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Radioimmunolocalization of murine mammary tumor virus proteins in gels
ANALYTICAL
BIOCHEMISTRY
119, 293-298 (1982)
Radioimmunolocalization of Murine Mammary Virus Proteins in Gels JAMESN. BAUSCH,K. S. Institute
of Cancer
Research,
PRASAD,
College of Physicians New York, New York
Tumor
AND S. SPIEGELMAN’ and Surgeons, 10032
Columbia
University,
Received June 10, 1981 A procedure for the immunological detection of antigens on sodium dodecyl sulfate-polyacrylamide gels using iodinated antibodies combined with an immunoconcentration step to enrich for the desired antigen(s) is described. This selective enrichment is achieved by separation of the antigen-antibody complexes with formalin-treated cells of Staphylococcus aureus and is followed by radioimmunolocalization on gels. This method is particularly useful for the characterization of antigens present at low concentrations in complex biological fluids. Its usefulness is exemplified here by the successful identification of mouse mammary tumor virus antigens in extracts of murine mammary tumors and in plasmas of tumor-bearing animals. In particular, the procedure permits the ready determination that the gp52 protein detected in the plasmas of tumor-bearing animals is in fact present as a 52,000-dalton species and not as a higher molecular weight precursor.
Methods have been developed to detect proteins on SDS’ gels by immunolocalization. Stumph et al. (1) localized nonhistone chromosomal proteins (NHCP) after SDSpolyacrylamide gel electrophoresis by incubating the gels with fluorescein-labeled antiNHCP antibodies. Olden and Yamada (2) detected proteins and glycoproteins on SDS gels by employing a sandwich technique of successive incubations with immune IgG and anti-y-globulin coupled to horseradish peroxidase. Using a similar technique, Van Raamsdonk et al. (3) detected as low as 1 ng of protein on very thin gel slices. Simi’ To whom correspondence should be addressed. ’ Abbreviations used: SDS, sodium dodecyl sulfate; IgG, immunoglobulin G; MMTV, mouse mammary tumor virus; gp52, 52,000-dalton glycoprotein of MMTV; RwMMTV IgG, IgG fraction of rabbit antiserum against MMTV; Ra-gp52 IgG, IgG fraction of rabbit antiserum against gp52; Ga-R IgG, IgG fraction of goat antiserum against rabbit IgG, NR IgG, normal rabbit IgG, SAF, formalinized Staphylococcus aureus cells; PBS, phosphate-buffered saline. 293
larly, yeast nonsense termination fragments in bacterial cell extracts were immunologically detected on gels using specific antibodies and [‘251]protein A (4). We report here the use of [iZSI]immunoglobulins to detect MMTV proteins in the virus and in mouse mammary tumor extracts and mouse plasma samples. In order to detect the viral proteins in plasma samples and extracts, we found it necessary to first immunoconcentrate the antigens as antibody-antigen complexes using Staphylococcus aureus cells (5). We achieved this enrichment by first treating the plasma or extracts with Ra-MMTV IgG and S. aureus cells. This step also reduces the amount of protein for gel electrophoresis. Following SDS-gel electrophoresis, we were successful in detecting the viral proteins either directly (by treating the gels with [‘251]Ra-MMTV IgG) or indirectly (Ra-MMTV IgG followed by [“‘I]Ga-R IgG or [‘251]protein A). The MMTV proteins identified in these studies were gp52, gp36, and ~27. The im0003-2697/82/020293-06$02.00/O Copyright Q 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
294
BAUSCH,
PRASAD, AND SPIEGELMAN
mediate goal was to develop a sensitive immunological detection system for these MMTV proteins in complex biological fluids. This research endeavor was spurred by recent immunological evidence (6-8) linking one of these (gp52) to a crossreactive protein found in human breast cancer. The method described here, however, should be of general use in a variety of situations requiring the characterization of interesting antigens. METHODS AND MATERIALS Virus and antisera preparations. MMTV, Ra-MMTV antiserum, and Ra-gp52 antiserum were prepared as described by Ritzi (9). The IgG fractions of the above antisera were isolated as described previously (6). Normal rabbit IgG (NR IgG and goat antirabbit IgG (Ga-R IgG) were purchased from Miles Laboratories. Preparation of adsorbent for immunoconcentration. Staphylococcus aureus cells
