Research paper
Cocaine binding to the Fab fragment of a humanized anti-cocaine mAb quantitated by dye absorption and fluorescence spectroscopy

https://doi.org/10.1016/j.jim.2021.113103Get rights and content
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Highlights

  • Cocaine binding of mAb Fab fragments quantitated by extrinsic visible dye absorption.

  • Fab small molecule ligand binding sites are easily and rapidly measured.

  • A novel DASPMI-based dye fluorescence antigen/ligand binding assay was developed.

  • These simple methods may be applicable to other small-molecule mAbs and Fab fragments.

Abstract

In this work, we establish that cocaine binding to the Fab fragment of a recombinant humanized anti-cocaine mAb (h2E2) can be directly and easily quantitated using simple and inexpensive absorption and fluorescence measurements, employing dyes typically used for differential scanning fluorimetry, DASPMI and SYPRO Orange. For concentrated samples of the Fab fragment, absorbance spectroscopy employing these dyes reveals the number of cocaine sites present, using either DASPMI (by measuring the increase in dye absorbance) or SYPRO Orange (by measuring the change in dye maximal absorbance wavelength). Interestingly, we observed that cocaine binding to the Fab fragment had a much different effect on the SYPRO Orange dye absorbance than previously reported for the intact h2E2 mAb, resulting in a large decrease in the total dye absorbance for the Fab fragment, in contrast to previous results with the intact h2E2 mAb. For dilute samples of Fab fragment, a dye fluorescence emission spectroscopy assay was developed to quantitate the number of cocaine (and other high affinity cocaine metabolites) binding sites via the ligand-induced decrease in fluorescence emission of both of these extrinsic dyes. The difference in the cocaine titrations for the high affinity (Kd < 30 nM) ligands, cocaine, cocaethylene and benzoylecgonine and the low affinity (Kd > 30 μM) ligands, norcocaine, ecgonine methyl ester, and ecgonine were obvious using this assay. These simple, direct, and inexpensive techniques should prove useful for evaluation of other small molecule antigen binding Fab fragments, enabling quantitation and rapid biochemical assessments necessary for determining Fab fragment suitability for in vivo uses and other assays and experiments.

Keywords

Monoclonal antibody
Fab fragment
Cocaine antigen binding
Absorption spectroscopy
DASPMI rotor dye
Fluorescence spectroscopy

Abbreviations

Fab
Fragment antigen-binding
mAb
Monoclonal antibody
h2E2
Humanized anti-cocaine monoclonal antibody
DASPMI
(4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide
MOPS
3-(N-morpholino)propanesulfonic acid
CE
Cocaethylene
COC
Cocaine
BE
Benzoylecgonine
NC
Norcocaine
EME
Ecgonine methyl ester
EG
Ecgonine

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