Browsing by Subject "D4 assay"

Laboratory enabled disease diagnosis is one of the cornerstones in patient care and greatly relies in immunological techniques to detect pathogens and quantify biomarkers related to a myriad of clinical conditions. The performance of immunoassays is directly dependent on the binding affinity of the molecules that enable capture and detection of analytical targets of interest and especially on the surfaces that interact with these sensing molecules and the complex elements present in biological samples. The lack of high-quality antibodies and nonspecific protein absorption (NSPA) on test substrates have historically hindered assay sensitivity and overall performance. While antibody development has witnessed a significant evolution over time, mostly driven by the interest in antibody-based drugs, major challenges still exist in the IVD reagent generation process. Regarding NSPA, only recently protein resistant surfaces found their way into immunoassay development applications with extremely promising results.

In this dissertation, we aimed to better understand the surface properties required to deliver a new generation of immunodiagnostics with high sensitivity and broad dynamic range. Our studies demonstrated that there are very specific physicochemical requirements a surface must present to enable inkjet based, simple fabrication of antigen detection tests. Interestingly, we determined that POEGMA based brushes, our prototypical surface, naturally presents a fine balance between protein resistance and hydrophilicity that enables their non-covalent biofunctionalization and use for IVD applications. Once we confirmed POEGMA as the ideal coating for the fabrication of antigen detection tests, our work evolved to address several of the challenges related to antibody generation for diagnostic test development. Towards this end, our work entailed the development of a new antibody pair that targets non-overlapping epitopes of ebolavirus secreted glycoprotein, a truncated version of the structural glycoprotein that is actively secreted from infected cells in early stages of the infection. The generation of these antibodies was achieved by associating scFv phage-display technology with the transient expression of promising scFv candidates as Fc fusions in mammalian cells followed by their seamless purification with an IsoTag based chromatography-free system. These elements when combined with a novel antibody pairing strategy, that leverages the D4 Assay’s multiplexing capabilities and ease of fabrication, warranted the rapid identification of optimal capture and detection reagents. Finally, employing these reagents, we developed and validated an ultra-sensitive ebolavirus detection test based on the D4 Assay test format, which was able to not only match but outperform the sensitivity of qRT-PCR. This exceptional sensitivity which can enable the deployment of life-saving treatment and containment efforts was demonstrated in two independent nonhuman primate models of the disease and attests to the success of this new IVD test development work-flow.

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.