Generation of specific monoclonal antibodies against the extracellular loops of human claudin-3 by immunizing mice with target-expressing cells

Bioscience, Biotechnology, and Biochemistry
Hiroshi AndoKazuyasu Nakamura

Abstract

Human claudin-3 (CLDN3) is a tetraspanin transmembrane protein of tight junction structures and is known to be over-expressed in some malignant tumors. Although a specific monoclonal antibody (MAb) against the extracellular domains of CLDN3 would be a valuable tool, generation of such MAbs has been regarded as difficult using traditional hybridoma techniques, because of the conserved sequence homology of CLDN3s among various species. In addition, high sequence similarity is shared among claudin family members, and potential cross-reactivity of MAb should be evaluated carefully. To overcome these difficulties, we generated CLDN3-expressing Chinese hamster ovary and Sf9 cells to use an immunogens and performed cell-based screening to eliminate cross-reactive antibodies. As a result, we generated MAbs that recognized the extracellular loops of CLDN3 but not those of CLDN4, 5, 6, or 9. Further in vitro studies suggested that the isolated MAbs possessed the desired binding properties for the detection or targeting of CLDN3.

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Citations

Dec 12, 2018·The Journal of Pharmacology and Experimental Therapeutics·Yosuke HashimotoMasuo Kondoh
Aug 21, 2019·International Journal of Molecular Sciences·Yosuke HashimotoMasuo Kondoh

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Methods Mentioned

BETA
phage display
transfection
electrophoresis
ELISA
confocal microscopy
flow cytometry
immunoprecipitation
immunoprecipitation assay
xenografts

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