 Today's lecture will be delivered by Mr. Sushil Vaidya who is an application scientist for Forte Bio Paul Life Sciences. He has worked in proteomics, lipidomics, small molecule characterization using mass spectrometry, HPLC, and LC-MS based method development for biomolecules and small molecules. He has thought understanding about the technical strategic planning in analysis of biosimilar characterization. In the next two lectures, Mr. Vaidya will have interaction with us about novel label-free biosensors, especially he will talk to you about biolayer interferometry based platform through a lecture and a demonstration sessions. So, let me welcome Mr. Vaidya for his lecture on BLI technology. Mr. Sushil Vaidya, I am an application scientist for the Paul Forte Bio instruments. I take care of these applications parts in the entire India where exactly the installation, the support, the training as well as the product promotions. This is the alternate technology where exactly the biolayer interferometry, the interference based technology. The SPR is the one the surface plasmon resonance, but this is the technology wise it is a very different and as well as the high throughput platform. So how this technology will be helpful in your the interactomics kind of workflows and I will go through the some what the technology behind the informations, how the principle behind this, how you can use this technology in your applications. So, I will go through that as you know that from the morning onwards we are discussing why the biomolecular interactions are very important. If you look at in our body systems all most of the living organisms whatever is happening in the systems all through the like a transcription factors binding to the DNA or the protein complex formations or in terms of the signal trans deductions where exactly the hormones and the growth factors and all what interactions happen those things and as well as the immune responses the antigen and antibody interactions. All this works on based on the interactions and even when it comes to the drug discovery where exactly we want to understand the mechanism of interactions, how the drugs is binding to the particular target when we are discovering such kind of a molecules we have look at the affinity. So when it comes to the dose I can give a simple example any drug if you take it like a some drugs you have to take it like a thrice set three times a day some drugs you have to take it a once in a week some drugs you have to take it in a like a once a once a day. So, how all these these dose actually decides. So, these dose dose are depends upon the interaction platforms. If you say you have a target when it is any molecule you have discovered when it is bind. So, how the affinity is how strong is the affinity? If it is that affinity is a strong then you required a less amount of a drug. If it is you required a more dose then you have to like a the action will be like a it is clearing from your body very fast then you required a more doses. So, how these will be helpful in your directly square by areas. So, in this actually when you look at all the systems interaction is the very important phenomena. So, we need to understand we need to characterize it. So, if you look at what are all the conventional technologies people are in routine they are using the ELISA based platforms where exactly people are using for the screening of the interactions. So, when it comes to the limitation of this ELISA is like that more it is a time consuming and you required a man pour more on to develop the assays and then you had a screen further and you have to select the right candidate for that. When it comes to this it takes a long long time the limitation and the reagent and the consumption of the reagents also more and it is a more laborious. The other technology if I look at is the ITC isothermcular rheumatismation where exactly the interactions when it when you can decide the stoichiometry of the interaction as well as the where exactly the parameters like delta A is where the when any reactions happens in the interactions where there may be heat released in the medium or heat absorbed in the medium. So, those are the parameters we can find out, but the what are the limitation with the ELISA and the ITC is like that you can determine based on the concentration versus the response then you can plot where you can read the steady state. But this will provide you the KD value where what we call it as a KD is nothing but the affinity constant, but it will not provide you the kinetics parameters like on rates and the off rates. This is the limitations with the ELISA or the ITC based technologies. So, why people are moving to the label free platforms is where you can get this is the typical example when there is a ligand say suppose the compound A and the protein B this is the this forms a complex and this is the forward direction when the complex formation the finally it will dissociates to the once again A plus B. So, these label free interaction platforms are applicable to the reversible kind of reactions. So, if you look at when any molecule binding say suppose ligand A it is binding to the analyte when you see this is the response this is this phase we called as the association phase. When the same complex when it dissociates back so, the complex will be dissociates then you can see the dissociation. So, the real time interactions will provide the both on rates and the off rates this is the kinetic information. So, none of the any other techniques like ITC or the ELISA it will give this information. So, that is why we this is a very important tool for the on rate and off rate determinations. So, if I look at the one of the example when it comes to the ELISA based based on the steady