Optimization of Radiolabeling methods of His-tagged single-chain antibody fragments (scFvs) with technetium tricarbonyl ^99mTc(CO)₃ as a molecular imaging agent

Single-chain antibody fragments (scFvs) are considered more valuable agents for clinical imaging compared with parent antibodies due to their rapid tumor uptake and high tumor-to-background ratios at early times.

A number of studies reported that biomolecules can be radiolabeled with 99mTc in a high yield using indirect labeling techniques or non-site-specific conjugations, but these approaches can reduce their biological activities. His-tags are specific sites for radiolabeling of scFvs with 99mTc-tricarbonyl ([^99mTc(CO)₃]⁺). According to the reported studies, radiolabeling efficiency of His-tag-containing biomolecules varies unpredictably and depends on a series of various factors including the structure of the biomolecule and the conditions of radiolabeling.

Therefore, in this study, we tried to radiolabel two His-tagged scFvs with precursor complex of ^99mTc(CO)₃ and evaluate their radiolabeling efficiencies in different conditions including type of buffer, volume, ionic strength, pH, the concentration of antibody, and activity, temperature, and time using Thin-layer chromatography (TLC) and gamma counter.

His-tagged scFvs were radiolabeled with [^99mTc(CO)₃] ⁺ in 100% radiochemical purity at a range of 480-600 µCi/µg for 2 h at 50°C. We demonstrated that the radiochemical purity of radiolabeled His-tagged scFvs increases with higher [^99mTc(CO)₃]⁺ activity, and antibody concentrations in a smaller volume, increasing the temperature and pH of the reaction medium. Moreover, the radiolabeled His-tagged scFvs showed high stability for 24 h in the reaction medium. In the present study, the optimal and efficient radiolabelling of His-tagged scFvs successfully obtained that they can be used as potential agents for in vivo imaging.