NDLI: Fluorescent labeled antibodies - balancing functionality and degree of labeling

Content Provider	PubMed Central
Author	Vira, Shaleen Elena, Mekhedov Humphrey, Glen Blank, Paul S.
Abstract	A critical assumption in utilizing labeled antibodies is that the conjugation reaction has no deleterious effects on antibody avidity. This study demonstrates that this assumption need not hold true and presents a methodology to quantitatively determine the degree of inactivation and/or changes in antibody-antigen binding that can occur with conjugation. Fluorescein isothiocyanate, FITC, was conjugated to a mouse monoclonal antibody (Fc125) against hemagluttinin (HA) using varying fluorophore:protein (F:P) labeling ratios. Antibody binding, as a function of the F:P labeling ratio, was evaluated using a kinetic ELISA assay and analyzed using global fitting. A two parameter adjustment of the antibody concentration and the maximum rate were sufficient to describe the rate changes. The concentration parameter dominated the rate changes consistent with the hypothesis that the coupling reaction inactivated an increasing fraction of the antibody population with a smaller change (~15 % at the highest F:P ratio) in antibody-antigen binding. An optimal F:P ratio that minimized both inactivation and unlabeled antibody was calculated. This procedure can be utilized to prepare functional, labeled antibody reagents with defined activity and can aid in quantitative applications in which the stoichiometry and functionality of the labeled antibody is critical.
Related Links	http://dx.doi.org/10.1016/j.ab.2010.03.036
Ending Page	150
Page Count	5
Starting Page	146
File Format	PDF
ISSN	00032697
e-ISSN	10960309
Journal	Analytical biochemistry
Issue Number	2
Volume Number	402
Language	English
Publisher Date	2010-07-01
Access Restriction	Open
Subject Keyword	Biophysics Cell Biology Biochemistry Molecular Biology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology Biophysics

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