Wisconsin Public Television University Place Wednesday Nite at the Lab  Dick Burgess Disease Diagnosis Using Oil and Water  November 9, 2011 UP 654  cc >> Tom Zinnen: Welcome, everyone, to Wednesday Nite at the Lab. I'm Tom Zinnen. I work here at the UW-Madison Biotechnology Center. I also work for UW-Extension Cooperative Extension. On behalf of our other sponsors, Wisconsin Public Television, Wisconsin Alumni Association, UW-Madison Science Alliance and PLATO, thanks again for coming to Wednesday Nite at the Lab. We do this every Wednesday night, 50 times a year. Tonight, I get to introduce to you a very special person for the Biotechnology Center. Dr. Richard Burgess, who was the founding director of the Biotech Center, and who was key in designing and building this building, which we entered back on October 16, 1995, not that it was a special day to me. Dr. Burgess is the James C. Watson Emeritus Professor of Oncology at the McArdle Lab of Cancer Research here at UW-Madison. He founded the Biotech Center in 1984, and was its director until 1996. 

He was born near Seattle, Washington, on September 8, 1942. He obtained his bachelor's in chemistry at Cal Tech in 1964, where he carried out undergraduate research on Phase M13 with Robert L. Sinsheimer. That is, he was doing that research when he wasn't playing varsity basketball for Cal Tech. He obtained his PhD in biochemistry and molecular biology with James C. Watson at Harvard in 1969, elucidating the subunit structure of E. coli, RNA polymerase, and discovering the first transcription factor, sigma. He was a Helen Hay Whitney Postdoctoral Fellow at the Institute for Molecular Biology with Alfred Tissieres in Geneva, Switzerland, from 1969 to 1971. Then he came here to Madison to join the faculty of the Department of Oncology in 1971 while on a Guggenheim Foundation Fellowship during the sabbatical year of '83 to '84, he was acting director of research in biochemistry at Genetics Systems Corporation back in Seattle. It was there where he learned how to make murine or mouse monoclonal antibodies for use in his biochemical studies. He's published more than 230 research papers on the biochemistry and molecular biology of RNA polymerase, transcription factors, and gene expression; on protein biotechnology and purification; and on target-based drug discovery. He has trained 32 PhD students, and nine master students. He was co-editor with Murray Deutscher of the Methods in Entomology Volume 463, entitled "A Guide to Protein Purification, Second Edition," published in October, 2009. 

Here at UW-Madison, Dr. Burgess teaches courses in protein purification, and characterization, and also topics in biotechnology. He is Editor-in-Chief of the journal called Protein Expression and Purification, and he is the chair and has been instructor since 1992 of a two-week lab course on protein purification held every two weeks each April at Cold Spring Harbor Laboratory on Long Island. He was cofounder of the UW-Madison Center for Biology Education, which serves to promote and improve biotechnology and modern biology education of the public. He served on the National Research Councils Committee on Bio-based Industrial Products from 1992 to 1995. He was the recipient of the 1982 Pfizer Award for Outstanding Contributions to Enzyme Chemistry and the 1999 Watson Medal for discovery of the first transcription factor sigma. He was awarded the United States Department of Agriculture Honor Award for work in developing bio pulping, an environmentally friendly technology in June, 1997. In 2003, he was elected a Fellow of the American Academy of Microbiology, was elected in 2007 to the Council of the Wisconsin Academy of Sciences, Arts and Letters, and was elected as Vice-President for Science in 2011. In 2008, he was elected a Fellow of the American Association for the Advancement of Science, otherwise known AAAS. In 2001, he co-founded Conjugon, a Madison, Wisconsin based Antimicrobial Biotech Company. He consults and serves on the Board of Directors of several biotech companies, and he has served on the Board of Directors for the Wisconsin Biotechnology and Medical Devices Association, which is now called Bioforward, from 2003 to 2006. 

He still plays an active role in translating basic university research into growing Wisconsin biotechnology businesses and corporations, and in educating the public about biotech. In addition to playing varsity basketball at Cal Tech, he still plays buckets three times a week. His hobbies include serious winemaking, rock climbing, and amateur archaeology, woodworking, wild flower gardening and fly fishing for trout. On a personal note, Dick is the person who hired me, for which I'll be grateful for a long, long time. And also he's the person who committed these two labs here to the outreach program and to the undergraduate lab course. These are really remarkable resources for biotech outreach for welcoming people to this campus. I think it's a great contribution to the Wisconsin Idea, here in the year of the Wisconsin Idea and I very much appreciate it. One of your parents was a teacher? >> Both. >> Both. And that shows and I think that's a tremendous commitment on the part of Dick and tremendous vision. 

