Analytical Sciences

Improved Detection Sensitivity and Selectivity Attained by Open-Sandwich Selection of an Anti-Estradiol Antibody

Xiaobo LIU,* Michael EICHENBERGER,** Yuuichiro FUJIOKA,*** Jinhua DONG,** and Hiroshi UEDA*,**

*Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8656, Japan
**Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8656, Japan
***Mitsubishi Chemical Medience Corporation, 4-2-8 Shibaura, Minato, Tokyo 108-8559, Japan

The development of a rapid and specific assay for 17β-estradiol (E2) will accelerate its in vitro diagnostics and/or environmental pollution control. Here, we employed an open-sandwich (OS) selection scheme to improve the sensitivity for E2 in an OS immunoassay, which is based on antigen-dependent stabilization of the antibody (Ab) variable region, Fv, where the two domains (V_H and V_L) dissociated in the absence of an antigen. The V_H domain of a cloned anti-E2 antibody displayed on M13 phage was randomly mutated, and after three OS biopanning rounds, a mutant that showed higher sensitivity in OS-ELISA for E2 was identified. Interestingly, compared with the wild-type V_H, the cross-reactivity of the mutant was significantly decreased for the analogous steroid testosterone, both in OS and competitive ELISAs. This is the first report concerning selection for an anti-hapten Ab without using any hapten-carrier conjugates, and the method will be especially suitable for selecting Ab fragments that show better performance in hapten OS immunoassays.

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