Protein Microarray-Guided Development of a Highly Sensitive and Specific Dipstick Assay for Glanders Serodiagnostics.

Burkholderia mallei, the causative agent of glanders, is a clonal descendant of Burkholderia pseudomallei, the causative agent of melioidosis, which has lost its environmental reservoir and has a restricted host range. Despite limitations in terms of sensitivity and specificity, complement fixation is still the official diagnostic test for glanders. Therefore, new tools are needed for diagnostics and to study the B. mallei epidemiology. We recently developed a highly sensitive serodiagnostic microarray test for human melioidosis based on the multiplex detection of B. pseudomallei proteins. In this study, we modified our array tests by using anti-horse IgG conjugate and tested sera from B. mallei-infected horses (n = 30), negative controls (n = 39), and horses infected with other pathogens (n = 14). Our array results show a sensitivity of 96.7% (confidence interval [CI] 85.5 to 99.6%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The reactivity pattern of the positive sera on our array test allowed us to identify a set of 12 highly reactive proteins of interest for glanders diagnosis. The B. mallei variants of the three best protein candidates were selected for the development of a novel dipstick assay. Our point-of-care test detected glanders cases in less than 15 min with a sensitivity of 90.0% (CI, 75.7 to 97.1%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The microarray and dipstick can easily be adopted for the diagnosis of both B. mallei and B. pseudomallei infections in different animals. Future studies will show whether multiplex serological testing has the potential to differentiate between these pathogens.

Carboxyl functionalized gold nanorods for sensitive visual detection of biomolecules.

Antibody-modified gold nanomaterials are central to many novel biosensing technologies for example the lateral flow assays technology. The combination of the specificity, provided by antibody-antigen interactions, and the unique optical properties of nanomaterials provide excellent properties for biosensors. Here, we present the use of gold nanorods (GNR) with the localized Surface Plasmon Resonance (LSPR) peak in the visible range for biomarker detection. The colour of the GNR can be tuned by the reaction conditions to provide multi-coloured gold nanorod conjugates. These antibody functionalized GNR have the potential to provide significant improvements in multiplexed analysis and sensitivity compared to conventional gold nanoparticle based lateral flow assays. However, a major challenge is the synthesis of stable conjugates that resist aggregation in samples with high ionic strength, (e.g. salt solutions) and allow highly sensitive detection of proteins. A detailed investigation of different reagents for the functionalization of gold nanorod materials are reported. An antibody modified GNR based lateral flow assay is validated for the determination of C-reactive Protein (CRP).