VIRAL IMMUNOLOGY Volume 5, Number 1, 1992 Mary Ann Lieben, Inc., Publishers

New Monoclonal Antibodies for the Detection of Immediate Early Antigens of Cytomegalovirus 3 BERNARD R. BRODEUR,1 2 MICHELE LUSSIER,1 YOLANDE LAROSE,1 EDMOND ROSSIER,2 HELEN MILLER,4 CAROL NAKAGAWA,3 and MIKE EVELEGH3

ABSTRACT Two new monoclonal antibodies, CIE-1 and CIE-2, were developed for the rapid detection of human cytomegalovirus (HCMV) infection. They were found to be reactive with immediate early protein of HCMV in the nuclei of infected fibroblasts, as early as 3 hours post-infection. By radioimmunoprecipitation, CIE-1 was found to react with a protein with an apparent molecular weight of 70,000, whereas CIE-2 precipitated 2 proteins of 70,000 and 72,000 daltons, respectively. Both monoclonal antibodies recognized three prototype strains of HCMV: AD-169, Towne, and Davis, and did not cross-react with other human herpes viruses. CIE-1 and CIE-2 were compared with four commercial anti-HCMV monoclonal antibodies (Clonab, Dupont, Sera-Lab and Syva) by testing 88 clinical isolates. Culture confirmation tests and shell vial assays showed that CIE-1 and CIE-2 were more sensitive than several of these reagents and equally sensitive to the Dupont reagent. Moreover, CIE-1 and CIE-2 produced a bright, sharp staining of the nuclei of infected cells. These monoclonal antibodies should thus be valuable in rapid diagnosis of HCMV.

INTRODUCTION and in congenitally is for individuals receiving such as and in Conventional antineoplastic therapy, and patients with acquired immunodeficiency syndrome methods for the detection of HCMV infections usually depend on viral isolation in human fibroblasts. Four weeks or more may be required before characteristic cytopathic effect (CPE) can be observed because of the low infectivity of many clinical specimens and because of the slow replicative cycle of HCMV. More rapid

mortality (HCMV) responsible significant morbidity Human cytomegalovirus immunocompromised patients infants, allograft recipients, (3,19,21). infected

'National Laboratory for Immunology, Laboratory Center for Disease Control, Ottawa, Ontario, Canada, Kl A 0L2; department of Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ontario, Canada, K1H 8M5; 3ADI Diagnostics Inc., Rexdale, Ontario, Canada, M9W 4Z7; ""Regional Virology Laboratory, Children's Hospital of Eastern Ontario, Ottawa, Ontario, Canada, K1H 8L1. 61


and sensitive diagnostic tests for HCMV infections are needed, since passive immunization and antivirals are increasingly being used for the prevention or therapy of HCMV infections in transplanted patients. Spin amplification prior to short-term culture followed by the detection of early nuclear antigens (EA), or shell vial assay (5,13,16), represents one of the rapid diagnostic methods for the identification of HCMV, and is widely used in clinical virology laboratories. The sensitivity of the assay has been shown to vary depending on the monoclonal antibody (MAb) used, the type of viral specimen being tested and the duration of the shell vial incubation (5,10,20,26). To increase the sensitivity of this assay, monoclonal antibodies (MAbs) specific to early or immediate-early proteins should be selected on the basis of maximal sensitivity and ease of detection with a wide range of HCMV isolates. We describe the production and characterization of two MAbs, CIE-1 and CIE-2, specific for the immediate early antigens (IEA) of HCMV. We evaluate their performance as diagnostic reagents in the shell vial assay for the early detection of HCMV, and compare them with commercially available MAbs.

