Fluorescent-labeled antibodies: Balancing functionality and degree of labeling

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.

Fluorescent IgG fusion proteins made inE. coli

“…Higher number of fluorophores will result in a dimmer reagent. 2,3 A recombinant fusion protein comprised of an antibody and a fluorescent moiety has the advantages of defined number and location of fluorophores. In the context of a fusion protein, the stoichiometry and location of fluorophores attached to the antibody are predetermined by the engineered DNA sequence, and as a result, are invariant.…”

“…Typically, no more than about three to five dyes can be attached to an antibody without self-quenching of fluorescence or inactivating the antibody. 2,3 A fusion protein comprising an antibody and a fluorescent protein could offer several advantages over the conventional labeling method. Fluorescent proteins have been genetically fused to many proteins in various species to produce stable chimeras that retain their original biological activity as well as retaining the fluorescent properties of the fluorescent protein.…”

High-Throughput Time-Resolved FRET Reveals Akt/PKB Activation as a Poor Prognostic Marker in Breast Cancer

“…Previously we have conjugated dyes directly to primary antibodies to obtain sufficient signal to monitor endogenous biomarker activation (24). However, we encountered several limitations: First, the conjugation process can result in the presence of multiple dye molecules at the antigen recognition site, with adverse consequences on antibody-antigen specificity (30). Second, the signal obtained is limited by the maximum number of dye molecules that can be bound to each antibody molecule while maintaining epitope recognition (22).…”

Selecting an Optimal Antibody for Antibody- Drug Conjugate Therapy

“…Alternatively, antibodies can be biotinylated with NHS-SS-biotin and subsequently complexed with streptavidin-linked fluorophore moieties (Nielsen et al 2006). While this process has a higher throughput than direct labeling, it also carries a risk of altering binding affinity of the antibody to its antigen (Vira et al 2010). The direct conjugation of a fluorophore to an antibody, while suitable only for a small number of antibodies in parallel, allows the user to monitor internalization and intracellular trafficking in real time and, importantly, also reduces the background noise that often arises from the use of secondary antibodies.…”