(Newman strain) were purchased from Sigma (St. Louis, MO.). These cells were formalinized and processed by the method of Kessler ( 10). This immunoadsorbent will henceforth be referred to as SAF. Zodination procedures. Immunoglobulins were radioiodinated by the method of Bolton and Hunter (I 1). Briefly, 60 gg of IgG in 100 ~1 of 0.1 M borate buffer, pH 8.5, was mixed with 1 mCi of Bolton-Hunter reagent (Amersham). After 20 min, the reaction was terminated by the addition of 100 cl1 0.2 M glycine in 0.1 M borate buffer, pH 8.5. The [1251]IgG was then separated from free iodine by passing the mixture through ACA34 (LKB, France) column (0.7 X 48 cm) equilibrated and run in PBS (0.15 M NaCl, 0.06 M phosphate, pH 7.4) containing 0.25% gelatin and 0.002% Thimerosol. The iodinated IgG samples had specific activities of approx 2-3 &i/pg. Adsorption of iodinared IgG. Iodinated IgG (approx 30 rg) preparations were adsorbed with insolubilized NIH/Swiss mouse liver and pooled normal human plasma (25
mg each) in 5 ml PBS. The suspension was stirred for 1 h at 37” and then for 3 h at 4”. After an overnight incubation at 4”, the suspension was centrifuged for 20 min at 10,OOOg and supernatant was collected. Tissue extracts. Mouse mammary tumor and mouse liver extracts were prepared using 3 M KC1 by the method of McCoy et al. ( 12). Briefly, the tissue was homogenized in 5- to 8-ml volumes of PBS. The homogenate was centrifuged for 20 min at 10,OOOg. The pellet obtained was stirred with 3 M KC1 (3- to 5ml volume) for 16 to 20 h at 4”. The mixture was then centrifuged at 40,OOOg for 1 h in a Sorvall RC2-B centrifuge. The supernatant was dialyzed for 48 h against PBS and concentrated using Aquacide G- 1. Immunoconcentration of MMTV proteins. Plasma from tumor-bearing mice (0.5-
1 ml) or tissue extracts (2-5 mg protein in 1 ml) were incubated with 100 pg RwMMTV IgG for 60 min at 37”. This was followed by the addition of SAF and incubated for 60 min at 37”. The complexes bound to the cells were pelleted by centrifugation at 3,000g for 15 min. The cellular pellet was washed twice with 2 ml PBS. The washed pellet was treated with SDS containing electrophoresis sample buffer, centrifuged, and the supernatant was analyzed by SDS-gel electrophoresis. Generally, the pretreatment of samples with normal rabbit IgG and SAF reduced any nonspecific background. Electrophoresis and immunolocalization on SDS-polyacrylamide gels. SDS-poly-
acrylamide gel electrophoresis of MMTV, immunoprecipitates of plasma samples or tissue extracts were carried out on 7.5% polyacrylamide gels according to the method of Shapiro et al. (13). The gels were fixed overnight in a 25% isopropanol, 10% acetic acid solution. This was followed by a series of 60-min incubations at 37” with 10% acetic acid, and 7% acetic acid. Finally, the gels were washed with several changes of PBS. The gels were cut lengthwise into four parts,
RADIOIMMUNOLOCALIZATION
permitting the examination of a single sample with four different reagents. In the direct technique, the longitudinally cut gel slices were incubated with 1 X lo6 cpm of the desired [ ‘251]IgG in 1 ml PBS containing 0.05% Tween-20 and 50 pg of unlabeled normal rabbit IgG for 36 h at 37” with gentle agitation. The unbound IgG was washed out with several changes of PBS at 37” for another 36 h. The gels were then sliced transversely (2 mm) and radioactivity was counted. In the indirect technique, the longitudinally sliced gels were first incubated with unlabeled Ra-MMTV or Rc+gp52 IgG for 36 h at 37”. The excess IgG was washed out as described above and gel slices were incubated with 1 X lo6 cpm of [‘251]Ga-R IgG. The gel slices were further processed in the same manner as the direct technique. RESULTS
