The effect of uniform capture molecule orientation on biosensor sensitivity: Dependence on analyte properties

Trilling, Anke K.; Harmsen, Michiel M.; Ruigrok, Vincent J. B.; Zuilhof, Han; Beekwilder, Jules

doi:10.1016/j.bios.2012.07.027

Cited by 82 publications

(70 citation statements)

References 40 publications

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“…The difference was totally due to the improved association rate. This result confirmed the binding functionality of the construct expressed in the cytoplasm and suggests that despite having similar overall mass, the relative length and/or folding of the different tags can influence the accessibility and actual binding of the two constructs to the antigen epitope, as already noticed by other authors [25,26]. We cannot rule out that the small observed differences in terms of antibody binding capacity were due to minimal stability variation of the different constructs.…”

Section: Resultssupporting

confidence: 87%

Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies

et al. 2014

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BackgroundThe isolation of recombinant antibody fragments from displayed libraries represents a powerful alternative to the generation of IgGs using hybridoma technology. The selected antibody fragments can then be easily engineered into (multi)-tagged constructs of variable mass and complexity as well as reconstituted into Camelidae IgG-like molecules when expressed fused to Fc domains. Nevertheless, all antibody constructs depend on an oxidizing environment for correct folding and consequently still belong to the proteins difficult to express in bacteria. In such organisms they are mostly produced at low yields in the periplasmic space.ResultsWe demonstrate that fusion constructs of recombinant antibodies in combination with multiple tags can be produced at high yields and totally functional in the cytoplasm of bacteria expressing sulfhydryl oxidase. The method was applied to structurally demanding molecules such as VHHs fused to SNAP and Fc domains and was validated using the antibody-derived reagents in a variety of immune techniques (FACS, ELISA, WB, IP, SPR, and IF).ConclusionsThe collected data demonstrate the feasibility of a method that establishes a totally new approach for producing rapidly and inexpensively functional Camelidae IgG-like monoclonal antibodies and antibody-based reagents containing multiple disulfide bonds and suitable for both basic research and clinical applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0140-1) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 87%

Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies

et al. 2014

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“…When different assay formats yield consistent results, this suggests that the biological activity of the assay reagents has not been compromised by immobilization. Although discrepant results from biosensor assays using different immobilization formats have been reported, 51,52,53 in their meta-analysis benchmarking study of optical biosensor data from over 1200 publications, Rich and Myszka 54 state that only a few investigators have directly compared multiple immobilization and capture methods to address the potential caveats associated with protein immobilization methods.…”

Section: Discussionmentioning

confidence: 99%

Impact of SPR biosensor assay configuration on antibody: Neonatal Fc receptor binding data

Wang¹,

McKay²,

Yee³

et al. 2016

mAbs

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Binding interactions with the neonatal Fc receptor (FcRn) are one determinant of pharmacokinetic properties of recombinant human monoclonal antibody (rhumAb) therapeutics, and a conserved binding motif in the crystallizable fragment (Fc) region of IgG molecules interacts with FcRn. Surface plasmon resonance (SPR) biosensor assays are often used to characterize interactions between FcRn and rhumAb therapeutics. In such assays, generally either the rhumAb (format 1) or the FcRn protein (format 2) is immobilized on a biosensor chip. However, because evidence suggests that, in some cases, the variable domains of a rhumAb may also affect FcRn binding, we evaluated the effect of SPR assay configuration on binding data. We sought to assess FcRn binding properties of 2 rhumAbs (rhumAb1 and rhumAb2) to FcRn proteins using these 2 biosensor assay formats. The two rhumAbs have greater than 99% sequence identity in the Fc domain but differ in their Fab regions. rhumAb2 contains a positively charged patch in the variable domain that is absent in rhumAb1. Our results showed that binding of rhumAb1 to FcRn was independent of biosensor assay configuration, while binding of rhumAb2 to FcRn was highly SPR assay configuration dependent. Further investigations revealed that the format dependency of rhumAb2-FcRn binding is linked to the basic residues that form a positively charged patch in the variable domain of rhumAb2. Our work highlights the importance of analyzing rhumAb-FcRn binding interactions using 2 alternate SPR biosensor assay configurations. This approach may also provide a simple way to identify the potential for non-Fc-driven FcRn binding interactions in otherwise typical IgGs.

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“…However, this method results in random orientation of the antibody. Orienting single domain antigen-binding fragments, also known as VHHs or nanobodies, on sensor surfaces by click chemistry leads to greatly improved sensitivity for biosensors (Trilling et al, 2013, 2014). The small size of VHHs (~15 kDa) and their excellent thermal and chemical stability profile make them suitable for numerous diagnostic and therapeutic applications (De Meyer et al, 2014; Muyldermans, 2013; Siontorou 2013).…”

Section: Introductionmentioning

confidence: 99%

“…The small size of VHHs (~15 kDa) and their excellent thermal and chemical stability profile make them suitable for numerous diagnostic and therapeutic applications (De Meyer et al, 2014; Muyldermans, 2013; Siontorou 2013). Since a large percentage of the VHH surface is involved in binding interactions, it is essential to create a uniform orientation using site-specific modifications (Trilling et al, 2013, 2014) to improve the biosensor’s performance. To achieve consistency in orientation, we previously used a combination of sortase-mediated trans-peptidation reactions and ‘click’ chemistry to site-specifically link VHHs with linkers coated onto GO (Agrawal et al, 2008; Chen et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Rapid capture and labeling of cells on single domain antibodies-functionalized flow cell

Chen

Duarte

et al. 2017

Biosensors and Bioelectronics

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Current techniques to characterize leukocyte subgroups in blood require long sample preparation times and sizable sample volumes. A simplified method for leukocyte characterization using smaller blood volumes would thus be useful in diagnostic settings. Here we describe a flow system comprised of two functionalized graphene oxide (GO) surfaces that allow the capture of distinct leukocyte populations from small volumes blood using camelid single-domain antibodyfragments (VHHs) as capture agents. We used site-specifically labeled leukocytes to detect and identify cells exposed to fungal challenge. Combining the chemical and optical properties of GO with the versatility of the VHH scaffold in the context of a flow system provides a quick and efficient method for the capture and characterization of functional leukocytes.

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The effect of uniform capture molecule orientation on biosensor sensitivity: Dependence on analyte properties

Cited by 82 publications

References 40 publications

Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies

Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies

Impact of SPR biosensor assay configuration on antibody: Neonatal Fc receptor binding data

Rapid capture and labeling of cells on single domain antibodies-functionalized flow cell

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