A SENSITIVE ENZYME-IMMUNOASSAY OF HUMAN THYROID-STIMULATING HORMONE (TSH) USING BISPECIFIC F(AB')(2) FRAGMENTS RECOGNIZING POLYMERIZED ALKALINE-PHOSPHATASE AND TSH

Bispecific F(ab')(2) fragments recognizing both human thyroid-stimulat ing hormone (TSH) and alkaline phosphatase (ALP) were prepared by disu lfide bond exchange between F(ab')(2) fragments of IgGI monoclonal ant ibodies (mAbs) against TSH and ALP, and were purified to homogeneity b y hydrophobic interaction HPLC, ALP was polymerized by glutaralde hyde , and a new sandwich enzyme-linked immunosorbent assay (ELISA) for TSH was developed by using the ALP polymers and bispecific F(ab')(2) frag ments against TSH and ALP. In this assay, the preparation of covalentl y 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 propo rtion to the degree of polymerization of ALP, and the lower detection limit obtained with the ALP trimer was 0.5 mu U/ml. The sensitivity wa s 30 times or more higher than that of the conventional ELISA using co valently linked enzyme-mAb conjugates. The use of bispecific F(ab')(2) permits the use of monomers and polymers of the signal enzyme and, th ereby, regulates the sensitivity of the assay system. (C) 1997 Elsevie r Science B.V.