Rapid detection of cytomegalovirus by 24-well plate centrifugation with the use of a monoclonal antibody to an early nuclear antigen

Two methods for the detection of cytomegalovirus (CMV) in 457 clinical specimens were compared: (1) centrifugal inoculation of MRC-5 cells seeded on coverslips in 24-well plates and staining with a monoclonal antibody to CMV early nuclear antigen after incubation for both 16-18 hours (EA-1) and four days (EA-4); and (2) conventional tube cell culture. CMV was identified in 50 (11%) specimens from 34 different patients. EA-1 and EA-4 had positive results for CMV in 32 (64%) and 36 (73%) of the specimens, respectively. Positive inclusions were present on only one coverslip in 31% of the cases by EA-1 and in 10% by EA-4. The number of inclusions was not necessarily predictive of tissue culture results. CMV was recovered by conventional tissue culture from 27 specimens (54%) after an average of 17 days (range, 6-26 days). One specimen, positive for CMV by EA-4, yielded herpes simplex virus (HSV), and from 9 of the 407 CMV-negative specimens, another virus was recovered: HSV from 6 specimens and varicella zoster virus, adenovirus, and enterovirus from one specimen each. CMV was detected in significantly more specimens by EA-4 than by tissue culture (P = 0.037). However, there was no significant difference in the detection of CMV between EA-1 and EA-4 or between EA-1 and conventional culture. The authors' data suggest that for maximum recovery of CMV from clinical specimens, both an early antigen assay and conventional tissue culture should be performed. For urine specimens it appears that inoculation of two coverslips followed by staining after overnight incubation is adequate. To optimize the yield of the early antigen assay when testing specimens other than urine, the authors recommend inoculating three coverslips, two of which should be stained after overnight incubation, and, if necessary, the third coverslip could be stained after a more prolonged incubation period.     

Detection of adenovirus by rapid 24-well plate centrifugation and conventional cell culture with dexamethasone

Two methods for rapid detection of adenovirus were tested: (i) 24-well plate centrifugation followed by staining with a monoclonal antibody after incubation for 24 h and 48 h, and (ii) pretreatment of A549 cells used in conventional cell culture and 24-well plate centrifugation with 10(-5)M dexamethasone. Twenty-seven clinical isolates of adenovirus and 12 specimens from which adenovirus had been recovered were included in the analysis. Both isolates and specimens had been frozen at -70 degrees C for up to 6 months. By 24-well plate centrifugation both with and without dexamethasone, 21 (78%) and 27 (100%) isolates were positive for adenovirus at 24 h and 48 h, respectively. Of the specimens, 6 (50%) and 8 (67%) were positive by 24-well plate centrifugation without dexamethasone at 24 h and 48 h, respectively, whereas with dexamethasone 3 (25%) were positive at 24 h and 7 (58%) were positive at 48 h. Overall, combining isolates and specimens, the sensitivity of 24-well plate centrifugation for detection of adenovirus at 24 h was 69% without dexamethasone and 62% with dexamethasone, and at 48 h the sensitivity was 90% without dexamethasone and 87% with dexamethasone. The specificity under all conditions tested was 100%. In conventional tissue culture dexamethasone inhibited recovery of adenovirus. Without dexamethasone, adenovirus was recovered from all 39 samples within 7 days after inoculation; however with dexamethasone pretreatment, the virus was detected in only 31 (79%) of the samples tested in the same period of time.     

Rapid detection of cytomegalovirus by tissue culture, centrifugation, and immunofluorescence with a monoclonal antibody to an early nuclear antigen

A rapid, sensitive and specific assay for the detection of cytomegalovirus (CMV) was developed utilizing MRC-5 cells in 24-well plates containing round coverslips. Centrifugation expedited the detection of CMV early antigen with monoclonal antibody. Immunofluorescent staining 16 h after inoculation with a stock CMV preparation (AD-169), demonstrated an 11-fold increase in the number of nuclear inclusions when the specimens were centrifuged (18 +/- 2.2) as compared to the non-centrifuged specimen (1.6 +/- 0.9). However, the number of nuclear inclusions depended on the age of the MRC-5 cells. They were more sensitive to CMV infection between 4 and 11 days after the cells were seeded into plates. Among 159 patient samples cultured for CMV, 23 (14%) were positive by the rapid method (mean of 32 h) and 18 (11%) by routine tissue culture (mean of 12 days). Cytomegalovirus in urine was detected within 1.3 days, whereas buffy coats (2.3 days) and bronchial washings (2.5 days) took longer. Staining for CMV inclusions at more than one time point was necessary for the optimal detection of CMV by the rapid method. We recommend using this assay system as it is rapid, specific, sensitive and versatile for the detection of CMV in many biological specimens.     