We were able to identify MMTV proteins by both the direct and indirect techniques of immunolocalization in SDS-polyacrylamide gels. Figure la illustrates the ability of [ ‘*‘I]Ra-gp52 to detect only gp52 in a gel containing all of the MMTV proteins. A lofold amplification of bound counts was obtained if treatment with unlabeled Ra-gp52 IgG was followed by [‘251]G-(r-R IgG (Fig. lb). However, the background in this case was appreciably higher than in the direct technique. In control gel slices, neither [ ‘2sI]normal rabbit IgG (Fig. la) nor [‘251]Ga-R IgG alone (Fig. 1b) bound to any of the proteins in the gels, We were also able to detect gp52, gp36, and p27 both directly (Fig. 2a) and indirectly (Fig. 2b) on gels containing MMTV proteins by using Ra-MMTV IgG which contained antibodies directed against these three proteins. It appears that the profile in this experiment (and others not dealt with here) is reflective not only of the amount of each protein present in the gel, but also the
OF PROTEINS
IO
295
20
30
40
50
FRACTION NUMBER FIG. 1. Immunolocalization of gp52 on SDS-polyacrylamide gel slices containing MMTV proteins (100 ng) using iodinated antibodies: (a) A, with [‘251]R~gp52 IgG (direct method); A, with [“‘I]NR IgG. (b) 0, with Ra-gp52 IgG followed by [‘251]Ga-R IgG (indirect method); 0, with only [‘2SI]Ga-R IgG. All the slices were part of the same gel. The four arrows indicate the electrophoretic migration of the molecular weight markers which were bovine serum albumin (BSA 68,000), ovalbumin (OVA 45,000), trypsin (TRY 26,000) and lysozyme (LYS 14,300).
quantity and qualities of antibodies present in the antiserum. In order to identify a minor protein in a complex mixture, we used an immunoconcentration step prior to radioimmunolocalization in gel. As described under Materials and Methods, mouse mammary tumor extracts or plasma samples from tumor-bearing mice were incubated with Ra-MMTV IgG ( 100 pg of IgG/ml of sample). The complexes formed were pelleted using SAF. After treatment of the bacterial pellet with SDS sample buffer, the complexes were subjected to electrophoresis and radioimmunolocalization in gel. By the addition of this immunoconcentration step, we detected gp52,
296
BAUSCH, PRASAD, AND SPIEGELMAN
resis analysis. When the immunoconcentration step in combination with the indirect technique was employed, [ ‘251]protein A (from S. aureus cells) was used in the place of [‘251]Ga-R IgG. The latter antibody was not employed since the binding of this iodinated probe to the heavy and light chains of rabbit IgG was found to severely interfere with the interpretability of the result. [“‘I]Protein A, however, did not bind to either the heavy or light chain of rabbit IgG after SDS-gel electrophoresis, and therefore was employed to amplify detection in the indirect mode. In general, we found a lofold amplification of bound counts could be obtained by employing unlabeled immune IgG followed by [ ‘*‘I]protein A or [‘25I ]GcxR IgG.
:k FRACkN N30UMBE4R0 50
FIG. 2. Immunolocalization of major MMTV proteins (gp52, gp36, and ~27) on SDS-polyacrylamide gel slices containing MMTV proteins (400 ng) using iodinated antibodies: (a) with [‘251]Rcx-MMTV IgG (direct method); (b) with Ra-MMTV IgG followed by [ ‘251]G~R IgG (indirect method). Both the slices were parts of the same gel.
gp36, and p27 in RI11 mouse mammary tumor extract using [‘2SI]R~-MMTV IgG (Fig. 3a). We also detected gp52 in plasma from tumor-bearing RI11 mice with [ ‘2SI]Rogp52 IgG (Fig. 3b). The specificity of the detection is supported by the following features. (a) No binding of [‘2’I]normal rabbit IgG was observed on these gels (Figs. 3a and b). (b) When SAF was treated alone with SDS sample buffer and the supernatant subjected to SDS-gel electrophoresis; no binding of [ ‘251]R~-MMTV IgG, [ “‘I]Ra-gp52 IgG or [ ‘251]normal rabbit IgG was detected. (c) No reaction was observed with normal RI11 mouse liver extracts or plasma from NIH/ Swiss mice. If the immunoconcentration step was omitted, the total protein concentration was prohibitively high for SDS-gel electropho-
FRACTION NUMBER FIG. 3. Immunolocalization of MMTV proteins in RI11 tumor extract and plasma from tumor-bearing RI11 mouse. Samples were treated with RWMMTV IgG. The immune complexes were bound to SAF and analyzed on SDS-gel electrophoresis as described under Materials and Methods. (a) RI11 tumor extract with (0) [‘251]R-MMTV IgG and (0) [‘*‘I]NR IgG; (b) RI11 tumor plasma with (0) [“‘I]R-gp52 IgG and (0) [‘251]NR IgG.
