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Four-layer antispecies radioimmunoassay (RIA) and enzyme immunoassay (EIA) procedures were developed for the detection of respiratory syncytial virus (RSV), parainfluenza type 2 virus, and adenovirus antigens in nasopharyngeal specimens from children hospitalized for acute respiratory disease. Polystyrene beads (RIA) or flat-bottomed polystyrene microtiter plates (EIA) were used as the solid phases, guinea pig anti-virus immunoglobulins were used as the captive antibodies, rabbit anti-virus immunoglobulins were used as the secondary antibodies, and 125I-labeled sheep anti-rabbit (RIA) or horseradish peroxidase-labeled swine anti-rabbit (EIA) immunoglobulins were used as the indicator antibodies. A comparison of the EIAs and RIAs with routinely used immunofluorescence (IF) techniques was made with 164 nasopharyngeal specimens collected from children with acute respiratory disease. Only 3 of 66 RSV IF-positive specimens were negative in RSV RIA, and of 83 RSV, parainfluenza type 2 virus, and adenovirus IF-negative specimens, 1 was positive in RSV RIA. Of 4 parainfluenza type 2 virus IF-positive and 11 adenovirus IF-positive specimens, each was positive in corresponding RIAs, and all 83 IF-negative specimens were negative in parainfluenza type 2 virus and adenovirus RIAs. The results of the RSV, parainfluenza type 2, and adenovirus EIAs confirmed the results of corresponding RIAs in each selected case tested. The RIAs and EIAs were found to be as specific and sensitive as IF techniques, and more practical in the rapid detection of respiratory viruses in nasopharyngeal secretions.

A sensitive method based on PCR followed by liquid-phase hybridization for detection of enterovirus and rhinovirus RNAs in clinical specimens and cell culture supernatants is described. RNA was extracted from stool samples, throat swabs, nasopharyngeal aspirates, cerebrospinal fluid, urine, and plasma with a commercial phenol-guanidinium-chloroform reagent and purified on a polysulfone membrane, on which the reverse transcriptase reaction was also done. Two sets of oligonucleotide primers from the 5' noncoding region of picornaviruses were selected for DNA amplification of 153-bp (enterovirus) and 120-bp (rhinovirus) regions. Double-stranded amplicons were digested into single strands with T7 gene 6 exonuclease and quantitated by an assay using a europium-labeled probe, streptavidin- and biotinylated probe-coated microtitration wells, and time-resolved fluorometry. The sensitivity of the assay was about one template molecule when purified coxsackievirus A9 RNA was used. All enterovirus prototype strains, except echoviruses 22 and 23, and clinical isolates grown in cell culture or suckling mice were strongly positive by the enterovirus PCR-hybridization, as were selected prototype strains and untyped isolates of rhinoviruses by the rhinovirus PCR-hybridization. In a series of 100 clinical specimens tested, the results for 92 agreed with virus culture results. The detection method described will be useful in etiopathogenic studies on enteroviruses and rhinoviruses.

Monoclonal antibodies (MAbs) to the M protein (M1) were used in the development of direct detection systems for type A influenza viruses in clinical specimens. Optimal detection by an enzyme-linked immunosorbent assay was achieved when MAbs were used as capture antibodies and rabbit polyclonal antibodies were used as sandwich antibodies. Detection by the enzyme-linked immunosorbent assay required amplification of the virus. direct detection in clinical specimens (nasopharyngeal aspirates) was accomplished when MAbs recognizing two distinct antigenic sites of M1 were used in a time-resolved fluoroimmunoassay. Type A influenza viruses could be detected equally well in specimens obtained during epidemics of both H3N2 and H1N1 influenza viruses.

