room temperature for 20 min: the reaction was terminated by adjusting the reaction mix to 0.025 M sodium azide and 0.05 M KI and the Sepharose beads pelleted by centrifugation. The supernatant was then dialyzed for 72 h against 4000 ml of buffer containing 25 mM Tris-HCl (pH 7.6), 25 mM KCl, and 10 mM KI with a change of buffer every 8 h. The iodinated a+amylase comigrated with an unlabeled a-amylase standard when subjected to electrophoresis on 12.5% SDS polyacrylamide gels (15) with a calculated MW of 56,000 + 500 in agreement with the MW calculated by others (16). Specific activity varied from 40,000 cpm/pg to 2,000,OOO cpm/pg. When specific activity of the a-amylase was greater than 450,000 cpmlpg, the labeled a-amylase was further processed by adsorption on immobilized antibody. Antibody was dialyzed against buffer containing 200 mM sodium citrate (pH 6.5), 25 mM NaCl, and 5 mM MgC12. Eight milligrams antibody (2 mg/ml final concentration) was added to 1 ml Sepharose 4B beads, activated with cyanogen bromide as described by Cuatrecasas (17) and allowed to react for 14 h at 4°C. The beads were then extensively washed with 100 mM glycine (pH 6.2). When used to purify [‘251]~-amylase, 0.1 ml of beads was agitated with labeled a-amylase in buffer A for 1 h at 4°C. The beads were then pelleted, washed five times with buffer, and the [ ‘ZjI]a-amylase eluted by addition of 100 mM glycine-HCl (pH 2.6) for 10 min at 4°C and dialyzed against buffer A prior to use. Solid-phase immwoassay procedure. All procedures were performed using autoclaved 10 X 75 disposable glass culture tubes. One milligram
178
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ROSENFELD
of albumin and 5-50 ng of [‘“jI]a-amylase were added to a volume of 0.5 ml buffer A. Samples to be assayed or known amounts of unlabeled a-amylase were added in an additional 0.5 ml of buffer A and the samples were agitated using a vortex mixer. Antibody was added at the indicated concentration (0.2-2.0 pug/ml), the samples were agitated and, following incubations at 4°C for 12 h, 0.1 M of the glutaraldehyde-fixed a-amylase slurry (0.1 mg) was added, the samples were agitated, and incubations continued for an additional 6 h. The cY-amylase slurry then was sedimented by centrifugation at 2000 rpm in an International Clinical Centrifuge and the supernatant was decanted and saved. The Pellet was washed three times with 2 ml of buffer A and the slurry was resuspended after each sedimentation. The supernatant and the pellet were then counted in a gamma counter with settings appropriate for “‘1. Immunoassay using goat anti-rabbit Zg. To further validate the solidphase immunoassay, the anti-amylase-cr-amylase complex was precipitated with goat anti-rabbit IgG. Reaction mixtures were identical to those described above except that 10h of a 1: 10 dilution of an IgG fraction of goat anti-rabbit IgG antisera (1 mglml) (previously determined to be the optimal concentration for maximal precipitation) was added: the tubes were agitated and then incubated for 26 h at 4°C. Following incubation, 2.5 ml of HZ0 was added and the tubes were spun at 3000 rpm in the IEC P-2 centrifuge for 30 min. The supernatant was decanted and the precipitate counted in a gamma counter. Under these conditions, less than 1% of the added [“51](Y-amylase was nonspecifically precipitated. Preparation of tissue for immunoassay. Fifty-pound pigs were anesthetized using pentabarbital and rapidly exsanguinated from the jugular arteries. Tissues to be assayed were immediately removed and placed in ice-cold saline. Tissues were minced and homogenized in 3 vol of buffer A per g of tissue using 20 strokes with a motor-driven Teflon pestle. The homogenates were adjusted with 0.1 M NaOH to pH 8.7 and Triton X- 100 was added to a final concentration of 0.1%. The homogenates were then sonicated at power 5 for 30 s using a Bronson sonicator and sedimented at 10,OOOg for 15 min in a Sorvall RC2. This procedure was designed to ensure quantitative release of a-amylase stored in zymogen granules or associated with nuclei and microsomes (18) in order that total cell a-amylase could be accurately determined. This procedure consistently released more than 98% of total cell a-amylase as determined by enzymatic assay of the 10,OOOg supernatant. Enzymatic assay for tissue a-amylase. a-Amylase-glycogen complexes were extracted from tissue homogenates as described by Loyter and Schramm (19), and enzymatic activity was measured by the method of Bernfeld (20). Displacement oj’[ 1251]cx-amylase by unlabeleda-amylase. Displacement
CY-AMYLASE
179
~D~O~MMUNOASSAY
0.1
1.0 IO AMYLASE ADDED
100 ingr'2.5)
IO00
FIG. 1. Fifty nanograms of [ lpjl] a-amylase (210,000 cpm/pg) was added to 1 ml of buffer A containing 1 mg albumin and varying amounts of unlabeled Lu-amylase (0.25 ng-2.5 pg). Purified anti-cr-amylase antibody (1 pg) was added and incubations continued at 4°C for 16 h. The n-amylase gel (0.3 mg protein} was then added and incubations at 4°C continued for 6 h with occasional agitation. The slurry was then pelleted and washed as described under Materials and Methods. Resuits are plotted as percentage maximal bound: free radioactivity against amount of unlabeled cu-amylase added as plotted on a log 10 scale. Similar results have been obtained in fifteen experiments of similar design.
