- Email: [email protected]
An easy and rapid method to screen large numbers of antibodies against internal cellular determinants
Journal of Immunological Methods, 106 (1988) 211-216 Else~aer
211
JIM 04618
An easy and rapid method to screen large numbers of antibodies against internal cellular determinants L o t h a r Stitz, H a n s H e n g a r t n e r , A l a n a Althage a n d R o l f M. Z i n k e r n a g e l lnstttute of Pathology, Unwersitv Hospital, 8091 Ziirich, Switzerland, and Institute of Virolog}', Justus-Ltebig Universio', 6300 Giessen, F. R. G. (Received 1 September 1987, accepted 14 September 1987)
A rapid and easy method is described to screen great numbers of antisera or antibodies against internal cellular antigens including viral antigens or to screen various target cells for proper expression of antigens; the method can also be applied to determine fluorescent foci to enumerate non-cytopathic viruses. Cells, infected with a particular virus or uninfected, adhering to flat-bottomed 96-well microtiter plates were fixed with conventional phosphate-buffered saline containing 4% formaldehyde for 10 min, alternatively, cells were first fixed with 3% paraformaldehyde for 10 min and were then treated with Trition X-100 for another 10 min. After two washes, either fluorescein-labelled antiviral antibodies or first antiviral antibodies followed by labelled anti-species antibodies were applied, incubated and washed off as usual. A few drops of a balanced salt solution were kept in the well and were drained off gently just before the plates were examined. The plates were viewed directly in an inverted UV microscope or were inspected and photographed bottoms up with a conventional UV microscope mounted with an old-fashioned uncorrected objective (20 x ) which, because of its shorter length, permitted proper focussing. For most cases studied, sensitivity was comparable to the fluorescence analysis method of cells on slides. The plates could be stored for several months in a dark refrigerator if kept moist. The method is rapid because it avoids individual handling of samples for the washing procedures and does not need growing and mounting of cells on slides; up to 1000 samples can be tested by one person in a day. Key words: Intracellular antigen: Viral antigen: Immunofluorescence; Screening of antibodies
Introduction
Screening of complex antisera or of cell culture supernatants containing monoclonal antibodies against internal cellular antigens or viral antigens is usually laborious and time consuming. In par-
('orrespondence to: R.M. Zinkernagel, Institut ftir Pathologic, Universitfitsspital, Sternwartstrasse 2, 8091 Ziirich, Switzerland. Abbreviations: BI)V, Borna disease virus; LCMV, lymphocytic choriomeningitis virus; MEM, minimal essential medium; i.p., post infection.
ticular the growing of cells or mounting of individual cell preparations on slides and subsequent washing procedures are cumbersome. We have successfully grown cells, infected and fixed them and then treated them with appropriate antibodies in 96-well flat-bottomed tissue culture trays. If an inverted UV microscope is not available, a conventional UV microscope cannot be used to screen for antibodies because of mechanical restraints: for focussing, the objective of a regular microscope had to be lowered more than the thick 96-well microtiter plates allowed for. Cutting off the rims of the wells with conventional methods
0022-1759/88/$03.50 ': 1988 Elsevier Science Publishers B.V. (Biomedical Division)
212
Fig. l. Photograph of the Leitz UV microscope used with the mounted 10 x 20 x obJectives from cheap student microscopes, note that the microscope table is devoid of the slide holder and that the 96-well plate is inverse so that the bottom is positioned close enough to the objective. (hot cutting wire) was not feasible. We exchanged the excellent objectives of our UV microscopes for uncorrected but much shorter objectives ( 1 0 × and 20 x ) from rather simple and cheap student microscopes. This change permitted screening of inverted 96-well plates for fluorescent antibodies against internal cellular or viral antigens.
Materials and methods
Virus', virus infection and fixation of cells The poorly cytopathic, lymphocytic choriomeningitis virus (LCMV) WE strain had been obtained originally from Dr. F. Lehmann-Grube, Hamburg, F.R.G. (Lehmann-Grube et al., 1977; Jacobson et al., 1979). Cells were grown in glass or plastic flasks and were infected at a multiplicity of 0.01/cell. At 48 h post infection (p.i.) the cells were trypsinized, washed, adjusted and dispensed in aliquots of 100 /~l/well. Borna disease virus (BDV) (Ludwig et al., 1973) was used as another non-cytopathic, tightly cell-associated infectious agent, causing a persistent, slowly productive infection spreading mainly by cell-to-cell contact.
