Super‐sensitive Enzyme Immunoassay for Thyroid Stimulating Hormone Using a New Synergistic Enhanced Chemiluminescent Endpoint

The enhancers 1,1′-biphenyl-4-yl boronic acid and 4-iodophenol act synergistically in the horseradish peroxidase-catalysed oxidation of luminol. This concentration-dependent effect reduces background, increases signal and hence improves signal/background for detection of peroxidase. The same type of synergistic effect was found when 1,1′-biphenyl-4-yl boronic acid was added to a commercial enhanced chemiluminescence signal reagent (Amerlite Signal Reagent). This synergistic enhanced chemiluminescent endpoint (Amerlite Signal Reagent containing 1,1′-biphenyl-4-yl boronic acid) for a horseradish peroxidase label has been tested in the Amerlite TSH and the Amerlite TSH-30 Ultrasensitive assays. The detection limit (mean of 20 replicates of the zero standard + 2SD) in the Amerlite TSH assay was 0.0029 mIU/L, and in the Amerlite TSH-30 Ultrasensitive assay the detection limit was 0.0005 mIU/L using the synergistic enhanced endpoint. Reassessment of the detection limit using a 1 : 40 dilution of the first standard (0.119 mIU/L) as the lowest assay standard gave a value of 0.0015 mIU/L for the Amerlite TSH-30 Ultrasensitive assay with the synergistic endpoint. A limited (n = 29) method comparison using samples from euthyroid, hyperthyroid and hypothyroid patients revealed excellent correlation between the conventional and synergistic TSH immunoassays.