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Enzyme-linked immunoassay for study of sarcoplasmic reticulum adenosine triphosphatase
Journal of Immunological Methods, 46 (1981) 289--298 Elsevier/North-Holland Biomedical Press
289
ENZYME-LINKED IMMUNOASSAY FOR STUDY OF SARCOPLASMIC RETICULUM ADENOSINE TRIPHOSPHATASE
ROMEO BETTO, ERNESTO DAMIANI, DONATELLA BIRAL and ISABELLA MUSSINI
National Research Council Unit for Muscle Biology and Physiopathology, Institute of General Pathology, University of Padova, via Loredan 16, 35100 Padua, Italy (Received 17 February 1981, accepted 22 June 1981)
The immunological reactivity of isolated sarcoplasmic reticulum from rabbit fast muscle was tested with antibody to the Ca2+-pump protein which is the predominant c o m p o n e n t of these membranes. Microplate enzyme-linked immunoassay (ELISA) gave highly reproducible results under the conventional conditions used for checkerboard titration of soluble antigens and antibody. Parallel electron microscope observation of the incubated SR vesicles, negatively stained with ammonium molybdate, shows that the immunologically reactive form of the Ca2+-pump protein is still present in membrane-bound form.
INTRODUCTION
The antigenic properties of sarcoplasmic reticulum (SR) Ca2*-activated ATPase (Ca2÷-pump protein), have been extensively investigated in several immunological studies (see Tada et al., 1979), by Outcherlony doublediffusion (Stewart et al., 1976), immunoprecipitation (Stewart et al., 1976; Sarzala and Michalak, 1978), quantitative complement fixation (De Foor et al., 1980) and by immuno-fluorescent staining of cryostat sections of skeletal muscle fibers with antibodies to ATPase (Jorgensen et al., 1979). Some of these techniques require previous solubilization of the isolated SR vesicles with detergents and may therefore modify the antigenic properties of the ATPase. Because of our previous experience with the ELISA assay with other muscle antigens (Biral et al., 1979), we have investigated the applicability of this procedure to the study of the SR Ca2÷-pump protein in derived membrane preparations from rabbit fast-twitch muscle, containing the ATPase as a markedly preponderant protein species. To the best of our knowledge, no study has so far appeared on the use of ELISA for characterization of antigens in membrane-bound form. Since the conditions of the assay could affect both the morphology and the membrane ultrastructural features of the SR vesicles, parallel observations were carried 0022-1759/81/0000--0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press
290
out using the electron microscope on the incubated 8R vesicles after negative staining. Our results show that ELISA can be adapted to quantitative determination of the SR ATPase protein, and that the immunologically reactive protein species is in membrane-bound form. M A T E R I A L S AND METHODS
Preparation of SR membranes and purification of immunogen The SR vesicles were isolated from rabbit adductor muscle, a fast-twitch muscle, as described by Salviati et al. (1979), except that they were sedimented from the mitochondria-free supernatant by a period of centrifugation between 15,000 × g for 20 min and 80,000 × g for 60 min. The SR vesicles were freed from contaminating phosphorylase and contractile proteins as follows. The isolated SR vesicles, after incubating overnight at 0--4°C in a 0.3 M sucrose-1 mM HEPES, pH 7.5 solution (10 mg protein/ ml), were diluted with an equal volume of 0.3 M sucrose-1 mM HEPES, pH 7.5, 1.2 M KC1, and centrifuged at 100,000 Xg for 60 min (Otswald and MacLennan, 1974). The sediment was washed once with 0.3 M sucrose1 mM HEPES, pH 7.5 and finally resuspended in the same medium. The Ca2+-pump protein was further purified by extracting the SR membranes with cholate (Meissner et al., 1973), under the conditions reported previously (Margreth et al., 1977).
Preparation of antibody Antisera against the SR Ca2+-pump protein were raised in adult hens according to Stewart et al. (1976), by 4 weekly intramuscular injections of 0.5 mg antigen in 0.5 ml 0.3 M sucrose solution emulsified with an equal volume of complete Freund's adjuvant (Difco Laboratories, Detroit, MI). Animals were bled 2 months after the start of immunization and a week after the last injection. The 7-globulin fraction of the antisera was isolated with an Na2SO4 precipitation method (Orlans et al., 1961).