RADIOIMMUNOLOCALIZATION
Our demonstration that the gp52 found in the plasma of tumored mice exists as free protein with a molecular weight of 52,000 (Fig. 3b) exemplifies the utility of the technique. Our results do not support the suggestion of McClelland (14) that the gp52like protein detected in the plasma is a precursor protein (gp70) or a mixture of gp52 with gp70. DISCUSSION
These experiments demonstrate the ability of the radioimmunological technique to immunospecifically localize MMTV proteins in SDS gels. The utility was significantly increased when immunoconcentration with SAF was included. A few of the advantages of the procedure described here as compared to procedures commonly employed in immunological detection may be briefly noted. Large volumes are not a deterrent in our procedures as they are in the standard radioimmunoassays. More importantly, immunolocalization is a noncompetitive reaction and hence may detect weakly crossreactive antigens that might not be detectable in a competitive radioimmunoassay. The converse of this technique was first reported by Kessler (15) in which he radiolabeled cell surface antigens, solubilized, and precipitated them with antibodies and S. aureus, then analyzed the precipitates by SDS-gel electrophoresis. More pertinently, researchers ( 16,17) have radiolabeled cell cultures producing MMTV and subsequently employed specific antisera to precipitate the proteins of interest. However, when the percentage of the relevant proteins in the sample is very small (less than 0.005%) this method introduces unacceptably large backgrounds. The use of labeled antibodies, rather than labeled antigens, circumvents this difficulty. It should be noted that this immunolocalization procedure also provides a very simple means of characterizing antisera produced in response to a mixture of antigens. This stems from the fact that the
297
OF PROTEINS
radioimmunolocalization does not require the use of purified radioactively labeled antigens for radioimmunoprecipitation, and offers the advantage of being able to simultaneously detect and identify antibodies being elicited in response to different proteins. Another point worth noting is that we found prior staining of the proteins with Coomassie blue did not affect their ability to bind with radioactive antibodies. This means that one can first visualize and roughly quantitate the proteins present and then use the same gel to immunologically identify the proteins with radiolabeled specific antibody. Finally, another attractive feature of the technique is the fact that one can ascertain the molecular weight of an unpurified antigen in a complex mixture since the antigen is subjected to SDS-gel electrophoresis analysis before incubation with labeled antibodies. The method described here combines the resolving power of SDS-gel electrophoresis with the high specificity of antibody-antigen reactions. By virtue of its simplicity it should be applicable to a wide variety of problems requiring the detection of small quantities of antigen in complex biological fluids, providing that the antigenic determinants can withstand SDS treatment. ACKNOWLEDGMENTS We thank Vincent D’Elia and Steven Moy for their excellent technical assistance. This investigation was supported by Grant CA-02332 and Contract NOl-CP’J1016 awarded by the National Cancer Institute. J. N. B. is a recipient of an NIH postdoctoral fellowship NOlF-32-CA 06141.
REFERENCES 1. Stumph, W. E., Elgin, S. (1974) J. Immunol. 113, 2. Olden, K., and Yamada, Biochem. 78,483-490. 3. Van Rammsdonk, W., Pool, (1977) J. Immunol. Meth. 4. Bigelis, R., and Burridge, K. phys. Rex Commun. 82,
C. R., and Hood, L. 1752-1756. K. M. (1977) Anal. C. W., and Heytig, C. 17, 337-348. (1978) Biochem. Bio322-327.
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5. Cullen, S. E., and Schwartz, B. D. (1976) J. Immunol. 117, 136-142. 6. Mesa-Tejada, R., Keydar, I., Ramanarayanan, M., Ohno, T., Fenoglio, C., and Spiegelman, S. (1978) Proc. Nat. Acad. Sci. USA 75, 15291533. 7. Zachrau, R. E., Black, M., Dion, A. S., Shove, B., Fine, D. L., Leis, H. P., Jr., and Williams, C. J. (1976) Cancer Res. 36, 3143-3146. 8. Black, M. M., Zachrau, R. E., Dion, A. S., Shove, B., Fine, D. L., L&s, H. P. Jr., and Williams, C. J. (1976) Cancer Res. 36,4137-4142. 9. Ritzi, E., Baldi, A., and Spiegelman, S. (1976) J. Viral. 25, 374-383. 10. Kessler,S. W. (1975)5. Immunol. 115,1617-1624.
