title: Direct construction of an open-sandwich enzyme immunoassay for one-step noncompetitive detection of thyroid hormone T4.
abstract: To establish a sensitive noncompetitive immunoassay for thyroxine (T4), we attempted to isolate anti-T4 antibodies from a phage display library based on a phagemid pDong1 ( Dong et al. Anal. Biochem.2009, 36, 386 ), which was designed to enable open-sandwich enzyme-linked immunosorbent assay (OS-ELISA) after selection on immobilized antigen. After the Fab-displaying phage library made from the splenocytes of T4-KLH immunized mice was subjected to biopanning on T4-BSA, two T4-specific clones were obtained. When they were assayed by indirect competitive ELISA, both clones showed low IC(50) (5-13 ng/mL), indicating their high affinity to T4. When they were used for OS-ELISA that detects antigen-dependency of the interaction between variable domains V(H) and V(L), a clone successfully detected 1 ng/mL of T4 with a working range superior to that of competitive IA. OS-ELISA was also performed with maltose binding protein (MBP)-fused V(H)/V(L) of this clone, which showed a detection limit less than 0.1 ng/mL T4. Moreover, the assay showed cross-reactivity with T3 similar to that of competitive ELISA, and also gave a reasonable total serum T4 concentration (90 ng/mL) from ethanol-extracted sample serum using the recombinant proteins. This is the first direct construction of an OS-ELISA system bypassing hybridoma, which will be applicable to the detection of many other small molecule antigens.
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan.
authors.forename: Kamrun Nahar
authors.initials: KN
authors.lastname: Islam
authors.forename: Masaki
authors.initials: M
authors.lastname: Ihara
authors.forename: Jinhua
authors.initials: J
authors.lastname: Dong
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Kasagi
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Mori
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
mesh_terms: D000818:Animals; D018076:DNA, Complementary / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D051379:Mice; D013974:Thyroxine / Q000032:analysis* / Q000276:immunology
chemical_list: D018076:DNA, Complementary; D007135:Immunoglobulin Variable Region; D013974:Thyroxine