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.     

Rapid detection of respiratory syncytial virus and influenza A virus in cell cultures by immunoperoxidase staining with monoclonal antibodies.

Peroxidase-labeled monoclonal antibodies against respiratory syncytial virus (RSV) and influenza A virus were used for immunoperoxidase staining (IPS) of cell cultures inoculated with nasopharyngeal aspirates. Cells were grown in 24-well plates, and specimens were inoculated by low-speed centrifugation. Cultures were incubated for 2 days at 37 degrees C and then fixed, stained, and observed by light microscopy. IPS was compared with standard virus isolation by using cultures of human diploid fibroblasts and Vero, HEp-2, and HeLa cell lines for RSV and Madin-Darby canine kidney cells for influenza A virus; these cultures were inoculated with specimens that were previously stored at -70 degrees C. Of 40 known RSV-positive specimens, 30 were found to be positive on reinoculation by both methods, and an additional 5 specimens were found to be positive by IPS only. Of 190 specimens tested for influenza A virus, 14 were positive by IPS and in tubes, and a further 8 specimens were positive by IPS only. IPS was also compared with direct detection of viral antigens in nasopharyngeal aspirates by a time-resolved fluoroimmunoassay (TR-FIA). Fresh nasopharyngeal aspirates were inoculated into human diploid fibroblasts and Madin-Darby canine kidney cells and tested for RSV and influenza A virus, respectively, by IPS. Of 110 specimens tested for RSV, 37 were positive in total, 32 were positive by IPS, and 33 were positive by TR-FIA. Of 150 specimens tested for influenza A virus, 39 were positive in total, 35 were positive by IPS, and 34 were positive by TR-FIA. IPS of cultures inoculated by centrifugation and incubated for 2 days is a sensitive method for the diagnosis of respiratory virus infections, and 24-well plates allow for the easy processing of a large number of specimens.     

呼吸道合胞病毒单克隆抗体的制备及鉴定

目的：建立能稳定分泌抗呼吸道合胞病毒（RSV-long株,国际标准株）单克隆抗体的杂交瘤细胞株。方法：杂交瘤技术制备单抗,鉴定各项特性。结果：培育出稳定分泌抗RSV蛋白的单克隆抗体的杂交瘤细胞5株。杂交瘤细胞株染色体数目在89～104条之间,其分泌的抗体分别属于鼠IgG1、IgG2a、IgG2b亚类,各种腹水单抗的荧光效价在1∶4000～1∶16000之间。一种单抗识别RSV基质蛋白（M）,两种单抗识别RSV融合蛋白（F）,另两种单抗识别RSV核衣壳蛋白（N）。单抗中和效价最高达1∶64。相加指数证实5种单抗识别不相关的抗原表位。用硫氰酸铵洗脱法比较了五种单抗相对亲和力的大小,依次为：1#〉2#〉4#〉3#〉5#。所有杂交瘤细胞株,经连续3个月培养及冻存半年后复苏,细胞生长良好,检测上清,其分泌抗体效价稳定。结论：已获得抗呼吸道合胞病毒的单克隆抗体,为RSV感染的早期诊断及进一步研究奠定了基础。     

Utility of a respiratory virus panel containing a monoclonal antibody pool for screening of respiratory specimens in nonpeak respiratory syncytial virus season.

An indirect immunofluorescence respiratory virus panel containing monoclonal antibodies directed against respiratory syncytial virus, parainfluenza virus types 1, 2, and 3, adenovirus, and influenza viruses A and B was used to screen specimens in the nonpeak respiratory syncytial virus seasons in 1989 and 1990. The results indicate that the respiratory virus panel is fairly sensitive (79%) and very specific (99%) for the detection of respiratory viruses directly in clinical specimens during these time periods.     

Rapid diagnosis of respiratory syncytial virus infection by antigen immunofluorescence detection with monoclonal antibodies and immunoglobulin M immunofluorescence test.

During a respiratory syncytial virus (RSV) infection outbreak in a pediatric hospital, diagnosis was made by immunofluorescence on smears by using an anti-RSV monoclonal antibody (IFm). Immunoglobulins M and G were titrated by indirect immunofluorescence on HEp-2 cells infected with an RSV strain. The IFm was sensitive (89%) and specific (75%) when compared with the cell culture method. We showed that the specimens which were found positive by IFm and negative by cell culture were truly positive. Under these conditions, the IFm test appears more sensitive and more specific than cell culture, particularly when no care is taken to maintain the specimens in the cold during transport. In this study the immunoglobulin M immunofluorescence test had a low sensitivity (34%), especially on serum samples taken on days 0 to 4 after the onset of illness.     