state analysis if you look at the KD parameters for this from the ELISA it looks like a both are same when it when you same when you performed on the label free platform if you look at the kinetic constant parameters see this example the blue trace if you look at it is dissociating very fast. When it comes to the pink one the dissociation is very slow ok. So, how why this is important is like the based on this the rate of the dissociations we can able to see the differentiation between the when you are selecting the right candidate. The SPR technologies the one the real time label free interaction systems where exactly on the gold chip you have a matrix to that you are quoting a one of the protein of interest and then you pass the analyte through the fluidic systems and then you can see the interaction change with respect to the angle. So, when it comes to the limitation in the SPR when we are doing you required a like a dedicated operator for this and apart from that the time it required for the initialization and then get to the data it is like a tedious it is almost like a day it will take. So, when you when you are doing a such kind of a interaction you required a more patients and to get the data it is a more time taking and all, but when and apart from that the one of the important is the microfluidics delivery systems when you are working with a the any kind of a samples which are like a cell culture based or if the sample from the body fluids and all that you have so much cell debris or any other impurities and all. There may be a possibility of that there is they clog the system the fluidic system. So, that is very expensive in in in case of the SPR if it is something get clogged it is a very the flow cell is a very hairy like structure here typically if it is clogged then we have to replace the the the assembly of that the that is the very expensive and the the maintenance cost will be a more, but when it comes to the the other technologies the BLI technology there is a more advantage I will discuss on that and apart from that the low throughput actually here it is in the SPR techniques you have a channel you have to inject one concentration over the other serial dilutions you have to inject over the surface, but when it when it comes to the BLI technology it is it is a very high throughput that is why the limitations comes into that when you when you are deciding the right candidates in case of the screening experiments it is the BLI is the more advantage in that. So, when it when it comes to the BLI technology I am going to talk on this this is the Forty Boyer is the parent company they invented this technology in the year 2003 onwards and and and the for the first system if you look at the 2005 it is come into the market they called as Octet QK and over a period of year we have really different instruments come into the market based on the throughput and all that. So, with this I can I can start what exactly the principle behind if you take it most of the interaction systems or any analytical techniques all are based on the light light is the except mass spectrometry rest of the analytical if you take it analytical any all are light based as you know that light have a property the wave property is the one and the the interference when it works the BLI comes the bio layer interferometry, but the interference patterns makes the signal pattern here. As I mentioned it is a its works on the wave property as you know that when it when the light passes through the the fiber optic there is a some kind of a matrix you have a obstacle the wave it is forward hitting that matrix and get reverse back. So, what happens is this is exactly the forward wave this is the reverse wave when it when it both forward and the reverse wave when it is superimposed together you can see there is a amplitude get increase this is we called as a constructive interference when the same wave this is the forward wave this is the reverse wave when it is this is the forward and the reverse wave when it is opposite together at 180 degree when what happens is that signal get cancelled here. So, this is we called as a destructive interference. So, constructive interference and the destructive interference makes the signal pattern. So, how we are doing that? So, we are using a biosensor here as as in the SPR they uses the chips the similar way we are using the biosensor here. The biosensor if you look at this is the sensor typically looks like a needle needle this is made up of a plastic and this is your a glass capillary which is nothing but the fiber optic. At the tip of this fiber optic we are coating it the biocompatible layer it is a optic layer to that optical layer you are attaching a one of the protein of your interest. So, suppose you have a two binding partners one of the protein you have to attach on this it is a nothing but the solution solid interface. Solution solution interfaces are the I can say that giving example ITC ELISA you can see as a solid solution solid interfaces because one of the thing you are putting on the plate. So, these label free platform also like a solution solid interfaces. So, one of the protein we are immobilizing on here and then dip into the well containing a corresponding binding partner. So, this sensor is a two dimensional binding surface the matrix what we have quoted at the tip is the biocompatible it is inert in nature you can work with any kind of your physiological systems buffers or the you can work from the pH ranges from 2 to 10 depending upon the application. So, most of your biomolecular interactions happens at the physiological pH and it is a uniform when we manufacture the sensors to sensors it is uniform across the lots and we test and then we release us and the whatever we have