Biomedical subjects
Publications and source records attributed to D Gallo.
A panel of monoclonal antibodies to herpes simplex virus glycoproteins was used for serological analysis of 130 strains. Based on specific immunological determinants, strains of each serotype clustered into subgroups. Monoclonal antibodies were suitable reagents for serotyping and have potential application to epidemiology of herpes simplex virus infections.
Seventy-seven clones of hybridomas selected for reactivity by immunofluorescence with human cytomegalovirus (CMV)-infected cells were produced by fusing mouse myeloma cells with the spleen cells of mice immunized with CMV strain AD169. The clones were classified into seven groups on the basis of the electrophoretic properties of the polypeptides immune precipitated from extracts of CMV-infected cells. Studies on the three groups of monoclonal antibodies directed against CMV surface membrane antigens showed the following. Clones in each group were differentiated by immunoglobulin subclass, neutralizing activity, and reactivity with the antigenic domains of proteins exposed on the surface membranes of intact CMV-infected cells. Monoclonal antibodies in each group precipitated one slowly migrating protein and multiple faster migrating forms which shared antigenic determinants. The first group of monoclonal antibodies precipitated four glycosylated polypeptides with apparent molecular weights of 130,000, 110,000, 100,000, and 60,000. Monoclonal antibody CH51 of this group neutralized infectious virus but failed to react with antigenic domains on the surfaces of infected cells. The second group of monoclonal antibodies precipitated four polypeptides with apparent molecular weights of approximately 66,000, 55,000, 50,000, and 46,000. Monoclonal antibodies CH65 and CH134 in this group had neutralizing activity and reacted with antigenic domains of proteins exposed on the surface of CMV-infected cells. The third group of monoclonal antibodies precipitated four polypeptides with apparent molecular weights of 49,000, 48,000, 34,000, and 25,000. Serological analysis of 15 naturally occurring CMV strains with a panel of monoclonal antibodies to surface membrane proteins showed that the antigenic determinants reactive with the antibodies tested were conserved in all of the strains. Monoclonal antibodies to surface membrane proteins on CMV-infected cells may prove to be valuable reagents for identification of virus isolates.
Epstein-Barr viral capsid antigen and nuclear antigen produced by modified procedures were evaluated for use in measuring viral capsid antigen immunoglobulin M and Epstein-Barr virus nuclear antigen antibody responses in sera from patients with suspected Epstein-Barr virus infections. Viral capsid antigen production was stimulated with a phorbol ester, and the Epstein-Barr virus nuclear antigen cells were fixed in suspension to eliminate loss of antigen during the drying process. Both preparations proved to be sensitive and reliable.
Monoclonal antibodies conjugated with fluorescein permitted rapid, strain-specific identification of influenza A isolates and type-specific identification of influenza B isolates by direct immunofluorescence staining. Identification of H1 influenza A strains could be accomplished by direct immunofluorescence on cell culture fluids lacking sufficient hemagglutinin activity to permit identification by hemagglutination inhibition.
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A microimmunofluorescence test was evaluated for use in measuring immunoglobulin M (IgM) antibodies in infant sera to five of the agents implicated in congenital and neonatal disease. Pen point dots of Toxoplasma gondii, cytomegalovirus, rubella virus, herpes simplex virus, and chlamydial cell culture antigens were applied to each circle of eight-circle printed slides. These multiple-antigen slides greatly facilitated the screening of 607 sera from infants and 117 sera from mothers for the presence of IgM antibody to these agents. Forty sera could be examined microscopically in approximately 30 min. All sera reacting with one or more antigens were tested for rheumatoid factor by the latex method, absorbed with glutaraldehyde-cross-linked human IgG, and retested for the presence of IgM antibody. IgM antibody to cytomegalovirus was demonstrated in sera from four newborns, but IgM antibody to rubella virus could not be detected until 21 days after birth, although rubella virus was isolated from sera from five younger infants. This may indicate that rubella IgM levels in many congenitally infected newborns are too low to be measured by the immunofluorescence method. Five percent of the sera from infants in this study possessed demonstrable IgM antibody to one of the antigens.
