Surface Plasmon Resonance - a Tool for Quantifying Binding Affinity of HLA Antibodies to HLA Proteins

Abstract

Introduction: The response of the kidney to antibody that may potentially bind and cause damage is critical. However, there is clearly a qualitative as well as a quantitative element to ABO specific and donor HLA specific antibodies that requires investigation before we can define the maximum acceptable or optimal level of donor-specific antibody present at the time of surgery in antibody incompatible transplantation. Surface plasmon resonance (SPR) is a powerful biophysical technique which will enable us to understand complex biochemical and kinetic parameters of the anti-HLA response. Method: Individual biotinylated HLA proteins derived from mammalian cell lines were coupled to separate channels of a Bio-rad XPR36 sensor chip. Monoclonal HLA-specific antibody and HLA-specific antibody derived from renal transplant patients were run over the chip and binding curves were obtained and analysed. Results: Binding of HLA-specific antibody to immobilised HLA proteins was observed in accordance with known patterns of HLA reactivity as defined by luminex microbead analysis and epitope theory. The conditions by which the chip can be regenerated and re-used have been optimised. We have previously shown with the binding of ABO antibodies to ABO bound onto SPR chips that estimates of binding affinity obtained from proprietary software were inaccurate and optimised fits were obtained using effective rate constant modelling based on heterogenous Langmuir with transplant modelling. Binding kinetics of HLA antibodies are currently being mathematically modelled to determine the net affinity derived from a complex polyclonal antibody population. Discussion: Surface plasmon resonance technology provides a rapid analysis and unique insight into the kinetics of the anti-HLA response. This will allow studies to define the affinity characteristics of the anti-HLA response in conventional and HLA-incompatible transplants.