A Point-Of-Care Immunoassay Platform for Measuring Antibody Avidity

Serological testing—detection of antibodies—plays a key role in the diagnosis, management, and surveillance of infectious diseases. Serological assays can detect both active and past infections which is essential in understanding epidemiological variables such as incidence and fatality rates. Another important metric, antibody avidity, provides additional insight into recency of infection, can be used to discriminate between closely related infectious species, assess vaccine efficacy and provides estimates of who is and who is not immune to certain infections. However, conventional methods of measuring antibody avidity are costly, time consuming, and utilize harsh denaturing reagents that negatively impact automated immuno-ELISA equipment. These challenges have deterred the development of point of care tests for antibody avidity. In this thesis, we investigated the performance of four assay formats for antibody detection developed by inkjet-printing assay reagents on glass surfaces coated with a non-fouling polymer brush. We then adopted the antibody detection formats to determine antibody avidity by measuring resistance of the antibody-antigen bonds to chaotropic agents. We further developed a new technique of measuring antibody avidity by reducing the concentration of capture antigen (cAg) on the immunoassay platform. In this new technique, avidity index was determined as the ratio of fluorescence intensity measured at a lower cAg concentration to intensity measured at a higher cAg concentration. This technique showed strong correlation (R > 0.8) with the conventional method of antibody avidity measurement (resistance to chaotropic agent) in three antibody-antigen systems. Additionally, we showed that the proposed platform can detect key biomarkers for identifying recent HIV1 infections. The targeted biomarkers were based on measuring titers and avidity of antibodies secreted against specific clades of HIV envelope proteins. They included clade C GP140 IgG3, transmitted/founder clade C GP140 IgG4 avidity, clade B GP140 IgG4 avidity, and GP41 immunodominant region (GP41-ID) IgG avidity. The proposed assay detected all four biomarkers with wide dynamic ranges (>103.6) and high sensitivity in diluted pooled human serum. The proposed platform for antibody avidity testing is rapid, easy to use and has high correlation with chaotropic resistance. It therefore has potential to enable measurement of antibody avidity at the point of care for clinical applications.