ELISA assay ELISA assay was carried out by the indirect method of Voller et al. (1976), under the general conditions described previously (Biral et al., 1979). Briefly, small aliquots (5--20 /A) of a suspension of purified SR vesicles in 0.3 M sucrose-1 mM HEPES, pH 7.5, were thoroughly dispersed in 0.1 M Na~arbonate solution, pH 9.6, containing 0.1% NAN3, to a final protein concentration of 5 pg/ml (for titration of antigen see Fig. 4). Antigen was adsorbed to microtiter wells (M29E, Cooke Microtiter) by incubating at 37°C for 1 h (step 1) and unbound antigen was removed by washing with 0.9% NaC1, 0.05% Tween 20 and 0.02% NaN3 (step 2). Step 3 (incubation with anti-(Ca2÷-pump protein)), step 4 (incubation with a conjugate prepared from purified antibodies to chicken immuno-
291
globulin labelled with alkaline phosphatase) as well as measurements o f phosphatase activity, were carried o u t as reported previously (Biral et al., 1979).
Gel electrophoresis ELISA )
derived enzyme-linked immunosorbent assay (GED-
This was derived from the original m e t h o d o f Lutz et al. {1978), with some modification. Disc gel electrophoresis of the SR membrane protein was carried o u t according to Weber and Osborn (1969) in 5% acrylamide gels. One o f the gels was stained with Coomassie Blue for densitometry. Parallel gels were sliced into 1.5 mm fractions. Each fraction was washed 3 times in distilled water and then incubated overnight in 0.2 ml of a 0.1 M Nacarbonate (pH 9.6)-0.1% NaN3 solution, in the plate well. ELISA assay was carried o u t as described above.
Determination and electrophoretic analysis of protein Protein was determined according to L o w r y et al. (1951). Electrophoretic analysis o f the SR membranes and o f purified Ca2+-pump protein, used as the immunogen, was according to Weber and Osborn {1969) or to Laemmli (1970). In the latter method an exponential polyacrylamide gradient (5-22.5%) was used.
Electron microscopy Negative staining of unincubated and incubated SR vesicles was done with 2% a m m o n i u m m o l y b d a t e , pH 7.2, b y m e t h o d s described earlier (Mussini et al., 1972), with some modifications. Collodium coated, carbon-baked grids were made more adhesive to the SR vesicles b y treating with a polylysine (1 #g/ml) solution (Mazia et al., 1975). A drop o f SR vesicles suspension (0.5 mg/ml) was placed on the grid, the excess liquid was withdrawn, and the SR vesicles were either immediately negatively stained or after adding a drop o f anti-(Ca2+-pump protein) ~,-globulin solution (2 pg/ml) and allowing it to react for 10 min. For examination o f the vesicles after incubation under the conditions o f the ELISA assay, the grids were m o u n t e d on the b o t t o m o f each microtiter well with a small drop of silicone and were removed at different steps o f the assay. Incubation was carried o u t at an antigen coating concentration o f 5 #g/ml, and an antibody concentration of 40 /~g/ml. Electron micrographs were taken on a Philips EM 301 electron microscope with double,condenser illumination, a 30 p m Au objective aperture and an acceleration voltage o f 80 kV. RESULTS
Characterization of the immunogen and of the antibody In SR vesicles isolated from rabbit fast muscle b y the procedures described in Materials and Methods, the Ca2÷-pump protein (Mr approxi-
Fig. 1. Protein pattern of derived SR vesicles from rabbit fast muscle, after electrophoresis (see Methods) according to Weber and Osborn (1969) (a), and to Laemmli (1970) (b, c). a, b: untreated vesicles. c: cholate-extracted SR vesicles. Key: Ca’+-pump: Ca’+ATPase, M, 100,000; Ms5: high affinity calcium binding protein, M, 55,000; CS, calsequestrin. In Weber and Osborn gel electrophoresis, CS shows a lower apparent Mr than in Laemmli’s method (45,000 and 65,000 respectively; see Michalak et al., 1980).
mately 100,000) constitutes about 80% of the total protein, on the basis of densitometric measurements of the stained electrophoretic gels (Fig. la, b). Following partial extraction with cholate, the SR loosely bound proteins, i.e. the M, 55,000 component and calsequestrin (cf., Meissner et al., 1973) appear to be completely removed indicating that Ca”-pump protein is virtually the only membrane component present (Fig. lc). As shown in Fig. 2, the antibodies elicited by immunization with cholateextracted SR vesicles are specifically reactive with the Ca”-pump protein, as judged by GED-ELISA of SR vesicles containing several protein components .