the coated material is a non denaturing it will not interfere with your interaction system. So, if you took a the diameter of this the optic fiber is just only 600 micrometer you required a very less amount of your sample to immobilize on the sensor surface. So, if you look at how this exactly the instrumentation inside in the on the the biolare interferometry the octet platforms this is the spectrophotometer here and it is connected with a robotic arm and these are the sensor tree I can if you look at this is a 96 well plate form. So, the robotic arm pickups the sensor and dip into your 96 well plate form it. So, this the sample plate we have a orbital shaker and the temperature control what is the difference between the SPR and the VLIs we do not have any microfluidics here just it is works on the dip and read it is pick up the sensor and dip into the well. So, everything happened the reaction whatever the happening at the tip of the sensor. So, in case of the microfluidic devices like SPR the flow assist in the binding, but here we have a orbital shaker which assist in the binding. So, when it comes to the high throughput we have a different channels like in SPR if you take it there is a 4 channel 2 channel instruments. So, where exactly the 4 channel means you can pass the 3 analytes and 1 acts as a reference and here we have an instrument we have a 8 channel here 8 interactions you can measure simultaneously that is a where the throughput comes we have a 96 channel here 96 interactions you can perform in 1 goal and we have a 16 channel 16 interactions you can perform in 1 goal we have a single channel also 2 channel as even we have 2 channel automators instrument. So, how exactly what the principle behind this as I mentioned it is works on the interference based. Sorry, go back to that previous slide. So, is the right hand the on rate and the left hand the off rate or how are you doing binding and then how are you going to get this associated. So, the sensor this is the sensor compartment the sensor actually pick up the robotic arm pick up the sensor and dip into the well containing the first buffer and I will show you the in the subsequent set how it is. So, what exactly the principle behind this as I mentioned it is the interference based we are passing a light it is just a white light get reflected back if you look at you can see the reflections coming from the one internal layer and one from your the ligand end. So, first I take it I had immobilized one of the protein of interest on the sensor surface then I dip into the well containing buffer. If you look at all this what exactly it is amplitudes different amplitudes we have constructive interference, destructive interference this is what we exactly we plot relative intensity versus the wavelength. So, what happens say if it is the one which have a higher amplitude where exactly the waves get superimposed then you can see the amplitude get increased where exactly there is a partially superimposed you can see this kind of and there is a destructive interference there is a completely signal get cancelled. So, what happens when the same sensor when you dip into the corresponding binding partner you can now you can see that earlier was just only light reflecting from the it is a blue colored layer. Now, you can see there is a one more orange layer if you see this now right which is reflecting from the orange layer. Once again if you look at all these waves are there earlier was the plot like this due to the molecule starts binding to the sensor surface you can see there is a shift in the interference pattern. This is what exactly as the molecule binds to the sensor surface you can see the shift. So, what happens exactly on the tip the molecules bind to the sensor surface it forms a bio layer it is depends upon the thickness of the bio layer more the molecules bind to the sensor surface sorry back more the molecules bind to the sensor surface you can see the relatively shift in your spectrum towards your right this is what exactly the principle. So, using this phenomenon as the more molecule you can bind you can see the correspondingly it relatively shifts towards on the right. So, you can do the quantitation using this phenomenon not only the kinetics you can you can determine the quantitation. So, in real what happens molecules bind to the sensor surface you can see the real time picture as the molecules binds to send then you can the it is leads to the equilibrium then the same sensor when you wash off the bound molecule you can see the dissociation. This is what exactly the real time you can look it the background is the this one the molecules bind to sensor surface then make a bio layer and relatively shifts and the once you washed off then it will come back. The octet system from Forte Bio provides a complete label-free solution for analyzing protein kinetics and quantitation. The disposable biosensors measure binding events directly in a standard 96-well microplate. A proprietary protein coating at the tip provides reproducible coupling of target molecules and a minimum of non-specific binding. Here we are using strip-tabbit encoded sensors to set up a five-step kinetic analysis where the first protein of the pair is biotinylated and we measure the binding and dissociation rates of a second molecule. With the sensors arrayed in a standard SBS format some steps can be performed outside the instrument like conditioning the sensors before the analysis. Up to eight sensors can be run in parallel and the menu-driven software assigns identities to each sensor to be used. In our experiment each column of the sample plate is loaded with reagent so that we can analyze eight samples in parallel. Firstly a buffer to establish the baseline then a loading