 Today we are going to understand another popular label-free technology which is based on biolayer interferometry. It is highly useful for studying biomolecular interactions. We discussed various applications based on surface plasmon resonance, SPR. This is another technology platform which is also label-free based on different physical principles. BLI is based on simple dip and reed system which is useful for measuring interaction between proteins, peptides, nucleic acid, small molecules and lipids. It is an optical technique that analyses the interference pattern of white light reflected from two surfaces, a layer of immobilized protein on a biosensor and an internal reference layer. Any change in the number of molecules bound to the biosensor trip causes a shift in the interference pattern that can be measured in real time. These biomolecular interactions are measured in a label-free environment with the ability to monitor binding specificity and kinetics. For example, association and dissociation rate constants and concentration analysis with precision and accuracy. Today we have Dr. Kau from Paul Life Sciences with us who will elaborate on the basics of biolayer interferometry technology and its applications in protein research. So let us welcome Dr. Kau for today's lecture. Hello everyone. Today I am going to give an introduction to biolayer interferometry and its application in protein research field. During the past 10 years, Photoball has developed a series of systems relying on the biolayer interferometry which is a label-free technology and allows you to perform real time analysis of biomolecule interactions and also to perform quantitation of the biomolecules in micro-volume sample sizes. Now let us have an understanding of the biolayer interferometry or BLI technology, now let us have to understand how it works in the real life. The core part of the biolayer interferometry or BLI technology lies with the biosensor. If you take a close look at the biosensor, it actually made by optical fiber in the end. So at the very end of the biosensor, it is a two-dimensional biocompatible matrix. This matrix allows you to immobilize the molecules on this surface to allow further analysis of biomolecule interactions. The benefit of using this patented biocompatible matrix is that it is very uniform, it minimizes the molecule binding to the membrane, and also it is non-denaturing. So what happens when we are doing experiments with biolayer interferometry? If we take a close look at the biosensor, we can actually observe two reflection layers. The internal reflection layer is engineered into the biosensor during the manufacturing process, and the second layer is an interface between the tip of the biosensor and the liquid. When the biosensor is put into the BLI instruments, the instruments will generate visible lights. These lights will go through the biosensor, and as we know, some of the lights will be reflected from the two layers of reflection surfaces. Since the white light has lights at different wavelengths, so the two reflected wavelengths will interact with each other. Some lights at certain wavelengths will show a constructive interference. Some will show a partially constructive interference, and some will see negative interference or destructive interference. No matter what, the system will monitor the reflected two beams and plotted the composite or composed two wavelengths at this x, y plot. And by plotting the wavelengths or wave pattern of these different waves on this x, y plot, we can get an original wave pattern. When an interaction happens, the molecule will bind to the end of the biosensor, and as a result, we will see the secondary or second reflection surface actually moves down, and this moving down increased the distance between the two reflection surfaces. And this increase will change the interference between the two reflected waves. As a result, what we will see is, originally we have wave pattern as shown here, but after interaction happens, because interference changes, so we will see a different wave pattern. It is kind of shift from the original wave pattern. The distance of the shift is proportional to the size of molecules on the membrane, the amount of molecules bound on the membrane, and also the density of the molecules bound on the membrane. So the system will monitor this wave pattern shift in real time, and from there we can get understanding about the details of the bar molecule interactions. Now, let us look at the example, if we are looking at the antibody-antigen interaction. As we know, the antibody will bind to antigen and form a complex. At the same time, the complex will go through a dissociation process and generate the antibody and antigen. As a result, if we have a biosensor, which already have immobilized antibody on the very end or on the bar matrix, and if we put this biosensor into the solution, contains antigen. The antigen will bind to the biosensor and result a shift of the wave pattern, which in turn generate the association curve between the antibody and antigen. Afterwards, if we move the biosensor to a buffer solution, the antigen will be dissociated from the antibody. Then from here, we can get a dissociation curve of the antibody-antigen interaction. So this is how the system works in the real time. Because the BLI technology is developed based on this biosensor, which can work with different sample types, it allowed us to analyze the interactions between bar molecules from molecules down to 150 Dalton to antibody to recombinant protein to virus and to bacteria. However, this system may not work directly with the intact mammalian cells because of the size of the cells has exceed the limit of the detection. And as another application, the BLI technology can work with different research purpose. We can use the technology to perform quantitation of the bar molecules. This can be achieved by immobilize a specific antibody or molecule onto the biosensor and use it to analyze the