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Molecular characterization of a membrane protein by a simple immunobinding procedure with monoclonal antibodies
J o u r n a l o f l m m u t ologicaIMethods, 72 (1984) 443-450
443
Elsevier JIM03175
Molecular Characterization of a Membrane Protein by a Simple Immunobinding Procedure with Monoclonal Antibodies A. Sonnenberg, G.J.C.M. Kolvenbag, E.J.M. A1 1 and J.
Hilgers
Di~ision of Tumor Biology, The Netherlands Cancer Institute, Plesrnanlaan 121, 1066 C X Amsterdam, and t Department of Biochemist£v, Unit~ersiO' ofA msterdam. Plantage Muidergracht 12, 1018 T V Amsterdam, The Netherlands
(Received 16 January 1984, accepted 30 April 1984)
A simple immunobinding procedure for the detection and molecular characterization of antigens is described. Antigen is adsorbed by immobilized antibodies, and this is followed by radiolabeling with iodine. Both adsorption and radioiodination are carried out in microtitcr wells. After gel electrophoresis and autoradiography the apparent molecular weight of the radiolabeled antigen may be estimated. With this procedure we show that 2 monoclonal antibodies, directed against different determinants, both detect a glycoprotein with an apparent molecular weight of 170,000. By a 2.-site sandwich immunoassay we demonstrate that these antibodies detect the same glycoprotein. Key words: monoclonal antibody - immunobinding
sandwich immunoassc 9,
glvcoprotein
Introduction M o n o c l o n a l a n t i b o d i e s allow the comparative investigation of antigenic determin a n t s on n o r m a l a n d neoplastic cells (Colcher et al., 1981; T a y l o r - P a p a d i m i t r i o u et al., 1981; Foster et al., 1982a, b; Hilkens et al., 1984). Quite often m o n o c l o n a l a n t i b o d i e s are p r o d u c e d that resemble each other in their i m m u n o h i s t o c h e m i c a l reaction patterns. A n t i b o d y ' b l o c k i n g ' a n d ' a d d i t i v e ' b i n d i n g assays have been developed to characterize such antibodies with respect to antigenic d e t e r m i n a n t s on purified molecules ( P a r h a m et al., 1982). However, the use of p r o t e i n mixtures in such assays makes it difficult to establish whether different antigenic d e t e r m i n a n t s are present on the same molecule. The 2-site sandwich i m m u n o a s s a y is a n elegant method of testing whether m o n o c l o n a l a n t i b o d i e s directed against different antigenic d e t e r m i n a n t s detect the same molecule. So far this assay has been used only to detect aqueous soluble proteins ( U o t i l a et al., 1981). 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.
444 In a previous report we described the production of a number of monoclonal antibodies against mouse mammary tumor antigens. Two of these antibodies were closely similar in their distribution on mouse mammary gland tissues, as well as on organs and tissues of neonatal mice, as tested with an immunoperoxidase test (Sonnenberg et al., 1984), We have now investigated the nature of the antigens detected with these 2 monoclonal antibodies. Using the 2-site sandwich immunoassay, we demonstrate that the 2 antibodies detect different antigenic determinants on 1 detergent solubilized glycoprotein. A simple procedure to determine the specificity of the 2 antibodies by gel electrophoresis is also described. This procedure is based on the sandwich immunoassay and performed in microtiter wells.
Materials and Methods
Characterization of monoclonal antibodies The IgG subclass of monoclonal antibodies MaB8, 50B8 and 45A9 was determined by a double diffusion test using specific rabbit and goat antibodies against rat IgG1, IgG2a, IgG2b and IgG2c (Pel Freeze Biologicals, Rogers, AR 72756). MaB8 and 50B8 were of the IgG2b class and 45A9 of the lgG2c class.
'Additive' binding assay Falcon polyvinylchloride microtiter plates were coated with crude membranes of mouse mammary tumors (10 /zg protein/well) by overnight drying (Sonnenberg et al., 1984). The plates were washed once with phosphate-buffered saline (PBS) and preincubated in 1% BSA in PBS for 60 min at room temperature, followed by 2 washes with PBS containing 5% fetal calf serum (FCS), 1% BSA, 0.05% Tween-20 and 0.05% NaN 3 (assay buffer). Serial dilutions of a culture medium containing monoclonal antibodies with different specificities were prepared in PBS and were added separately or 2 at a time in different combinations to each well. Incubation proceeded for 60 rain at room temperature. After incubation, the plates were washed 3 times with assay buffer and 2 × 105 cpm in 50/zl of 1251-labeled rabbit anti-rat IgG (specific activity about 20/~Ci//~g) was added to each well. The rabbit anti-rat IgG was immunopurified and absorbed on a glutaraldehyde cross-linked mouse serum column to prevent high background due to crossreaction with mouse IgGs. The plates were incubated at room temperature for another 60 rain and then washed extensively with assay buffer. Wells were cut individually and the bound radioactivity in each well was counted in a gamma counter.