When cytopathic viruses (e.g., vaccinia or vesicular stomatitis virus) were used, cells were usually infected after they had adhered to the microtiter wells because, if infected beforehand, they usually failed to adhere a n d / o r tended to come off the plastic too readily during the washing procedures. Cells in microwells were fixed at room temperature in 4% formaldehyde or 3% paraformaldehyde in phosphate-buffered saline for 10.-30 min followed by 10-20 rain treatment with 3~ Triton X-100 in phosphate-buffered saline. After fixation, the cell preparations can be kept for weeks in the refrigerator before they are used for analysis.
Tissue culture Adherent primary peritoneal macrophages or established fibroblast cell lines (MC57G and 1,929 cells) were employed in studies on LCMV and cytopathic viruses; for BDV either persistently infected M D C K cells or acutely infected primary rabbit embryonal brain tissue cultures were used. They were grown in minimal essential medium (MEM) with 10% heat-inactivated fetal calf serum and penicillin plus streptomycin. Cells were seeded at 1 5 × 104 cells/well and allowed to adhere for
213 2- 20 h. Cells were either infected prior to or after transfer to the microwells.
Antibodies Pooled hyperimmune mouse anti-LCMV sera were used at a dilution of 1 in 100 and 25 ~l/well. Several monoclonal antibody preparations against LCMV protein were also used as undiluted culture supernatant, diluted 1 in 10, or as ascites fluid diluted 1 in 1000, A fluorescent-labelled goat anti-mouse Ig antibody was purchased from Tago, Burlingame, CA ( F I T C goat (Fab')2 anti-mouse IgG + IgM, code 4343) and used at 1 in 50 or 1 in 100 diluted 25 ~l/well. For BDV studies, sera from infected rats or mice and rat monoclonal antibodies against an intracellular antigen were used. All antibodies were applied at room temperature or at 3 7 ° C for 30 rain,
Microscopes Plates can be easily viewed through an inverted UV microscope; we have used the Leitz Diavert model with a 32 × objective (Phaco 170/-1 L32/0.40) in the bright field. If such a microscope is not available, a conventional microscope, e.g., Ortholux II from Leitz, may be easily used if one notes the following changes. First, the plate has to be viewed in an inverted position (i.e., bottom up) and, second, instead of the usual objectives of 45 m m length one has to use simple and cheap ones which are considerably shorter i.e., 35-37 ram; only these short objectives allow for appropriate focussing. We have routinely used 20 × objectives from student microscopes, e.g., Wild, Heerbrugg, Switzerland (objective 20 x / 0 . 4 5 / N o . 29200) or objectives from simple Olympus inverted microscopes CK (objective: C 2 0 / 0 . 4 0 / 1 . 2 / n o . 404157).
Results and discussion
The procedure used is a modification of a method originally described by Rapp et al. (1959) and Philipson (1961) and as reviewed by Spendlove (1967) and Klement et al. (1977). For the tests we used flat-bottomed 96-well microtiter plates which are very convenient to handle. Medium from the microtiter wells was flicked off
leaving the cells in the wells. To prevent detachment of cells, aspiration of fluids with Pasteur pipettes was considered worthwhile, whereby care was taken to always point the pipette at the same area to limit loss of cells. The wells were then washed with 100 ~1 of a balanced salt solution using a multichannel pipette, the tips of which had been cut off to guarantee a more gentle flow. Next, 100 p~l of phosphate-buffered 4%, formaldehyde was added to the wells and left for 10-30 min at room temperature. Alternatively, especially in experiments with BDV, cells were fixed with 3% paraformaldehyde for 10 rain and additionally treated for another 10 rain with 3%, Triton X-100 in order to achieve good permeability. After two washes the first antibody was added. Ideally, this was carried out using a short Pasteur pipette with a rather narrow bore or a calibrated pipette from which a drop of about 25-30/xl was delivered. For antibody titrations we routinely prepared serial dilutions in separate microtiter plates with a muhichannel pipette and transferred the test samples in the same way to the test wells, After 30 min at either room temperature or at 37 °C, the wells were washed twice as described and the second antibody (if required) was added for 30 rain and incubated at room temperature or 3 7 ° C for 30 rain. If short of time, we found that the incubation periods for both antibodies could be reduced to 10 or 15 rain with good results. After two washes, 50 pA or 100 ~1 of the balanced salt solution were added to the wells and plates were kept in ~ dark refrigerator ( 4 ° C ) until examined; they could be stored for several days up to 2 months (the longest period checked) under these conditions. Before examination the supernatant was either aspirated off or most easily, simply drained off by inverting the plates on to a water absorbing paper. We have used the same protocol, but without using 4%, formaldehyde to fix the cells to screen for cell surface antibodies on adherent cells. One has to use pre-warmed media and take special care to avoid washing off the cells. Although feasible, we found this application not very reliable. The method has been used successfully for enumeration of poor or non-cytopathogenic viruses (e.g., lymphocytic choriomeningitis virus (LCMV) (Fig. 2a)) and Borna disease virus (Fig. 2b). For
214
Fig. 2a: Examples of immunofluorescence on LCMV infected and fixed MC57G fibroblast monolayers and using a mouse anti-LCMV hyperimmune serum (1 in 100 diluted 25 p.I/well) as first antibody and a goat anti-mouse Ig (25 #1 1 in 50 diluted per well) as second antib~xty. (Original magnifications 20 × ). The inverted plates were viewed and pathographed with a conventional Ortholux II UV microscope.
