Intensely luminescent immunoreactive conjugates of proteins and dipicolinate-based polymeric Tb (III) chelates

Partial alkylation of polylysine with 4-(iodoacetamido)-2,6-dimethylpyridine dicarboxylate (IADP), followed by exhaustive reaction with succinic anhydride, yielded polymers (PLDS, polymer of lysine, dipicolinate, and succinate) containing large numbers (50-100) of 4-substituted dipicolinic acid moieties per molecule, with the remaining lysyl side chains succinylated. Competition experiments showed that PLDS binds Tb(III) ions with much higher affinity than does EDTA and strongly enhances the visible luminescence they emit when excited with ultraviolet light. Carbodiimide-mediated coupling to proteins, including bovine serum albumin, ovalbumin, and protein A, yielded PLDS-protein conjugates whose Tb(III) chelates displayed intense green luminescence and millisecond excited state lifetimes. These conjugates retained sufficient immunoreactivity to allow their use in sensitive luminescence-based immunodetection schemes for proteins immobilized on nitrocellulose. The presence of 10 ng of ovalbumin could be easily visualized by eye when probed with rabbit anti-ovalbumin and PLDS-protein A-Tb(III). The ease of preparation of PLDS-protein-Tb(III) conjugates, and their favorable luminescence properties, make them promising reagents for use in time-resolved luminescence immunoassays and other ultrasensitive detection schemes for macromolecules.