293
~
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M55
2 o o
o° ~°oo°ee°o°.°°..°°,
Fig. 2. Protein densitometric profile (upper tracing) and GED-ELISA (lower tracing) o f SR vesicles after 5% polyacrylamide SDS-gel electrophoresis according to Weber and Osborn (1969). Four gels, loaded with 40 pg protein o f unextracted SR vesicles, were run in parallel. After electrophoresis, one gel was stained with Coomassie Blue and was scanned at 550 nm in a Gilford spectrophotometer. The other gels were sliced and the corresponding fractions (1.5 ram) pooled. ELISA assay was carried out at a concentration o f anti-(Ca2+-pump protein) o f 20 pg/ml. (For details see Methods.)
Ultrastructural aspects o f the antibody reaction with SR vesicles In preliminary experiments it was found that SR vesicles suspended in the isolation medium and examined on polylysine-coated grids, after negative staining with ammonium molybdate {Fig. 3a), had the same ultrastructural appearance described for SR vesicles adsorbed onto conventional carbon-coated grids (Mussini et al., 1972), such as the presence o f characteristic 3--4 nm outer projections (Stewart and MacLennan, 1974). The stepwise addition o f SR vesicles and o f antibody in PBS solution, resulted in the formation o f a fuzzy coat at the outer membrane profile (Fig. 3b, arrow), presumably corresponding to antibody bound to antigenic sites o f the Ca2+-pump protein at the level o f the 3--4 nm particles. No such formation was observed when normal serum was added. When the SR vesicles were immobilized on the polylysine substrate during 60 min incubation under the conditions of the ELISA assay (step
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Coating concentration of antigen (mg
1
/ml)
2
4
10
20
Antibody concentration (mg
40
/rnl)
Fig. 4. Antigen titration. Microtiter wells were coated with SR vesicles at the protein concentrations indicated, and incubated with a fixed amount of antibody to Ca2+-pump protein (20 pg/ml). Fig. 5. Titration of anti-(rabbit Ca2+-pump protein) antibody. Microtiter wells were coated with 5 pg/ml of SR membrane protein. Absorbance values are given as means + S.E.M.; S.E.M. values smaller than the symbols are not drawn.
1), and were then washed as described for step 2 (see Methods), relatively small morphological changes occurred, such as flattening and distortion of the vesicles. However, the basic membrane ultrastructure appeared to be retained (Fig. 3c, d). Subsequent incubation of the immobilized SR vesicles with anti-(Ca2*-pump protein) antibody and with anti-IgG (steps 3--4) (Fig. 3e, f) was accompanied by the formation of a peripheral coat as with unincubated vesicles. This was slightly more evident at step 4 (arrow).
Titration of coating concentration of SR membrane protein An exponential dose-response curve was obtained as a function of the coating concentration of SR membrane protein, as described for ELISA assay with a variety of antigens under similar experimental conditions. By using a semilogarithmic plot, the response was found to be approximately linear over the concentration range 0.5--10 ~g/ml SR vesicle coating protein for a fixed concentration of antibody (Fig. 4). Fig. 3. Electron micrographs of negatively stained SR vesicles from rabbit fast muscle. a: unincubated SR vesicles, b: SR vesicles reacted with anti-(Ca2+-pump protein) by successive dropwise addition directly on the grid. c--f: incubated SR vesicles from steps 1 to 4 of ELISA assay (see Methods). a: × 210,000; b: × 200,000; c: × 200,000; d: × 200,000; e: X 180,000; f: X 180,000.
296 0.75
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20
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Antibody concentration (rng /ml) Fig. 6. Comparison between the SR vesicles from rabbit adductor muscle (o), and from rat tibialis anterior muscle (o), with respect to immunological reactivity with anti-(rabbit Ca2+-pump protein) antibody. Both antigens were tested at a coating concentration of 5 pg/ml.