AND SPIEGELMAN 11. Bolton, A. E., and Hunter, W. M. (1973) J. Biochem. 133, 529-539. 12. McCoy, J. L., Jerome, L. F., Dean, J. H., Cannon, G. B., Alford, T. C., Doering, T., and Herberman, R. B. (1974) J. Nat. Cancer Inst. 53, 1 l17. 13. Shapiro, A. L., Vinuela, E., and Maize], J. V., Jr. (1967) Biochem. Biophys. Res. Commun. 28, 815-820. 14. McClelland, A. J. (1979) Nature (LondonJ 277, 13. 15. Kessler, S. W. (1976)J. Immunol. 117,1482-1490. 16. Racevskis, J., and Sarkar, N. H. (1978) Viral. 75, 188-197. 17. Schochetman, G., Fine, D. L., and Massey, R. J. (1978) Virol. 88, 379-383.
BIOCHEMISTRY
119, 293-298 (1982)
Radioimmunolocalization of Murine Mammary Virus Proteins in Gels JAMESN. BAUSCH,K. S. Institute
of Cancer
Research,
PRASAD,
College of Physicians New York, New York
Tumor
AND S. SPIEGELMAN’ and Surgeons, 10032
Columbia
University,
Received June 10, 1981 A procedure for the immunological detection of antigens on sodium dodecyl sulfate-polyacrylamide gels using iodinated antibodies combined with an immunoconcentration step to enrich for the desired antigen(s) is described. This selective enrichment is achieved by separation of the antigen-antibody complexes with formalin-treated cells of Staphylococcus aureus and is followed by radioimmunolocalization on gels. This method is particularly useful for the characterization of antigens present at low concentrations in complex biological fluids. Its usefulness is exemplified here by the successful identification of mouse mammary tumor virus antigens in extracts of murine mammary tumors and in plasmas of tumor-bearing animals. In particular, the procedure permits the ready determination that the gp52 protein detected in the plasmas of tumor-bearing animals is in fact present as a 52,000-dalton species and not as a higher molecular weight precursor.
Methods have been developed to detect proteins on SDS’ gels by immunolocalization. Stumph et al. (1) localized nonhistone chromosomal proteins (NHCP) after SDSpolyacrylamide gel electrophoresis by incubating the gels with fluorescein-labeled antiNHCP antibodies. Olden and Yamada (2) detected proteins and glycoproteins on SDS gels by employing a sandwich technique of successive incubations with immune IgG and anti-y-globulin coupled to horseradish peroxidase. Using a similar technique, Van Raamsdonk et al. (3) detected as low as 1 ng of protein on very thin gel slices. Simi’ To whom correspondence should be addressed. ’ Abbreviations used: SDS, sodium dodecyl sulfate; IgG, immunoglobulin G; MMTV, mouse mammary tumor virus; gp52, 52,000-dalton glycoprotein of MMTV; RwMMTV IgG, IgG fraction of rabbit antiserum against MMTV; Ra-gp52 IgG, IgG fraction of rabbit antiserum against gp52; Ga-R IgG, IgG fraction of goat antiserum against rabbit IgG, NR IgG, normal rabbit IgG, SAF, formalinized Staphylococcus aureus cells; PBS, phosphate-buffered saline. 293
larly, yeast nonsense termination fragments in bacterial cell extracts were immunologically detected on gels using specific antibodies and [‘251]protein A (4). We report here the use of [iZSI]immunoglobulins to detect MMTV proteins in the virus and in mouse mammary tumor extracts and mouse plasma samples. In order to detect the viral proteins in plasma samples and extracts, we found it necessary to first immunoconcentrate the antigens as antibody-antigen complexes using Staphylococcus aureus cells (5). We achieved this enrichment by first treating the plasma or extracts with Ra-MMTV IgG and S. aureus cells. This step also reduces the amount of protein for gel electrophoresis. Following SDS-gel electrophoresis, we were successful in detecting the viral proteins either directly (by treating the gels with [‘251]Ra-MMTV IgG) or indirectly (Ra-MMTV IgG followed by [“‘I]Ga-R IgG or [‘251]protein A). The MMTV proteins identified in these studies were gp52, gp36, and ~27. The im0003-2697/82/020293-06$02.00/O Copyright Q 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
