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Protein A-coated erythrocyte binding to cell surface antigens: Application to quantitate retrovirus infectivity in vitro
655
SHORT COMMUNICATIONS TABLE FOCI OF INFECTIOUS
CENTERS
1
INDUCED
BY F-MuLV
IN
Virus dilution (1.0 ml/flask)
Calculated titer from S+L- assay* (FFU/ml)
Observed foci/flask
Control l/1600 l/800 l/400 l/266 l/200 l/160
0 25 50 100 150 200 250
0 16 30 65 90 127 145
a Infectious focus assay performed as described in the text. * Calculated titers are based on F-MuLV titer of 40.000 FFU/ml of the method of Bassin et al. (4).
ing rosetting, the monolayers are washed twice with prewarmed PBS and are immediately fixed and stained by flooding the flask with staining solution “B.” After 10 min the stain is removed and the flasks are rinsed with distilled water and air dried. Macroscopic rosette foci are visible to the unaided eye (Fig. lA), but counting accuracy is improved with a dissecting microscope (10X). Foci are defined unambiguously by clumps of attached erythrocytes (Fig. 1B) when viewed under phasecontrast or bright field microscopy. The dose response for F-MuLV in SC-1 cells is linear over a range normal for other plaque assays (Table 1) and yields numbers and variances among replicate samples comparable to measurements made 2
Virus dilution (1.0 ml/flask)
Observed foci/flask
Control l/1,600,000 l/800,000 l/400,006 l/267,006 1/200,000 1/100,000
0 15 42 50 118 121 333
’ Infectious focus assay performed the text.
Titer estimates FFU/ml 2.4 3.4 2.0 3.2 2.4 3.3
0 X x x X X x
1.
2. 3.
6. 7. 8. 9.
10’ lo1 lo7 10’ lo7 lo7
as described in
2.6 2.4 2.6 2.4 2.5 2.3
0 X X X X X X
ld 104 10’ ld lo4 10’
obtained on S+L- cells by a variation
(18)
ACKNOWLEDGMENTS The research was supported by Grant PCM791372 from the National Science Foundation and in part by Grant CA23032 from the U. S. Public Health Service. T.F. was supported by a predoctoral training grant from the U. S. Public Health Service.
5.
FOCI OF INFECTIOUS CENTERS INDUCED BY XENOTROPICBALB v-2 VIRUS IN CCL64 CELLSO
Titer estimates (FFU/ml)
with the SL- method. The rosette assay is applicable to other virus-host systems as shown by the foci observed with xenotropic Balb-2 virus in CCL64 mink lung fibroblasts (Fig. 1B and Table 2).
4. TABLE
SC-1 CELLS’
10. 11. 12. 13.
REFERENCES HARTLEY, J. W., ROWE, W. P., CAPPS, W. I., and HUEBNER, R. J., J. Viral. 3, 126-132 (1969). SCOLNICK, E. M., PARKS, W. P., and LIVINGSTON, D. M., J. Immune.!. 109.570-577 (1972). DECLEVE, A., NIWA, O., HILGERS, J., and KAPLAN, H. S., Virology 57, 491-502 (1974). BASSIN, R. H., TUTTLE, N., and FISCHINGER, P. J., Nature (London) 229, 564-566 (1971). ROWE, W. P., PUGH, W. E., and HARTLEY, J. W., Virology 42, 1136-1139 (1970). NEXO, B. A., Virology 77, 849-852 (1977). REMBAUM, A., and DREYER, W. J., Science 208. 364-368 (1980). RUTA, M., and KABAT, D., J. Viral. 35, 844-853 (1980). GODING, J. W., J. Immunol. Methods 10. 61-66 (1976). O’BRIEN, B. R. A., Exp. Cell. Res. 21, 226-228 (1960). MURRAY, M. J., and KABAT, D., J. Biol. Chem. 254, 1340-1348 (1979). DRESLER, S., RUTA, M., MURRAY, M. J., and KABAT, D., J. Virol. 30, 564-575 (1979). SHERTON, C. C., EVANS, L. H., POLONOFF, E., and KABAT, D., J. Viral. 19, 118-125 (1976).
