High-Specific-Activity 111ln-labeled Anticarcinoembryonic Antigen Monoclonal Antibody: Improved Method for the Synthesis of Diethylenetriaminepentaacetic Acid Conjugates1

A new method has been developed for conjugating diethylenetriaminepentaacetic acid (DTPA) to proteins using the Nhydroxysuccinimide active ester of DTPA. The DTPA-active ester was prepared using diisopropylcarbodiimide in a simple single step synthesis. DTPA-conjugated proteins were prepared by adding the DTPA-active ester reaction mixture to protein solu tions (5 mg/ml) buffered at pH 7.0 and purified by Sephadex G50 chromatography. A monoclonal antibody directed against carcinoembryonic antigen was reacted with four different amounts of the DTPA-active ester. Solid-phase enzyme immunoassay showed that the ¡mmunologicalactivity of the antibody conjugate was not altered when the active esterantibody molar ratio was 36:1 or 72:1; however, it decreased when the ratio was 180:1 or 360:1. The antibody heavy and light chains had slightly decreased electrophoretic mobilities when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a result consistent with the covalent attachment of DTPA to the protein. Sephadex G-200 chromatography showed that the na tive and conjugated antibodies were the same size. When the DTPA-conjugated antibody was incubated with 10, 50, and 100 /¿Ci of "Mn/^g of protein, specific activities of 9.8, 43.1, and 56.3 nC'\/u.g were obtained. Enzyme immunoassay and radioimmunoassay of the '"In-labeled antibody showed that it retained its full immunological activity. The high specific activity of the 111lnlabeled antibody makes it suitable for imaging carcinoembryonic antigen-bearing tumors using low doses of antibody.