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Resources Making AAV Viral Genome Titer for In-process Samples a Breeze Accurate measurement of AAV genome titer throughout the development and production for gene therapy is important to ensure the effectiveness of the final product. Conventionally, this task has been accomplished using qPCR and more recently digital PCR (ddPCR) methods. Each has its own benefits and downsides. While qPCR offers a 4 logs dynamic range, it has several drawbacks – significant variability with CV (coefficients of variation) reaching up to 30%, susceptibility to inhibitory factors such as PCR inhibitors, adverse impact of extraneous DNA, lengthy analysis times and the necessity to establish a standard curve. The ddPCR on the other hand presents several advantages viz. it eliminates the need for a standard curve, is accurate and provides high precision ranging from 3% to 20%. However, this impressive accuracy and precision is attainable within a limited quantitative dynamic range of approx. 2logs. The ddPCR’s multiplexing capability facilitates the examination of genome integrity, thus making it very useful. That said, the ddPCR process involves extended assay times, a relatively intricate multi-step workflow, and vulnerability to PCR limitations, posing challenges for routine application. Notably, 100-to-1,000,000-fold dilution is needed for samples to fall within the working range and to minimize matrix effects seen in in-process samples, introducing error and extended sample processing time. The cost per sample can also be substantial. The above observations motivated us to develop a fast, easy, and accurate assay for in-process samples based on a novel method that excels in precision, speed, accuracy and most importantly simplicity. Our groundbreaking approach revolves around DNA hybridization, immunochemistry and biolayer interferometry, encompassing a streamlined two-step procedure involving lysis and hybridization within a single tube, succeeded by detection using a biosensor. Central to the methodology are two oligo probes—an approximate 40 nt fluorescein-labelled probe and a 40 nt SuPlex probe. The SuPlex oligo includes a 30 Thymine oligonucleotide bearing biotin at both the 5′ and 3′ ends. The target region for these probes can be located within the Gene of Interest (GOI) or the promoter/enhancer regions. The lysis and hybridization steps involve heating at 950 C, promptly followed by rapid cooling to 50 C. The resultant hybridized sample is quantitated employing an anti-fluorescein biosensor. The assay offers a quantitative dynamic range of 5E+9 to 5E+12 and delivers precision ranging from 3% to 9%. The assay time is mere 30 minutes. The inherent robustness of this method makes it compatible with DNase1, proteinase K, extraneous DNA, PCR inhibitors and in-process buffers, rendering it highly suitable for both research and manufacturing. Notably, another unique feature is the ability to quantify individually both positive and negative strands of the packed genome. Also, our preliminary data using oligo probes for GFP, CMV promoter, CMV enhancer and SV40 regions shows the potential for determining genome integrity. So, while QPCR and digital PCR are currently the main tools for AAV genome titer assay, the DNA hybridization-biolayer interferometry approach described here shows significant benefits over the PCR methods. Its ability to characterize packed genome content deeper by providing strand specific titers, and robustness against various inhibitory factors positions it as an asset in both research and manufacturing contexts. Further, we have applied the same methodology for direct RNA titer without reverse transcription in Lentivirus.
