Hi,
We've been using EDC/NHS activation to covalently attach proteins to a carboxylated gold surface with fair success. We'd like to begin immobilizing IgG's, and I'm worried about controlling the orientation. Can you recommend a covalent immobilization method that might do a better job of controlling antibody orientation? Do you think EDC/NHS is sufficient?
Most info I can find on the subject is locked up in proprietary kits.
Thanks
EDC/NHS will result in a random orientation of the IgG. If you want to orient the IgG with the complementarity-determining regions accessible, start by attaching protein A or G to the surface with EDC/NHS. These proteins will bind to the Fc region of the IgG.
Before you go with Protein A or G, check that it binds your species IgG strongly. For instance, Rat IgG1 is poorly bound by both Protein A and G (according to spec sheets). Rat IgG1 happens to be a common antibody clone for mouse antigens.
I'm pretty sure Luminex uses EDC/SulfoNHS, and BD Biosciences beads react with sulfhydryl groups on antibodies as well.
Dear Adam,
a possibility is to immobilize first by EDC/NHS a Fc specific Anitbody (for example rabbit anti-mouse IgG). After that you can use this Ab to capture your specific one. This system has the advantage that your Ab is correctly orientated and can be easily regenerated.
The key for immunoglobulins covalent immovilization is to have a low activated matrix in order to have less reactive sites and do not bind strongly to the immunoglobulins by different places. It is the usual way for IgG immovilization in divinyl sulfone activated agarose (Mini-Leak, from Kem-En-Tec).
Hi Adam, you might want to look into first attaching a spacer arm to the carboxylated Au surface. A spacer will give the Ab more flexibility to bind antigens.
For example, you can first attach 11-aminoundecanoic acid
http://www.sigmaaldrich.com/catalog/product/aldrich/a82605?lang=en®ion=US
to the Au through EDC/NHS chemistry. After that, you can attach your Ab using the same EDC/NHS chemistry.
In hindsight, attaching undecanoic acid to your surface will make it very hydrophobic, which might not be good for your Ab, but Pierce has a line of PEG compounds that have a NH2 and a COOH group:
http://www.piercenet.com/product/carboxy-peg-amine-compounds
These compounds might be more compatible with Ab stability.
Thanks I an actually using these and you're right they outperform the others.
Do not look for complication, just bind your IgG to a low activated matrix. Many of the IgG reactive sites will still be free for binding to your interest molecules.
Although it is recommended in the literature to place -COOH on the surface and let amines be soluble to make kinetics faster, other way around might be somehow in favor of better orientation, since most of Aspartic Acids and Glutamic Acids happen to be in Fc region.
Years ago a matrix of agarose with divynilsulphonate activation was developed by a small company in Denmark (Kem-En-Tec). It was used to immobilized different molecules, including antibodies (Abs). For Abs the less activated matrix was used in order to keep binding for obtained the highest activity after immobilization. Isolation of Immunoglobulins
Europa EP 0921855 B1
Udstedt november 2003
Opfindere:
Allan Lihme, Marie Bendix Hansen
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