Radiometric multiplexed immunoassay for the simultaneous detection of thyroxine and thyroid stimulating hormone in serum samples

65-70This paper reports the development of a multiplexed immunoassay (MI) for the detection of thyroxine (T4) and thyroid-stimulating hormone (TSH) in human serum samples. The MI was performed by immobilizing ~ 1 mm spots of polyclonal anti-T4 and monoclonal anti-TSH antibodies at different locations on a very thin (~ 20 micron), highly microporous polycarbonate (PC) track-etched membrane (TEM). Both the competitive assay for T4 and non-competitive assay for TSH were performed on the same membrane using a mixture of 125I-T4 and 125I-anti-TSH monoclonal antibodies for detection. The signal from the antibody spots was detected and quantified using a Phosphor-Imager. There was no interference between the antibodies for their respective analytes. The MI was sensitive (T4: 0.12 µg/dL, TSH: 0.03 µIU/mL) and reproducible with intra-assay and inter-assay coefficients of variation (CV) less than 20%. Thirty-four patients’ serum samples were analyzed by MI, and the results correlated very significantly with the radioimmunoassay (RIA) for T4 and immunoradiometric assay (IRMA) for TSH done routinely (r = 0.92, p p < 0.001 for TSH). Thus, by multiplexing immunoassays for the simultaneous detection of multiple analytes of interest, significant reduction in assay time and cost required for each analysis can be achieved