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

Area of Science:

• Biochemistry
• Immunotechnology
• Molecular Diagnostics

Background:

• L-thyroxine (T4) quantification is vital for metabolic regulation, disease diagnosis, and therapy monitoring.
• Conventional immunoassays struggle with small molecule quantification and are time-consuming.
• A need exists for rapid, on-site T4 detection methods.

Purpose of the Study:

• To develop a novel, rapid immunoassay for L-thyroxine (T4) detection.
• To express and characterize a recombinant anti-T4 single-chain variable fragment (scFv).
• To create a Quenchbody (Q-body)-based assay for swift T4 quantification.

Main Methods:

• Expression of recombinant anti-T4 scFv in E. coli.
• Development of a sandwich ELISA for T4 detection.
• Labeling scFv with fluorescent dye for a Q-body based one-pot immunoassay.
• Comparative analysis of ELISA and Q-body assay performance.

Main Results:

• Recombinant anti-T4 scFv demonstrated high binding efficiency.
• Sandwich ELISA detected T4 in the nanogram range.
• Q-body assay provided a T4 concentration-dependent fluorescent response within 3 minutes.
• Q-body assay is faster, while ELISA is more sensitive.

Conclusions:

• A novel anti-T4 Q-body system enables convenient in situ immunoassay of T4.
• Both ELISA and Q-body systems are valuable, chosen based on experimental needs.
• The Q-body assay offers a significant advantage in speed for T4 detection.