Titration of antibody Titration of antibody concentration was carried out with microtiter wells coated with 5 pg/ml o f SR membrane (Fig. 5), with highly reproducible results. It was also found that the antibody concentration for halfmaximal saturation o f antigen (20 ~g/ml) was the same at coating concentrations of SR membrane protein b e t w e e n 2 and 5 ~zg/ml (not shown in Fig. 5). The dose response as a function of antibody concentration also appeared to be similar for SR vesicles isolated from fast muscle of the rabbit and the rat, at identical coating concentrations o f SR membrane (Fig. 6). The latter result is in agreement with earlier comparative studies of SR Ca2+-pump protein b y De F o o r et al. (1980), using a microcomplement fixation test. DISCUSSION
Our results show that ELISA is a suitable assay for determination of membrane-bound protein antigens. As far as the SR Ca2÷-pump protein is concerned, the procedure used is sensitive enough to detect this antigen at coating concentrations of total membrane protein as low as 2 ~g/ml, the optimal concentration being a b o u t 5 ~g/ml, equivalent to 4/~g/ml o f antigen in highly purified SR membrane preparations from rabbit fast muscle. Specificity o f this assay system is indicated by its ability to reveal a close immunological relationship between this protein and the homologous protein in the isolated SR from fast muscle of the rat, as borne o u t b y the previous study o f De F o o t et al. (1980), with the more
297
laborious quantitative complement fixation test. Our electron microscope observations support the interpretations that the basic membrane ultrastructure of the SR vesicles is not too adversely affected by high alkaline pH and by the inclusion of non-ionic detergent Tween 20, during the ELISA assay. Although it is likely that under these conditions the SR vesicles are freely permeable to antibody, the membrane ultrastructure of our SR vesicles indicates that they are 'right-side' vesicles with outwardly projecting 3--4 nm particles. These particles, which are believed to comprise the antigenic determinant of the Ca2+-pump protein (Stewart et al., 1976; Tada et al., 1979) and to correspond to the hydrophilic portion of the ATPase monomer, are shown here to be the sites of antibody binding. On the other hand, we found the immunological reactivity of the SR vesicles was unchanged after mild trypsin treatment (data not shown) that resulted in cleavage of the ATPase protein into two peptides of 55,000 daltons and 45,000 daltons (Margreth et al., 1975, 1980). It was reported that the antigenic determinants of the Ca2+-pump protein are localized in the larger peptide fragment {Stewart et al., 1976). Since it has been suggested that clusters of 3 or 4 ATPase molecules constitute the oligomeric, functional form of the ATPase enzyme (Scales and Inesi, 1976) in SR membranes, our results do not answer the question whether specific antibody may cross-link adjacent ATPase molecules. Furthermore, it cannot be decided with any certainty at present whether the antigenic determinants of the Ca2+-pump protein are configurational or sequence determinants. However, the observation of a high immunological reactivity of the Ca2÷-pump protein under the denaturing conditions of the GED-ELISA procedure (see Results), perhaps suggests that sequence determinants might be involved. ACKNOWLEDGEMENTS
This work was supported by funds from Consiglio Nazionale delle Ricerche. We are grateful to Prof. Alfredo Margreth for helpful criticism and for revising the manuscript before submission for publication. We also wish to thank Dr. G. Salviati and Dr. S. Salvatori for participation in some of the experiments. REFERENCES Biral, D., L. Dalla Libera, C. Franeeschi and A. Margreth, 1979, J. Immunol. Methods 31, 93. De Foor, P.H., D. Levitsky, T. Biryukova and S. Fleischer, 1980, Arch. Biochem. Biophys. 200, 196. Jorgensen, A.O., V. Kalnins and D.H. MacLennan, 1979, J. Cell. Biol. 80, 372. Laemmli, U.K., 1970, Nature 227,680. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265.
298 Lutz, H., M. Ermini, E. Jenny, S. Brugmann, F. Joris and H. Weber, 1978, Histochemistry 57,223. Margreth, A., G. Salviati, S. Salvatori and L. Dalla Libera, 1975, in: Calcium Transport in Contraction and Secretion, ed. E. Carafoli (North-Holland Publishing Company, Amsterdam) p. 383. Margreth, A., G. Salviati and S. Salvatori, 1977, in: Membranes Element and Movements of Molecules, Vol. 6, Methodological Surveys in Biochemistry, edo E. Reid (Ellis Hotwood, Chichester) p. 25. Margreth, A., R. Betto, S. Salvatori, G. Salviati, P. Volpe and E. Damiani, 1980, in: Muscular Dystrophy Research: Advances and New Trends, ed. C. Angelini et al. (Excerpta Medica, Amsterdam) p. 53. Mazia, D., G. Schatten and W. Sale, 1975, J. Cell. Biol. 66, 198. Meissner, G., G.E. Conner and S. Fleischer, 1973, Biochim. Biophys. Acta 298, 246. Michalak, M., K.P. Campbell and D.H. MacLennan, 1980, J. Biol. Chem. 255, 1317. Mussini, I., A. Margreth and G. Salviati, 1972, J. Ultrastruct. Res. 38,459. Orlans, E., E. Rose and S. Marrack, 1961, Immunology 4, 262. Otswald, T.J. and D.H. MacLennan, 1974, J. Biol. Chem. 249,974. Salviati, G., R. Betto, S. Salvatori and A. Margreth, 1979, Biochim. Biophys. Acta 574, 280. Sarzala, M.G. and M. Michalak, 1978, Biochim. Biophys. Acta 513, 221. Scales, D. and G. Inesi, 1976, Biophys. J. 16, 735. Stewart, P.S. and D.H. MacLennan, 1974, J. Biol. Chem. 249, 985. Stewart, P.S., D.H. MacLennan and A.E. Shamoo, 1976, J. Biol. Chem. 251,712. Tada, M., T. Yamamoto and Y. Tonomura, 1979, Physiol. Rev. 58, 1. Voller, A., D. Bidwell and A. Bartlett, 1976, in: Manual of Clinical Immunology, eds. N.R. Rose and H. Friedman (American Society for Microbiology, Washington, DC) p. 506. Weber, K. and M. Osborn, 1969, J. Biol. Chem. 244, 4406.
