Facile generation of monoclonal antibodies suitable for conjugation

Monoclonal antibodies (mAb) are unique tools in therapeutics and immunodiagnostics applications but many of these applications rely on conjugated mAbs. Whether conjugating drugs or tracers, the conjugation process, frequently taking advantage of primary amines on lysine residues, may affect the binding activity of the antibodies. Furthermore, due to the sticky nature of many mAbs, unfavorable interactions may become eminent, with the result of high background signals. The workload associated with producing mAbs, able to withstand conjugation, preserving stability and affinity and avoiding off-target interactions, is comprehensive and related with only incidental success. We designed a method, where uncloned hybridomas were pre-selected for secretion of mAbs with the above characteristics. Using human collectin K1 (CL-K1, alias CL-11, Colec11) as a model antigen, mAbs present in culture supernatant from uncloned hybridomas were immobilized on Protein A beads, followed by solid phase biotinylation and subsequent elution. ELISA was employed to compare the binding activity of conjugated vs. unconjugated mAbs, and furthermore for their application in combination with other antibodies. From a group of 96 uncloned hybridomas we accomplished in obtaining five suitable mAbs, among which, two mAbs were superior. The successful conjugation of the selected mAbs with fluorophores and subsequent applications in microscopy and flow cytometry were further demonstrated. In conclusion, pre-selection of uncloned hybridomas, by testing of their mAbs' ability to withstand conjugation with tracers or drugs, is a successful strategy to avoid a huge workload of cloning numerous hybridomas, in order to obtain conjugatable mAbs.