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Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry. - Clinical proteomics
In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trolâ„¢) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells.Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry.The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45â€‰Â±â€‰0.09)â€‰Ã—â€‰10(5) and (0.85â€‰Â±â€‰0.11)â€‰Ã—â€‰10(5), respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC.Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.
Unfolding properties of recombinant human serum albumin products are due to bioprocessing steps. - Biotechnology progress
We have used differential scanning calorimetry (DSC) to determine the unfolding properties of commercial products of human serum albumin (HSA) prepared from pooled human blood, transgenic yeast, and transgenic rice. The initial melting temperatures (Tm1 ) for the unfolding transitions of the HSA products varied from 62Â°C to 75Â°C. We characterized the samples for purity, fatty acid content, and molecular weight. The effects of adding fatty acids, heat pasteurization, and a low pH defatting technique on the transition temperatures were measured. Defatted HSA has a structure with the lowest stability (Tm of âˆ¼62Â°C). When fatty acids are bound to HSA, the structure is stabilized (Tm of âˆ¼64-72Â°C), and prolonged heating (pasteurization at 60Â°C) results in a heat-stabilized structural form containing fatty acids (Tm of âˆ¼75-80Â°C). This process was shown to be reversible by a low pH defatting step. This study shows that the fatty acid composition and bioprocessing history of the HSA commercial products results in the large differences in the thermal stability.Â© 2014 American Institute of Chemical Engineers.
Mouse cell line authentication. - Cytotechnology
The scientific community has responded to the misidentification of human cell lines with validated methods to authenticate these cells; however, few assays are available for nonhuman cell line identification. We have developed a multiplex polymerase chain reaction assay that targets nine tetranucleotide short tandem repeat (STR) markers in the mouse genome. Unique profiles were obtained from seventy-two mouse samples that were used to determine the allele distribution for each STR marker. Correlations between allele fragment length and repeat number were determined with DNA Sanger sequencing. Genotypes for L929 and NIH3T3 cell lines were shown to be stable with increasing passage numbers as there were no significant differences in fragment length with samples of low passage when compared to high passage samples. In order to detect cell line contaminants, primers for two human STR markers were incorporated into the multiplex assay to facilitate detection of human and African green monkey DNA. This multiplex assay is the first of its kind to provide a unique STR profile for each individual mouse sample and can be used to authenticate mouse cell lines.
Breast cancer biomarker measurements and standards. - Proteomics. Clinical applications
Cancer is a heterogeneous disease characterized by changes in the levels and activities of important cellular proteins, including oncogenes and tumor suppressors. Genetic mutations cause changes in protein activity and protein expression levels that result in the altered metabolism, proliferation, and metastasis seen in cancer cells. The identification of the critical biochemical changes in cancer has led to advances in its detection and treatment. An important example of this is the measurement of human epidermal growth factor receptor 2 (HER2), where increased expression occurs in approximately 20-30% of breast cancer tumors. HER2 is a member of the epidermal growth factor receptor family and is an important biomarker expressed on the cell surface. Measurement of the HER2 levels in tumor cells provides diagnostic, prognostic, and treatment information, because a targeted therapeutic is available. The most common methods to measure HER2 levels are immunohistochemistry and in situ hybridization assays. The accurate and reliable measurements of the specific changes in protein biomarkers for detection and treatment of cancer are important challenges. This review is focused on efforts to improve the quantitation and reliability of cancer biomarkers by using standards and reference materials.Â© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Human CD4+ lymphocytes for antigen quantification: characterization using conventional flow cytometry and mass cytometry. - Cytometry. Part A : the journal of the International Society for Analytical Cytology
To transform the linear fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale, a biological cell reference material is needed. Optimally, this material should have a reproducible and tight ABC value for the expression of a known clinical reference biomarker. In this study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) and two lyophilized PBMC preparations, Cyto-Trol and PBMC-National Institute for Biological Standard and Control (NIBSC) relative to freshly prepared PBMC and whole blood samples. It was found that the ABC values for CD4 expression on cryopreserved PBMC were consistent with those of freshly obtained PBMC and whole blood samples. By comparison, the ABC value for CD4 expression on Cyto-Trol is lower and the value on PBMC-NIBSC is much lower than those of freshly prepared cell samples using both conventional flow cytometry and CyTOFâ„¢ mass cytometry. By performing simultaneous surface and intracellular staining measurements on these two cell samples, we found that both cell membranes are mostly intact. Moreover, CD4(+) cell diameters from both lyophilized cell preparations are smaller than those of PBMC and whole blood. This could result in steric interference in antibody binding to the lyophilized cells. Further investigation of the fixation effect on the detected CD4 expression suggests that the very low ABC value obtained for CD4(+) cells from lyophilized PBMC-NIBSC is largely due to paraformaldehyde fixation; this significantly decreases available antibody binding sites. This study provides confirmation that the results obtained from the newly developed mass cytometry are directly comparable to the results from conventional flow cytometry when both methods are standardized using the same ABC approach.Published 2012 Wiley Periodicals, Inc.