MATERIALS AND METHODS Human diploid cells and viruses. Human foreskin fibroblast cells (HFF) and human embryo lung fibroblast cells (HEL) were used throughout this study. Fibroblasts were grown in basal medium Eagle modified (BME; Gibco Laboratories, Grand Island, NY) or Eagle's minimal essential medium (MEM; Gibco), supplemented with 2 or 10% heat-inactivated fetal calf serum (FCS; Gibco), 2 mM L-glutamine, and 20 |xg/ml gentamicin. The Towne and Davis prototype strains were obtained from the culture collection of the Laboratory Centre for Disease Control (Health and Welfare, Ottawa, Canada). Cell-free stocks of the prototype strains were prepared as previously described (17). Commercial slides of varicella-zoster virus (VZV) and Epstein-Barr virus (EBV) infected cells were obtained from Electro-nucleonics (Columbia, MD). Slides of herpes simplex virus (HSV), respiratory syncytial virus (RSV), measles virus, and Toxoplasma gondii were obtained from Gull Laboratories (Salt Lake City, UT). Virus Titration. HCMV infection was carried out in 24-well tissue culture plates and the titration performed using a standard plaque assay (18) with the following modifications. Confluent HFF cells were infected in quadruplicate and overlaid 1 h later with 0.5% agar (Oxoid) in BME supplemented with 5% FCS. Eight to ten days post-infection (p.i.) a neutral red solution (1:10,000) was added. Following overnight incubation, plaques were counted at 40x magnification. The AD-169 reference stock had a titer of approximately 1 X 106PFU/ml. Immunization of mice and monoclonal antibody production. Two HCMV antigen preparations were used successfully for the immunization of Balb/c mice. The first antigen preparation was obtained by infection of a confluent fibroblast monolayer at a high multiplicity of infection (MOI) with the AD-169 prototype strain. In order to increase the yield of IEA, a second preparation was obtained from infected HFF cells treated with viral translation and transcription inhibitors. Briefly, infected cells were cultivated for 3 h in the presence of 50 u-g/ml cycloheximide (Sigma Chemical Company, St. Louis, MO) which inhibited the translation of IE transcripts. The cycloheximide was then removed and the cells were cultivated for a further 40 h with 10 |xg/ml actinomycin D (Sigma). This inhibitor allowed the accumulation of IE proteins, but inhibited the transcription of EA proteins (6). MAb CL-1 was routinely used to monitor the efficacy of drug treatment in preventing the synthesis of late viral proteins. This MAb is directed against the gp55/130 complex, a late HCMV antigen, and produced a strong fluorescence throughout the cytoplasm of infected cells from 24 h onwards (17). Both antigen preparations were sonicated and clarified by low speed centrifugation. Mice received intraperitoneal injections of 50 p-g of protein in complete Freund's adjuvant, followed by two injections in incomplete Freund's adjuvant, at weekly intervals, and an intravenous injection 3 to 4 days before fusion. Spleen cells from two mice showing the presence of specific antibodies to nuclear antigens in their sera, as detected by an indirect immunofluorescence assay (IFA), were fused with the SP2/0 myeloma cell line as previously described (7). Hybridomas were screened by IFA on HCMV-infected and uninfected HFF cells. Hybridomas secreting MAbs reacting with the nucleus of infected cells only were selected for further 62