Tonight, you get to hear about One Second Suffices. He's got a very interesting rapid oil and water method of purifying proteins. It's a great pleasure to be able to welcome Dick Burgess to Wednesday Nite at the Lab. [applause]  >> Richard Burgess: Thank you, Tom. When we hired Tom, we were trying very hard to increase outreach to the general public about science, particularly biology, because we thought biotech was maybe a little too narrow almost. But he's done a wonderful job, and all these programs, like this one, are his ways of trying to introduce to you what goes on around here. Those of us who are on the faculty have been here just a little over 40 years, 40 years and two months. We're used to seeing exciting things all the time. And I admire your determination to come out on a lousy night and hopefully I'll give you something that you can go home and say that was interesting. Only my mother-in-law calls me Richard. 

My name is Dick Burgess. I think you heard all the introductory stuff. I didn't know Tom was going to read it all. I want to just right up front say that this work I'm talking about is actually-- I played a secondary role in it. The real discovery came from Dave Beebe's lab in biomedical engineering and from his post-doc Scott Berry and graduate student Lindsey Strotman; and from my senior scientist, Nancy Thompson; and then another professor in oncology, Shigeki Miyamoto, and his student, Shawn Jackson. So one of the messages I want to get across is that these days it's hard to do research by yourself. And collaboration among groups is common. And in my opinion it's really fun to talk to engineers. Engineers speak a somewhat different language than biologists, although really I was trained as a chemist, so I'm sort of in between somewhere, but this kind of a project doesn't happen if you sit in your office and talk to people who think just like you do. This is the kind of a project that happens when you start to brainstorm with people who think a little different. 

And so, I just turned 69. I thought I would be closing my lab and riding off into the sunset about now. But as you'll find out, not only did we sort of get a new lease on my research career, but we just recently got a grant from the Gates Foundation to develop this technology to hopefully help find low-cost diagnostics for developing countries. But what I want to tell you is sort of a story, a story about how our protein work sort of blended into our interaction with Dave Beebe's lab, and how that has developed over the last year or so.  So my passions are the what work I did my thesis work on in graduate school, which was the enzyme RNA polymerase, which makes copies of genes from DNA into RNA, but I'm not going to talk about that. In the last 20 years, a lot of my effort has gone into teaching about and learning about, and writing about how to purify proteins, enzymes or proteins. Insulin is a protein. All the things that are important are proteins, except for maybe a little bit about DNA. But I love proteins. I do a lot of work related to proteins and teaching people about proteins. 

Another thing I really love is interdisciplinary research and brainstorming, as I said. I really like to be stimulated by people who think about similar, general problems, but from a completely different perspective. I also like to think about, occasionally think about how what I'm doing can be used. The year I spent on sabbatical in a biotech company, that was one of the first companies that started to make a special kind of antibodies called monoclonal antibodies. They were diagnosing diseases, thinking about treatments for cancer. It was really exciting. I got to thinking, you know, here I am. I'm a professor of oncology in the McArdle Laboratory for Cancer Research, but if I discovered something, no one would ever be able to be treated by that discovery until there was a company involved in developing it, and making a product and going through the clinical trials that are needed. So I became very friendly to the idea that scientists need to really think about, is there a way I can use what I do to create products that can help people? So I'm very interested in the technology transfer, how do you take research, basic research results, and convert them into potentially commercial entities that can hire people and pay taxes, etc. I'm also very interested, as Tom said, about education. 

When I started being Director of the Biotech Center in 1984, everybody was afraid of biotechnology. It was scary, because people didn't really understand. Is there a problem...?  People were afraid of the science because they didn't understand it. And I gave something like 40 public lectures a year, just trying to help people understand what biotechnology was. I've many times, had people come up to me afterwards and say, I came in here against what you're doing and now I think it's great, you know. So it was a wonderful thing to see them change their attitude once they learned a little. And to me that's an important thing. So I think letting-- We also, in a good society, base our public policy on facts. And in fact, for the public to use science to make good public policy, they need to understand a little bit of it, and that's why I think it's impressive that you're all here trying to learn something about the science. And finally I am very enthusiastic about what I do, and I just love to share my enthusiasm with people who want to hear it. 

