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ABSTRACT: The diagnosis of respiratory infections by detecting viral antigens has received considerable attention using immunofluorescent assays (IFA) and enzyme immunoassays (EIA). Time-resolved fluoroimmunoassay (TR-FIA) has been developed for several viruses.
To prepare monoclonal antibodies to coronavirus strains, to incorporate them into a TR-FIA, and test the assay on clinical specimens.
Monoclonal antibodies were prepared to the N nucleoprotein of the two human respiratory coronaviruses, HCV strains 229E and OC43. Monoclonals to both viruses were completely type-specific; they did not cross-react between themselves or with multiple strains of other respiratory viruses. These antibodies were configured into optimized EIA and TR-FIA tests. The all-monoclonal tests were then compared to polyclonal EIA tests in terms of their ability to detect virus in clinical specimens.
The all-monoclonal TR-FIA was uniformly the most sensitive, detecting virus in all 13 229E-positive specimens compared to 69% for the monoclonal EIA and 54% for the polyclonal EIA test. Similar results were obtained for 10 OC43-positive specimens: 100% in TR-FIA, 90% in monoclonal EIA, and 80% in polyclonal EIA. For 229E in TR-FIA, mean positive/negative (P/N) ratios were 143 for 229E-positive human embryonic lung fibroblast (HLF) cell culture fluids and 10 for positive nasopharyngeal aspirate specimens; for OC43 in TR-FIA, mean P/N values were 964 for OC43-positive rhabdomyosarcoma (RD) cell culture fluids and 174 for positive NPA specimens. The sensitivities of the TR-FIA were determined with purified virions to be 0.308 ng virus per well for HCV-229E and 0.098 ng virus per well for HCV-OC43.
This rapid and sensitive test appears to be much more sensitive than traditional antigen detection assays but will require more extensive field testing on clinical specimens.
Clinical and Diagnostic Virology 07/1994; 2(3):165-79.
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ABSTRACT: Strains of respiratory syncytial virus from 3 major areas of Australia and Papua New Guinea (PNG) were analyzed for variations in their antigenic and biological properties and in the molecular weights of their major structural proteins. Seventy-eight strains from infants and young children with LRI were collected from 1981-1984. The RSV season in the Australian cities lasted from April through September, with major peaks in July of each year, while the RSV season in tropical PNG was year-round, with small peaks in March and October of each year coinciding with excessive rainfall. Fifty-six strains were analyzed in detail; 40 were typed by time-resolved fluoroimmunoassay with monoclonal antibodies as group A strains and 16 were group B; both groups were concurrent. Three children of one family had sequential RSV infections 13 months apart, and the etiologic group A strain was identical both years in terms of growth and antigenic properties with strain-specific ferret antisera; the second infection was milder in all three children. On average, the group A strains replicated considerably better than group B strains in HEp2 cells, producing 53% more syncytia and 99% higher infectious virus titers in 31% less time in culture. Ten group A and B reference strains exhibited the same growth patterns as the A and B regional strains, respectively. Differences in antigenicity as measured with hyperimmune antisera to prototype Long strain were even greater. Group A strains exhibited a mean 68% greater IFA staining than B strains, a 71% greater EIA reaction, and were neutralized to 69% higher serum titers than B strains. Again, the reference A and B strains included as controls gave patterns identical to those of the regional strains. Finally, the P phosphoprotein had consistently higher molecular weight in A strains (mean 35,900) than B strains (mean 33,100). Small variations in the sizes of the F and G glycoproteins were not sufficient to suggest grouping on this basis.
Archives of Virology 02/1994; 136(1-2):133-47. · 2.11 Impact Factor
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ABSTRACT: Monoclonal antibodies were prepared to the F and M proteins of parainfluenza 4A and 4B and to mumpsvirus to obtain reagents that could be configured into type-specific yet broadly-reactive IFA, EIA, and TR-FIA tests. Several antibodies to parainfluenza 4A also detected subtype 4B, although to a somewhat lower signal, and thus were well suited to generic parainfluenza type 4 tests that were comparable to similar tests previously described for parainfluenza types 1, 2, and 3. Monoclonals to subtype 4B were less able to detect 4A because of high background problems in one or another test. Monoclonals to mumpsvirus F protein were completely type-specific. These antibodies were screened by IFA and EIA for broad reactivity with diverse strains of each virus and were configured into optimized EIA and TR-FIA tests. The all-monoclonal tests were then compared to polyclonal tests in terms of their ability to detect virus in clinical specimens. The all-monoclonal TR-FIA was uniformly the most sensitive, detecting virus in 80% of culture-positive parainfluenza 4A specimens, 67% of parainfluenza 4B specimens, and 90% of mumps specimens, compared to 40-67% for the monoclonal EIA tests and 33-60% for the polyclonal EIA tests. For parainfluenza 4 TR-FIA, mean P/N values were 379 for subtype 4A cell culture fluids (228 for subtype 4B cultures) and 57 for 4A clinical specimens (43 for 4B specimens). For mumpsvirus TR-FIA, mean P/N values were 27 for culture fluids and 32 for clinical specimens. The sensitivities of the TR-FIA were determined with purified virus to be 0.28 ng virus per well for parainfluenza 4A and 0.70 ng virus per well for mumpsvirus.