294
BAUSCH,
PRASAD, AND SPIEGELMAN
mediate goal was to develop a sensitive immunological detection system for these MMTV proteins in complex biological fluids. This research endeavor was spurred by recent immunological evidence (6-8) linking one of these (gp52) to a crossreactive protein found in human breast cancer. The method described here, however, should be of general use in a variety of situations requiring the characterization of interesting antigens. METHODS AND MATERIALS Virus and antisera preparations. MMTV, Ra-MMTV antiserum, and Ra-gp52 antiserum were prepared as described by Ritzi (9). The IgG fractions of the above antisera were isolated as described previously (6). Normal rabbit IgG (NR IgG and goat antirabbit IgG (Ga-R IgG) were purchased from Miles Laboratories. Preparation of adsorbent for immunoconcentration. Staphylococcus aureus cells
(Newman strain) were purchased from Sigma (St. Louis, MO.). These cells were formalinized and processed by the method of Kessler ( 10). This immunoadsorbent will henceforth be referred to as SAF. Zodination procedures. Immunoglobulins were radioiodinated by the method of Bolton and Hunter (I 1). Briefly, 60 gg of IgG in 100 ~1 of 0.1 M borate buffer, pH 8.5, was mixed with 1 mCi of Bolton-Hunter reagent (Amersham). After 20 min, the reaction was terminated by the addition of 100 cl1 0.2 M glycine in 0.1 M borate buffer, pH 8.5. The [1251]IgG was then separated from free iodine by passing the mixture through ACA34 (LKB, France) column (0.7 X 48 cm) equilibrated and run in PBS (0.15 M NaCl, 0.06 M phosphate, pH 7.4) containing 0.25% gelatin and 0.002% Thimerosol. The iodinated IgG samples had specific activities of approx 2-3 &i/pg. Adsorption of iodinared IgG. Iodinated IgG (approx 30 rg) preparations were adsorbed with insolubilized NIH/Swiss mouse liver and pooled normal human plasma (25
mg each) in 5 ml PBS. The suspension was stirred for 1 h at 37” and then for 3 h at 4”. After an overnight incubation at 4”, the suspension was centrifuged for 20 min at 10,OOOg and supernatant was collected. Tissue extracts. Mouse mammary tumor and mouse liver extracts were prepared using 3 M KC1 by the method of McCoy et al. ( 12). Briefly, the tissue was homogenized in 5- to 8-ml volumes of PBS. The homogenate was centrifuged for 20 min at 10,OOOg. The pellet obtained was stirred with 3 M KC1 (3- to 5ml volume) for 16 to 20 h at 4”. The mixture was then centrifuged at 40,OOOg for 1 h in a Sorvall RC2-B centrifuge. The supernatant was dialyzed for 48 h against PBS and concentrated using Aquacide G- 1. Immunoconcentration of MMTV proteins. Plasma from tumor-bearing mice (0.5-
1 ml) or tissue extracts (2-5 mg protein in 1 ml) were incubated with 100 pg RwMMTV IgG for 60 min at 37”. This was followed by the addition of SAF and incubated for 60 min at 37”. The complexes bound to the cells were pelleted by centrifugation at 3,000g for 15 min. The cellular pellet was washed twice with 2 ml PBS. The washed pellet was treated with SDS containing electrophoresis sample buffer, centrifuged, and the supernatant was analyzed by SDS-gel electrophoresis. Generally, the pretreatment of samples with normal rabbit IgG and SAF reduced any nonspecific background. Electrophoresis and immunolocalization on SDS-polyacrylamide gels. SDS-poly-
acrylamide gel electrophoresis of MMTV, immunoprecipitates of plasma samples or tissue extracts were carried out on 7.5% polyacrylamide gels according to the method of Shapiro et al. (13). The gels were fixed overnight in a 25% isopropanol, 10% acetic acid solution. This was followed by a series of 60-min incubations at 37” with 10% acetic acid, and 7% acetic acid. Finally, the gels were washed with several changes of PBS. The gels were cut lengthwise into four parts,