1500 1400 1300 1200 g Ii00 8 1000 z 900 -:
800 -
t g
700 600
E 500 :: 400 300 -
'\ '-\v
200 100 IO
100
1000
1 10,000
AMYLASE ADDED
Frc. 2. Immunoassay for cY-amylase using IgG fraction of goat anti-rabbit IgG to precipitate for antigen-antibody complex. One milligram albumin, 25 ng ru-amylase (11 1,250 cpm/pg), varying concentrations of unlabeled a-amylase, IO ~1 at 1: 10 dilution of the IgG fraction of goat anti-rabbit IgG were added to 0.5 ml of buffer A. Incubations were for 36 h; the precipitate was collected and counted as described in Methods. Results are the average of duplicates differing by less than 2% and the a-amylase added is plotted on a log 10 scale.
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WEDNER,
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I I 0.1 I PROTEIN
AND
ROSENFELD
I 1.0 100 ADDED ("g/2.5)
I 1000
FIG. 3. Specificity of competition of binding of [ ‘251]a-amylase. The assay was designed as described in Fig. 1 except that several proteins were added in the concentrations indicated on the log 10 scale of the abscissa. @---a, cY-amylase: O---O, albumin; n---n, ovalbumin; O---O, hemoglobin.
of [ 1251]a-amylase binding to antibody by unlabeled cr-amylase, as determined by a-amylase gel precipitation, was linear over a three to four log concentration of a-amylase (Fig. 1). Duplicates differed by less than 4%. The binding occurred rapidly: however, consistent duplicates were obtained with long incubations. Incubation at 4°C was as effective as incubations at room temperature. Amounts of a-amylase gel added could be varied from 0.10-0.5 mg per assay without alteration in results. Interassay variation differed by less than 10%. Comparison of solid-phase immunoassay with precipitation by second antibody. The displacement of labeled cu-amylase by known amounts of cr-amylase using precipitation by goat anti-rabbit IgG is shown in Fig. 2. The displacement curve was similar to that obtained using gelled (Yamylase. Specificity of reaction. The specificity of the assay was confirmed by failure of other proteins to compete for the binding of [‘251]a-amylase (Fig. 3). In order to document the fact that the immunoassay had appropriate specificity to allow assay of tissue without initial purification, known amounts of purified porcine a-amylase were added to homogenates of porcine kidney, liver, and muscle. The tissue homogenates had no effect on the competition of [ 1251]~-amylase binding by the added unlabeled a-amylase (Table 1). Pancreatic homogenate did compete with [ 1251]a-amylase binding and, as shown in Fig. 4, the assay was linear over a wide range of homogenate added. The specificity of the immunoassay was additionally tested by comparing the enzymatic assay and radioimmunoassay using tissues which contain little a-amylase activity or which are believed to have entirely
a!-AMYLASE
181
RADIOIMMUNOASSAY
TABLE
1
EFFECTOFTISSUEHOMOGENATESONIMMUNOASSAYOFOL-AMYLASE"
Tissue homogenate added
Unlabeled cu-amylase added
None
2.5 pg
Salivary gland Salivary gland Liver Liver Kidney Kidney
2.5 yg -
[‘251]ol-Amylase bound (cpm) 1.235 539 1,179 539 1,129 542 1.138 532
2.5 Pi2 -
2.5 pg
’ Tissue homogenates were prepared as described in Methods and, where indicated, 1Opg of each homogenate was added to 1 ml of buffer A, 250 ng [1251]ol-amylase (8200 cpm/pg), and antibody (500 @ml) was added to each reaction mix, and the bound [*251]ol-amylase was quantitated as described in Methods. Results are the average of duplicates differing by less than 3%.