Archives of Virology 02/1993; 130(3-4):335-52. · 2.11 Impact Factor
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ABSTRACT: An all-monoclonal antibody, time-resolved fluoroimmunoassay was compared with several enzyme immunoassays for the detection of respiratory syncytial virus and parainfluenza virus type 1, 2, and 3 antigens in clinical specimens. The most sensitive enzyme immunoassay for parainfluenza virus type 1 was an all-monoclonal antibody assay with biotin-labeled detector antibody and streptavidin-peroxidase conjugate, but for respiratory syncytial virus and parainfluenza virus types 2 and 3 the most sensitive assay was a polyclonal antibody assay with horse capture antibodies and bovine or rabbit detector antibodies with anti-species peroxidase. All tests were evaluated with nasopharyngeal aspirate specimens from respiratory illnesses and with cell culture harvests of multiple strains of each virus isolated over many years. The time-resolved fluoroimmunoassay detected respiratory syncytial virus antigen in 92% of the specimens positive by culture, which was a decidedly higher sensitivity than either the monoclonal or polyclonal antibody enzyme immunoassay format (62 and 76%, respectively). For the parainfluenza viruses the time-resolved fluoroimmunoassay detected type-specific antigen in 94 to 100% of culture-positive specimens and again was more sensitive than the all-monoclonal antibody enzyme immunoassays (75 to 89%) or all-polyclonal antibody enzyme immunoassays (66 to 95%). Combined with results from a previously reported adenovirus time-resolved fluoroimmunoassay, these tests identified respiratory antigens in large numbers of clinical specimens.
Journal of Clinical Microbiology 07/1989; 27(6):1243-9. · 4.15 Impact Factor
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ABSTRACT: To characterize the specificity of monoclonal antibodies against respiratory syncytial virus, we developed a technique which combined the biotin-avidin system, immunoprecipitation, and transblot electrophoresis. The viral proteins were first biotinylated and then immunoprecipitated with a monoclonal antibody or respiratory syncytial virus immune serum, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and electrophoretically transferred to nitrocellulose paper. The protein bands were located on the paper with avidin-peroxidase plus a precipitable substrate. This procedure gave reproducible results qualitatively similar to those obtained by radioimmunoprecipitation but without the cost, risk of radiation exposure, or disposal problems of radioisotopes. This procedure should have widespread applications.
Journal of Clinical Microbiology 07/1984; 19(6):934-6. · 4.15 Impact Factor
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ABSTRACT: We describe here a microneutralization procedure for conveniently testing large numbers of specimens for antibodies to enterovirus 70. The test utilized human rhabdomyosarcoma cells and was read by staining with crystal violet after 4 days of incubation. The test compares well with other serological assays, being more sensitive than the standard tube neutralization test and the complement fixation test, but less sensitive than the hemagglutination inhibition test. However, the hemagglutination inhibition test required concentrated, partially purified virus as antigen, as did the complement fixation test, and was difficult to read, so that its greater sensitivity may not be of practical significance. By all four test procedures, a recent isolate of enterovirus 70 was a more sensitive antigen than the prototype strain, as shown by greater geometric mean titers in sera of patients from various epidemics.
Journal of Clinical Microbiology 07/1984; 19(6):826-30. · 4.15 Impact Factor
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ABSTRACT: A procedure is described for spectrophotometrically measuring the position and intensity of ELISA-stained protein bands after western blot analyses. Partially purified virus proteins separated by acrylamide gel electrophoresis and electrophoretically transblotted onto nitrocellulose paper were identified by ELISA reactions according to established procedures. The nitrocellulose paper strips were then rendered transparent on glass slides by treatment with a plastics solvent (Gelman Sepra-Clear). The slides were then scanned at 480 nm to obtain the precise migration distance and area under the curve (i.e., quantity) of each band, thus allowing accurate comparison of the proteins from different virus strains.
Journal of Virological Methods 06/1984; 8(3):265-8. · 2.01 Impact Factor
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ABSTRACT: An immunofluorometric assay (IFMA) has been evaluated as a screening assay to detect monoclonal antibodies to the group-specific antigen of the adenovirus hexon component. The antibodies were produced as mouse ascitic fluids from hybridoma cells generated from Balb/C mice immunized with purified adenovirus type 2 hexon component and crude adenovirus type 3 culture supernatants. The purified IgG fractions from all monoclonal ascitic fluids tested were identified as the IgG1 K mouse isotype. Antibody titers ranged from 102,400 to 204,800 by the IFMA, from 200 to 12,800 by indirect FA, and were generally nonreactive in counterelectrophoresis, complement fixation, hemagglutination-inhibition, serum neutralization, and immune electron microscopy titrations. The IFMA is a reliable method for quantitating low levels of specific antibody in large numbers of test samples, and is therefore ideal as a screening assay for monoclonal antibody in tissue culture fluids and in mouse ascitic fluids.
Archives of Virology 02/1984; 80(1):1-10. · 2.11 Impact Factor