So that's some of my passions. So I was told by a professor of mine one time that some people in the audience will know a lot of what we're talking about, and some people won't know anything. If you talk to the few who know a lot, you're going to lose everybody else. But if you talk to the people who don't know very much about it, you're going to, it's going to be a little boring to the people who know more. So the strategy is to teach people as much as you can as quickly as you can, so that you can get people to a stage where they can follow what you're saying. So I'm going to try to do that. I really don't know your backgrounds, but I have a feeling that you have active minds and that you are interested in learning. So the first part of my talk is really, it's a short part, but it's about the work my lab does on making monoclonal antibodies and their use in protein purification. So who knows what a monoclonal antibody is? Who's ever heard of it? Okay. Few people. 

Have you heard of an antibody? Okay. So if you inject a rabbit or mouse with a protein, it will make an immune response. That's what happens when you get vaccinated against the flu. You're making an immune response. You're making antibodies against the flu virus. Well, you can immunize a rabbit or a mouse against a protein that you're interested in, and it will mount an immune response, and make antibodies, it will react with that. The thing you inject, the vaccine, is the antigen. That's the thing that causes the animal to make the antibody. The antibody is a protein molecule that can specifically bind to the additive. Monoclonal antibodies were a major development that occurred, I don't know, in the early '80s, or late '70s, which allowed, it's just a better way to make antibodies. 

You can make lots of them. You can make very specific homogeneous antibodies, but it's really immaterial for this talk. We found a way of using what we call gentle release antibodies. This little thing is what I call monoclonal antibody. We found a way to make antibodies that would bind onto a protein, but then release it easily. And we call these things polyol responsive, because they respond to a polyol, like propylene glycol, a couple carbons with a O-Hs on it. That's what a polyol is. Glycerol is a polyol. But, again, it's really not important, except to say that this class of antibodies can bind tightly, but it can release under gentle conditions. If you have an antibody, a monoclonal antibody that reacts with a protein, it doesn't react just any old place, it reacts to one specific place. 

That's called the epitope, that site that the antibody bonds to. So if I know that this antibody binds to this epitope, I can take that little piece of protein and I can hook it on to another protein, and now my antibody will bind to that other protein, because it has that little epitope on it. That's called an epitope tag. Finally, there's a thing called green fluorescent protein. You might have heard of it. Jellyfish have this protein that allows them to be fluorescent. A Nobel Prize was given a few years ago for the people who discovered this, because it's been used widely in research. And I'll show you how we use it. If you have any questions, don't hesitate to ask, because if you have a question, I'm sure other people have a question. But I will try to define my terms. 

Okay, so there's something called Immunoaffinity Chromatography. This is where you use the specific binding of an antibody to pull out of a mixture of proteins, the protein that has the epitope. So in a cell, there may be 10,000 or 100,000 different proteins, but only one of them has this little site that your antibody that you've got will bind to. So you can actually purify that protein out of a whole mixture of proteins. That's called immunoaffinity. Basically, you have a little bead that has antibodies. And antibodies are sometimes represented as a little Y. Each end of the Y is a site that will bind to an epitope. Okay, so here's a mixture of proteins. So you put them on. 

Only the yellow protein, which is recognized by the antibody on the column, binds to the column. Everything else flows right through. So you add it, then you wash it, and you wash off some proteins that might have been stuck by accident, or even just trapped somehow. But you wash it. And then finally, you elute it off with something that will weaken the interaction between the protein and the antibody. So we use the antibody as a very specific tool to grab a very small subset of the total proteins in an extract. That's called "immuno," from antibody; "affinity," which means it has an affinity or a binding specificity for it; "chromatography," means that we've used a column to separate our protein from everything else. So that's what immunoaffinity chromatography is. Okay. This is green fluorescent protein. 

It's a beautiful, beautiful structure. It's called a beta barrel. It just has amino acid, a polypeptide chain that wraps around and makes this beautiful barrel. And inside there's a little part of the protein can actually become fluorescent. So when a certain wavelength of light hits it, it will then give off a wavelength of light and that fluorescence is what you see. And it happens to be green. There's another related protein. It's called red fluorescent protein. Guess what color it gives off? We took the epitope, the little spot on our original protein that our antibody bound to, and we grafted it on to GFP. So we had a GFP that was epitope tagged. 

