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.

A Few Notes on Antibodies: Part 1

During my doctorate research, I shifted my research focus from more traditional biosensor technologies, such as immunosensors, to sensing applications of molecularly imprinted polymers.  However, I still dabble in immunosensors fairly often, and my expertise and previous experience in antibody-based sensors comes in handy, particularly for other researchers who come to me with questions and guidance in this field.  Here at LU, my research group is currently waiting to receive a fluorescent microscope that we recently ordered.  When the new scope arrives, I will be using it to conduct a study examining targeted antibody immobilization onto gold electrodes.  The plan is to block specific areas of our sensor substrate so that antibodies will only be able to bind to certain regions.  When fluorescent bacteria are introduced, they'll bind specifically to the antibodies, and this phenomenon should be clearly visible under the fluorescent microscope.  So in the spirit of beginning this work, I thought I'd discuss some of the basics of antibodies and antibody immobilization.  I'm going to be brief, so I may have to turn this into a series of posts.

First off, I need to clarify one thing:  when I say antibody, I am referring solely to immunoglobulin G (IgG) antibodies.  Next, I guess I should describe what antibodies are and what they do.  The IgG antibody is a relatively high molecular weight protein.  It's produced by our immune cells (B cells) in response to infection.  When the antibodies are produced during infection, they bind to the bacterium or virus or whatever, coating its surface and acting as a sort of signaling beacon.  Other immune cells are able to detect this beacon and attack and destroy the infection.  So in a very general sense, that's the natural function of antibodies.

This natural function of the IgG antibody is made possible by one of its most important properties; selectivity.  The IgG antibody is a large Y-shaped protein that looks something like this:

At the top ends of the two 'arms' of the antibody are binding sites that are capable of binding to one single antigen, which is the target bacteria or virus or whatever, and only that one antigen.  This is what is referred to as selectivity.

Researchers like me who are interested in using the properties of antibodies for biosensors and other applications must be able to readily produce or purchase these antibodies.  As it turns out, there is a fairly effective way of harvesting the antibodies that are selective for a particular antigen of interest.  Let's say, for instance, that you want an antibody that is selective for the flu virus.  To produce these antibodies, you would take a syringe that contained the flu virus and inject it into the lumen of the gut - or some other space where the antigen would not leak out into the bloodstream - of a mammal.  The mammal used is commonly a mouse, rabbit, or goat.  The B cells infiltrate the gut lumen of the animal and begin churning out antibodies that are selective for the flu virus.  Then you would go back and draw out the fluid from the lumen of the gut and purify the antibodies that were produced.  And there you have it - anti-influenza antibodies.

Having only touched the surface of this topic, there will definitely be more to come.