Rapid detection of respiratory syncytial virus antigens in nasopharyngeal secretions

The combined use of fluorescein-labelled monoclonal antibody and a cytocentrifuge for preparation of cell spots greatly reduced the time for rapid diagnosis, and improved the sensitivity and ease of detection of respiratory syncytial (RS) virus antigen in specimens of nasopharyngeal secretions.     

Rapid detection of respiratory syncytial virus in nasopharyngeal aspirates by a commercial enzyme immunoassay.

A commercial enzyme immunoassay (EIA) for the rapid detection of respiratory syncytial virus (RSV) in respiratory secretions was evaluated by comparison with both virus isolation in HEp-2 cells and indirect immunofluorescence (IFA) staining of exfoliated respiratory cells. Initial examination of 80 nasopharyngeal aspirates collected from infants with acute respiratory illness showed that the RSV EIA was positive for 21 of 24 specimens positive by virus isolation or IFA (87.5% sensitivity) and negative for 53 of 56 specimens negative by virus isolation and IFA (95% specificity). The EIA appears to be an acceptable and more rapid test than virus isolation for the detection of RSV, especially for laboratories in which prompt inoculation of specimens is not always possible. IFA staining with commercial bovine anti-RSV serum was found to be the most sensitive and rapid test for the detection of RSV. However, three of four specimens positive by IFA and negative by virus isolation were not cultured under optimal conditions. In addition, the IFA test requires a highly trained technologist to interpret the staining results.     

Rapid immunoperoxidase assay for detection of respiratory syncytial virus in nasopharyngeal secretions.

Samples of nasopharyngeal secretions obtained from 70 infants and young children with acute respiratory disease were examined for the presence of respiratory syncytial virus by immunoperoxidase assay (IPA). The IPA was compared with the immunofluorescence assay and with cell culture isolation. Respiratory syncytial virus antigen-positive cells were detected by both IPA and immunofluorescence assay in 28 specimens; 25 samples were positive in cell culture. The agreement between virus isolation and IPA and IFA was 89%. The applicability of IPA to rapid viral diagnosis of respiratory syncytial virus infection is discussed.     

Rapid detection of respiratory syncytial virus in nasopharyngeal specimens obtained with the rhinoprobe scraper.

We compared the Rhinoprobe scraping technique for collection of superficial nasal mucosa epithelial cells and rapid detection of respiratory syncytial virus by immunofluorescence with paired, swab-collected specimens for virus culture from 1,257 infants and children with acute respiratory infections. Compared with viral culture as the reference test, the sensitivity, specificity, and accuracy of the immunofluorescence test were 83.6, 93.6, and 91.3%, respectively. We found the Rhinoprobe method safe, easy to use, and helpful in obtaining large quantities of epithelial cells for detection of respiratory syncytial virus and other respiratory viruses.     

Rapid detection of respiratory syncytial virus in nasopharyngeal secretions by enzyme-linked immunosorbent assay.

A new enzyme-linked immunosorbent assay (ELISA) respiratory syncytial virus antigen detection kit (Ortho Diagnostics, Inc., Raritan, N.J.) was compared with virus culture and with the indirect fluorescent antibody test (FAT) by using fresh nasal washings from children with suspected respiratory syncytial virus infection. Both uncentrifuged nasal washings and pellets from centrifuged split specimens were examined by ELISA. The ELISA was considered positive when the optical density was greater than the mean background optical density plus 0.15. Specimens positive by ELISA but negative by culture and FAT were reexamined in an ELISA blocking assay. Of 337 uncentrifuged specimens, 124 (37%) were positive by culture, 107 (32%) were positive by FAT, and 166 (49%) were positive by ELISA. Blocking assays showed that 21 of 30 (70%) of the seemingly false-positive ELISA tests were, in fact, true-positives and that the cultures and FATs were falsely negative. A patient specimen was considered positive when it was positive by virus culture, FAT, or blocking assay. The sensitivity, specificity, and positive predictive value of the ELISA test were 88, 94, and 95%, respectively. Centrifugation of nasal washings raised the sensitivity from 88 to 92% but reduced the specificity from 94 to 81%. We conclude that the Ortho ELISA test of uncentrifuged nasal washings is more sensitive than virus culture or indirect FAT and is highly specific.     

Isolation and characterization of a chimpanzee monoclonal antibody to the G glycoprotein of human respiratory syncytial virus.

