title: A simple hybridoma screening method for high-affinity monoclonal antibodies using the signal ratio obtained from time-resolved fluorescence assay.
abstract: 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-alpha-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-alpha-fetoprotein hybridoma screening with this assay, it was possible to effectively select high-affinity monoclonal antibodies with KD values below 1x10(-8) M. High-sensitivity sandwich enzyme-linked immunosorbent assay which detects domain III of alpha-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.
authors.affiliation: Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Daigo
authors.forename: Sui
authors.initials: S
authors.lastname: Sugita
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Mochizuki
authors.forename: Hiroko
authors.initials: H
authors.lastname: Iwanari
authors.forename: Kanae
authors.initials: K
authors.lastname: Hiraishi
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Miyano
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Kodama
authors.forename: Takao
authors.initials: T
authors.lastname: Hamakubo
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D000937:Antigen-Antibody Reactions; D015374:Biosensing Techniques; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D005453:Fluorescence; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D051379:Mice; D000509:alpha-Fetoproteins / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000509:alpha-Fetoproteins