Rapid detection of cytomegalovirus in cell culture by indirect immunoperoxidase staining with monoclonal antibody to an early nuclear antigen.

A method for the rapid detection of cytomegalovirus (CMV) in MRC-5 cells 48 h after inoculation with clinical specimens was developed. A commercially available monoclonal antibody to a CMV early nuclear antigen was used in an indirect immunoperoxidase (IPA) staining procedure performed directly on acetone-fixed cell monolayers in standard tubes (16 by 125 mm). Of 190 clinical specimens tested, 30 specimens produced CMV cytopathic effect in tissue culture (TC-CPE) within 14 days after inoculation and, of these 30, 28 were positive for CMV after 48 h by the IPA staining procedure (sensitivity, 93%). Of the remaining 160 clinical specimens negative by TC-CPE within 14 days, 7 were positive by the IPA stain (specificity, 96%). However, three of these seven specimens were positive by TC-CPE upon subculture after the initial 14-day incubation period, and one specimen was overgrown by herpes simplex virus type 2 before CMV cytopathic effect could develop. The mean time to appearance of cytopathic effect for the 30 specimens positive by TC-CPE within 14 days was 6.7 days. These findings indicate that this IPA staining is a useful method for the rapid detection of CMV in cell monolayers inoculated with clinical specimens.     

Detection of cytomegalovirus in shell vial cultures by using a DNA probe and early nuclear antigen monoclonal antibody.

An in situ biotinylated DNA probe assay was evaluated as an adjunct to anti-cytomegalovirus early nuclear antigen indirect immunofluorescence and cytopathic effect on cytomegalovirus-infected monolayers in shell vial cultures. Viral infection was detected by early nuclear antigen indirect immunofluorescence at 24 h and by DNA probe assay and shell vial cytopathic effect at 5 days.     

Detection of cytomegalovirus in clinical specimens by virus isolation and by a monoclonal antibody against the early nuclear antigen

A commercially available monoclonal antibody against the 72000 Dalton early nuclear protein (EA) of cytomegalovirus (CMV) strain AD169 was used in an indirect immunofluorescence staining procedure (IF) for rapid detection of CMV-infected cells in tissue cultures inoculated with clinical specimens (200 urines, 22 throat washings, 5 stools, 4 bronchoalveolar lavage fluids). The results obtained by this method were compared with those obtained by virus isolation with and without centrifugal enhancement of viral infectivity. In 66 (28.6%) of the 231 samples, CMV was detected by at least one of the methods used. Of 59 specimens producing CMV-specific cytopathic effect (CPE) in tissue culture, 46 (78%) were also positive in the EA test 16 hours after inoculation. Seven CPE-negative samples were, however, positive in the EA test. Five (38%) of the false negative EA test results were due to CMV strains that did not react with the monoclonal antibody used.     

Rapid (24h) neutralization assay for the detection of antibodies to human cytomegalovirus using a monoclonal antibody to an HCMV early nuclear protein

Neutralizing antibodies to human cytomegalovirus (HCMV) may play an important role during the course of an HCMV infection but yet detection with conventional methods remains difficult and time consuming. A new neutralization test has been developed basically corresponding to the known microtitre technique. A monoclonal antibody to an HCMV early nuclear protein is utilized to detect HCMV (AD169) infected fibroblasts by biotin/streptavidin-enhanced immunoperoxidase staining. The test procedure was thereby confined to only 24 h. Evaluation of the new neutralization test (24h-NT) was done in comparison to the conventional method (NT). Preselected serum samples (n = 217), of which n = 169 were HCMV-seropositive in ELISA (reciprocal titre greater than or equal to 40), were tested for neutralizing antibodies (NAbs) to HCMV. ELISA titres were measured by a commercially available kit (Behring, F.R.G.). NAbs could be detected in 107 sera by 24h-NT (reciprocal titre greater than or equal to 5) and in 110 sera determined by NT (reciprocal titre greater than or equal to 5). Those sera were exclusively positive in ELISA. ELISA negative sera (n = 48; reciprocal titre less than 40) yielded no detectable neutralizing titres. This specific and rapid test should facilitate HCMV-NAbs detection for a wide variety of applications including routine virology diagnostics, evaluation of HCMV hyperimmunoglobulins and medical research.     