215
Fig. 2b: Examples of Borna virus-infected MDCK fibroblasts stained with rat anti-Borna serum (diluted 1 in 100, 25 p.l/well) and a rabbit anti-rat FITC labelled antibody (diluted 1 in 40). The original magnifications were 25 x (right) and 32 x (left). Note the typical intense intranuclear staining of inclusion bodies characteristic of Borna virus infection.
LCMV it is apparently not even necessary to use an agar or methylcellulose overlay because the medium seems to be sufficiently stabilizing to prevent virus from spreading far away from the infectious center. Also LCMV fluorescent plaques can be read after 2 days, whereas conventional and rather difficult plaquing procedures take 4-5 days. This easily renders the method very practicable and useful. Recently, we have successfully adapted this method for the quantitation of a persistent virus infection causing absolutely no cytopathic effet in vitro, using BDV. Additionally to the already mentioned advantages of this method, we were able to reduce incubation time for BDV titration on rabbit embryonal brain tissue culture from 12-14 days to about half the time. We have been using the described method to regularly screen all our adherent target cells used in 51Cr-release assays for proper degrees of infection. To this end we plate 2 - 4 x 104 target cells into flat-bottomed 96-well plates, centrifuge them, let them adhere for 1-2 h and then proceed as described. The method is efficient, quick and cheap for screening internal cellular antigens, including viral antigens. It has the advantage of using very few cells (1-5 x 104/sample) and little antisera (25 ~l/well). It permits screening of either various cells for antigen expression or large numbers of
hybridoma supernatants or antisera for relevant antibodies within a short period of time. The most time-consuming step remains the preparation and dispensing of the test samples. One person can easily screen up to 1000 samples in 1 day. This study was supported by Grants 3.323-0.82 and 3.259-0.85 from the Swiss National Science Foundation, United States Public Health Service Grant AI-17285-0.5, grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 47) and the Kanton of ZLirich.
References Jacobson, S., Dutko, F.J. and Pfau, C.J. (1979) Determinants of spontaneous recovery and persistence in MDCK cells infected with lymphocytic choriomeningitis virus. J. Gen. Virol. 44, 113. Klement, V. and Nicolson, M.O. (1977) Methods for assays of RNA tumor viruses. In: K. Maramorosch and H. Koprowski (Fxts.), Methods in Virology. Academic Press, New York, p. 59. Lehmann-Grube, F. and Ambrassat. J. (1977) A new method to detect lymphocytic choriomeningitis virus-specific antibody in human sera. J. Gen. Virol. 37, 85. Ludwig, H., Becht, H. and Groh, L. (1973) Borna-disease (BD). a slow virus infection: Biological properties of the virus. Med. Microbiol. Immunol. 158. 275. Philipson, L. (1961) Adenovirus assay by the fluorescent cell counting procedure. Virology 15, 263.
216 Rapp, F., Seligman, S.J., Jaross, L.B. and Gordon, 1. (1959) Quantitative determination of infectious units of measles virus by counts of immunofluoreseent loci. Proc. Soc. Exp. Biol. Med. 101,289.
Spendlove. R.S. (1967) Search of virus infection or asynchronous appearance of antigen in infected cells. In: K. Maramorosch and H. Koprowski (Eds.), Methods in Virology. Academic Press, New York, p. 475.
