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. Through intricate networks and complex feedback mechanisms, cytokines modulate each other as well as a multitude of cellular events and play an important role in health and disease. The simultaneous measurement of multiple cytokines in a single biological sample has tremendous potential value to further our understanding of the role of these immunomodulators in health and disease. This chapter describes a multiplexed, microsphere-based flow cytometric method to quantitate and compare multiple cytokines simultaneously in human serum. Serum poses a challenge for multiplexed cytokine analysis owing to the presence of numerous proteins and other potentially interfering factors. Assessment of the "real world" performance of the multiplexed, microsphere-based flow cytometry assay with clinically relevant sample types is critical for determining the proper application of these methods in deciphering the role of cytokines in disease pathogenesis and for evaluating drug action.

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. We evaluated the kinetics of cell surface marker expression and cytokine release from PBMCs stimulated in parallel with various activating agents over a time course. These stimulating agents induced early (CD69 and CD71) and late (CD25 and HLA-DR) activation markers to varying antigen densities, indicated different cytokine profiles, and showed differential inhibition with dexamethasone (DEX), an inhibitor of early signaling events. Based on the association or correlation of the kinetics of activation marker expression and secreted cytokines, the results of our study indicate the appropriate time points for the simultaneous measurement of both these activation products. This study defines the kinetics for both measures of T cell activation and provides a comprehensive review with various polyclonal activators that can serve as a reference for monitoring lymphocyte function in clinical study samples.

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. In the present study, we have used the microsphere-based multiplex method to simultaneously quantitate and compare six analytes, encompassing a representation of the Th1/Th2 cytokine panel (interleukin (IL)-2, IL-4, IL-5, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and IL-10), in both serum and culture supernatants from peripheral blood mononuclear cells (PBMCs). A detailed validation procedure for these determinations is described along with a comparative analysis of the performance of the multiplexed assay in serum and culture supernatant matrices. Our results indicate that precision of the multiplexed assay is comparable in both culture supernatant and serum. However, the accuracy of quantification of cytokines in the serum matrix but not in culture supernatant may be compromised depending upon the cytokine being analyzed. Therefore, one must use caution when interpreting data from such complex matrices. Nevertheless, this assay format is appropriate to profile cytokines in clinical trial samples.

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). Typically, these methods have been limited to the measurement of a single analyte, require large sample volume and are time and cost involved. The LabMAP (Luminex) system has been previously used to quantify cytokines in tissue culture supernatants and in animal serum. In the present study, the LabMAP technology was used for quantifying for the first time, pro-inflammatory cytokines such as, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta), IL-6 and IL-8 levels in lipopolysaccharide (LPS)-stimulated human plasma samples. Both single-cytokine and two-cytokine (biplexed) panel formats were evaluated and the performance in the two formats was compared. A detailed validation procedure for these determinations is described along with a side-by-side comparison with ELISA results. Our results indicate that the LabMAP system can be used to measure cytokine levels in LPS-stimulated human plasma samples and that the levels obtained by this technique are comparable with ELISA results. It is therefore feasible to use this optimized technology to detect and quantify cytokines and other potential biomarkers in a complex milieu such as human plasma in support of clinical studies.