Resources How AAV Process Development can Benefit from Automated High Sensitivity Quantitation Adeno-associated virus (AAV), a prominently used vector in human gene therapy is a small, non-enveloped parvovirus consisting of a single-stranded DNA genome enclosed in a protein capsid. AAV vector is used to transfer therapeutic genes to humans for the treatment of monogenic diseases, ocular diseases, and clinical trials of deficiencies like hemophilia and thalassemia. Its non-pathogenic nature, low immunogenicity, ability to retain long-term gene expression, and low likelihood to induce an inflammatory response make it very promising gene therapy vector. This blog post provides a brief outlook on the importance of quantitation in AAV process development and how the innovative solutions from Gator Bio fill this gap in AAV characterization. Importance of quantitation Accurate quantitation of the AAV genome and capsid titer is an important step in AAV process development to ensure safe and efficacious dosing. High vector doses of AAV due to inaccurate quantitation can lead to an inflammatory response that is dangerous for sensitive organs such as the eyes. Conventional AAV quantitation include PCR-based methods (qPCR). The major drawback of PCR-based quantitation is that it does not provide any information on the loss of integrity in viral particles. Moreover, PCR assays are prone to contamination by salts, proteins, or plasmid DNAs, and thus pose a risk of inaccurate quantitation. Other established immunoassays such as ELISA, and Dot blot for capsid titer quantitation are time-consuming, labor-intensive, and thus significantly impede the AAV process development cycle and its manufacturing. Gator Bio’s solutions for accurate quantitation Gator Bio has developed Gator® Plus, unique plug-n-play, cost-effective high-throughput bioanalytical tool for accurate, high-sensitivity serotype quantitation in a highly parallelized, automated manner. It is based on Biolayer Interferometry (BLI) technology and uses CaptureSelect AAVX nanobody probes to bind and quantitate AAV capsid serotypes including AAV 1-8 and 10 with a dynamic range of 5×106 – 1×1013 vp/mL. The probes are compatible with various matrices used in process development The assay developed using these probes can be used for quantitation of AAVs from harvesting stage to final product QC Eight channels are operational in parallel and can process up to 96 samples within less than 30 minutes, resulting in high throughput quantification. The probes render an impressive regeneration performance and are reusable up to 10 times without any loss in binding, thus saving a lot of process time and operating costs. Integration with GatorOne® software allows easy data analysis and sample AAV concentration measurement. It allows saving multiple standard curves needed to quantify different AAV serotypes, thereby saving the time to generate standard curves repeatedly, thus enhancing the process efficiency.Drop us a line if you would like to know more about our quantitation solutions.References: Martinez-Fernandez de la Camara, C. et al. Genes 2021, 12, 601. https://doi.org/10.3390/genes12040601Shmidt, A.A. et al. Pharmaceuticals 2022, 15, 23. https://doi.org/10.3390/ph15010023Cui, M. et al. Molecules 2019, 24, 3973; doi:10.3390/molecules24213973https://www.gatorbio.com/wp-content/uploads/2022/04/AAVX-vs-Progen-App-Note.pdf
Resources The Challenges and Opportunities of AAV Quantitation AAVs are common, harmless viruses that readily infect humans, therefore being very effective gene therapy vectors. Precise and accurate AAV titer quantitation is crucial to AAV based gene therapy, and as of now there is a lack of standardized quantification method across different production systems and laboratories. What are AAVs and how are they used? Adeno-associated viruses (AAVs) are a group of viruses that infect humans and primates. AAVs are very small (around 20 nm in diameter) and cannot replicate without the help of other viruses. They score low on a scale of virus complexity: just consisting of a protein shell with a small single-stranded DNA payload. While AAVs produce a mild immune response in most humans, they’re not known to cause any disease. They also appear to be very common in human populations, with around 50% – 80% of human populations exhibiting seropositivity for antibodies directed against AAV capsid proteins.1 These qualities make AAV serotypes (in particular AAV2) excellent candidates for gene therapy, which is used to treat disease and genetic disorders by replacing ‘faulty’ copies of a gene with ‘healthy’ ones delivered by a viral vector. 