PHARMACOKINETICS BY FLOW CYTOMETRY: RECOMMENDATIONS FOR DEVELOPMENT AND VALIDATION OF FLOW CYTOMETRIC METHOD FOR PHARMACOKINETIC STUDIES - Flow Cytometry in Drug Discovery and Development - page 224

11.2.4 Selection of Secondary Ab Detection Reagent
11.2.4.1 Considerations for Selection of Secondary Ab as Detection
Reagent Nonspecific binding to cells can be problematic. To reduce nonspecific
binding of the secondary Ab to cells via Fc receptor, the use of antidrug IgG F(ab) 0 2 is
highly recommended. In addition, isotype-specific secondary Ab (e.g., antihuman
IgG1 for a therapeutic human IgG1 MAb) may be useful to minimize background.
These approaches would also be appropriate for animal studies. If the PK method is
intended for preclinical studies, the use of antispecies-specific antibodies should be
evaluated against the relevant immunoglobulin species (e.g., mouse IgG or monkey
IgG). In general, either fluorochromes FITC or PE are typically used due to their
stability.
The useof both commercial secondaryAb andcustomconjugatedAb is suitable. For
custom-made secondary Ab, conjugation methods should be optimized to achieve an
appropriate conjugation ratio (FITCor PE/drug). If the ratio is too high, binding to drug
maybeaffected,whilehavinga ratio that is toolowwouldresult inpoor assaysensitivity.
It is recommended that the binding affinity of conjugated secondary Ab with different
conjugation ratio should be evaluated and compared to unconjugated secondary Ab. In
addition, binding capabilities of the secondary Ab (particularly if it is monoclonal)
should also be evaluated and not compete with binding of Ab drug-target Ag.
Multiple secondary detection antibodies from several vendors should be evaluated
to select those with high binding affinity, high specificity, and low background. Once
the detection Ab is selected, additional lots, if available, should be evaluated to assess
lot-to-lot variability.
11.2.4.2 Optimization of the Ab Binding Condition It is critical that all aspects of
binding conditions be optimized including, but not limited to, incubation time,
temperature, and so on. For high affinity binding, relatively shorter incubation
time (30-60 min) would be sufficient. Low affinity binding will need longer time
and lower temperature such as 4 C. The use of DOE (design of experiment) or
fractional factorial design would help optimize assay conditions and assess
interaction of various factors.
11.2.4.3 Detection Ab Titration A broad titration of the detection Ab should be
performed to ensure optimal signal and minimize background. Full dose-response
curves (secondary detection Ab concentration or dilution versus MFI median) with
drug at high concentrations would promote appropriate selection of secondary
detection Ab concentration or dilution. In general, using an Ab concentration or
dilution generating a maximal signal is recommended. The concentration or dilution
should not be used close to the inflection point where small concentration changes
could result in significant signal changes, thereby impacting assay precision.
11.2.4.4 Specificity This is an important aspect of reagent characterization. The
secondary detection Ab specificity should be assessed in a number of ways. First,
detection Ab should detect drug binding to cell surface target antigen (such as
CHO-Ag). In addition, the detection antibody should not demonstrate irrelevant