characterization. These specific hybrids were cloned by sequential limiting dilutions, expanded, and frozen in liquid nitrogen. Ascitic fluid was produced in Fl hybrid mice (Swiss Webster x Balb/c) as described (1). The class, subclass, and light chain type of each MAb were determined (11). Clinical isolates. Twenty-six fresh HCMV isolates (most from urine samples from heart and renal transplant patients) showing typical CPE in conventional tube culture, were obtained from the Regional Virology Laboratory in Ottawa. These clinical isolates are referred to as "fresh isolates-Ottawa". Twentyeight clinical isolates frozen at first pass level since 1989 ("sub-cultures-Ottawa") and eight HCMV-positive frozen urine samples ("frozen urines-Ottawa) were obtained from the same laboratory. The presence of HCMV was reconfirmed in conventional cell cultures in the "sub-cultures" but not in the frozen urine samples. One autopsy specimen, positive in conventional culture, was a direct smear of lung tissue and is referred to as "lung smear-Ottawa". Fifteen clinical isolates were also obtained from the Department of Microbiology, Laval University, Quebec. These isolates, collected between 1984 and 1987, had been sub-cultured 2 to 8 times before being used in this study. They are referred to as "sub-cultures-Quebec". Finally, 10 clinical isolates, already fixed in shell vials, were obtained from the Wellesley Hospital in Toronto. Of these, 6 were bronchoalveolar lavages (BAL), 2 were urine samples, 1 was a stomach mucosa biopsy, and 1 was a throat wash. These are referred to as "fresh isolâtes-Toronto." Conventional cell cultures were examined 3 times per week over a 4-week period for the appearance of CPE. Commercial monoclonal antibodies and conjugates. For comparison purposes, commercial M Abs used in this study were diluted in 1% BSA (w/v) and 0.004% Evans blue (final concentration) unless otherwise stated. Sera-Lab MAb E-13 specific to an IEA of 68,000 daltons (Sera-Lab, Cedarlane Laboratories, Hornby, Ont.) was used at a dilution of 1/500 or 1/1000. Dupont chromatographically purified MAb specific to IEA (Product no. 9221, mfg. by Biotech Research Inc. for Dupont Specialty Diagnostics, Billerica, MA), was used at a 1/20 dilution. Clonab reagent specific to an IEA of 70,000 daltons (pool of MAbs C10 and Cl 1; Biotest Diagnostics, Dreieich, W. Germany, obtained through Gelman, Montreal, Que.) was used at a 1/5 dilution. Reagent 1 of Syva Microtrak CMV Culture Identification Test (Syva Company, Palo Alto, CA), containing two MAbs, one specific to an IEA of 72,000 daltons and the second to an EA of 50,000 daltons, was supplied ready to use in protein-stabilized buffer and Evans blue counterstain. Affinity-purified goat anti-mouse immunoglobulins (IgA + IgG + IgM, heavy and light chain specific) conjugated to FITC were obtained from Cappel (Organon Teknica Corp., West Chester, PA) and were used at a 1/100 dilution. This conjugate was used for all MAbs in this study, except for the Syva kit which was

supplied with its own ready-to-use conjugate.

Indirect immunofluorescence assay. The IFA was carried out as described (24) with slight modifications. Briefly, HFF cells grown on multitest slides (ICN Biochemicals' Inc, CA) were infected with AD-169 at a MOI of 0.5. Approximately 5 days post-infection (p.i.), the cells were fixed in ice cold acetone for 10 min, air-dried, and stored at —70CC until use for screening hybridomas. Conventional tube cultures of HEL cells were infected with HCMV clinical isolates from Ottawa by spin amplification as described (14). Clinical isolates from Quebec were passaged once on HFF cells grown in 25 cm2 flasks. At + 1 to +4 CPE, the infected cells were trypsinized, dispensed on multitest slides, and incubated for 90 min. at 37°C. The slides were then fixed and stored at 70°C until use for characterization of the hybridomas. IFA staining incubation steps were carried out at 37CC for 30 min. IFA microscopy was carried out by epifluorescence using a Leitz UV microscope, model Ortholux II, fitted with a standard FITC filter set and Fluotar 25x and 40x —


Shell vial cultures. Early detection of HCMV antigen in shell vial cultures was performed as described by Gleavesetal. (4), and Lautenschlager et al. (9). Shell vials (Allied Corporation, Fisher Scientific, Pittsburgh, PA) containing sterile round cover slips (12 mm diameter), were seeded with 2.5 x 104 HEL cells, and incubated for 3 days at 37°C. Following inoculation with 300 to 500 |xl of clinical specimen, shell vials were centrifuged at 800X g for 40 min. The inoculum was replaced with 1 ml of maintenance medium consisting of MEM supplemented with 2% FCS. After incubation for 3 days at 37°C in the presence of 5% C02, the medium was removed by aspiration. The cover slips were rinsed twice with PBS, once with acetone, and then fixed in acetone for 10 min at room temperature. The cover slips were rinsed once with PBS before incubation with 150 (xlof M Ab for 30 min at 37°C, and further processed as described for IFA. A positive result consisted of the observation of one or more positively fluorescing nuclei. 63