VIROLOGY
111,656-661
(1981)
Protein A Assay for Mouse Mammary Tumor Virus gp52 Determinants Human Mammary Tumor Cells
on Murine and
ANDREA H. CALLIS AND EARL M. RITZ? Department
of Microbiology
and Immunology, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38165 Accepted February
9, 1981
An isotopic staphylococcal protein A test (ISPAT) has been used to detect determinants of the mouse mammary tumor virus (MMTV) envelope antigen (gp52) on murine mammary tumor cells and to search for an immunologically related protein on the surface of established human mammary tumor cell lines. The substitution of fluorescein isothiocyanate-conjugated staphylococcal protein A (SPA) in assay for iodinated SPA demonstrated that gp52 detected on murine cells by adherent cell assay was predominantly membrane associated rather than cytoplasmic in nature. Swiss mouse embryo cells producing a C-type retrovirus (Moloney murine leukemia virus) were negative when assayed for gp52. Comparative assays (ISPATs) for gp52 cell surface determinants on human and murine mammary tumor cells did not demonstrate gp52-specific binding on five cell lines derived from human breast tumors and one derived from the milk of a lactating woman; however, parallel control GR mammary cultures, assayed on the same day, demonstrated the capability of the assay to detect gp52 determinants. Results indicated that this assay could have detected gp52 determinants on human cells at levels 1111th to 1160th of the amounts detected on murine cells.
The presence of a protein immunologically related to the mouse mammary tumor virus (MMTV) envelope glycoprotein (gp52) has been detected by immunohistochemical assays in tissue sections of 39% of human mammary tumors tested (1, .Q, and a human protein purified from milk of normal donors has demonstrated immunological and sequence relatedness to MMTV gp52 (3). In addition, reports of serological and cellular reactivity of breast cancer patients with MMTV and/or MMTV gp52 suggested that human mammary tumor cells might also possess cell surface determinants reactive with antigp52 serum (4-9). The majority of these studies, however, have demonstrated that less than 50% of patients were reactive with MMTV antigen. Although MMTV gp52 has been detected by RIA in plasma of tumor-bearing mice (X)-12), gp52 determinants were not similarly detected by RIA in sera of 89 breast cancer patients (11). ’ To whom
reprint
requests
0042-6822/81/080656-06$02.00/O
should
be addressed.
A protein A assay of cell monolayers (13) was adopted to determine if parallel comparative assays of human and mouse tumor cells would specifically demonstrate the presence of human cell surface determinants related to gp52. Since gp52 determinants have been detected even on the surface of cloned murine MMTV nonproducer tumor cells (Y), this study has focused on the cell surface. Furthermore, the envelope glycoprotein of MMTV is the major MMTV protein detected on the surface of murine mammary tumor cells (13, 14), and the envelope glycoprotein of a similar “putative” human retrovirus might, by analogy, be expected to be present as a cell surface antigen (CSA). The results obtained herein with fluorescein-labeled SPA and murine cells have indicated that gp52 detected under routine isotopic assay conditions is predominantly membrane associated. Although control murine cultures were positive for gp52 antigen and indicated that this assay could have detected gp52 determinants on 656
VIROLOGY
111.653-655
(1981)
Protein A-Coated
Erythrocyte Quantitate
Binding to Cell Surface Antigens: Retrovirus Infectivity in Vitro
THOMAS FITTING Department
of
Biochemistry,
AND DAVID
School of Medicine,
University
Application
to
KABAT’ of
Oregon Health Sciences Center,
Portland, Oregon 97201 Accepted February
5, 1981
Fibroblasts infected with murine leukemia virus (MuLV) bind erythrocytes coated with protein A to form rosettes in the presence of MuLV-specific antisera. This method, which is potentially applicable to any retrovirus and susceptible cell, has been specifically adapted as a focus assay for quantitating both ecotropic and xenotropic MuLV.
