Sensitivity and reproducibility enhancement in enzyme immunosorbent assays based on half fragment antibodies

The free sulfhydryl groups of the hinge region of monovalent antibody fragments (rIgG) allow the orientation of rIgG on functionalized surfaces in immunosensors. To evaluate the contribution of reduction and orientation on signal enhancement we compared the performance of whole antibodies and their rIgG in ELISA performed on polystyrene or maleimide-functionalized microplates. Monoclonal anti-horseradish peroxidase (anti-HRP) and monoclonal anti-fPSA antibodies (1 mg/mL) were reduced with 2-mercaptoethylamine (53 mM). Western blot confirmed the presence of rIgG as a band at 75 kDa, detectable only by anti-heavy chain but not by anti-light chain antibodies, suggesting a possible folding rearrangement. Using anti-HRP we confirmed the retention of the antigen binding capacity of rIgG. Moreover, we observed a signal enhancement for rIgG even if randomly absorbed on polystyrene [linear regression slope (95%CI): rIgG 0.524 (0.434-0.614), IgG 0.370 (0.430-0.399); P = 0.0016] suggesting that chemical reduction might affect the antigen binding capacity of antibodies. ELISA with anti-fPSA rIgG coated on polystyrene confirmed these observations. Oriented anti-fPSA rIgG on a maleimide surface showed comparable signals to the assay performed on polystyrene for each analyzed concentration of antigen (P_ANOVA = 0.1980), anyway, with a significant improvement of the repeatability likely providing a more homogeneous capturing surface (SD rIgG_maleimide-rIgG_polystirene: fPSA 0.725 ng/mL:0.74-2.89; 1.45 ng/mL:1.56-8.69; 3.625 ng/mL:3.52-15.03; 7.25 ng/mL:7.78-18.44).