A Few Notes on Antibodies: Part 2

Now that I've covered the basic function of antibodies and how they're made, I think I'll turn my attention to immobilization techniques.  There are a number of different ways to stick antibodies onto a solid surface, and the strategy that is used is mostly dependent on the type of surface you're working with.  Gold, for example, is pretty easy.  This is because the cystein residues that are present all over the antibody structure will bind - albeit fairly weakly - to gold.  There is a natural attraction between the thiol group of the cystein residue and the gold.  So when you expose IgG antibodies to gold at a nice comfortable pH of 7.5-8.5, the antibodies will adsorb onto the gold surface.  Although this method is easy and relatively effective, it does not create a very stable bond.

To immobilize antibodies with a stronger binding scheme, the protein must be covalently attached to the surface.  This type of covalent linkage between a surface and a protein is often used when the surface is glass.  Glass is a material whose surface is surprisingly easy to modify using a class of compounds called silanes.  Silane molecules are most often based around a single silicon atom.  The silicon atom has three ethoxy or methoxy groups.  These groups will covalently bind to glass, creating extremely stable bonds that are also able to crosslink with other nearby silanes to further stabilize the silane layer.  The fourth valence electron is bound to an organic species - usually a functional group connected to the silicon via a short hydrocarbon linker.  One of the more common silanes used in immobilization techniques is mercaptopropyl(triethoxysilane), and its structure looks like this:

By looking at the structure, you can clearly see the three ethoxy groups (O-CH3) bound directly to the Si atom, and the one mercapto group (SH) connected to the Si atom by a three-carbons (propyl) bridge.

So once the surface of the glass is functionalized with a silane layer, it is much more reactive than the fairly inert native glass surface.  The next step would then be to connect the functional layer of the modified glass surface to one of the amino acids of the IgG antibody.  This is accomplished through the use of a crosslinker.  To give give an example, the crosslinker that I have the most experience with is GMBS (long chemical IUPAC name:  4-Maleimidobutyric acid N-hydroxysuccinimide ester).  And this is what it looks like:

GMBS is known as a heterobifunctional crosslinker because the two ends of the molecule are different and are reactive towards different type of functional groups.  In this case, the maleimide group on the left binds covalently to the mercapto group of the functionalized glass.  The succinimidyl ester on the right then binds to amine groups found on the peptide chains that make up the antibody protein.  Once this reaction successfully completes - which happens fairly quickly - you end up with a glass surface that is coated in IgG antibodies.  And therefore, the surface is now capable of selectively binding the antigen of interest.

One last note about antibody immobilization: the the steric position of the antibodies on the glass is important.  By simply crosslinking the protein directly to the glass surface, you have no way of controlling the position of the antibody.  For instance, the crosslinkage could occur at or near the antigen binding site of antibody.  This would mean that this particular antibody would end up immobilized 'upside down,' with the antigen binding sites so close to the glass surface that the antigen would be unable to bind.  To remediate this problem, you can first crosslink special proteins, such as Protein A or Protein G, to the surface.  Protein A and Protein G have a binding site that is specific to a highly conserved region near the 'bottom' of the antibody, on the opposite side of the IgG from the antigen binding sites.  After immobilization of Protein A or G, you can introduce the antibody, it will bind to the Protein A or G, and you end up with a surface in which all of the antibodies are pointed 'up' with the antigen binding sites exposed and available.