Methods to quantitate mammalian retroviruses by in vitro plaque or focus assays have largely supplanted relatively costly and cumbersome methods which depend on in vivo infectivity or on measurement of virus antigens synthesized by cultured cells (1-S). However, many retroviruses do not cause cytopathic changes in cells and will not rescue defective transforming viruses [as in the S’L- technique (4)] or induce syncytia with XC cells (5). Furthermore, it is important to realize that the virus titers obtained with any of these tissue culture assays are only relative numbers which are functions of the adsorption time and temperature, the culture conditions (e.g., the presence of DEAE-dextran), and the target cells employed. For example, we have observed that MuLV titers measured in SL- cells cannot be accurately employed for estimating multiplicities of virus infection into BALB/c or normal rat kidney fibroblasts. For these reasons it would be extremely useful to have a generally applicable retrovirus assay method which could utilize any target cell which is most relevant to a particular investigation. Immunological methods for detecting cell surface antigens that are expressed by infected cells have been adapted to titer virus and are, in principle, applicable to many different virus-host culture systems (6, 7). These approaches utilize “tagging” 1 To whom reprint
strategies which depend on having an antibody specific for a virus-encoded cell surface antigen. Recently, we described an erythrocyte rosette method for detection of cell surface antigens expressed by MuLV-infected fibroblasts (8). In this method, erythrocytes coated with protein A bind to fibroblast monolayers only in the presence of antibodies specific for cell surface antigens. Furthermore, adherence of erythrocytes to fibroblasts in the absence of antibodies is extremely low so that ratios of specific to nonspecific binding are routinely greater than 103. In this communication we describe an application of this technique in a focus assay for ecotropic MuLV and for assay of a xenotropic MuLV growing in mink lung fibroblasts. To conduct the rosette focus assay, two special reagents are required in addition to a source of virus, susceptible fibroblasts, and an appropriate antiserum. (A) Sheep erythrocytes are coupled to Staphylococcus aureus protein A using a modification of Goding’s CrC13 method (9). Briefly, 100 ~1 (sedimented volume) of sheep erythrocytes are washed five times in 0.9% NaCl and suspended at 25” in 700 ~1 0.9% NaCl containing 100 kg protein A (Sigma Chemical Co.). Then, 500 ~1 of 0.004% CrCl3 (prepared as described by Goding (9) and diluted immediately before use) is added dropwise while continuously vortexing the erythrocyte suspension. After 10 min, the suspension is diluted five-
requests should be addressed. 653
0042-6822/81/030653-03$0290/O Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
SHORT COMMUNICATIONS TABLE FOCI OF INFECTIOUS
CENTERS
1
INDUCED
BY F-MuLV
IN
Virus dilution (1.0 ml/flask)
Calculated titer from S+L- assay* (FFU/ml)
Observed foci/flask
Control l/1600 l/800 l/400 l/266 l/200 l/160
0 25 50 100 150 200 250
0 16 30 65 90 127 145
a Infectious focus assay performed as described in the text. * Calculated titers are based on F-MuLV titer of 40.000 FFU/ml of the method of Bassin et al. (4).
ing rosetting, the monolayers are washed twice with prewarmed PBS and are immediately fixed and stained by flooding the flask with staining solution “B.” After 10 min the stain is removed and the flasks are rinsed with distilled water and air dried. Macroscopic rosette foci are visible to the unaided eye (Fig. lA), but counting accuracy is improved with a dissecting microscope (10X). Foci are defined unambiguously by clumps of attached erythrocytes (Fig. 1B) when viewed under phasecontrast or bright field microscopy. The dose response for F-MuLV in SC-1 cells is linear over a range normal for other plaque assays (Table 1) and yields numbers and variances among replicate samples comparable to measurements made 2
Virus dilution (1.0 ml/flask)
Observed foci/flask
Control l/1,600,000 l/800,000 l/400,006 l/267,006 1/200,000 1/100,000
0 15 42 50 118 121 333
’ Infectious focus assay performed the text.
Titer estimates FFU/ml 2.4 3.4 2.0 3.2 2.4 3.3
0 X x x X X x
1.
2. 3.
6. 7. 8. 9.
10’ lo1 lo7 10’ lo7 lo7
as described in
2.6 2.4 2.6 2.4 2.5 2.3
0 X X X X X X
ld 104 10’ ld lo4 10’
obtained on S+L- cells by a variation
(18)
ACKNOWLEDGMENTS The research was supported by Grant PCM791372 from the National Science Foundation and in part by Grant CA23032 from the U. S. Public Health Service. T.F. was supported by a predoctoral training grant from the U. S. Public Health Service.
