The monocyte activation test detects potentiated cytokine release resulting  from the synergistic effect of endotoxin  and non-endotoxin pyrogens

Solati, Shabnam; Zhang, Ting; Timman, Shahrzad

doi:10.1177/17534259221097948

Cited by 6 publications

(3 citation statements)

References 16 publications

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“…Our standard approach to interrogating these responses is to gradient purify mononuclear cells from whole blood and incubate the cells for 24 h in RPMI containing 1% human serum and various pattern recognition receptor agonists. For this study we exposed cells to a pan-bacterial stimulus comprised of 1 µg/mL lipopolysaccharide (LPS) and 10 8 copies/mL heat-killed Staphylococcus aureus (HKSA) 20 . We found that after 24 h at 37 °C, unstimulated gradient-purified mononuclear cells spontaneously released several cytokines and chemokines (Fig.…”

Section: Resultsmentioning

confidence: 99%

Peripheral blood mononuclear cell phenotype and function are maintained after overnight shipping of whole blood

Johnson

Overlee

Sagen

et al. 2022

Sci Rep

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Same day processing of biospecimens such as blood is not always feasible, which presents a challenge for research programs seeking to study a broad population or to characterize patients with rare diseases. Recruiting sites may not be equipped to process blood samples and variability in timing and technique employed to isolate peripheral blood mononuclear cells (PBMCs) at local sites may compromise reproducibility across patients. One solution is to send whole blood collected by routine phlebotomy via overnight courier to the testing site under ambient conditions. Determining the impact of shipping on subsequent leukocyte responses is a necessary prerequisite to any experimental analysis derived from transported samples. To this end, whole blood was collected from healthy control subjects and processed fresh or at 6, 24 and 48 h after collection and handling under modeled shipping conditions. At endpoint, whole blood was assessed via a complete blood count with differential and immunophenotyped using a standardized panel of antibodies [HLADR, CD66b, CD3, CD14, CD16]. PBMCs and neutrophils were isolated from whole blood and subjected to ex vivo stimulation with lipopolysaccharide and heat-killed Staphylococcus aureus. Stimulated release of cytokines and chemokines was assessed by cytometric bead array. RNA was also isolated from PBMCs to analyze transcriptional changes induced by shipping. The complete blood count with differential revealed that most parameters were maintained in shipped blood held for 24 h at ambient temperature. Immunophenotyping indicated preservation of cellular profiles at 24 h, although with broadening of some populations and a decrease in CD16 intensity on classical monocytes. At the transcriptional level, RNAseq analysis identified upregulation of a transcription factor module associated with inflammation in unstimulated PBMCs derived from whole blood shipped overnight. However, these changes were limited in both scale and number of impacted genes. Ex vivo stimulation of PBMCs further revealed preservation of functional responses in cells isolated from shipped blood held for 24 h at ambient temperature. However, neutrophil responses were largely abrogated by this time. By 48 h neither cell population responded within normal parameters. These findings indicate that robust immunophenotyping and PBMC stimulated response profiles are maintained in whole blood shipped overnight and processed within 24 h of collection, yielding results that are representative of those obtained from the sample immediately following venipuncture. This methodology is feasible for many patient recruitment sites to implement and allows for sophisticated immunological analysis of patient populations derived from large geographic areas. With regard to rare disease research, this meets a universal need to enroll patients in sufficient numbers for immunoprofiling and discovery of underlying pathogenic mechanisms.

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Section: Resultsmentioning

confidence: 99%

Peripheral blood mononuclear cell phenotype and function are maintained after overnight shipping of whole blood

Johnson

Overlee

Sagen

et al. 2022

Sci Rep

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“…Furthermore, contaminants, such as microbial species and endotoxins, must be prohibited properly, or completely removed. On the other side, the application of additional analysis, e.g., a monocyte activation test, have been attempted to eradicate the presence of non-endotoxin pro-inflammatory contaminants (peptidoglycan) [ 21 ].…”

Section: Characterization Of Mabsmentioning

confidence: 99%

Analytical Techniques for the Characterization and Quantification of Monoclonal Antibodies