A new solid-phase immunoassay, time-resolved fluoroimmunoassay (TR-FIA), for rubella antibody was developed. The test used polystyrene beads coated with rubella antigen as the solid phase and a chelate of the rare earth metal europium as fluorescent label. A fast light pulse from a xenon flash lamp was used to excite the label, and after a 400-mus delay time the emission fluorescence was measured for 500-mus at 1-ms intervals during a total counting time of 1 s. Background fluorescence of short duration caused by fluorescent serum components and scattering could be eliminated by including the delay time. The TR-FIA was compared with hemagglutination inhibition, single radial hemolysis, and two types of radioimmunoassay (RIA) (a commercial RIA [GammaCoat] and a noncommercial RIA [T-RIA]) by using 60 serum specimens from patients with remote rubella infection. Overall agreement of TR-FIA with hemagglutination inhibition and GammaCoat was 96.7%, with single radial hemolysis 98.3%, and with T-RIA 100%. Linear regression coefficients varied from 0.83 to 0.94, the best being obtained with single radial hemolysis and T-RIA. TR-FIA was also found to be suitable for the diagnosis of acute infections, as significant increases of antibody level were detected in all 30 paired serum specimens tested from patients with an acute rubella infection. Sensitivity and specificity comparable to those of RIA and enzyme immunoassay were obtained with TR-FIA. Furthermore, the advantage that TR-FIA has over RIA is that it incorporates a nonisotopic and stable label; its advantage over EIA is that it is easier to standardize because no additional reaction with substrate is required.

A four-layer antispecies radioimmunoassay (RIA) was developed for the detection of adenovirus in stool specimens. Polystyrene beads were used as the solid phase, anti-adenovirus guinea pig immunoglobulin (1 microgram per bead) was used as the primary antibody, anti-adenovirus rabbit immunoglobulin (16 micrograms/ml) was used as the secondary antibody, and 125I-labeled sheep anti-rabbit immunoglobulin was used as the indicator antibody. A highly purified, crystallized adenovirus type 2 hexon antigen was used as the immunizing antigen for the production of hyperimmune sera. The sensitivity of the test was 1 ng of hexon protein per ml. Each of the 13 stool specimens positive for adenovirus by electron microscopy was positive for adenovirus by the RIA. Of 200 nonconcentrated stool specimens negative by electron microscopy, 14 additional specimens were positive by the RIA, increasing the detection rate from 6% by electron microscopy to 13% by the RIA. A confirmatory test was done on the RIA-positive, electron microscopy-negative specimens, and the test indicated a true specific result with each specimen. A confirmatory test was also done on each specimen with a low positive counts per minute value. The specificity of the RIA was further demonstrated by the fact that a positive result was found with only 3 of 295 specimens positive by the rotavirus RIA. In two of these three specimens, adenovirus and rotavirus were also detected simultaneously by electron microscopy, and the third specimen was from a patient with serological evidence for a dual infection. The adenovirus and rotavirus RIAs are now in a routine diagnostic laboratory, and in the 307 stool specimens tested during the first 5 months, the positive rate was 32% for rotavirus and 9.5% for adenovirus.

A sensitive method based on competitive PCR was developed to detect and quantitate enteroviral RNA in clinical specimens, with special emphasis on controlling contamination and the presence of potential inhibitory factors in the specimens. Oligonucleotide primers from the conserved parts of the 5' untranslated and VP2 capsid protein-coding regions were selected to differentiate between enteroviruses and rhinoviruses on the basis of the length of the cDNA amplicons. RNA transcribed from a truncated cDNA copy of the echovirus 11 genome was used as an internal control for the reverse transcription reaction and PCR. This allowed simple differentiation of the control and viral PCR products from each other by agarose gel electrophoresis and nonradioactive quantitation of the viral RNA in the clinical specimens. By direct sequencing of the PCR products and subsequent computer analysis of the data, potential laboratory contaminations could be monitored and enteroviruses from clinical samples could be grouped into four distinct clusters, thus enabling genetic typing of the viruses. The described method can be applied to the diagnosis and epidemiology of enteroviral infections.

