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

Kenji Daigo, Sui Sugita, Yasuhiro Mochizuki, Hiroko Iwanari, Kanae Hiraishi, Kenjiro Miyano, Tatsuhiko Kodama, Takao Hamakubo

Research output: Contribution to journalArticle

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-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.

Original languageEnglish
Pages (from-to)219-228
Number of pages10
JournalAnalytical Biochemistry
Volume351
Issue number2
DOIs
Publication statusPublished - Apr 15 2006

Fingerprint

Hybridomas
Assays
Fetal Proteins
Screening
Fluorescence
Monoclonal Antibodies
Antibodies
Antigens
Immunosorbents
Antibody Affinity
Enzyme-Linked Immunosorbent Assay
Kinetics
Enzymes

Keywords

  • α-Fetoprotein
  • High-affinity monoclonal antibody
  • Hybridoma screening
  • Time-resolved fluorescence assay

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

A simple hybridoma screening method for high-affinity monoclonal antibodies using the signal ratio obtained from time-resolved fluorescence assay. / Daigo, Kenji; Sugita, Sui; Mochizuki, Yasuhiro; Iwanari, Hiroko; Hiraishi, Kanae; Miyano, Kenjiro; Kodama, Tatsuhiko; Hamakubo, Takao.

In: Analytical Biochemistry, Vol. 351, No. 2, 15.04.2006, p. 219-228.

Research output: Contribution to journalArticle

Daigo, Kenji ; Sugita, Sui ; Mochizuki, Yasuhiro ; Iwanari, Hiroko ; Hiraishi, Kanae ; Miyano, Kenjiro ; Kodama, Tatsuhiko ; Hamakubo, Takao. / A simple hybridoma screening method for high-affinity monoclonal antibodies using the signal ratio obtained from time-resolved fluorescence assay. In: Analytical Biochemistry. 2006 ; Vol. 351, No. 2. pp. 219-228.
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