Effect of dexamethasone on detection of herpes simplex virus in clinical specimens by conventional cell culture and rapid 24-well plate centrifugation.

During a 4-month period, two methods for rapid detection of herpes simplex virus (HSV) were examined: (i) pretreatment of A549 cells with dexamethasone for conventional tissue culture (277 specimens) and (ii) 24-well plate centrifugation using A549 cells with and without dexamethasone pretreatment and staining with serotype-specific monoclonal antibodies (Syva Co., Palo Alto, Calif.) after incubation for 16 to 18 h (153 specimens). By conventional tube cell culture, both with and without dexamethasone, HSV was identified in 88 of 277 (32%) specimens. Significantly more specimens were positive for HSV at 24 h (46 versus 27 specimens) and at 48 h (a total of 72 versus 59 specimens) (P less than 0.0001) in dexamethasone-treated A549 cells. Of the 153 specimens tested by conventional culture and 24-well plate centrifugation, HSV was detected in 44 (29%) by conventional culture, and by 24-well plate centrifugation with and without dexamethasone, HSV was detected in 32 (21%) and 30 (20%) specimens, respectively. The sensitivity, specificity, and positive and negative predictive values of 24-well plate centrifugation with A549 cells for detection of HSV were 73 (71% without dexamethasone), 100, 100, and 90%, respectively. In conventional tube cell culture, pretreatment of A549 cells with dexamethasone results in more rapid detection of HSV. Centrifugal inoculation of dexamethasone-treated and untreated A549 cells in 24-well plates and staining with monoclonal antibodies after incubation for 16 to 18 h is an insensitive means to detect HSV in clinical specimens and should not replace conventional tube cell culture.     

Rapid detection of cytomegalovirus in MRC-5 cells inoculated with urine specimens by using low-speed centrifugation and monoclonal antibody to an early antigen.

A commercially available monoclonal antibody directed against an early nuclear protein of cytomegalovirus was used with low-speed centrifugation for the rapid detection of this virus from urine specimens inoculated onto MRC-5 cells. A total of 19 of 162 (11.7%) urine specimens inoculated were positive by both immunofluorescence and peroxidase-antiperoxidase procedures (sensitivity, 100%), whereas only 18 of the samples produced cytopathic effects in conventional cell culture (specificity, 94.7%). All specimens were positive by immunofluorescence and peroxidase-antiperoxidase procedures at 36 h postinfection, whereas an average of 9 days was required for cytopathic effects to develop in cell cultures.     

Production and characterisation of a human monoclonal antibody to cytomegalovirus and its use in an early nuclear fluorescence assay

A human monoclonal antibody to cytomegalovirus (CMV) was produced by transforming peripheral blood mononuclear cells of a patient with recent CMV infection. It is directed against a late antigen located in the nucleus of CMV infected fibroblasts at 24-72 hours postinfection and immuneprecipitates 65K and 48K proteins from 35S-labelled CMV infected cells. Results of its use in an early nuclear fluorescence assay for rapid diagnosis are presented.     

Early detection of cytomegalovirus in cell culture by a monoclonal antibody

A commercially available monoclonal antibody directed against early cytomegalovirus (CMV) antigen was used for the demonstration of CMV by immunofluorescence (IF) in cell culture within 2 days. The results were compared with the appearance of CMV-specific cytopathogenic effect (CPE). Urine specimens from 31 healthy children in day-care centers were inoculated on human embryonic fibroblasts. In addition, 45 CMV strains that had been stored at -70 degrees C were reinoculated. CMV was detected in 8/31 urine specimens by IF and 7 of these gave a specific CPE at an average of 16 days post-inoculation. One specimen was negative by IF but specific CPE was found at day 13. After reinoculation, CMV was detected in 76% by IF while 44 specimens developed CPE within a 6-week period. Demonstration of early CMV antigen in cell culture was found to be a rapid method for early diagnosis of CMV. Since the conventional cell culture with detection of CPE was more sensitive it may be useful to combine the two methods.     

