All ETDs from UAB

Advisory Committee Chair

Roderick Fullard

Advisory Committee Members

John Laurent

Tammy Than

Document Type

Thesis

Date of Award

2011

Degree Name by School

Master of Science (MS) School of Optometry

Abstract

Cytokines in tears can be biomarkers for ocular inflammation in dry eye subjects. Multiplex bead-based assays measure the concentration of multiple cytokines in a single tear sample. Bio-Rad® polystyrene bead-based assay kits have provided acceptable results for tear samples in this lab. Recently, Bio-Rad decided to replace polystyrene beads with magnetic beads. In earlier experiments in this laboratory, magnetic bead-based as-says did not produce results equivalent to polystyrene bead-based assays. The purpose of this investigation was to modify the magnetic bead-based tear cytokine assay to produce equivalent results to those obtained with the polystyrene bead-based assay. The modifications included running numerous bead-based assays that used different wash systems, plate types, and buffers in order to produce the least interference. Manual and automatic wash systems produced similar results but the automatic was superior because of its automation. The magnetic beads on the plastic plate produced more bead aggregation and worse linearity of dilution than on the filter plate. Tears were stored in proprietary and serum-containing buffers, such as Teknova assay buffer, Millipore assay buffer, and Brookwood Biomedical serum buffer, in an attempt to reduce interference in bead-based tear assays. Overall, the results showed the automatic wash system, the filter plate, and Teknova assay buffer with anti-protease to be optimal for tear bead-based assays. Using these optimal conditions, the final comparison of polystyrene and magnetic bead-based assays showed similar linearity of dilution, which is crucial for small tear samples of dry eye subjects, and better spike-recovery, which measures interference from tear matrix effects, with polystyrene bead-based assays. The results of the final comparison made the polystyrene assay more reliable for future tear assays. Direct comparisons of the two assay types showed the magnetic bead-based assay to be underestimating the polystyrene bead-based assay results. The conclusion was the magnetic bead-based assay was unable to produce results equivalent to polystyrene bead-based assay results. These results led to the decision to use polystyrene bead kits from a company other than Bio-Rad and explore aggressive buffers to reduce interference for future tear assays.

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