Radioimmunoprecipitation assay (RIPA). HCMV viral proteins were labelled as described previously (8) with the following modifications. HFF cells in 75 cm2 flasks were infected with AD-169 at an MOI of 0.1. When CPE reached 75%, the cells were radiolabelled for 18 h with 35S-methionine: L-cysteine (30 u.Ci/ml)(sp. act. 1037 Ci/mmol; ICN) in methionine-free medium containing 0.1% dialysed FCS. Radiolabelled infected and mock-infected cells were extracted in Tris-HCl lysis buffer pH 8.0 (50 mM Tris, 150 niM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 100 u.g/ml phenylmethylsulfonylfluoride (PMSF; Sigma), and 2 p-g/ml aprotinin (Sigma) for 30 min on ice. The infected and mock-infected cell lysates were then clarified by centrifugation for 1 min at 10,000 rpm. The supernatants

stored at —70°C in 200 p.1 aliquots. Unless otherwise specified, incubation steps were performed at 4°C. Immunoprecipitation of labelled antigen preparations involved a preclearing step with 2% normal rabbit serum. Cleared labelled antigens (5 x 106 cpm) were mixed with 50 p.1 of ascitic fluid. After 3 h of incubation, 20 p.1 of a 10% suspension of protein G Sepharose beads (Pharmacia Fine Chemicals, Sweden), previously adsorbed for 2 h with 10% BSA (w/v), was added to the antigen-antibody solution, and allowed to react an additional hour with constant mixing. The protein G-antibody-antigen complexes were then pelleted by centrifugation and washed 3 times in Tris HCl-buffer pH 8.0 (10 mM Tris, 140 mM NaCl, 0.1% NP40 and 100 u.g/ml PMSF). The antigens were electrophoresed under reducing conditions on a 10% SDS-polyacrylamide resolving gel using the Mini Protean system (Bio-Rad). The gel was fixed, then washed successively in dimethyl-sulfoxide (DMSO), in DMSO containing 22% PPO (2.5-Diphenyloxazole; Fisher), and in distilled water. The gel was dried and exposed at —70°C for 24 h to X-ray film (Cronex film; Dupont, Wilmington, DE) in a Kodak X-omatic cassette fitted with intensifying screens. Standard proteins labelled with ,4C (BRL, Gaithersburg, MD) were used as molecular weight markers. Neutralizing activity of MAbs. Neutralization assays were performed in 24-well tissue culture plates by a plaque reduction assay (18) with the following modifications. Ascitic fluids were mixed with an equal volume of cell-free HCMV (AD-169)-stock suspension, containing 100 to 150 PFU and 10% normal fresh-frozen rabbit serum as a source of complement. After 2 h at 37°C, 150 p.1 of the virus-antibodycomplement mixture was added in quadruplicate to confluent HFF cells and incubated for an additional hour at 37°C. HCMV neutralizing antibody titers were expressed as the highest dilution of antibody producing a 50% or greater reduction in plaque counts, compared with controls in which the antibody was replaced by medium. The highly neutralizing murine MAb CL-1 was used as positive control. Purified CL-1 (3.7 mg/ml) has a neutralizing titer greater than 1:128,000 in the presence of complement. were

RESULTS Characterization of MAbs CIE-1 and CIE-2. The immunoglobulin class, subclass and light chain type of the two MAbs were determined. CIE-1 is an IgG,« and CIE-2 is an IgG2aK. Both MAbs recognized the 3 prototype strains of HCMV, AD-169, Towne and Davis. No reactivity was observed in cells infected with HSV type 1 or 2, VZV, EB V, RSV, measles virus or T. gondii or with uninfected human fibroblasts. None of the MAbs, either alone or as a pool, showed neutralizing activity. Kinetic studies and metabolic inhibitors. When ascitic fluids, diluted 1:1000 for CIE-1 and 1:650 for CIE-2, were reacted with untreated HCMV infected cells, the typical fluorescence pattern for IEA and EA was observed, with homogeneous nuclear staining (Fig. 1). To extend this observation, kinetic studies were performed with infected cells fixed at 1, 2, 3, 6, 8, 12, 18, 24, 48 h and 7 days p.i. These studies showed that the antigens recognized by the MAbs appeared as early as 3 h p.i., and persisted in the nuclei of infected cells throughout the infectious cycle. At 3 h p.i., the fluorescence was very faint, consisting of dots inside the nucleus (Fig. 1, arrow). The fluorescence gradually became stronger and more uniform, reaching its maximum at 18 h p.i., and remaining constant thereafter. In order to define more precisely the temporal class of viral proteins recognized by MAbs CIE-1 and CIE-2, transcription and translation inhibitors were used. Cells expressing HCMV IEA were produced as described for the immunizing preparation. Cells expressing IEA and EA were produced by culturing HFF cells for 48 h in the presence of 75 p-g/ml cytosine arabinoside, an inhibitor of DNA polymerase, suppressing the expression 64


