Development of bead injection methodology for immunoassays

Abstract

The research presented in this thesis develops Bead Injection techniques for application in immunological research and with the aim of developing rapid screening methods for use in clinical settings.A method is introduced, called label dilution, which is analogous to the well-established isotope dilution method. The principle is tested on a model system of commercially available antibodies and protein-coated Sepharose beads and implemented using Bead Injection in the lab-on-valve format. This micro-scale method uses a labeled form of the target molecule as an internal standard. Label dilution employs ratiometric measurements using the absorbance signals from the label and the target molecules for quantitative determination of an analyte. The label dilution method is shown to discriminate between selective and non-selective binding and provides a means for monitoring bioligand interactions in real time. This Bead Injection method provides a sensitive, automated technique for the determination of low-level analytes in complex samples.The second phase of this work introduces an analytical method for the detection and study of GAD65 autoantibodies, which have been implicated in the onset of type 1 diabetes. There is a clinical need for a rapid and automated assay for GAD65 autoantibodies. Therefore, this method has been designed to exploit the advantages of Bead Injection analysis for enzyme-linked immunosorbent assays (ELISA). BI ELISA is a microscale technique that uses enzyme-labeled secondary antibodies to detect the capture of target antibodies on antigen-coated Sepharose beads. BI ELISA offers the same signal amplification and indirect detection as traditional ELISA protocols, but has the advantages of reduced assay time, fewer sample preparation steps, and complete automation of solution handling and detection.