Ligand binding to a humanized anti-cocaine mAb measured by dye absorption spectroscopy.
In: Biochemical & Biophysical Research Communications, Jg. 535 (2021-01-08), S. 93-98
academicJournal
Zugriff:
Here we demonstrate that the antigen binding function of a humanized anti-cocaine mAb (h2E2) can be directly and easily determined using simple and inexpensive absorption spectroscopy and dyes commonly used for differential scanning fluorimetry, such as DASPMI and SYPRO Orange. Therapeutic monoclonal antibodies are commonly formulated in buffers which can interfere with necessary functional assays, containing additives and excipients such as mild detergents. Using the undiluted therapeutic product h2E2 mAb in its formulation buffer containing 0.01% polysorbate 80, the number of antigen/cocaine binding sites can be determined by the increase in absorbance (for DASPMI dye) or by the decrease in absorbance maximum wavelength (for SYPRO Orange dye), confirming proper function of the therapeutic mAb product. This ligand-induced visible dye absorption change can also be used to qualitatively evaluate the relative affinities of various metabolites of cocaine. These results are confirmed and extended by binding data obtained in the same formulation buffer using intrinsic tyrosine and tryptophan fluorescence quenching by cocaine, as well as by differential scanning fluorimetry. Interestingly, the binding of the cocaine metabolite norcocaine was demonstrated to be differentially affected by the pH 6 formulation buffer used for this mAb, presumably due to the differential ionizability of the demethylated norcocaine tropane ring nitrogen. This simple, direct, and inexpensive technique should prove useful for evaluation of other small molecule binding mAbs directly in their formulation buffers containing detergent, allowing rapid functional assessment of the produced therapeutic proteins. Image 1 • Cocaine binding of clinically formulated mAb is quantitated by visible dye absorption. • Functional ligand binding sites easily and rapidly measured by dye absorbance changes. • Cocaine metabolite relative affinities are qualitatively assessed using DASPMI dye. • This simple and direct method may be applicable to other small molecule directed mAbs. [ABSTRACT FROM AUTHOR]
Titel: |
Ligand binding to a humanized anti-cocaine mAb measured by dye absorption spectroscopy.
|
---|---|
Autor/in / Beteiligte Person: | Kirley, Terence L. ; Norman, Andrew B. |
Link: | |
Zeitschrift: | Biochemical & Biophysical Research Communications, Jg. 535 (2021-01-08), S. 93-98 |
Veröffentlichung: | 2021 |
Medientyp: | academicJournal |
ISSN: | 0006-291X (print) |
DOI: | 10.1016/j.bbrc.2020.12.034 |
Schlagwort: |
|
Sonstiges: |
|