A sensitive enzyme immunoassay of human thyroid-stimulating hormone (TSH) using bispecific F(ab′) 2 fragments recognizing polymerized alkaline phosphatase and TSH

Abstract

Bispecific F(ab′) 2 fragments recognizing both human thyroid-stimulating hormone (TSH) and alkaline phosphatase (ALP) were prepared by disulfide bond exchange between F(ab′) 2 fragments of IgG1 monoclonal antibodies (mAbs) against TSH and ALP, and were purified to homogeneity by hydrophobic interaction HPLC. ALP was polymerized by glutaraldehyde, and a new sandwich enzyme-linked immunosorbent assay (ELISA) for TSH was developed by using the ALP polymers and bispecific F(ab′) 2 fragments against TSH and ALP. In this assay, the preparation of covalently linked enzyme–mAb conjugates was not needed, and the interaction of mAb with non-specific proteins was greatly reduced by the use of F(ab′) 2 fragments. The sensitivity for TSH was shown to increase in proportion to the degree of polymerization of ALP, and the lower detection limit obtained with the ALP trimer was 0.5 μU/ml. The sensitivity was 30 times or more higher than that of the conventional ELISA using covalently linked enzyme–mAb conjugates. The use of bispecific F(ab′) 2 permits the use of monomers and polymers of the signal enzyme and, thereby, regulates the sensitivity of the assay system.