concentration of other molecules. It can also be used to analyze the kinetics of the bar molecule interactions for us to get a KON, K-O, KD. And in the pharmaceutical industry, it can also be used to perform functional testing the epitobene analysis and also perform isotyping of the antibodies. In the next couple of slides, I will give you some example about the application of the BLI technology in different field. Firstly, let's look at the application of the BLI technology in kinetic application. When we are doing a kinetic experiment, we firstly need to coat the biosensor with a ligand. In this experiment, we are using the biosensors with stripped avidine immobilized on it. So the ligand has already been biotelinated and we will first put the biosensor into the buffer to remove the protective agent from the biosensor. After that, the biosensor is moved into the wells that contains the ligand. The biotelinated ligand will bind to the stripped avidine and generate a loading curve as we can see here. Afterwards, the biosensor is then moved back to the buffer, then we can get the baseline before the real interaction happens. As a fourth step of the interaction, the biosensor is moved into the wells which contains the analyte of interest. The analyte of interest will bind to the ligand which has already been immobilized on the biosensor and from here, we can get the association curve between the analyte and the ligand. And as a last step of this analysis, the biosensor is moved back to the wells which contains the buffer, then in the buffer wells, the analyte will dissociate from the ligand and we can generate the dissociation curve. One thing we need to pay attention here is all the interactions, the curves are generated in a real time. We can get many details from this analysis. Depends on the system you are using, we are allowed to analyze either 8 samples or 16 samples or even 96 samples at the same time, which dramatically speed up the analysis process. Now let us look at one of the publications which use the biolayer interferometry to analyze the binding between the two proteins. In this particular research, we are studying the binding of the MHC class 1 with another protein called CPXY203. So in this particular study, the researchers performed a very stringent analysis between the two molecule interactions. So they firstly use the SPR technology, which is another technology used to analyze barbed molecule interactions, then they get the KD value. And in another experiment, they then performed the analysis using the BLI technology. And the data shown here supports that the kinetic constants achieved on the BLI technology is the same as one achieved with SPR technology. So the main purpose of this study is to show us that it is very important for us to validate our results in different testing platforms. So this will help us to be sure that results we get is not false positive. I believe this theory or this kind of thinking will be very useful when you are designing your own experiments. As another example of the application of the biolayer interferometry, let us look at quantitative analysis of BLI. When we are doing a quantitative analysis, the first step is to generate a standard curve. So during the setup, the biosensors is firstly deep into the wells, which contains the standards. The standard will bind to the biosensor and by calculating the initial binding rate of the molecules to the biosensor, we can get a standard curve with a binding rate as a y-axis and a concentration of the molecules as the x-axis. After we get the standard curve, we can then regenerate the biosensors by putting the biosensors into the regeneration buffer followed by the normal buffer to neutralize the biosensor. Afterwards, the biosensors are dipped into the wells, which contains the samples at unknown concentration. From the binding curve, we can also get the initial binding rate of this molecules at unknown concentration and by plotting this initial binding rate onto the standard curve, we can get to know the concentration of these unknown samples. One advantage of using BI technology to analyze the concentration of the molecules is that it is able to analyze multiple samples at the same time and also it only takes you about 15 to 30 minutes to finish analyzing 96 samples. Depends on the assay you are using, you can actually regenerate the biosensors, which means that you only need 16 or 8 biosensors to analyze the whole 96 well plate or 384 well plates. So this dramatically facilitates the application of the BI technology in the pharmaceutical industry as well as in the academic research field. Here is one of the examples, which shows that how we can use BI technology to detect the protein in a crude cell lysate. In this particular experiment, we actually analyze the samples with a system called blitz. The blitz is a single channel system developed using BI technology. So in the experiments, we are expressing two molecules or two proteins in E. coli. The research have shown that these two proteins will bind to each other and they can form a tight complex in E. coli. Between the two proteins, one of them has been heat tagged. So we can actually use anti-heat antibody to detect the particular fragment. We express these two proteins simultaneously in E. coli and we round the cell lysate on the SDS page gel. From this gel, we can see that we actually cannot tell whether the two proteins are expressed in the E. coli or not because there are very high background on the SDS page gel. So how can we do that? The researchers in this particular study used blitz platform to analyze the concentration, the expression of the molecules in the E. coli. What they have done is, from the cultured E. coli samples, they just take out 4 microliters of the cultured bacteria and then dip these 4 microliters samples onto the blitz system. Then we also use anti-heat biosensor attached to the blitz system and the anti-heat biosensor is then dipped into the sample which is only 4 