A novel nano-immunoassay method for quantification of proteins from CD138-purified myeloma cells: biological and clinical utility

Misiewicz-Krzemińska, Irena; Corchete, Luis A.; Rojas, Elizabeta A.; Martínez‐López, Joaquín; Oriol, Albert; Bladé, Joan; Lahuerta, Juan José; Miguel, Jesús F. San; Mateos, María Victoria; Gutiérrez, Norma C.

doi:10.3324/haematol.2017.181628

Cited by 14 publications

(18 citation statements)

References 47 publications

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“…Proteins were extracted simultaneously with genomic DNA and RNA by ice‐cold acetone precipitation, as previously described 20 . The total protein assay was used to quantify protein concentration using WES™ system (ProteinSimple).…”

Section: Methodsmentioning

confidence: 99%

Expression of p53 protein isoforms predicts survival in patients with multiple myeloma

et al. 2022

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Loss and/or mutation of the TP53 gene are associated with short survival in multiple myeloma, but the p53 landscape goes far beyond. At least 12 p53 protein isoforms have been identified as a result of a combination of alternative splicing, alternative promoters and/or alternative transcription site starts, which are grouped as α, β, γ, from transactivation domain (TA), long, and short isoforms. Nowadays, there are no studies evaluating the expression of p53 isoforms and its clinical relevance in multiple myeloma (MM). We used capillary nanoimmunoassay to quantify the expression of p53 protein isoforms in CD138-purified samples from 156 patients with newly diagnosed MM who were treated as part of the PET-HEMA/GEM2012 clinical trial and investigated their prognostic impact.Irena Misiewicz-Krzeminska and Norma C. Gutiérrez contributed equally to this study.

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Section: Methodsmentioning

confidence: 99%

Expression of p53 protein isoforms predicts survival in patients with multiple myeloma

et al. 2022

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“…Whole‐cell lysates were obtained from two samples of CD34 + cells with or without incorporation of MSC‐EV. Capillary Electrophoresis Immunoassay or Simple Western analyses were performed using the WES machine (ProteinSimple, Santa Clara, CA), according to the manufacturer's protocols . The data were analyzed with inbuilt Compass software (ProteinSimple).…”

Section: Methodsmentioning

confidence: 99%

The Incorporation of Extracellular Vesicles from Mesenchymal Stromal Cells Into CD34+ Cells Increases Their Clonogenic Capacity and Bone Marrow Lodging Ability

et al. 2019

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Mesenchymal stromal cells (MSC) may exert their functions by the release of extracellular vesicles (EV). Our aim was to analyze changes induced in CD34 + cells after the incorporation of MSC-EV. MSC-EV were characterized by flow cytometry (FC), Western blot, electron microscopy, and nanoparticle tracking analysis. EV incorporation into CD34 + cells was confirmed by FC and confocal microscopy, and then reverse transcription polymerase chain reaction and arrays were performed in modified CD34 + cells. Apoptosis and cell cycle were also evaluated by FC, phosphorylation of signal activator of transcription 5 (STAT5) by WES Simple, and clonal growth by clonogenic assays. Human engraftment was analyzed 4 weeks after CD34 + cell transplantation in nonobese diabetic/severe combined immunodeficient mice. Our results showed that MSC-EV incorporation induced a downregulation of proapoptotic genes, an overexpression of genes involved in colony formation, and an activation of the Janus kinase (JAK)-STAT pathway in CD34 + cells. A significant decrease in apoptosis and an increased CD44 expression were confirmed by FC, and increased levels of phospho-STAT5 were confirmed by WES Simple in CD34 + cells with MSC-EV. In addition, these cells displayed a higher colony-forming unit granulocyte/macrophage clonogenic potential. Finally, the in vivo bone marrow lodging ability of human CD34 + cells with MSC-EV was significantly increased in the injected femurs. In summary, the incorporation of MSC-EV induces genomic and functional changes in CD34 + cells, increasing their clonogenic capacity and their bone marrow lodging ability. STEM CELLS 2019;37:1357-1368 SIGNIFICANCE STATEMENTIn the current study, the authors validate for the first time that preincubating human CD34 + cells with extracellular vesicles derived from human mesenchymal stromal cells not only modifies the gene expression of the recipient cells (inducing a downregulation of proapoptotic genes and overexpression of genes involved in colony formation and JAK-STAT pathway) but also significantly increases their in vitro clonogenic ability and, most importantly, increases their 4-week bone marrow lodging ability in vivo in a standard xenotransplantation model. This strategy could potentially be exploited to increase the hematopoietic engraftment in the clinical setting.

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“…A new approach may be useful to this end. Protein Simple (PS) offers a system, “Capillary‐Electrophoresis Western blotting (Simple Western)” that was described as showing specificity, sensitivity, and precision required for testing protein targets in the low picogram levels in different fields of application . According to PS, the assay takes place in an ultrathin nano‐capillary (5 cm x 100 μm, 400 nL) where the proteins are first separated by electrophoresis, then immobilized to the capillary wall via a photo‐chemical reaction and finally probed with specific primary and secondary horseradish peroxidase (HRP)‐linked antibodies.…”

Section: Introductionmentioning

confidence: 99%

Detection of erythropoiesis stimulating agents in urine samples using a capillary Western system

Desharnais

Naud

Ayotte

2018

Drug Testing and Analysis

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The presence of erythropoiesis stimulating agents (ESAs) in the urine samples collected from athletes is detected using traditional Western blotting following either size-based separation (SDS/SAR-PAGE) or isoelectric focusing (IEF). Although there is an important testing effort, there is little doubt that ESAs are still abused in sports and that reducing the costs of the tests might increase the number of tests and improve deterrence. The capillary electrophoresis system developed by Protein Simple may be useful to this end. This platform is fully automated and could be easily implemented in anti-doping laboratories, which would contribute to the improvement of the overall assay performance and standardization of the method. Such an automated system could be of interest during major sports events, such as the Olympic Games, where a high number of samples needs to be analyzed in a short period of time. From the experiments conducted so far, we conclude that the technique is promising, with the sensitivity and reproducibility needed to screen ESAs in human urine samples.

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A novel nano-immunoassay method for quantification of proteins from CD138-purified myeloma cells: biological and clinical utility

Cited by 14 publications

References 47 publications

Expression of p53 protein isoforms predicts survival in patients with multiple myeloma

Expression of p53 protein isoforms predicts survival in patients with multiple myeloma

The Incorporation of Extracellular Vesicles from Mesenchymal Stromal Cells Into CD34+ Cells Increases Their Clonogenic Capacity and Bone Marrow Lodging Ability

Detection of erythropoiesis stimulating agents in urine samples using a capillary Western system

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