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

• Published in: Analytical biochemistry Vol. 616; p. 114090
• Main Authors: Susini, Vanessa, Caponi, Laura, Rossi, Veronica Lucia, Sanesi, Antonio, Romiti, Nadia, Paolicchi, Aldo, Franzini, Maria
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2021
• Subjects: Half-fragment antibodies; High-sensitive immunoassays; Reduced antibodies; Half-fragment antibodies; High-sensitive immunoassays; Reduced antibodies
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2020.114090

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 (PANOVA = 0.1980), anyway, with a significant improvement of the repeatability likely providing a more homogeneous capturing surface (SD rIgGmaleimide-rIgGpolystirene: 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). [Display omitted] •Monovalent antibody is obtained by reduction of disulfide bridges of hinge region.•Monovalent antibodies maintain antigen binding capacity.•Chemical reduction enhances the antigen binding capacity of monovalent antibodies.•Chemical reduction induces structural rearrangements in reduced monovalent antibodies.•The orientation on monovalent antibodies in ELISA assay improves the reproducibility.