Detection of cytomegalovirus in urine samples by enzyme‐linked immunosorbent assay

McKeating, Jane A.; Stagno, Sergio; Stirk, P. R.; Griffiths, Paul

doi:10.1002/jmv.1890160410

Cited by 37 publications

(21 citation statements)

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“…McKeating et al (1985) reported that their ELISA for the detection of HCMV failed to detect the virus in fresh urine samples, but that upon storage at 4 °C for 1 to 2 weeks initially negative samples became ELISA-positive. They postulated the presence of an inhibitory substance in fresh urine which might be destroyed upon storage, and subsequently McKeating et al (1986) identified fl2m as such an inhibitor.…”

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confidence: 99%

“…One factor unexplained by the electron microscopy evidence regarding non-involvement of envelope proteins in fl2m binding was the report by McKeating et al (1985) of failure to detect HCMV in fresh urine samples by means of their ELISA system which utilized monoclonal antibodies said to be directed against HCMV glycoproteins. Their assumption was that fl2m in urine bound to the viral glycoproteins thus inhibiting their attachment to the solid phase.…”

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confidence: 99%

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beta2 Microglobulin Binds to the Tegument of Cytomegalovirus: an Immunogold Study

Stannard

1989

Journal of General Virology

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SUMMARYPrevious reports have provided evidence for the ability of human cytomegalovirus (HCMV) to bind the host protein/~z microglobulin (fl2m) from body fluids or culture mediung, and thus enhance infectivity of the virus, both by evasion of immune neutralization and the capacity to employ the bound flzm for attachment to the host cell. Immunocytochemical techniques and negative stain electron microscopy were used to identify the ultrastructural components of HCMV involved in its interaction with f12 m. Probes comprising colloidal gold coupled to//zm were seen to bind not to the envelope as previously suspected, but to material closely surrounding the nucleocapsids. It is postulated that the tegument proteins of HCMV, via their capacity to bind f12 m, play an important role in the preservation of infectivity of disrupted virions by enabling unenveloped capsids to bind to cells and gain entry by a pathway other than that normally taken by intact virions.In recent years, interesting information has come to light regarding the affinity of human cytomegalovirus (HCMV) for f12 microglobulin (fl/m). McKeating et al. (1985) reported that their ELISA for the detection of HCMV failed to detect the virus in fresh urine samples, but that upon storage at 4 °C for 1 to 2 weeks initially negative samples became ELISA-positive. They postulated the presence of an inhibitory substance in fresh urine which might be destroyed upon storage, and subsequently McKeating et al. (1986) identified fl2m as such an inhibitor. Furthermore, flzm was shown to bind to HCMV strain AD169 grown in cell culture when fl2m had been added to the culture fluids (Grundy et al., 1987a), but only after the release of the virions from the cell. McKeating et al. (1987) demonstrated that the binding of fl2m to urinary HCMV was also able to prevent neutralization of the virus by various neutralizing antisera, and because the presence of flzm could be demonstrated only in the Triton X-100-soluble fraction of HCMV harvested from urine, and not in the Triton-insoluble fraction, they concluded that fl2m was associated with the envelope, and possibly masked antigenic sites necessary for neutralization. Further studies (Grundy et al., 1987 b) demonstrated that the binding of fl2m to HCMV enhanced the infectivity of the virus and the authors postulated that bound fl2m assisted in the attachment of the virus to the host cell. It was also shown that fl2m and HCMV compete for binding sites on fibroblasts, where fl2m is normally non-covalently bound to the heavy chain of class I HLA molecules. As a result of evidence which showed that fl2m-coated HCMV could bind more efficiently to Raji cells (which express class I HLA molecules) than to Daudi cells (which lack these determinants), it was postulated that HCMV could use class I HLA molecules as a virus receptor and displace fl2m from the fl2m-HLA heavy chain dimers present on the cell surface.Although many of these earlier findings were based on the assumption that fl2m was able to bind to viral glycoproteins on the v...

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confidence: 99%

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confidence: 99%

beta2 Microglobulin Binds to the Tegument of Cytomegalovirus: an Immunogold Study

Stannard

1989

Journal of General Virology

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confidence: 99%

“…Second, it is well recognized but to our knowledge not previously published, that CMV in urine cannot be neutralized by antisera with good neutralizing activity against CMV grown in cell culture. Third, we have recently reported that CMV in urine specimens cannot be captured onto a solid phase by CMV-specific monoclonal antibodies which can capture CMV grown in vitro (McKeating et al, 1985), suggesting some differences in surface antigens between viruses from the two sources.We have shown that urine contains a host protein, f12 microglobulin (flzm) which, at physiological concentrations, inhibits the capture of cell culture-grown CMV by virus-specific monoclonal antibodies (McKeating et al, 1986). We postulated that this inhibition was due to the binding of fl2m to the virus masking the viral antigenic determinants.…”

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confidence: 99%

Cytomegalovirus in Urine Specimens Has Host beta2 Microglobulin Bound to the Viral Envelope: A Mechanism of Evading the Host Immune Response?