211
JIM 04618
An easy and rapid method to screen large numbers of antibodies against internal cellular determinants L o t h a r Stitz, H a n s H e n g a r t n e r , A l a n a Althage a n d R o l f M. Z i n k e r n a g e l lnstttute of Pathology, Unwersitv Hospital, 8091 Ziirich, Switzerland, and Institute of Virolog}', Justus-Ltebig Universio', 6300 Giessen, F. R. G. (Received 1 September 1987, accepted 14 September 1987)
A rapid and easy method is described to screen great numbers of antisera or antibodies against internal cellular antigens including viral antigens or to screen various target cells for proper expression of antigens; the method can also be applied to determine fluorescent foci to enumerate non-cytopathic viruses. Cells, infected with a particular virus or uninfected, adhering to flat-bottomed 96-well microtiter plates were fixed with conventional phosphate-buffered saline containing 4% formaldehyde for 10 min, alternatively, cells were first fixed with 3% paraformaldehyde for 10 min and were then treated with Trition X-100 for another 10 min. After two washes, either fluorescein-labelled antiviral antibodies or first antiviral antibodies followed by labelled anti-species antibodies were applied, incubated and washed off as usual. A few drops of a balanced salt solution were kept in the well and were drained off gently just before the plates were examined. The plates were viewed directly in an inverted UV microscope or were inspected and photographed bottoms up with a conventional UV microscope mounted with an old-fashioned uncorrected objective (20 x ) which, because of its shorter length, permitted proper focussing. For most cases studied, sensitivity was comparable to the fluorescence analysis method of cells on slides. The plates could be stored for several months in a dark refrigerator if kept moist. The method is rapid because it avoids individual handling of samples for the washing procedures and does not need growing and mounting of cells on slides; up to 1000 samples can be tested by one person in a day. Key words: Intracellular antigen: Viral antigen: Immunofluorescence; Screening of antibodies
Introduction
Screening of complex antisera or of cell culture supernatants containing monoclonal antibodies against internal cellular antigens or viral antigens is usually laborious and time consuming. In par-
('orrespondence to: R.M. Zinkernagel, Institut ftir Pathologic, Universitfitsspital, Sternwartstrasse 2, 8091 Ziirich, Switzerland. Abbreviations: BI)V, Borna disease virus; LCMV, lymphocytic choriomeningitis virus; MEM, minimal essential medium; i.p., post infection.
ticular the growing of cells or mounting of individual cell preparations on slides and subsequent washing procedures are cumbersome. We have successfully grown cells, infected and fixed them and then treated them with appropriate antibodies in 96-well flat-bottomed tissue culture trays. If an inverted UV microscope is not available, a conventional UV microscope cannot be used to screen for antibodies because of mechanical restraints: for focussing, the objective of a regular microscope had to be lowered more than the thick 96-well microtiter plates allowed for. Cutting off the rims of the wells with conventional methods
0022-1759/88/$03.50 ': 1988 Elsevier Science Publishers B.V. (Biomedical Division)
212
Fig. l. Photograph of the Leitz UV microscope used with the mounted 10 x 20 x obJectives from cheap student microscopes, note that the microscope table is devoid of the slide holder and that the 96-well plate is inverse so that the bottom is positioned close enough to the objective. (hot cutting wire) was not feasible. We exchanged the excellent objectives of our UV microscopes for uncorrected but much shorter objectives ( 1 0 × and 20 x ) from rather simple and cheap student microscopes. This change permitted screening of inverted 96-well plates for fluorescent antibodies against internal cellular or viral antigens.
Materials and methods
Virus', virus infection and fixation of cells The poorly cytopathic, lymphocytic choriomeningitis virus (LCMV) WE strain had been obtained originally from Dr. F. Lehmann-Grube, Hamburg, F.R.G. (Lehmann-Grube et al., 1977; Jacobson et al., 1979). Cells were grown in glass or plastic flasks and were infected at a multiplicity of 0.01/cell. At 48 h post infection (p.i.) the cells were trypsinized, washed, adjusted and dispensed in aliquots of 100 /~l/well. Borna disease virus (BDV) (Ludwig et al., 1973) was used as another non-cytopathic, tightly cell-associated infectious agent, causing a persistent, slowly productive infection spreading mainly by cell-to-cell contact.