So we can do that. You can make the gene for the GFP, and you can make the gene encode for this little stretch of 15 amino acids. Now you will make that protein. It will have that extra little tag of amino acids. Okay, and this GFP will bind to this antibody, which we call 8RB13. It's just a name we gave it when we made it. It's not a very catchy name, but that's what it is. This is work that was done in my lab. The first author was an undergraduate, a student helper who came in and loved to do research and we gave her this project, and ended up writing a beautiful paper about it and she's the first author. So one of the beauties of being an undergrad at a research university like Madison is you have the opportunity to work in a lab and actually do something. 

Okay, so I don't really want to go into too much detail here, but this antibody, this kind of antibody I mentioned before, it's called a polyol responsive. It binds tightly to the epitope and in this particular assay, called an ELISA, you get a strong signal if it's bound. If it starts to come off, the signal goes down. I really don't want to spend the time it takes to describe all the details of this assay base because it's not super important except to say that by increasing the propylene glycol, and increasing the salt, we can go from a condition where the interaction between the antibody and the epitope is strong and it stays that way, to a situation where it comes right off. So we can affect how tightly something binds by just changing the salt. And that's actually pretty cool. I mean, even at this 40% propylene glycol, when there's no salt around, it binds tightly. But when we start adding salt, it binds weaker and weaker. Okay, and so, here's the visual. So here's a column. 

It's about this long. It holds two milliliters of beads, which have our antibody on it. So that's an immunoaffinity column. We put an extract on which had this epitope tag GFP. So it binds to the column because the antibody that's on the beads binds to the epitope that's on the GFP. And it stays there. You can wash it, and wash it, and wash it. It never comes off. If you wash it with no polyol and high salt, it doesn't come off. If you wash it with high propylene glycol and no salt it doesn't come off. 

But if you take a combination of salt and polyol, then it comes right off. So these are one mil fractions. There's one here, one here, one here. If you start putting on this buffer, which is a buffer that will cause the protein to release from the antibody, you'll see, about after two mils, you see your protein come off. As that buffer runs through this zone, this fluorescence moves right down the column and it comes off. And, again, this is an experiment to show that by going down from half molar to quarter molar to eighth molar to sixteenth molar salt, what happens is it takes longer for that protein to come off, meaning it's actually still binding a little bit, and it slowed down as it moves down the column. The upshot of this is that this is an antibody which can bind epitope tag GFP, but we can vary how tightly it binds. If we add this buffer, it comes right off. If we add this buffer, it's bound weakly. If we add one of these buffers, it doesn't come off at all. 

It binds very tightly. So this is where we were about a year and a half ago. We had been working on this. And then I got a phone call from Dave Beebe, who's a professor, as I said, in biomedical engineering, and they had developed a technology. Since then, they've published a couple papers on this using it to purify RNA and to purify cells. Dave knew me because he had taken my course at Cold Spring Harbor. So he was my student. Even though he was a professor, he came to this course for two weeks, 14-hour-a-day lab course. He knew I loved to purify proteins. And he said, would you be interested in helping us demonstrate that our new technology works with proteins? So I'll tell you about the technology. 

And this is really what the interesting story is. But I had to give you a little bit of background about the antibodies. So this was in May of 2010. We had this thing, which we now call IFAST. We're going to write a grant to the Gates Foundation to try to get some money to do research to develop our technology into a diagnostic, so that we can treat people in Africa under a tree. I mean, a very primitive clinic with no electricity, no equipment, no refrigeration. And we thought this technology might actually be able to be used that way. So we did some experiments and it turned out we got great results and we put in the grant and we got it. $3 million grant. I think it's $3 million. 

$2 million, or something. A lot of money. It was enough to help me support my senior scientist for three years, which means that my lab will stay open for three years. And it just turned out that this antibody, 8RB13 and this epitope tag GFP was perfect as a model system for studying purification of proteins. Okay, that's the background. Okay, so a few more concepts. Epitope tag GFP, I've already talked to you about. It's a GFP molecule, a green fluorescent protein that has this amino acid tag that our antibody binds to. Affinity reagent is actually the antibody. It's the thing that has some affinity, in this case, for the tag. 

