A sensitive heterogeneous electrochemical enzyme immunoassay has been developed for thyroid stimulating hormone (TSH) by modifying a commercially available two-site immunoenzymometric assay. p-Aminophenyl phosphate (PAPP) was used as the substrate of alkaline phosphatase, and hydrolysed to p-aminophenol (PAP). The amount of PAP produced from the assay was proportional to the amount of TSH in the sample. Detection of PAP was done by oxidative amperometry in a flow injection system. The working electrode was a glassy carbon electrode whose potential was held at +325 mV (vs Ag/AgCl). The amperometric detection of PAP required only 1 microliter of sample (the range of linearity: 50.0 fmol-100 pmol PAP, the limit of detection: 10.9 fmol PAP). Intra-assay precision over the assay range of linearity (0.02-60 mIU l-1 or 0.02-60 pIU TSH) showed a maximum RSD of 8.0%, and a low detection limit of 0.01 mIU l-1 or 0.01 pIU TSH. The study also indicates that this two-site electrochemical enzyme immunoassay correlates well with the Bio-Rad's immunoradiometric assay currently used in our medical center (r = 0.992, slope = 1.53, n = 43) and a highly sensitive immunochemiluminometric assay in the Nichols Institute (r = 0.986, slope = 0.499, n = 23).

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