Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Yang, Danlin; Singh, Ajit Pratap; Wu, Helen; Kroe‐Barrett, Rachel

doi:10.3791/55659

Cited by 19 publications

(16 citation statements)

References 18 publications

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“…The application of BLI technology to a variety of biological and drug discovery problems is expanding. [76][77][78] As an early discovery research screening tool, BLI can be used to more quickly eliminate candidates from the pipeline and can be beneficial in diversifying the types of assays used in discovery research. The approach described here is an important tool that can be used in conjunction with other biophysical methods, such as NMR and AUC, to better investigate crowded solution phenomena.…”

Section: Discussionmentioning

confidence: 99%

Measuring the effects of macromolecular crowding on antibody function with biolayer interferometry

Kim

Yao

Vanam

et al. 2019

mAbs

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Biotherapeutic proteins are commonly dosed at high concentrations into the blood, which is an inherently complex, crowded solution with substantial protein content. The effects of macromolecular crowding may lead to an appreciable level of non-specific hetero-association in this physiological environment. Therefore, developing a method to characterize the diverse consequences of nonspecific interactions between proteins under such non-ideal, crowded conditions, which deviate substantially from those commonly employed for in vitro characterization, is vital to achieving a more complete picture of antibody function in a biological context. In this study, we investigated non-specific interactions between human serum albumin (HSA) and two monoclonal antibodies (mAbs) by static light scattering and determined these interactions are both ionic strength-dependent and mAbdependent. Using biolayer interferometry (BLI), we assessed the effect of HSA on antigen binding by mAbs, demonstrating that these non-specific interactions have a functional impact on mAb:antigen interactions, particularly at low ionic strength. While this effect is mitigated at physiological ionic strength, our in vitro data support the notion that HSA in the blood may lead to non-specific interactions with mAbs in vivo, with a potential impact on their interactions with antigen. Furthermore, the BLI method offers a high-throughput advantage compared to orthogonal techniques such as analytical ultracentrifugation and is amenable to a greater variety of solution conditions compared to nuclear magnetic resonance spectroscopy. Our study demonstrates that BLI is a viable technology for examining the impact of non-specific interactions on specific biologically relevant interactions, providing a direct method to assess binding events in crowded conditions.

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Section: Discussionmentioning

confidence: 99%

Measuring the effects of macromolecular crowding on antibody function with biolayer interferometry

Kim

Yao

Vanam

et al. 2019

mAbs

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“…All these instruments have their advantages and disadvantages regarding throughput, consistency of data with high quality, and fluidics. Details are given in [414]; company links are provided in [415]. The second study compares Biacore 3000, Octet RED96, IBIS MX96, and 2 imaging RIfS instruments [416].…”

Section: Detection Methods Reviewed and Comparedmentioning

confidence: 99%

Critical assessment of relevant methods in the field of biosensors with direct optical detection based on fibers and waveguides using plasmonic, resonance, and interference effects

Gauglitz

2020

Anal Bioanal Chem

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Direct optical detection has proven to be a highly interesting tool in biomolecular interaction analysis to be used in drug discovery, ligand/receptor interactions, environmental analysis, clinical diagnostics, screening of large data volumes in immunology, cancer therapy, or personalized medicine. In this review, the fundamental optical principles and applications are reviewed. Devices are based on concepts such as refractometry, evanescent field, waveguides modes, reflectometry, resonance and/or interference. They are realized in ring resonators; prism couplers; surface plasmon resonance; resonant mirror; Bragg grating; grating couplers; photonic crystals, Mach-Zehnder, Young, Hartman interferometers; backscattering; ellipsometry; or reflectance interferometry. The physical theories of various optical principles have already been reviewed in detail elsewhere and are therefore only cited. This review provides an overall survey on the application of these methods in direct optical biosensing. The "historical" development of the main principles is given to understand the various, and sometimes only slightly modified variations published as "new" methods or the use of a new acronym and commercialization by different companies. Improvement of optics is only one way to increase the quality of biosensors. Additional essential aspects are the surface modification of transducers, immobilization strategies, selection of recognition elements, the influence of non-specific interaction, selectivity, and sensitivity. Furthermore, papers use for reporting minimal amounts of detectable analyte terms such as value of mass, moles, grams, or mol/L which are difficult to compare. Both these essential aspects (i.e., biochemistry and the presentation of LOD values) can be discussed only in brief (but references are provided) in order to prevent the paper from becoming too long. The review will concentrate on a comparison of the optical methods, their application, and the resulting bioanalytical quality.

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“…Pipette serial dilutions of purified Fab into 200 µL of 1x kinetics buffer, with a high concentration of 500 nM, and subsequent serial dilutions of 250 nM, 125 nM, and 62.5 nM.Aliquot reagents into black flat bottom polypropylene 96-well microplate as shown in Figure 5 A , where each well contains 200 µL of the indicated solution.Collect data using the Kinetics Assays in the Data Acquisition software, as described previously383977 (Figure 5 A ).

Briefly, transfer the biosensors into wells containing 1x kinetics buffer to baseline for 60 s before loading 25 ng µL -1 of glycoprotein for 240 s (or until a threshold of 1.0 nm is reached) at 1,000 rpm.

After a second baseline of 60 s in 1x kinetics buffer, transfer the biosensors into wells containing the serial dilution of Fab.

…”

Section: Protocolmentioning

confidence: 99%

“…Collect data using the Kinetics Assays in the Data Acquisition software, as described previously383977 (Figure 5 A ).

After a second baseline of 60 s in 1x kinetics buffer, transfer the biosensors into wells containing the serial dilution of Fab.

…”

Section: Protocolmentioning

confidence: 99%

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Ereño‐Orbea

Sicard

Cui

et al. 2018

JoVE

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Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

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Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Cited by 19 publications

References 18 publications

Measuring the effects of macromolecular crowding on antibody function with biolayer interferometry

Measuring the effects of macromolecular crowding on antibody function with biolayer interferometry

Critical assessment of relevant methods in the field of biosensors with direct optical detection based on fibers and waveguides using plasmonic, resonance, and interference effects

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

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