step with the biotinylated protein and a second buffer for a new baseline. The binding protein is being presented in a crude cellular lysate and the dissociation step will be measured in buffer. Once the sample plate is loaded the experiment is defined in software so that an appropriate analysis will be made at each step. Sample identities and whole method files can be stored and imported to simplify or standardize routine analysis procedures. Data files and results will be stored in a predefined location to be analyzed at the instrument or at a remote terminal. The sample plate is heated to the selected temperature and a flow across the sensor surface is established to overcome mass transport effects. Using optical fibers octet feeds broad spectrum white light down the sensor and collects the reflections. One reflection comes from an internal optical layer and one from the interface between the streptavidin layer and the surrounding solution. Most of the light scatters in the surrounding matrix. In a process that we call biolayer interferometry the two reflections generate a spectral pattern that is collected and analyzed at the spectrometer. Constructive interference gives intensity peaks and destructive interference causes intensity troughs right across the visible spectrum. As a calibration the position of the interference pattern is measured for all eight sensors and then shifts in the pattern are plotted in real time. The streptavidin sensors are loaded by transferring into the wells with biotinulated protein. Biolayer interferometry is insensitive to changes in refractive index or pH of the matrix. When more molecules bind to the streptavidin layer the optical thickness of the layer changes and as the thickness changes the interference pattern shifts. One nanometer of protein binding to the sensor produces a one nanometer shift to the blue in the interference pattern and a one nanometer change in optical thickness is recorded for that sensor in real time. The change in thickness relative to the initial calibration is plotted for all eight sensors. With real time monitoring and eight samples run in parallel it's easy to optimize protein loading so that it can be performed outside of the instrument. Once the biosensors are loaded they are transferred to a buffer solution to establish a new baseline. There is no dissociation observed from the tightly bound biotinulated protein. Biolayer interferometry is relatively insensitive to matrix effects and octet has no microfluidic constraints so measurements can be made in very crude samples. Culture media with serum, paraplasmic extracts or cell lysates with particulates can all be measured easily and accurately with minimal interference from the matrix. These crude protein preparations exhibit different binding rates and the real time data output allows you to monitor the behavior of the proteins as well as the overall progress of the experiment. The process is so simple that it provides a convenient tool for protein quantitation in complex mixtures. The dissociation step may be a five minute off-rate for a quick screening experiment or two hours or more of data collection to determine a KD for tightly binding pairs. Octet is simple enough to use as a screening tool and five minute off-rates can be measured for 96 samples in little over an hour providing ranking data for clone selection. Alternatively, a tightly binding pair can be monitored for over two hours limited only by the evaporation of the sample. After the dissociation, the biosensors can be ejected and new sensors selected without any need to run regeneration methods or run cleaning protocols. All of the sample solutions are recoverable or they can be reused in the same format with new targets depending on your experimental protocol. Because octet is so easy to use, assay development is rapid and you can quickly generate the data you need to make clonal selections or optimize a purification process. For accurate kinetic determination, single use sensors provide precise answers without the need to work up regeneration protocols. For higher throughput screening and yes-no determinations, reuse protocols provide a cost effective solution. And once you have developed an experimental protocol, you can automate the whole process all the way through to data analysis. At the end of the experiment, select data analysis and you have access to a complete suite of software tools. Curve fitting algorithms from Origin allow for the reporting of binding and dissociation rates together with display of residuals and data tables. Or you can export results into your favorite program for further analysis. With a range of biosensor surfaces including streptavidin, protein A, and amine reactive sensors for custom protein coupling, octet provides a complete solution for protein kinetics and quantitation. So what exactly information you can get from this? So as I mentioned, the one based on the shift, you can determine the concentrations. You can do the direct one step kind of binding sandwich. If you want to convert ELISA platform onto the instrumentation, you can convert it. So depending upon, you can determine the micromolar concentration you can go as low as the nanogram or the mg per ml to picogram per ml. So the quantitation you can determine and the kinetics. The kinetics as I mentioned on rates offer it in the affinity constant you can determine irrespective of whatever the biomolecules it's maybe a protein to protein, protein to small molecules, protein DNA, protein RNA and all that. Apart from the specificities where exactly you can do the functional testings, functional testing with respect to FC gamma interactions where the people are doing in the drug discovery. The rank ordering, when you're doing the hybridoma