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Early detection of cytomegalovirus in cell culture by a monoclonal antibody
65
Journal of Virological Methods, 14 (1986) 65-69
Elsevier JVM 00511
Early detection of cytomegalovirus in cell culture by a monoclonal antibody Benita
Zweygberg
Department of Clinical Microbiology,
Wirgart
and Lena Grillner’
Section of Virology, Karolinska Hospital, S-104 01 Stockholm, Sweden
(Accepted 25 March 1986)
A commercially available monoclonal antibody directed against early cytomegalovirus (CMV) antigen was used for the demonstration of CMV by immunofluorescence (IF) in cell culture within 2 days. The results were compared with the appearance of CMV-specific cytophatogenic effect (CPE). Urine specimens from 31 healthy children in day-care centers were inoculated on human embryonic fibroblasts. In addition, 45 CMV strains that had been stored at -70°C were reinoculated. CMV was detected in 8/31 urine specimens by IF and 7 of these gave a specific CPE at an average of 16 days post-inoculation. One specimen was negative by IF but specific CPE was found at day 13. After reinoculation, CMV was detected in 76% by IF while 44 specimens developed CPE within a 6-week period. Demonstration of early CMV antigen in cell culture was found to be a rapid method for early diagnosis of CMV. Since the conventional cell culture with detection of CPE was more sensitive it may be useful to combine the two methods. early antigen, monoclonal, cytomegalovirus,
antigen detection
Introduction
Cytomegalovirus (CMV) produces, before viral DNA is synthesized, a number of early antigens in infected cells. Early protein synthesis can be detected 2-20 h after infection and is preceded by synthesis of immediate early proteins which appear within 4 h (Stinski, 1978). Antibodies against these early antigens can be used for the demonstration of CMV early after infection in cell culture by immunofluorescence (IF). There is a special need for rapid diagnostic procedures for demonstrating CMV since the development of typical cytopathogenic effects (CPE) in cell culture is a slow process often requiring several weeks. Monoclonal antibodies against early CMV proteins have been used in combination with fluorescein and peroxidase conjugates and biotin-avidin indirect fluorescent stain for the demonstration of CMV in cell culture (Gleaves et al., 1984; * To whom correspondence
should be addressed.
0166~0934/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)
Swenson and Kaplan, 1985; Alpert et al., 1985). In the present study we used a commercially available monoclonal antibody for early detection of CMV in cell culture by immunofluorescence. CMV was isolated from urine specimens from healthy children. The results were compared with demonstration of CPE in cell culture.
Materials and Methods Clinical specimens
Urine samples were collected from 31 healthy children in two day-care centers for the isolation of CMV. In addition, 45 CMV strains that had been stored at -70°C were reinoculated on fibroblasts. CMV monoclonal
antibody
A monoclonal antibody (2 H 2.4) directed against early nuclear CMV protein (72,000 dalton) obtained from Biotech Research Laboratories, Inc., Rockville, MD was used for the demonstration of CMV in cell culture by IF. A fluorescein isothiocyanate-labeled goat anti-mouse IgG (Gibco Laboratories, Chagrin Falls, OH) was used as secondary antibody. Virus isolation Cell cultures. CMV was isolated on human embryonic lung fibroblasts (local strain). The fibroblasts were cultured with Eagle’s minimum essential medium with 10% fetal bovine serum (FBS) for outgrowth and 2% FBS for maintenance with penicillin (100 IE/ml), streptomycin (100 &ml) and neomycin (50 kg/ml). The fibroblasts were grown to confluence on circular coverslips (12 mm) in 24well plastic plates (Nunc, Roskilde, Denmark) in a humidified 5% CO, incubator. Inoculation of samples. Each sample was treated with penicillin (5,000 IE/ml) and
streptomycin (5 mg/ml) and inoculated into 8 wells (0.1 ml/well); 2 wells in one plate for demonstration of CPE and 6 wells in another plate for detection of early CMV antigen (see below). The plates were then centrifuged at 700 x g for 1 h at 20°C in a Beckman J 6-M centrifuge. Detection of CMV cytopathogenic
effects. The plates were kept in the CO, incubator and observed daily for typical CMV CPE for at least 6 weeks. Detection of early CMV antigens. Two coverslips were removed 1, 2 and 7 days after inoculation. Specimens negative at day 1 were retested at day 2 and if still negative also at day 7. After washing in phosphate-buffered saline (PBS), pH 7.5, and fixation with cold acetone for 10 min, the coverslips were stored at -70°C until stained by the 2 H 2.4 CMV monoclonal antibody. The coverslips were incubated with 50 ~1 of the monoclonal antibody (5 tqjrnl)
61
for 60 min at 37°C and then washed three times in PBS. After that, the coverslips were incubated with the conjugate (diluted l/40) for 60 min at 37°C washed three times and stained with Evans blue for 5 min. The coverslips were mounted with Entellan (Merck) on glass slides and examined with a Leitz fluorescence microscope at 400 X magnification.