Authentication of African green monkey cell lines using human short tandem repeat markers. - BMC biotechnology
Tools for authenticating cell lines are critical for quality control in cell-based biological experiments. Currently there are methods to authenticate human cell lines using short tandem repeat (STR) markers based on the technology and procedures successfully used in the forensic community for human identification, but there are no STR based methods for authenticating nonhuman cell lines to date. There is significant homology between the human and vervet monkey genome and we utilized these similarities to design the first multiplex assay based on human STR markers for vervet cell line identification.The following STR markers were incorporated into the vervet multiplex PCR assay: D17S1304, D5S1467, D19S245, D1S518, D8S1106, D4S2408, D6S1017, and DYS389. The eight markers were successful in uniquely identifying sixty-two vervet monkey DNA samples and confirmed that Vero76 cells and COS-7 cells were derived from Vero and CV-1 cells, respectively. The multiplex assay shows specificity for vervet DNA within the determined allele range for vervet monkeys; however, the primers will also amplify human DNA for each marker resulting in amplicons outside the vervet allele range in several of the loci. The STR markers showed genetic stability in over sixty-nine passages of Vero cells, suggesting low mutation rates in the targeted STR sequences in the Vero cell line.A functional vervet multiplex assay consisting of eight human STR markers with heterozygosity values ranging from 0.53-0.79 was successful in uniquely identifying sixty-two vervet monkey samples. The probability of a random match using these eight markers between any two vervet samples is approximately 1 in 1.9 million. While authenticating a vervet cell line, the multiplex assay may also be a useful indicator for human cell line contamination since the assay is based on human STR markers.
Past and ongoing shifts in Joshua tree distribution support future modeled range contraction. - Ecological applications : a publication of the Ecological Society of America
The future distribution of the Joshua tree (Yucca brevifolia) is projected by combining a geostatistical analysis of 20th-century climates over its current range, future modeled climates, and paleoecological data showing its response to a past similar climate change. As climate rapidly warmed approximately 11 700 years ago, the range of Joshua tree contracted, leaving only the populations near what had been its northernmost limit. Its ability to spread northward into new suitable habitats after this time may have been inhibited by the somewhat earlier extinction of megafaunal dispersers, especially the Shasta ground sloth. We applied a model of climate suitability for Joshua tree, developed from its 20th-century range and climates, to future climates modeled through a set of six individual general circulation models (GCM) and one suite of 22 models for the late 21st century. All distribution data, observed climate data, and future GCM results were scaled to spatial grids of approximately 1 km and approximately 4 km in order to facilitate application within this topographically Complex region. All of the models project the future elimination of Joshua tree throughout most of the southern portions of its current range. Although estimates of future monthly precipitation differ between the models, these changes are outweighed by large increases in temperature common to all the models. Only a few populations within the current range are predicted to be sustainable. Several models project significant potential future expansion into new areas beyond the current range, but the species' historical and current rates of dispersal would seem to prevent natural expansion into these new areas. Several areas are predicted to be potential sites for relocation/ assisted migration. This project demonstrates how information from paleoecology and modern ecology can be integrated in order to understand ongoing processes and fuiture distributions.
U.S. Government engagement in support of global disease surveillance. - BMC public health
Global cooperation is essential for coordinated planning and response to public health emergencies, as well as for building sufficient capacity around the world to detect, assess and respond to health events. The United States is committed to, and actively engaged in, supporting disease surveillance capacity building around the world. We recognize that there are many agencies involved in this effort, which can become confusing to partner countries and other public health entities. This paper aims to describe the agencies and offices working directly on global disease surveillance capacity building in order to clarify the United States Government interagency efforts in this space.
Development of a candidate secondary reference procedure (immunoassay based measurement procedure of higher metrological order) for cardiac troponin I: I. Antibody characterization and preliminary validation. - Clinical chemistry and laboratory medicine : CCLM / FESCC
In this study, the first steps in the development of a secondary reference measurement procedure (RMP) 'higher metrological order measurement procedure' to support the cardiac troponin I (cTnI) standardization initiative is described. The RMP should be used to assign values to serum-based secondary reference materials (RMs) without analytical artifacts causing bias. A multiplexed bead-based assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to identify the optimum monoclonal antibody pair (clones 560 and 19C7) for the RMP. Using these antibodies, an ELISA-based procedure was developed to accurately measure the main cTnI forms present in blood. The proposed RMP appears to show no bias when tested on samples containing various troponin complexes, phosphorylated and dephosphorylated forms, and heparin. The candidate assay displayed suitable linearity and sensitivity (limit of detection, 0.052 Î¼g/L) for the measurement of the proposed cTnI secondary RMs. Preliminary comparison data on patient samples with a commercial cTnI assay are also provided to support the suitability of RMP for value assignment to RMs. Full validation and final assessment of the RMP will be performed through transferability and inter-comparison studies.
An immunoprecipitation coupled with fluorescent Western blot analysis for the characterization of a model secondary serum cardiac troponin I reference material. - Clinica chimica acta; international journal of clinical chemistry
Cardiac troponin I (cTnI) is considered the 'gold standard' cardiac biomarker. However, the result comparability of commercial cTnI immunoassays is still lacking despite the availability of NIST Standard Reference Material, SRM 2921 (human cardiac troponin). To facilitate the standardization of the cTnI immunoassays, a secondary reference material consisting of a panel of three cTnI-positive human serum pools is proposed by the IFCC Working Group on Standardization of Troponin I. The objective of this study is to develop measurement procedures for the characterization of the future secondary reference material using a pooled cTnI-positive serum sample as a development model.We used magnetic beads coupled with 6 different anti-cTnI monoclonal antibodies that bind specifically to different amino acid sequence regions of the cTnI molecule to immunoprecipitate cTnI proteins from the pooled cTnI-positive serum sample followed by sensitive detection using a fluorescent Western blot.The degradation of cTnI in the pooled sample was detected and the concentration of cTnI was determined.We demonstrated the utility of this measurement procedure in support of the development of the proposed secondary cTnI-positive, serum-based reference material.Copyright Â© 2010 Elsevier B.V. All rights reserved.
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