Assessment of the Effect of Increased Fluorophore Labelling on the Binding Ability of an Antibody

McCormack, Teresa A.; O'Keeffe, Gerard; Craith, Brian Mac; O’Kennedy, Richard

doi:10.1080/00032719608001447

Cited by 16 publications

(14 citation statements)

References 6 publications

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“…The affinity of one of the antibodies (Mab528) seemed to be relatively resistant to labeling with the AlexaFluor dyes, whereas the dissociation constant of the other four antibodies substantially increased upon labeling (Table 1). While the data corroborate previous evidence (6,10,11) showing that conjugation of fluorescent dyes decreases antibody affinity in most cases, it also reveals that conjugation with AlexaFluor647 affects antibody affinity more significantly than labeling with AlexaFluor546.…”

Section: Determination Of the Effect Of Fluorescence Labeling On The supporting

confidence: 89%

“…This simplification is based on the assumption that the distribution of labeling ratios in the stock is identical to that in the bound fraction. However, anecdotal and indirect evidence suggests that conjugation of multiple fluorophores to monoclonal antibodies deteriorates the affinity of antibodies (6,10,11). Unfortunately, none of these investigations has directly measured the properties of the bound fraction.…”

Section: Introductionmentioning

confidence: 99%

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The Effect of Fluorophore Conjugation on Antibody Affinity and the Photophysical Properties of Dyes

Szabó

Szendi-Szatmári

Ujlaky-Nagy

et al. 2018

Biophysical Journal

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Because the degree of labeling (DOL) of cell-bound antibodies, often required in quantitative fluorescence measurements, is largely unknown, we investigated the effect of labeling with two different fluorophores (AlexaFluor546, AlexaFluor647) in a systematic way using antibody stock solutions with different DOLs. Here, we show that the mean DOL of the cell-bound antibody fraction is lower than that of the stock using single molecule fluorescence measurements. The effect is so pronounced that the mean DOL levels off at approximately two fluorophores/IgG for some antibodies. We developed a method for comparing the average DOL of antibody stocks to that of the isolated, cell-bound fraction based on fluorescence anisotropy measurements confirming the aforementioned conclusions. We created a model in which individual antibody species with different DOLs, present in an antibody stock solution, were assumed to have distinct affinities and quantum yields. The model calculations confirmed that a calibration curve constructed from the anisotropy of antibody stocks can be used for determining the DOL of the bound fraction. The fluorescence intensity of the cell-bound antibody fractions and of the antibody stocks exhibited distinctly different dependence on the DOL. The behavior of the two dyes was systematically different in this respect. Fitting of the model to these data revealed that labeling with each dye affects quantum yield and antibody affinity differentially. These measurements also implied that fluorophores in multiply labeled antibodies exhibit self-quenching and lead to decreased antibody affinity, conclusions directly confirmed by steady-state intensity measurements and competitive binding assays. Although the fluorescence lifetime of antibodies labeled with multiple fluorophores decreased, the magnitude of this change was not sufficient to account for self-quenching indicating that both dynamic and static quenching processes occur involving H-aggregate formation. Our results reveal multiple effects of fluorophore conjugation, which must not be overlooked in quantitative cell biological measurements.

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Section: Determination Of the Effect Of Fluorescence Labeling On The supporting

confidence: 89%

Section: Introductionmentioning

confidence: 99%

The Effect of Fluorophore Conjugation on Antibody Affinity and the Photophysical Properties of Dyes

Szabó

Szendi-Szatmári

Ujlaky-Nagy

et al. 2018

Biophysical Journal

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“…Often, a succinimidyl-ester functional group is attached to a fluorophore core and this functionality confers reaction specificity with primary amines to form fluorophore-antibody conjugates. The presence of multiple primary amines, especially primary amines in the antibody active site, can result in fluorophore conjugation that changes antigen binding characteristics and in the extreme, completely inactivates the antibody [3; 4]. Steric hindrance and the absence of additional reactive sites on the fluorophore are presumed to limit the degree of antibody modification by the conjugation reaction.…”

Section: Introductionmentioning

confidence: 99%

Fluorescent-labeled antibodies: Balancing functionality and degree of labeling

Vira

Mekhedov

Humphrey

et al. 2010

Analytical Biochemistry

165

149

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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.

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“…Alternatively, as fluorochrome conjugation can reduce the avidity of a given antibody [22,23], the decreased retrieval of 1A8-FITC + neutrophils might be explained by a reduced binding ability of 1A8-FITC to Ly6G. Alternatively, as fluorochrome conjugation can reduce the avidity of a given antibody [22,23], the decreased retrieval of 1A8-FITC + neutrophils might be explained by a reduced binding ability of 1A8-FITC to Ly6G.…”

Section: Resultsmentioning

confidence: 99%

“…The decreased detectability of the 1A8-FITC + neutrophils may result from a degradation of the FITC molecule as a result of its lower thermal stability in vivo. Alternatively, as fluorochrome conjugation can reduce the avidity of a given antibody [22,23], the decreased retrieval of 1A8-FITC + neutrophils might be explained by a reduced binding ability of 1A8-FITC to Ly6G. Therefore, we tested labeling of neutrophils with fluorescent 1A8 under physiologic conditions.…”

Section: A8-fitc Labeling Reduces Retrieval Of Neutrophils Followingmentioning

confidence: 99%

Fluorescent Ly6G antibodies determine macrophage phagocytosis of neutrophils and alter the retrieval of neutrophils in mice

Bucher¹,

Schmitt²,

Autenrieth

et al. 2015

Journal of Leukocyte Biology

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Fluorescently labeled Ly6G antibodies enable the tracking of neutrophils in mice, whereas purified anti-Ly6G rapidly depletes neutrophils from the circulation. The mechanisms underlying neutrophil depletion are still under debate. Here, we examined how identical Ly6G antibodies coupled to different fluorochromes affect neutrophil fate in vivo. BM cells stained with Ly6G antibodies were injected into mice. The number of retrieved anti-Ly6G-FITC(+) cells was reduced significantly in comparison with anti-Ly6G-APC(+) or anti-Ly6G-PE(+) cells. Flow cytometry and multispectral imaging flow cytometry analyses revealed that anti-Ly6G-FITC(+) neutrophils were preferentially phagocytosed by BMMs in vitro and by splenic, hepatic, and BM macrophages in vivo. Direct antibody injection of anti-Ly6G-FITC but not anti-Ly6G-PE depleted neutrophils to the same degree as purified anti-Ly6G, indicating that the FITC-coupled antibody eliminates neutrophils by a similar mechanism as the uncoupled antibody. With the use of a protein G-binding assay, we demonstrated that APC and PE but not FITC coupling inhibited access to interaction sites on the anti-Ly6G antibody. We conclude the following: 1) that neutrophil phagocytosis by macrophages is a central mechanism in anti-Ly6G-induced neutrophil depletion and 2) that fluorochrome-coupling can affect functional properties of anti-Ly6G antibodies, thereby modifying macrophage uptake of Ly6G-labeled neutrophils and neutrophil retrieval following adoptive cell transfer or injection of fluorescent anti-Ly6G.

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Assessment of the Effect of Increased Fluorophore Labelling on the Binding Ability of an Antibody

Cited by 16 publications

References 6 publications

The Effect of Fluorophore Conjugation on Antibody Affinity and the Photophysical Properties of Dyes

The Effect of Fluorophore Conjugation on Antibody Affinity and the Photophysical Properties of Dyes

Fluorescent-labeled antibodies: Balancing functionality and degree of labeling

Fluorescent Ly6G antibodies determine macrophage phagocytosis of neutrophils and alter the retrieval of neutrophils in mice

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