Establishment of Open Sandwich Immunoassay Using Antibody Fragments Derived from Combinatorial Libraries

Abstract

Open sandwich immunoassay (OS-IA) is a novel immunoassay principle that enables antigen measurement utilizing antigen-dependent stabilization of an antibody variable region (Fv=VH+VL). For example in OS-ELISA, an antigen is sandwiched between immobilized VL and enzyme-labeled VH, and antigendependent formation of stable ternary complex is detected by the colorimetric reaction of the enzyme. In spite of many merits due to noncompetitive mode such as high sensitivity and wide dynamic range, until now, each variable region fragment has only been prepared by cloning the corresponding gene from a hybridoma cell-line, and by expressing it in E. coli hosts. To bypass these laborious steps and to further expand its possibility, here we tried to establish OS-IA systems from scratch, through the selection of single chain VH -VL (scFv) or VH -VH libraries derived from immunized mice.