When cytopathic viruses (e.g., vaccinia or vesicular stomatitis virus) were used, cells were usually infected after they had adhered to the microtiter wells because, if infected beforehand, they usually failed to adhere a n d / o r tended to come off the plastic too readily during the washing procedures. Cells in microwells were fixed at room temperature in 4% formaldehyde or 3% paraformaldehyde in phosphate-buffered saline for 10.-30 min followed by 10-20 rain treatment with 3~ Triton X-100 in phosphate-buffered saline. After fixation, the cell preparations can be kept for weeks in the refrigerator before they are used for analysis.
Tissue culture Adherent primary peritoneal macrophages or established fibroblast cell lines (MC57G and 1,929 cells) were employed in studies on LCMV and cytopathic viruses; for BDV either persistently infected M D C K cells or acutely infected primary rabbit embryonal brain tissue cultures were used. They were grown in minimal essential medium (MEM) with 10% heat-inactivated fetal calf serum and penicillin plus streptomycin. Cells were seeded at 1 5 × 104 cells/well and allowed to adhere for
213 2- 20 h. Cells were either infected prior to or after transfer to the microwells.
Antibodies Pooled hyperimmune mouse anti-LCMV sera were used at a dilution of 1 in 100 and 25 ~l/well. Several monoclonal antibody preparations against LCMV protein were also used as undiluted culture supernatant, diluted 1 in 10, or as ascites fluid diluted 1 in 1000, A fluorescent-labelled goat anti-mouse Ig antibody was purchased from Tago, Burlingame, CA ( F I T C goat (Fab')2 anti-mouse IgG + IgM, code 4343) and used at 1 in 50 or 1 in 100 diluted 25 ~l/well. For BDV studies, sera from infected rats or mice and rat monoclonal antibodies against an intracellular antigen were used. All antibodies were applied at room temperature or at 3 7 ° C for 30 rain,
Microscopes Plates can be easily viewed through an inverted UV microscope; we have used the Leitz Diavert model with a 32 × objective (Phaco 170/-1 L32/0.40) in the bright field. If such a microscope is not available, a conventional microscope, e.g., Ortholux II from Leitz, may be easily used if one notes the following changes. First, the plate has to be viewed in an inverted position (i.e., bottom up) and, second, instead of the usual objectives of 45 m m length one has to use simple and cheap ones which are considerably shorter i.e., 35-37 ram; only these short objectives allow for appropriate focussing. We have routinely used 20 × objectives from student microscopes, e.g., Wild, Heerbrugg, Switzerland (objective 20 x / 0 . 4 5 / N o . 29200) or objectives from simple Olympus inverted microscopes CK (objective: C 2 0 / 0 . 4 0 / 1 . 2 / n o . 404157).
Results and discussion
The procedure used is a modification of a method originally described by Rapp et al. (1959) and Philipson (1961) and as reviewed by Spendlove (1967) and Klement et al. (1977). For the tests we used flat-bottomed 96-well microtiter plates which are very convenient to handle. Medium from the microtiter wells was flicked off
leaving the cells in the wells. To prevent detachment of cells, aspiration of fluids with Pasteur pipettes was considered worthwhile, whereby care was taken to always point the pipette at the same area to limit loss of cells. The wells were then washed with 100 ~1 of a balanced salt solution using a multichannel pipette, the tips of which had been cut off to guarantee a more gentle flow. Next, 100 p~l of phosphate-buffered 4%, formaldehyde was added to the wells and left for 10-30 min at room temperature. Alternatively, especially in experiments with BDV, cells were fixed with 3% paraformaldehyde for 10 rain and additionally treated for another 10 rain with 3%, Triton X-100 in order to achieve good permeability. After two washes the first antibody was added. Ideally, this was carried out using a short Pasteur pipette with a rather narrow bore or a calibrated pipette from which a drop of about 25-30/xl was delivered. For antibody titrations we routinely prepared serial dilutions in separate microtiter plates with a muhichannel pipette and transferred the test samples in the same way to the test wells, After 30 min at either room temperature or at 37 °C, the wells were washed twice as described and the second antibody (if required) was added for 30 rain and incubated at room temperature or 3 7 ° C for 30 rain. If short of time, we found that the incubation periods for both antibodies could be reduced to 10 or 15 rain with good results. After two washes, 50 pA or 100 ~1 of the balanced salt solution were added to the wells and plates were kept in ~ dark refrigerator ( 4 ° C ) until examined; they could be stored for several days up to 2 months (the longest period checked) under these conditions. Before examination the supernatant was either aspirated off or most easily, simply drained off by inverting the plates on to a water absorbing paper. We have used the same protocol, but without using 4%, formaldehyde to fix the cells to screen for cell surface antibodies on adherent cells. One has to use pre-warmed media and take special care to avoid washing off the cells. Although feasible, we found this application not very reliable. The method has been used successfully for enumeration of poor or non-cytopathogenic viruses (e.g., lymphocytic choriomeningitis virus (LCMV) (Fig. 2a)) and Borna disease virus (Fig. 2b). For
214
Fig. 2a: Examples of immunofluorescence on LCMV infected and fixed MC57G fibroblast monolayers and using a mouse anti-LCMV hyperimmune serum (1 in 100 diluted 25 p.I/well) as first antibody and a goat anti-mouse Ig (25 #1 1 in 50 diluted per well) as second antib~xty. (Original magnifications 20 × ). The inverted plates were viewed and pathographed with a conventional Ortholux II UV microscope.