FIG. 1. Indirect immunofluorescence photomicrograph of human foreskin fibroblasts infected with HCMV strain AD-169 for 3 days and reacted with CIE-1 MAb. Arrow indicates a pattern of immunofluorescence similar to the pattern observed when the cells are infected for 3 h (magnification, x250).

of late antigen (6). The fluorescence obtained with cycloheximide and actinomycin D-treated cells was similar to but weaker than that obtained with untreated infected cells. Cytosine arabinoside-treated cells produced the same nuclear fluorescence as untreated infected cells (not shown). HCMV-infected cells treated with inhibitors to block the expression of late viral proteins were tested with late MAb CL-1. No cytoplasmic fluorescence could be observed in infected cells treated with cycloheximide and actinomycin D or cytosine arabinoside. Radioimmunoprecipitation. HCMV proteins recognized by each MAb were visualized by analyzing the immune complexes of CIE-1 and CIE-2 with radiolabelled HCMV polypeptides (Fig. 2). CIE-1 MAb recognized a viral antigen with an apparent molecular weight of 70,000 (lane 1 ). CIE-2 MAb immunoprecipitated two proteins with apparent molecular weights of 70,000 and 72,000 (lane 3). The 70,000 Da protein was also immunoprecipitated by the mouse anti-HCMV serum (lane 5). No proteins were immunoprecipitated in mock infected cell lysates (lanes 2, 4, and 6). Detection of HCMV clinical isolates. The efficiency of CIE-1 and CIE-2 for the detection of HCMV IE antigens was compared with that of different commercial MAbs. This was performed by IFA on HEL cells grown in conventional tube cultures (confirmatory assay) or in shell vials (pre-CPE test). Table 1 shows the reactivity of CIE-1, CIE-2, Clonab, Dupont, Sera-Lab and Syva MAbs against a variety of HCMV clinical isolates tested in conventional cell cultures. There was a complete agreement between the presence of typical CPE and IFA detection of HCMV using CIE-1 and CIE-2, whether the isolates originated from urine, autopsy specimen, or sub-cultures. Most isolates had a very low infectivity and produced only a small number of fluorescent nuclei. Syva, Sera-Lab and Dupont MAbs also showed complete agreement between CPE and IFA. The Clonab MAb did not detect three sub-cultures from Ottawa although typical CPE was observed. Table 2 illustrates the reactivity of the same MAbs in a pre-CPE test, against a variety of HCMV clinical isolates cultured for 3 days in shell vials. Each MAb failed to detect some isolates: CIE-1 (1/53), CIE-2 (2/53), Clonab (2/20), Dupont (1/35), Sera-Lab (4/22), and Syva (1/27). Only the groups of "fresh isolates" and "sub-cultures" were confirmed positive in conventional culture. The frozen urine samples inoculated in shell vials and the shell vials obtained from Toronto were not tested in parallel by conventional culture.












FIG. 2. Radioimmunoprecipitation of HCMV polypeptides. Human foreskin fibroblast cells were infected with HCMV (lanes l, 3 and 5) or mock-infected (lanes 2, 4 and 6). Lanes l and 2: MAb CIE-1 ; lanes 2 and 3: MAb CIE-2; lanes 5 and 6: mouse anti-HCMV serum. [I4C] standard proteins are identified with their kilodalton values on the left.

DISCUSSION In recent years, because of the availability of antiviral drugs and CMV hyperimmune globulins, there has been an increased demand for rapid diagnosis of HCMV infection in immunocompromised patients. Several reports have described the use of spin amplification in shell vials followed by the detection of HCM V-IEA and EA by IFA, using MAbs. The sensitivity of the assay has been shown to depend on the MAb, the type of specimens, and the incubation period for the shell vials (5,10,20,26). To optimize the shell vial assay, the selected MAbs should be specific to IE and/or E nuclear proteins and display a high sensitivity with a wide spectrum of isolates. The fluorescent nuclei should also be easily detectable. The MAbs CIE-1 and CIE-2 discussed in this paper reacted with nuclear viral antigen in cells infected with AD-169 as early as 3 h p.i., and as early as 16 h following infection with clinical specimens. Specific antigens appeared and persisted in the nucleus for up to 7 days post-infection without migration into the cytoplasm. The reactivity of MAbs CIE-1 and CIE-2 with the nuclei of cycloheximide and actinomycin D-treated infected cells was also indicative of the IE nature of the antigens recognized. Indeed, by radioimmunoprecipitation Table 1. Reactivity