RADIOIMMUNOLOCALIZATION
permitting the examination of a single sample with four different reagents. In the direct technique, the longitudinally cut gel slices were incubated with 1 X lo6 cpm of the desired [ ‘251]IgG in 1 ml PBS containing 0.05% Tween-20 and 50 pg of unlabeled normal rabbit IgG for 36 h at 37” with gentle agitation. The unbound IgG was washed out with several changes of PBS at 37” for another 36 h. The gels were then sliced transversely (2 mm) and radioactivity was counted. In the indirect technique, the longitudinally sliced gels were first incubated with unlabeled Ra-MMTV or Rc+gp52 IgG for 36 h at 37”. The excess IgG was washed out as described above and gel slices were incubated with 1 X lo6 cpm of [‘251]Ga-R IgG. The gel slices were further processed in the same manner as the direct technique. RESULTS
We were able to identify MMTV proteins by both the direct and indirect techniques of immunolocalization in SDS-polyacrylamide gels. Figure la illustrates the ability of [ ‘*‘I]Ra-gp52 to detect only gp52 in a gel containing all of the MMTV proteins. A lofold amplification of bound counts was obtained if treatment with unlabeled Ra-gp52 IgG was followed by [‘251]G-(r-R IgG (Fig. lb). However, the background in this case was appreciably higher than in the direct technique. In control gel slices, neither [ ‘2sI]normal rabbit IgG (Fig. la) nor [‘251]Ga-R IgG alone (Fig. 1b) bound to any of the proteins in the gels, We were also able to detect gp52, gp36, and p27 both directly (Fig. 2a) and indirectly (Fig. 2b) on gels containing MMTV proteins by using Ra-MMTV IgG which contained antibodies directed against these three proteins. It appears that the profile in this experiment (and others not dealt with here) is reflective not only of the amount of each protein present in the gel, but also the
OF PROTEINS
IO
295
20
30
40
50
FRACTION NUMBER FIG. 1. Immunolocalization of gp52 on SDS-polyacrylamide gel slices containing MMTV proteins (100 ng) using iodinated antibodies: (a) A, with [‘251]R~gp52 IgG (direct method); A, with [“‘I]NR IgG. (b) 0, with Ra-gp52 IgG followed by [‘251]Ga-R IgG (indirect method); 0, with only [‘2SI]Ga-R IgG. All the slices were part of the same gel. The four arrows indicate the electrophoretic migration of the molecular weight markers which were bovine serum albumin (BSA 68,000), ovalbumin (OVA 45,000), trypsin (TRY 26,000) and lysozyme (LYS 14,300).
quantity and qualities of antibodies present in the antiserum. In order to identify a minor protein in a complex mixture, we used an immunoconcentration step prior to radioimmunolocalization in gel. As described under Materials and Methods, mouse mammary tumor extracts or plasma samples from tumor-bearing mice were incubated with Ra-MMTV IgG ( 100 pg of IgG/ml of sample). The complexes formed were pelleted using SAF. After treatment of the bacterial pellet with SDS sample buffer, the complexes were subjected to electrophoresis and radioimmunolocalization in gel. By the addition of this immunoconcentration step, we detected gp52,
296
BAUSCH, PRASAD, AND SPIEGELMAN
resis analysis. When the immunoconcentration step in combination with the indirect technique was employed, [ ‘251]protein A (from S. aureus cells) was used in the place of [‘251]Ga-R IgG. The latter antibody was not employed since the binding of this iodinated probe to the heavy and light chains of rabbit IgG was found to severely interfere with the interpretability of the result. [“‘I]Protein A, however, did not bind to either the heavy or light chain of rabbit IgG after SDS-gel electrophoresis, and therefore was employed to amplify detection in the indirect mode. In general, we found a lofold amplification of bound counts could be obtained by employing unlabeled immune IgG followed by [ ‘*‘I]protein A or [‘25I ]GcxR IgG.