In addition to tests for the group-specific hexon antigen of adenoviruses, adenoviruses can be detected in clinical specimens by hybridization assays utilizing the widely shared base sequences of the region of the hexon gene that codes for the group-reactive determinants. We have developed a liquid-phase hybridization system with biotin- and europium-labeled probes which are reacted after DNA amplification of a 161-bp region of the hexon gene and which are quantitated by time-resolved (TR) fluorometry in streptavidin-coated microtiter wells. Polymerase chain reaction (PCR)-TR fluorometry is not a rapid test in the usual sense, but it is highly useful for specimens with inherent toxicity or with low virus yield, such as organ minces and specimens obtained late in the course of an illness. In a survey of 103 specimens tested by this method, including urine, stool, and tissue suspensions, the agreement with the hexon-specific TR fluoroimmunoassay antigen test for positive specimens was 100% and the sensitivity compared with that of virus culture was 91%. The PCR-TR fluorometry system was also shown to be advantageous as a quantitative measure of PCR products.

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.

An antibody capture enzyme immunoassay (EIA) was adapted for the detection of immunoglobulin M (IgM) antibody to Sindbis (SIN) virus. Sera from humans with a febrile illness characterized by rash and arthralgia in eastern Finland (Pogosta [POG] disease) and Sweden (Ockelbo disease) and from humans with western equine encephalitis (WEE) virus infection in the United States were tested for IgM antibodies by EIA. Seroconversions were documented in patients with POG disease and with WEE virus infections by using SIN virus as antigen and rabbit anti-SIN virus immunoglobulin; this confirms previous observations that POG disease is caused by a virus closely related to SIN virus and that IgM antibodies to WEE complex alphaviruses are not type specific. This IgM EIA provided a sensitive diagnostic and research tool applicable to epidemiologic problems posed by POG disease.

A solid-phase radioimmunoassay for detecting respiratory syncytial virus and adenovirus serum immunoglobulin A (IgA) antibodies was developed. An antigen consisting of purified adenovirus type 2 hexons or a crude lysate of respiratory syncytial virus-infected cells was first adsorbed onto polystyrene beads. The coated beads were then incubated with dilutions of serum, and IgA antibodies which attached to the solid-phase virus antigen were subsequently detected with 125I-labeled anti-human alpha antibodies. The anti-human alpha antibodies used were isolated by immunosorbent chromatography from rabbit antiserum produced by immunization with IgA purified from serum of an IgA myeloma patient. A total of 46 serum specimens from 13 patients with respiratory syncytial virus infections and 10 patients with adenovirus infections were tested. Complement fixation, homologous IgG and IgM radioimmunoassay, and heterologous IgA radioimmunoassay testing were also done. Specific values higher than 10,000 cpm were often reached with convalescent serum specimens, and positive-to-negative serum binding ratios of 50 or more were frequently obtained with lower serum dilutions. IgA titers of convalescent sera were from 1,000 to 16,000, and with few exceptions a fourfold or greater rise in the IgA titer was detected in the homologous IgA radioimmunoassay.

A solid-phase radioimmunoassay method has been developed for the detection of rubella virus-specific immunoglobulin G (IgG) and IgM antibodies in human serum specimens. Purified rubella virus was adsorbed onto polystyrene balls, and antibodies that attached to the virus-treated balls were detected by subsequent binding of 125I-labeled anti-human gamma or anti-human mu immunoglobulins. A total of 77 serum specimens were tested. Binding ratios between positive and negative sera were as high as 22 in the IgG assay but rarely exceeded 3 in the IgM assay. The sensitivity of the IgG assay was found to be 16 to 256 times higher than that of the rubella virus hemagglutination inhibition test. The IgG radioimmunoassay can be readily adopted for routine diagnostic use. The IgM radioimmunoassay, however, due to its lower sensitivity, must be modified before being routinely applied.