Clinical Isolates


Conventional Cell Culture














7/7 6/9

7/7 9/9 1/1


7/7 12/12 1/1 35/35



7/7 9/9 nd 29/29

6/6 12/12 1/1 30/30







Quebec Sub-cultures Ottawa Fresh isolates" Sub-cultures


12/12 1/1




aMostly urine specimens. bNot determined.


MAb DETECTION OF CMV Table 2. Reactivity of MAbs

Toronto Fresh isolates Ottawa Fresh isolates Sub-cultures Frozen urines Total


Clinical Isolates

by the

Shell Vial Assay










19/19 16/16 7/8 52/53

19/19 16/16 8/8 51/53

13/15 5/5 nd 18/20

14/14 9/10 1/1 34/35

12/16 5/5 1/1 18/22

5/6 26/27










9/9 11/11

"Not determined. were shown to react with a protein of apparent molecular weight of 70,000, the immediate to major early protein of the AD-169 strain (12). Therefore, CIE-1 and CIE-2 are corresponding IE to present proteins exclusively in the nuclei of HCMV-infected cells. specific Furthermore, CIE-1 and CIE-2 MAbs were shown to recognize all three prototype strains of HCMV. They did not react with uninfected cells or with cells infected with other human herpesviruses, measles, RSV, or T.


(RIPA), both MAbs

gondii. Randazzo and Michalski (16) recommended that new HCMV diagnostic reagents should be evaluated with high-titer stocks of HCMV as well as low-titer clinical specimens, to determine their ability to detect small amounts of virus. In the present study, both MAbs were tested against a panel of clinical isolates (many of which were of low titer), and were compared with 4 different commercial MAbs for the early detection of HCMV antigens. CIE-1 and CIE-2 performed at least as well as the other MAbs in terms of the number of positive specimens detected by conventional cultures. Indeed, all MAbs, except the Clonab reagent, detected all clinical isolates in this confirmatory assay (Table 1). Shell vials were inoculated 3 days after seeding since it has been shown (22) that fibroblast cells in shell vials are more susceptible to HCMV infection if used before day 11. Shell vials were processed at 3 days p.i. for increased sensitivity (9). Results showed that CIE-1 and CIE-2 MAbs performed at least as well as commercially available MAbs (Table 2). Some of the false negative results may be related to the low infectivity of several clinical isolates, as well as to the random distribution of infective particles and/or infected cells in the different aliquots of the same specimens. Furthermore, clinical samples containing low concentrations of virus may have failed to produce detectable amounts of early antigens within short incubation periods (26). In the latter study, 43% of urine samples negative for the early antigen of HCMV showed characteristic CPE as late as 4 to 6 weeks after inoculation in conventional cell cultures. In our study, CIE-2 failed to detect two isolates from Toronto while CIE-1 and Syva reagents each missed one strain in the "Ottawa frozen urines" group. The infectivity of these samples probably was low, as suggested by the low

number of fluorescent nuclei observed when stained with the other MAbs. On the other hand, because of minor genomic and antigenic differences among HCMV strains (2), different MAbs may detect different HCMV isolates. Failure of one single MAb to detect all HCMV strains has been reported previously (15), and combinations of IEA and/or EA MAbs have been used (4,20,23) to provide increased sensitivity. The Syva and Clonab reagents used in this study each contained two MAbs. Since the isolates that CIE-1 and CIE-2 failed to detect were different, it is possible that a pool of CIE-1 and CIE-2 might have detected all the clinical strains tested. Interestingly, differences between MAbs were more pronounced when we compared their fluorescence patterns. CIE-1 and CIE-2 gave a strictly nuclear bright fluorescence similar to that obtained with the Dupont MAb. The Syva reagent produced a fluorescence that was often non-typical with nuclear and diffused cytoplasmic staining. This could result in false negative readings, particularly in samples with low infectivity. Similar results were reported by Woods et al. (25). The Sera-Lab and the Clonab MAbs often displayed a weak fluorescence; this may be the reason for the false negative results obtained with these reagents (Tables 1 and 2). Other investigators in England (D. Pillay, H. Charman, J. Lok and P.D. Griffiths, Dept. of Virology, 67


