Direct construction of an open-sandwich enzyme immunoassay for one-step noncompetitive detection of thyroid hormone T4.

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.

Micro open-sandwich ELISA to rapidly evaluate thyroid hormone concentration from serum samples.

BACKGROUND: Thyroxine (T4) is the most commonly measured thyroid hormone for the diagnosis of thyroid function. To elucidate a rapid and sensitive assay for T4, we made a microfluidics-based noncompetitive immunodetection chip system using anti-T4 antibody fragments obtained from a phage display library. RESULTS: Based on the open-sandwich ELISA principle that detects antigen-dependency of the interaction between the two antibody variable regions V(H) and V(L), we could detect less than 1 ng/ml of T4. The assay was also successfully applied to evaluate total T4 concentration in the serum of healthy individuals. CONCLUSION: This would be the first micro open-sandwich ELISA constructed with antibody fragments directly selected from immunized mice. The system will be applied to the sensitive detection of many diagnostic markers.

One-pot and sequential organic chemistry on an enzyme surface to tether a fluorescent probe at the proximity of the active site with restoring enzyme activity.

A new and simple method to tether a functional molecule at the proximity of the active site of an enzyme has been successfully developed without any activity loss. The one-pot sequential reaction was conducted on a surface of human carbonic anhydrase II (hCAII) based on the affinity labeling and the subsequent hydrazone/oxime exchange reaction. The reaction proceeds in a greater than 90% yield in the overall steps under mild conditions. The enzymatic activity assay demonstrated that the release of the affinity ligand from the active site of hCAII concurrently occurred with the replacement by the aminooxy derivatives, so that it restored the enzymatic activity from the completely suppressed state of the labeled hCAII. Such restoring of the activity upon the sequential modification is quite unique compared to conventional affinity labeling methods. The peptide mapping experiment revealed that the labeling reaction was selectively directed to His-3 or His-4, located on a protein surface proximal to the active site. When the fluorescent probe was tethered using the present sequential chemistry, the engineered hCAII can act as a fluorescent biosensor toward the hCAII inhibitors. This clearly indicates the two advantages of this method, that is (i) the modification is directed to the proximity of the active site and (ii) the sequential reaction re-opens the active site cavity of the target enzyme.

Molecular recognition in a supramolecular hydrogel to afford a semi-wet sensor chip.

This communication describes a new molecular recognition chip using a semi-wet microenvironment provided by a self-assembled hydrogel. On the basis of the evidence that the molecular recognition capability of artificial chemosensors are practically retained even in the hydrogel compared to those in aqueous solution, we miniaturized the functionalized hydrogel to produce an unprecedented molecular recognition chip. We believe that the present noncovalent immobilization method is generally applicable to many chemosensors, which leads to a unique semi-wet sensor chip suitable to convenient and high-throughput assay to plural analytes.