Respiratory syncytial virus (RSV) is the most common cause of serious lower respiratory tract disease in infants and young children. In this study a hybridoma line secreting a chimpanzee monoclonal antibody that neutralizes RSV was isolated. Two chimpanzees were immunized with recombinant vaccinia viruses that express the RSV F or G surface glycoprotein and 1 month later were infected intranasally with the wild-type RSV strain A2. Peripheral blood lymphocytes obtained from the animals were transformed with Epstein-Barr virus, and lymphoblastoid cell lines that secreted anti-RSV antibodies were identified by an RSV antigen-binding enzyme-linked immunosorbent assay. Supernatants from RSV antibody-secreting lymphoblastoid cell lines were tested for in vitro virus neutralization before being fused to the heteromyeloma cell GLI-H7. A chimpanzee antibody [immunoglobulin G3(lambda) subclass] produced from a hybridoma line designated E1.4/2 was shown to bind to the RSV G glycoprotein and neutralize a panel of subgroup A viruses, but not subgroup B viruses, at low (nanomolar) concentrations. Mice passively immunized with this antibody were partially resistant to RSV strain A2 challenge. The usefulness of such antibodies in immunoprophylaxis and immunotherapy of RSV infection is discussed.     

Commercial monoclonal antibodies for rapid detection of respiratory syncytial virus by direct immunofluorescence

A commercially available direct immunofluorescence (IF) test using a reagent consisting of a pool of two monoclonal antibodies against selected surface and internal proteins of respiratory syncytial virus (RSV) was evaluated in comparison with the indirect IF technique using a commercial bovine anti-RSV hyperimmune serum for the rapid detection of RSV in nasopharyngeal aspirates from 228 hospitalized children. Overall agreement between the two IF methods was 95%. The direct IF test was quicker to perform and easier to interpret than the indirect IF test.     

Immunoglobulin G antibody avidity in patients with respiratory syncytial virus infection.

The titer and avidity of respiratory syncytial virus-specific antibodies were measured in 196 serum specimens from 93 children with an acute, laboratory-confirmed respiratory syncytial virus infection. An enzyme immunoassay method based on the ability of urea to dissociate the bound antibodies with low avidity from the antigen was used. Three patterns of immune responses were observed. Children less than 6 months of age usually had low titers of antibodies with high avidity in their acute-phase serum samples. These antibodies were concluded to be of maternal origin, since their reaction pattern was similar to that of healthy adults. During the next few weeks, a slight increase in titers with a concurrent decrease in antibody avidity was observed. All children 6 to 24 months of age had low-avidity antibodies in their acute-phase serum samples, which matured to high avidity during the follow-up. On the contrary, about half of the children greater than 24 months of age had high-avidity antibodies already in the acute-phase serum samples. We conclude that the former children were experiencing primary infections with respiratory syncytial virus and the latter were experiencing reinfections. All adults with remote immunity had antibodies with high avidity.     

Detection of antibody to bovine syncytial virus and respiratory syncytial virus in bovine fetal serum.

Batches of commercial fetal bovine serum, described by the suppliers as antibody-free, all contained antibody to bovine syncytial virus (BSV) when tested by indirect immunofluorescence. Antibody to bovine respiratory syncytial virus (RSV) was not detected in these sera. Twenty-four percent of individual fetal bovine sera contained antibody to BSV, and 14% contained antibody to RSV when tested by indirect immunofluorescence. BSV antibody titers in fetal sera from dams with high BSV antibody levels were variable but always higher than RSV antibody titers. Radial immunodiffusion studies with BSV-positive sera revealed the presence of immunoglobulin M (IgM), IgG, and IgA, but the quantity of these immunoglobulins was not directly related to the BSV antibody titers. The evidence suggests that the antibody present in fetal sera arose as the result of infection rather than from maternal transfer across the placenta.     

Detection of antibody to respiratory syncytial virus by membrane fluorescence.

An indirect membrane fluorescent antibody technique was established to study HEp 2 cells infected with respiratory syncytial (RS) virus. It was possible to detect IgG and IgM antibody to RS virus in the sera of patients with respiratory infections using this technique. The technique was further applied to the detection of IgA antibody to the same virus in colostrum.     

Rapid diagnosis of respiratory syncytial virus infections in immunocompromised adults.

