In the field of drug discovery, two important metrics of candidate drugs are their binding affinity and kinetics to target receptors. Dr. Moses Rodriguez and his colleagues at the Mayo Clinic have found monoclonal IgM antibodies exhibiting therapeutic effects for multiple sclerosis and amyotrophic lateral sclerosis in animal models, and therefore desired to obtain the kinetic profiles of these antibodies to their targets. Dr. Sang-Hyun Oh’s lab at the University of Minnesota specializes in designing and fabricating plasmonic devices, and have developed a nanohole array sensor coated with silicone dioxide which permits formation of cell mimicking supported lipid bilayers. The focus of this dissertation has been to build these devices and develop assays to measure the binding between these antibodies and receptors in cell extracts and supported lipid bilayers. The first antibody to measure was rHIgM22, which binds to myelin membrane. We did not know the receptor, so we used myelin extracts which would include the unknown receptors, and attached these particles to the sensor surface by passive immobilization. To reduce particle size into the sensor detection window, we extruded the particles through pores of known dimensions. After immobilization we measured binding with antibodies. Unfortunately, binding with rHIgM22 was undetectable, but a similar antibody, mouse IgM O4, which also binds to myelin and has a therapeutic effect, did bind consistently and gave KD, apparent = 2.6 ± 3.6 nM, ka = 2.5 ± 0.0l × 10^4 M^-1^s-1, and kd,slow = 6.6 ± 0.3 × 10^-5 s^-1. The second antibody to measure was rHIgM12, which binds to neuronal membranes. We found rHIgM12 binds to the gangliosides GT1b and GD1a, but not GM1. These gangliosides were incorporated into supported lipid bilayers (5 mol %) and binding to the antibodies was measured. Binding of rHIgM12 to GT1b gave KD, apparent = 24.8 ± 7.9 nM, ka = 2.19 ± 0.196 × 10^4 M^-1s^-1, and kd,slow = 4.72 ± 1.15 × 10^-4 s^-1. Binding of rHIgM12 to GD1a gave KD, apparent = 42.3 ± 20.6 nM, ka = 1.79 ± 0.516 × 10^4 M^-1s^-1, and kd,slow = 4.43 ± 1.38 × 10^-4 s^-1.
University of Minnesota Ph.D. dissertation. 2016. Major: Biomedical Engineering. Advisor: Sang-Hyun Oh. 1 computer file (PDF); 112 pages.
Measuring Binding Kinetics of Therapeutic Antibodies to Membrane Receptors Using Nanohole Array SPR Biosensors.
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