NOVEL PHOTOCHEMISTRY FOR BIOTINYLATION OF ANTIBODIES

We recently discovered that antibodies can be photolabeled with photoreactive adenosine probes with high efficiency while maintaining full antigen binding capacity. The photoreaction takes place at micromolar concentration under physiological conditions and in short time. Labeled tryptic peptides were isolated and identified to be from CDR and FR regions of both chains. Computer model generated using this data shows the intercalation of heterocyclic compounds such as purine nucleotides and nucleosides to conserved tryptophan (103) of the heavy chain. Two biotin photoprobes were designed, synthesized and tested to link biotin to antibodies. Monoclonal and polyclonal antibodies were successfully photo-biotinylated and tested in ELISA using enzyme linked avidine. The photo-biotinylation is highly efficient and produces antibodies that are as sensitive as commercially available and exhibit full antigen binding and specificity. Three major goals of this revised phase I proposal are to 1) synthesize several second generation photo-biotinylation reagents which will produce more sensitive and specific biotinylated antibodies, 2) validate specificity and sensitivity in pilot ELISAs using monoclonal and polyclonal antibodies, and 3) demonstrate the general applicability of the method. The Phase I experiment are designed to enter phase II to complete the validation of general applicability of the method, to produce biotinylated antibodies with high specific activity for commercial use, and to develop specific biotinylation in complex mixtures such as sera.