2 What is AAV quantitation? The use of AAVs in gene therapy applications has increased steadily over the last 20 years, and today AAVs are one of the most actively investigated gene therapy vehicles. Scientists have successfully used “recombinant” AAV (i.e., engineered versions of AAV that lack viral DNA) as essentially protein-based DNA delivery systems to treat a variety of conditions via gene therapy.The development of safe and effective, AAV gene therapies can only be accomplished if AAVs are accurately characterized and quantitated.5 The commercial demise of the first European-approved AAV-based gene therapy product due in part to cost issues highlighted the need for efficient AAV vector manufacturing and downstream processing.6 , which needs standardized methods to quantitate and characterize AAV vectors. Challenges in AAV Quantitation Multiple methods have been – and still are – employed to quantitate AAV capsid and genome titer. These include enzyme-linked immunosorbent assay (ELISA), quantitative polymerase chain reaction (qPCR), droplet digital PCR (ddPCR), and high-pressure liquid chromatography (HPLC).7,8 These techniques are not only time-consuming; but the lack of standardized methods for AAV quantification makes it difficult to compare yields from different production systems and across laboratories..9 AAV Quantitation from Gator Bio Gator Bio has developed a unique solution to the challenge of accurate and efficient AAV quantitation. Gator AAV probes are high-specificity antibody-based biosensors enabling the direct capture and rapid quantitation of different AAV serotypes in crude lysates, column eluates, and cell culture supernatants. Offering high accuracy and wide dynamic range (109 – 1013 VP/mL for most AAV serotypes); Gator AAV probes provide a fast and efficient alternative to relatively time-consuming and labor-intensive traditional methods like ELISA. The reusability and crude sample tolerance of these probes make the method cost-effective and suitable for deployment at multiple stages in the development and manufacture of AAV-based gene therapies. Gator Bio is a world-leading developer of BLI systems and solutions. To find out more about AAV quantitation solutions from Gator Bio, get in touch with us today. References and Further Reading Grieger, J. C. & Samulski, R. J. Adeno-associated Virus as a Gene Therapy Vector: Vector Development, Production and Clinical Applications. in Gene Therapy and Gene Delivery Systems (eds. Schaffer, D. V. & Zhou, W.) vol. 99 119–145 (Springer-Verlag, 2005). Zinn, E. & Vandenberghe, L. H. Adeno-associated virus: fit to serve. Curr Opin Virol 8, 90–97 (2014). Naso, M. F., Tomkowicz, B., Perry, W. L. & Strohl, W. R. Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs 31, 317–334 (2017). How does gene therapy work?: MedlinePlus Genetics. https://medlineplus.gov/genetics/understanding/therapy/procedures/. Dobnik, D. et al. Accurate Quantification and Characterization of Adeno-Associated Viral Vectors. Front. Microbiol. 10, 1570 (2019). Ai, J., Ibraheim, R., Tai, P. W. L. & Gao, G. A Scalable and Accurate Method for Quantifying Vector Genomes of Recombinant Adeno-Associated Viruses in Crude Lysate. Human Gene Therapy Methods 28, 139–147 (2017). D’Costa, S. et al. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR. Mol Ther Methods Clin Dev 5, 16019 (2016). Dorange, F. & Le Bec, C. Analytical approaches to characterize AAV vector production & purification: Advances and challenges. Cell and Gene Therapy Insights 4, 119–129 (2018). Aucoin, M. G., Perrier, M. & Kamen, A. A. Critical assessment of current adeno-associated viral vector production and quantification methods. Biotechnology Advances 26, 73–88 (2008).
