A bispecific F(ab')2 fragment recognizing both human thyroid-stimulating hormone (TSH) and alkaline phosphatase (AP) was prepared by disulfide bond exchange between F(ab')2 fragments of IgG1 mAb against TSH and AP. AP was polymerized by glutaraldehyde, and a sandwich enzyme-linked immunosorbent assay for TSH was developed by using the AP polymers and the bispecific F(ab')2 fragment. In this assay, the preparation of covalently linked AP-mAb conjugates was not needed, and the interaction of mAb with non-specific proteins was greatly reduced. The sensitivity for TSH increased in proportion to the degree of AP polymerization, and the lower detection limit obtained with the AP trimer was 0.5 muU/ml. The use of the bispecific F(ab')2 fragment allows us to use monomers and polymers of AP and thereby regulates the sensitivity of the assay.