'Blocking' assay Polyvinylchloride plates coated with crude membranes were incubated with serial dilutions of a culture medium containing monoclonal antibodies with different specificities. The plates were incubated for 60 min at room temperature and washed 3 times with assay buffer, and 50 /zl of ~2SI-labeled purified monoclonal antibody (105 cpm) were added to each well. After 60 rain at room temperature the plates were washed 3 times with assay buffer, and the bound radioactivity in each well counted in a gamma counter.
445 Purification and radioiodination of monoclonal antibodies Monoclonal antibodies were purified from culture media by affinity chromatography on rabbit anti-rat IgG Sepharose. Bound antibodies were eluted from the rabbit anti-rat IgG adsorbent with 1 M glycine HC1 (pH 2.8) and immediately neutralized by the addition of 5% NaHCO 3. The eluted monoclonal antibodies were dialyzed against PBS and concentrated by ultrafiltration with a PM10 filter (Amicon). Antibody concentrations were determined spectrophotometrically assuming an E2~0 1% of 14.0. Fifty microgram portions of purified IgG were labeled with ~25I according to Hunter and Greenwood (1962). Immunoreactivities of labeled and unlabeled antibodies were compared in a competitive binding assay with 10/~g crude membranes coated to microtiter wells. Isolation of glycoproteins from mouse mammary tumors Crude membranes of mouse mammary tumors were solubilized in 1% NP40, 50 mM Tris-HC1 (pH 7.5), 150 mM NaC1 and 1 mM CaCI 2 for 2 h at 4°C. Non-solubilized material was removed by pelleting at 100,000 × g for 1 h. The solubilized membrane proteins were passed over lentil-lectin Sepharose and the bound glycoproteins eluted with 0.25 M 1-O-methyl-a-D-glucopyranoside in 0.5% NP40, 50 mM Yris HC1 (pH 7.5), 150 mM NaC1 and 1 mM CaC12. Two-site sandwich radioimmunoassay Polyvinylchloride microtiter plates were coated overnight at 4°C with purified monoclonal antibodies with different specificities (0.5 /~g in 50 ~1 0.1 M NaHCO 3 per well). The coating of the wells with the different antibodies was tested in a radioimmunoassay with ~25I-labeled rabbit anti-rat IgG. If necessary antibody concentrations were adjusted to give comparable amounts of coated antibodies. Serial dilutions of a lentil-lectin binding glycoprotein fraction were prepared in PBS, containing 0.1% NP40. Fifty microliter portions were added to each well coated with the different antibodies; the highest concentration of the lentil-lectin binding glycoprotein fraction used was 10 #g. The plate was incubated for 2 h at room temperature and washed 3 times with PBS containing 0.1% NP40. Binding of 125I-labeled purified monoclonal antibody to the immunoadsorbed antigen(s) was assayed as described. Immunobinding and radiolabeling Antigens immunopurified as described for the 2-site sandwich immunoassay were iodinated in the microtiter well by the chloramine-T method (Hunter and Greenwood, 1962). Ten microliters 0.1% NP40, 10 ptl 0.5 M KzHPO 4 (pH 7.5) and 10/~1 Na1251 (100/~C) were added in this order to each well. The iodination reaction was started by the addition of 10 /~1 chloramine-T (1 mg/ml). After incubation for 5 min at room temperature the reaction was stopped with 10/~1 K2S205 (1 mg/ml). The plate was then washed 5 × with PBS containing 0.1% NP40. Wells were cut individually and transferred into microfuge tubes. To each microtiter well, 50/~1 sample buffer (1% SDS, 10 mM iodoacetamide, 10% glycerol and 0,05 M Tris-HC1 (pH 6.8)) was added and the microfuge tubes containing the microtiter wells were incubated for 5
446 min at 95°C. After incubation the supernatants were centrifuged and electrophoresed on SDS polyacrylamide gels with the discontinuous buffer system of Laemmli (1970). Analysis of antigens under reducing conditions was accomplished by replacement of iodoacetamide with mercaptoethanol in the sample buffer.
Metabolic labeling of mouse mammary tumor cells and immunoprecipitation of antigens D5-C mouse mammary tumor cells, grown to confluency, were labeled with [35S]methionine (200 /~Ci/ml) in Dulbecco's modified Eagle's medium with the methionine concentration reduced to 10% of the normal level and supplemented with 10 /~g/ml insulin and 50 n g / m l hydrocortisone and incubation continued for 16 h at 37°C in a CO 2 incubator. After incubation, the cells were washed twice with PBS on the culture dish and then solubilized in PBS containing 1% NP40 and 0.005% PMSF (phenylmethylsulfonyl fluoride) for 2 h at 4°C. Non-solubilized material was removed by centrifugation at 10,000 × g for 15 rain in an Eppendorf microfuge. The radiolabeled membrane proteins (1 x 107 cpm in 200 /~1 PBS containing I ci NP40 and 0.005% PMSF) and antibody (1 5 /~g in 200 /~l supernatant) were incubated for 60 min at room temperature. Fifty micrograms affinity purified rabbit anti-rat (7 m g / m l ) were added to each immunoprecipitate and incubation continued for 1 h. Finally, 100 /xl 10% Protein A-Sepharose (Pharmacia) suspension in PBS containing 0.1% NP40 and 1% BSA were added, and incubation continued for 2 h at room temperature. The Sepharose beads were pelleted by centrifugation for 1 rain at 10,000 × g and washed 4 times with PBS containing 0.1% NP40. The pellet was suspended in 50/~1 sample buffer, incubated for 5 min at 95°C and centrifuged for 2 min at 10,000 × g. The supernatant was electrophoresed on an SDS-polyacrylamide gel, employing the discontinuous buffer system of Laemmli (1970).
