Selective UV Crosslinking of Peptides and Functional Moieties to Antibodies


 A method for covalently attaching peptides and small molecules to antibodies through a highly conserved binding domain located on the antibody Fab fragment.  This conserved domain has been observed across all species and all immunoglobulin isotypes an provides for an excellent insertion location for near limitless possible functional moieties.  Through an in silico screening of small molecules and experimental verification we were able to select for a small molecule that selectively binds to this conserved binding domain.  Once associated, a stable covalent bond is formed through a photo-chemical reaction in a UV energy dependent manner.  This results in a unique and consistent covalent insertion of a target molecule selectively to the antibody light chains that does not disrupt  the antibodies ability to bind its respective antigen nor impact Fc recognition.  Using this method we have been able to develop a immunosensor that maintains nearly 100% antibody activity through the oriented immobilization of antibodies onto an ELISA surface.  Current immunosensors often rely on the random orientation of antibodies nonspecifically adsorbed to the surfaces which renders up to 90% of antibodies inactive through sterically hindering access to their binding sites or egatively impacting antibody conformation.  Validation of this initial proof of concept demonstrates the potential for this process to enhance the detection abilities of many currently employed immunosensor technologies as well as detection modalities currently being developed for lab-on-a-chip application.  This method of crosslinking also has implications in the development of next generation pharmaceutical antibodies by providing a site specific insertion of function moieties (without disrupting natural antibody immune activation)  such as: chemotherapeutics, cell penetrating peptides, targeting sequences, imaging specific molecules, and facilitates oriented immobilization of antibodies onto a number of nanoparticle drug delivery platforms (such as liposomes).



Key Features and Benefits:

1. UV coupling of fluorescent molecules to antibodies for immunocytochemistry,

immunohistochemistry, fluorescent activated cell sorting (FACS), and flow



2. UV coupling of biotinylated small molecules and peptides for Western Blot and

enzyme-linked immunosorbent assays (ELISA) to be implemented as primary



3. UV coupling of functional peptides that include but are not limited to: homophilic

peptide sequences, cell internalization sequences, receptor targeting sequences, and



4. Oriented immobilization of antibodies onto surfaces that results in nearly 100%

antibody activity while maintaining Fc recognition for diagnostic applications on

various platforms.


5. Oriented immobilization on nanoparticle drug delivery systems such as: liposomes,

micelles, and dendrimers.


6. UV coupling of reactive moieties to antibodies to facilitate the secondary coupling of

UV sensitive molecules selectively to the nucleotide binding site. Chemistries:

maleimide/thiol, click chemistry, and other orthogonal chemistries to primary amine

reactivity.  Eg. UV coupling a heterobifunctional ligand containing an indole and maleimide

reactive group to then react a thiol containing peptide or small molecule to the

antibody OR react the maleimide containing antibody to a suiface/nanoparticle.


7. UV coupling of molecules that are specific for imaging selective cell populations in



8. UV coupling of chemotherapeutics to antibodies to be implemented in targeted drug

delivery systems.

Patent Information:
For Information, Contact:
Karen Deak
University of Notre Dame
574 631-6695
Zihni (Basar) Bilgicer
Nathan Alves
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