Novel unconventional binding site in the variable region of immunoglobulins. Rajagopalan K, Pavlinkova G, Levy S, Pokkuluri PR, Schiffer M, Haley BE, Kohler H Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, Stanford University School of Medicine, CA 94305, USA. Proc Natl Acad Sci U S A 1996 Jun 11;93(12):6019-24 The variable immunoglobulin (Ig) domains contain hypervariable regions that are involved in the formation of the antigen binding site. Besides the canonical antigen binding site, so-called unconventional sites also reside in the variable region that bind bacterial and viral proteins. Docking to these unconventional sites does not typically interfere with antigen binding, which suggests that these sites may be a part of the biological functions of Igs. Herein, a novel unconventional binding site is described. The site is detected with 8-azidopurine nucleotide photoaffinity probes that label antibodies efficiently and under mild conditions. Tryptic peptides were isolated from photolabeled monoclonal antibodies and aligned with the variable antibody domains of heavy and light chains. The structure of a variable Ig fragment was used to model the binding of the purine nucleotide to invariant residues in a hydrophobic pocket of the Ig molecule at a location distant from the antigen binding site. Monoclonal and polyclonal antibodies were biotinylated with the photoaffinity linker and used in fluorescence-activated cell sorter and ELISA analyses. The data support the utility of this site for tethering diagnostic and therapeutic agents to the variable Ig fragment region without impairing the structural and functional integrity of antibodies. PNAS On-Line: http://www.pnas.org/cgi/content/abstract/93/12/6019 PubMed: http://www.ncbi.nlm.nih.gov:80/entrez/PubMed&list_uids=8650212
Site-specific photobiotinylation of antibodies, light chains, and immunoglobulin fragments. Pavlinkova G, Lou D, Kohler H Immpheron Inc., Lexington, Kentucky, 40509, USA. Methods 2000 Sep;22(1):44-8 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. Copyright 2000 Academic Press. http://www.ncbi.nlm.nih.gov:80/entrez/PubMed&list_uids=11020316
Site-specific photobiotinylation of immunoglobulins, fragments and light chain dimers. Pavlinkova G, Rajagopalan K, Muller S, Chavan A, Sievert G, Lou D, O'Toole C, Haley B, Kohler H Department of Microbiology and Immunology, University of Kentucky, Lexington 40436, USA. J Immunol Methods 1997 Feb 14;201(1):77-88 Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved site on the Ig structure using a mild photochemical activation step. This site resides in the Fv fragment and involves invariant residues which provide base stacking interactions to the purine ring of ATP (Rajagopalan et al. (1996) Proc. Natl. Acad. Sci. USA 93, 6019-6024). Biotin was coupled to either the phosphate or the ribose of the 8-azidopurine nucleotide or nucleoside photoaffinity probe and shown to insert into the affinity site efficiently. Several monoclonal and polyclonal antibodies, as well as enzymatic and recombinant antibody fragments and light chain dimers were photoaffinity biotinylated and used in ELISA, FACS 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 purification. 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 classical non-site specific random biotinylation chemistry. http://www.ncbi.nlm.nih.gov:80/entrez/PubMed&list_uids=9032411
Enhanced molecular mimicry of CEA using photoaffinity crosslinked C3d peptide. Lou D, Kohler H Immpheron, Inc., Lexington, KY 40509, USA. Nat Biotechnol 1998 May;16(5):458-62 Antigen mimicry of using anti-idiotypic antibodies for use as cancer vaccines has been disappointing due to the weak immunogenicity of immunoglobulin variable domains. To enhance the immunogenicity of an anti-idiotype vaccine we incorporated a molecular adjuvant peptide into the antibody. The peptide is derived from the C3d region known to bind CR2 receptors on B-cells. A photoreactive peptide is synthesized that affinity-labels a single site in the antibody variable domain. The molecular adjuvant peptide is crosslinked to the anti-idiotype mimetic by chemical means without modifying other sites on the antibody. The C3d-conjugated anti-idiotype antibody induces a strong idiotype and antigen-specific response in mice. http://www.ncbi.nlm.nih.gov:80/entrez/PubMed&list_uids=9592395 http://www.nature.com/nbt/wilma/v16n5.894290857.html
|
For
the full pdf version of this article see 