Resources What is Biomolecular Interaction Analysis? Biomolecular interaction analysis seeks to understand the interactions between biomolecules and the results of these interactions. This type of analysis is critical for medical research and pharmaceuticals. Here we explain some of the key principles of biomolecular interaction analysis and its applications. Why Study Biomolecular Interaction? Interactions between biomolecules are a critical aspect of many biological processes. Whilst some proteins function on their own, most proteins are only active in complex forms. This means that their relationships with other biomolecules such as metals, lipids, proteins, and nucleic acids are extremely important. Biomolecular interaction analysis looks at the physical contact between protein partners as well as the functional trends in signaling and metabolic pathways. Protein interactions and molecular interactions are intrinsic to all areas of cellular process such as: Regulation of metabolic pathways Cellular motion Signal transduction Environment sensing With so many critical processes being informed by biomolecular interaction, the right form of analysis must be chosen. Surface Plasmon Resonance SPR SPR is a label-free method of biomolecular interaction analysis that works by using incident light to stimulate oscillating conduction electrons between positive and negative permittivity material. SPR measures adsorption onto planar metal or onto the surface of metal nanoparticles. SPR is well-suited to measuring the affinity and selectivity of biomolecular interactions. It can be used for investigating association and dissociation rate constants and presenting the kinetics of biomolecular interactions. This technique can also be used for equilibrium binding analysis. SPR does not require additional reagents, sample preparation, or assays and has high precision for classifying protein-protein interactions.[1] Isothermal Titration Calorimetry ITC is a method used for the quantitative study of a range of biomolecular interactions. This technique works by measuring the heat directly that is absorbed or released in a biomolecular binding event. The main benefit of ITC is that it can determine each binding parameter concurrently in just one experiment and no modification of binding partners is required. Bio Layer Interferometry BLI BLI (biolayer interferometry) is an optical biosensing technology used for biomolecular interaction analysis. One of the key benefits of BLI is that it offers real-time label-free measurements for data analysis. BLI studies macromolecular interactions, focusing on the interference patterns from white light reflected from the surface of the biosensor tip. It is a simple technique that measures Kon, Koff, and KD in just one rapid assay. BLI is robust and easy to set up, it can be used for a range of samples including both crude and purified samples. This means that it can be used for a variety of investigations without needing to swap between systems for samples. Its small footprint means that it does not take up valuable space in the lab, making it more versatile than many other technologies. BLI also allows for a broad variety of affinity values with the ability to investigate the following: Nanobodies Antibodies AAV Small molecules Nucleic acid Membrane proteins Biomolecular Interaction Analysis from Gator Bio Gator Bio stocks a range of BLI models suitable for biomolecular interaction analysis. It is a highly flexible, accessible, and sensitive technology that can characterize large varieties of proteins and molecules. If you would like to find out more about how our products could help with your biomolecular interaction analysis, read our dedicated page here. [1] https://pubmed.ncbi.nlm.nih.gov/23533209/
Resources Small Molecule Analysis via Biolayer Interferometry Why is small molecule analysis important? In pharmacology and molecular biology, “small molecules” have a molecular mass below around 900 Daltons.1 This distinction is far from arbitrary: The smaller a molecule is, the more easily it can permeate living systems. Small molecules are better able to diffuse across cell membranes, enabling them to reach intracellular action sites and giving them generally much higher bioavailability than larger “macromolecules” such as RNA or proteins.2 For this reason, the vast majority of pharmaceutical drugs are “small” molecules (with a few notable exceptions, such as insulin, which is a protein and consequently classed as a macromolecule). Small molecule analysis is therefore a key concern of pharmacology and molecular biology. In this context, “small molecule analysis” generally refers not only to the identification and quantification of small molecules themselves but to the analysis of their interactions with other molecules of interest (such as receptors). Yielding information on concentration, kinetics, binding and interaction affinities, small molecule analysis is fundamental to drug discovery and biomolecular research. What is biolayer interferometry and how does it work? First developed in the early 21st century, biolayer interferometry (BLI) is a relatively new technique for small molecule analysis.3–5 BLI uses the principle of optical interference to monitor binding activity between a pair of unique biomolecules.6 One of these molecules – the ligand – is bound (immobilized) to the surface of a fiber optic tip which has been coated with a biocompatible matrix to enable selective binding. Once the tip is prepared, it is “dipped” into a solution containing the other molecule in the pair (the analyte). As the analyte begins to interact with the ligand on the sensor tip, the optical thickness of the biological layer (consisting of ligand and analyte) on the surface of the sensing tip increases. Small molecule analysis is enabled by monitoring the precise thickness of this layer of interacting molecules using white light interferometry, which compares the spectral shift to that of a reference surface. How can biolayer interferometry be used for small molecule analysis? BLI provides real-time label-free monitoring of molecular interactions on the sensor surface by continuously measuring the thickness of the layer formed by ligand-analyte interactions. This is why BLI is such a powerful tool for small molecule analysis. Alongside surface plasmon resonance, BFI is one of only a few widely available biosensing techniques which doesn’t require labeling. However, BFI offers a few unique advantages for small molecule analysis. Crucially, only molecules binding to or dissociating from the sensor can shift the produced interference pattern and generate a response profile. This means that unbound molecules, changes in flow rate, or changes in the refractive index of the surrounding medium don’t influence measurements. BLI is also fluidics-free and can carry out small molecule analysis for either crude or purified samples. BLI is sensitive enough for peptide and small molecule analysis; and, thanks to its simple operation and the use of reusable fiber-optic tips, is well-suited to parallelized high-throughput applications. BLI Small Molecule Analysis from Gator Bio Founded by the inventors of biolayer interferometry, Gator Bio is a world-leading producer of BLI platforms for small molecule analysis applications.7 Combining a 1 mm glass rod with patented optical layers and specialized biosensor surface chemistry, Gator® BLI systems allow for high-capacity immobilization of biotinylated proteins for a wide range of molecular weights. Following immobilization, Gator® systems enable rapid and accurate small molecule analysis, determining critical interaction parameters such as kon, koff, and kD for molecules down to 150 Da. Are you interested in finding out more about the Gator BLI platform for small molecule analysis? Get in touch with us today. References and Further Reading 1. Dougherty, T. J. & Pucci, M. J. Antibiotic Discovery and Development. (Springer Science & Business Media, 2011). 2. Veber, D. F. et al. Molecular Properties That Influence the Oral Bioavailability of Drug Candidates. J. Med. Chem. 45, 2615–2623 (2002). 3. Label-free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization – PubMed. https://pubmed.ncbi.nlm.nih.gov/19758119/. 4. Biosensor-based small molecule fragment screening with biolayer interferometry – PubMed. https://pubmed.ncbi.nlm.nih.gov/21660516/. 5. Biolayer Interferometry | Gator Bio. https://www.gatorbio.com/technology. 6. Apiyo, D. O. Chapter 10:Biolayer Interferometry (Octet) for Label-free Biomolecular Interaction Sensing. in Handbook of Surface Plasmon Resonance 356–397 (2017). doi:10.1039/9781788010283-00356. 7. About | Gator Bio. https://www.gatorbio.com/about.
Resources Protein Purification, Expression Optimization & More Quantitation Applications Quantification of Biomolecules Quantitation applications are fundamental to numerous biological research protocols and in the production of pharmaceutical-grade biomolecules. It is frequently desirable to confirm the purification of antibodies or other biomolecules to characterize biologic processes or ensure the quality of starting materials1,2. Quantitation applications are thus key to a range of research and therapeutic protocols. Protein Purification With ever-growing biotechnology capabilities, protein engineering is an increasingly important aspect of both research and biological therapies. Quantitation applications are paramount in providing confidence in protein purification methodologies before protein stduies3. Similarly, quantitation applications ensure that proteins destined for pharmaceutical use conform to rigorous quality parameters to ensure patient safety. Expression Optimization The majority of molecular biology experiments start with cloned DNA, which is then transferred to E.coli or a similar expression vehicle to obtain the protein of interest. To maximize the expression of the desired protein, the DNA construct must be designed to optimize the efficiency of expression by the vehicle selected. Furthermore, the purity of the DNA must be ensured through quantitation applications to increase the output of the target protein. Adeno-associated virus (AAV) titer determination AAV is widely used as the