The paramagnetic particle is sometimes also called the magnetic bead, teeny little bead that if you put a magnet next to a tube, the beads will all move through solution and stick to the side. A paramagnetic bead that has a protein G on it, is a bead that is coated with a covalently attached protein that binds antibodies. So what we do is we mix the protein G bead with our monoclonal antibody, and now the antibody binds to the protein T, and now we have an affinity bead that is specific for binding the epitope, in this case the GFP. An analyte in the parlance of diagnostics is the thing you're trying to measure. You know, you have a blood sample, you want to know whether the person has malaria. People who have malaria have a certain protein in their blood. If you can grab that protein, separate it away from blood, you're in a position to determine whether it's there and how much is there. And that's the concept of a diagnostic, is to actually measure, determine whether a certain thing is present. And finally an immiscible phase in this case is some oil. Remember, oil and water don't mix. 

Well, that's the whole principle of this technology. The term IFAST, which is the name we've given to this technology is Immiscible Filtration Assisted by Surface Tension. It's pretty obscure. IFAST is easy to remember, but we'll talk about it some more. So the basic idea is the following. This is a device. This device, there are five of them on this. It's basically a thin, glass slide with sort of a silicone plastic with little wells, three little wells in it. And they're interconnected. So you put a buffer over here. 

That's called the output. And over here you put a sample, like blood or a cell, an extract or whatever it is that you're starting with. That's the input. In between, you put a droplet of olive oil. Now, you don't put balsamic vinegar in this one! [laughter] But the olive oil, it can be other kinds of oil. Anything that is immiscible with water. In other words, they don't dissolve in each other. They stay separate. If you put the oil in here, because this is so teeny, surface tension holds the interface flat. Now, normally if you pour oil on water, it floats, because it's lighter than water, right? But if you put it into this little thing, because it's so small, the oil doesn't float on the water. 

It just stays right next to it. And one of the things that Dave Beebe is-- In my opinion, his strength is that he thinks small. I mean, he thinks about micro channels and very small volumes. And this is a consequence of thinking small, is that you can actually design something where the oil will stay in this middle well and not mix with the water buffer on either side. So you put your crude sample in this side. You put in some magnetic beads that have an antibody. Then you take a little magnet, a teeny little magnet, and you just drag it across like that and you pull the magnetic beads that were here through the oil and over to this other side. Now, that's just, you know, it's not rocket science. I mean, this is just pulling some little magnetic beads through a little droplet of oil. These are eight microliters. 

A typical drop out of an eye dropper is 50 microliters. So we're talking about something that's less than a sixth of a volume of a drop. But, anyway, so we have a sample, an oil phase, and then an output phase. And by this simple process of pulling the magnet and beads across into here, we've purified whatever that antibody bound to. So this is, you know, if the blue is the on-put or the crude sample, the white in between is the oil, and the pink is the output, then this is five devices. Here's a configuration. It's easy to manufacture these with different shapes and stuff. Here's one with a sample, oil, a wash well, another oil phase, and then an output. They're all basically the same. They're a way of taking a crude sample of a magnetic bead carrying an affinity reagent, letting it bind its analyte, pulling it through the oil to the other side. 

So in that one second or so that it takes to drag those beads across, you've purified your sample. So this is pretty astounding, in my opinion. I mean, and I have to say, I just sort of step back a minute. Scientists live for the time when something happens where you now have all of a sudden you can see all kinds of possibilities. And, you know, it was just, it was a very exciting time when we got these results that I'm going to show you now because we could just think of all kinds of ways you might be able to use this simple technology of drawing magnetic beads through a droplet of olive oil, something as simple as that, and yet this is something that people really hadn't done before and in a way that allowed the kind of flexibility that we have with multiple wells, and so on. So this process again is called Immiscible Filtration Assisted by Surface Tension It really means that the oil stays separate from the water and you can draw the beads right through, from the water, from the aqueous phase through the oil into the other side. I want to just show you a little movie. So here is a little video of that process. So let's see, if I can go to... This thing you see right here is the magnet. 

This magnet. And when you put down on it the little device, and this has several, you see the dark things? Those are the magnetic beads. You pull it. It pulls them through. Now it goes into the oil and comes out the other side. That's it. I'll do it again. [laughs] Don't blink this time.  So there's the little-- the on-put well with the beads. Having bound something, now you pull it through. 

Now it's in the oil. And then it's back into the output well. Now, it turns out that when-- There's lots of little beads there. When they go through the oil, they sort of ball up into a blob. It turns out that the liquid on the outside of the blob is just sort of squeegeed right off by the oil. And so you carry through very little of trapped liquid. As a result, this is very effective. For those of you who do experiments in the lab, this turns out to be as effective as three washes. So normally you take, I mean, the traditional method, if you suppose this is a little tube, you take your magnetic beads and you mix them with your sample, just like we mixed our beads with our sample in that on-put well. Then you hold the magnet to one side. 

