IMMUNOGENICITY TESTING USING FLOW CYTOMETRY - Flow Cytometry in Drug Discovery and Development - page 201

and/or altered reactivity to the target and therefore characterization of the reagent,
particularly its binding activity, is important to select the most appropriate reagent and
ensure the quality of long-term supply [6]. A common consideration when developing
neutralizing antibody (NAb) assays that measure drug binding to cell surface proteins
is the direct labeling of the drug. As loss of functional binding may occur as a result of
label conjugation, site-directed labeling may be necessary to avoid conjugation of
sites essential for target binding. If direct labeling strategies are unsuccessful, then use
of secondary conjugated reagents to detect the bound drug may also be used.
10.2.1 Fluorescent Dye Selection
Multiple fluorescent dyes are commercially available, including organic dyes,
phycolipoproteins, tandem dyes, and (more recently) nanocrystals, all with distinct
excitation and emission spectra. Matching the fluorescent dye, brightness and
photostability to the intended assay use, required sensitivity, and the optical char-
acteristics of the detection system are all important parameters for successful assay
development [7, 8]. Consideration of the target/signal event marker abundance may
lead the user to a brighter fluorochrome for the detection of rare targets/signal event
markers, while more abundant targets/signal event markers may be detected with
fluorochromes that are less bright. In addition, when using several fluorochromes at
the same time, it is critical to adjust the instrument and correct for spectral overlap to
ensure accurate interpretation of the signal output [9, 10].
10.2.2 Assay Detection Reagent
10.2.2.1 Selection and Characterization The detection reagent in the assay may
be one of the following types of reagent: antibody, soluble receptor, drug, ligand, or
enzyme. An antibody reagent may be an intact whole molecule or a fragment of the
antibody (Fab), which may offer the advantage of reduced Fc receptor binding on
target cells during the staining procedure. During evaluation of candidate molecules
for suitability as assay detection reagents, the specificity and selectivity of the reagent,
the intended use, and the mode of action are all considered. Also, during the assay
reagent selection process, it is highly recommended that long-term availability be
considered to ensure availability throughout the assay life cycle.
10.2.2.2 Labeling the Detection Reagent with Fluorescent Dye Unless com-
mercially available, labeling the assay detection reagent with fluorescent dye(s) at
several different molar coupling ratios (MCRs) allows the assessment of dye selection
incorporation level and with predefined assay performance criteria. Susceptibility of
the reagent binding site to fluorochrome conjugation and interferencewith subsequent
interactions with the target are important to assess during the optimization phase of
assay development (Figure 10.1a). In addition, comparison of the binding affinities of
the unlabeled and labeled assay reagents can be used to assess the degree to which the
addition of the fluorochrome or the conjugation procedure influences the binding
activity of the conjugate (Figure 10.1b). In some cases, increasing the fluorochrome