Early evidences of structural rearrangements in antibodies induced by chemical reduction of the hinge region
The experiment is aimed to verify the integrity and functionality of the antigen binding site in reduced antibodies. The data allow the estimation of the activity of HRP bound by whole and reduced antibodies, in such a way it is possible to estimate the antigen binding capacity of antibodies directed against HRP. The data reveals that not only the antigen binding capacity of reduced antibodies was retained, but the signal generated by these ones is higher than the signal generated by whole antibody.
Steps to reproduce
ELISA assay: Nunc MaxiSorp™ microplates (Thermo Fisher Scientific, Waltham, MA, USA) were rinsed with 200 µl of PBS-T and were coated with 100 µl of intact or reduced antibody anti-horseradish peroxidase (HRP) 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 as previously described. Different amount of the antigen, the enzyme HRP (0.25 ng; 1 ng; 2 ng in water; 100 µl/well) were incubated for 1 h followed by three washing steps. To assess the not specific binding, one well for each concentration of HRP was not coated with antibodies. 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. Data were collected at 450 nm using the plate reader Multiskan GO (Thermo Fisher Scientific, Waltham, MA, USA). All incubation steps were carried out at room temperature. Statistical data analysis of the results was carried out using GraphPad Prism 8.