microliters. The heat stack proteins, if they are present in the samples, they will bind to the heat stack biosensor and generate a binding curve. So by looking at the generation of the binding curve, we can actually tell where the protein of interest is expressed in the E. coli or not. So we can see here by using only 4 microliters samples without any sample purification, we can easily perform the analysis to tell the presence of our protein of interest in the E. coli which saves really a lot of time from 30 seconds compared with one day of the western blot. Now let's look at another application of the BRI technology in the vaccine title determination. As I mentioned earlier, because of the unique feature of the BRI technology, we can actually use the technology to bind to the virus directly without further sample purification or process. Traditionally in the vaccine industry, we are relying on SRID method to perform the quantitation of the vaccine virus title. However, this method is very time consuming. It takes up to three days to analyze the virus title and also it has very limited sample throughput where it is only able to analyze maybe 15 or 16 samples at the same time. Thirdly, the sensitivity and accuracy of the SRID method is limited and the interpretation of the final results is actually subject to personal interpretation. So this method may not be a very perfect method for you to determine the virus title in the sample. People have also been using ELISA method to analyze the vaccine title. However, similar to SRID, ELISA takes a very long time to finish the whole experiment process and it has very low precision and to determine the title of the virus and has very limited dynamic range. To overcome all these shortcomings, the researchers are using the BRI technology to determine the virus title in the real experiments and as we can see here, from the sample preparation to the time we get the final results, it only takes about 3 hours for you to finish the whole process. I would like to show you how to use the BRI technology to select the right construct for protein crystallization studies. In these experiments, so the researchers would like to crystallize some constructs at different truncations. What they have done is they have attached the heat tag together with the protein as a heat tag protein. So the experiment is carried out based on two assumptions. Firstly, if the protein is folded correctly, the tag will be exposed on the surface which allows it to bind to the biosensor. On the other hand, if the protein is not folded correctly, the tag will be buried inside the protein complex and as a result, the misphotoprotein are not able to bind to the biosensor with anti-heat antibody coated on it. Also the misphotoprotein tends to form aggregates. So in this particular research, our researchers firstly perform SEC size exclusion chromatography on a crude cell lysate. From there, they can get a pattern distribution of the different constructs. And also, by using SEC analysis, they can identify which portion is a soluble portion and which portion is the aggregated portion. As a second step of the study, the researchers then express the different constructs in the E. coli and use the B. r.i. technology to analyze the binding of the expressed protein to the biosensors with anti-heat antibody immobilized on it. So the researchers measured the initial binding rate of these constructs to the biosensor. And by calculating the initial binding rate, they can plot this data on this x, y plot as you can see on the upper right corner of the slide. It's very interesting to notice that the constructs with the highest initial binding rate actually corresponds to the constructs with the highest soluble portion. So these two constructs is selected for further crystallization study and they have the researchers then get the crystals of these two constructs. The main learning point from this study is that by performing a one-minute assay using B. r.i. technology on the octet platform allows you to correctly prioritize the protein constructs which is suitable for downstream crystallization which helps you to simplify your research process and speed up the process for you to get the crystals for further structural analysis. Because there are so wide applications of the BR technology, we have developed a wide range of the biosensors for you to analyze antibody concentration or interaction or you can use the biosensors to analyze the heat tag proteins or GST tag proteins or if your protein is not tagged or they are not antibodies, you can also use other biosensors such as AR2G biosensors which has amine group immobilized on the biosensor or the strupter avidin biosensors which includes the assay biosensor, assay biosensor and assay ex biosensor for you to analyze the strupter avidin or boutininated molecule interaction. So as a summary of my lecture, we kind of go into the basic mechanisms of the BR technology and we also included some of the examples of using BR technology in different research field which including protein-protein interaction, protein quantitation or even protein crystallization. The benefit of using the BR technology or even the octet platform in your research field is that it allows you to perform your analysis in a short time. The platform is very straightforward and it is very easy to use. Lastly, if you are working with a small molecule samples, this system allows you to analyze samples in DMSO up to 5 percent of the concentration. So here I will wish that after going through this lecture, you have get a better understanding of the BRI technology and also to understand how this technology will help you in your protein research. Thank you. So today we have learnt about the basics of BLI or biolayer interferometry technology and how it is used for different applications in research. In our next lectures, we will discuss and demonstrate in detail about this label-free technology BLI to perform the protein-protein interactions. Thank you.