Establishment of a Quenchbody-based L-thyroxine detection method and its comparison with ELISA systems

The quantification of L-thyroxine (T4) is crucial for regulating metabolism, diagnosing diseases, and monitoring the efficacy of T4 replacement therapy. However, because T4 is a hapten biomarker with a molecular weight of 777 g/mol, conventional immunoassay approaches, including Western blotting and some types of ELISA, have limited accuracy in the quantification of small molecules, including T4. Furthermore, these methods are time-consuming and involve multiple incubation and reaction steps. Therefore, a novel immunoassay method is required for simple and rapid on-site detection of T4. In this study, we expressed a recombinant anti-T4 single-chain variable fragment (scFv) in soluble form using Escherichia coli. The scFv exhibited high T4-binding efficiency, and T4 concentration-dependent titration curves indicated that the sandwich ELISA could detect T4 in the nanogram range. We labeled the scFv using a fluorescent dye for a Quenchbody (Q-body)-based one-pot immunoassay, which yielded a T4 concentration-dependent fluorescent response in 3 min. A comparison of the Q-body-based T4 detection system with ELISA-based methods demonstrated that the ELISA system was more sensitive but the Q-body assay was more rapid. Therefore, both ELISA and Q-body systems can be used depending on the experimental purpose, with the newly developed anti-T4 Q-body system being applicable for convenient in situ immunoassay of T4.