screenings with the large map platforms where you can provide the which is the best antibody will bind to the target. Based on, you can give the rank order on the off rate of the molecules. And the epitope binding, we have a dedicated software for the epitope binding. You can do the screening of your epi, where exactly your molecules binds to that. And the isotoping also, subtyping of your, the IgG's. So, we have a lot of applications on this. I will go through later, those applications. So, one of the important parameter is, most of the people are doing the quantitations based on the like Bradford assays or the total protein content. When you say, suppose you have a protein in a kind of a matrix where exactly you're expressing protein of interest in the cell culture or the want to determine in the patient samples for how much my protein of interest which is there or not. So, you can easily, because it works on the dip and read, there is a no such kind of say, even though if it is cell debris are there, impurities are there, anything has there, it is purely and affinity based interactions you can easily quantitate using this phenomenon. So, that's why the advantage is like that, you can go with any kind of sample matrix. It is need not like that, you have to go for the purified one. When it comes to the SPR, you have to go with the more purified samples. And the second advantage is like that, you can go with the pH ranges easily like a two to 10. Depending upon the application, you can screen quickly because it's have a high throughput eight channel, you can quickly screen the samples, which pH is the favor for the binding. So, these are the platforms we have, these are the 16 channel instrument, this is the two channel instrument, this is the eight channel instrument, and this is the 96 channel instrument. Depending upon the high throughput, what is required, you can choose the instrument in that. And the more advantages like that, as I mentioned, there's no clogging, it's works on the dependent rate. And the high throughput, you can save your time, you can quickly screen the experiments. And the good thing is like that, when it comes to the interactions where exactly your samples have a DMSO or the glycerol. When it comes to SPR, presence of the DMSO and the glycerol, sometimes you can see the bulk effects, because SPR is a very sensitive to those changes. But with a dependent rate, you can easily go with such kind of a, there is no interference from the glycerol or the DMSO in that. And the good advantage is like that, it's typically the software is so user friendly, just if I train for a half an hour, you can start your experiments, no dedicated operator required for this. Typically the programming is like your ELISA operator. So what are all the ranges? What kind of molecules we can go? Except cells and atoms, we can go in the blue areas, all these biomolecules we can go with the interactions. I can show you the cells recently, we have got a one year of the publication from the GenMap. I will discuss with those things. So these are the different applications we have segregated, kinetic applications, quantitative, screenings and development, assesses, developments and all that. We have publications wise, we have more than 1,500 from the past 10 years we crossed, all published in the different impact factor journals. So how exactly it works? If you look at the animation, if I say this is a typically a protein quantitation, if I take it any affinity based, so suppose you are working with one of the HISTAC protein, how much is expressed in your culture? So suppose if I have a standard in that, if I dilute in the plate, 9612 plates, lower to higher dilutions, take a 8 sensors when I dip in this, you can get a curves like this. Based on the binding rate versus the concentrations, the higher concentration have bigger and the lower ones are this. When the same sensor, if I go for the regeneration, so where the bound analyte get washed off here and then when it dip into the unknown solutions where your cells means your protein is expressed, you can easily quantitate using this plot. So very quick experiment in a just in the 15 minutes you can determine the concentration of your protein of interest. So how exactly works the kinetics workflows? First the sensor will dip into the well-containing buffer, you get a baseline here and next material comes to dip into the well which have the loading and then goes back to the well-containing buffer, there exactly the unbound material get washed off and then corresponding analyte it is binding, you can see the signal and the goes back to dissociation. The very simple it's just, you have to the robotic arm back and forth it will move. Just I will explain once the buffer, the sensor, the sensor dip into the well-containing, a first line of buffer, you can get it just a baseline. There is no any molecule binding to that, that's why you get a baseline. When the same sensor when it moved to the next well containing a capture molecule, one of the protein of interest you have to immobilize on the sensor surface. You are putting in the second well, then the sensor dip into that well, you can see the loading response. As I mentioned some molecules point to sensor surface, you can see the change, the phase shift. So you can see the loading response and the same sensor move back to the well-containing a buffer, here just a baseline, there is no molecules binding, unbound molecules get washed off here and the sensor next move to well-containing a buffer, sorry the analyte, you can see the response. And the same sensor goes back to the well-containing a buffer, bound analyte get washed off. Very simple to operate this experiment, you can quickly screen just in 15 to 20 minutes, you