Quantification of the immunoreactive fraction (IRF) of radioactive isotope-labeled antibodies or their fragments is necessary to assess the specific activity of radiopharmaceuticals. Traditionally, cells expressing the target molecules on their surface are used to determine IRF, but such analysis is time-consuming and has difficulties with standardization. The aim of the study was to develop a fast and reliable method for quantitative determination of IRF by 68Ga-labeled VHH antibodies to PD-L1 based on the use of magnetic particles coated with antigen molecules.
Materials and methods: Commercially available magnetic particles coated with protein A have been used in our study. The antigen conjugated with the Fc fragment (PD-L1-Fc) was immobilized on the particles. The IRF value of 68Ga radionuclide-labeled nanobodies (VHH) against PD-L1 (68Ga-VHH-PD-L1) was determined using magnetic particles coated with antigen molecules and cells expressing the antigen on their surface. When VHH antibodies were conjugated to 68Ga radionuclide, protein molecules were modified using bifunctional chelating agents: tetraazacyclododecanetetraacetic acid (DOTA) or deferoxamine (DFO). The magnitude of IRF was defined as the ratio of radioactivity specifically bound to particles or cells to the total radioactivity added to the sample.
Results: The specificity of the 68Ga-VHH-PD-L1 radioimmunoconjugate binding to the antigen-coated magnetic particles has been proved. Some special aspects, which should be taken into consideration when using this method, have been established. The comparison of the IRF estimates using the antigen-expressing cells and magnetic particles has not revealed any significant differences in the results obtained in our study. Nevertheless, the presented method based on magnetic particles with immobilized antigen molecules requires only 15 min to determine the radioimmunoconjugate IRF, which is of fundamental importance for the routine assessment of the specificity of radiopharmaceuticals containing short-lived isotopes.
Keywords: VHH antibodies; immunoreactive fraction; magnetic particles; radioimmunoconjugate.