vector to introduce functional genes to humans with a disorder that stems from a genetic aberration. Such gene therapy is controlled by strict regulatory standards, including specifications related to vector purity and potency. The ability to accurately determine the dose of therapeutic AAV vector is thus critical to the gene therapy process. Accurate quantification of viral genome is fundamental to demonstrating purity and confirming dose4. Serological testing The determination of antigen-specific antibodies in plasma samples is an important diagnostic tool and provides important serosurveillance information during pandemic infections5. Analytic tests that provide quantitative results usually require complex and labor-intensive immunoassays. There is a need for a rapid, highly sensitive technique for antibody quantitation applications. Biolayer Interferometry These quantitation applications and more are satisfied by a novel label-free technique using biosensors — biolayer interferometry (BLI) 6,7,8. BLI measures the interference pattern of white light reflected from the surface of a biosensor, which indicates the presence of biomolecular interactions. Used in conjunction with a standard curve of known concentrations, BLI provides a suitable tool for rapid data acquisition across numerous quantitation applications. BLI technology is suitable for quantitation applications involving complex matrices. It can be used for quantitation applications in samples that are crude supernatants or cell lysates, as well as purified proteins. High sensitivity is achieved across numerous quantitation applications from small molecules to large biologic complexes. Furthermore, measurements are made in real-time, so changes in biomolecule concentration can be monitored with high precision. Only target biomolecules are measured, making BLI a powerful tool for a wide range of quantitation applications. Gator Products for BLI Quantitation Applications Gator offers a range of products to facilitate the use of BLI in quantitation applications. GatorPrime™ is a robust and maintenance-free BLI instrument for label-free analysis9. With automated biosensor loading and automated regeneration of biosensors, it offers a hands-free alternative to labor-intensive ELISA methodologies for quantitation applications. GatorPrime incorporates eight independent spectrometers that enable quantitation applications to be conducted simultaneously on eight samples. With the capacity for assays sizes up to 168 samples per run, the GatorPrime system represents an efficient solution for quantitation applications. The system includes quick-start modes to support quantitation applications that are carried out by non-specialist operatives. GatorPlus is a high-throughput device used in quantitation, kinetics, and epitope binning. Based on bio-layer interferometry (BLI) technology, this instrument provides rapid results. In addition, Gator provides specialized solutions to further facilitate quantitation applications10. For example, Gator™ AAVX enables the direct capture and quantitation of different serotypes of AAV in crude lysates, column eluates, cell lysates and cell culture supernatants using the CaptureSelect™anti-AAVX ligand. The Gator™ Ni-NTA Kit enables rapid and continuous quantification of His-tagged proteins without the need for Ni2+ recharging. The Gator BLI system uses software that complies with the FDA’s code of Federal Regulations (CFR) Title 2111. This is critical for quantitation applications involved in the monitoring and manufacturing of biological drugs. All data acquired is time-stamped and traceable and its enhanced audit trails, including recorded user sessions, comply with FDA guidance. The Gator system is thus suitable for use in environments where adherence to good manufacturing/laboratory practice is paramount and analyses must be CFR21-compliant. If you would like to find out more about how Gator Bio can help with your quantitation applications, you can read more on our website. References Westbrook JA, et al. Proteomics Clin Appl. 2015 Apr;9(3-4):295-300. Doi: 10.1002/prca.201400120. Heegaard NH, et al. Electrophoresis. 1999 Oct;20(15-16):3122-33. Lillehoj EP, Malik VS. Adv Biochem Eng Biotechnol. 1989;40:19-71. Jungmann A, et al. Hum Gene Ther Methods. 2017; 28(5):235-246. Dzimianski JV, et al. Sci Rep. 2020;10(1):21738. Qiao SP, et al. Chembiochem. 2021 Jun 2;22(11):1974-1984. Noy-Porat T, et al. STAR Protoc. 2021 Sep 15;2(4):100836. Dysinger M, et al. J Immunol Methods. 2012 May 31;379(1-2):30-41. Gator. https://www.gatorbio.com/products/gatorprime Gator. https://www.gatorbio.com/products/biosensors-and-reagents Gator. https://www.gatorbio.com/products/software