The beads move over. Then you suck away the liquid, because you've removed whatever you want. You put some buffer in. You do like this for a couple minutes. Then you take the magnet, you put it like this. The beads all move over to one side. You suck off the liquid that doesn't contain the beads. You put some more buffer in. You mix it up. You do that three times. 

It turns out that all of that washing is equivalent to passing those beads through a droplet of olive oil. And that is the really important part about this, in a way, because it really means that in a one second, you can accomplish what normally takes ten minutes. Now, that's cool, but, you know, in itself-- It turns out we found a reason why that was much cooler than we realized at first. And I'll try to explain that. So let me go back to my other thing here.  Okay, so we've got our little setup here. So I'm going to go through this fast because I really introduced a lot of this. The device itself is this silicone type plastic. There's an on-put, an oil phase, and an output chamber that are about eight microliters. We have a little magnet. 

We have a buffer. We have oil, which can be olive oil or it can be some other thing like a fluorocarbon oil. We have a paramagnetic bead to which we can bind a monoclonal antibody. In this case our analyte is the green fluorescent protein with the epitope tag on it. So we can actually see this happening. The contaminant, you know, what you want to do, in this kind of an assay, you want to see if it's selective, so a key term is selective, meaning it grabs what you want, but it doesn't take anything else. And you want it to be efficient. You want it to grab everything that's there and pull it across. So you want a high yield of your analyte that's captured and pulled across and purified. And you don't want to pull anything else. 

So those are the two things that are important. So we took red fluorescent protein, which is red, but it doesn't have the epitope tag on it. It shouldn't bind to our bead, because it doesn't have the epitope that the antibody can grab onto. We mixed it in with this broken E. coli, just a cell lysate, a mass of proteins, and we did the experiment. So, here's the beginning. Now, here's a schematic. The beads are in the input well with the crude lysate. There's green fluorescent protein. You can photograph this under a light that will show the green fluorescence, and you can photograph it under a light that shows red fluorescence. So these are two different pictures. 

So they're both over here. We move it across. Now the green fluorescent protein is over here, but the red fluorescent protein stays behind. It turns out that something like less than 1%, of the red fluorescent protein gets carried across, and it turns out that something like 95% of the GFP is captured and pulled across. It turns out that the blue here is conventional, three, you know, magnet on the side of a tube with three washes that take about ten minutes, and the red is the IFAST thing where we basically took one second. So you can see that the results are very similar between the red and the green. This is how much GFP is left behind, and this is how much GFP is pulled across; and this is how much RFP is left behind, and this is how much RFP is pulled across. So very similar property, performance, only instead of washing for ten minutes, we've washed for one second. So that was the thing. This is a log scale. 

So we put some fluorescent dye, not even on a protein, and we showed that there's this amount if you do one, two, three, or four conventional washes. You're down to like .2% of this. If you do one or two passages through the oil phase, you're down even lower than four. So one passage through is between two and three washes, and two passages is better than four washes. So very effective washing, very simply. Just pull the beads from here, through oil, through a little wash and on through the oil to your final destination. So we were saying, this is great, this is great. We can write a paper. We can show that this works. We can purify proteins. 

Then we got to thinking, wow, you know, this is so much faster than people normally can do this, maybe there's something more to this that we can take advantage of. So this is a little sidetrack for a minute here. If you have two things, A and B, and they bind to form AB, they also can dissociate. So the measure of how tightly they're bound is something called Equilibrium Dissociation Constant, or Kd. Even I don't understand this as well as I should, so I'm not going to try to explain it, except to say if this is a very small number, it means that it's very tight. So ten to the minus ninth is a very small number, and things that bind that tightly will dissociate with a half-life of about 30 minutes. That means that if I have pure AB, and I put it into a tube, about half of it will dissociate it into A plus B in 30 minutes. It stays together really well. If it's 1,000 times weaker, then its half-life is two seconds. That means, that if you put it in and you wash it, and you hook one of these on, you know, onto an antibody on a column, and you start to wash it, in two seconds, half of B has fallen off. 

In another two seconds another half has fallen off. In another two seconds another half as fallen off. And so in a minute there's nothing left. And so that's exactly-- What we realized was that even if something had a very short half-life, let's say ten seconds, and if we only take one second to separate it away, there's no time for B to fall off of A. And therefore we can capture very weak interactions. You may not fully appreciate why that's so important, but I'll try to explain. Because I didn't appreciate it. Nobody appreciated this a year ago. This is a relatively new concept. It turns out that many things in the cell are really multi sub-unit complexes, the enzymes that make RNA, the enzymes that make DNA, the enzymes that repair DNA damage. 

