 Today's lecture will be delivered by Dr. Uma Sinha Dutta, she is a Global Training Manager at G Healthcare. Dr. Uma's research experience includes molecular virology, molecular and structure characterization of segmented RNA virus, early detection kit, both for immunodiagnostics and RT-PCR. She is an expert in surface plasma resonance technology, especially GE's Bayakur certified trainer and in cell analyzer GE's high content analyzer. In the next 2 lectures, Dr. Uma will explain the concept of surface plasma resonance technology by using Bayakur technology platform. She is going to provide you not only basic understanding of how SPR works and how the Bayakur technology platform works, but also a brief overview of applications possible and how to process the data, analyze data and interpret in meaningful manner. So let us have Dr. Uma Dutta Sinha's lecture today. I had a chance to look at the Bayakur, we talked about some of the SPR technology, how it actually determines the interaction based on the surface plasma resonance technology. You know what's the association phase, what's the dissociation phase, regeneration's required to run the cycles. So post that I would like to talk about a little bit on the assay development part. When you are ready to do your, or start up with your Bayakur experiments, what are the things that you would like to take care of when you have to interactance and you would like to see the interactions, what are the things that you would like to optimize, which one should go as a ligand, which one should go as an analyte, you know, various other things, what would be the reference service like, you know, things like that. So let's start. So this is the basic assay development. Before I do that, I just, I would like to spend a minute to talk about the organization that I have worked for. So I am part of GE Healthcare and I work for a small group called FastTrack. FastTrack is actually a group which offers services to the customers. There are two parts to it. First is the process development. The other is the training and education. And I am currently the global FastTrack training manager. So I take care of all the trainings that happen globally. And FastTrack, like I said, it's a global organization. It is located strategically over the world and, you know, we do all kinds of process development and training. So coming to the objectives of the lecture. So like I said, we will talk about on the assay development part, what you would do when you first would like to set up a beer core assay, you know, what are the optimizations that you would like to do, what, you know, things like which one to, what, first of all, how are the, you know, assay, first assay, how does it should look like, then which one can be a ligand, what can be an analyte, what are the different reference surfaces that you can do, you know, and very important also, we talked about regeneration. How do you optimize the regeneration condition? Because, you know, if your regeneration is not perfect, your runs typically do not go so well, right? So the first, so let's look at some of the assay formats. The first one is the direct binding assay format, which is very simple, you know, your interactant is actually immobilized on the surface, like you can see here. So we talked about in beer core that you have a ligand and an analyte, right? Whatever goes on the surface is called the ligand and whatever is flown on the, on the flow cell is called the analyte. So in the direct binding, your ligand is actually covalently linked to the surface and your analyte is moving on top, right? You also have another direct format, it's nothing, we call it an enhancement or it is also similar to the capture method, right, what we were talking about. So the first, the capturing molecule is immobilized, then you actually bind your ligand and then your actual analyte comes and binds, right? So this is, you can also call this an enhancement molecule, sometimes if you're, if you're treating this as a direct binding, if this molecule is extremely small, then you can use an enhancer, you know, a specific molecule that it binds to, read it. In the direct binding cases, your sensorograms would look something like this. In this type, you, this is where your baseline is, this is your association phase and this is your dissociation and if you're looking at, and after that you can actually do a regeneration for this type only, but if you're looking at this enhancement, what you do is actually, first this is where your red molecule is binding to your ligand and then your enhancer is binding. So that's your final response that you look at, right? There are certain indirect bindings that you can do where we use it, we utilize the competition. So the binding doesn't really happen on the surface, you're having the binding happening on the, in the solution, right? So this is called the solution competition. What you do is here, you mix the analyte and the detecting molecule in a particular portion. The analyte is kept constant, where in with increasing concentration of detecting molecule, okay? And you mix them. So with the increasing concentration of detecting molecule, what will happen is you, you will, with increasing concentration of detecting molecule, you will have free