Epitope bias in antibody affinity assays arising from charge-dependent orientation in carbodiimide-mediated protein immobilization using β ₂ -microglobulin as a model antigen

Carbodiimide reactions are widely employed for protein immobilization. Although coupling to primary amines is theoretically random, the charge on carbodiimide reactive intermediates can induce a preferential orientation of proteins during immobilization. The phenomenon of epitope bias has been observed during the screening of high-affinity antibodies using such immobilized proteins.