Results
Monoclonal antibodies MaB8 and 50B8 were generated by fusing mouse myeloma cells (SP2/0) with spleen cells from rats immunized with mouse mammary tumor cells (Sonnenberg et al., 1984). Both antibodies were of the IgG2b antibody class. The specificity of MaB8 and 50B8 was determined in 2 different binding assays. The results of an 'additive' binding assay are shown in Fig. l. Compared with the maximal binding obtained with saturating concentrations of only one of the antibodies, more radiolabeled rabbit anti-rat IgG was bound when the 2 monoclonal antibodies were used simultaneously. Thus the 2 monoclonal antibodies either bind to different antigenic determinants on the same molecule or are directed against different antigens. The different specificities of MaB8 and 50B8 are also demonstrable in a direct 'blocking' assay. As may be seen in Fig. 2, binding of radiolabeled monoclonal antibody 50B8 was blocked by preincubation of immobilized mouse mammary tumor membranes with unlabeled 50B8; binding was not affected by unlabeled MaB8. Because specific binding activity of MaB8 is lost during the iodination
447
procedure, confirmation by the reciprocal experiment could not be obtained. The possibility that the 2 antibodies detect different determinants on the same molecule was tested in a 2-site sandwich radioimmunoassay, In this assay, microtiter wells are coated with monoclonal antibodies and subsequently incubated with a lentil-lectin binding glycoprotein fraction and a radiolabeled antibody. Fig. 3 shows the results with 3 antibodies with different specificities (MaB8 and 50B8 and 45A9) used for coating and radiolabeled 50B8. Monoclonal antibody 45A9 does not react with an antigen in the glycoprotein fraction and was included in this experiment as a negative control. It could be shown that the coated monoclonal antibody MaB8 immunoadsorbed an antigen also detectable with radiolabeled 50B8. The antigen could be sandwiched to a much lesser extent between the 50B8 monoclonal antibodies themselves, suggesting that only 1 determinant was available for binding with 50B8. The specificity of the 2 antibodies was further tested by radiolabeling of the immunoadsorbed antigen in microtiter wells followed by gel electrophoresis. Autoradiography of gels electrophoresed under reducing conditions showed a specific band with an a p p a r e n t m o l e c u l a r w e i g h t o f 170,000 for 50B8 a n d M a B 8 , n o t d e t e c t a b l e w i t h 4 5 A 9
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Fig. 1. Additive binding assay with monoclonal antibodies MaB8 and 50B8 for determination of epitope specificity. Microtiter plates coated with crude mammary tumor membranes were incubated with serial dilutions of monoclonal antibodies MaB8 and 50B8, added either separately or in combination. The amount of antibody bound was determined by subsequent incubation with labeled rabbit anti-rat IgG and counting the bound label in a gamma counter. O O, Monoclonal antibody 50B8 alone; • •, monoclonal antibody MaB8 alone; [] C3, combination of monoclonal antibodies MaB8 and 50B8. Fig. 2. Blocking assay with monoclonal antibodies MaB8 and 50B8 for determination of epitope specificity. Serial dilutions of monoclonal antibodies MaB8 and 50B8 were incubated with crude mouse mammary tumor membranes coated to microtiter wells. The blocking of antibody binding was determined by subsequent incubation with labeled monoclonal antibody 50B8 and counting the bound label in a gamma counter. [] n, Preincubation with unlabeled MaB8; • a, preincubation with unlabeled 50B8.
448 (Fig. 4). N o specific b a n d was d e m o n s t r a b l e when the gel was el ect r o p h o r esed under n o n - r e d u c i n g conditions. T h e a p p a r e n t m o l e c u l a r weight of the MaB8 and 50B8 m o n o c l o n a l a n t i b o d y d ef i n ed antigen was also d e t e r m i n e d in a r a d i o i m m u n o p r e c i p i t a t i o n , using [35S]methionine-labeled antigens of a m o u s e m a m m a r y t u m o r cell line. Th e a p p a r e n t m o l e c u l a r weight of the i m m u n o p r e c i p i t a t e d antigen was again d e t e r m i n e d under r e d u c i n g an d n o n - r e d u c i n g conditions. The results (Fig. 5) showed for both antibodies 1 b a n d with an a p p a r e n t m o l e c u l a r weight of 170,000, c o n f i r m i n g the results o b t a i n e d by the i m m u n o r a d i o l a b e l i n g procedure. As in the case of the imm u n o r a d i o l a b e l i n g p r o c e d u r e a higher b a c k g r o u n d was o b t a i n e d with the MaB8 a n t i b o d y than with 50B8.
