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.

Learning Curve

Whoa...Blogger has made some changes to their interface, so hopefully my confusion won't result in any illegible posts.  I must say, though, that they've simplified things nicely.  Perhaps too simple, as it's been difficult for me to figure out how to do tasks that used to be almost second nature. 

For example, the old interface had a navigation bar at the top of the page, with one of the nav buttons displaying the word 'STATS.'  When clicked, you were taken to a page in which you could view various statistics and information on your readership (number of blog and individual post views, redirect URLs, etc.).  To get to that same stats page with the new interface is a little less intuitive.  There is a button near the top of the page with an icon that resembles a piece of paper with text on it.  Next to that is an upside down triangle.  When you click on the upside down triangle, a drop-down box opens that contains a list of link options.  One of the options is 'Stats,' and when you click on it, you are directed to the statistics page that I described previously.

Don't get me wrong, the interface is very sleek and I think I'll come to prefer it to the old one.  It's just going to take some getting used to.  I hope.

For Your Viewing Pleasure

As I've said before, and will probably say again, I've always been fascinated by the workings of our universe and the magnificence of space. I try to make it over to NASA's website to check out their Image of the Day Gallery once in a while, and I'm always amazed at how awesome the featured pictures are.

Today, I ran across this article at Wired Science. A Rice University researcher took 14 years of images of high-energy jets released from forming stars from the Hubble Space Telescope and pieced them together into a series of seamless animations. The result? I would call it staggering, eerie, beautiful. But you're welcome to come up with your own adjectives.

The video below includes a small selection of the animations. The rest can be found here.

Seeing Red, In More Ways Than One

As the co-owner of a small high-tech business, Emergent Sensor Technologies (EST), I have a vested interest in the federal Small Business Innovation Research (SBIR) program, and to a lesser degree, the Small Business Technology Transfer Research (STTR) program. These programs were originally designed to give small businesses kick-start funding to pursue highly advanced and innovative research projects that might be too risky to undertake otherwise. The end goal of the program? To create jobs. And this makes a lot of sense considering national job growth is fueled almost solely by small businesses. In fact, when EST is awarded its first SBIR, we plan to hire new employees nearly immediately, with additional employees to be added within 1.5-2 years after the date of the award.

Unfortunately, the SBIR program, like many other federal science-related programs, is on the chopping block. According to Rick Shindell of SBIR Insider,

The news on SBIR reauthorization is not good. In fact, at this time, the odds for obtaining reauthorization by the September 30, 2011 deadline is grim, and congress seems to have no taste for what would be the 13th continuing resolution (CR or extender) of the program. In short, the SBIR/STTR programs are likely to lapse, at least for a time, but perhaps permanently.

I don't think I should have to state explicitly that this is an utter travesty, but I'm going to anyways: This is an utter travesty.

It's become clear that in Washington, D.C., job growth and economic development has taken a back seat to deficit reduction. This, despite high unemployment and a depressed economy for the foreseeable future. It's a shame that balancing our federal budget, a matter which has very little effect on the majority of Americans, is being touted as some sort of pressing problem, all at the expense of our country's future. It shows a real lack of foresight and vision. And it's paving the way for us to be overcome as the world's most powerful nation, leaving us whimpering and grabbing at China's coattails.

You Say Scientific Reference, I Say Reprobate

As an avid bicyclist, bicycle aficionado, and all-around bike-lover, I have a great respect for the bicycle helmet. Beyond general self-awareness and following the rules of the road, the bicycle helmet is one of the few ways a cyclist can help ensure his or her own safety. That's why I was taken aback when I came across a supposed bicycle advocacy website that purposefully denounced the use of helmets, citing their "scientific reference on helmets." Their reference is for the so-called 'Bicycle Helmet Research Foundation.' I should warn you that, should you decide to visit this reference website, your head might explode due to the sheer volume of stupid that will be racing over the internet directly into your brain.

Here's what the 'research institute' is all about. They've compiled a bunch of research studies that show that bicycle helmets make cyclists safer. They take these studies and tear them apart, looking for any gaps or holes that can be used to decry the findings and conclusions of the study. Then they look at a bunch research studies that show that helmet-wearing cyclists are not statistically significantly safer than non-helmeted cyclists, which they take as gospel. This is, of course, because these studies back the conclusion that they had obviously settled upon beforehand: helmets may or may not make bicyclists safer, so no one should use them.