The ribosomes that make proteins from messenger RNA. Almost everything you can think about that goes on in the cell is carried out by a machine made of multiple proteins. Now, when we purify one of those machines, we go through many steps of purification, many times where it's bound to a column where we wash it for a long time. So we're only isolating those sub units that hang together really tightly. But it's probably the case, and I would bet each of you a pitcher of beer that there are proteins that are very important parts of those complexes, which we've never seen, because when we grab onto that complex and wash it, we wash off all those things that dissociate with a half-life of two seconds. We lose them. And if we look at what's left, we see what's tightly bound, but we don't see the things that were weakly bound and came off. So we think that we have a method for isolating complexes that nobody's ever been able to see before. These turn out to be-- It's starting to become apparent that this kind of interaction may play a very important role in biology. It's just that it's been impossible to study. 

So here's a this simple thing, drawing magnetic beads through a droplet of olive oil, and all of a sudden you have a new way to study the biochemistry of cellular processes. That's what makes one get up in the morning and go to work, is the chance that something like that will happen, and you will all of a sudden say, this is really neat, and think of all the ways we can use it. After I gave a talk to my department about a year ago, and there's 22 faculty in my department other than me, 20 of them came up to me right after my talk and said, I've got to have this. I need to do experiments where I can study weak interactions. How do I get these things? So, anyway, that's the response I've gotten by all scientists, by chemical scientists, that I've talked to. When they hear about this, they immediately think of ways they can use this for their own research. So here's an example, just more of an illustration. Here's the bead. There's an antibody. It's binding the yellow molecule. 

But there is another weakly-bound red molecule. When you wash it, the red molecule falls off until when you finally elute the yellow off the antibody, there's very little red left. It's dissociated. Where with our case, we have some red bound to the yellow, but since there are no washes involved, we can-- when we elute off the yellow, along comes the red. So we can identify that. There are ways of determining what that protein is. That's a whole other story using mass spectroscopy. So it has the potential to see things that were previously unseen. That's what we titled our NIH grant that we just put in. It's called "Seeing the Unseen." It sounds a little mysterious. 

But to me, it's incredibly exciting to realize that-- It turns out that in biology, there's lots of things that were there and nobody ever saw them, because they didn't have the right tools. The history of any science is that way. But when the right tool comes along that allows you to detect something, now all of a sudden you see it and you understand that, you know, you've made just a huge leap forward in understanding how things work when you can start to better understand what are the critical components of this complex.  I'm not sure, this may be rubbing it in, but again, remember our antibody that was sensitive to salt? Very low salt, it bound very tightly, and at very high salt, it bound very weakly? Well, here we took the epitope tag GFP and we pulled it through with the antibody bead at different salts. What we found was that our IFAST method captured over 90% of this stuff. When you got to the salt where the binding was getting pretty weak, it came down. So if it's too weak, there's nothing bound to pull across. Using the conventional method, much less sensitive right off the bat. So if you go into this range right here where this protein is dissociating quickly, you can hardly see it with the conventional method, yet we see it extremely well with our method. We've used this now, there must be a half a dozen or more examples of where we've identified weak interactions that have been extremely difficult to detect before. 

But these are known interactions. But we can see them, where other people could barely see them. What we're now wanting to do is look for things that nobody's even barely seen. So the conclusions are that this IFAST technology, this basically fabricating a little linear device that involves oil and water phases, pulling an affinity bead through the oil to wash, has very good yield. We've gotten 95% recovery. Very good selectivity. We can discriminate against the things that shouldn't be bound. And it has extraordinary speed. A single phase is as good as two to three conventional washes and we can use a double phase device that gives us better than four washes. We took advantage of a system we had been working on, not with this in mind. 

We didn't even know that this technology existed. But when Dave came to me and said we want you to help us show that our technology will work to purify proteins, we had the perfect system ready. It was just one of those serendipity things, where you just have the right thing in the right place at the right time and things happened very quickly. We can therefore isolate these weakly-interacting proteins in amounts that are quite detectable. We can see them on gels. We can see them on something called a western blot, which is a very sensitive immunological detection. I'm sure there's plenty there to see in a mass spectroscopy, which are the three highly sensitive ways that you can look for things that were bound. So we're now trying to develop and show that it works well with mass spec, and it can be used to detect unknown, but physiologically important proteins, and that's the basis of the NIH grant that we're working on. We've used this, and other people have used it to find things that were weak, and now we can see them. And coming back to the Gates' grant, we are working with Dave's lab on what I would call an extremely simple, low cost protein isolation that's part of a point of care diagnostics system for use in developing countries. 

