title: Insulin receptor-insulin interaction kinetics using multiplex surface plasmon resonance.
abstract: Type 2 diabetes affects millions of people worldwide, and measuring the kinetics of insulin receptor-insulin interactions is critical to improving our understanding of this disease. In this paper, we describe, for the first time, a rapid, real-time, multiplex surface plasmon resonance (SPR) assay for studying the interaction between insulin and the insulin receptor ectodomain, isoform A (eIR-A). We used a scaffold approach in which anti-insulin receptor monoclonal antibody 83-7 (Abcam, Cambridge, UK) was first immobilized on the SPR sensorchip by amine coupling, followed by eIR-A capture. The multiplex SPR system (ProteOn XPR36™, Bio-Rad Laboratories, Hercules, CA) enabled measurement of replicate interactions with a single, parallel set of analyte injections, whereas repeated regeneration of the scaffold between measurements caused variable loss of antibody activity. Interactions between recombinant human insulin followed a two-site binding pattern, consistent with the literature, with a high-affinity site (dissociation constant K(D1)  = 38.1 ± 0.9 nM) and a low-affinity site (K(D2)  = 166.3 ± 7.3 nM). The predominantly monomeric insulin analogue Lispro had corresponding dissociation constants K(D1)  = 73.2 ± 1.8 nM and K(D2)  = 148.9 ± 6.1 nM, but the fit to kinetic data was improved when we included a conformational change factor in which the high-affinity site was converted to the low-affinity site. The new SPR assay enables insulin-eIR-A interactions to be followed in real time and could potentially be extended to study the effects of humoral factors on the interaction, without the need for insulin labeling.
authors.affiliation: Biomolecular Interaction Centre and Department of Chemical and Process Engineering, University of Canterbury, Private Bag 4800, Christchurch, New Zealand, 8041.
authors.forename: Kannan
authors.initials: K
authors.lastname: Subramanian
authors.forename: Conan J
authors.initials: CJ
authors.lastname: Fee
authors.forename: Rayleen
authors.initials: R
authors.lastname: Fredericks
authors.forename: Richard S
authors.initials: RS
authors.lastname: Stubbs
authors.forename: Mark T
authors.initials: MT
authors.lastname: Hayes
mesh_terms: D006801:Humans; D007328:Insulin / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism*; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D007328:Insulin; D011972:Receptor, Insulin
keywords: Lispro; insulin; insulin receptor; interactions; kinetics; surface plasmon resonance