can get the kinetics data. So we have a, on the shelf, we have a different biosensors chemist with us, depending upon your protein of interest, you have a tag or you can choose or if you don't have any tag, you can go with some kind of modifications like biotinulations or the amine coupling, you can immobilize. So these are the pre-coated for the antibody platforms, we have protein A, protein G, protein L and some FC capture sensors. And for the HIST tag-based proteins, we have a nickel NTA, we have anti-HIST sensors, you have GST tag bases, we have anti-GST sensors. And if there is no any tag, your expressions, then you can biotinulate your protein of interest and then you can couple to the striptavidin sensor, the biotinulated protein. We have a APA sensors, a minopropyl saline for the hydrophobic interaction, your protein is more hydrophobic, no need, no need, any tag or anything, just you can just bind to the protein of interest on that. And for the striptavidin, we have a, this is the quartz-based sensors, especially for the small molecule protein interactions. So come to the application side, where, how we are going to use, the one of the example here is, this is the one of the group they had, they are interested in developing the vaccine-based on the influenza. They identified the two antibodies which are broadly neutralizing capacity to the influenza. In this experiments, what the author did is, he isolated a 15 group, one and the 44 group different hemagglutininin, he had taken from these groups and then he did a biotinulation and then he immobilized using the striptavidin sensors, all the 59 hemagglutininin to that and then screened with the respective, the antibodies. Among that, if you look at these, you can see the binding interactions of the different antibodies to the respective hemagglutinins. From this, they had developed the broadly neutralizing of the two antibodies in that. So very quickly, we can screen this kind of applications using this. So next example I can show is the, where we can understand the protein structure and the function. Here, what is the LED7 is the one of the important tumor suppression microRNA. So in this, what author did is the KSRP, KSRP is the protein which is binding to the LED7 precursor. So what happens is here, the KSRP and the LED7 is your microRNA which is biotinulated and immobilized onto the striptavidin sensor. And this KSRP have a four domains. So among that, he did a mutation. In one of the protein, the domain KH3, if he did a mutation, it is not binding. So with a wild type, he had showed that it's the KSRP binding. In the four domains, among the one KH3, if he did a mutation, it is not showing a binding. So using this, he concluded that with respect to this, KH3 domain is the one of the important parameter for the binding of the KSRP protein to the LED7. So it is one of the important, the transcriptional factors for the cell differentiation and very important in case of the recruiting the oncogenes for the, where exactly the cell differentiation happens. If it is a mutation happens in that particular gene, there may be chance of the carcinoma and all. So one more, where exactly facilitated binding interaction studies here. So this is one of the example where exactly the fuse system is the one which is important in the cell differentiation, seismic is the important regulatory gene. So this sick means the expression of this particular gene is more in case of the cell differentiation. So how this happens, this regulation? So the author concluded using this, there is a called as a FBP protein, presence of this FIR only interacting to the FBP protein. So in this experiment, what had done? The biotinylated the fuse for the DNA, he immobilized onto the striptavidin sensor and then he performed with the binding of the FIR. So in presence of the FBP protein only the FIR is binding, without this proteins it's not binding. So he concluded like that. So very important tool when you do the, in case of the basic research, you can quickly screen those. So one more example I'm showing here is the folding and unfolding patterns of the proteins. So in this example, the sensors were exactly the biotinylated, the growl is immobilized on the sensor surface. The unfolded protein will be binds very far, then you can see the response. The folded protein will not bind. So using the chafferone model you can use for the screening whether the folded or the unfolded patterns can easily study. And the one more example, in case of the diagnostic immunosys, giving example here is like that the kinetics parameter 15 anti HRP to antibodies they measure. They compare with the ELISA where they had, comparison they had done. You can quickly screen the which of the pairing, the antibody pairing which have a better diagnostic for the diagnostic applications. When you are developing any ELISA kits or, you required a primary antibody and the secondary antibody. You can quickly screen using that. So which are the best based on the, you were the kinetics profiles. So after most screenings. So Aptamer is one of the area now is picking up comparison to your antibodies. Most of the many laboratories in India they are working on the Aptamer's. So Aptamer's also similar, they have a different size and the shape. When it binds to the particular, the target you can see the response. So Aptamer's also have like a major application in diagnostic industries. So one of the example you can quickly screen using the Aptamer interaction studies using the BLI technology. So the one more important where exactly when it comes to drug discovery applications, small molecule protein interactions, if you look at with a one concentration different, you can screen as many as compounds as possible. And then you can look