Results Detection of CMV in urine from healthy children
CMV was detected in 8 out of 31 urine specimens (26%) within 2 days after inoculation by IF (6 specimens on day 1, and 2 on day 2) using the 2 H 2.4 CMV monoclonal antibody. Typical CMV CPE also developed in 7 of these 8 specimens (mean time 16 days). CMV had been isolated some months previously from the child whose specimen was positive only by IF. One specimen gave a CMV CPE 13 days after inoculation but no early antigen was detected with IF. The sensitivity % CMV positive samples 100
q
IF monoclonal
ab
CPE
75
SO-
25 -
1 Day
1
Fig. I. Comparison between immunofluorescence (IF), using a monoclonal antibody against early CMV antigen, and cytopathogenic effect (CPE) for detection of CMV in cell culture after reinoculation of 45 CMV strains, stored at -70°C. Percentage of positive specimens by the two methods at different days post-inoculation is shown.
and specificity for IF in comparison to demonstration spectively.
of CPE is 88 and 99%, re-
Reisolation of CMV
CMV was demonstrated with IF within 2 days after inoculation in 31 of the 4.5 specimens (69%) (26 specimens on day 1, and 5 on day 2) that had been stored at -70°C. At this time, CPE had developed in only 17 specimens (38%) (Fig. 1). At day 7, 3 additional specimens were positive by IF, in total 34 samples out of 45. However, CMV CPE was detected in 44 of the 4.5 specimens after the 6-week observation period. One sample (negative by IF) demonstrated a typical CPE as late as 45 days after inoculation. Finally, CMV could not be reisolated from one specimen.
Discussion
Different methods for rapid detection of CMV, apart from the demonstration of early CMV proteins in cell culture, have been published during the last years. Enzyme-linked immunosorbent assays have been used (Sundqvist and Wahren, 1981; McKeating et al., 1985) but the sensitivity has been low in comparison to virus isolation. DNA hybridization has been found to be a valuable method for early and rapid detection of CMV in urine specimens but has required concentration of urine by ultracentrifugation or extraction with 2-butanol to obtain an acceptable sensitivity (Chou and Merigan, 1983; Virtanen et al., 1984). However, used for diagnosis of cytomegaloviral pneumonia in situ hybridization was as sensitive as viral culture and antigen detection (Myerson et al., 1984). The results of the present study confirm that monoclonal antibodies against early CMV proteins can be used for demonstration of CMV in cell culture within 24-48 h after inoculation. Thus, we could detect CMV within this period in 7 out of 8 isolation positive specimens as well as in one sample from which CMV could not be isolated but had been some months previously. Conventional virus isolation was more sensitive when the two methods were compared for reisolation of CMV from frozen strains. On the other hand, the observation period was 6 weeks for the detection of CPE compared to 48 h for early antigens. Gleaves et al. (1984) showed that low-speed centrifugation of urine specimens onto cell culture monolayers increased the sensitivity of IF. They found that IF with the same monoclonal antibody as we have used, detected CMV 36 h after inoculation in 19/162 urine specimens of which only 18 were positive by virus isolation. In a later report, they found that even when used 16 h post-inoculation, this method was as specific and more sensitive than conventional tube cell culturing (Gleaves et al., 1985). Swenson and Kaplan (1985) used the same monoclonal antibody but no centrifugation and they had a sensitivity of 93% by immunoperoxidase staining and a specificity of 96%. Virus isolation was in the present study obviously more sensitive than IF for detection of CMV in stored specimens. This can be explained by the fact that we
69
used a long observation period (6 weeks) and low-speed centrifugation also for virus isolation. On the contrary, Gleaves et al. (1984) did not use low-speed centrifugation for virus isolation and Swenson and Kaplan (1985) observed the cell cultures for CPE only during 14 days and subcultured only specimens with discordant results. Rapid diagnosis of active CMV infection in immunocompromised patients was obtained with a pool of monoclonal antibodies against early antigens in 80% compared with conventional virus isolation (Griffiths et al., 1984). In that study, experiments with the AD 169 strain showed that lo3 TCID 50 were required to give a positive reaction by IF with monoclonal antibodies against early antigens. In most studies, specimens from immunocompromised patients and congenitally infected children have been used for evaluation of the new diagnostic methods. This group of patients usually excrete high titers of virus which are easily detectable with most methods. However, we could show that IF with the monoclonal antibody also is useful for demonstration of CMV excretion in healthy children. The availability of rapid methods for the diagnosis of CMV infections in immunocompromised patients is essential since different therapeutic measurements can be more readily evaluated and compared. Since the sensitivity seems to be lower than that for virus isolation it may be useful to combine the two methods to obtain a maximal rapidity and sensitivity, especially when therapeutic trials are performed.