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Fig. 3. Two-site sandwich immunoassay with monoclonal antibodies MaB8, 50B8 and 45A9 for determination of antigen specificity. Microtiter plates coated with monoclonal antibodies were incubated with a serial dilution of a lentil-lectin binding glycoprotein fraction. Immunoadsorbed antigens were detected by subsequent incubation with labeled monoclonal antibody 50B8 and counting the bound label in a gamma counter. • •, Coated monoclonal antibody MaB8: O O. coated monoclonal antibody 50B8; • A, coated monoclonal antibody 45A9. Fig. 4. NaDodSO4/polyacrylamide gel analysis of antigens immunoadsorbed from a lentil-lectin binding glycoprotein fraction by monoclonal antibodies MaB8, 50B8, 45A9 coated to microtiter wells. Immunoadsorbed antigens were labeled with 125I, solubilized in sample buffer containing 5% (v/v) 2-mercaptoethanol and analyzed on a 10% polyacrylamide gel. Exposure of the autoradiograph was for 6 h. A and B, immunobinding with monoclonal antibody 45A9; C and D, immunobinding with monoclonal antibody MaB8: E and F, immunobinding with monoclonal antibody 50B8.
449
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Fig. 5. NaDodSO4/polyacrylamide gel analysis of immunoprecipitates prepared with monoclonal antibodies MaB8 and 50B8 from ['~SS]methionine-labeled lysates of P3MTD5 mouse m a m m a r y tumor cells. Culture media from S p 2 / 0 mouse myeloma cells was used as a control, hnmunoprecipitates were analyzed on a 10% polyacrylamide gel in the prescence of 2-mcrcaptoethanol (A C) and on a 7% polyacrylamide gel in the prescence of iodoacetamide ( D - F ) . Exposure of the autoradiograph was for 7 days. A and D, immunoprecipitation with culture media from S P 2 / 0 myeloma cells; B and E, immunoprecipitation with culture media from MaB8 antibody producing hybridoma: C and F, imnmnoprecipitation with culture media from 50B8 antibody producing hybridoma.
Since no major differences were found in the apparent molecular weight of the antigen under reducing and non-reducing conditions, it was concluded that the antigen is a monomeric protein. Discussion
We describe here a simple and rapid immunobinding procedure for characterization of membrane proteins. Antigens are labeled after adsorption to immobilized antibodies, offering the advantage that the antigenic site is protected against destruction by radioiodination. Therefore, the procedure allows detection of practically all antigens. Only antigens that cannot be radioiodinated or antigens that lose the antigenic site after solubilization remain undetectable. A disadvantage of the immunobinding procedure, seen also in this study, is that in SDS gels specific bands are lost due to comigration or due to poor resolution with labeled IgG. While in gels electrophoresed under reducing conditions the MaB8 and 50B8 antibodies detect a specific band of 170 kDa, no specific band was demonstrable under non-reducing conditions.
450
In the immunobinding procedure, the high background with the MaB8 antibody as compared with the 50B8 antibody is not caused by the quality of the affinity purified antibodies. This conclusion was drawn because it made no difference whether immunoprecipitates were prepared with culture medium of the MaB8 antibody producing hybridoma or with affinity purified antibodies. The origin of the high background with MaB8 is not clear. Possibly, the affinity of the monoclonal antibody for the epitopes decreases after solubilization of the antigen, resulting in an effect on the background.
Acknowledgments We thank Ir. J. Hilkens for useful discussions. This work was supported by Grant NKI 79-6 from the Queen Wilhelmina Foundation for the Fight against Cancer in the Netherlands.
References Colcher, D., P. Horan Hand, M. Nuti and J. Schlom. 1981, Proc. Natl. Acad. Sci. U S A . 78, 3199. Foster, C.S., P.A.W. Edwards, E.A. Dinsdale and A.M. Neville, 1982a, Virchows Arch. Abt. A. Pathol. Anat. 394, 279.
Foster, C.S., E.A. Dinsdale, P.A.W. Edwards and A.M. Neville, 1982b, Virchows Arch. Abt. A. Pathol. Anat. 394, 295. Hilkens, J., J. Hilgers, F. Buys, Ph. Hageman, D. Schol, G. Van Doornewaard and J. Van den Tv, cel, 1984, Proc. Biol. Fluids 31, 1013. Hunter, W.M. and F.C. Greenwood, 1962, Nature (London) 194, 495. Laemmli, U.K., 1970, Nature (London) 227, 680. Parham, P., M.J. Androlewicz, F.M. Brodsky, N.J. Holmes and J.P. Ways, 1982, J. Immunol. Methods 53, 133. Sonnenberg, A., J. Daams, J. Hilkens and J. Hilgcrs, 1984, submitted. Taylor-Papadirnitriou, J., J. Peterson, J. Arklie, J. Burchell, R.L. Ceriani and W.F. Bodmer, 1981, Int. J. Cancer 28, 17. Uotila, M., E. Ruoslahti and E. Engvall, 1981, J. Immunol, Methods 42, 11.
