Site-specific photobiotinylation of antibodies, light chains, and immunoglobulin fragments.
The high affinity of biotin for avidin has been exploited for many antibody-based assays. This requires that biotin is covalently conjugated to the antibody molecule. Several chemically reactive biotinylation reagents are commercially available. Except for the attachment via sulfhydryl groups in the immunoglobulin (Ig) molecule, these reagents attach biotin randomly to various amino acid side chains. Although non-site-specific modification of antibodies does not interfere in most immunoassays, specific application and sensitive antibodies would benefit from site-specific biotinylation. Here we describe an affinity biotinylation technique based on a photoreactive biotin reagent. The design of this reaction was possible from the discovery of a conserved binding site in the variable Ig domain for nucleotides and nucleosides. The described photoaffinity biotinylation offers the advantages of ease, convenience, and production of a reproducible and defined biotinylated antibody preparation.
Pavlinkova
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G
Immpheron Inc., Lexington, Kentucky 40509, USA. gpavlink@unmc.edu
Lou
D
D
Kohler
H
H
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001360:Avidin / Q000378:metabolism; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D019921:Biotinylation / Q000379:methods; D002846:Chromatography, Affinity; D017124:Conserved Sequence; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008027:Light; D019920:Photoaffinity Labels / Q000378:metabolism*; D010782:Photolysis; D015203:Reproducibility of Results; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D001386:Azides; D007140:Immunoglobulin Fab Fragments; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019920:Photoaffinity Labels; D001360:Avidin; C022294:2-azidoadenosine; D001710:Biotin; D000241:Adenosine