Combining the Benefits of Biotin-Streptavidin Aptamer Immobilization with the Versatility of Ni-NTA Regeneration Strategies for SPR.

The high affinity of the biotin-streptavidin interaction has made this non-covalent coupling an indispensable strategy for the immobilization and enrichment of biomolecular affinity reagents. However, the irreversible nature of the biotin-streptavidin bond renders surfaces functionalized using this strategy permanently modified and not amenable to regeneration strategies that could increase assay reusability and throughput. To increase the utility of biotinylated targets, we here introduce a method for reversibly immobilizing biotinylated thrombin-binding aptamers onto a Ni-nitrilotriacetic acid (Ni-NTA) sensor chip using 6xHis-tagged streptavidin as a regenerable capture ligand. This approach enabled the reproducible immobilization of aptamers and measurements of aptamer-protein interaction in a surface plasmon resonance assay. The immobilized aptamer surface was stable during five experiments over two days, despite the reversible attachment of 6xHis-streptavidin to the Ni-NTA surface. In addition, we demonstrate the reproducibility of this immobilization method and the affinity assays performed using it. Finally, we verify the specificity of the biotin tag-streptavidin interaction and assess the efficiency of a straightforward method to regenerate and reuse the surface. The method described here will allow researchers to leverage the versatility and stability of the biotin-streptavidin interaction while increasing throughput and improving assay efficiency.

Strategies for Mitigating Commercial Sensor Chip Variability with Experimental Design Controls.

Surface plasmon resonance (SPR) is a popular real-time technique for the measurement of binding affinity and kinetics, and bench-top instruments combine affordability and ease of use with other benefits of the technique. Biomolecular ligands labeled with the 6xHis tag can be immobilized onto sensing surfaces presenting the Ni2+-nitrilotriacetic acid (NTA) functional group. While Ni-NTA immobilization offers many advantages, including the ability to regenerate and reuse the sensors, its use can lead to signal variability between experimental replicates. We report here a study of factors contributing to this variability using the Nicoya OpenSPR as a model system and suggest ways to control for those factors, increasing the reproducibility and rigor of the data. Our model ligand/analyte pairs were two ovarian cancer biomarker proteins (MUC16 and HE4) and their corresponding monoclonal antibodies. We observed a broad range of non-specific binding across multiple NTA chips. Experiments run on the same chips had more consistent results in ligand immobilization and analyte binding than experiments run on different chips. Further assessment showed that different chips demonstrated different maximum immobilizations for the same concentration of injected protein. We also show a variety of relationships between ligand immobilization level and analyte response, which we attribute to steric crowding at high ligand concentrations. Using this calibration to inform experimental design, researchers can choose protein concentrations for immobilization corresponding to the linear range of analyte response. We are the first to demonstrate calibration and normalization as a strategy to increase reproducibility and data quality of these chips. Our study assesses a variety of factors affecting chip variability, addressing a gap in knowledge about commercially available sensor chips. Controlling for these factors in the process of experimental design will minimize variability in analyte signal when using these important sensing platforms.

Application of the Nicoya OpenSPR to Studies of Biomolecular Binding: A Review of the Literature from 2016 to 2022.

The Nicoya OpenSPR is a benchtop surface plasmon resonance (SPR) instrument. As with other optical biosensor instruments, it is suitable for the label-free interaction analysis of a diverse set of biomolecules, including proteins, peptides, antibodies, nucleic acids, lipids, viruses, and hormones/cytokines. Supported assays include affinity/kinetics characterization, concentration analysis, yes/no assessment of binding, competition studies, and epitope mapping. OpenSPR exploits localized SPR detection in a benchtop platform and can be connected with an autosampler (XT) to perform automated analysis over an extended time period. In this review article, we provide a comprehensive survey of the 200 peer-reviewed papers published between 2016 and 2022 that use the OpenSPR platform. We highlight the range of biomolecular analytes and interactions that have been investigated using the platform, provide an overview on the most common applications for the instrument, and point out some representative research that highlights the flexibility and utility of the instrument.

Individual recombinant repeats of MUC16 display variable binding to CA125 antibodies.

BACKGROUND: Despite its importance in the clinical management of ovarian cancer, the CA125 biomarker - located on the mucin protein MUC16 - is still not completely understood. Questions remain about MUC16's function and structure, specifically the identity and location of the CA125 epitopes. OBJECTIVE: The goal of this study was to characterize the interaction of individual recombinant repeats from the tandem repeat domain of MUC16 with antibodies used in the clinical CA125 II test. METHODS: Using E. coli expression, we isolated nine repeats from the putative antigenic domain of CA125. Amino acid composition of recombinant repeats was confirmed by high-resolution mass spectrometry. We characterized the binding of four antibodies - OC125, M11, "OC125-like," and "M11-like" - to nine recombinant repeats using Western blotting, indirect enzyme-linked immunosorbent assay (ELISA), and localized surface plasmon resonance (SPR) spectroscopy. RESULTS: Each recombinant repeat was recognized by a different combination of CA125 antibodies. OC125 and "OC125-like" antibodies did not bind the same set of recombinant repeats, nor did M11 and "M11-like" antibodies. CONCLUSIONS: Characterization of the interactions between MUC16 recombinant repeats and CA125 antibodies will contribute to ongoing efforts to identify the CA125 epitopes and improve our understanding of this important biomarker.

Individual recombinant repeats of MUC16 display variable binding to CA125 antibodies.

BACKGROUND: Despite its importance in the clinical management of ovarian cancer, the CA125 biomarker-located on the mucin protein MUC16-is still not completely understood. Questions remain about MUC16's function and structure, specifically the identity and location of the CA125 epitopes. OBJECTIVE: The goal of this study was to characterize the interaction of individual recombinant repeats from the tandem repeat domain of MUC16 with antibodies used in the clinical CA125 II test. METHODS: Using E. coli expression, we isolated nine repeats from the putative antigenic domain of CA125. Amino acid composition of recombinant repeats was confirmed by high-resolution mass spectrometry. We characterized the binding of four antibodies-OC125, M11, "OC125-like," and "M11-like"-to nine recombinant repeats using Western blotting, indirect enzyme-linked immunosorbent assay (ELISA), and localized surface plasmon resonance (SPR) spectroscopy. RESULTS: Each recombinant repeat was recognized by a different combination of CA125 antibodies. OC125 and "OC125-like" antibodies did not bind the same set of recombinant repeats, nor did M11 and "M11-like" antibodies. CONCLUSIONS: Characterization of the interactions between MUC16 recombinant repeats and CA125 antibodies will contribute to ongoing efforts to identify the CA125 epitopes and improve our understanding of this important biomarker.