443
Elsevier JIM03175
Molecular Characterization of a Membrane Protein by a Simple Immunobinding Procedure with Monoclonal Antibodies A. Sonnenberg, G.J.C.M. Kolvenbag, E.J.M. A1 1 and J.
Hilgers
Di~ision of Tumor Biology, The Netherlands Cancer Institute, Plesrnanlaan 121, 1066 C X Amsterdam, and t Department of Biochemist£v, Unit~ersiO' ofA msterdam. Plantage Muidergracht 12, 1018 T V Amsterdam, The Netherlands
(Received 16 January 1984, accepted 30 April 1984)
A simple immunobinding procedure for the detection and molecular characterization of antigens is described. Antigen is adsorbed by immobilized antibodies, and this is followed by radiolabeling with iodine. Both adsorption and radioiodination are carried out in microtitcr wells. After gel electrophoresis and autoradiography the apparent molecular weight of the radiolabeled antigen may be estimated. With this procedure we show that 2 monoclonal antibodies, directed against different determinants, both detect a glycoprotein with an apparent molecular weight of 170,000. By a 2.-site sandwich immunoassay we demonstrate that these antibodies detect the same glycoprotein. Key words: monoclonal antibody - immunobinding
sandwich immunoassc 9,
glvcoprotein
Introduction M o n o c l o n a l a n t i b o d i e s allow the comparative investigation of antigenic determin a n t s on n o r m a l a n d neoplastic cells (Colcher et al., 1981; T a y l o r - P a p a d i m i t r i o u et al., 1981; Foster et al., 1982a, b; Hilkens et al., 1984). Quite often m o n o c l o n a l a n t i b o d i e s are p r o d u c e d that resemble each other in their i m m u n o h i s t o c h e m i c a l reaction patterns. A n t i b o d y ' b l o c k i n g ' a n d ' a d d i t i v e ' b i n d i n g assays have been developed to characterize such antibodies with respect to antigenic d e t e r m i n a n t s on purified molecules ( P a r h a m et al., 1982). However, the use of p r o t e i n mixtures in such assays makes it difficult to establish whether different antigenic d e t e r m i n a n t s are present on the same molecule. The 2-site sandwich i m m u n o a s s a y is a n elegant method of testing whether m o n o c l o n a l a n t i b o d i e s directed against different antigenic d e t e r m i n a n t s detect the same molecule. So far this assay has been used only to detect aqueous soluble proteins ( U o t i l a et al., 1981). 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.
444 In a previous report we described the production of a number of monoclonal antibodies against mouse mammary tumor antigens. Two of these antibodies were closely similar in their distribution on mouse mammary gland tissues, as well as on organs and tissues of neonatal mice, as tested with an immunoperoxidase test (Sonnenberg et al., 1984), We have now investigated the nature of the antigens detected with these 2 monoclonal antibodies. Using the 2-site sandwich immunoassay, we demonstrate that the 2 antibodies detect different antigenic determinants on 1 detergent solubilized glycoprotein. A simple procedure to determine the specificity of the 2 antibodies by gel electrophoresis is also described. This procedure is based on the sandwich immunoassay and performed in microtiter wells.
Materials and Methods
Characterization of monoclonal antibodies The IgG subclass of monoclonal antibodies MaB8, 50B8 and 45A9 was determined by a double diffusion test using specific rabbit and goat antibodies against rat IgG1, IgG2a, IgG2b and IgG2c (Pel Freeze Biologicals, Rogers, AR 72756). MaB8 and 50B8 were of the IgG2b class and 45A9 of the lgG2c class.
'Additive' binding assay Falcon polyvinylchloride microtiter plates were coated with crude membranes of mouse mammary tumors (10 /zg protein/well) by overnight drying (Sonnenberg et al., 1984). The plates were washed once with phosphate-buffered saline (PBS) and preincubated in 1% BSA in PBS for 60 min at room temperature, followed by 2 washes with PBS containing 5% fetal calf serum (FCS), 1% BSA, 0.05% Tween-20 and 0.05% NaN 3 (assay buffer). Serial dilutions of a culture medium containing monoclonal antibodies with different specificities were prepared in PBS and were added separately or 2 at a time in different combinations to each well. Incubation proceeded for 60 rain at room temperature. After incubation, the plates were washed 3 times with assay buffer and 2 × 105 cpm in 50/zl of 1251-labeled rabbit anti-rat IgG (specific activity about 20/~Ci//~g) was added to each well. The rabbit anti-rat IgG was immunopurified and absorbed on a glutaraldehyde cross-linked mouse serum column to prevent high background due to crossreaction with mouse IgGs. The plates were incubated at room temperature for another 60 rain and then washed extensively with assay buffer. Wells were cut individually and the bound radioactivity in each well was counted in a gamma counter.