:k FRACkN N30UMBE4R0 50
FIG. 2. Immunolocalization of major MMTV proteins (gp52, gp36, and ~27) on SDS-polyacrylamide gel slices containing MMTV proteins (400 ng) using iodinated antibodies: (a) with [‘251]Rcx-MMTV IgG (direct method); (b) with Ra-MMTV IgG followed by [ ‘251]G~R IgG (indirect method). Both the slices were parts of the same gel.
gp36, and p27 in RI11 mouse mammary tumor extract using [‘2SI]R~-MMTV IgG (Fig. 3a). We also detected gp52 in plasma from tumor-bearing RI11 mice with [ ‘2SI]Rogp52 IgG (Fig. 3b). The specificity of the detection is supported by the following features. (a) No binding of [‘2’I]normal rabbit IgG was observed on these gels (Figs. 3a and b). (b) When SAF was treated alone with SDS sample buffer and the supernatant subjected to SDS-gel electrophoresis; no binding of [ ‘251]R~-MMTV IgG, [ “‘I]Ra-gp52 IgG or [ ‘251]normal rabbit IgG was detected. (c) No reaction was observed with normal RI11 mouse liver extracts or plasma from NIH/ Swiss mice. If the immunoconcentration step was omitted, the total protein concentration was prohibitively high for SDS-gel electropho-
FRACTION NUMBER FIG. 3. Immunolocalization of MMTV proteins in RI11 tumor extract and plasma from tumor-bearing RI11 mouse. Samples were treated with RWMMTV IgG. The immune complexes were bound to SAF and analyzed on SDS-gel electrophoresis as described under Materials and Methods. (a) RI11 tumor extract with (0) [‘251]R-MMTV IgG and (0) [‘*‘I]NR IgG; (b) RI11 tumor plasma with (0) [“‘I]R-gp52 IgG and (0) [‘251]NR IgG.
RADIOIMMUNOLOCALIZATION
Our demonstration that the gp52 found in the plasma of tumored mice exists as free protein with a molecular weight of 52,000 (Fig. 3b) exemplifies the utility of the technique. Our results do not support the suggestion of McClelland (14) that the gp52like protein detected in the plasma is a precursor protein (gp70) or a mixture of gp52 with gp70. DISCUSSION
These experiments demonstrate the ability of the radioimmunological technique to immunospecifically localize MMTV proteins in SDS gels. The utility was significantly increased when immunoconcentration with SAF was included. A few of the advantages of the procedure described here as compared to procedures commonly employed in immunological detection may be briefly noted. Large volumes are not a deterrent in our procedures as they are in the standard radioimmunoassays. More importantly, immunolocalization is a noncompetitive reaction and hence may detect weakly crossreactive antigens that might not be detectable in a competitive radioimmunoassay. The converse of this technique was first reported by Kessler (15) in which he radiolabeled cell surface antigens, solubilized, and precipitated them with antibodies and S. aureus, then analyzed the precipitates by SDS-gel electrophoresis. More pertinently, researchers ( 16,17) have radiolabeled cell cultures producing MMTV and subsequently employed specific antisera to precipitate the proteins of interest. However, when the percentage of the relevant proteins in the sample is very small (less than 0.005%) this method introduces unacceptably large backgrounds. The use of labeled antibodies, rather than labeled antigens, circumvents this difficulty. It should be noted that this immunolocalization procedure also provides a very simple means of characterizing antisera produced in response to a mixture of antigens. This stems from the fact that the
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OF PROTEINS
radioimmunolocalization does not require the use of purified radioactively labeled antigens for radioimmunoprecipitation, and offers the advantage of being able to simultaneously detect and identify antibodies being elicited in response to different proteins. Another point worth noting is that we found prior staining of the proteins with Coomassie blue did not affect their ability to bind with radioactive antibodies. This means that one can first visualize and roughly quantitate the proteins present and then use the same gel to immunologically identify the proteins with radiolabeled specific antibody. Finally, another attractive feature of the technique is the fact that one can ascertain the molecular weight of an unpurified antigen in a complex mixture since the antigen is subjected to SDS-gel electrophoresis analysis before incubation with labeled antibodies. The method described here combines the resolving power of SDS-gel electrophoresis with the high specificity of antibody-antigen reactions. By virtue of its simplicity it should be applicable to a wide variety of problems requiring the detection of small quantities of antigen in complex biological fluids, providing that the antigenic determinants can withstand SDS treatment. ACKNOWLEDGMENTS We thank Vincent D’Elia and Steven Moy for their excellent technical assistance. This investigation was supported by Grant CA-02332 and Contract NOl-CP’J1016 awarded by the National Cancer Institute. J. N. B. is a recipient of an NIH postdoctoral fellowship NOlF-32-CA 06141.
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