289
ENZYME-LINKED IMMUNOASSAY FOR STUDY OF SARCOPLASMIC RETICULUM ADENOSINE TRIPHOSPHATASE
ROMEO BETTO, ERNESTO DAMIANI, DONATELLA BIRAL and ISABELLA MUSSINI
National Research Council Unit for Muscle Biology and Physiopathology, Institute of General Pathology, University of Padova, via Loredan 16, 35100 Padua, Italy (Received 17 February 1981, accepted 22 June 1981)
The immunological reactivity of isolated sarcoplasmic reticulum from rabbit fast muscle was tested with antibody to the Ca2+-pump protein which is the predominant c o m p o n e n t of these membranes. Microplate enzyme-linked immunoassay (ELISA) gave highly reproducible results under the conventional conditions used for checkerboard titration of soluble antigens and antibody. Parallel electron microscope observation of the incubated SR vesicles, negatively stained with ammonium molybdate, shows that the immunologically reactive form of the Ca2+-pump protein is still present in membrane-bound form.
INTRODUCTION
The antigenic properties of sarcoplasmic reticulum (SR) Ca2*-activated ATPase (Ca2÷-pump protein), have been extensively investigated in several immunological studies (see Tada et al., 1979), by Outcherlony doublediffusion (Stewart et al., 1976), immunoprecipitation (Stewart et al., 1976; Sarzala and Michalak, 1978), quantitative complement fixation (De Foor et al., 1980) and by immuno-fluorescent staining of cryostat sections of skeletal muscle fibers with antibodies to ATPase (Jorgensen et al., 1979). Some of these techniques require previous solubilization of the isolated SR vesicles with detergents and may therefore modify the antigenic properties of the ATPase. Because of our previous experience with the ELISA assay with other muscle antigens (Biral et al., 1979), we have investigated the applicability of this procedure to the study of the SR Ca2÷-pump protein in derived membrane preparations from rabbit fast-twitch muscle, containing the ATPase as a markedly preponderant protein species. To the best of our knowledge, no study has so far appeared on the use of ELISA for characterization of antigens in membrane-bound form. Since the conditions of the assay could affect both the morphology and the membrane ultrastructural features of the SR vesicles, parallel observations were carried 0022-1759/81/0000--0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press
290
out using the electron microscope on the incubated 8R vesicles after negative staining. Our results show that ELISA can be adapted to quantitative determination of the SR ATPase protein, and that the immunologically reactive protein species is in membrane-bound form. M A T E R I A L S AND METHODS
Preparation of SR membranes and purification of immunogen The SR vesicles were isolated from rabbit adductor muscle, a fast-twitch muscle, as described by Salviati et al. (1979), except that they were sedimented from the mitochondria-free supernatant by a period of centrifugation between 15,000 × g for 20 min and 80,000 × g for 60 min. The SR vesicles were freed from contaminating phosphorylase and contractile proteins as follows. The isolated SR vesicles, after incubating overnight at 0--4°C in a 0.3 M sucrose-1 mM HEPES, pH 7.5 solution (10 mg protein/ ml), were diluted with an equal volume of 0.3 M sucrose-1 mM HEPES, pH 7.5, 1.2 M KC1, and centrifuged at 100,000 Xg for 60 min (Otswald and MacLennan, 1974). The sediment was washed once with 0.3 M sucrose1 mM HEPES, pH 7.5 and finally resuspended in the same medium. The Ca2+-pump protein was further purified by extracting the SR membranes with cholate (Meissner et al., 1973), under the conditions reported previously (Margreth et al., 1977).
