Development of a quantitative assay to measure EBV viral load in patients with autoimmune type 1 diabetes and healthy subjects

Clinical studies of immune modulating agents such as otelixizumab, an anti-CD3 monoclonal antibody that is being investigated as a possible treatment for type 1 diabetes, required a sensitive, specific, reproducible method of quantifying Epstein-Barr virus (EBV) load in healthy adults and in patients with conditions not generally associated with increased EBV levels. An assay was developed using a commercial kit and was fully validated. To enhance assay sensitivity and reproducibility, viral load was quantified per 106 peripheral blood mononuclear cells (PBMCs) rather than per ml blood. The lower limit of quantitation (LLQ) was approximately 1000 EBV copies per 106 PBMCs, while the effective LLQ of an assay using whole blood was 5000 EBV copies per ml blood. Based on results from 39 healthy subjects, a reference range of up to 10 000 EBV copies per 106 PBMCs was established. Of 126 subjects with type 1 diabetes, none had viral loads greater than 10 000 EBV copies per 106 PBMCs.