It would be really great if people could just share organs and tissues, and even better, if we could just grow them in the lab. Trust me, we're working on it. However, as Edward Djerejian of the Baker Policy Institute once said, "Vision without funding is hallucination." The NIH and NSF funding has taken a big hit in budgets in the last decade or so. Over 100,000 people are waiting for a solid organ, and that doesn't count tissues like bone marrow or corneas or skin. A new person gets added about every 10 minutes to this list. Behind me is an image of the National Donor Monument in Naarden in the Netherlands. This honors people who have donated organs and tissues to help other people live. As of the last time I pull stats up, 18 people were dying on a wait list each day. So, preventing rejection and therefore wastage of precious organs is another example of how understanding immune response is critical to providing good medical care. The problem is even more acute for people of African ancestry, because of the greater genetic diversity on the African continent. Africans and their descendants in other countries have a much broader range of MHC types. So, finding a good match especially for bone marrow, which needs a perfect match, is harder for them. They would be much more likely to benefit from increased knowledge in providing tolerance. One of our most important tools in preventing transplant rejection is to match the organ to the donor with respect to blood type and their MHC I and II alleles. So, we're going to take a brief detour to look at how we use the immune system to develop a variety of kinds of very specific assays. Then, how we can apply some of these assays to identifying specific tissue components. This assays all depend on the technology used to make monoclonal antibodies, something we discussed in detail in lecture four. So, the important points that we need right now is a reminder that once you have a plasma cell, it's going to make all of the antibodies with the same CDR. But the sad part of that is it's only going to last for two weeks. So, we need to fuse it with a B cell cancer cell, a myeloma. When we do that, we have something that we call a hybridoma that will last indefinitely and keep chucking out those antibodies. Again, the monoclonal antibodies. So, this will continue to divide, continue to chuck out antibodies, and we have essentially a money tree. The neat thing of all of this, is that it's going to make a ton of antibodies that are all identical. So, the plasma cells are engineered for immortality by fusing them with myeloma cells. The few cells are selected for in high medium which has two DNA-based precursors hypaxanthine and tydamine, and the aminopyrine or methotrexate that we saw before, which prevents de novo synthesis of DNA. So, only the hybridoma can survive in this environment and make all these wonderful antibodies. So, what are we going to do with them? Well, we want to select a hybridoma line that makes an antibody to an antigen we want to test for. So, this is one that we're going to use hypothetically that binds to TSH. This particular assay is still used to assay for TSH today. Again, a reminder you have to go through this process for every single antibody, you want different antibody, you want to make, so somebody did this one for TSH. If we want a different antibody that does say HCG, we need to go through this procedure all over again. Once we have the antibody we need, we now need to radioactively label a bunch of TSH. We're going show the radioactive label TSH in this nice yellow and orange combo. So, usually these are now labeled with iodine-125, this has a half-life of 60 days. So, the bad news is you have to keep generating it from a nuclear reactor and then attaching it. But the good news is when you're done with this assay, you can stick these in the back closet and in a couple of years the radioactivity will be essentially gone. So, what I'm going to do then is put these two together. So, here are a bunch of the TSH antibodies with radioactive TSH attached. In the current scheme of things, each one of these things is going to be attached to a surface, so that they can't float away. Now, the next thing I want to do is add a sample of serum, and I want to test the TSH levels in this serum. So, I put the serum in here with the TSH and what the TSH it's going to do is displace some of those radioactive TSHs. Now, the next thing I do is wash this off and now I have removed all the radioactive TSHs. So, you can see that what I have done is essentially lowered the radioactive reporter signal in this particular sample using the TSH from my testing serum. Okay. Now, in this case we had relatively little TSH. What happens, if we have more TSH. So, here's a sample with a lot of TSH. You can see it's going to displace the TSH. It will displace even more of it and I will have even less radioactivity remaining. So, I can graph this. I'm going to have sort of an arbitrary measure of radioactivity on the y-axis and I can see what happens to that with an increase in concentration of antigen in the blood on the x-axis. Now, this particular one is for TSH, the physiological range of TSH is typically around one to three or so nanograms per milliliter. The whole idea is I'm going to set this up so that I have a linear change in the radioactivity in the range of the probable range of the antigen that I have supplied it. Obviously, I would also have to calibrate this differently for different antigens, different things that we're testing. There are a number of technical tweaks that we have to do with this. One that I reminded you of before is that we now stick the antibodies onto a surface, so that it makes it easier to add a sample and then wash off any radioactive TSH that's displaced. So, I'm making this sound way easier than it was to develop. So, as it turns out the person who developed this Rosalyn Sussman Yalow, actually won in 1977 Nobel Prize in Physiology or Medicine for having done this. Again, she had to go through a whole lot of really technical manipulations to get it to work. I want to say also though that she was trying to do this by the available of monoclonal antibodies, and that once she did this, she established the principle that you could use monoclonal antibodies in a very sensitive and specific test for antigens. In particular in this case, these were in most cases hormones or other biological substances in the blood that were difficult to measure. But since then, this principle has been expanded to the ELISA reactions, which don't take radioactivity, and can actually be put into over-the-counter testing kits. This has also been expanded to check for a variety of other environmental pollutants and hazards. So, I want to thank Rosalyn Yalow, for doing the kind of cutting edge work that leads to solutions to problems she wasn't even thinking about at the time.