Preparation of antibody Antisera against the SR Ca2+-pump protein were raised in adult hens according to Stewart et al. (1976), by 4 weekly intramuscular injections of 0.5 mg antigen in 0.5 ml 0.3 M sucrose solution emulsified with an equal volume of complete Freund's adjuvant (Difco Laboratories, Detroit, MI). Animals were bled 2 months after the start of immunization and a week after the last injection. The 7-globulin fraction of the antisera was isolated with an Na2SO4 precipitation method (Orlans et al., 1961).
ELISA assay ELISA assay was carried out by the indirect method of Voller et al. (1976), under the general conditions described previously (Biral et al., 1979). Briefly, small aliquots (5--20 /A) of a suspension of purified SR vesicles in 0.3 M sucrose-1 mM HEPES, pH 7.5, were thoroughly dispersed in 0.1 M Na~arbonate solution, pH 9.6, containing 0.1% NAN3, to a final protein concentration of 5 pg/ml (for titration of antigen see Fig. 4). Antigen was adsorbed to microtiter wells (M29E, Cooke Microtiter) by incubating at 37°C for 1 h (step 1) and unbound antigen was removed by washing with 0.9% NaC1, 0.05% Tween 20 and 0.02% NaN3 (step 2). Step 3 (incubation with anti-(Ca2÷-pump protein)), step 4 (incubation with a conjugate prepared from purified antibodies to chicken immuno-
291
globulin labelled with alkaline phosphatase) as well as measurements o f phosphatase activity, were carried o u t as reported previously (Biral et al., 1979).
Gel electrophoresis ELISA )
derived enzyme-linked immunosorbent assay (GED-
This was derived from the original m e t h o d o f Lutz et al. {1978), with some modification. Disc gel electrophoresis of the SR membrane protein was carried o u t according to Weber and Osborn (1969) in 5% acrylamide gels. One o f the gels was stained with Coomassie Blue for densitometry. Parallel gels were sliced into 1.5 mm fractions. Each fraction was washed 3 times in distilled water and then incubated overnight in 0.2 ml of a 0.1 M Nacarbonate (pH 9.6)-0.1% NaN3 solution, in the plate well. ELISA assay was carried o u t as described above.
Determination and electrophoretic analysis of protein Protein was determined according to L o w r y et al. (1951). Electrophoretic analysis o f the SR membranes and o f purified Ca2+-pump protein, used as the immunogen, was according to Weber and Osborn {1969) or to Laemmli (1970). In the latter method an exponential polyacrylamide gradient (5-22.5%) was used.
Electron microscopy Negative staining of unincubated and incubated SR vesicles was done with 2% a m m o n i u m m o l y b d a t e , pH 7.2, b y m e t h o d s described earlier (Mussini et al., 1972), with some modifications. Collodium coated, carbon-baked grids were made more adhesive to the SR vesicles b y treating with a polylysine (1 #g/ml) solution (Mazia et al., 1975). A drop o f SR vesicles suspension (0.5 mg/ml) was placed on the grid, the excess liquid was withdrawn, and the SR vesicles were either immediately negatively stained or after adding a drop o f anti-(Ca2+-pump protein) ~,-globulin solution (2 pg/ml) and allowing it to react for 10 min. For examination o f the vesicles after incubation under the conditions o f the ELISA assay, the grids were m o u n t e d on the b o t t o m o f each microtiter well with a small drop of silicone and were removed at different steps o f the assay. Incubation was carried o u t at an antigen coating concentration o f 5 #g/ml, and an antibody concentration of 40 /~g/ml. Electron micrographs were taken on a Philips EM 301 electron microscope with double,condenser illumination, a 30 p m Au objective aperture and an acceleration voltage o f 80 kV. RESULTS
Characterization of the immunogen and of the antibody In SR vesicles isolated from rabbit fast muscle b y the procedures described in Materials and Methods, the Ca2÷-pump protein (Mr approxi-
Fig. 1. Protein pattern of derived SR vesicles from rabbit fast muscle, after electrophoresis (see Methods) according to Weber and Osborn (1969) (a), and to Laemmli (1970) (b, c). a, b: untreated vesicles. c: cholate-extracted SR vesicles. Key: Ca’+-pump: Ca’+ATPase, M, 100,000; Ms5: high affinity calcium binding protein, M, 55,000; CS, calsequestrin. In Weber and Osborn gel electrophoresis, CS shows a lower apparent Mr than in Laemmli’s method (45,000 and 65,000 respectively; see Michalak et al., 1980).
