Tuesday, August 30

Blood Cytokines Measurement With Multiplex Immunoassays

Cytokines, generated by both immune and non-immune cells, are thought to play a role in the genesis and progression of mental diseases. Indeed, numerous researchers have compared blood cytokine levels in psychiatric patients to those of healthy controls or observed their levels in patients during disease development to uncover biomarkers.

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Nonetheless, few studies have proven that such cytokines stay constant across weeks and months in healthy people. Before employing blood cytokine levels as biomarkers of disease characteristics, disease status, or treatment response, this is an essential topic to examine. Although Luminex multiplex immunoassay technology represents an advancement in biomarker identification because it allows for the simultaneous examination of large panels of analytes from a small sample volume, it is necessary to verify whether these assays yield sufficient sensitivity and reproducibility when applied to the blood of neuropsychiatric patients.

As a result, over 30 weeks, we compared two multiplex immunoassays, the bead-based Luminex® (Bio-Rad) and the electro-chemiluminescence-based V-plex® (MesoScaleDiscovery), for the detection and quantification of 31 cytokines, chemokines, and growth factors in the sera and plasma of patients with major depressive episodes (MDE) and age- and sex- Although both platforms had low coefficients of variability (CV) across duplicates in the calibration curves, the V-PLEX® platform had more excellent overall linearity.

Neither platform, however, was able to identify the absolute levels for all of the analytes examined. The intra-assay reproducibility of the 16 analytes identified by both tests was generally more remarkable with the V-PLEX® platform. Although it is not a general rule that sera and plasma findings will be associated, consistent results were more familiar with the V-PLEX® platform.

Furthermore, the V-PLEX® findings were more consistent with the gold standard ELISA simplex test for IL-6 in both sera and plasma. The intra-individual variability of the measures was low for Eotaxin, G-CSF, IL-4, IL-7, IL-9, IL-12p40, IL-12p70, IL-15, MIP-1, PDGF-BB, TNF, TNF-, and VEGF, but intermediate or high for IFN-, IL-6, IL-8, IL-10, and IP10, but medium or high for IFN-, IL-6, IL-8 These findings show that significant caution should be exercised when converting the results of multiplex cytokine profiling into biomarker development in psychiatry. Custom Luminex assay is developed to examine antibodies, analyte,  species, etc. It is an early-stage drug development process.

John L. Fahey is a pioneer in the detection of circulating cytokines and immune-activation indicators and a leader in the quality assessment/control of circulating cytokine assays. Calculating circulating cytokine levels can give valuable information in the research of illness pathophysiology. This paper presents insights into the detection of circulating cytokines, including the use of multiplex assays.

Recent advances in laboratory technologies such as flow cytometry, Luminex based assays, Meso Scale Discovery assays, planar multiplex assays, and procedural improvements such as the use of high-quality reagents, a susceptible assessment format, and the use of a smaller specimen volume have enabled the measurement of a more comprehensive panel of cytokines in serum/plasma in a short period.

The accuracy and precision of these multiplex assays using novel technologies and platforms are dependent on the selection of a specific high-quality pair of cytokine capture and detection antibodies, operator experience, standardization of multiplex cytokine measurement, and performance of the assay under restricted in-house quality control and well-established national quality assurance programs. All these steps of drug discovery research are included in the drug discovery CRO.

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