Direct Construction of an Open-Sandwich Enzyme Immunoassay for One-Step Noncompetitive Detection of Thyroid Hormone T4

• Published in: Analytical chemistry (Washington) Vol. 83; no. 3; pp. 1008 - 1014
• Main Authors: Islam, Kamrun Nahar, Ihara, Masaki, Dong, Jinhua, Kasagi, Noriyuki, Mori, Toshihiro, Ueda, Hiroshi
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.02.2011
• Subjects: Analytical chemistry; Animals; Antigens; Binding sites; Biochemistry; Chemistry; Cloning; DNA, Complementary - genetics; Enzyme-Linked Immunosorbent Assay - methods; Enzymes; Exact sciences and technology; Hormones; Immunoassay; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - immunology; Mice; Miscellaneous; Proteins; Rodents; Thyroid gland; Thyroxine - analysis; Thyroxine - immunology; Chemical analysis; Selection; Immobilization; Immunological method; Design; Molecules; Binding protein; Phage display; Sample preparation; Detection limit; Serum; Recombinant protein; Clone; Construction; Ethanol; Enzyme; Sample; Use; Rodentia; One step method; Hybridoma; Concentration; Maltose; Enzyme immunoassay; Antigen; Vertebrata; Mammalia; Mouse; Affinity; Thyroid hormone; Library; Detection; ELISA assay
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac102801r

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 IC50 (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 VH and VL, 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 VH/VL 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.