'Blocking' assay Polyvinylchloride plates coated with crude membranes were incubated with serial dilutions of a culture medium containing monoclonal antibodies with different specificities. The plates were incubated for 60 min at room temperature and washed 3 times with assay buffer, and 50 /zl of ~2SI-labeled purified monoclonal antibody (105 cpm) were added to each well. After 60 rain at room temperature the plates were washed 3 times with assay buffer, and the bound radioactivity in each well counted in a gamma counter.
445 Purification and radioiodination of monoclonal antibodies Monoclonal antibodies were purified from culture media by affinity chromatography on rabbit anti-rat IgG Sepharose. Bound antibodies were eluted from the rabbit anti-rat IgG adsorbent with 1 M glycine HC1 (pH 2.8) and immediately neutralized by the addition of 5% NaHCO 3. The eluted monoclonal antibodies were dialyzed against PBS and concentrated by ultrafiltration with a PM10 filter (Amicon). Antibody concentrations were determined spectrophotometrically assuming an E2~0 1% of 14.0. Fifty microgram portions of purified IgG were labeled with ~25I according to Hunter and Greenwood (1962). Immunoreactivities of labeled and unlabeled antibodies were compared in a competitive binding assay with 10/~g crude membranes coated to microtiter wells. Isolation of glycoproteins from mouse mammary tumors Crude membranes of mouse mammary tumors were solubilized in 1% NP40, 50 mM Tris-HC1 (pH 7.5), 150 mM NaC1 and 1 mM CaCI 2 for 2 h at 4°C. Non-solubilized material was removed by pelleting at 100,000 × g for 1 h. The solubilized membrane proteins were passed over lentil-lectin Sepharose and the bound glycoproteins eluted with 0.25 M 1-O-methyl-a-D-glucopyranoside in 0.5% NP40, 50 mM Yris HC1 (pH 7.5), 150 mM NaC1 and 1 mM CaC12. Two-site sandwich radioimmunoassay Polyvinylchloride microtiter plates were coated overnight at 4°C with purified monoclonal antibodies with different specificities (0.5 /~g in 50 ~1 0.1 M NaHCO 3 per well). The coating of the wells with the different antibodies was tested in a radioimmunoassay with ~25I-labeled rabbit anti-rat IgG. If necessary antibody concentrations were adjusted to give comparable amounts of coated antibodies. Serial dilutions of a lentil-lectin binding glycoprotein fraction were prepared in PBS, containing 0.1% NP40. Fifty microliter portions were added to each well coated with the different antibodies; the highest concentration of the lentil-lectin binding glycoprotein fraction used was 10 #g. The plate was incubated for 2 h at room temperature and washed 3 times with PBS containing 0.1% NP40. Binding of 125I-labeled purified monoclonal antibody to the immunoadsorbed antigen(s) was assayed as described. Immunobinding and radiolabeling Antigens immunopurified as described for the 2-site sandwich immunoassay were iodinated in the microtiter well by the chloramine-T method (Hunter and Greenwood, 1962). Ten microliters 0.1% NP40, 10 ptl 0.5 M KzHPO 4 (pH 7.5) and 10/~1 Na1251 (100/~C) were added in this order to each well. The iodination reaction was started by the addition of 10 /~1 chloramine-T (1 mg/ml). After incubation for 5 min at room temperature the reaction was stopped with 10/~1 K2S205 (1 mg/ml). The plate was then washed 5 × with PBS containing 0.1% NP40. Wells were cut individually and transferred into microfuge tubes. To each microtiter well, 50/~1 sample buffer (1% SDS, 10 mM iodoacetamide, 10% glycerol and 0,05 M Tris-HC1 (pH 6.8)) was added and the microfuge tubes containing the microtiter wells were incubated for 5
446 min at 95°C. After incubation the supernatants were centrifuged and electrophoresed on SDS polyacrylamide gels with the discontinuous buffer system of Laemmli (1970). Analysis of antigens under reducing conditions was accomplished by replacement of iodoacetamide with mercaptoethanol in the sample buffer.
Metabolic labeling of mouse mammary tumor cells and immunoprecipitation of antigens D5-C mouse mammary tumor cells, grown to confluency, were labeled with [35S]methionine (200 /~Ci/ml) in Dulbecco's modified Eagle's medium with the methionine concentration reduced to 10% of the normal level and supplemented with 10 /~g/ml insulin and 50 n g / m l hydrocortisone and incubation continued for 16 h at 37°C in a CO 2 incubator. After incubation, the cells were washed twice with PBS on the culture dish and then solubilized in PBS containing 1% NP40 and 0.005% PMSF (phenylmethylsulfonyl fluoride) for 2 h at 4°C. Non-solubilized material was removed by centrifugation at 10,000 × g for 15 rain in an Eppendorf microfuge. The radiolabeled membrane proteins (1 x 107 cpm in 200 /~1 PBS containing I ci NP40 and 0.005% PMSF) and antibody (1 5 /~g in 200 /~l supernatant) were incubated for 60 min at room temperature. Fifty micrograms affinity purified rabbit anti-rat (7 m g / m l ) were added to each immunoprecipitate and incubation continued for 1 h. Finally, 100 /xl 10% Protein A-Sepharose (Pharmacia) suspension in PBS containing 0.1% NP40 and 1% BSA were added, and incubation continued for 2 h at room temperature. The Sepharose beads were pelleted by centrifugation for 1 rain at 10,000 × g and washed 4 times with PBS containing 0.1% NP40. The pellet was suspended in 50/~1 sample buffer, incubated for 5 min at 95°C and centrifuged for 2 min at 10,000 × g. The supernatant was electrophoresed on an SDS-polyacrylamide gel, employing the discontinuous buffer system of Laemmli (1970).
