Efficient conjugation of DTPA to an IgM monoclonal antibody in ascites fluid

We have developed a simple, rapid, and reproducible method for conjugating the bifunctional metal chelator diethylenetriaminepentaacetic acid (DTPA) to an IgM monoclonal antibody (MoAb) without first isolating the MoAb from the ascites fluid. Treatment of the protein mixture in the ascites fluid with cyclic DTPA anhydride (cDTPAA) followed by HPLC purification on a size exclusion column allowed isolation of the DTPA-IgM conjugate which could then be labeled with 111In in greater than or equal to 80% yield. Over the range of total protein concentrations used (11-44 mg/mL), the number of DTPA molecules per molecule of IgM was approximately one-half the molar ratio of cDTPAA to total protein. We have used this method to prepare an 111In labeled anti-Thy 1.2 IgM, a MoAb with specificity for a murine cell-surface antigen found on normal and malignant T cells and neuroectodermal tissues. Analysis of the DTPA-IgM conjugate prior to and after 111In labeling using indirect immunofluorescence flow cytometry and a target-cell binding assay showed that the antigen specificity of this anti-Thy 1.2 MoAb is not substantially altered by the presence of up to 8 DTPA molecules per IgM molecule.