detecting molecule in the solution, right? And then when you put this mixture here, so with the increasing concentration of analyte, you actually see drop in response unit, right? So it's, it's actually a reverse, you know? With increasing, usually in the direct binding with the increasing concentration of analyte, you see increase in response unit. Here you're seeing just the opposite, right? This is another one. It's a similar format. So here, you're actually having a competition in the, in the term that you are, you're having a competition in the solution. The bound ones, the bound ones will not come and bind here. Only the free detecting molecule are available to come and bind, right? Now here, when you, you're mixing it with, so this is where we call, it is a surface competition. The competition is happening at the surface. You have the analyte as well as you attach or link your analyte with a competing analyte, in the sense it's, it's a high, high molecular weight analog where you link it with. And the key is the high molecular weight analog when you attach it. The, the size of this should be considerably larger than your analyte. You mix them. Here they actually, in earlier case, the binding was happening in solution. But here, of course, there is no binding. When you put this mixture on, on, on your surface where your ligands are bound, you see the binding only because of your competing analyte, okay? When you, these analytes are so small and negligible, you do not get to see your binding. So here too, with increasing concentrations of your analyte, you actually see less and less of binding. So it's again a reverse plot. So if you summarize a response versus analyte concentration in direct binding, you see increase in RU with analyte, whereas in indirect, you actually see a decrease in binding with your, so these are basically two types of, yes, sir. So on the direct enhancement, is that, how long do you get the rate constant of that? Is, or isn't that dependent on the rate of the enhancing moment, why there's that? That's, that's right. So you, you probably will not get the rate constants there. You can only do only certain applications, like concentration determination. Yeah, that's a very good point. Yes, so you, so these are different various formats that are offered, but then again, it is limited to what applications you are using to, yeah. Okay. Coming to the general steps of Birkor assay, I think I don't need to explain that. You are all now quite comfortable with this. You understand this, but we'll talk about now in detail on the surface preparation. Why, you know, so surface preparation. So when you have a set of two interactumers, right? You would like to use it in Birkor to see and bind it, right? So you need to first understand why you would like to, which one you would like to choose as a ligand, which one you would like to choose it as a analyte, things like that, okay? So first of all, what is immobilization? We all know, right? How do we stick it to the surface? We covalently link it, right? It is not just any attraction, it's covalently linkage, right? So once you immobilize, that is immobilized for good. You cannot strip it out, okay? So once you immobilize something on the surface, you can't strip it out, right? So points to consider, you know, first when you have it, which one you would like to immobilize? Then how you would like to immobilize? Which chemistry are you going to use? Whether you're going to use the direct binding approach, you're going to use the indirect binding approach, all these things, right? What is the immobilization level you would like to use, okay? Because when different applications require different immobilization techniques, amounts, right? So for example, in kinetics, you know, adding too much of ligand is extremely detrimental. You do not get the right kinetic data. Your ligands have to be immobilized very, very low. Whereas if you're doing a concentration analysis, you need to have a high ligand concentration. So these knowledge is very, very important, okay? And which sensor chip is suitable for your assay? We talked about various sets of sensor chip, like, you know, CM5, CM3, C1, when you have a lot of non-specification, hydrophobic for HPA, you know? So all these things, NTA for nickel tagged, if you have something like that, okay? What to immobilize? You come back to the question of which one to immobilize. So the first thing that you would like to look at is actually the molecular weight of the interaction. Which one would you think should go as a ligand if you have two interactants? And one of them is large and one of them is small. Which one would you like it to go as a ligand? The small one, absolutely. Because you can use the larger one as an analyte, so you get to use a, you know, higher response, you know? But having said that, with the Biocode T200, the sensitivity it offers, even if you have a smaller, you know, small molecules can still be used as an analyte, but you do see some amount of background, you know? A noise. But then it is still enough to do a, you know, kinetic analysis, but not with the earlier versions like X100 where the sensitivity was not as high with the T200. Tagging of the interactants, if you have a tag, you would obviously like to use a capture