Two monoclonal antibodies against two distinct conserved epitopes of the respiratory syncytial virus (RSV) nucleocapsid protein were used in a direct time-resolved fluoroimmunoassay (TR-FIA) for the detection of RSV antigens in nasopharyngeal aspirates. The capture antibody was adsorbed to the solid phase of microdilution strip wells, and the indicator antibody was labeled with a europium chelate. Specimens and label were incubated simultaneously for 1 h at 37 degrees C in the coated wells. After the test samples were washed, fluorescence enhancement solution was added, strips were shaken, and the time-resolved fluorescence was measured. The test procedure took only 75 min, and the total time for 20 specimens, with pretreatment by sonication, was 2 to 3 h. We prospectively evaluated the detection of RSV in nasopharyngeal aspirates of pediatric patients by TR-FIA and by virus isolation in human diploid fibroblasts. TR-FIA detected 40 of 42 isolation-positive specimens. Nine additional isolation-negative specimens were positive by TR-FIA; all proved to be true positives by a blocking-type confirmatory assay. The sensitivity, specificity, positive predictive value, and negative predictive value for TR-FIA were 95, 96, 82, and 99%, respectively, of the values obtained by virus isolation and 96, 100, 100, and 99%, respectively, of the values obtained by virus isolation and the confirmatory assay.

A sensitive time-resolved fluoroimmunoassay (TR-FIA), adapted from TR-FIA procedures already described, was developed with monoclonal antibodies and compared with several enzyme immunoassays (EIAs) for detecting adenovirus antigens in clinical specimens. The most sensitive EIA was an all-monoclonal assay with biotin-labeled detector antibody and streptavidin-peroxidase conjugate. All tests were evaluated with nasopharyngeal aspirate specimens from respiratory illness, with tissue homogenates from patients with systemic infection, and with stool specimens from gastrointestinal illnesses. For respiratory and tissue specimens, the TR-FIA detected adenovirus in 85% of the specimens positive by culture, which was a sensitivity similar to those of the all-monoclonal biotin-avidin EIA (79%) and the polyclonal-capture biotin-avidin EIA (88%). For stool specimens, the TR-FIA detected adenovirus in 100% of the specimens positive by culture, which was a decidedly higher sensitivity than either EIA format (78 and 75%, respectively). The TR-FIA was shown to be an efficient, flexible, and specific test for large numbers of clinical specimens.

Monoclonal antibodies that are broadly reactive with either influenza A or influenza B viruses were used to develop a 2- to 3-h antigen capture time-resolved fluoroimmunoassay (TR FIA) for detecting influenza viral antigens in both original nasopharyngeal aspirate specimens and in tissue cultures inoculated with nose or throat swab specimens. The lower limit of sensitivity of the assay was about 10 pg of protein as determined with purified influenza A nucleoprotein expressed by recombinant DNA. When the TR FIA was performed with 96 nasopharyngeal aspirate specimens collected during outbreaks of influenza A (H3N2) virus and the results were compared with serodiagnosis results with paired sera, the specificity and sensitivity of TR FIA for the demonstration of influenza A infections were 95 and 85%, respectively. In culture confirmation assays, more than 80% of the swab specimens that grew influenza A or B virus within 7 days could be identified by the TR FIA within 48 h of the inoculation of cells. The results are consistent with those previously reported for respiratory syncytial virus and extend the applicability of monoclonal antibody-based TR FIA for the rapid diagnosis of acute respiratory viral infections.

A direct antibody capture enzyme immunoassay (EIA) for the detection of rubella-specific immunoglobulin M (IgM) antibody was developed. Polystyrene microtiter strips coated with rabbit anti-human IgM were used as solid phase, and a semipurified rubella antigen labeled with horseradish peroxidase was used as indicator. By testing a panel of 238 serum specimens (not including sera from newborns with congenital rubella), the direct EIA was compared with the indirect EIA used routinely in our diagnostic unit and with a commercial IgM capture EIA (RubEnz M II) that employs a horseradish peroxidase-labeled anti-rubella monoclonal antibody as indicator. Overall agreement of direct EIA with indirect EIA and RubEnz M II was 95%, whereas agreement between direct and indirect EIA was 96.2%, and agreement between direct EIA and RubEnz M II was 97.8%. Sensitivity of the direct assay was higher than that of the indirect EIA and similar to that of the RubEnz M II assay. Specific IgM antibody could always be detected in serum specimens taken from patients with primary acute rubella infection between days 4 and 56 after the onset of rash. The assay was highly specific, and it was not affected either by rheumatoid factor or by high levels of specific IgG in sera. Another important advantage that the direct EIA has over the indirect EIA is that it requires 10 to 20 times less antigen per serum specimen tested.