Arlo D. Wold

Monoclonal Antibody for Rapid Laboratory Detection of Cytomegalovirus Infections: Characterization and Diagnostic Application

Monoclonal antibodies to early (2H2.4, molecular weight 72,000 daltons) and late (2F3.0, molecular weight 68,000 daltons) antigens of the AD-169 strain of cytomegalovirus (CMV) were prepared by fusing mouse spleen cells with NS-1 mouse myeloma cells. The 2H2.4 monoclonal antibody produced a dense immunofluorescence with prominent lobular staining within the nucleus of CMV-infected substrate cells, whereas the reaction of 2F3.0 was more diffuse and generally involved the entire nucleus of the cells. Both monoclonal antibodies had little or no neutralizing activity against CMV in plaque-reduction assays. No cross-reactions were observed between these monoclonal antibodies and other members of the herpesvirus group. The 2H2.4 monoclonal antibody to early CMV antigen was used in a shell vial assay with a low-speed centrifugation step for the rapid (within 16 hours after inoculation) diagnosis of CMV infections. Optimal conditions for the test included centrifugation of shell vials at 700 X g for 45 minutes at 36 degrees C. An inoculum volume of 0.2 ml provided a reasonable balance between the optimal sensitivity for detecting specific viral fluorescence and the easy discrimination of the specific immunofluorescence from the background debris. Because of the commercial availability of the monoclonal antibody and the simplicity of the procedures used in the shell vial assay and subsequent fluorescence techniques, this rapid assay can be done in any laboratory that is familiar with cell culture manipulations.

Detection of Herpes Simplex Virus DNA in Genital and Dermal Specimens by LightCycler PCR after Extraction using the IsoQuick, MagNA Pure, and BioRobot 9604 Methods

ABSTRACT We evaluated two automated systems, MagNA Pure (Roche Molecular Biochemicals, Indianapolis, Ind.) and BioRobot 9604 (Qiagen, Inc., Chatsworth, Calif.) as effective replacements for the manual IsoQuick method (Orca Research, Inc., Bothell, Wash.) for extraction of herpes simplex virus (HSV) DNA from dermal and genital tract specimens prior to analysis by LightCycler PCR. Of 198 specimens (152 genital, 46 dermal), 92 (46.2%) were positive for HSV DNA by LightCycler PCR after automated extraction of specimens with either the MagNA Pure or BioRobot 9604 instrument. The manual IsoQuick method yielded HSV DNA (total n = 95) from three additional specimens that were negative by the automated method (P = 0.25, sign test). Although the mean numbers of LightCycler PCR cycles required to reach positivity differed statistically significantly among all three of the methods of extraction, the estimated means differed by no more than 1.5 cycles (P < 0.05). Seventy (76%) of the 92 specimens that were LightCycler PCR positive by all three extraction methods were also positive by shell vial cell culture assay. HSV DNA was detected by a lower LightCycler PCR cycle number (26.1 cycles) in specimens culture positive for the virus than in culture-negative samples (33.3 cycles) (P < 0.0001). The manual IsoQuick and automated MagNA Pure and BioRobot 9604 methods provide standardized, reproducible extraction of HSV DNA for LightCycler PCR. The decision to implement a manual versus an automated procedure depends on factors such as costs related to the number of specimens processed rather than on the minimal differences in the technical efficiency of extraction of nucleic acids among these methods.

Bacitracin differentiation for presumptive identification of group A β-hemolytic streptococci: Comparison of primary and purified plate testing

The detection of group A beta-hemolytic streptococci by bacitracin susceptibility was examined, with the presumptive identification on primary blood agar plates compared with that on pure subcultured plates. A total of 638 group A streptococcus isolates was recovered from 4,632 throat swab specimens. Only about half of these isolates were identifiable on the primary plate, compared with 96% identifiable on pure subcultured plates. The unreliable results with bacitracin disk testing on primary plates were due to (1) too few colonies present in the vicinity of the disk or (2) colonies overgrown by oropharyngeal flora.

Development, Implementation, and Optimization of LightCycler PCR Assays for Detection of Herpes Simplex Virus and Varicella-Zoster Virus from Clinical Specimens

For over 35 years, viruses from clinical specimens have been detected by cell culture techniques; this basic method has been the “gold standard” for the laboratory diagnosis of virus infections [1–4]. Conventionally, specimens were inoculated into cell cultures seeded in tubes; many groups of viruses were recognized by characteristic cytopathic effects induced generally after several days of incubation. In the early 1980s, the incubation time for detection of viruses was significantly reduced by introduction of the shell vial cell culture assay in which early antigens of the virus were detected with monoclonal antibodies after centrifugation and incubation of the vessels for 1–3 days [5, 6].

Changing Trends of Diagnostic Virology in a Tertiary Care Medical Center

Mayo Clinic (MC) has 290,000 new patient registrations each year in a multidiscipline tertiary care practice located in a small community with a population of 75,000. Many of the patients are immunosuppressed such as those undergoing treatment for neoplastic or rheumatologic diseases. Others are admitted with acquired immunodeficiency syndrome (AIDS) or receive organ transplants (cornea, kidney, bone marrow, liver, pancreas, cardiothoracic) in a program that has involved 2,362 procedures. The increasing numbers of immunosuppressed patients, compared to 10–15 years ago, has had a profound effect on both the frequency and types of viruses recovered in the clinical laboratory. This communication compares the detection of viruses during a period from 1974–1982 with our experience in 1988.

Monoclonal antibodies for rapid detection of viral antigens in shell vials

Diagnosis of Cytomegalovirus Infections and Development of Monocional Antibodies Cytomegalovirus (CMV) is a common infection that can cause serious disease in infants who have acquired the virus in utero, in patients immunosuppressed for organ transplantation, and in other individuals who receive chemotherapy or high levels of corticosteroids for treatment of neoplastic and other medical processes. Only 4 years ago, the standard method for detecting CMV infection in the laboratory was the inoculation of diploid fibroblast cell cultures in tubes with subsequent identification of typical cytopathic effects that required an average time period of 7 8 days for recognition (3). Immunologic methods for rapid detection of CMV in clinical specimens waned for lack of high quality homotypic antisera that could produce sensitive and specific results and, importantly, be commercially available. Subsequently, two monoclonal antibodies were prepared against an early and a late antigen of the ADI69 strain of CMV by Shuster et al. at the Mayo Clinic. These monoclonal antibodies provided the key to the development of a rapid test for detection of the virus (10). Since 1973, we have used shell vials to diagnose Chlamydia trachomatis infections. This bacterium is .only 3 4 times larger than the CMV virion; nevertheless, centrifugation of the specimen inoculum at low speed (700 x g) has been shown to enhance the rate and ultimate infectivity of this organism and viruses in susceptible cell cultures. Because we wanted to be able to detect CMV as rapidly as possible after inoculation of cell cultures in the laboratory, we used the monoclonal antibody prepared to an early antigen (72,000 d) to assay for the viral components synthesized after 16 hr incubation by the indirect immunofluorescence test (monoclonal antibody available from DuPont Specialty Diagnostics, Wilmington, DE).