Resources Why are Binding Kinetics Important? Binding kinetics have been studied for many years and make up the basis of pharmacological theory today. Whilst all pharmaceutical drugs are different, the binding kinetics have many similarities. The term binding kinetics refers to how quickly a compound binds to its target and the speed at which it dissociates from it. Binding kinetics measure the on-rate and the off-rate but have historically been challenging to measure. This article will explain the fundamentals of binding kinetics and how they work. The Importance of Binding Affinity The binding affinity of a drug is inextricably linked to binding kinetics. The binding affinity refers to the levels of drugs needed to occupy 50% of the target molecules at equilibrium. This number is the target occupancy, used to predict in-vivo efficacy. The residence time is a quantitative value referring to how long it takes for drug-receptors to reach target occupancy. The Importance of Binding Kinetics in Affinity Measurements The importance of binding kinetics is based on the fact that the time to reach equilibrium is dependent on the dissociation time. When the opposing rates are equal, a binding interaction is at equilibrium. Because association takes place first for dissociation to occur. This is why full binding kinetics data is required to effectively measure binding affinity. When using binding kinetics, meeting an equilibrium isn’t required and therefore it can be a much faster method. Binding Kinetics in Drug Discovery Accurate and fast measurement of binding affinity is critically important in the drug discovery process. If the actual affinity is above what is measured then patients may risk overdose in clinical drug trials. Similarly, if it is lower than recorded potentially life-saving drugs could be missed because of under-dosing. Binding kinetics differentiate medication by informing dose-response relationships. This means that physiological effectors can be appropriately counteracted by drug concentrations. Binding kinetics are intrinsically linked to the translation of drug action to safety, efficacy, and duration of response outcomes. How to Measure Binding Kinetics Establishing an understanding of binding kinetics is critical for developing drug molecules. To do this, equilibrium constants must be established. Koff is the dissociation constant in min-1. Kon is the association constant in inverse minutes multiplied by inverse concentration. KD is computed from Koff/Kon. Expressed in Molar units Gator Bio BLI provides a simple means of measuring kinetics. Using real-time data, binding kinetics (Kon and Koff) and affinity values (KD) can be measured. Because BLI offers insight into dissociation, association rates, and affinity value it can help predict the activity of biomolecules in vivo.
Resources 4 Key Benefits of Using BLI for Binding Kinetics Measurements Binding kinetics make up the foundation of pharmacological theory in the modern era. Binding kinetics is a term that is concerned with the speed at which a compound binds to a target and how quickly it dissociates from it. BLI (bio-layer interferometry) is an optical biosensing technology used in analyzing biomolecular interactions without requiring fluorescent labeling. BLI is one of the few widely available biosensing technologies that are label-free. BLI measures macromolecular interactions by analyzing the patterns of interference from white light reflected from a biosensor tip surface. This article discusses the benefits of BLI for measuring binding kinetics. What Parameters of Binding Kinetics can be Determined Using BLI? BLI allows for real-time determination of association rate (kon) and dissociation rate (koff) in molecular interaction and as such the binding kinetics. These rates provide the opportunity to understand the overall affinity (KD) of the interaction to be calculated. This value can also be ascertained directly from the formation of concentration-dependent molecular complexes. Benefits of BLI Simplicity One key benefit of using BLI for binding kinetics measurements is that Kon, Koff, and KD can be measured in one rapid assay. Detection takes place in real-time without marking biological molecules. This makes the measurement quicker and simpler than processes that require multiple assays. Wide Ranges Using BLI for binding kinetics allows for a wide range of affinity values. BLI can be used for many investigations such as nanobodies, membrane proteins, antibodies, AAV, small molecules, nucleic acids, and more. Compatible with a Range of Samples BLI is compatible with both crude and purified samples. This means it can be used in a range of investigations and there is no need to swap between systems for samples. No Labels Required Label-free techniques can monitor responses in real-time, meaning kinetic information regarding dissociation and association is accessible. They can also monitor bio-molecular interactions with native binding partners without any impediment from modified analytes and therefore more accurate biochemical data is acquired. BLI for Binding Kinetics Gator Bio software provides a range of models for data analysis. Reference subtraction, processing, and data fitting can be performed quickly. Biosensors from Gator Bio combine a 1mm diameter glass rod with patented optical layers and surface chemistry specifically built at the distal end of the biosensor. To find out more about how this technology can be used in binding kinetics applications, contact the team at Gator Bio today.