ArticleJournal of immunological methods2020
A novel differential scanning fluorimetry analysis of a humanized anti-cocaine mAb and its ligand binding characteristics.
Article in Journal of immunological methods, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed, 25 citations in OpenAlex.
- Synthesis and characterization of a functional benzoylecgonine - bovine serum albumin conjugate for quantification of a humanized monoclonal anti-cocaine antibody.Biochemistry and biophysics reports · 2025Article
- Selective real-time disulfide reduction with simultaneous fluorescent labeling of a humanized anti-cocaine mAb.Biochemistry and biophysics reports · 2025Article
- Decreased solubility and increased adsorptivity of a biotinylated humanized anti-cocaine mAb.Analytical biochemistry · 2025Article
- Novel partial reduction of the humanized anti-cocaine mAb h2E2 for selective cysteine labeling.Biochemical and biophysical research communications · 2024Article
- Characterization and optimization of fluorescein isothiocyanate labeling of humanized h2E2 anti-cocaine mAb.Biochemistry and biophysics reports · 2023Article
- Reformulation and Thermal Stability of a Therapeutic Anti-Cocaine mAb.Journal of pharmaceutical sciences · 2023Article
- Isothermal titration calorimetry determination of thermodynamics of binding of cocaine and its metabolites to humanized h2E2 anti-cocaine mAb.Biochemistry and biophysics reports · 2022Article
- Advancing humanized monoclonal antibody for counteracting fentanyl toxicity towards clinical development.Human vaccines & immunotherapeutics · 2022Article
- Tyrosine nitration of a humanized anti-cocaine mAb differentially affects ligand binding of cocaine and its metabolites.Biochemistry and biophysics reports · 2022Article
- Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability.International journal of molecular sciences · 2022Article
- Cocaine binding to the Fab fragment of a humanized anti-cocaine mAb quantitated by dye absorption and fluorescence spectroscopy.Journal of immunological methods · 2021Article
- Ligand binding to a humanized anti-cocaine mAb measured by dye absorption spectroscopy.Biochemical and biophysical research communications · 2021Article
- Multi-domain unfolding of the Fab fragment of a humanized anti-cocaine mAb characterized by non-reducing SDS-PAGE.Biochemical and biophysical research communications · 2020Article
- Ligand binding to a humanized anti-cocaine mAb detected by non-reducing SDS-PAGE.Biochemistry and biophysics reports · 2020Article
Corrections and comments
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
An anti-cocaine monoclonal antibody (mAb) designated h2E2 will soon enter clinical trials for the treatment of cocaine abuse disorders. Importantly, this antibody selectively binds cocaine and its active metabolite, cocaethylene, with high affinity, while binding inactive metabolites with substantially lower affinities. Here, we used differential scanning fluorimetry (DSF) to characterize the stability and ligand binding properties of this antibody and its cocaine-binding Fab fragment. The Sypro orange dye commonly used for DSF revealed multiple overlapping thermal protein denaturation transitions for both the mAb and the Fab fragment, making quantitative analysis of ligand binding by thermal stabilization problematic. However, by using the "rotor" dye, DASPMI (4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide), which measures the rotational restriction of the fluorescent dye (as opposed to the Sypro orange dye which measures the hydrophobicity of the dye microenvironment), a simple two state thermal denaturation transition that is stabilized by ligand binding was observed for the h2E2 mAb, enabling Boltzmann fitting and quantitative thermodynamic analysis of the DASPMI DSF mAb cocaine and metabolite binding data. The computed affinities were consistent with ligand binding affinities determined using other techniques. Thus, this novel DASPMI DSF method can simply, inexpensively, and very rapidly generate ligand binding constants for the h2E2 mAb, despite the presence of multiple, overlapping, thermally unfolding protein domains characteristic of all mAbs. This approach is likely applicable to other mAbs currently in use for many research and therapeutic applications.
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