The anticomplement immunofluorescence (ACIF) test was compared with the fluorescent antibody-to-membrane antigen (FAMA) test for determining varicella-zoster virus antibody levels as a measure of varicella-zoster virus immunity status. The ACIF test was found to be comparable to the FAMA test in sensitivity and could be used for examining sera at low dilutions of 1:2 and 1:4. In addition, the ACIF method proved to be a more economical procedure in terms of antigen required and personnel time necessary to perform the test. Heterologous varicella-zoster virus antibody titer rises were demonstrated by the FAMA test with 10 serum pairs from patients with clinically diagnosed genital herpes simplex virus infection, indicating that the FAMA test is no more suitable than other serological methods for serodifferentiation of those herpes simplex virus and varicella-zoster virus infections in which antibody increases occur to both antigens.
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An anti-complement immunofluorescence test utilizing cytomegalovirus immune hamster sera specifically identified cytomegalovirus isolates showing an early, typical cytopathic effect. Inclusion of a control conjugate against adenovirus group antigen permitted correct identification, as adenoviruses, of a few isolates initially suspected of being cytomegalovirus on the basis of cytopathic effect.
Direct immunofluorescence (IF) staining was compared with virus isolation for detection of herpes simplex viruses (HSV) and varicella-zoster virus (VZV) directly in clinical materials. These included 199 vesicular lesion specimens and 280 tissue specimens. Correspondence between IF and isolation results was 88% in testing for HSV in lesion specimens and 98% in testing for HSV in various tissue (mostly brain) specimens. Overall, IF was positive for 82% of the specimens in which HSV was demonstrated, and virus was isolated from 89% of the HSV-positive specimens. IF was markedly more sensitive than isolation for demonstrating VZV in lesion and tissue specimens, detecting all of the specimens positive for VZV, whereas isolation detected only 23%. IF detected VZV antigen in a number of lesion specimens taken late after onset, past the time when they would be expected to yield infectious virus. Specificity of positive IF reactions for HSV or VZV in the absence of virus isolation was supported by the facts that (i) staining was obtained with only a single, presumably homologous, immune conjugate, (ii) clinical symptoms were compatible with infection with the virus for which positive IF findings were obtained, and (iii) positive electron microscopy findings for herpesviruses or positive serological results for VZV were also obtained in some instances. Factors to be considered in achieving specificity of IF staining for these human herpesviruses are discussed.
An anti-complement immunofluorescence (ACIF) test that detects human cytomegalovirus (CMV) antigen in the nuclei of infected cells was used for assay of CMV antibodies in human sera. Various factors influencing the sensitivity and specificity of the ACIF test system were investigated, and results were applied to the development of a procedure which could be completed in a relatively short length of time and gave reproducible results. Results obtained in the ACIF test were compared with those obtained in complement fixation, indirect hemagglutination, and neutralization tests, and the ACIF test was shown to be suitable for detection of significant antibody titer rises and stationary levels of CMV antibody. Heterotypic antibody responses were not seen with sera from other human herpesvirus infections. The nonspecific cytoplasmic staining that occurs in indirect immunofluorescence tests for CMV did not occur in the ACIF system, and sera that were anti-complementary in complement fixation tests could be examined satisfactorily by ACIF. Thus, the test is a valuable supplemental or back-up procedure for the serodiagnosis of CMV infection.
A new thin-layer chromatographic (TLC) method is described for measurement of conjugated (CB) and unconjugated (UCB) bilirubin in bile. Bile is streaked on a silica-gel plate and developed with chloroform. Bilirubin in the mobile UCB band and stationary CB band is quantitated by diazotization from the scraped gel with p-iodoaniline reagent. Both CB and UCB in bile yielded identical azopigment absorbance. There was no contamination of the UBC band with either CB or oxidative derivatives of bilirubin, and over 98 per cent added 14C-UBC migrated with the mobile band. Recoveries from TLC averaged 98.2 +/- 4.3 per cent of the applied pigment, duplicated agreed within 10 per cent, and the method accurately detected the enzymatic and alkaline hydrolysis of CB to UCB. Concentrations of UCB in nine samples of normal human gallbladder bile were found to be less than 1.9 mg./dl. and never exceeded 1.2 per cent of the total bilirubin.
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