method to put it as a ligand, right? Functional groups. The few things. Functional groups and binding activity of the immobilized, they go together, actually. Because if you are using a functional group to immobilize, which is actually in the active pocket, then, you know, you destroy the activity. Then you, you know, then there's no point, right? So, you need to have some amount of information of, you know, the functional group that you are using and that is not being used to, you know, immobilize it on the surface, right? Purity. The most pure one should go as a ligand, you know? You cannot have an impure ligand. The more junk you immobilize, the data becomes more dirty. You can still use it as an analyte, you know? So, of course, again, when I talk about analytes being impure, it's only limited to applications like binding experiments. If you have to do kinetic, you cannot have, you cannot afford to have a impure analyte. But if you're doing a concentration analysis, if you're doing a binding, it's fine to have a slightly impure analyte. Valancies. The more number of valencies, where do you think it should go? It should, you know, so like an antibody and an analyte. So, you use anti-beta-2 microglobulin today, right? And beta-2 microglobulin as an analyte. Which one did it go on the ligand? The anti, the valency was 2. You put that on the surface, right? So, but if you had done the opposite, and, okay. So the question is, if you have an antibody on the surface and have an analyte on the, flowing on the surface, it is actually a one-to-one binding because we call it as, you know, the binding is considered with respect to the analyte. But if you have the antibody on the, flowing on the thing and your analyte, the same detecting molecule is on your surface, then it becomes bivalent analyte, right? The bivalent analyte. So your mode would change. PI of the protein. PI of the protein, I would like to slightly stop by out here and we will explain it in greater detail in the upcoming slides. Because that you will see is an extremely important when you're immobilizing your ligand, okay? Amount available. Obviously, if your amount is very available, is very little, then, you know, you would rather use it as a ligand because when you're using it as an analyte, you have to, you know, run various number of cycles leading to more number of, you know, requirement of analyte. And then assay requirements. Of course, assay requirement is very, very important. What actually do you want to get out of the result? That is very important too. Surface preparation, I think you saw this slide. You know, surface can be prepared in two ways. One, you directly immobilize the ligand, right? In the other one, you actually capture your ligand. So in capture, your actually capturing molecule is actually immobilized directly on the surface. Now the difference would be is that, you know, in this case, you lose the directionality of your ligand like we were discussing. So it is actually immobilized at random using, you know, any of the free amine groups or the thiol groups on the surface. Whereas if you're using a capture molecule, you maintain a directionality of your ligand. If you, if your assay requires so. This one that you're looking at is actually the step of your immobilization. So how do we immobilize is like, you know, first we activate our surface using EDC NHS. And don't ask me what's the full form because I really cannot remember ever the EDC NHS, it what it does, it actually activates your surface into a, the carboxyl groups into a reactive ester group, okay? And that's where you're seeing the EDC NHS is being pushed. After that, you know, at this point, your surface is actually activated. And then you push your ligand, okay? With, which has the free amine groups or the free other groups. And at this point, all your proteins are getting covalently attached or linked to the surface, okay? And then finally, you do a blocking with ethanolamine. The, this blocking is to block all the activated ester groups which are not, which has not formed a covalent link. Because if you do not block at that stage when you're actually passing your analyte, they may come and bind there, right? So this blocking step is extremely important. And the difference from here to here is actually your immobilization level, okay? Choice of immobilization strategy, it'll depend on your ligand again. Amine coupling is very widely used. You know, in most cases, you know, more than 90% of the cases we typically use amine coupling, particularly in proteins when we are talking about because there's huge amount of amine groups available in most proteins, right? So if your ligand is actually unstable, then you would actually use a capture method, right? If you're using a covalent chemistry to immobilize and your ligand loses its activity, then you would rather use a capture method. If it's an impure ligand, like remember we said that your ligand needs to be very, very pure to be put on the covalently linked on the surface. But if it is an impure ligand, then you can do is capture it, right? So you can only capture your specific ligands. If your covalent linking actually loses the, you know, results in loss of activity, say for example, you have done amine, then you, and it loses the