based on the off rate, you can select the right candidates. This is the one of the work from the University of Michigan. They had done a publication on this. They have developed the three heats with the complete kinetics characterization using this, the bromodamine one protein. And the one more example I can show is the characterization of this AMPK protein to the two compounds thaladazine and the one more here. So thaladazine and the structure isomer and the hemindazine, it's like compared to this, the thaladazine is the more binding compared to the other platforms. You can quickly screen using this tool. And one more, the next very important when it comes to the ligand fishing experiments, I think it is more relevant when it comes to the interactomic studies. Because what is exactly the ligand fishing is, so suppose you don't know what is the protein is interacting to your one of the protein of interest. So suppose you take it, the protein of interest when you can immobilize on the sensor surface and then put it into the cell extracts or a lysates or anything, which is the protein is binding to the sensor surface, you can elute it and then you can go for the mass spec studies. So one of the example I'm here I'm showing is the interaction you dip it and then you can elute those the binding combinations. And you can go for the electron microscope or you can go for the mass spec studies. So here one of the example I'm showing is Montenegro Anthropoxyne prepoor. This example they activated the tip and they had immobilized the protein of interest, the anthrax prepoor and then they had processed with the one molar urea at 37 degree. The prepoor to pore conversion happens and when they dip into the lipid collet mixtures, you can see the micelle formation between the pore and the lipid. So what they had done, removing the biosensor tip and they dip in a buffer and decoupling reagent then preparing for the EMI grid, you can look at the electron microscope, how the complexes are. So you can easily you can perform such kind of experiments because when it comes to the SPR, when you elute the complex mixture and when it comes to when you are going to collect that because your sample is more diluted, then you have to concentrate it, then you have to perform the further studies. You can enrich in the dip in the well itself here. So one more example is the, it is an industrial work, the erythropoietin they were expressing the Novoda-Nordis company where exactly happens when they are doing the quantitation, they are getting the concentration very less. Then they thought that what happening in that? So they identified some host cell histones blocking the epobinding. So they eluted those mixtures and they perform the mass spec with that. Then they identify that the protein of interest is binding to the, it's blocking the binding of the epobinding to that. The GenMap recently has published this data. They immobilized the cell, the entire cell on the sensor surface. And they had tried a different combinations here. One of the example I can show you is they had taken a collagen here. They immobilized the collagen on the amine coupling sensor and then the cell which is the A431 cells which is expresses the EGFR and the beta adrenoreceptors, orexpress receptors. They put it the cell on the sensor surface and then they perform the interaction studies with respect to the EGF here. You can see this, the binding interaction. It is not exactly the kinetics. It is orthogonal to your cell-based assays. So what happens is, say suppose it is a such a cell, it's a big cell you are immobilizing on the sensor surface. Then a tinny like some protein is binding to sensor surface. You can't see a biolayer change on the sensor surface. But what happens is when any molecules bind to the respective target on the cell surface, you can see in the cell, you can see there is some events happens. There is a actin modulations or any other path phase. So these changes will leads to the signal change on the sensor surface. We call it as a dynamic remodulation, the word. So using this phenomenon, they had done the quantitation kind of stuff, the cell businesses orthogonal to that. And very important when it comes to the ELISA and the HPLC comparisons, where most the industry people, map platforms people are working with, they are using this platform for the tighter determinations for their antibody productions. Very quickly, the good advantage is like that, 96 samples you can screen just in a 40 minutes or I can say the quantity, the 40 minutes. But in comparison to your protein HPLC or the ELISA based methodologies, it will takes a long time. ELISA takes around 3 to 4 hours, but even protein HPLC for the 96 samples, it's like 8 to 10 hours it will take. But you can just in a 40 minutes, you can quickly the titrate the protein of interest in this. So one of the comparison here they had done, the early clone selection cell line developments, they had done a correlation with respect to the HPLC versus the octet platform. It's on comparable with the other methodologies. So depending upon the applications, you can choose the right sensor. You can put into different all applications, either it may be protein protein, protein DNA or protein RNA, octamers or cell waste gases or the nanoparticles, we have a lot of publications in that. So I hope you got a better understanding about this label free biosensor, Biolayer Interferometry Technology BLI. The next lecture will include a demonstration session that was conducted during this workshop. I'm sure you will be now able to understand these concepts much better and you will also understand how to perform these experiments for your biological samples of interest, for your biological problems of interest. Thank you.