Simultaneous Measurement of Thyroxine and Thyrotropin from Newborn Dried Blood-Spot Specimens Using a Multiplexed Fluorescent Microsphere Immunoassay

[摘要] All newborns in the United States are screened by state-sponsored programs for congenital hypothyroidism. Traditionally, this has involved an initial screen for thyroxine (T4), followed by testing thyrotropin (TSH) concentrations of infants with low T4 values. This algorithm or an alternative whereby only TSH is assayed does not detect all hypothyroid newborns (1). A simultaneous assay that measures both T4 and TSH would be preferred for screening. Luminex MultiAnalyte Profiling Technology (LabMAP) was used to develop an immunoassay that measures T4 and TSH simultaneously from a single blood-spot sample. The method uses a mixture of two distinct sets of uniquely fluorescent microspheres, which are identified by distinct red and orange fluorescent dyes by the Luminex100 flow analyzer (2). Quantification is accomplished with a green fluorescent reporter molecule. At present, 100 distinct sets of fluorescent microspheres are available, permitting the simultaneous measurement of as many as 100 different analytes.Uniquely, LabMAP provides the ability to combine different immunoassay formats in a single detection system: a competitive-inhibition format for T4 measurement and a sandwich-capture format for measurement of TSH. Using two Luminex spectral-addressable fluorescent microsphere sets, we covalently coupled T4-BSA and anti-TSH capture antibody to two distinct microsphere sets. The anti-TSH monoclonal capture antibody (100 μg), clone 204-12410 (OEM Concepts), and 25 μg of the T4-BSA antigen (Fitzgerald Industries International) were separately covalently attached to the carboxylate groups …

[学科分类] 过敏症与临床免疫学