5.
FOCI OF INFECTIOUS CENTERS INDUCED BY XENOTROPICBALB v-2 VIRUS IN CCL64 CELLSO
Titer estimates (FFU/ml)
with the SL- method. The rosette assay is applicable to other virus-host systems as shown by the foci observed with xenotropic Balb-2 virus in CCL64 mink lung fibroblasts (Fig. 1B and Table 2).
4. TABLE
SC-1 CELLS’
10. 11. 12. 13.
REFERENCES HARTLEY, J. W., ROWE, W. P., CAPPS, W. I., and HUEBNER, R. J., J. Viral. 3, 126-132 (1969). SCOLNICK, E. M., PARKS, W. P., and LIVINGSTON, D. M., J. Immune.!. 109.570-577 (1972). DECLEVE, A., NIWA, O., HILGERS, J., and KAPLAN, H. S., Virology 57, 491-502 (1974). BASSIN, R. H., TUTTLE, N., and FISCHINGER, P. J., Nature (London) 229, 564-566 (1971). ROWE, W. P., PUGH, W. E., and HARTLEY, J. W., Virology 42, 1136-1139 (1970). NEXO, B. A., Virology 77, 849-852 (1977). REMBAUM, A., and DREYER, W. J., Science 208. 364-368 (1980). RUTA, M., and KABAT, D., J. Viral. 35, 844-853 (1980). GODING, J. W., J. Immunol. Methods 10. 61-66 (1976). O’BRIEN, B. R. A., Exp. Cell. Res. 21, 226-228 (1960). MURRAY, M. J., and KABAT, D., J. Biol. Chem. 254, 1340-1348 (1979). DRESLER, S., RUTA, M., MURRAY, M. J., and KABAT, D., J. Virol. 30, 564-575 (1979). SHERTON, C. C., EVANS, L. H., POLONOFF, E., and KABAT, D., J. Viral. 19, 118-125 (1976).
VIROLOGY
111,656-661
(1981)
Protein A Assay for Mouse Mammary Tumor Virus gp52 Determinants Human Mammary Tumor Cells
on Murine and
ANDREA H. CALLIS AND EARL M. RITZ? Department
of Microbiology
and Immunology, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38165 Accepted February
9, 1981
An isotopic staphylococcal protein A test (ISPAT) has been used to detect determinants of the mouse mammary tumor virus (MMTV) envelope antigen (gp52) on murine mammary tumor cells and to search for an immunologically related protein on the surface of established human mammary tumor cell lines. The substitution of fluorescein isothiocyanate-conjugated staphylococcal protein A (SPA) in assay for iodinated SPA demonstrated that gp52 detected on murine cells by adherent cell assay was predominantly membrane associated rather than cytoplasmic in nature. Swiss mouse embryo cells producing a C-type retrovirus (Moloney murine leukemia virus) were negative when assayed for gp52. Comparative assays (ISPATs) for gp52 cell surface determinants on human and murine mammary tumor cells did not demonstrate gp52-specific binding on five cell lines derived from human breast tumors and one derived from the milk of a lactating woman; however, parallel control GR mammary cultures, assayed on the same day, demonstrated the capability of the assay to detect gp52 determinants. Results indicated that this assay could have detected gp52 determinants on human cells at levels 1111th to 1160th of the amounts detected on murine cells.
The presence of a protein immunologically related to the mouse mammary tumor virus (MMTV) envelope glycoprotein (gp52) has been detected by immunohistochemical assays in tissue sections of 39% of human mammary tumors tested (1, .Q, and a human protein purified from milk of normal donors has demonstrated immunological and sequence relatedness to MMTV gp52 (3). In addition, reports of serological and cellular reactivity of breast cancer patients with MMTV and/or MMTV gp52 suggested that human mammary tumor cells might also possess cell surface determinants reactive with antigp52 serum (4-9). The majority of these studies, however, have demonstrated that less than 50% of patients were reactive with MMTV antigen. Although MMTV gp52 has been detected by RIA in plasma of tumor-bearing mice (X)-12), gp52 determinants were not similarly detected by RIA in sera of 89 breast cancer patients (11). ’ To whom
reprint
requests
0042-6822/81/080656-06$02.00/O
should
be addressed.