So what does that mean? Well, if you're sick and you live in a little village in Africa somewhere, and you walk a day and a half to a clinic and they say, well, you're clearly sick, but we're not quite sure what you have, but let's take a blood sample and we'll send it in to Johannesburg. And in a month we'll get the results back, and then you come back to the clinic and then we'll be able to treat it, maybe. Well, the person may not come back. Or they may have died in the meantime. I mean, what you'd really like to do is to be able to say, okay, take this blood sample, okay, put it into these well. We're going to put antibodies that will bind to antigens associated with various viruses and pathogens. We're going to do this, and then we're going to determine how much of those, whether those pathogens were present in that blood sample, or that urine sample, or that sputum sample. Then we're going to, right away, know what you have, and right now, we're going to give you the treatment. So it's point of care. That means that right at the place where you're going to be giving the treatment, you diagnose what it is. 

There's no lag for a month while somebody, the patient proceeds to get worse and worse. But the challenges are that I guess a clinic underneath the tree next to the river has no electricity, or very little. Maybe it has a solar panel, maybe not. But it has no $500,000 pieces of equipment that a normal hospital might have for diagnosing, for doing blood chemistry. It has no refrigeration. All this stuff has to be able to withstand temperatures that get up into the 100s, 110s, 120s, high humidity, not ideal conditions for storing biological materials. Anything that you would come up with would have to be simple. It would have to be cheap, I mean inexpensive. I mean, it can't cost $25 per device if it's going to be used in that situation. It's got to cost and be much cheaper than that. 

You have to, never the less, be accurate. It's got to be able to tell you, is that antigen there or not. And it has to be robust. It can't require that you deliver your blood droplet highly precisely. You just take a little thing you put in, whether you put six microliters, or eight microliters, or 10 microliters, it shouldn't matter. It should be insensitive to that kind of variation, because you don't expect equipment to be available to deliver things that precisely. So it's a challenge. It's a real challenge, but the idea would be, and so what the Gates Foundation did was to say, okay, we're going to give some grants for people who are collecting samples. What do you? You have sputum. It's viscous. 

Do you add an enzyme that will break down the viscosity so that the sample is easier to handle? How do you take and pre-treat the sample? How do you do this? This is all part of the equation. How do you analyze what comes out? If its nucleic acid, you can do something called a polymerize chain reaction, which amplifies it. But how are we going to sensitively determine whether there's a malaria antigen there or not? Then finally, after you've done that, you've got to integrate those results in a way that can be downloaded, sent to a cell phone, to someplace they can be stored and where the person at the clinic can use that information to decide on what would be the appropriate treatment. It turns out that the Gates Foundation funded 22 grants that cover these various areas. The Grant Council in Canada has had a similar project, and we decided to join forces. So they've actually got another one here, which is in communication. It turns out, even if we had the ideal thing, it's not clear that it would actually be widely disseminated and used. Most of these countries don't have any policy for certifying diagnostics. It's sort of sad. So there's a whole political thing here, which is how do you get people, even if you have the ideal thing, how do you get people to use it widely? How do you get the physicians to trust it? This is true of all new diagnostics, but it might be particularly true there. 

But the impact could be very large. It's sort of exciting to think that starting out, thinking about how do we purify proteins in a very abstract way, that we've led to a situation where maybe we can provide an important component that might allow rapid diagnostics and treatment of disease, even in places that do not have the expensive facilities needed that we have in a modern hospital. So this device that was invented in Dave Beebe's lab, recently a company has been started called Elaion, which is the Greek word for oil. This is to answer the question when my colleagues say, well, how can I get this. Well, this company is going to be making these things. Bill Checovich is sitting in the third row there. He's the person who's been doing most of the business development work for Elaion. I think he's been getting a very positive response as he talks to both scientists and companies who want to start to use this. We have the market here, potentially in diagnostics, but there's also clearly a market in the research arena, in the drug development arena. So it's maybe the pharmaceutical companies who will want to use this. 

We'll see. Anyway, thank you. [applause]