Quantitative immunofluorescence is routinely used in flow cytometric assay of cell surface antigens. Intracellular antigens have not been as tractable. Recent publications (Proc Natl Acad Sci 80:5573-5577, 1983; Cytometry 6: [208][209][210][211][212][213][214] 1985) and the results presented here demonstrate that highly specific staining and subsequent quantitative analysis are not only possible but rather easily achieved. High purity antibodies and optimized fixing and staining technique are required. Under conditions presented in this paper, 97% of fluorescein specific signal is specific to the T antigen of SV40 when monoclonal antibody to this antigen is used with a transformed cell line. Three levels of quantitative analysis are discussed: 1) estimation of the fraction of positive cells in a mixed +I-population, 2) estimation of the average content of antigen in a population of cells, and 3) measurement of the distribution of antigen content within a population of cells. Results are presented that demonstrate that relatively low specific signal (measured as percentage of total signal) can be tolerated to achieve the first level and that the current methods available that produce a high specific signal are sufficient to achieve the second level. The third level will require further research aimed at lowering the variation introduced by the method of measurement.
Key terms: DNA content changes, permissive infection, viral antigens, T-antigen, V-antigen Considerable interest has been generated in the analysis of gene products obtained from both viral and cellular oncogenes, since these genes may be responsible for such activities as cell proliferation, differentiation, and the acquisition of the neoplastic phenotype (1). Simian Virus 40 (SV40) has been an important model system for studies of viral replication, viral transformation, and gene regulation (14,20,22). The early viral gene products large T and small t antigen are thought to be responsible for events in viral replication in permissive cells and also for the initiation and establishment of the neoplastic phenotype in susceptible nonpermissive cells (4). The SV40 T antigen is known to bind viral DNA at the origin of replication and initiate viral DNA synthesis in the permissive cell (19,26). Further, the T antigen activates late viral transcription and inhibits early viral transcription (2). The part that the T antigen plays in transformation may be at a number of levels which include gene regulation and the interactions with the plasma membrane and cellular proteins such as p53 (15,25,27). The molecular approach to the study of viral expression has provided much information concerning the role of these proteins in permissive and nonpermissive cells.One further method of investigation would utilize flow cytometry to quantitatively assay the appearance of nity to assay various parameters of a population of cells, such as DNA and specific viral and cellular proteins, and to correlate these parameters on a single-cell basis (3,5,17,29). The instrumentation also allows quantitation of each of these parameters singly and in concert. In previous studies, a technique was developed that allowed simultaneous staining of cellular DNA content and the viral T antigen using a specific monoclonal antibody (13). This technique has been applied to study SV40 infection of permissive CV-1 cells. Changes in host cell cycle distribution and the synthesis of both T antigen and the late viral proteins that encapsulate the viral DNA were followed in time. MATERIALS AND METHODSCells and Virus The cells utilized in this study were the CV-1 line of African green monkey kidney, passages 26 through 38, CV-1 cells were grown in modified Eagle's medium (MEM, GIBCO) with 2 x amino acids and vitamins. MEM with 10% fetal bovine serum (FBS) was used for growth and 5% FBS for maintenance. The CHE lines were grown in MEM with 5% FBS. Cultures were kept at 37°C in humidified 5% C 0 2 incubator (17).The SV40 stock virus used was RH911, grown and plaque-assayed for titer on CV-1 cells. All cells and virus stock tested negative for mycoplasma contamination (17).Infection was performed on 100-mm plastic petri dishes containing CV-1 cells that were maintained for 1-2 days after reaching confluence. At this time, media was removed and 1.0 ml of virus, diluted in MEM, was added to the cultures. The cells were infected with varying multiplicities of infecti...
Human diploid fibroblasts (HDF) have a finite life span in cell culture which can be extended when transformed with simian virus 40 (SV40). Flow cytometric analysis of SV40-HDF transformation allowed DNA content changes to be correlated with the appearance, quantity, and distribution of T antigen, p53, and V antigen, three proteins associated with this process. These studies demonstrated a shift in the DNA content to tetraploidy, which was correlated with the age of the SV4O-HDF but not the time of infection. A significant increase of the epitope recognized by PAbl22 to host p53 and the epitope PAblOl to SV40 T antigen occurred at the same time the tetraploid population appeared. However, an antigen reactive with SV40 V antibody was present at high levels in most of the population early after infection, but the levels declined with time. The percentage of PAblOl-T antigen-positive cells increased more rapidly in cells infected at a late passage, and this was concomitant with the shift in DNA content to tetraploid. Analysis of the mean fluorescence of total, gated populations (GI, G2, and > Gt) demonstrated that a threshold level of p53 and T antigen was reached in each compartment of the cell cycle. As the transformed phenotype appeared, a population of cells was continually released into the supernatant, and although these cells had a DNA pattern similar to the monolayer cells, the T antigen and p53 levels were 3-5 times higher in the tetraploid 6 2 cells.These studies correlated the expression of proteins associated with viral transformation in HDF which vary with time and shift in DNA content.
An early cytomegalovirus (CMV) antigen was detected with a monoclonal antibody by two-color fluorescent flow cytometry. With the aid of a human diploid fibroblast cell strain, FLOW 2000, infected with the AD169 strain of CMV, the viral antigen and the DNA content of infected or uninfected cells were measured. There was no evidence of change in the cell-cycle distribution of the infected cells. The viral antigen was detected within 30 minutes following virus adsorption at 0.1 and 1.0 plaque-forming unitskells; and the percentage of positive cells increased with time and viral dosage. All stages of the cell cycle were susceptible to viral infection and the average fluorescence was greater than the background fluorescence. Flow cytometry detected the viral antigen earlier than conventional immunofluorescent microscopy and cell culture for CMV cytopathological effect (CPE). Ten bronchoalveolar lavages assayed by flow cytometry and conventional diagnostic procedures demonstrated that flow cytometry might be useful in early diagnosis for CMV infection.Key terms: Monoclonal antibody, early virus antigen (CMV) detection of virus-infected cells Cytomegalovirus (CMV) infection has been recognized as a significant cause of morbidity and mortality in bone marrow, cardiac, and renal transplant patients as well as in patients with human immunodeficiency virus (HIV) infections (1,(3)(4)(5)10,19,21,22). Traditionally, CMV infection has been demonstrated by virus isolation and identification in cell culture or by the finding of pathognomonic inclusion bodies in biopsy specimens (9). Serologic testing has been used to confirm exposure to CMV but requires acute and convalescent sera demonstrating a fourfold rise in titer and is less reliable since immunosuppressed patients may not have a change in antibody titer in the presence of active CMV infection (2,8,13,20). Although the combination of viral replication in tissue culture, a rising antibody titer, and inclusion bodies on histopathology is a sensitive and accurate indicator of virus infection, as long as 8 weeks may be required for virus identification and confirmation of diagnosis (9). With the advent of effective antiviral chemotherapy for human CMV infections, more rapid and sensitive techniques are required to identify CMV infections during the acute stage of the illness (12,151. Recently, methods employing the techniques of monoclonal antibodies that recognize CMV specific proteins synthesized shortly after infection and of DNA hybridization using labeled DNA probes specific for CMV have been used to identify CMV infection either directly on the clinical specimen or after viral amplification in cell culture (6,7,9,(16)(17)(18). The advantage of these newer techniques is that an identification of virus infection can be made in a number of hours to a few days. These techniques, however, can be labor intensive and may not be useful for screening the large number of samples received in a clinical microbiology laboratory. In addition, quantitative data, which may be potentiall...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.