A protein A assay of cell monolayers (13) was adopted to determine if parallel comparative assays of human and mouse tumor cells would specifically demonstrate the presence of human cell surface determinants related to gp52. Since gp52 determinants have been detected even on the surface of cloned murine MMTV nonproducer tumor cells (Y), this study has focused on the cell surface. Furthermore, the envelope glycoprotein of MMTV is the major MMTV protein detected on the surface of murine mammary tumor cells (13, 14), and the envelope glycoprotein of a similar “putative” human retrovirus might, by analogy, be expected to be present as a cell surface antigen (CSA). The results obtained herein with fluorescein-labeled SPA and murine cells have indicated that gp52 detected under routine isotopic assay conditions is predominantly membrane associated. Although control murine cultures were positive for gp52 antigen and indicated that this assay could have detected gp52 determinants on 656
VIROLOGY
111.653-655
(1981)
Protein A-Coated
Erythrocyte Quantitate
Binding to Cell Surface Antigens: Retrovirus Infectivity in Vitro
THOMAS FITTING Department
of
Biochemistry,
AND DAVID
School of Medicine,
University
Application
to
KABAT’ of
Oregon Health Sciences Center,
Portland, Oregon 97201 Accepted February
5, 1981
Fibroblasts infected with murine leukemia virus (MuLV) bind erythrocytes coated with protein A to form rosettes in the presence of MuLV-specific antisera. This method, which is potentially applicable to any retrovirus and susceptible cell, has been specifically adapted as a focus assay for quantitating both ecotropic and xenotropic MuLV.
Methods to quantitate mammalian retroviruses by in vitro plaque or focus assays have largely supplanted relatively costly and cumbersome methods which depend on in vivo infectivity or on measurement of virus antigens synthesized by cultured cells (1-S). However, many retroviruses do not cause cytopathic changes in cells and will not rescue defective transforming viruses [as in the S’L- technique (4)] or induce syncytia with XC cells (5). Furthermore, it is important to realize that the virus titers obtained with any of these tissue culture assays are only relative numbers which are functions of the adsorption time and temperature, the culture conditions (e.g., the presence of DEAE-dextran), and the target cells employed. For example, we have observed that MuLV titers measured in SL- cells cannot be accurately employed for estimating multiplicities of virus infection into BALB/c or normal rat kidney fibroblasts. For these reasons it would be extremely useful to have a generally applicable retrovirus assay method which could utilize any target cell which is most relevant to a particular investigation. Immunological methods for detecting cell surface antigens that are expressed by infected cells have been adapted to titer virus and are, in principle, applicable to many different virus-host culture systems (6, 7). These approaches utilize “tagging” 1 To whom reprint
strategies which depend on having an antibody specific for a virus-encoded cell surface antigen. Recently, we described an erythrocyte rosette method for detection of cell surface antigens expressed by MuLV-infected fibroblasts (8). In this method, erythrocytes coated with protein A bind to fibroblast monolayers only in the presence of antibodies specific for cell surface antigens. Furthermore, adherence of erythrocytes to fibroblasts in the absence of antibodies is extremely low so that ratios of specific to nonspecific binding are routinely greater than 103. In this communication we describe an application of this technique in a focus assay for ecotropic MuLV and for assay of a xenotropic MuLV growing in mink lung fibroblasts. To conduct the rosette focus assay, two special reagents are required in addition to a source of virus, susceptible fibroblasts, and an appropriate antiserum. (A) Sheep erythrocytes are coupled to Staphylococcus aureus protein A using a modification of Goding’s CrC13 method (9). Briefly, 100 ~1 (sedimented volume) of sheep erythrocytes are washed five times in 0.9% NaCl and suspended at 25” in 700 ~1 0.9% NaCl containing 100 kg protein A (Sigma Chemical Co.). Then, 500 ~1 of 0.004% CrCl3 (prepared as described by Goding (9) and diluted immediately before use) is added dropwise while continuously vortexing the erythrocyte suspension. After 10 min, the suspension is diluted five-
requests should be addressed. 653
0042-6822/81/030653-03$0290/O Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.