Results
Monoclonal antibodies MaB8 and 50B8 were generated by fusing mouse myeloma cells (SP2/0) with spleen cells from rats immunized with mouse mammary tumor cells (Sonnenberg et al., 1984). Both antibodies were of the IgG2b antibody class. The specificity of MaB8 and 50B8 was determined in 2 different binding assays. The results of an 'additive' binding assay are shown in Fig. l. Compared with the maximal binding obtained with saturating concentrations of only one of the antibodies, more radiolabeled rabbit anti-rat IgG was bound when the 2 monoclonal antibodies were used simultaneously. Thus the 2 monoclonal antibodies either bind to different antigenic determinants on the same molecule or are directed against different antigens. The different specificities of MaB8 and 50B8 are also demonstrable in a direct 'blocking' assay. As may be seen in Fig. 2, binding of radiolabeled monoclonal antibody 50B8 was blocked by preincubation of immobilized mouse mammary tumor membranes with unlabeled 50B8; binding was not affected by unlabeled MaB8. Because specific binding activity of MaB8 is lost during the iodination
447
procedure, confirmation by the reciprocal experiment could not be obtained. The possibility that the 2 antibodies detect different determinants on the same molecule was tested in a 2-site sandwich radioimmunoassay, In this assay, microtiter wells are coated with monoclonal antibodies and subsequently incubated with a lentil-lectin binding glycoprotein fraction and a radiolabeled antibody. Fig. 3 shows the results with 3 antibodies with different specificities (MaB8 and 50B8 and 45A9) used for coating and radiolabeled 50B8. Monoclonal antibody 45A9 does not react with an antigen in the glycoprotein fraction and was included in this experiment as a negative control. It could be shown that the coated monoclonal antibody MaB8 immunoadsorbed an antigen also detectable with radiolabeled 50B8. The antigen could be sandwiched to a much lesser extent between the 50B8 monoclonal antibodies themselves, suggesting that only 1 determinant was available for binding with 50B8. The specificity of the 2 antibodies was further tested by radiolabeling of the immunoadsorbed antigen in microtiter wells followed by gel electrophoresis. Autoradiography of gels electrophoresed under reducing conditions showed a specific band with an a p p a r e n t m o l e c u l a r w e i g h t o f 170,000 for 50B8 a n d M a B 8 , n o t d e t e c t a b l e w i t h 4 5 A 9
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Fig. 1. Additive binding assay with monoclonal antibodies MaB8 and 50B8 for determination of epitope specificity. Microtiter plates coated with crude mammary tumor membranes were incubated with serial dilutions of monoclonal antibodies MaB8 and 50B8, added either separately or in combination. The amount of antibody bound was determined by subsequent incubation with labeled rabbit anti-rat IgG and counting the bound label in a gamma counter. O O, Monoclonal antibody 50B8 alone; • •, monoclonal antibody MaB8 alone; [] C3, combination of monoclonal antibodies MaB8 and 50B8. Fig. 2. Blocking assay with monoclonal antibodies MaB8 and 50B8 for determination of epitope specificity. Serial dilutions of monoclonal antibodies MaB8 and 50B8 were incubated with crude mouse mammary tumor membranes coated to microtiter wells. The blocking of antibody binding was determined by subsequent incubation with labeled monoclonal antibody 50B8 and counting the bound label in a gamma counter. [] n, Preincubation with unlabeled MaB8; • a, preincubation with unlabeled 50B8.
448 (Fig. 4). N o specific b a n d was d e m o n s t r a b l e when the gel was el ect r o p h o r esed under n o n - r e d u c i n g conditions. T h e a p p a r e n t m o l e c u l a r weight of the MaB8 and 50B8 m o n o c l o n a l a n t i b o d y d ef i n ed antigen was also d e t e r m i n e d in a r a d i o i m m u n o p r e c i p i t a t i o n , using [35S]methionine-labeled antigens of a m o u s e m a m m a r y t u m o r cell line. Th e a p p a r e n t m o l e c u l a r weight of the i m m u n o p r e c i p i t a t e d antigen was again d e t e r m i n e d under r e d u c i n g an d n o n - r e d u c i n g conditions. The results (Fig. 5) showed for both antibodies 1 b a n d with an a p p a r e n t m o l e c u l a r weight of 170,000, c o n f i r m i n g the results o b t a i n e d by the i m m u n o r a d i o l a b e l i n g procedure. As in the case of the imm u n o r a d i o l a b e l i n g p r o c e d u r e a higher b a c k g r o u n d was o b t a i n e d with the MaB8 a n t i b o d y than with 50B8.