Detection of Ebola viral antigen by enzyme-linked immunosorbent assay using a novel monoclonal antibody to nucleoprotein

With the increase in international traffic, the risk of introducing rare but severe infectious diseases like Ebola hemorrhagic fever is increasing all over the world. However, the system for the diagnosis of Ebola virus infection is available in a limited number of countries. In the present study, we developed an Ebola virus antigen-detection enzyme-linked immunosorbent assay (ELISA) system using a novel monoclonal antibody (MAb) to the nucleoprotein (NP). This antibody recognized an epitope defined by a 26-amino-acid stretch near the C terminus of NP. In a sandwich ELISA system with the MAb, as little as 30 ng of purified recombinant NP (rNP) was detected. Although this MAb was prepared by immunization with rNP of subtype Zaire, it also reacted to the corresponding region of NP derived from the Reston and Sudan subtypes. These results suggest that our ELISA system should work with three of four Ebola subtypes. Furthermore, our ELISA system detected the NP in subtype Reston-infected monkey specimens, while the background level in noninfected specimens was very low, suggesting the usefulness of the ELISA for laboratory diagnosis with clinical specimens.     

Rapid 24-well plate centrifugation assay for detection of influenza A virus in clinical specimens

Two methods for detection of influenza virus in 234 clinical respiratory specimens were compared: (i) a 24-well plate-centrifugation assay using Madin Darby canine kidney (MDCK) cells and staining with monoclonal antibody pools to influenza A and B (Centers for Disease Control, Atlanta, GA) after incubation for 16 h and 40 h, and (ii) conventional tube cell culture using MDCK cells and primary rhesus monkey kidney cells. Influenza A was identified in 23 specimens (10%). No influenza B was recovered. The rapid centrifugation and tissue culture methods were positive for influenza A in 21 (91%) and 16 (70%) of the 23 specimens, respectively. Fourteen specimens were positive by both methods, 2 were positive by tissue culture alone, and 7 were positive by rapid centrifugation only. Of the 21 specimens positive by rapid centrifugation, 16 (76%) were detected after overnight incubation, and 5 (24%) were positive only after incubation for 40 h. Cytopathic effect was observed in 13 (81%) of the 16 isolates identified by tissue culture after an average of 6 days, and 3 (19%) were identified only by hemadsorption and staining with monoclonal antibodies at day 10. Compared with conventional tissue culture, the 24-well plate centrifugation assay is a more rapid and more sensitive method for detecting influenza virus in clinical specimens.     

Detection of cytomegalovirus from clinical specimens in centrifugation culture by in situ DNA hybridization and monoclonal antibody staining.

An in situ DNA hybridization kit for cytomegalovirus (CMV) was evaluated for the detection of CMV in centrifugation culture. Of 61 clinical specimens, 17 (27.8%) were positive for CMV by monoclonal antibody staining following centrifugation. Of the 17 positive specimens, 15 were detected by DNA hybridization (24.5%). However, the earliest that CMV could be detected by DNA hybridization was 58 h as compared with 16 h with monoclonal antibodies following centrifugation. DNA hybridization remains of great interest for the study and detection of CMV infection. However, current DNA hybridization techniques are not sufficiently rapid to replace the use of monoclonal antibodies in centrifugation culture.     

Detection of specific immunoglobulin M antibodies to cytomegalovirus by using monoclonal antibody to immunoglobulin M in an indirect immunofluorescence assay.

The detection of immunoglobulin M (IgM) antibodies to cytomegalovirus-induced late antigens by an indirect immunofluorescence assay was improved by the use of monoclonal antibodies to human IgM. Nonspecific background fluorescence was absent, facilitating the reading of the slides and the detection of a specific fluorescence reaction in sera with low levels of specific IgM. Moreover, the indirect immunofluorescence assay with monoclonal antibodies to IgM proved more sensitive than the indirect immunofluorescence assay with polyclonal antibodies to IgM. The absorption of human sera on staphylococcal protein A avoided false reactions due to the presence of rheumatoid factor and high levels of specific IgG in test sera.     