Acknowledgement
We want to thank Ingegerd Neideman for skilful help with typing of the manuscript.
References Alpert., G., M.-C. Mazeron, R. Coliman and S. Plotkin, 1985, J. Infect. Dis. 152, 631. Chou, S. and T.C. Merigan, 1983, N. Engl. J. Med. 308, 921. Fiacco, V., Y.J. Bryson and D.A. Bruckner, 1984, J. Clin. Microbial. 19, 928. Gleaves, C.A., T.F. Smith, E.A. Shuster and G.R. Pearson, 1984, J. Clin. Microbial. 19, 917. Gleaves, C.A., T.F. Smith, E.A. Shuster and G.R. Pearson, 1985, J. Clin. Microbial. 21, 217. Griffiths, P.D., D.D. Panjwani, P.R. Stirk, M.G. Ball, M. Ganczakowski, H.A. Blacklock and H.G. Prentice, 1984, Lancet 2, 1242. McKeating, J.A., S. Stagno, P.R. Stirk and P.D. Griffiths, 1985, J. Med. Virol. 16, 367. Myerson, D., R.C. Hackman and J.D. Meyers, 1984, J. Infect. Dis. 150, 272. Stinski, M.F., 1978, J. Viral. 26, 686. Sundqvist, V.-A. and B. Wahren, 1981, J. Viral. Methods 2, 301. Swenson, P.D. and M.H. Kaplan, 1985, J. Clin. Microbial. 21, 669. Virtanen, M., A.-C. Syvanen, J. Oram, H. Soderlund and M. Ranki, 1984, J. Clin. Microbial. 20, 1083.
Journal of Virological Methods, 14 (1986) 65-69
Elsevier JVM 00511
Early detection of cytomegalovirus in cell culture by a monoclonal antibody Benita
Zweygberg
Department of Clinical Microbiology,
Wirgart
and Lena Grillner’
Section of Virology, Karolinska Hospital, S-104 01 Stockholm, Sweden
(Accepted 25 March 1986)
A commercially available monoclonal antibody directed against early cytomegalovirus (CMV) antigen was used for the demonstration of CMV by immunofluorescence (IF) in cell culture within 2 days. The results were compared with the appearance of CMV-specific cytophatogenic effect (CPE). Urine specimens from 31 healthy children in day-care centers were inoculated on human embryonic fibroblasts. In addition, 45 CMV strains that had been stored at -70°C were reinoculated. CMV was detected in 8/31 urine specimens by IF and 7 of these gave a specific CPE at an average of 16 days post-inoculation. One specimen was negative by IF but specific CPE was found at day 13. After reinoculation, CMV was detected in 76% by IF while 44 specimens developed CPE within a 6-week period. Demonstration of early CMV antigen in cell culture was found to be a rapid method for early diagnosis of CMV. Since the conventional cell culture with detection of CPE was more sensitive it may be useful to combine the two methods. early antigen, monoclonal, cytomegalovirus,
antigen detection
Introduction
Cytomegalovirus (CMV) produces, before viral DNA is synthesized, a number of early antigens in infected cells. Early protein synthesis can be detected 2-20 h after infection and is preceded by synthesis of immediate early proteins which appear within 4 h (Stinski, 1978). Antibodies against these early antigens can be used for the demonstration of CMV early after infection in cell culture by immunofluorescence (IF). There is a special need for rapid diagnostic procedures for demonstrating CMV since the development of typical cytopathogenic effects (CPE) in cell culture is a slow process often requiring several weeks. Monoclonal antibodies against early CMV proteins have been used in combination with fluorescein and peroxidase conjugates and biotin-avidin indirect fluorescent stain for the demonstration of CMV in cell culture (Gleaves et al., 1984; * To whom correspondence
should be addressed.
