Multiplexed cytokine sandwich immunoassays: clinical applications.

Flow cytometric, microsphere-based immunoassays have been developed for the simultaneous detection of soluble analytes in a variety of sample types. The ability to discriminate between individual microspheres on the basis of size, fluorescent intensity, and/or wavelength has allowed the simultaneous analysis of multiple analytes from the same sample. Cytokines are particularly good candidates for multiplexed analysis.

Comparative analysis of lymphocyte activation marker expression and cytokine secretion profile in stimulated human peripheral blood mononuclear cell cultures: an in vitro model to monitor cellular immune function.

Activation of lymphocytes is a complex, yet finely regulated cascade of events that results in the expression of cytokine receptors, production and secretion of cytokines and expression of several cell surface molecules that eventually lead to divergent immune responses. Assessing the qualitative and quantitative nature of lymphocyte function following immunotherapy provides valuable information about the immune responses mediated by a therapeutic agent. To facilitate evaluation of the immunomodulatory activity of therapeutic agents, we have established a platform of in vitro immunoassays with normal human peripheral blood mononuclear cells (PBMCs) treated with several polyclonal activators that are known to exhibit different modes of action.

Validation and comparative analysis of a multiplexed assay for the simultaneous quantitative measurement of Th1/Th2 cytokines in human serum and human peripheral blood mononuclear cell culture supernatants.

There is increasing evidence suggesting a relationship between cytokine levels and disease pathogenesis, which has led to interest in analyzing multiple cytokines in biological fluids and culture supernatants for various research and clinical studies. The introduction of methodologies allowing simultaneous measurement of interrelated biomarkers/cytokines has further revolutionized this process. In contrast to tissue culture supernatant, the measurement of cytokines in serum has proven to be difficult to characterize in multiplexed formats because of the presence of large dynamic concentration ranges of proteins and other interfering factors that are present in this matrix.

Simultaneous quantification of proinflammatory cytokines in human plasma using the LabMAP assay.

There is an emerging trend in the pharmaceutical industry to evaluate a variety of surrogate biomarkers in Phase I/II clinical studies with the intention of determining potential activity of drugs early in clinical development. A number of cytokines expressed in pathological conditions are currently being considered as potential surrogates of disease and/or drug activity. The quantitative measurement of such analytes (biomarkers) in biological fluids has traditionally been performed by bioassays, enzyme-linked immunosorbent assay (ELISA), ribonuclease protection assay (RPA) and polymerase chain reaction (PCR).