A sensitive enzyme immunoassay of human thyroid-stimulating hormone (TSH) using bispecific F(ab')2 fragments...

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 microU/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.