activity, then try and use a thiol coupling or aldehyde coupling. For acidic ligands, typically, you know, capture chemistry is mostly used. And if regeneration condition is also, you know, difficult, like if you have not found out a regeneration condition, sometimes it is very difficult to find out a regeneration condition for some cases. In those cases, also you use capture chemistry, okay? Coming to the PI point, which is extremely important here when you're immobilizing your ligand. And I think I don't need to reiterate this, you know, we all know what's the, you know, what happens when your protein is put in a pH less than your PI, your protein is actually positively charged, right? And if it is more than your PI, your protein is actually negatively charged. So when we use this, it's the same thing in a more schematic thing. Now, the PKA of the surface, the chip, the PKA of the surface is actually close to 3.5, okay? So if you are going less than pH 3.5, so I'm talking about a scenario where I would like to immobilize my ligand and I would like to put my ligand or the protein in a certain buffer where it is in a particular charge, positive or negative, let's decide that, right? So if I put my protein in a pH 3.5, say, most likely if the protein, of course, PI is higher than that, it'll be positively charged, but the surface actually loses its charge, okay? It has absolutely no charge. So in that case, you know, there is no attraction between the protein and the surface. And why are we talking about the attraction between the protein and the surface? Because we talked about covalent linkage, right? But the covalent linkage to happen, the protein has to come close enough to your surface so that your covalent bond can be formed, okay? So this scenario where your pH is very low, typically it's not a good scenario to immobilize your ligand. You do not see immobilization. So it's too low for immobilization. Now at a pH higher than 3.5, you know, your surface actually attends a net negative charge, okay? And if your PI is higher than that pH in that particular pH and you're able to keep the protein positive, you know, then your attraction happens. Okay, and this is the ideal scenario where your, you know, covalent linking can happen very ideally. Again, if your pH is higher than your PI, you know, then there is no attraction because both of them becomes negative, right? So that's what we call it as pre-concentration also. So when we are doing and ligand immobilization and sometimes we see that we are not attaining the amount of RU that we are expecting, this is something that is extremely important apart from the chip quality and EDC quality and things like that. So this is also something very important to keep. And this is the same thing that we talked about. Now, you know, not all the time you may have information about the correct PI, right? So we have a tool called scouting, pH scouting, which actually tells you which buffer would be most convenient or ideal to use it for immobilization buffer, okay? For immobilization purpose for that particular protein. So you can take a little bit of your protein before you do your actual immobilization and run into this pH scouting experiment, okay? Take a small amount and then mix it with various buffers. Typically the buffer that we supply is from pH 5.5 to pH 4, okay? Mix them in all different, same concentrations but different buffers and then run them. And then try to compare and see what is the peak like. Remember, here there is no immobilization happening. It is just the pre-concentration, only the attraction that is happening and then you see the increase in IO out here, right? So in this case that you can see that pH 5.5 is the lowest and the highest is pH 4. So what does it, what do you think it means? That does that mean that pH 4 is the best for immobilization? So most people think that increasing the lower the pH we go, we can attract the protein more and eventually we can get the best. And well, there is some amount of truth in that but not always, you know? So if you see out here in pH 5.5 which is actually kind of like the highest pH here, you attend quite large amount of RUs, okay? You go to around something like, I can't even see 10,000 RUs which is an extremely huge amount of RUs. You don't need that much amount of proteins to, you know, if you are reaching around 4,000 or 3,000 it's more than enough to, you know, do any of your applications that we are talking about. Now, so we actually did not get enough information from there, so we took the same thing and we ran it through the whole immobilization process, right? And you see in pH 5.5, you get to around 13,000 RUs, right? And pH 5, you get to around close or maybe similar. I don't think there's, I mean, 13.5 and 12.6, I think I would say it's very close to each other. 