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Fig. 3. Two-site sandwich immunoassay with monoclonal antibodies MaB8, 50B8 and 45A9 for determination of antigen specificity. Microtiter plates coated with monoclonal antibodies were incubated with a serial dilution of a lentil-lectin binding glycoprotein fraction. Immunoadsorbed antigens were detected by subsequent incubation with labeled monoclonal antibody 50B8 and counting the bound label in a gamma counter. • •, Coated monoclonal antibody MaB8: O O. coated monoclonal antibody 50B8; • A, coated monoclonal antibody 45A9. Fig. 4. NaDodSO4/polyacrylamide gel analysis of antigens immunoadsorbed from a lentil-lectin binding glycoprotein fraction by monoclonal antibodies MaB8, 50B8, 45A9 coated to microtiter wells. Immunoadsorbed antigens were labeled with 125I, solubilized in sample buffer containing 5% (v/v) 2-mercaptoethanol and analyzed on a 10% polyacrylamide gel. Exposure of the autoradiograph was for 6 h. A and B, immunobinding with monoclonal antibody 45A9; C and D, immunobinding with monoclonal antibody MaB8: E and F, immunobinding with monoclonal antibody 50B8.
449
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Fig. 5. NaDodSO4/polyacrylamide gel analysis of immunoprecipitates prepared with monoclonal antibodies MaB8 and 50B8 from ['~SS]methionine-labeled lysates of P3MTD5 mouse m a m m a r y tumor cells. Culture media from S p 2 / 0 mouse myeloma cells was used as a control, hnmunoprecipitates were analyzed on a 10% polyacrylamide gel in the prescence of 2-mcrcaptoethanol (A C) and on a 7% polyacrylamide gel in the prescence of iodoacetamide ( D - F ) . Exposure of the autoradiograph was for 7 days. A and D, immunoprecipitation with culture media from S P 2 / 0 myeloma cells; B and E, immunoprecipitation with culture media from MaB8 antibody producing hybridoma: C and F, imnmnoprecipitation with culture media from 50B8 antibody producing hybridoma.
Since no major differences were found in the apparent molecular weight of the antigen under reducing and non-reducing conditions, it was concluded that the antigen is a monomeric protein. Discussion
We describe here a simple and rapid immunobinding procedure for characterization of membrane proteins. Antigens are labeled after adsorption to immobilized antibodies, offering the advantage that the antigenic site is protected against destruction by radioiodination. Therefore, the procedure allows detection of practically all antigens. Only antigens that cannot be radioiodinated or antigens that lose the antigenic site after solubilization remain undetectable. A disadvantage of the immunobinding procedure, seen also in this study, is that in SDS gels specific bands are lost due to comigration or due to poor resolution with labeled IgG. While in gels electrophoresed under reducing conditions the MaB8 and 50B8 antibodies detect a specific band of 170 kDa, no specific band was demonstrable under non-reducing conditions.
450
In the immunobinding procedure, the high background with the MaB8 antibody as compared with the 50B8 antibody is not caused by the quality of the affinity purified antibodies. This conclusion was drawn because it made no difference whether immunoprecipitates were prepared with culture medium of the MaB8 antibody producing hybridoma or with affinity purified antibodies. The origin of the high background with MaB8 is not clear. Possibly, the affinity of the monoclonal antibody for the epitopes decreases after solubilization of the antigen, resulting in an effect on the background.
Acknowledgments We thank Ir. J. Hilkens for useful discussions. This work was supported by Grant NKI 79-6 from the Queen Wilhelmina Foundation for the Fight against Cancer in the Netherlands.
References Colcher, D., P. Horan Hand, M. Nuti and J. Schlom. 1981, Proc. Natl. Acad. Sci. U S A . 78, 3199. Foster, C.S., P.A.W. Edwards, E.A. Dinsdale and A.M. Neville, 1982a, Virchows Arch. Abt. A. Pathol. Anat. 394, 279.
Foster, C.S., E.A. Dinsdale, P.A.W. Edwards and A.M. Neville, 1982b, Virchows Arch. Abt. A. Pathol. Anat. 394, 295. Hilkens, J., J. Hilgers, F. Buys, Ph. Hageman, D. Schol, G. Van Doornewaard and J. Van den Tv, cel, 1984, Proc. Biol. Fluids 31, 1013. Hunter, W.M. and F.C. Greenwood, 1962, Nature (London) 194, 495. Laemmli, U.K., 1970, Nature (London) 227, 680. Parham, P., M.J. Androlewicz, F.M. Brodsky, N.J. Holmes and J.P. Ways, 1982, J. Immunol. Methods 53, 133. Sonnenberg, A., J. Daams, J. Hilkens and J. Hilgcrs, 1984, submitted. Taylor-Papadirnitriou, J., J. Peterson, J. Arklie, J. Burchell, R.L. Ceriani and W.F. Bodmer, 1981, Int. J. Cancer 28, 17. Uotila, M., E. Ruoslahti and E. Engvall, 1981, J. Immunol, Methods 42, 11.