Royal Free Hospital, School of Medicine, London NW3, U.K., Third International Cytomegalovirus Workshop, 1991, Bologna, Italy, Abstract page 80) have used the shell vial assay to prospectively compare the performance of a pool of CIE-1 and CIE-2 with that of another IEA MAb, in 1500 consecutive clinical samples from a wide range of sources. The sensitivity of the pool was 90% versus 89% for the control IEA MAb. There was no evidence of false positive signals. Significantly, the fluorescence pattern obtained with the CIE- l/CIE-2 pool was brighter and easier to read. In conclusion CIE-1 and CIE-2 MAbs appear to be excellent candidates for the early detection of HCMV infections by IFA, since the typical shape and size of the brightly stained nuclei are preserved and provide a rapid differentiation from non-specific staining. In that regard, they are comparable to the Dupont reagent and have similar sensitivities. Work is in progress to optimize the shell vial assay using MAbs CIE-1 and CIE-2 as a pool and to evaluate them with larger numbers of HCMV-positive and negative specimens from different sources.

REFERENCES 1. Brodeur, B.R. and P.S.




High yield monoclonal antibody production in ascites.

2. Chou, S. 1990. Differentiation of cytomegalovirus strains clinical specimens. J. Infect. Dis. 162:738-742.

J. Immunol. Methods

by restriction analysis of DNA sequences amplified from

3. Drew, L. 1988. 4. 5.

Cytomegalovirus infection in patients with AIDS. J. Infect. Dis. 158:449-456. Gleaves, C.A., E.C. Reed, R.C. Hackman, and J.D. Meyers. 1987. Rapid diagnosis of invasive cytomegalovirus infection by examination of tissue specimens in centrifugation culture. Am. J. Clin. Pathol. 88:354-358. Gleaves, C.A., T.F. Smith, E.A. Shuster, and G.R. Pearson. 1984. Rapid detection of cytomegalovirus in MRC-5 cells inoculated with urine specimens by using low-speed centrifugation and monoclonal antibody to an early antigen. J. Clin. Microbiol. 19:917-919.

6. Griffiths, P.D. and J.E. 241:313-324.


1987. Molecular






Biochem. J.

7. Hamel, J., B.R. Brodeur, Y. Larose, P.S. Tsang, A. Belmaaza, and S. Montplaisir. 1987. A monoclonal antibody directed against a serotype-specific outer-membrane protein of H'aemophilus influenzae type b. J. Med. Microbiol. 23:163-170.

8. Larose, Y., E.S. Tackaberry, and B.R. Brodeur. 1991. Human monoclonal antibodies to cytomegalovirus recognize viral epitopes on the surface of virus-infected cells. Human Antibodies Hybridomas 2:143-149. 9.

Lautenschlager, I., J. Suni, J. Ahonen, C. Grönhagen-Riska, P. Ruutu, T. Ruutu, and P. Tukiainen. 1989. Detection of cytomegalovirus by the early-antigen immunofluorescence test versus conventional tissue culture. Eur. J. Clin. Microbiol. Infect. Dis. 8:610-613.

10. Lucas, G., J.M. Seigneurin, J. Tamalet, S. Michelson, M. Baccard, J.F. Delagneau, and P. Deletoile. 1989. Rapid diagnosis of cytomegalovirus by indirect immunofluorescence assay with monoclonal antibody F6b in a commercially available kit. J. Clin. Microbiol. 27:367-369. 11.


Lussier, M., B.R. Brodeur, and S. Winston. 1989. Detection of Neisseriagonorrhoeae by dot-enzyme immunoassay

using monoclonal antibodies. J. Immunoassay 10:373-394. Mach, M., T. Stamminger, and G. Jahn. 1989. Human cytomegalovirus: recent aspects from molecular biology. J.

Gen. Virol. 70:3117-3146.