4.5, there's a reduction, it's much less and then again, four is even less. So whereas our pre-concentration earlier showed, you know, with 4.5 and 4, it was still increasing. But here, actually, you are actually attending much lesser RU than considered to pH 5 and pH 5.5. The logic out here is actually, so when you are actually attracting so much in pH 5.5, increasing the pH, lowering the pH even further and attracting doesn't really help for covalent linkage. So that's where you start getting your steric hindrance and where your, immobilizing, your covalent linkage is not again happening in an ideal scenario. So the key thing, like she said, is to have your proteins in a comfortable environment close to where your proteins will be comfortable, not stress them out in harsher condition. If required, if you do not see any rise, like for example, in these cases, if you do not see any rise in pH 5.5 and 5 and suddenly you see a pH rise in 4.5, that's where you go into a lower pH like 4.4, 4.5 or 4. Otherwise, stay close to, you know, more comfortable environment. Does it make sense? Okay. Now, I think somebody was asking about the immobilization level. So this is, so how do we find out the immobilization level and in in Biacorne term and how much should we immobilize actually when we are doing a Biacorne assay? So we have this formula, which we call it as, it's kind of like a, what do you say? Vedvake in Biacorne. Our max is equal to molecular weight of analyte by molecular weight of ligand multiplied by RL. Into the stoichiometry. So R max, we haven't talked about R max yet. R max is called the maximum binding capacity of your surface. Once you immobilize your surface, the capacity of the surface to bind maximally your analyte, that's your R max. RL is the ligand immobilized. So when you immobilize your ligand, you get a particular R U, right? Say for example you immobilize 500 R U's or 1000 R U's, right? So that's your RL. A stoichiometry of your binding, whether it's one is to one or if it's a bi-vand it's two, right? So typically theoretical R max, what we find out from here is higher than your experimental R max. Usually when you actually immobilize your ligand, you tend to, you know, lose some activity, you know? Or your ligand to begin with may not have 100% activity. But in some cases when you see much higher R max, you know, inexperimentally as compared to theoretical, immediately it should strike a bell that, a bell should ring in your mind and something is wrong. Either the stoichiometry that we considered was not right, or there is some non-specific activity, or there is aggregates happening, right? So there's a small exercise, if you would like to do, hopefully it'll wake you up if you guys are sleeping. So say for example if we have a ligand that I would like to immobilize, and I would like to work at an R max of 100, okay? And the molecular weight of your analyte and ligand are given. So your ligand looks like a map, like 150 kilo Dalton. Your analyte is 25 KDA. The stoichiometry is one, and I would like to reach an R max of 500, right? So how much RL, how much of ligand should I immobilize? Anybody? 600, right? So if you get 600, then typically I would, since your theoretical R max is usually higher than your, you know, experimental R max, we typically go ahead and if it's 600 from your theoretical I would go ahead and immobilize 700 or 800 to compensate a little bit and then you know, start your experiment, okay? Is it okay? So the circle of R max? Right, yeah. So if you're doing a kinetic analysis, you have to be, even 100 R use is quite high sometimes. Sometimes you have to go as low as 50, 20, or you know, T 200 actually allows you to work as low as five R use, R max, okay? So you can still get a very decent, nice, without noise graph, which we can perfectly do an evaluation on and get a K, KD value of it. If it's a concentration or affinity determination, then it has to be on a higher range. Like you know, when I say higher range, it can be around say 3000 to 5000, ranging on that. So you find out the surface saturation. Say for example, you know, if you're immobilizing a 150 KDA molecule, so your surface saturation comes at close to around 10,000 to 15,000 R use, okay? That's the surface saturation. Yeah, roughly. So and if it is say around 25 KDA, then it is around 2000 or 2500. That's the surface saturation. And when you're doing a kinetics, you should never be in a saturation mode. You know, there is something called mass transport limitation that happens, which actually affects your concentration of your analyte. So you would have to keep your ligand concentration extremely low. So otherwise you get erroneous kinetic results. How much will come? When you say 600, 700, what's the unit? R use, resonance unit, response unit. Resonance intensity. Yes, exactly. So like I said, right, it is related to the refractive index. So the change in refractive, the change in mass will change the refractive index and that is related to your response unit, response unit. Because the analyte molecular weight is not too high. No analyte molecular weight is there. Yeah, okay. It can allow a lot. I mean, and then it's not about T 200 allowing it. It's the analyte and the ligand molecular weight which will contribute to it. Yes. First is five uses. Five R use of R max. Yes. Okay, so I'm sure this was very informative lecture by Dr. Uma. You are convinced that she's able to convey the very hard ideas and principles involved in this technology in a very lucid manner. In the next class, Dr. Uma will continue to explain some more detail of Bayer-Core technology. She'll also provide a demonstration and working of how to do SPR experiment on Bayer-Core platform in the next class. Thank you.