Ligand binding to a humanized anti-cocaine mAb detected by non-reducing SDS-PAGE
Monoclonal antibodies are very useful tools in experimental biology, as well as being valuable and effective therapeutic drugs. They can be targeted against proteins with varied functions, or against small molecules of interest to both researchers and clinicians, such as drugs of abuse, including co...
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| Format: | Article |
| Language: | English |
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Elsevier
2020-09-01
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| Series: | Biochemistry and Biophysics Reports |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405580820301047 |
| _version_ | 1818250043593850880 |
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| author | Terence L. Kirley Andrew B. Norman |
| author_facet | Terence L. Kirley Andrew B. Norman |
| author_sort | Terence L. Kirley |
| collection | DOAJ |
| description | Monoclonal antibodies are very useful tools in experimental biology, as well as being valuable and effective therapeutic drugs. They can be targeted against proteins with varied functions, or against small molecules of interest to both researchers and clinicians, such as drugs of abuse, including cocaine. Since there is no currently FDA approved pharmacological treatment for cocaine abuse, our laboratory has developed an anti-cocaine mAb for the treatment of cocaine use disorders. This humanized anti-cocaine antibody, named h2E2, has been thoroughly characterized both functionally and structurally, in preparation for the start of clinical development. We previously showed that this mAb could be characterized by sequential thermal unfolding of antibody domains using non-reducing SDS-PAGE. We also demonstrated that ligand-induced protein stabilization can be used to quantitatively measure cocaine and cocaine metabolite binding to the h2E2 mAb, utilizing differential scanning fluorimetry. Here, we demonstrate the utility of non-reducing SDS-PAGE for the qualitative assessment of binding of cocaine and some of its metabolites, both to the intact mAb, as well as to fragments containing the antigen binding site (Fab and F(ab’)2 fragments). These results clearly show a ligand concentration dependence of the stabilization of the cocaine binding domain in non-reducing SDS-PAGE, as well as visually differentiating the relative binding affinities of various cocaine metabolites. Thus, non-reducing SDS-PAGE is a simple and widely available technique that is useful as a measure of binding of cocaine and its metabolites to the h2E2 mAb, and it is likely that this technique will also be applicable to other small molecule-directed mAbs. |
| first_indexed | 2024-12-12T15:46:07Z |
| format | Article |
| id | doaj.art-3668bcb4789144a4bc48d2a7dead9315 |
| institution | Directory Open Access Journal |
| issn | 2405-5808 |
| language | English |
| last_indexed | 2024-12-12T15:46:07Z |
| publishDate | 2020-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Biochemistry and Biophysics Reports |
| spelling | doaj.art-3668bcb4789144a4bc48d2a7dead93152022-12-22T00:19:45ZengElsevierBiochemistry and Biophysics Reports2405-58082020-09-0123100795Ligand binding to a humanized anti-cocaine mAb detected by non-reducing SDS-PAGETerence L. Kirley0Andrew B. Norman1Corresponding author.; Department of Pharmacology and Systems Physiology, College of Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH, 45267-0575, USADepartment of Pharmacology and Systems Physiology, College of Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH, 45267-0575, USAMonoclonal antibodies are very useful tools in experimental biology, as well as being valuable and effective therapeutic drugs. They can be targeted against proteins with varied functions, or against small molecules of interest to both researchers and clinicians, such as drugs of abuse, including cocaine. Since there is no currently FDA approved pharmacological treatment for cocaine abuse, our laboratory has developed an anti-cocaine mAb for the treatment of cocaine use disorders. This humanized anti-cocaine antibody, named h2E2, has been thoroughly characterized both functionally and structurally, in preparation for the start of clinical development. We previously showed that this mAb could be characterized by sequential thermal unfolding of antibody domains using non-reducing SDS-PAGE. We also demonstrated that ligand-induced protein stabilization can be used to quantitatively measure cocaine and cocaine metabolite binding to the h2E2 mAb, utilizing differential scanning fluorimetry. Here, we demonstrate the utility of non-reducing SDS-PAGE for the qualitative assessment of binding of cocaine and some of its metabolites, both to the intact mAb, as well as to fragments containing the antigen binding site (Fab and F(ab’)2 fragments). These results clearly show a ligand concentration dependence of the stabilization of the cocaine binding domain in non-reducing SDS-PAGE, as well as visually differentiating the relative binding affinities of various cocaine metabolites. Thus, non-reducing SDS-PAGE is a simple and widely available technique that is useful as a measure of binding of cocaine and its metabolites to the h2E2 mAb, and it is likely that this technique will also be applicable to other small molecule-directed mAbs.http://www.sciencedirect.com/science/article/pii/S2405580820301047Monoclonal antibodyNon-reducing SDS-PAGECocaine bindingCocaine metabolitesElectrophoretic migrationAntibody domain unfolding |
| spellingShingle | Terence L. Kirley Andrew B. Norman Ligand binding to a humanized anti-cocaine mAb detected by non-reducing SDS-PAGE Biochemistry and Biophysics Reports Monoclonal antibody Non-reducing SDS-PAGE Cocaine binding Cocaine metabolites Electrophoretic migration Antibody domain unfolding |
| title | Ligand binding to a humanized anti-cocaine mAb detected by non-reducing SDS-PAGE |
| title_full | Ligand binding to a humanized anti-cocaine mAb detected by non-reducing SDS-PAGE |
| title_fullStr | Ligand binding to a humanized anti-cocaine mAb detected by non-reducing SDS-PAGE |
| title_full_unstemmed | Ligand binding to a humanized anti-cocaine mAb detected by non-reducing SDS-PAGE |
| title_short | Ligand binding to a humanized anti-cocaine mAb detected by non-reducing SDS-PAGE |
| title_sort | ligand binding to a humanized anti cocaine mab detected by non reducing sds page |
| topic | Monoclonal antibody Non-reducing SDS-PAGE Cocaine binding Cocaine metabolites Electrophoretic migration Antibody domain unfolding |
| url | http://www.sciencedirect.com/science/article/pii/S2405580820301047 |
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