O010 A novel microfluidic immunoassay for in-solution quantification of alloantibody affinity and concentration in transplantation and beyond

Abstract Introduction Antibody characterisation is fundamental in transplantation and infectious diseases, but current immunoassays cannot determine two antibody properties, affinity (KD) concentration ([Ab]). We aimed to overcome these limitations allow in-depth profiling of antibodies directly sera provide insights into clinical translation. Methods Using a microfluidic diffusional sizing-based strategy, we developed (MAAP), novel in-solution immunoassay that simultaneously determines KD [Ab] serum. MAAP was validated using the HLA-alloantibody system applied HLA Ab-incompatible (HLAi) anti-SARS-CoV-2 immunity. Results enabled quantification (KD [Ab]) alloantibody-HLA interactions both purified alloantibody-spiked sera. demonstrated transplant single-HLA-bead (SAB) were avidity [Ab]-dependent, cellular (flow-cytometry complement-dependent-cytotoxicity) antibody-mediated cytotoxicity proportional antibody-HLA KD, micromolar functionally insignificant despite high SAB signal. In HLAi transplants, differentiated clinically significant donor-specific-alloantibodies (leading rejection) from those tolerated similar assay output provided memory re-activation immune-monitoring post-transplantation. SARS-CoV-2, showed wide variation anti-RBD convalescent (n=34), good correlation with serum neutralisation capacity (p<0.001), evidence maturation 3-months post-infection (despite reduction). vaccinated (n=17), significantly weaker against Omicron than wild-type providing variant immune-escape strategies. Conclusion This work outlines path towards demonstrates importance abundance relevant humoral