0166~0934/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)
Swenson and Kaplan, 1985; Alpert et al., 1985). In the present study we used a commercially available monoclonal antibody for early detection of CMV in cell culture by immunofluorescence. CMV was isolated from urine specimens from healthy children. The results were compared with demonstration of CPE in cell culture.
Materials and Methods Clinical specimens
Urine samples were collected from 31 healthy children in two day-care centers for the isolation of CMV. In addition, 45 CMV strains that had been stored at -70°C were reinoculated on fibroblasts. CMV monoclonal
antibody
A monoclonal antibody (2 H 2.4) directed against early nuclear CMV protein (72,000 dalton) obtained from Biotech Research Laboratories, Inc., Rockville, MD was used for the demonstration of CMV in cell culture by IF. A fluorescein isothiocyanate-labeled goat anti-mouse IgG (Gibco Laboratories, Chagrin Falls, OH) was used as secondary antibody. Virus isolation Cell cultures. CMV was isolated on human embryonic lung fibroblasts (local strain). The fibroblasts were cultured with Eagle’s minimum essential medium with 10% fetal bovine serum (FBS) for outgrowth and 2% FBS for maintenance with penicillin (100 IE/ml), streptomycin (100 &ml) and neomycin (50 kg/ml). The fibroblasts were grown to confluence on circular coverslips (12 mm) in 24well plastic plates (Nunc, Roskilde, Denmark) in a humidified 5% CO, incubator. Inoculation of samples. Each sample was treated with penicillin (5,000 IE/ml) and
streptomycin (5 mg/ml) and inoculated into 8 wells (0.1 ml/well); 2 wells in one plate for demonstration of CPE and 6 wells in another plate for detection of early CMV antigen (see below). The plates were then centrifuged at 700 x g for 1 h at 20°C in a Beckman J 6-M centrifuge. Detection of CMV cytopathogenic
effects. The plates were kept in the CO, incubator and observed daily for typical CMV CPE for at least 6 weeks. Detection of early CMV antigens. Two coverslips were removed 1, 2 and 7 days after inoculation. Specimens negative at day 1 were retested at day 2 and if still negative also at day 7. After washing in phosphate-buffered saline (PBS), pH 7.5, and fixation with cold acetone for 10 min, the coverslips were stored at -70°C until stained by the 2 H 2.4 CMV monoclonal antibody. The coverslips were incubated with 50 ~1 of the monoclonal antibody (5 tqjrnl)
61
for 60 min at 37°C and then washed three times in PBS. After that, the coverslips were incubated with the conjugate (diluted l/40) for 60 min at 37°C washed three times and stained with Evans blue for 5 min. The coverslips were mounted with Entellan (Merck) on glass slides and examined with a Leitz fluorescence microscope at 400 X magnification.
Results Detection of CMV in urine from healthy children
CMV was detected in 8 out of 31 urine specimens (26%) within 2 days after inoculation by IF (6 specimens on day 1, and 2 on day 2) using the 2 H 2.4 CMV monoclonal antibody. Typical CMV CPE also developed in 7 of these 8 specimens (mean time 16 days). CMV had been isolated some months previously from the child whose specimen was positive only by IF. One specimen gave a CMV CPE 13 days after inoculation but no early antigen was detected with IF. The sensitivity % CMV positive samples 100
q
IF monoclonal
ab
CPE
75
SO-
25 -
1 Day
1
Fig. I. Comparison between immunofluorescence (IF), using a monoclonal antibody against early CMV antigen, and cytopathogenic effect (CPE) for detection of CMV in cell culture after reinoculation of 45 CMV strains, stored at -70°C. Percentage of positive specimens by the two methods at different days post-inoculation is shown.
and specificity for IF in comparison to demonstration spectively.
of CPE is 88 and 99%, re-
Reisolation of CMV
CMV was demonstrated with IF within 2 days after inoculation in 31 of the 4.5 specimens (69%) (26 specimens on day 1, and 5 on day 2) that had been stored at -70°C. At this time, CPE had developed in only 17 specimens (38%) (Fig. 1). At day 7, 3 additional specimens were positive by IF, in total 34 samples out of 45. However, CMV CPE was detected in 44 of the 4.5 specimens after the 6-week observation period. One sample (negative by IF) demonstrated a typical CPE as late as 45 days after inoculation. Finally, CMV could not be reisolated from one specimen.
