Sensitivity enhancement and the role of orientation in enzyme immunosorbent assay 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 (1mg/ml) were reduced with 2-mercaptoethylamine (53mM). 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, anyway, with a significant improvement of the repeatability.
Steps to reproduce
Antibody reduction 1 mg/ml antibody was reduced by 53 mM 2-mercaptoethylammine in Dulbecco's PBS for 90 minutes at 37°C in mild agitation Assessment of the antigen binding capacity of whole and reduced anti-HRP The antigen binding capacity of whole or reduced anti-HRP was assessed by enzyme-linked immunosorbent assay (ELISA). Microplates were coated with 100 µl of intact or reduced anti-HRP antibody at the concentration of 50 µg/ml in 10 mM PBS-EDTA. Plates were incubated for 2 hours and then washed threefold with 200 µl of PBS-T. Nonspecific binding was blocked with 5% (w/v, 200 µl) non-fat milk/PBS-T for 1 h, and then washed. Antigen, the enzyme HRP (100 µl/well) were incubated for 1 h followed by three washing steps. The presence of HRP were revealed using 1 mg/ml OPD in 50 mM phosphate-citric acid pH 5 containing 1 µl/ml of 33% hydrogen peroxide. Absorbance was detected at 450 nm using the plate reader Multiskan GO. All incubation steps were carried out at room temperature. Assessment of the antigen binding capacity of physisorbed or oriented antibodies The antigen binding capacity of whole or reduced antibodies was tested both on polystyrene and maleimide activated microplates by an enzyme-linked fluorescent assay (ELFA). Bio-Plex Pro™ Flat Bottom Plates 96-well microplates or Pierce™ Maleimide Activated Plates were rinsed with 200 µl of PBS containing TWEEN® 20 0.01% w/v (PBS-T) and then were coated with 100 µl of 50 µg/ml whole or reduced anti-FPSA in PBS containing 10 mM EDTA. Plates were incubated for 2 hours, and excess coating solution was removed by three washing steps with 200 µl of PBS-T. Nonspecific binding of polystyrene microplates was blocked with 200 µl of 5% (w/v) non-fat milk solubilized in PBS-T for 1 h, and then washed as described; to inactivate excess maleimide groups, plates were then incubated for 1 hour at room temperature with cysteine solution at 10µg/ml. Cysteine solution were freshly prepared before each experiment. The calibrator S1 of the VIDAS® FPSA assay diluted at different concentrations in PBS (0,072 ng/ml; 0,725 ng/m; 1,45 ng/ml; 3,62 ng/ml and 7,25 ng/ml) was used as sample. PBS was used as negative control. Samples were incubated for 1 h at room temperature followed by three washing steps to remove the unbound antigen. All the samples were tested in quadruplicate. After 1 h incubation at room temperature and three washing steps, 100 µl/well of a secondary antibody conjugated with ALP were added. After a further 1 h incubation, 200 µl/well of VIDAS® optical substrate were added. The fluorescence generated was measured after 20 minutes on Fluoroskan Ascent (Thermo Fisher Scientific, Waltham, MA, USA) with 20 msec integration time, excitation wavelength at 390 nm and emission at 460 nm. All incubation steps were carried out at room temperature.