Sensitivity enhancement and the role of orientation in enzyme immunosorbent assay based on half fragment antibodies

The site-directed antibody immobilization on the capture surface of immunosensor by free thiols represents a promising innovation for immunochemical methods due to simplicity of accomplishment and low cost compared to other techniques. The aim of the study was to investigate the effect of antibody reduction, combined or not with orientation, on the enhancement of the sensitivity of the solid-phase immunoassays. Antigen binding capacity of reduced antibodies was tested by immunosorbent assays performed on polystyrene or maleimide activated microplates: these surfaces allowed a random or oriented binding or reduced antibodies, respectively. Whole antibodies were used as control in all experiments. Western blot highlighted that reduction induced some structural rearrangements. Reduced antibodies showed a greater antigen-binding capacity in comparison to the whole antibodies even when randomly physisorbed on polystyrene surfaces. Orientation of reduced antibodies on maleimide surface improved the reproducibility of the assay decreasing the CV% to 0.2%- 0.7% in comparison to 0.9%-15% of reduced antibodies on polystyrene. In conclusion, results highlighted the enhancement of immunoassay sensitivity with a synergy of reduction and orientation with an increase of affinity for the antigen and a decrease of CV, respectively.