title: In vitro evaluation of concentration, labeling effectiveness and stability for131I-labeled radioimmunoassay ligand using real-time detection technology.
abstract: Radioimmunoassay belongs to the analytical method enabling highly specific and sensitive quantification of molecules. The verification of the real-time radioimmunoassay technology usefulness for ligand-quality characteristics evaluation such as concentration, influence of radiolabeling on binding affinity and stability was estimated. The anti-epidermal growth factor receptor antibody131I-cetuximab was employed as the ligand antibody. The concentration of131I-cetuximab was derived from the shape of binding curves coming from the ligand-receptor interaction. The binding curves also allowed the estimation of131I-cetuximab binding affinity for different radiolabeling procedures (incubation times 1, 5, and 10 minutes) in stability testing up to 96 hours at 4°C. The stability testing also included comparative analysis by size exclusion high-performance liquid chromatography. The assessment of cetuximab concentrations using real-time method showed acceptable accordance between real and calculated values. The real-time method revealed that 1-minute radiolabeling proved to be the optimal incubation time for direct radioiodination of cetuximab. Stability testing showed the significant change in radioligand affinity by one order at the longest incubation times (72 and 96 hours). Characterization of stability and binding behavior of radiolabeled monoclonal antibodies by the verified real-time method before use in other assays may be employed to eliminate variability and suboptimal antibody performance.
authors.affiliation: Department of Biophysics and Physical Chemistry, Faculty of Pharmacy, Charles University, Hradec Kralove, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Barta
authors.affiliation: Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Hradec Kralove, Czech Republic.
authors.forename: Jiri
authors.initials: J
authors.lastname: Janousek
authors.affiliation: Department of Nuclear Medicine, Faculty Hospital Hradec Kralove, Czech Republic.
authors.forename: Katerina
authors.initials: K
authors.lastname: Zilkova
authors.affiliation: Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Hradec Kralove, Czech Republic.
authors.forename: Frantisek
authors.initials: F
authors.lastname: Trejtnar
mesh_terms: D000074322:Antineoplastic Agents, Immunological / Q000737:chemistry* / Q000494:pharmacology; D045744:Cell Line, Tumor; D000068818:Cetuximab / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D007457:Iodine Radioisotopes / Q000737:chemistry* / Q000494:pharmacology; D008024:Ligands; D011863:Radioimmunoassay / Q000379:methods / Q000592:standards; D011869:Radioligand Assay / Q000379:methods* / Q000592:standards; D019275:Radiopharmaceuticals / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D000074322:Antineoplastic Agents, Immunological; D007457:Iodine Radioisotopes; D008024:Ligands; D019275:Radiopharmaceuticals; D000068818:Cetuximab
keywords: anti-EGFR antibody; cetuximab; concentration through kinetics; ligand-receptor interaction; radioimmunoassay; radioiodination