TY - JOUR
T1 - A simple hybridoma screening method for high-affinity monoclonal antibodies using the signal ratio obtained from time-resolved fluorescence assay
AU - Daigo, Kenji
AU - Sugita, Sui
AU - Mochizuki, Yasuhiro
AU - Iwanari, Hiroko
AU - Hiraishi, Kanae
AU - Miyano, Kenjiro
AU - Kodama, Tatsuhiko
AU - Hamakubo, Takao
PY - 2006/4/15
Y1 - 2006/4/15
N2 - 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.
AB - 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.
KW - α-Fetoprotein
KW - High-affinity monoclonal antibody
KW - Hybridoma screening
KW - Time-resolved fluorescence assay
UR - http://www.scopus.com/inward/record.url?scp=33645283348&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33645283348&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2005.12.007
DO - 10.1016/j.ab.2005.12.007
M3 - Article
C2 - 16455037
AN - SCOPUS:33645283348
SN - 0003-2697
VL - 351
SP - 219
EP - 228
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -