Since immobilized metal ion affinity chromatography (IMAC) was first reported, several modifications have been developed. Among them, Ni(2+) immobilized by chelation with nitrilotriacetic acid (NTA) ...bound to a solid support has become the most common method for the purification of proteins carrying either a C- or N-terminal histidine (His) tag. Despite its broad application in protein purification, only little is known about the binding properties of the His-tag, and therefore almost no thermodynamic and kinetic data are available. In this study, we investigated the binding mechanism of His-tags to Ni(2+)-NTA. Different series of oligohistidines and mixed oligohistidines/oligoalanines were synthesized using automated solid-phase peptide synthesis (SPPS). Binding to Ni(2+)-NTA was analyzed both qualitatively and quantitatively with surface plasmon resonance (SPR) using commercially available NTA sensor chips from Biacore. The hexahistidine tag shows an apparent equilibrium dissociation constant (K(D)) of 14 plus/minus 1 nM and thus the highest affinity of the peptides synthesized in this study. Furthermore, we could demonstrate that two His separated by either one or four residues are the preferred binding motifs within hexahis tag. Finally, elongation of these referred motifs decreased affinity, probably due to increased entropy costs upon binding.
•Biosensor SPR measures affinity and kinetics in real time.•Binding interaction between diamidines compounds with RNA sequences by using SPR.•A detailed RNA immobilization steps have described on ...Biacore sensor chip.•SPR data analyze in Biaevaluation software with specific examples.•SPR assay for developing new drugs that target biological molecules.
Biosensor surface plasmon resonance (SPR) is a highly sensitive technique and is most commonly used to decipher the interactions of biological systems including proteins and nucleic acids. Throughout the years, there have been significant efforts to develop SPR assays for studying protein-protein interactions, protein-DNA interactions, as well as small molecules to target DNAs that are of therapeutic interest. With the explosion of discovery of new RNA structures and functions, it is time to review the applications of SPR to RNA interaction studies, which have actually extended over a long time period. The primary advantage of SPR is its ability to measure affinities and kinetics in real time, along with being a label-free technique and utilizing relatively small quantities of materials. Recently, developments that use SPR to analyze the interactions of different RNA sequences with proteins and small molecules demonstrate the versatility of SPR as a powerful method in the analysis of the structure-function relationships, not only for biological macromolecules but also for potential drug candidates. This chapter will guide the reader through some background material followed by an extensive assay development to dissect the interactions of small molecules and RNA sequences using SPR as the critical method. The protocol includes (i) fundamental concepts of SPR, (ii) experimental design and execution, (iii) the immobilization of RNA using the streptavidin-biotin capturing method, and (iv) affinities and kinetics analyses of the interactions using specific example samples. The chapter also contains useful notes to address situations that might arise during the process. This assay demonstrates SPR as a valuable quantitative method used in the search for potential therapeutic agents that selectively target RNA.
The Octet biosensors provide a high-throughput alternative to the well-established surface plasmon resonance (SPR) and SPR imaging (SPRi) biosensors to characterize antibody-antigen interactions. ...However, the utility of the Octet biosensors for accurate and reproducible measurement of binding rate constants of monoclonal antibodies (mAbs) is limited due to challenges such as analyte rebinding, and mass transport limitation (MTL). This study focuses on addressing these challenges and provides experimental conditions to reliably measure kinetics of mAb-antigen interactions. The mAb capture density of less than 0.6 nm was found to be optimal to measure a wide range of binding affinities on Octet HTX biosensor. The titration kinetic and single cycle kinetic assays performed on Octet HTX generated reproducible binding kinetic parameters and correlated with the values measured on Biacore 4000 and MASS-1. Kinetic assays performed on 0.1 nm density mAb surfaces significantly reduced MTL and enabled characterization of picomolar affinity mAbs. Finally, kinetic analysis performed on 150 antibodies to 10 antigens with molecular weights ranging from 21kD to 105kD showed concordance between Octet HTX, Biacore 4000 and MASS-1 (R2 > 0.90). The data presented in this study suggest that under optimal experimental conditions, Octet biosensor is capable of generating kinetic values comparable to SPR/SPRi biosensors.
•High density antibody capture surface exhibited analyte rebinding and mass transport limitation (MTL).•Kinetic assays performed on 0.1 nm density antibody surface significantly reduced MTL and enabled characterization of picomolar affinity interactions.•A total of 165 fully human antibodies binding to 14 different antigens with molecular weight ranging from 14kD to 105kD were characterized on Octet HTX, Biacore 4000 and MASS-1 and the results revealed that measured binding kinetic values were comparable across different biosensors.
The acquisition of reliable kinetic parameters for the characterization of biomolecular interactions is an important component of the drug discovery and development process. While several benchmark ...studies have explored the variability of kinetic rate constants obtained from multiple laboratories and biosensors, a direct comparison of these instruments' performance has not been undertaken, and systematic factors contributing to data variability from these systems have not been discussed. To address these questions, a panel of ten high-affinity monoclonal antibodies was simultaneously evaluated for their binding kinetics against the same antigen on four biosensor platforms: GE Healthcare's Biacore T100, Bio-Rad's ProteOn XPR36, ForteBio's Octet RED384, and Wasatch Microfluidics's IBIS MX96. We compared the strengths and weaknesses of these systems and found that despite certain inherent systematic limitations in instrumentation, the rank orders of both the association and dissociation rate constants were highly correlated between these instruments. Our results also revealed a trade-off between data reliability and sample throughput. Biacore T100, followed by ProteOn XPR36, exhibited excellent data quality and consistency, whereas Octet RED384 and IBIS MX96 demonstrated high flexibility and throughput with compromises in data accuracy and reproducibility. Our results support the need for a “fit-for-purpose” approach in instrument selection for biosensor studies.
Surface plasmon resonance (SPR) is an optical phenomenon being used to monitor molecular binding events. With the advantages of being label-free, real-time, and sensitive, SPR assays have become one ...of the most commonly used techniques to measure binding kinetics, affinity, specificity, and concentration of molecular interactions. In an SPR experiment to measure small molecule-protein interactions, the protein is immobilized on the biosensor surface, while the small molecule flows through the surface of the sensor chip. The interactions between the small molecules and proteins are monitored by subsequent changes in the refractive index and quantified with resonance units. In this chapter, we have utilized an SPR assay to study the interaction of flavonoids and the glucose-regulated protein 78. Assay steps are detailed for immobilization optimization, SPR instrument setup, operation, sample injection, and affinity data analysis.
Surface plasmon resonance (SPR) analysis provides important binding characteristic information for an antibody to its binding partner, such as binding specificity and affinity (KD). In recent years, ...SPR has been increasingly used in biosimilar development as part of the comparative analytical similarity assessment. Although there is no systematic study describing how to qualify SPR assays, there are various SPR result types (outputs) that have been used for assay qualification in publicly available regulatory documents. The mixed usage of SPR output can cause confusion and can be misleading when comparing binding attributes among antibody molecules. In this report, using a recombinant huIgG1 (mAb 1) antibody as an example, we performed assay qualification strictly based on the nature of the biomolecular interaction. We recommend that KD should be used as the output of assay qualification when the KD can be measured accurately by SPR. When KD cannot be accurately determined in a SPR setting, sensorgram comparison and Parallel Line Analysis (PLA) can be used to qualify the assay. We emphasize the importance of setting up appropriate SPR assay conditions for target and/or Fc receptor interactions to ensure the assay qualification parameters, such as accuracy and repeatability, to meet the criteria acceptable for regulatory filings. With increasing numbers of biotherapeutics being developed, the methods and guidelines provided here can help to align SPR application between the drug development industry and regulatory authorities which will benefit the scientific communities involved in biotherapeutic drug development.
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•Binding activity of mAb 1 to its target and four of human FcgRs using SPR method optimized for each interaction.•Assay qualification was performed using KD values to calculate accuracy, repeatability and intermediate precision.•When KD cannot be determined, sensorgram comparison and Parallel Line Analysis (PLA) maybe used to qualify the assay.•We emphasize the importance of setting up appropriate SPR assay conditions to ensure the assay qualification parameters to meet the criteria.•The methods and guidelines provided may help to align SPR applications among the industry and regulatory authorities.
