Spectrophotometric quantitation of peroxidase-stained protein bands following gel electrophoresis and the western blot transfer technique with respiratory syncytial virus.

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.

Time-resolved fluoroimmunoassays with monoclonal antibodies for rapid identification of parainfluenza type 4 and mumps viruses.

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.

Use of the biotin-avidin system to study the specificity of antibodies against respiratory syncytial virus.

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.

Comparison of monoclonal antibody time-resolved fluoroimmunoassay with monoclonal antibody capture-biotinylated detector enzyme immunoassay for respiratory syncytial virus and parainfluenza virus antigen detection.

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.

Biotransformation of zeranol: disposition and metabolism in the female rat, rabbit, dog, monkey and man.

The disposition of [3H]zeranol has been studied in the female Wistar rat, New Zealand rabbit, beagle dog, rhesus monkey and man. The blood elimination half-life of total radioactivity in rabbit was 26 h, monkey 18 h and man 22 h. In all species studied the drug was absorbed, oxidized and/or conjugated, and was extensively excreted via the bile in all species except rabbit and man, in which urinary excretion predominated. Blood total radioactivity in man probably consisted entirely of conjugates of zeranol and/or its metabolites. Urinary metabolites in all species included conjugates (beta-glucuronides and/or sulphates) of zeranol and the major metabolite zearalanone. A more polar minor metabolite was isolated from human urine and was shown to be hydroxy-zeranol. Taleranol (7 beta-zearalanol, the lower-melting diastereoisomer), a probable metabolite of zeranol (7 alpha-zearalanol, the higher-melting diastereoisomer) in animals and in man, was shown to be a urinary metabolite in a female New Zealand white rabbit which had received [3H]zeranol (8 mg/kg per day) for seven days. A reverse isotope dilution method was developed for the quantification of both diastereoisomers of zearalanol, and also zearalanone, in urine.

Metabolism of butalbital, 5-allyl-5-isobutylbarbituric acid, in the dog.

Butalbital, 5-allyl-5-isobutylbarbituric acid, labeled in the 2-position with 14C, was administered to dogs. Ninety-two percent of the radioactivity of the dose was excreted in the urine. The drug and three major urinary metabolites were identified in the urinary excretion of the dog. The major metabolite was 5-isobutyl-5-(2,3-dihydroxypropyl)barbituric acid, which accounted for 50.2% of the dose. Smaller amounts of the unchanged drug (2.6% of the dose) and urea (8.6% of the dose) were present. 5-Allyl-5-(3-hydroxy-2-methyl-1-propyl)barbituric acid, formed by omega-hydroxylation, accounted for 10.1% of the dose; the optical rotation of the 1,3-diethyl derivative was [alpha]D20 = +10.5. Five minor and unidentified metabolities accounted for an additional 10.7% of the dose. A total of 82.2% of the dose was accounted for.

Biotransformation of mazindol. I. Isolation and identification of some metabolites from rat urine.

Three metabolites of tritium-labeled mazindol were isolated from rat urine by the inverse isotope-dilution technique in which the labeled metabolites were synthesized by a second, smaller group of rats. These metabolites were isolated by Amberlite XAD-2 chromatography and silica gel column and preparative thin-layer chromatography. The major metabolite (II) was shown by mass spectrometry of its trimethylsilyl derivative. NMR spectroscopy, and degradation studies to be 5-(p-chlorophenyl)-2,5-dihydro-5-hydroxy-3H-imidazol(2,1-a)isoindol-3-one. A comparison of its mass spectrum with that of an authentic sample prepared from 1-(p-chlorophenyl)-3-ethoxy-1-methoxy-1H-isoindole and glycine ethyl ester confirmed the assignment. Metabolite III was shown by its mass spectrum, NMR spectrum, degradation, and analogy with metabolite II to be 5-(p-chlorophenyl)-2,5-dihydro-2,5-dihydroxy-3H-imidazo (2,1-a)isoindol-3-one. Only a small amount of metabolite IV was isolated as an artifact, 3-(p-chlorophenyl)-2-glycyl-3-methoxy-1-isoindolinone, as shown by its mass spectrum and degradation to 2-(p-chlorobenzoy)benzoic acid. The metabolite IV is believed to be the corresponding 3-hydroxy compound. Synthesis of IV by base-catalyzed hydrolysis of metabolite II supports the structural assignment. In addition, the facile conversion of synthetic IV into the corresponding 3-methoxy derivative by acidic methanol was also observed.

Quantitation of enterovirus 70 antibody by microneutralization test and comparison with standard neutralization, hemagglutination inhibition, and complement fixation tests with different virus strains.

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.

Application of a solid-phase immunofluorometric assay to the selection of monoclonal antibody specific for the adenovirus group-reactive hexon antigen.

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.

Antigen detection in human respiratory Coronavirus infections by monoclonal time-resolved fluoroimmunoassay.

BACKGROUND: 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. OBJECTIVES: To prepare monoclonal antibodies to coronavirus strains, to incorporate them into a TR-FIA, and test the assay on clinical specimens. STUDY DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.

Subgrouping of respiratory syncytial virus strains from Australia and Papua New Guinea by biological and antigenic characteristics.

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.