SITE-SPECIFIC PHOTOBIOTINYLATION OF IMMUNOGLOBULINS, FRAGMENTS AND LIGHT-CHAIN DIMERS

Herein we report a new method to rapidly photoinsert biotin into a spe cific and highly conserved site on the Ig structure using a mild photo chemical activation step. This site resides in the Fv fragment and inv olves invariant residues which provide base stacking interactions to t he purine ring of ATP (Rajagopalan et al. (1996) Proc, Natl. Acad. Sci , USA 93, 6019-6024), Biotin was coupled to either the phosphate or th e ribose of the 8-azidopurine nucleotide or nucleoside photoaffinity p robe and shown to insert into the affinity site efficiently. Several m onoclonal and polyclonal antibodies, as well as enzymatic and recombin ant antibody fragments and light chain dimers were photoaffinity bioti nylated and used in ELISA, FAGS and Western blots. The selectivity of this site-specific biotinylation method also allows for biotinylation of antibodies in culture supernatants and immune sera without prior pu rification. Because the biotinylation takes place under physiological conditions and within a short time period, photobiotinylation would be the preferred method for antibodies which are easily damaged by class ical non-site specific random biotinylation chemistry.