Application of forced enzyme complementation technology: The detection of IgG antibodies to Herpes Simplex Virus in a homogeneous immunoassay. De Las Heras R, Li J, Fry S, McCourt J,

Abstract 13 Application of forced enzyme complementation technology: The detection of IgG antibodies to Herpes Simplex Virus in a homogeneous immunoassay. De Las Heras R, Li J, Fry S, McCourt J, Huang C, Arel E, Hazell S, Kachab E. Panbio Ltd, Brisbane, Australia. Forced Enzyme Complementation (FEC) has been used effectively for the detection of protein-protein interactions in vivo (1, 2). This technology is based on the premise that a reporter enzyme can be split into two complimentary enzyme fragments (α fragment and ω fragment) that are each attached to analyte binding reagents (disease and antibody specific reagents respectively). Presence of an analyte (in this case an antibody) cross-links the interacting moieties thereby forcing the enzyme fragments into close proximity, resulting in the formation of an active reporter enzyme. Using this technology we have developed of an in vitro homogeneous assay platform for the detection and differentiation IgG antibodies to HSV 1 and HSV 2 in patient samples. Three enzyme fragments were developed, an IgG specific enzyme fragment and a second enzyme fragment linked to antigens that are specific for either HSV1 or HSV2. The reagents and assay system were constructed and assessed using a panel of 150 patient samples. All assay components for each specific assay were placed in a single master mix. The patient sample was added directly to the master mix followed by the substrate. This mix was then incubated for 15 minutes prior to a kinetic read-out for 60 minutes. The data were normalised by reference to a nominated calibration sample. The HSV 1 assay had 98% assay sensitivity and 100% specificity with reference to previously characterised samples. The specificity of the HSV1 assay based on its cross-reactivity to HSV 2 Immunoglobulin G positive samples was 100%. The HSV 2 assay had 94% assay sensitivity and 100% specificity with reference to previously characterised samples. The specificity of the HSV 2 assay based on its cross-reactivity to HSV 1 Immunoglobulin G positive samples was 100%. These results show excellent correlation with those obtained using the current ELISA technology in terms of assay sensitivity, specificity and cross-reactivity. Exploiting Forced Enzyme Complementation technology in a homogeneous assay format paves the way for the consolidation of clinical chemistry assays and immunoassays, as well as assays for critical biomarkers onto a single instrument. This consolidation will significantly impact the workflow of the clinical laboratory eliminating system multiplication and the need for specialised instrumentation, thus maximising efficiency and productivity for the end user. 1. Galarneau A, Primeau M, Trudeau L-E, Michnick SW. β-lactamase protein fragment complementation assays as