Although rapid antigen detection methods for the documentation of respiratory syncytial virus (RSV) infections are widely used with pediatric patients, these tests have not been prospectively evaluated in immunocompromised (IC) adults. For bone marrow transplant recipients and adult patients undergoing chemotherapy for leukemia who had recent onset of respiratory symptoms, respiratory samples (combined nasal wash [NW]-throat swab [TS], endotracheal tube [ET] aspirate, or bronchoalveolar lavage [BAL] samples) were collected for simultaneous culture and rapid antigen detection with the Directigen test kit (Becton Dickinson, Cockeysville, Md.). NW specimens from hospitalized pediatric patients with suspected RSV infection were also evaluated. Viral quantitation was performed on aliquots of the original specimens. A total of 539 samples from 372 adult patients were evaluated. RSV was isolated from 56 specimens (40 NW-TS, 7 ET aspirate, and 9 BAL specimens). By using culture as the "gold standard," rapid antigen detection had a sensitivity of 15% for adult NW-TS specimens, 71.4% for ET aspirate specimens, and 88.9% for BAL specimens; the specificity was > or = 97% for all specimen types. Significantly greater viral quantities were present in pediatric NW specimens than in adult NW specimens. In adults, more virus was present in BAL and ET aspirate specimens than in NW-TS specimens. Rapid detection of antigen respiratory samples obtained from the lower respiratory tracts of IC adults is sensitive and specific, but detection in upper respiratory tract samples is insensitive. The lower sensitivity of antigen detection in NW-TS specimens may be due to decreased viral load. A BAL specimen is more sensitive than an NW-TS specimen for the rapid diagnosis of RSV disease in IC adults.     

Evaluation of five methods for respiratory syncytial virus detection.

A total of 117 nasal aspirates were cultured for respiratory syncytial virus (RSV) and tested for RSV antigen by a direct fluorescent-antibody (DFA) test (Bartels Immunodiagnostic Supplies, Inc., Bellevue, Wash.), the Directigen enzyme immunoassay (EIA; Becton Dickinson Microbiology Systems, Cockeysville, Md.), the TestPack EIA (Abbott Laboratories, North Chicago, Ill.), and RSV EIA (Abbott). Agreement of two of five methods or a positive RSV culture were required to validate a result. A total of 57 of 117 (48.7%) specimens were culture positive in HEp-2 cells, A549 cells, or both. A total of 5 of 117 (4.3%) additional specimens met the criteria of a positive specimen; i.e., 62 of 117 (53.0%) specimens were positive. Results obtained from 77 of 117 (65.8%) specimens were concordant for all five methods. The sensitivities, specificities, and positive and negative predictive values for the culture and DFA methods were 91.9, 100, 100, and 91.7% and 91.9, 96.4, 96.6, and 91.4%, respectively. The sensitivities, specificities, and positive and negative predictive values for the three EIA procedures, Directigen, TestPack, and RSV EIA, were 75.8, 80.0, 81.0, and 74.6%; 93.6, 100, 100, and 93.2%; and 71.0, 100, 100, and 75.3%, respectively. New self-contained EIA configurations and the DFA method offer attractive alternatives to the culture method. Technical simplicity, rapid turnaround time, performance, and cost must all be considered when selecting a system for RSV detection.     

呼吸道合胞病毒 L 蛋白单克隆抗体的制备

目的：制备人呼吸道合胞病毒L蛋白单克隆抗体,并对其进行性质鉴定。方法：选取RSV-A2 L蛋白上具有亲水性可能的loop区段,将这些目的片段展示在HBc149上进行原核表达并纯化。重组蛋白免疫6~8周BALB/c雌性小鼠,根据免疫后血清的ELISA和WB分析结果选择L3935和L4099组小鼠进行脾脏免疫。融合后,用间接ELISA法筛选和多次克隆化,获得稳定分泌抗L蛋白单克隆抗体细胞株,并结合ELISA、免疫荧光和免疫印迹对所获得的单抗进行鉴定。结果：获得11株L蛋白特异性单克隆抗体,亚类鉴定结果IgG1、IgG2a和IgG3单抗各3株,IgG2b单抗2株。结论：成功制备了L蛋白特异性单克隆抗体,也为RSV感染的免疫和发病机制研究奠定基础。     

Rapid diagnosis of respiratory syncytial virus infection by using Pernasal swabs.

The sensitivity and specificity of direct immunofluorescence microscopy performed on Pernasal swab specimens and compared with those of nasopharyngeal aspirates were 93 and 99%, respectively. Posterior nasopharyngeal swabs applied immediately to microscope slides allow a rapid and simple screening procedure for the diagnosis of acute respiratory syncytial virus infections.