mately 100,000) constitutes about 80% of the total protein, on the basis of densitometric measurements of the stained electrophoretic gels (Fig. la, b). Following partial extraction with cholate, the SR loosely bound proteins, i.e. the M, 55,000 component and calsequestrin (cf., Meissner et al., 1973) appear to be completely removed indicating that Ca”-pump protein is virtually the only membrane component present (Fig. lc). As shown in Fig. 2, the antibodies elicited by immunization with cholateextracted SR vesicles are specifically reactive with the Ca”-pump protein, as judged by GED-ELISA of SR vesicles containing several protein components .
293
~
Ca- Pump
M55
2 o o
o° ~°oo°ee°o°.°°..°°,
Fig. 2. Protein densitometric profile (upper tracing) and GED-ELISA (lower tracing) o f SR vesicles after 5% polyacrylamide SDS-gel electrophoresis according to Weber and Osborn (1969). Four gels, loaded with 40 pg protein o f unextracted SR vesicles, were run in parallel. After electrophoresis, one gel was stained with Coomassie Blue and was scanned at 550 nm in a Gilford spectrophotometer. The other gels were sliced and the corresponding fractions (1.5 ram) pooled. ELISA assay was carried out at a concentration o f anti-(Ca2+-pump protein) o f 20 pg/ml. (For details see Methods.)
Ultrastructural aspects o f the antibody reaction with SR vesicles In preliminary experiments it was found that SR vesicles suspended in the isolation medium and examined on polylysine-coated grids, after negative staining with ammonium molybdate {Fig. 3a), had the same ultrastructural appearance described for SR vesicles adsorbed onto conventional carbon-coated grids (Mussini et al., 1972), such as the presence o f characteristic 3--4 nm outer projections (Stewart and MacLennan, 1974). The stepwise addition o f SR vesicles and o f antibody in PBS solution, resulted in the formation o f a fuzzy coat at the outer membrane profile (Fig. 3b, arrow), presumably corresponding to antibody bound to antigenic sites o f the Ca2+-pump protein at the level o f the 3--4 nm particles. No such formation was observed when normal serum was added. When the SR vesicles were immobilized on the polylysine substrate during 60 min incubation under the conditions of the ELISA assay (step
t~ ',D
295 0.75
0.50
E e-
E c
'~
0.50
0 0
0.25
0.25
S 1
10
Coating concentration of antigen (mg
1
/ml)
2
4
10
20
Antibody concentration (mg
40
/rnl)
Fig. 4. Antigen titration. Microtiter wells were coated with SR vesicles at the protein concentrations indicated, and incubated with a fixed amount of antibody to Ca2+-pump protein (20 pg/ml). Fig. 5. Titration of anti-(rabbit Ca2+-pump protein) antibody. Microtiter wells were coated with 5 pg/ml of SR membrane protein. Absorbance values are given as means + S.E.M.; S.E.M. values smaller than the symbols are not drawn.
1), and were then washed as described for step 2 (see Methods), relatively small morphological changes occurred, such as flattening and distortion of the vesicles. However, the basic membrane ultrastructure appeared to be retained (Fig. 3c, d). Subsequent incubation of the immobilized SR vesicles with anti-(Ca2*-pump protein) antibody and with anti-IgG (steps 3--4) (Fig. 3e, f) was accompanied by the formation of a peripheral coat as with unincubated vesicles. This was slightly more evident at step 4 (arrow).
Titration of coating concentration of SR membrane protein An exponential dose-response curve was obtained as a function of the coating concentration of SR membrane protein, as described for ELISA assay with a variety of antigens under similar experimental conditions. By using a semilogarithmic plot, the response was found to be approximately linear over the concentration range 0.5--10 ~g/ml SR vesicle coating protein for a fixed concentration of antibody (Fig. 4). Fig. 3. Electron micrographs of negatively stained SR vesicles from rabbit fast muscle. a: unincubated SR vesicles, b: SR vesicles reacted with anti-(Ca2+-pump protein) by successive dropwise addition directly on the grid. c--f: incubated SR vesicles from steps 1 to 4 of ELISA assay (see Methods). a: × 210,000; b: × 200,000; c: × 200,000; d: × 200,000; e: X 180,000; f: X 180,000.