Evaluation of a direct fluorescein-conjugated monoclonal antibody for detection of cytomegalovirus in centrifugation culture.

A fluorescein-conjugated murine monoclonal antibody (MAb) reactive with cytomegalovirus (CMV) was evaluated for the detection of CMV in centrifugation culture. Of 188 specimens, 90 were positive for CMV in centrifugation culture. The fluorescein-conjugated MAb detected CMV in 86 of 90 (95%) specimens at 16 h postinoculation, and 88 of 90 (98%) were positive at 36 h. The fluorescein-conjugated MAb can be used in a direct immunofluorescence assay that can be completed in 15 min following cover slip fixation. Use of this antibody in centrifugation culture provides a convenient and rapid assay for the identification of CMV.     

Detection of nuclear cytomegalovirus antigen in cell culture as compared to classical virus isolation.

Rapid detection of human cytomegalovirus early nuclear protein in human diploid cells by immunofluorescence (IF) using commercial (Du Pont) monoclonal antibody was compared with the classical virus isolation in these cells. From 30 specimens tested early nuclear protein was detected in 16 cases but the virus was isolated out of 4 samples only.     

Detection of cytomegalovirus in urine samples by an enzyme-linked immunosorbent assay using a monoclonal antibody against the viral 150-kilodalton protein.

McKeating et al. (J.A. McKeating, P.D. Griffiths, and J.E. Grundy, J. Gen. Virol. 68:785-792, 1987; J. A. McKeating, J. E. Grundy, Z. Varghese, and P. D. Griffiths, J. Med. Virol. 18:341-348, 1986; J. A. McKeating, S. Stagno, P. R. Stirk, and P. D. Griffiths, J. Med. Virol. 16:367-373, 1985) reported previously that beta 2 microglobulin inhibits the detection of human cytomegalovirus (CMV) in urine specimens by an enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody against the glycoprotein of CMV. They postulated that beta 2 microglobulin binds to the viral glycoproteins and masks the antigenic determinants. We developed here an ELISA method for the detection of CMV in urine by using a monoclonal antibody against the viral 150-kDa protein to capture the viral antigen. This assay detected CMV both in culture medium and in urine specifically at concentrations higher than 10(3) PFU/ml and quantitatively at concentrations higher than 10(4) PFU/ml. The sensitivity of the ELISA increased about 10-fold when peroxidase-labeled F(ab')2 from goat anti-human immunoglobulin G was used as a secondary detecting antibody in combination with concentration of the virus in urine samples by ultracentrifugation. The inhibition of ELISA by beta 2 microglobulin was not observed in this ELISA system. When 56 urine specimens from renal transplant recipients were examined for CMV antigens, the ELISA system had a sensitivity of 78% and a specificity of 97%. The positive and negative predictive values of the assay were 95 and 86%, respectively. Furthermore, CMV antigens in urine were quantitated by the assay during the course of typical CMV disease of renal transplant recipient. These results suggest strongly that the measurement of CMV antigens in urine by our rapid and quantitative ELISA system provides very useful data for the monitoring of CMV infections in renal transplant recipients and making decisions about therapy.     

Detection of both hepatitis B e antigen and antibody in a single assay using monoclonal reagents

Monoclonal antibodies raised against HBeAg were used to develop a HBeAg and anti-HBe detection assay. Monoclones containing anti-HBe were used both for the coating of the solid phase and for the fluid phase label in a sandwich type assay. The percentage binding of 125I-labelled anti-HBe to serum HBeAg was much greater than that seen in a similar assay using only polyclonal reagents. Therefore it was possible to add a small quantity of HBeAg for neutralising any anti-HBe present in a test serum without affecting HBeAg detection. This small amount of serum HBeAg was incorporated into each test sample thus allowing the determination of the e status of a patient using only one aliquot of test serum. This single test assay could be performed either as a radioimmunoassay or as an ELISA. The sensitivity of these assays was found to be greater than the conventional polyclonal assay particularly with regard to sera containing anti-HBe.