Conjugation of biotin-coated luminescent quantum dots with single domain antibody-rhizavidin fusions

Liu, Jinny L.; Walper, Scott A.; Turner, Kendrick B.; Lee, P. Audrey Brozozog; Medintz, Igor L.; Susumu, Kimihiro; Oh, Eunkeu; Zabetakis, Dan; Goldman, Ellen R.; Anderson, George P.

doi:10.1016/j.btre.2016.03.001

Cited by 18 publications

(17 citation statements)

References 40 publications

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“…Rhizavidin is a dimeric biotin binding protein derived from the symbiotic nitrogen-fixing bacterium Rhizobium etli CFN42 ( Helppolainen et al, 2007 ; Meir et al, 2009 ). Previously, we demonstrated the first genetic fusions of sdAbs with rhizavidin; we showed ricin binding sdAbs prepared as fusions with rhizavidin provided improved capture reagents and could be used to functionalize biotin-conjugated quantum dots ( Liu et al, 2014 ; Liu et al, 2016 ). Because the anti-ricin sdAb monomers have sub nM affinity, we attributed the better toxin detecion using rhizavidin fusions to oriented immoblization.…”

Section: Resultsmentioning

confidence: 99%

Bglbrick strategy for the construction of single domain antibody fusions

Goldman

Broussard

Anderson

et al. 2017

Heliyon

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Single domain antibodies, recombinantly expressed variable domains derived from camelid heavy chain antibodies, are often expressed as multimers for detection and therapeutic applications. Constructs in which several single domain antibodies are genetically fused serially, as well as those in which single domain antibodies are genetically linked with domains that naturally form multimers, yield improvement in apparent binding affinity due to avidity. Here, using a single domain antibody that binds envelope protein from the Dengue virus, we demonstrated the construction of single domain antibody dimers using the Bglbrick cloning strategy. Constructing single domain antibodies and multimerization domains as Bglbrick parts enables the easy mixing and matching of parts. The dimeric constructs provided enhanced fluorescent signal in assays for detection of Dengue virus like particles over the monomeric single domain antibody.

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Section: Resultsmentioning

confidence: 99%

Bglbrick strategy for the construction of single domain antibody fusions

Goldman

Broussard

Anderson

et al. 2017

Heliyon

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“…Additionally, several strategies have been described for increasing the stability of sdAb [37], and have been shown to increase melting temperatures by as much as 20 °C [43]. Similarly, sdAb can be engineered to tailor them for specific assay formats [28,32,55].…”

Section: Discussionmentioning

confidence: 99%

“…SdAb combine the sensitivity and specificity of conventional antibodies with advantages that come from being comprised of only a single domain, such as high physical-chemical stability including heat-resistance, the ability to refold after denaturation, excellent solubility in water, and the capacity to be produced using recombinant technology in good yield [21,22,23,24,25,26]. SdAb that are produced using recombinant technology, most often in Escherichia coli , are amenable to the formation of fusion constructs to tailor their integration into a variety of assay formats and sensor systems [27,28,29,30,31,32]. They can also be modified to improve their biophysical properties; mutagenesis has led to variants with improved protein production and stability, as assessed by the protein’s melting point [33,34,35,36,37].…”

Section: Introductionmentioning

confidence: 99%

Selection of Single-Domain Antibodies towards Western Equine Encephalitis Virus

et al. 2018

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In this work, we describe the selection and characterization of single-domain antibodies (sdAb) towards the E2/E3E2 envelope protein of the Western equine encephalitis virus (WEEV). Our purpose was to identify novel recognition elements which could be used for the detection, diagnosis, and perhaps treatment of western equine encephalitis (WEE). To achieve this goal, we prepared an immune phage display library derived from the peripheral blood lymphocytes of a llama that had been immunized with an equine vaccine that includes killed WEEV (West Nile Innovator + VEWT). This library was panned against recombinant envelope (E2/E3E2) protein from WEEV, and seven representative sdAb from the five identified sequence families were characterized. The specificity, affinity, and melting point of each sdAb was determined, and their ability to detect the recombinant protein in a MagPlex sandwich immunoassay was confirmed. Thus, these new binders represent novel recognition elements for the E2/E3E2 proteins of WEEV that are available to the research community for further investigation into their applicability for use in the diagnosis or treatment of WEE.

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“…Despite the size of this conjugate, which could be limiting in some applications, usability in cell biology has already been mentioned. 27,28 Besides the possibility to prepare a "lab-made" quantum dotbased conjugate by one of the methods mentioned above, there is also a commercially available labeling kit (SiteClick™ Antibody Labeling Kit) enabling specic conjugation of QDs with IgG molecules via modied carbohydrate domains. However, this antibody labeling system brings a few disadvantages such as the limited amount of QDs per 1 mol of IgG molecules and contamination of the nal conjugation product by free nonconjugated QDs.…”

Section: Introductionmentioning

confidence: 99%