296 0.75
E
0.50
c
o 0
'¢1" 0.25
1
2
4
10
20
40
Antibody concentration (rng /ml) Fig. 6. Comparison between the SR vesicles from rabbit adductor muscle (o), and from rat tibialis anterior muscle (o), with respect to immunological reactivity with anti-(rabbit Ca2+-pump protein) antibody. Both antigens were tested at a coating concentration of 5 pg/ml.
Titration of antibody Titration of antibody concentration was carried out with microtiter wells coated with 5 pg/ml o f SR membrane (Fig. 5), with highly reproducible results. It was also found that the antibody concentration for halfmaximal saturation o f antigen (20 ~g/ml) was the same at coating concentrations of SR membrane protein b e t w e e n 2 and 5 ~zg/ml (not shown in Fig. 5). The dose response as a function of antibody concentration also appeared to be similar for SR vesicles isolated from fast muscle of the rabbit and the rat, at identical coating concentrations o f SR membrane (Fig. 6). The latter result is in agreement with earlier comparative studies of SR Ca2+-pump protein b y De F o o r et al. (1980), using a microcomplement fixation test. DISCUSSION
Our results show that ELISA is a suitable assay for determination of membrane-bound protein antigens. As far as the SR Ca2÷-pump protein is concerned, the procedure used is sensitive enough to detect this antigen at coating concentrations of total membrane protein as low as 2 ~g/ml, the optimal concentration being a b o u t 5 ~g/ml, equivalent to 4/~g/ml o f antigen in highly purified SR membrane preparations from rabbit fast muscle. Specificity o f this assay system is indicated by its ability to reveal a close immunological relationship between this protein and the homologous protein in the isolated SR from fast muscle of the rat, as borne o u t b y the previous study o f De F o o t et al. (1980), with the more
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laborious quantitative complement fixation test. Our electron microscope observations support the interpretations that the basic membrane ultrastructure of the SR vesicles is not too adversely affected by high alkaline pH and by the inclusion of non-ionic detergent Tween 20, during the ELISA assay. Although it is likely that under these conditions the SR vesicles are freely permeable to antibody, the membrane ultrastructure of our SR vesicles indicates that they are 'right-side' vesicles with outwardly projecting 3--4 nm particles. These particles, which are believed to comprise the antigenic determinant of the Ca2+-pump protein (Stewart et al., 1976; Tada et al., 1979) and to correspond to the hydrophilic portion of the ATPase monomer, are shown here to be the sites of antibody binding. On the other hand, we found the immunological reactivity of the SR vesicles was unchanged after mild trypsin treatment (data not shown) that resulted in cleavage of the ATPase protein into two peptides of 55,000 daltons and 45,000 daltons (Margreth et al., 1975, 1980). It was reported that the antigenic determinants of the Ca2+-pump protein are localized in the larger peptide fragment {Stewart et al., 1976). Since it has been suggested that clusters of 3 or 4 ATPase molecules constitute the oligomeric, functional form of the ATPase enzyme (Scales and Inesi, 1976) in SR membranes, our results do not answer the question whether specific antibody may cross-link adjacent ATPase molecules. Furthermore, it cannot be decided with any certainty at present whether the antigenic determinants of the Ca2+-pump protein are configurational or sequence determinants. However, the observation of a high immunological reactivity of the Ca2÷-pump protein under the denaturing conditions of the GED-ELISA procedure (see Results), perhaps suggests that sequence determinants might be involved. ACKNOWLEDGEMENTS
This work was supported by funds from Consiglio Nazionale delle Ricerche. We are grateful to Prof. Alfredo Margreth for helpful criticism and for revising the manuscript before submission for publication. We also wish to thank Dr. G. Salviati and Dr. S. Salvatori for participation in some of the experiments. REFERENCES Biral, D., L. Dalla Libera, C. Franeeschi and A. Margreth, 1979, J. Immunol. Methods 31, 93. De Foor, P.H., D. Levitsky, T. Biryukova and S. Fleischer, 1980, Arch. Biochem. Biophys. 200, 196. Jorgensen, A.O., V. Kalnins and D.H. MacLennan, 1979, J. Cell. Biol. 80, 372. Laemmli, U.K., 1970, Nature 227,680. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265.
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