Immunoassay of blood spot TSH; development of a rapid two-site immunoradiometric assay and comparison with radioimmunoassay as a screening method for neonatal hypothyroidism

The development of a two-site immunoradiometric assay (IRMA) for thyrotropin (TSH) eluted from dried blood filter paper discs is described and compared with a conventional TSH radioimmunoassay (RIA) as a screening procedure for neonatal hypothyroidism. The iodinated antibody for the IRMA is prepared by a reproducible four step procedure. Specific antibody is selected from whole TSH antiserum with TSH covalently linked to microparticulate cellulose, iodinated as the solid phase antigen antibody complex, and eluted by repeated changes in pH from 3 to 10. The pooled eluted material is finally purified by Sepharose 6-B gel chromatography to recover labelled monomeric anti-TSH IgG. Solid phase antibody for the IRMA is prepared by covalently linking a partially purified IgG fraction of TSH antiserum to carbonyldiimidazole-activated microparticulate cellulose. Typically yields of 2–3 mg protein per 100 mg cellulose (2–3%, w/w) are obtained. The two-site IRMA involves a primary incubation of excess labelled TSH antibody and the blood disc for 16–18 h at pH 8 and a secondary 3 h incubation under agitation, with solid phase TSH antibody. Bound and free fractions are separated by a semi-automated washing procedure. Compared with the conventional RIA, the two-site IRMA is quicker (results available within 24 h compared to 3 days for RIA), more precise at clinically important TSH levels (25–50 mU TSH/l), and more sensitive (detection limit < 2 mU/l compared to 6 mU/l for RIA). The two-site IRMA is technically simpler than RIA and has proved rugged in routine practice. TSH levels in filter paper blood spots prepared from whole blood with levels of standard TSH (MRC 68/38) within the range 6 to 200 mU/l, correlated well in both assays ( r = 0.969). Recoveries of 98% and 104% were demonstrated for the two-site IRMA and RIA, respectively. It is concluded that the two-site TSH IRMA has advantages over conventional RIA in speed, sensitivity, precision and ruggedness and can be recommended as an efficient screening procedure for neonatal hypothyroidism.