215
Fig. 2b: Examples of Borna virus-infected MDCK fibroblasts stained with rat anti-Borna serum (diluted 1 in 100, 25 p.l/well) and a rabbit anti-rat FITC labelled antibody (diluted 1 in 40). The original magnifications were 25 x (right) and 32 x (left). Note the typical intense intranuclear staining of inclusion bodies characteristic of Borna virus infection.
LCMV it is apparently not even necessary to use an agar or methylcellulose overlay because the medium seems to be sufficiently stabilizing to prevent virus from spreading far away from the infectious center. Also LCMV fluorescent plaques can be read after 2 days, whereas conventional and rather difficult plaquing procedures take 4-5 days. This easily renders the method very practicable and useful. Recently, we have successfully adapted this method for the quantitation of a persistent virus infection causing absolutely no cytopathic effet in vitro, using BDV. Additionally to the already mentioned advantages of this method, we were able to reduce incubation time for BDV titration on rabbit embryonal brain tissue culture from 12-14 days to about half the time. We have been using the described method to regularly screen all our adherent target cells used in 51Cr-release assays for proper degrees of infection. To this end we plate 2 - 4 x 104 target cells into flat-bottomed 96-well plates, centrifuge them, let them adhere for 1-2 h and then proceed as described. The method is efficient, quick and cheap for screening internal cellular antigens, including viral antigens. It has the advantage of using very few cells (1-5 x 104/sample) and little antisera (25 ~l/well). It permits screening of either various cells for antigen expression or large numbers of
hybridoma supernatants or antisera for relevant antibodies within a short period of time. The most time-consuming step remains the preparation and dispensing of the test samples. One person can easily screen up to 1000 samples in 1 day. This study was supported by Grants 3.323-0.82 and 3.259-0.85 from the Swiss National Science Foundation, United States Public Health Service Grant AI-17285-0.5, grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 47) and the Kanton of ZLirich.
References Jacobson, S., Dutko, F.J. and Pfau, C.J. (1979) Determinants of spontaneous recovery and persistence in MDCK cells infected with lymphocytic choriomeningitis virus. J. Gen. Virol. 44, 113. Klement, V. and Nicolson, M.O. (1977) Methods for assays of RNA tumor viruses. In: K. Maramorosch and H. Koprowski (Fxts.), Methods in Virology. Academic Press, New York, p. 59. Lehmann-Grube, F. and Ambrassat. J. (1977) A new method to detect lymphocytic choriomeningitis virus-specific antibody in human sera. J. Gen. Virol. 37, 85. Ludwig, H., Becht, H. and Groh, L. (1973) Borna-disease (BD). a slow virus infection: Biological properties of the virus. Med. Microbiol. Immunol. 158. 275. Philipson, L. (1961) Adenovirus assay by the fluorescent cell counting procedure. Virology 15, 263.
216 Rapp, F., Seligman, S.J., Jaross, L.B. and Gordon, 1. (1959) Quantitative determination of infectious units of measles virus by counts of immunofluoreseent loci. Proc. Soc. Exp. Biol. Med. 101,289.
Spendlove. R.S. (1967) Search of virus infection or asynchronous appearance of antigen in infected cells. In: K. Maramorosch and H. Koprowski (Eds.), Methods in Virology. Academic Press, New York, p. 475.