12,000 I I.000 10.000 9000
G -5 is ?I g < z LL
8000 7000 6000 5000 4000
,_____
--------
30
I
60
TISSUE
90
_------z
a--__-----------I
120
I
I
I
160 190 210 240
HOMOGENATE
I
270
ASSAYED Cpg)
4. Radioimmunoassayable a-amylase as a function of the amount of tissue homogenate added to the assay. Pancreatic and liver tissue homogenates were prepared as described in Methods and radioimmunoassay was accomplished as described in Fig. 1 varying the pg of protein of homogenate added. Duplicate determinations differed by less than 4%. a-0, pancreatic homogenate; O-O, liver homogenate. FIG.
182
WEDNER,
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TABLE COMPARISON
OF ENZYMATIC
AND
ROSENFELD
2
AND RADIOIMMUNOASSAYABLE
(Y-AMYLASE~
Tissue homogenate assayed
o-Amylase enzymatic activity (Uimg protein) % maximal activity
RIAo-amylase (ng/lO~g total protein)
Liver Salivary gland Kidney Muscle Parotid gland Pancreas
1.5 3.4 8.3 35.5 100.0 68.2
90%, are obtained when binding to gelled CYamylase is used as the criterion for purity. The latter estimate is probably more accurate since the antibodies with highest affinity for txamylase may not be eluted from the gel, resulting in an underestimation of the purity of the antibody. DISCUSSION It is well established that CAMP serves as a second messenger and mediates many of the intracellular effects produced by polypeptide hormones and biogenic amines (19,20). The mechanism by which hormones acting via CAMP rapidly induce synthesis of specific proteins in their evidence target tissues remains unresolved. There is inconclusive suggesting that new RNA synthesis may not be required for induction of proteins in several model systems (l-6). In order to study this question, model systems have been selected in which CAMP rapidly induces synthesis of a specific protein. In pancreatic minces, CAMP, PGEI, and pancreozymin induce a rapid increase in the synthesis of cu-amylase
a-AMYLASE
RADIOIMMUNOASSAY
185
(M.G.R., unpublished data). In isolated cell suspensions from parotid gland, epinephrine or CAMP stimulate synthesis of cY-amylase (5,6). These studies require a highly purified antibody to porcine a-amylase and a sensitive radioimmunoassay to permit its quantitation. Rabbit anti-a-amylase antibody was prepared by standard techniques and purified by affinity chromatography of the IgG fraction of the rabbit antiserum with at least an 18- to 20-fold increase in specific activity. The antibody preparation was further purified by DEAE-cellulose, CMcellulose chromatography to remove traces of contaminating ribonuclease in order to permit use of the antibody for additional studies involving immuno-precipitation of the polyribosomes bearing nascent cY-amylase chains. The immunoassay which was devised permits reproducible quantitation of a-amylase in the range of 10 ng- 10 pg of ac-amylase. The immunoassay demonstrates a linear a-amylase content over a broad range of concentrations of tissue homogenates assayed and has been successfully utilized to quantitate porcine pancreatic a-amylase levels. The sensitivity of the assay is entirely adequate to permit quantitation of QIamylase in minces or suspension cultures of pancreatic acinar cells. In addition, it appears that the antibody to pancreatic cu-amylase is not applicable to studies of the porcine parotid gland. Studies in other species have been interpreted to suggest that pancreatic and parotid CYamylase are not immunologically identical, although a weak cross reactivity was seen with anti-pancreatic a-amylase serum and parotid tissue homogenates (16). Other studies have suggested pancreatic and salivary a-amylases were immunologically very similar (21). The apparently low estimates of a-amylase content of porcine parotid gland using competition with [‘““I] pancreatic a-amylase as the assay could mean (i) that only a small percentage of the total parotid a-amylase is identical to that in the pancreas, or (il’) that there is limited immunological cross reactivity between the parotid and pancreatic forms with the apparently low levels of parotid a-amylase reflecting competition with pancreatic CYamylase which was used to generate the standard curve. Resolution of this question will be achieved when an antibody to porcine parotid (Yamylase is available for study. The assay described appears to be specific for a-amylase since it detects a-amylase only in tissues which are known to contain the enzyme and the protein precipitated by the immunoassay technique comigrates with a porcine a-amylase standard when analyzed by SDS-polyacrylamide gel electrophoresis. The use of glutaraldehyde gelled cY-amylase to precipitate antigen-antibody complexes in the immunoassay was chosen because of the desirability of this method in preparation of the polysomes bearing a-amylase chains. A double antibody precipitation method for immunoassay of cy-amylase is an effective alternative.