13. Martin, W.J. and T.F. Smith. 1986. Rapid detection of cytomegalovirus in bronchoalveolar lavage monoclonal antibody method. J. Clin. Microbiol. 23:1006-1008.

specimens by a

14. Miller, H., B. McCulloch, M.P. Landini, and E. Rossier. 1989. Comparison of immunoblotting with other serological methods and virus isolation for the early detection of primary cytomegalovirus infection in allograft recipients. J. Clin. Microbiol. 27:2672-2677. 15.

Popow-Kraupp, T. and C. Kunz. 1988. Detection of cytomegalovirus in clinical specimens by virus isolation and by monoclonal antibody against the early nuclear antigen. J. Med. Virol. 24:275-282.



MAb DETECTION OF CMV 16. Randazzo, D.N. andF.J. Michalski. 1988. Microbiol. 26:369-370.

Comparison of antibodies for rapid detection of cytomegalovirus. J. Clin.

17. Rossier, E., K. Dimock, D. Taylor, Y. Larose, P.H. Phipps, and B.R. Brodeur. 1987. Sensitivity and specificity of enzyme immunofiltration and DNA hybridization for the detection of HCMV-infected cells. J. Virol. Methods 15:109-120. 18. Schmidt, N.J., J. Dennis, and E.H. Lenett. 1976. Plaque reduction neutralization test for human cytomegalovirus based upon enhanced uptake of neutral red by virus-infected cells. J. Clin. Microbiol. 4:61-66. 19. Stagno, S., R.F. Pass, M.E. Dworsky, R.E. Henderson, E.G. Moore, P.D. Walton, and CA. Alford. 1982. Congenital cytomegalovirus infection: the relative importance of primary and recurrent maternal infection. N. Engl. J. Med. 306:945-949. 20. Stirk, P.R. andP.D. Griffiths. 1987. Use of monoclonal antibodies for the diagnosis of cytomegalovirus infection the detection of early antigen fluorescent foci (DEAFF) in cell culture. J. Med. Virol. 21:329-337.


21. Sullivan, J.L. and J.B. Hanshaw. 1982. Human cytomegalovirus infections, pp. 57-83 In: R. Glaser, T. Gotlieb-Stematsky eds: Human Herpesvirus Infections: Clinical Aspects, Marcel Dekker, New York. 22.

Thiele, G.M., M.S. Bicak, A. Young, J. Kinsey, R.J. White, and D.T. Purtilo. 1987. Rapid detection of cytomegalovirus by tissue culture, centrifugation, and immunofluorescence with a monoclonal antibody to an early nuclear antigen. J. Virol. Methods 16:327-338.


der Bij, W., R. Torensma, W.J. van Son, J. Anema, J. Schirm, A.M. Tegzess, and T.H. The. 1988. Rapid immunodiagnosis of active cytomegalovirus infection by monoclonal antibody staining of blood leukocytes. J. Med.


Virol. 25:179-188. 24. 25.


Wiley, J.A., Brodeur, B.R., Dimock, K.D. and Sattar, S.A. 1990. Neutralizing monoclonal antibody against Enterovirus-70 reacts with viral proteins 1C and ID. Viral Immunol. 3:137-146. Woods, G.L., A.M. Johnson, and G.M. Thiele. 1990. Clinical comparison of two assays for rapid detection of cytomegalovirus early nuclear antigen. Am J. Clin. Pathol. 93:373-377. Zweygberg Wirgart, B., M. Landqvist, I. Hökeberg, B.M. Eriksson, E. Olding-Stenkvist, and L. Grillner. 1990. Early detection of cytomegalovirus by a new monoclonal antibody, CCH2. J. Virol. Methods 27:211-220. Address reprint requests to: Bernard R. Brodeur, Ph.D., Chief National Laboratory for Immunology, Laboratory Centre for Disease Control,

Tunney's Pasture, Ottawa, Ontario, Canada, K1A 012.


New monoclonal antibodies for the detection of immediate early antigens of cytomegalovirus.

Two new monoclonal antibodies, CIE-1 and CIE-2, were developed for the rapid detection of human cytomegalovirus (HCMV) infection. They were found to b...
3MB Sizes 0 Downloads 0 Views