Discussion
Different methods for rapid detection of CMV, apart from the demonstration of early CMV proteins in cell culture, have been published during the last years. Enzyme-linked immunosorbent assays have been used (Sundqvist and Wahren, 1981; McKeating et al., 1985) but the sensitivity has been low in comparison to virus isolation. DNA hybridization has been found to be a valuable method for early and rapid detection of CMV in urine specimens but has required concentration of urine by ultracentrifugation or extraction with 2-butanol to obtain an acceptable sensitivity (Chou and Merigan, 1983; Virtanen et al., 1984). However, used for diagnosis of cytomegaloviral pneumonia in situ hybridization was as sensitive as viral culture and antigen detection (Myerson et al., 1984). The results of the present study confirm that monoclonal antibodies against early CMV proteins can be used for demonstration of CMV in cell culture within 24-48 h after inoculation. Thus, we could detect CMV within this period in 7 out of 8 isolation positive specimens as well as in one sample from which CMV could not be isolated but had been some months previously. Conventional virus isolation was more sensitive when the two methods were compared for reisolation of CMV from frozen strains. On the other hand, the observation period was 6 weeks for the detection of CPE compared to 48 h for early antigens. Gleaves et al. (1984) showed that low-speed centrifugation of urine specimens onto cell culture monolayers increased the sensitivity of IF. They found that IF with the same monoclonal antibody as we have used, detected CMV 36 h after inoculation in 19/162 urine specimens of which only 18 were positive by virus isolation. In a later report, they found that even when used 16 h post-inoculation, this method was as specific and more sensitive than conventional tube cell culturing (Gleaves et al., 1985). Swenson and Kaplan (1985) used the same monoclonal antibody but no centrifugation and they had a sensitivity of 93% by immunoperoxidase staining and a specificity of 96%. Virus isolation was in the present study obviously more sensitive than IF for detection of CMV in stored specimens. This can be explained by the fact that we
69
used a long observation period (6 weeks) and low-speed centrifugation also for virus isolation. On the contrary, Gleaves et al. (1984) did not use low-speed centrifugation for virus isolation and Swenson and Kaplan (1985) observed the cell cultures for CPE only during 14 days and subcultured only specimens with discordant results. Rapid diagnosis of active CMV infection in immunocompromised patients was obtained with a pool of monoclonal antibodies against early antigens in 80% compared with conventional virus isolation (Griffiths et al., 1984). In that study, experiments with the AD 169 strain showed that lo3 TCID 50 were required to give a positive reaction by IF with monoclonal antibodies against early antigens. In most studies, specimens from immunocompromised patients and congenitally infected children have been used for evaluation of the new diagnostic methods. This group of patients usually excrete high titers of virus which are easily detectable with most methods. However, we could show that IF with the monoclonal antibody also is useful for demonstration of CMV excretion in healthy children. The availability of rapid methods for the diagnosis of CMV infections in immunocompromised patients is essential since different therapeutic measurements can be more readily evaluated and compared. Since the sensitivity seems to be lower than that for virus isolation it may be useful to combine the two methods to obtain a maximal rapidity and sensitivity, especially when therapeutic trials are performed.
Acknowledgement
We want to thank Ingegerd Neideman for skilful help with typing of the manuscript.
References Alpert., G., M.-C. Mazeron, R. Coliman and S. Plotkin, 1985, J. Infect. Dis. 152, 631. Chou, S. and T.C. Merigan, 1983, N. Engl. J. Med. 308, 921. Fiacco, V., Y.J. Bryson and D.A. Bruckner, 1984, J. Clin. Microbial. 19, 928. Gleaves, C.A., T.F. Smith, E.A. Shuster and G.R. Pearson, 1984, J. Clin. Microbial. 19, 917. Gleaves, C.A., T.F. Smith, E.A. Shuster and G.R. Pearson, 1985, J. Clin. Microbial. 21, 217. Griffiths, P.D., D.D. Panjwani, P.R. Stirk, M.G. Ball, M. Ganczakowski, H.A. Blacklock and H.G. Prentice, 1984, Lancet 2, 1242. McKeating, J.A., S. Stagno, P.R. Stirk and P.D. Griffiths, 1985, J. Med. Virol. 16, 367. Myerson, D., R.C. Hackman and J.D. Meyers, 1984, J. Infect. Dis. 150, 272. Stinski, M.F., 1978, J. Viral. 26, 686. Sundqvist, V.-A. and B. Wahren, 1981, J. Viral. Methods 2, 301. Swenson, P.D. and M.H. Kaplan, 1985, J. Clin. Microbial. 21, 669. Virtanen, M., A.-C. Syvanen, J. Oram, H. Soderlund and M. Ranki, 1984, J. Clin. Microbial. 20, 1083.