A simple hybridoma screening method for high-affinity monoclonal antibodies using the signal ratio obtained from time-resolved fluorescence assay

In hybridoma screening, quantitative kinetic evaluation is difficult since the concentration of each antibody in the hybridoma supernatant is unknown. From modeling calculations, we hypothesized that the ratio of two different antigen–antibody concentrations might allow discrimination of high-affinity monoclonal antibodies irrespective of the antibody concentration. Using anti-α-fetoprotein monoclonal antibodies of known affinity, we set the signal ratio of a time-resolved assay at >0.1, in which the antigen concentrations were 10 and 100 ng/mL. From anti-α-fetoprotein hybridoma screening with this assay, it was possible to effectively select high-affinity monoclonal antibodies with KD values below 1 × 10−8 M. High-sensitivity sandwich enzyme-linked immunosorbent assay which detects domain III of α-fetoprotein has been established using selected high-affinity monoclonal antibodies. This screening method is useful for selection of high-affinity monoclonal antibodies of potential diagnostic value.