Abstract:
:Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.
journal_name
Hum Vaccin Immunotherjournal_title
Human vaccines & immunotherapeuticsauthors
Kirley TL,Norman ABdoi
10.4161/21645515.2014.990856subject
Has Abstractpub_date
2015-01-01 00:00:00pages
458-67issue
2eissn
2164-5515issn
2164-554Xjournal_volume
11pub_type
杂志文章abstract::The Advisory Committee on Immunization Practices recommends administering diphtheria, tetanus and acellular pertussis (DTaP) vaccines to children at 2, 4, 6, 15-18 months, and 4-6 y of age; preferably with the same-brand vaccine for the whole series. We estimated age-appropriate DTaP dose completion and the proportion...
journal_title:Human vaccines & immunotherapeutics
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doi:10.4161/21645515.2014.985506
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journal_title:Human vaccines & immunotherapeutics
pub_type: 杂志文章,评审
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journal_title:Human vaccines & immunotherapeutics
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doi:10.4161/hv.20178
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journal_title:Human vaccines & immunotherapeutics
pub_type: 杂志文章,评审
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pub_type: 杂志文章,meta分析
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journal_title:Human vaccines & immunotherapeutics
pub_type: 杂志文章,评审
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更新日期:2012-04-01 00:00:00
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journal_title:Human vaccines & immunotherapeutics
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journal_title:Human vaccines & immunotherapeutics
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journal_title:Human vaccines & immunotherapeutics
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pub_type: 杂志文章
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journal_title:Human vaccines & immunotherapeutics
pub_type: 杂志文章
doi:10.1080/21645515.2015.1057363
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pub_type: 杂志文章,评审
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pub_type: 杂志文章,评审
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journal_title:Human vaccines & immunotherapeutics
pub_type: 杂志文章
doi:10.4161/hv.25096
更新日期:2013-09-01 00:00:00
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doi:10.1080/21645515.2016.1264547
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