Biomedical Engineering Reference
In-Depth Information
MCR may directly impact
the binding ability of the reagent
to the target
(Figure 10.1c).
10.2.2.3 Characterization and Long-Term Usage Long-term use of any reagent
requires characterization, stability assessment, lot-to-lot reproducibility studies,
investigation of temperature and buffer storage conditions, effects of freeze-thaw
cycles, and so on. Standardization of the preparation procedure and monitoring
the characterization profile of new lots of assay reagent can minimize disruption
of sample testing and maintain assay performance consistency (Figure 10.1d).
Fluorescently conjugated reagents require the same level of characterization as
other ligand binding reagents [6]. In addition, assessment of potential reagent
photobleaching stability may be incorporated into the characterization profile,
though in our collective experience this has not been shown to be an issue. As
with all ligand binding assays, tracking and trending assay performance offers
confidence in the quality and stability of the reagent in terms of both binding
ability and fluorescent intensity over time and with different lots of reagents [11].
10.2.3 Positive Control
10.2.3.1 Selection and Characterization Antidrug and neutralizing antibody
assays require positive controls (PCs) that both bind the drug and neutralize its
activity, respectively. Assay positive controls may consist of antibodies (whole or
fragments, monoclonal or polyclonal), receptors, ligands, or natural antagonists.
Purposeful immunization of animals with a biotherapeutic to generate antibodies may
also be successful at generating neutralizing antibodies, although maturation of the
antibody response [12] to include neutralizing antibodies may take significantly
longer time than the nonneutralizing antibodies.
Immunogenicity assays are designed to detect antibodies to the biotherapeutic
molecule present in complex biological matrices that may be of nonclinical or human
origin. Dilutions of the biological sample matrix can minimize matrix interferences
from either the sample matrix or the positive control if using a polyclonal serum.
Purification of the PC may be necessary when the neutralizing activity is low in order
to enrich for neutralizing antibodies. Assessment of the effects of matrix interferences
from the sample and the positive control biological matrix may be investigated as
shown in Figure 10.2. Similar neutralizing activity was observed using either the
hyperimmunized rabbit sera or the ligand affinity-purified PC. The data indicate that
the rabbit sera did not interfere with the receptor-mediated uptake of the fluorescently
labeled biotherapeutic.
10.2.4 Cells
10.2.4.1 Selection and Characterization Cell lines used in assays for
immunogenicity testing can be obtained from different sources such as research
labs, central repositories, or scientific collaborators. Investigation of cell binding or
intracellular signal response measured in biological sample matrix determines
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