Basically the authors have decided that they don't like helmets, so they dig up a bunch of literature to justify themselves, often failing to understand or purposefully misconstruing the conclusions of the papers that they've cited. I should probably be a little more fair and honest about this; the authors are actually against mandatory bicycle helmet legislation. No matter their underlying goals, though, it really is quite despicable.

How about this instead: Helmet use has been estimated to reduce head injury risk by 85 percent, according to the Insurance Institute for Highway Safety (IIHS). Oh and by the way, I'd say the biggest difference between the IIHS and these turds who run the anti-helmet website is that the IIHS isn't made up of a bunch of quacks trying to come to a preconceived conclusion.

Overall, the website reminded me a lot of the websites of climate change skeptics. Obviously subjective and including only the evidence required to illuminate their own narrow viewpoint. And I think it's rather pathetic that this is what these people have devoted their time to. Not wearing bicycle helmets seems to have caused them to take a few too many blows to the head, if you know what I mean. But I guess we're all entitled to our point of view, no matter how idiotic it may be.

Toot My Horn

My most recent manuscript submission has been published online. As I promised in a previous post to link to the paper on my blog, you can find the article here (probably requires a subscription). This paper doesn't include any mind-blowing discoveries, so I'm not going to spend a whole lot of time hashing out all the details of the study. But here's a brief picture of what was done...

The paper is titled, "Comparison of molecular imprinted particles prepared using precipitation polymerization in water and chloroform for fluorescent detection of nitroaromatics." First off, I've described the process of molecular imprinting and how the molecular imprinted polymers work; you can find this information here if you'd like to know more. In essence, a molecularly imprinted polymer is a plastic material that starts as a liquid-phase precursor solution containing the template molecule that you want to imprint. When the material polymerizes, creating a rigid solid, the template is physically and chemically bound within the material. A chemical extraction process is then used to remove the template molecules within the material. This leaves behind binding sites in the polymer that, when re-exposed to the template, are able to specifically rebind the molecule. In this case, I used two different template molecules, TNT (the explosive) and its little brother DNT. Both of these compounds are nitroaromatics and are important in the detection of bombs, IEDs, landmines, etc.

For this study, I prepared the imprinted polymer particles using precipitation polymerization. In precipitation polymerization, the liquid precursor of the imprinted polymer contains an excess of solvent. When there is enough solvent, one section of forming polymer in the solution is unable to link up to another forming section of polymer in the solution because of all the solvent that is between them. Because of this, discrete particles of imprinted polymer are formed within the solution.

The selection of what type of solvent to use for this process is very important, as it determines how effectively the imprinted polymer binding sites will bind to the template molecule. In previous work, I experimentally determined that chloroform was the best solvent for imprinting nitroaromatic molecules. But here's the catch: molecularly imprinted polymers bind the template molecule best when they are exposed to the molecule in the same solvent that was used to form the imprinted polymer, and I wanted to expose the imprinted polymer to the template molecule in a water environment. So there's a trade-off going on. On the one hand, I already knew that chloroform was the most effective solvent for imprinting. But on the other hand, water would be the ideal solvent if I planned to expose the imprinted polymer to the template in water.

So I set out to compare the two solvents, chloroform and water, but a funny thing happened along the way. I used scanning electron microscopy (SEM) images to look at the polymer particles and found that they were nothing alike. In the image below, (a) is the SEM of the particles produced in chloroform and (b) is of the particles produced in water. The images showed us that not only was the chemistry of the two polymers different because of the solvents, but the entire morphology was different as well.

To test which polymer would bind the TNT and DNT templates best, I doped the imprinted polymer particles with a fluorescent dye. When the template bound to the binding sites of the imprinted polymer, it would then quench the fluorescence of nearby dye molecules, which could be detected using a spectrometer. By carefully analyzing the data from these studies, I found that chloroform was the most effective solvent. This meant that the increased imprinting efficiency of the chloroform-based polymer was more important than using the same solvent for polymerization and rebinding. These results weren't much of a surprise, but they could be very beneficial for other researchers working in this field, as they can now refer to my paper rather than conducting this fairly time-consuming study on their own.