Detection of genital herpes simplex infections by a tissue culture-fluorescent-antibody technique with biotin-avidin

Abstract

Several cell lines were evaluated for their suitability for rapid detection of herpes simplex virus (HSV) from clinical genital specimens. Human foreskin fibroblast (Flow 7000) cells were found to be most suitable in terms of sensitivity and adherence characteristics. HSV in clinical specimens was isolated by a standard tissue culture method by monitoring the cytopathic effect, and the titers of the HSV-positive specimens were determined. More than 65% of the HSV-positive genital specimens showed titers of less than or equal to 10(4) 50% tissue culture infective doses per ml. The standard tissue culture-cytopathic effect method required 3 to 10 days for detection of HSV in clinical specimens of low infectivity. A more rapid technique was developed which involved a short-term tissue culture (24 h) on Lab-Tek chambers followed by staining with biotin-linked HSV antibody and avidin-fluorescein conjugate. Because of the high binding affinity of this system due to multiple binding of biotin to avidin and multiple attachment of biotin to the antibody molecule, the biotin-avidin fluorescent-antibody technique produced a quality of fluorescence far superior to that of the conventional fluorescent-antibody techniques. The tissue culture-biotin-avidin fluorescent-antibody method was as sensitive as the tissue culture-cytopathic effect test. This method provides an improved, more rapid test (26 h) for detecting HSV in clinical specimens.

Rapid detection of herpes simplex virus in clinical specimens by use of a capture biotin-streptavidin enzyme-linked immunosorbent assay

“…The tissue culture method is generally considered standard, but it requires up to 7 days for completion. We have reported that short-term tissue culture followed by staining is as sensitive as and more rapid (24 h) than tissue culture alone (12,13). Direct staining of tissue smears is rapid, but not as sensitive or specific as tissue culture (13), and electron microscopy is rapid, but less available and less sensitive than tissue culture (9).…”

“…Some of the methods currently available to detect HSV include conventional tissue culture techniques (15), shortterm tissue culture followed by staining (12,13) or electron microscopy (9), and direct staining of tissue smears taken from the lesions of the patient (3,7,17,18). The tissue culture method is generally considered standard, but it requires up to 7 days for completion.…”

“…(Parts of this work were presented at the Second Annual Meeting of the Society for Virology, East Lansing, Mich., [10][11][12][13][14] July 1983, and at the Workshop on New and Useful Methods in Rapid Viral Diagnosis, organized by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and Pan American Group for Rapid Viral Diagnosis in Bethesda, Md., 29-30 September 1983. ) MATERIALS AND METHODS Preparation of stock virus.…”

“…Determination of infectivity of HSV. The infectivity of HSV in stock virus preparations and in clinical specimens was determined as described previously (12). In brief, Flow 7000 monolayer cultures were infected with 0.2-ml volumes of serial 10-fold dilutions of the virus preparations and observed for cytopathic effects.…”

Microbiological Quality Control for Laboratory Rodents and Lagomorphs

“…Protein A and protein G, derived from the cell wall of Staphylococcus aureus and certain streptococci, respectively, bind certain IgG subclasses from various species and thus can sometimes be substituted for species-specific secondary antibodies (DeLellis, 1981;Hrapchak, 1980;Mahoney and Chernesky, 1999). Other popular modifications of the indirect method that amplify signal by increasing the concentration of enzyme bound to the solid phase are the peroxidaseantiperoxidase (PAP) and avidin-biotin-enzyme complex (ABC) techniques (Hsu et al, 1980;Milios and Leong, 1988;Nerurkar et al, 1982). The ABC system makes use of the strong interaction between avidin, an egg-white protein, or streptavidin from Streptomyces avidinii, and the low-molecular-weight vitamin biotin coupled to antibodies and enzymes (Wilchek and Bayer, 1984).…”