Encephalitis caused by the alphaherpes viruses HSV 1, HSV 2 and VZV can be devastating and rapid, accurate diagnosis is required. Whilst existing molecular techniques are invaluable in diagnosing acute disease, detection of antibody is needed to confirm infection and to make a diagnosis after the acute stage or during post-infectious encephalitis. Current immunoassays are limited by the volume of sample required. The aim of this project was to develop a rapid, accurate, low sample volume assay to improve diagnosis using Luminex technology.The immunodominant proteins of HSV and VZV, glycoprotein D (gD) and glycoprotein E (gE), were expressed in insect cells using a baculovirus expression vector. Expressed proteins were purified, characterised and used to develop in-house enzyme-linked immunosorbent assays (ELISA) to detect HSV and VZV type-specific antibodies. The performance of each newly developed in-house ELISA was compared with commercial ELISA assays using well characterised serum panels. An excellent correlation between the in-house ELISAs and the commercial ELISA assays (100% for HSV gD and 99% for VZV gE) was observed. To differentiate between HSV-1 and HSV-2 a new commercial ELISA assay (Omega) utilising a branched chain peptide (peptide 55 which provides immune selection of HSV-2 specific antibody) was evaluated against two commercially available HSV-2 ELISA assays. The Omega assay showed an overall agreement of 97.6% with Western blot and other ELISA assays. The two expressed proteins, together with peptide 55, were used to develop a triplex fluorescent microbead immunoassay for the simultaneous detection and quantitation of anti-viral antibody in human sera. Initially a monoplex assay for each analyte was developed and optimised individually and then the three assays were mixed together in a triplex assay. Results for HSV-1 gD and VZV gE obtained from the triplex assay showed a 100% agreement with HSV-1 and VZV in-house ELISA results. In the case of peptide 55, the triplex assay results showed better sensitivity than the Omega ELISA assay with an overall agreement with Western blot and other assays of 98.4%. In addition, in order to facilitate the diagnosis of alphaherpesviruses CNS infections the triplex assay was joined together with a biplex fluorescent microbead immunoassay designed for detecting and measuring human IgG and albumin in CSF and serum samples. The sensitivity and reproducibility of the resultant five-analyte multiplex immunoassay and the previous triplex assays were compared and found to have equivalent sensitivity and specificity. The sensitivity and minimal sample requirements of the new assay suggests that it will be a powerful tool for the diagnosis and study of both acute and post-infectious viral encephalitis.
|Date of Award||1 Aug 2011|
- The University of Manchester
|Supervisor||Pamela Vallely (Supervisor) & Paul Klapper (Supervisor)|
- Luminex technology