186
WEDNER,
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AND
ROSENFELD
The preparation of a highly purified ribonuclease-free antibody to porcine pancreatic cY-amylase and its use in a highly sensitive and specific radioimmunoassay permits documentation of the CAMP stimulation of a-amylase synthesis in the porcine pancreas and parotid gland. Exploration of the mechanism of this induction will be reported elsewhere. ACKNOWLEDGMENTS The authors gratefully acknowledge the aid of Dr. Gary David for iodination techniques and Dr. Charles Parker for advice and review of the manuscript. This investigation was supported by PHS Research Grant No. AM13 149 from the National Institute of Arthritis, Metabolism & Digestive Diseases and by Grant No. BC169 from the American Cancer Society.
REFERENCES 1. Ferguson, J. J. Jr., (1963) J. Biok Chem. 238, 2754-2759. 2. Garren, L. D., Ney, R. L., and Davis, W. W. (1965) Proc. Nat. Acad. Sci. [/,%I 53, 1443-1450. 3. Grand, R. J., and Gross, R. P. (1970) Proc. Nat. Acad. Sci. USA 65, 1081-1088. 4. Wicks, W. D. (1971) J. Biol. Chem. 246, 2 17-223. 5. Labrie. F., Beraud, G.. Gauthier, M., and Lemay, A. (1971) J. Biol. Chem. 246, 1902-1908. 6. Grand, R. J., and Gross, P. R. (1969)J. Biol. Chem. 244, 5608-5615. 7. Comstock. J. P., Rosenfeld, G. C., O’Malley. B. W., and Means, A. R. (1972) Proc. Nat. Acad. Sci. USA 69, 2377-2380. 8. O’Malley, B. W., Rosenfeld, G. C., Comstock. J. P., and Means, A. R. (1972) Nature New Biol. 240, 45-47. 9. Palacios, R., and Schimke, R. T. (1973) J. Biol. Chem. 248, 1424-1430. 10. Palmiter, R. D.. Palacios, R., and Schimke, R. T. (1972) J. Biol. Chem. 247, 3296-3304. 11. Ouchterlony. 0. (1948) Acta Pathol. Microbial. Scnnd. 25, 186- 19 1. 12. Fahey. J. L. (1971) in Methods in Immunology and Immunochemistry (Williams, C. A.. and Chase, M. W., eds), Vol. I, p. 321, Academic Press, New York. 13. Avrameas, S., and Ternynck, T. (1969) Immunochemistry 6, 53-66. 14. David, G. S., and Reisfeld, R. A. (1974) Biochemistry 13, 1014-1021. 15. Neville, D. M.. Jr. (1971) J. Biol. Chem. 246, 6328-6334. 16. Sanders. T. G.. and Rutter, W. J. (1972) Biochemistry 11, 130-136. 17. Cuatrecasas, P. (1970) /. Biol. Chem. 245, 3059-3065. 18. Schramm. M., and Danon, D. (1961) Biochim. Biophys. Acta 50, 102-I 12. 19. Loyter. A.. and Schramm, M. (1962) Biochim. Biophys. Acta 65, 200-206. 20. Bernfeld, P. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds). Vol. 1, p. 149, Academic Press, New York. 21. McGeachin. R. L., Pavord. M. W., Widmer, D. N.. and Prell, P. A. (1966) Comp. Biochem. Physiol. 18, 767-772.