Plasmonic optical fiber-based biosensors are currently in their early stages of development as practical and integrated devices, gradually making their way towards the market. While the majority of ...these biosensors operate using white light and multimode optical fibers (OFs), our approach centers on single-mode OFs coupled with tilted fiber Bragg gratings (TFBGs) in the near-infrared wavelength range. Our objective is to enhance surface sensitivity and broaden sensing capabilities of OF-based sensors to develop in situ sensing with remote interrogation. In this study, we comprehensively assess their performance in comparison to the gold-standard plasmonic reference, a commercial device based on the Kretschmann-Raether prism configuration. We present their refractive index sensitivity and their capability for insulin sensing using a dedicated microfluidics approach. By optimizing a consistent surface biotrapping methodology, we elucidate the dynamic facets of both technologies and highlight their remarkable sensitivity to variations in bulk and surface properties. The one-to-one comparison between both technologies demonstrates the reliability of optical fiber-based measurements, showcasing similar experimental trends obtained with both the prismatic configuration and gold-coated TFBGs, with an even enhanced limit of detection for the latter. This study lays the foundation for the detection of punctual molecular interactions and opens the way towards the detection of spatially and temporally localized events on the surface of optical probes.
Surface plasmon resonance (SPR) is an optical technique that is utilized for detecting molecular interactions that occur in direct protein-protein interactions. Binding of a mobile molecule (analyte) ...to a molecule immobilized on a thin metal film (ligand) changes the refractive index of the film. The angle of extinction of light that is completely reflected, after polarized light impinges upon the surface, is altered and monitored as a change in detector position for a dip in reflected intensity (the surface plasmon resonance phenomenon). Because the method strictly detects mass, there is no need to label the interacting components, thus eliminating possible changes of their molecular properties. One of the advantages in SPR is its high sensitivity, compatible with the need for purification of small amounts of protein for analysis. This chapter concentrates on practical methodologies for performing surface plasmon resonance analysis.
HLA-DM is now known to have a major contribution to the selection of immunodominant epitopes. A better understanding of the mechanisms controlling epitope selection can be achieved by examination of ...the biophysical behavior of MHC class II molecules upon binding of antigenic peptides and of the effect of DM on the interactions. Using purified soluble molecules, in this chapter we describe several in vitro methods for measuring peptide binding to HLA-DR molecules and the effects of HLA-DM on this interaction. A simple qualitative method, Gentle SDS-PAGE Assay assesses the ability of peptides to form tight complexes with MHC class II molecules. Measuring binding kinetics is among the most informative approaches to understanding molecular mechanisms, and here we describe two different methods for measuring binding kinetics of peptide-MHC complexes. In one method, rates of association and dissociation of fluorescently labeled peptides to soluble MHC class II molecules can be determined using G50 spin columns to separate unbound peptides from those in complex with MHC molecules. In another method, association and dissociation of unlabeled peptides and MHC class II molecules can be determined in real time using BIAcore Surface Plasmon Resonance (SPR). We also describe an intrinsic tryptophan fluorescence assay for studying transient interactions of DM and MHC class II molecules.
•Non-invasive diagnostic technique for β-thalassemia point mutations by SPR biosensor.•SPR biosensor based on novel hybridization format for point mutations identification.•Diagnostic approach ...reliable, reproducible and with non-invasive sampling.
Optical biosensors based on Surface Plasmon Resonance (SPR), such as the Biacore™ X100, are widely used to study in real-time and in label-free mode bio-molecular interactions, including those allowing the identification of single point mutations responsible of genetic diseases, such as thalassemia and cystic fibrosis. The aim of this study was to verify whether the Biacore™ X100 can be proposed for the real-time detection of four mutations of the human β-globin gene causing β-thalassemia, a genetic blood disorder associated with absence (β0) or reduction (β+) of adult hemoglobin and severe anemia. In particular we analyzed the most frequent thalassemia point mutations present in the Mediterranean area (β039, β0IVSI-1, β+IVSI-110 and β+IVSI-6) using a novel SPR-based interaction format where two oligonucleotide probes (one complementary to the normal sequence and the other to the mutated one) were immobilized on sensor chips and asymmetric PCR targets obtained from genomic DNA of analyzed subjects were injected. For the development of the diagnostic approach, genomic DNAs of different genotypes for each mutation were obtained from blood samples or salivary swabs of 71 subjects, including healthy individuals, heterozygous β-thalassemia carriers and homozygous β-thalassemia patients. The results obtained allow proposing a new non-invasive diagnostic SPR-based protocol for thalassemia single point mutations using blood samples and salivary swabs as a source of genomic DNA.
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