Alhazmi

Albratty

2023

Pharmaceuticals

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Monoclonal antibodies (mAbs) are a fast-growing class of biopharmaceuticals. They are widely used in the identification and detection of cell makers, serum analytes, and pathogenic agents, and are remarkably used for the cure of autoimmune diseases, infectious diseases, or malignancies. The successful application of therapeutic mAbs is based on their ability to precisely interact with their appropriate target sites. The precision of mAbs rely on the isolation techniques delivering pure, consistent, stable, and safe lots that can be used for analytical, diagnostic, or therapeutic applications. During the creation of a biologic, the key quality features of a particular mAb, such as structure, post-translational modifications, and activities at the biomolecular and cellular levels, must be characterized and profiled in great detail. This implies the requirement of powerful state of the art analytical techniques for quality control and characterization of mAbs. Until now, various analytical techniques have been developed to characterize and quantify the mAbs according to the regulatory guidelines. The present review summarizes the major techniques used for the analyses of mAbs which include chromatographic, electrophoretic, spectroscopic, and electrochemical methods in addition to the modifications in these methods for improving the quality of mAbs. This compilation of major analytical techniques will help students and researchers to have an overview of the methodologies employed by the biopharmaceutical industry for structural characterization of mAbs for eventual release of therapeutics in the drug market.

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“…Therefore, the detection of LPS becomes imperative in order to assure food safety as well as to prevent fatal illnesses [5] . Due to the substantial risk posed to human health and the highly toxic nature of LPS, numerous analytical techniques have been devised for LPS detection, one of which is the rabbit pyrogen test, [6] the Limulus‐based test, [7] the monocyte activation test, [8] and LPS sensors utilizing LPS binding proteins, peptides, and aptamers to detect the presence of LPS [9–11] . The rabbit pyrogen test holds the distinction of being the initial method approved by the US Food and Drug Administration (FDA).…”

Section: Introductionmentioning

confidence: 99%

Direct Electrochemical Sensing of Lipopolysaccharide by Graphene Nanoplatlets@Cytochrome c Hydrogel Using a Microfluidic Chip

Kumar,

Pathania

et al. 2024

ChemistrySelect

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Contamination by lipopolysaccharide (LPS), an endotoxin which is present in the outer membrane of Gram‐negative bacteria causes adverse toxic effects on human health. Herein, we report a facile graphene nanoplatelets@cytochrome c (GNP@Cyt c) hydrogel based voltammetric electrochemical sensor for LPS. Firstly, the resultant hydrogel sensor facilitated a large electroactive surface coverage for electron transfer, and secondly provided a 3D porous microenvironment for interaction of abundant electrocatalytic sites in Cyt c with LPS. Under optimal incubation conditions, the heme iron of Cyt c in its reduced form (Fe2+) can directly bind to highly anionic carbohydrate groups of LPS, thereby giving rise to specific interaction. Moreover, the binding of Cyt c (Fe2+) with LPS served as a electrocatalytic unit, generating a strong oxidation peak current at 0.019 V. Importantly, no separate redox mediator was required and the electrochemical signal obtained by LPS‐Cyt c (Fe2+) complex sufficed for detection purposes. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were employed to investigate the electrochemical aspects of GNP@Cyt c hydrogel modified screen‐printed carbon electrode (SPCE). Further, the GNP@Cyt c hydrogel/SPCE demonstrated LPS dose‐dependent changes in oxidation current which enabled selective quantitative detection. This method exhibited a detection limit as low as 8.4×10−4 ng mL−1 with a wide linear range of 0.01–2.0 ng mL−1. For continuous on‐chip monitoring of LPS, a customized microfluidic electrochemical device approach was established which endorses the practical utility of the developed sensor. Further, the integrated microfluidic electrochemical device showed excellent recovery upon spiking LPS in lemon juice and tap water samples. Overall, the fabricated sensor holds great promise for point‐of‐care screening of trace LPS contamination, especially for food safety.

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The monocyte activation test detects potentiated cytokine release resulting from the synergistic effect of endotoxin and non-endotoxin pyrogens

Cited by 6 publications

References 16 publications

Peripheral blood mononuclear cell phenotype and function are maintained after overnight shipping of whole blood

Peripheral blood mononuclear cell phenotype and function are maintained after overnight shipping of whole blood

Analytical Techniques for the Characterization and Quantification of Monoclonal Antibodies

Direct Electrochemical Sensing of Lipopolysaccharide by Graphene Nanoplatlets@Cytochrome c Hydrogel Using a Microfluidic Chip

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