Precise construction of oligonucleotide–Fab fragment conjugate for homogeneous immunoassay using HaloTag technology

Päkkilä, Henna; Peltomaa, Riikka; Lamminmäki, Urpo; Soukka, Tero

doi:10.1016/j.ab.2014.11.018

Cited by 3 publications

(5 citation statements)

References 45 publications

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“…In addition, HaloTag has been employed for the investigation of protein–DNA interactions using high-throughput methods for functional analysis of human genes 73 and conjugation of DNA oligonucleotides to Fab fragments as a potential cancer diagnostic tool. 74 …”

Section: Analyzing Protein–protein and Protein–dna Interactionsmentioning

confidence: 99%

“…Values obtained from this study were adapted for computation simulations to assist in analyzing possible competitive effects in signal transduction pathways. In addition, HaloTag has been employed for the investigation of protein–DNA interactions using high-throughput methods for functional analysis of human genes and conjugation of DNA oligonucleotides to Fab fragments as a potential cancer diagnostic tool …”

Section: Analyzing Protein–protein and Protein–dna Interactionsmentioning

confidence: 99%

“…In addition, HaloTag has been employed for the investigation of protein−DNA interactions using high-throughput methods for functional analysis of human genes 73 and conjugation of DNA oligonucleotides to Fab fragments as a potential cancer diagnostic tool. 74 ■ PROTEOMIC ANALYSIS USING PROTEIN ASSAYS Dismal survival rates of specific diseases can often be linked to insufficient tools for their early detection. 75 To overcome this pitfall, researchers are investigating novel diagnostic assays as a route for detecting disease at earlier stages, when treatment is optimal.…”

Section: ■ Evaluation Of Protein Functionmentioning

confidence: 99%

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HaloTag Technology: A Versatile Platform for Biomedical Applications

2015

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Exploration of protein function and interaction is critical for discovering links between genomics, proteomics, and disease state; yet the immense complexity of proteomics found in biological systems currently limit our investigational capacities. While affinity and autofluorescent tags are widely employed for protein analysis, these methods have limited success as they lack specificity and require multiple fusion tags and genetic constructs. As an alternative approach, the innovative HaloTag protein fusion platform allows for comprehensive analysis of protein function and interaction using a single genetic construct with multiple capabilities. This is accomplished using a simplified process, in which a variable HaloTag ligand binds rapidly to the HaloTag protein (usually linked to the protein of interest) with high affinity and specificity. In this review article, we examine all current applications of the HaloTag technology platform for biomedical applications such as the study of protein isolation and purification, protein function, protein-protein and protein-DNA interactions, biological assays, in vitro cellular imaging, and in vivo molecular imaging. In addition, novel uses of the HaloTag platform are briefly discussed with potential future applications.

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Section: Analyzing Protein–protein and Protein–dna Interactionsmentioning

confidence: 99%

Section: Analyzing Protein–protein and Protein–dna Interactionsmentioning

confidence: 99%

Section: ■ Evaluation Of Protein Functionmentioning

confidence: 99%

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HaloTag Technology: A Versatile Platform for Biomedical Applications

2015

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“…21 Additionally, site-specific DNA–Fab conjugation has recently been used to develop an extremely sensitive homogeneous immunoassay, detecting PSA at concentrations of 0.27 ng/mL. 22 The availability of genetically encoded unnatural amino acids with unique chemical reactivity can provide a solution to these challenges. Previously, we have site-specifically incorporated p -acetylphenylalanine (pAcF) and p -azidophenylalanine (pAzF) into proteins in response to amber nonsense codons in bacteria and mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

“…In the context of immuno-PCR, there was a significant difference in signal when comparing site-specific and random DNA conjugation . Additionally, site-specific DNA–Fab conjugation has recently been used to develop an extremely sensitive homogeneous immunoassay, detecting PSA at concentrations of 0.27 ng/mL . The availability of genetically encoded unnatural amino acids with unique chemical reactivity can provide a solution to these challenges.…”

Section: Introductionmentioning

confidence: 99%

Antibody Microarrays Utilizing Site-Specific Antibody–Oligonucleotide Conjugates

Wold¹,

McBride²,

Axup³

et al. 2015

Bioconjugate Chem.

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Protein arrays are typically made by random absorption of proteins to the array surface, potentially limiting the amount of properly oriented and functional molecules. We report the development of a DNA encoded antibody microarray utilizing site-specific antibody–oligonucleotide conjugates that can be used for cell immobilization as well as the detection of genes and proteins. This technology allows for the facile generation of antibody microarrays while circumventing many of the drawbacks of conventionally produced antibody arrays. We demonstrate that this method can be used to capture and detect SK-BR-3 cells (Her2+ breast cancer cells) at concentrations as low as 102 cells/mL (which is equivalent to 10 cells per 100 µL array) without the use of microfluidics, which is 100- to 105-fold more sensitive than comparable techniques. Additionally, the method was shown to be able to detect cells in a complex mixture, effectively immobilizing and specifically detecting Her2+ cells at a concentration of 102 SK-BR-3 cells/mL in 4 × 106 white blood cells/mL. Patients with a variety of cancers can have circulating tumor cell counts of between 1 and 103 cells/mL in whole blood, well within the range of this technology.

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Nanomaterial bio-activation and macromolecules functionalization: The search for reliable protocols

Marco

2018

Protein Expression and Purification

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Precise construction of oligonucleotide–Fab fragment conjugate for homogeneous immunoassay using HaloTag technology

Cited by 3 publications

References 45 publications

HaloTag Technology: A Versatile Platform for Biomedical Applications

HaloTag Technology: A Versatile Platform for Biomedical Applications

Antibody Microarrays Utilizing Site-Specific Antibody–Oligonucleotide Conjugates

Nanomaterial bio-activation and macromolecules functionalization: The search for reliable protocols

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