Hi all,
I recently did a project where I did carboxyl-to-amine immobilisation with a monoclonal interferon gamma antibody (human ELISA development kit, Mabtech). I had carboxyl groups on a carbon surface which I activated using EDC/NHS. This was successful, but I am unsure about the location of the amine groups on my antibodies.
Most antibody driven immobilisation documents give an image of an antibody with a carboxyl group located at the end of the Fc region. This obviously cannot be the location of the "linker" as I am looking for amine groups on my antibodies.
Would you be able to help me get a source that could give me the relevant information regarding the above-mentioned interaction or a more detailed explanation of the antibodies that I used.
Kind regards,
Stephan
Amino groups from lysine residues are the most likely to react. Hermanson's book has a lot of information about your concern:
https://www.sciencedirect.com/book/9780123822390/bioconjugate-techniques
Hi Stephan,
You performed an immobilization in random orientation. More information is probably not possible. Perhaps you want to try a more specifc and directed approach:
Article Preactivation Crosslinking—An Efficient Method for the Orien...
Omar Gonzalez-Ortega, thanks for your reply. I checked the textbook in our resources and it does discuss many immobilisation techniques and principles relating to these.
Michael G. Weller, I do appreciate the answer, but I feel that it does not help me with the question I have.
I think I can simplify it by just asking where I can find more detailed information on antibody structures, specifically IgG? From this I can probably find what I am looking for. I have read some text where they mentioned lysine on (or in?) the Fc region of the antibodies that might be where my antibodies bind.
Hi Stephan Petri Schoeman . I dont know if this is what you are looking for but there are glycosyl groups in the "armpits" of most IgGs. that can be specifically modified.
Article Targeting Antibodies to Carbon Nanotube Field Effect Transis...
I am not sure what you are really looking for. Perhaps this structural database is what you need? https://www.rcsb.org/ There you can find some nice IgG structures and their respective complexes.
IgG molecule is ~1200 amino acids. Some time ago I got a sample sequence of a Fab, and there were at least 5-6 lysine and as many of aspartic/glutamic residues. So, when you conjugate IgG using EDC/NHS, the attachment is random and can easily happen on Fab. That is actually one of the many problems associated with protein attachment to surfaces using EDC/NHS protocol, such as: improper orientation; denaturation due to strong covalent forces that create stress on the molecule; steric hindrance - when molecules are too close to each other and Fabs are blocked by neighboring IgG. There is a whole field of study dealing with protein attachment to surfaces (biotin/streptavidin, mixed SAMs, IDA/Ni/6xHis, etc.). I personally prefer Protein G/A approach. Hope that helps.
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