A highly sensitive sandwich enzyme immunoassay of human thyroid-stimulating hormone in dried blood on filter paper discs for mass-screening of neonatal hypothyroidism

A sensitive sandwich enzyme immunoassay of human thyroid-stimulating hormone (hTSH) in dried blood on filter paper discs is described for mass-screening of hypothyroidism in the neonates. One 3 mm filter paper disc per assay, on which 2.7 μl of neonatal blood had been dried, was incubated in buffer to extract hTSH. One monoclonal anti-hTSH β-subunit IgG1-coated polystyrene ball per assay was incubated with the extract and, after washing, with affinity-purified rabbit antihuman chorionic gonadotropin Fab′-β-D-galactosidase conjugate. Bound enzyme activity was assayed by fluorimetry using 4-methylumbelliferyl-β-D-galactoside as a substrate. The assay range of hTSH was 0.037–370 mU/l of blood, and blood hTSH levels in normal neonates were 1.33 ± 0.95 (SD) mU/l (range: 0.18–6.45 mU/l; n = 652). Therefore, this assay with a single protocol may detect not only primary hypothyroidism with abnormally high blood hTSH levels, but also secondary (pituitary) and tertiary (hypothalamic) hypothyroidism with abnormally low blood hTSH levels in the neonates, whereas the previously described enzyme immunoassays can detect only primary hypothyroidism. And this assay may be easily automated with one-step immunoreaction.