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Dually Labeled Biomolecules for Characterizing Biotinylated Species through Competitive Binding Reactions.

ABSTRACT: The capability of biotinylated molecules to bind streptavidin may be a more functional measure of the success of target biotinylation than titration of total bound biotins per molecule. It was demonstrated that the binding capability could be assessed by a competitive assay, in which a biotinylated antibody (BA) (or protein, ligand, receptor etc.) of interest competed with a reference antibody (or a protein) dually labeled with biotin and electrochemiluminescence (ECL) moieties for the binding sites of streptavidin coated on the surface of magnetic beads. Inversely related to the ECL signal, the binding capability of a biotinylated antibody can be reproducibly evaluated by multiple sets of easily acquired data series rather than by a single measurement. This method can be employed in an ordinary laboratory with an automated ECL analyzer or other readout instruments for routine characterization of any biotinylated species, such as proteins, ligands, receptors, and polypeptides.

SUBMITTER: Xu Y

PROVIDER: S-EPMC7758948 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Dually Labeled Biomolecules for Characterizing Biotinylated Species through Competitive Binding Reactions.

Xu Yan Y Pan Yuli Y Li Lingnuo L Zhou Ming M

ACS omega 20201211 50

The capability of biotinylated molecules to bind streptavidin may be a more functional measure of the success of target biotinylation than titration of total bound biotins per molecule. It was demonstrated that the binding capability could be assessed by a competitive assay, in which a biotinylated antibody (BA) (or protein, ligand, receptor etc.) of interest competed with a reference antibody (or a protein) dually labeled with biotin and electrochemiluminescence (ECL) moieties for the binding s ...[more]

PMID: 33376896

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Project description:Magnetic resonance image-guided focused ultrasound has emerged as a viable non-invasive technique for the treatment of central nervous system-related diseases/disorders. Application of mechanical and thermal effects associated with focused transcranial ultrasound has been studied extensively in pre-clinical models, which has paved the way for clinical trials. However, in vivo treatment evaluation techniques on drug delivery application via blood-brain barrier opening has not been fully explored. Current treatment evaluation techniques via magnetic resonance imaging are hindered by systemic toxicity resulting from free gadolinium delivery. Here we propose a novel treatment evaluation strategy to overcome limitations by (i) synthesizing liposomes that are dually labeled with gadolinium, a magnetic resonance imaging (MRI) contrast agent, and rhodamine, a fluorophore; (ii) applying a focused ultrasound (FUS)-mediated BBB opening technique to deliver the liposomes across vascular barriers, achieving local gadolinium enhancement while reducing systemic and unwanted regional toxic effects associated with free gadolinium; and (iii) utilizing the MRI modality to confirm the delivery as it is already included in the FUS treatment in clinic. Liposomes were secondarily labeled with a fluorescent marker to confirm results obtained by MRI quantification postmortem. Two different sizes, 77.5 nm (group A) and 140 nm (group B), of gadolinium- and fluorescence-labeled liposomes were fabricated using thin-film hydration followed by extrusion methods and determined their stability up to 6 h under physiologic conditions. Gadolinium signal was detected on contrast-enhanced T1-weighted MRI 5 h after the delivery of liposomes via the BBB opening approach with an ultrasound pulse of 0.42 MPa (estimate in water) combined with microbubbles. MRI contrast was enhanced significantly in sonicated regions compared with non-sonicated regions of the brain. This was due to the accumulation of labeled liposomes, which was confirmed by detection of rhodamine fluorescence in histologic sections. The relative increase in MRI signal intensity was greater for smaller liposomes (mean diameter = 77.5 nm) than larger liposomes (mean diameter = 140 nm), which suggested a greater accumulation of the smaller liposomes in the brain after ultrasound-mediated opening of the BBB. Our findings suggest that the dual-labeled nanocarrier platform can be established, the FUS-mediated BBB opening approach can be used to deliver it through vascular barriers and MRI can be used to evaluate the extent of nanocarrier delivery.

Dataset Information

Dually Labeled Biomolecules for Characterizing Biotinylated Species through Competitive Binding Reactions.

Publications

Dually Labeled Biomolecules for Characterizing Biotinylated Species through Competitive Binding Reactions.

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