A semi-automated neutral red based chemosensitivity assay for drug screening.

A semi-automated colorimetric chemosensitivity assay was developed. The assay utilizes the vital stain neutral red for the rapid screening of potential anticancer agents using solid tumor cell lines. The cell lines used in this assay and presented in this report are CX-1 colon adenocarcinoma and A549 lung carcinoma. The assay, performed in 96 well tissue culture plates, allows for short drug exposure times (3 hrs.) followed by quantitation of cell number (neutral red absorbance) following four cell doubling times. Cell number directly correlated with absorbance of eluted neutral red at 540 nm. However, optimal amounts of dye and staining times varied between cell lines. IC50 concentrations (for inhibition of cell growth) determined using this assay were in good agreement with results from clonogenic assays using similar drug treatment conditions. The assay technique was determined to be capable of detecting antineoplastic compounds operating by a wide variety of mechanisms.

Relationship of in vitro immune responses to Epstein-Barr herpesvirus and severity of infectious mononucleosis.

Immune responses to Epstein-Barr herpesvirus (EBV) and EBV-related antigens were studied serially in 18 patients with heterophil antibody-positive infectious mononucleosis and in 18 control subjects. Enhanced cellular immune responses to EBV particles and to EBV intracellular soluble antigens were found in the patients at convalescence, suggesting that the development of specific cellular immune responses was associated with apparent control of the virus infection. In addition, a correlation between severity of disease and specific cellular immune response was found. Patients with severe clinical signs were found to have a more active cellular immune response to EBV intracellular soluble antigens early in the infection compared with patients with mild disease. This suggests that an increased immune reactivity to intracellular antigens during the early part of the illness is related to the severity of clinical manifestations in infectious mononucleosis. Serum antibody to viral capsid antigen and early antigen was not related to the severity of clinical disease.

Infection of rhesus monkeys and chimpanzees with Epstein-Barr virus.

Seventy-two nonhuman primates were entered into a long-term study to evaluate the pathogenicity of Epstein-Barr virus (EBV). Infectious virus was inoculated into 42 rhesus monkeys, 4 chimpanzees and 1 cynomolgus monkey. Immunostimulation or immunosuppression was attempted in 34 of these animals to enhance the oncogenic potential of the virus. Eleven inoculated animals were followed for more than 3 years and two were observed for 8 years. No tumors were observed in any of the animals; however, serological evaluation of the 47 inoculated primates and 25 matched controls indicated that at least 14 rhesus monkeys and the cynomolgus monkey were successfully infected with EBV. The potential use of rhesus monkeys as a model for EBV-induced disease in humans is discussed.

Simultaneous radioimmunoassay for specific antibodies to members of the human herpesvirus group.

A method of described for the simultaneous radioimmunoassay (RIA) for antibody to members of the human herpesvirus group. The RIA is compared with some of the conventional serologic techniques used to quantitate antibody to these viruses (Epstein-Barr virus, cytomegalovirus, herpesvirus type 1 and varicella-zoster virus). Color-coded beads, each coated with the antigens of a different herpesvirus, were similtaneously placed in a well which contained a human serum to be assayed for antibody to each of these 4 viruses. The results of this test were compared with the results obtained when the serum was assayed for antibody to the 4 viruses in 4 separate tests. We conclude that the antigen-antibody reactions do not significantly interfere with each other when a serum is assayed for antibody to the 4 viruses simultaneously. A comparison of the RIA with conventional serologic techniques shows excellent correlation in the antibody titers obtained. Features of the solid-phase RIA allow significant savings of time, reagents and space, and thus make it feasible for the small laboratory to screen large numbers of sera for antibody to a variety of antigens.

Detection of Epstein-Barr virus antigens with enzyme-conjugated antibody.

Monospecific conjugated (fluorescein isothiocynate and horseradish peroxidase) goat antisera, prepared against three human immunoglobulin classes, IM (mu), IgG (gamma) and IgA (alpha), were compared for their ability to detect human Ig classes possessing specificity for Epstein-Barr virus (EBV) viral capsid antigens (VCA) in a chronically infected human lymphoid cell line, P3J-HRIK. It was determined that the enzyme system was significantly more sensitive than immunofluorescence in detecting most of the immunoglobulins in sera from cancer patients. Some patients with nasopharyngeal carcinomas (NPC) had extremely high levels of EBV-specific IgA, whereas cancers other than NPC may have lower EBV-specific IgA titers.