Evaluation of iodogen-coated tubes for 125I-iodination of monoclonal antibodies

Full text: 125I-iodine labelled antibodies such as anti-CD-20, Rituximab, Mabthera(r): are required for various initial immunoreactivity experiments. For this purpose a progressive method is the usage of IODOGEN precoated iodination tubes. Iodogen has become a wide spread mild and effective solid phase oxidation agent in radiochemistry for the past years. There are different techniques to coat either the reaction vial or the antibody itself, yet for getting acquainted with radioiodination ready to use coated tubes are feasible. Iodogen is a 1,3,4,6-tetrachloro-3α,6α-diphenylglycouril, insoluble in aqueous solutions. Per tube 50μg of this agent are coated inside onto the glass wall of the bottom of the vessel. The labelling procedure is as follows: 0.1 ml antibody - solution (containing l mg MoAb) and 40 to 60 MBq of 125I - NaI in PBS (0.6 m1) are injected through the septum of the tube. After an incubation time of 15 min at room temperature (gently shaking) the reaction is stopped by adding 0.1 ml of a solution of l mg potassium iodide in l ml saline, and simultaneously BioRad AG1-X8, 100-200 mesh, anion exchange resin (20 % in saline). After l min of ion exchange the reaction mixture is passed through a 0.22 μm membrane filter. Further purification can be obtained by gel filtration through PD-10 columns with PBS as eluent. Radiochernical purity is surveyed by ITLC (0.9 % saline); the 0.5 m1-fractions of the eluate of the gelfiltration are analyzed by γ-scintillation-counting. Overall yields are between 30 and 40 %, yet both yield and reaction velocity are declining with enlarging the reaction volume. The anion exchange resin traps any possible 125I-iodide very well yet the following 0.22 μm-filtration is consecuted by a retention of up to 23 % of total activity. In the PD-10 purification step the first 2 ml must be discarded whereas the antibody elutes with the next 3 to 4 ml. 125I-Iodide would elute afterwards. The labelling reaction can be performed in the described way very easily yet attention has to be payed to keep the reaction volume as small as possible since otherwise the Na 125I would not get into contact with the iodogen reaction zone at the bottom of the tube. (author)