McKeating

Griffiths

Grundy

1987

Journal of General Virology

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SUMMARYWe have previously reported that human cytomegalovirus (CMV) from urine specimens cannot be captured onto a solid phase by CMV-specific monoclonal antibodies that can capture CMV grown in vitro. We report here that CMV exists in vivo in body fluids such as urine as f12 microglobulin (flzm)-coated particles. We have demonstrated the presence of fl2m on CMV purified directly from urine by Western blotting and have shown that the fl2m was associated with the viral envelope. Urinary CMV could be specifically bound by an affinity column comprising a monoclonal antibody specific for fl2m bound to Sepharose. The flzm-coated urinary CMV could not be neutralized by hyperimmune globulin, human immune sera or murine monoclonal antibodies that could neutralize CMV grown in cell culture. We conclude that the binding of fl,m by CMV masks the important antigenic sites necessary for neutralization which are recognized by man's immune response. We propose that CMV has evolved this mechanism of coating itself in a host protein as a mechanism of evading the host immune response and facilitating transmission between individuals. INTRODUCTIONHuman cytomegalovirus (CMV) in clinical specimens appears to differ from cell culturegrown virus in several respects. Firstly, the virus in urine samples appears to be much more stable than that grown in cell culture (Feldman, 1968;Stagno et al., 1980), which is reported to be thermolabile (Vonka & Benyesh-Melnick, 1966). Second, it is well recognized but to our knowledge not previously published, that CMV in urine cannot be neutralized by antisera with good neutralizing activity against CMV grown in cell culture. Third, we have recently reported that CMV in urine specimens cannot be captured onto a solid phase by CMV-specific monoclonal antibodies which can capture CMV grown in vitro (McKeating et al., 1985), suggesting some differences in surface antigens between viruses from the two sources.We have shown that urine contains a host protein, f12 microglobulin (flzm) which, at physiological concentrations, inhibits the capture of cell culture-grown CMV by virus-specific monoclonal antibodies (McKeating et al., 1986). We postulated that this inhibition was due to the binding of fl2m to the virus masking the viral antigenic determinants. In an accompanying paper (Grundy et al., 1987a), we have shown that indeed cell culture-grown CMV exhibits a strong binding capacity for fl2m in vitro. We predicted that in vivo, in body fluids such as urine, CMV exists as fl2m-coated particles, and the aim of the present study was to verify that prediction using virus preparations obtained directly from infected patients.

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“…For the rapid diagnosis of active CMV infection, several attempts have been made. Those included (i) the detection of virus particles by electron microscopy (20), (ii) the detection of viral antigens by enzyme-linked immunosorbent assay (ELISA) (17,24), immunofluorescence staining (1,8,13,14,22,26,31,33), iminunoperoxidase staining (12,25,30), or dot immunoassay (23,34), and (iii) the detection of viral nucleic acid by DNA hybridization (4,12,22,25,27,28,32) or polymerase chain reaction (PCR) (11). Among those attempts, the detection of viral antigens in cell culture with monoclonal antibodies against IEA or EA is a rapid, sensitive and the most practical method to identify the CMV isolates.…”

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confidence: 99%

Rapid Diagnosis of Cytomegalovirus Infections by Direct Immunoperoxidase Staining with Human Monoclonal Antibody against an Immediate‐Early Antigen

Eizuru

Minematsu

Minamishima

et al. 1991

Microbiology and Immunology

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Direct immunoperoxidase technique using a horseradish peroxidase (HRP)-conjugated Fab' fragment of human monoclonal antibody (humab C7), designated HRP-C7, was evaluated as a rapid diagnosis of cytomegalovirus (CMV) infection. A total of 138 clinical specimens consisting of 124 urine samples and 14 oral swabs were examined for CMV by the direct HRP-C7 staining in comparison with conventional virus isolation. The number of CMV-positive samples by each method was 40 (29.0%) for the former and 37 (26.8%) for the latter, respectively. By HRP-C7 staining, CMV was identifiable within 24 hr after inoculation. By conventional isolation method, an average of 10.3 days had passed before cytopathic effect characteristic of CMV appeared in the cell culture. Some false-positive and false-negative cases were discussed in relation to toxicity of urine samples, storage of the samples, and amount of CMV in the sample. The sensitivity and specificity of HRP-C7 method against conventional isolation method were 89.2% and 93.1%, respectively. Thus, HRP-C7 staining is useful for a rapid diagnosis of CMV infections, Human cytomegalovirus (CMV) is an important pathogen not only for fetuses, but also for immunocompromised hosts such as patients with organ transplantation, acquired immunodeficiency syndrome (AIDS), or neoplastic diseases. Those patients are at high risk for active CMV infection, with CMV pneumonitis being critical event (18). In addition, through its own immunosuppressive effect, active CMV infection itself predisposes the patients to secondary infections by bacteria, fungi or protozoa (3). Therefore, the rapid confirmation of active CMV infections in those patients is essential for early initiation of anti-CMV treatments (21). The most reliable diagnosis of active CMV infections is virus isolation. However, CMV grows so slowly in cell culture that detection of CMV by its typical cytopathic effect (CPE) requires a long incubation for weeks.CMV produces a number of early antigens (EA) in the infected cells before viral 1015

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Detection of cytomegalovirus in urine samples by enzyme‐linked immunosorbent assay

Cited by 37 publications

References 10 publications

beta2 Microglobulin Binds to the Tegument of Cytomegalovirus: an Immunogold Study

beta2 Microglobulin Binds to the Tegument of Cytomegalovirus: an Immunogold Study

Cytomegalovirus in Urine Specimens Has Host beta2 Microglobulin Bound to the Viral Envelope: A Mechanism of Evading the Host Immune Response?

Rapid Diagnosis of Cytomegalovirus Infections by Direct Immunoperoxidase Staining with Human Monoclonal Antibody against an Immediate‐Early Antigen

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