Isotachophoresis Enhanced Point-of-Care Immunoassays for Clinical Diagnostics

Thesis (Ph.D.)--University of Washington, 2017-06Lateral flow diagnostics are the most common and most commercially successful form of point-of-care diagnostic. The first and most prevalent lateral flow assay is the pregnancy test. Lateral flow assays are low cost, rapid, and simple to operate. However, lateral flow immunoassays have been limited in their success due to poor performance related to an inability to detect low analyte concentrations. In this thesis, we aim to create a high-performance point-of-care immunoassay by integrating sample concentration into a sandwich immunoassay. We use a method called isotachophoresis (ITP) to enhance the detection of low analyte concentrations in the lateral flow format. We present the performance characteristics for a lateral flow enhanced with ITP diagnostic (LID) assay for the detection of strep throat and the detection of chlamydia. We additionally put forth a method for investigating parameters that are important for the performance of LID assays. Lastly, we validate the LID format in clinically relevant samples. We show a limit-of-detection of strep throat cells in clean buffer to be 2000 CFU/mL, an improvement of more than 100-fold over a commercial lateral flow assay. This assay also demonstrates an improvement in the analytical sensitivity when compared to the commercial assay. We also present the detection of chlamydia in the LID format demonstrating a 3-fold improvement over a commercial lateral flow assay. The dramatic difference in improvement when comparing the streptococcus assay to chlamydia motivated an investigation into the parameters that are important when using isotachophoresis to enhance the performance of a lateral flow assay. For designing the chemistry of a LID assay, it is important to investigate the ionic strength (IS), pH and lysis chemistry. Each of these parameters affect the binding of antibody to antigen, as well as the performance of the ITP. Designing with both the binding and ITP performance in mind can be difficult. We present guidelines for obtaining and utilizing these parameter effects in the chemistry design. We develop a model to calculate the limit-of-detection for lateral flow and LID assays from antibody and assay parameters. This model shows that the benefit of a LID assay can be limited by non-specific binding between label and capture antibodies. The model finds that ITP is the most beneficial to antibody pairs with low non-specific affinities. Finally, we test the strep throat LID assay in contrived clinical samples to validate the compatibility of the format with clinical samples. Our assay is able to successfully differentiate positive from negative samples in these clinically relevant samples. Our assay identifies all positive samples in the expected clinical range