Preliminary validation of two novel immunoassays for detecting VEGF in human and mouse plasma using single molecule counting technology

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e22099-e22099
Author(s):  
R. Puskas ◽  
D. Held ◽  
D. Sheets ◽  
B. K. Klein ◽  
E. Macy ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Human Plasma ◽  
Plasma Cell ◽  
Cross Reactivity ◽  
Conditioned Media ◽  
Elisa Assay ◽  
Plasma Samples ◽  
Preliminary Validation ◽  
Tissue Specimens ◽  
Human And Mouse

e22099 Background: Growth in biomarkers as therapeutic targets and as surrogate markers for efficacy presents a need for increasingly sensitive immunoassays to expand biomarker applicability. Improved immunoassays will provide: (1) better evaluation and validation of new drug candidates, (2) better matching of patients to new therapies, (3) accelerated drug approval (4) earlier diagnosis of at-risk patients, and (5) a deeper understanding of cancer biology. Towards this end, Singulex has developed two ultra- sensitive VEGF Immunoassays for human and mouse vascular endothelial growth factor (VEGF). Here we report the preliminary validation of these two novel assays. Methods: Two novel assays were developed with the Erenna Immunoassay System for detecting VEGF: human (hVEGF) and mouse (mVEGF). Analytical sensitivity, cross-reactivity and precision were determined and compared to an ELISA based VEGF assay. Both the Singulex assay and ELISA assay were used to test a range of specimen types (plasma, cell lysates, conditioned media, and tissue specimens) from humans and mice. Preliminary assays with human plasma and tissue specimens were conducted to compare hVEGF levels between normal and breast cancer samples. Results: The Singulex hVEGF assay had an LOD of 0.1 pg/mL, an LLOQ of 0.3 pg/mL, and 84–107% spike recovery; 90X more sensitive than the ELISA assay. Human VEGF concentrations were quantified in all specimens tested compared to the ELISA, which quantified VEGF in only 8% of plasma samples, but all of the cell lysate samples. The Singulex mVEGF assay had an LOD of 3.5 pg/mL, LLOQ of 5 pg/mL, and 68–111% spike recovery; 3X more sensitive than the ELISA assay. Cross-reactivity for the two assays was minimal for all specimen types tested, except for human plasma samples where the mVEGF assay demonstrated 80–100% CR. Conclusions: We show that the Singulex hVEGF and mVEGF Immunoassays can detect VEGF at or below pg/mL levels, and can effectively quantify VEGF levels in plasma, cell lysates, conditioned media, and tissue samples from mice and humans. These novel assays are an important tool when used to assess tumor and normal breast cancer tissue and plasma. [Table: see text]

Radioimmunoassay of Sodium Cromoglycate in Human Plasma

1983 ◽  
Vol 20 (1) ◽  
pp. 31-36 ◽  
Author(s):  
Kevan Brown ◽  
John J Gardner ◽  
William J S Lockley ◽  
John R Preston ◽  
David J Wilkinson
Keyword(s):  
Sodium Cromoglycate ◽  
Human Plasma ◽  
Direct Analysis ◽  
Cross Reactivity ◽  
Plasma Samples ◽  
Antibody Technique ◽  
Human Volunteers ◽  
Iodine 125 ◽  
Therapeutic Doses ◽  
Quantifiable Concentration

A sensitive radioimmunoassay method for sodium cromoglycate in human plasma is described. The lowest quantifiable concentration of sodium cromoglycate is 0·93 nmol/l when 0·1 ml plasma samples are analysed. Direct analysis of sodium cromoglycate concentrations in plasma samples collected up to several hours after the administration of single therapeutic doses of the compound is possible. An antiserum raised in a sheep, radioligand heterogeneously labelled with iodine-125, and a second antibody technique for the separation of bound and free radioligand are employed. The inter-assay coefficient of variation is less than 14% (n=23). The range of the method is limited; both 0·01 and 0·1 ml volumes of plasma must be analysed to encompass the concentration range 0·93–139 nmol/l which may be encountered in plasma samples from patients and human volunteers. The method is specific for sodium cromoglycate as indicated by a low cross-reactivity of the anti-cromoglycate antiserum with a number of drugs.

scholarly journals Detection of Brevetoxin in Human Plasma by ELISA

2021 ◽  
Author(s):  
Brady R Cunningham ◽  
Rebecca M Coleman ◽  
Adam M Schaefer ◽  
Elizabeth I Hamelin ◽  
Rudolph C Johnson
Keyword(s):  
Human Plasma ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Quantitative Detection ◽  
Red Tides ◽  
Plasma Samples ◽  
Detection Range ◽  
Paralytic Shellfish ◽  
Test Kit ◽  
Relative Standard

Abstract Florida red tides have become more common and persistent in and around the Gulf of Mexico. When in bloom, red tides can produce brevetoxins in high concentrations, leading to human exposures primarily through contaminated food and ocean spray. The research described here includes adapting and validating a commercial brevetoxin water test kit for human plasma testing. Pooled plasma was fortified with a model brevetoxin, brevetoxin 3, at concentrations from 0.00500 to 3.00 ng/mL to generate calibration curves and quality control samples. The quantitative detection range was determined to be 0.0400–2.00 ng/mL brevetoxin 3 equivalents with inter- and intraday accuracies ranging from 94.0% to 109% and relative standard deviations <20%, which is within the US Food and Drug Administration guidelines for receptor-binding assays. Additionally, cross-reactivity was tested using 4 of the 10 known brevetoxins and 12 paralytic shellfish toxins. The cross-reactivity varied from 0.173% to 144% for the commercially available brevetoxin standards and 0% for the commercially available paralytic shellfish toxin standards. Fifty individual unexposed human plasma samples were measured to determine the limit of detection and endogenous interferences to the test. The validated method was used to test 31 plasma samples collected from humans potentially exposed to brevetoxins, detecting 11 positives. This method has been proven useful to measure human exposure to brevetoxins and can be applied to future exposure events.

Gp130 and ras mediated signaling in human plasma cell line INA-6: a cytokine-regulated tumor model for plasmacytoma

2001 ◽  
Vol 2 (1) ◽  
pp. 42-53 ◽  
Author(s):  
Renate Burger ◽  
Andreas Guenther ◽  
Frank Bakker ◽  
Marc Schmalzing ◽  
Sabrina Bernand ◽  
...  
Keyword(s):  
Cell Line ◽  
Human Plasma ◽  
Plasma Cell ◽  
Tumor Model

scholarly journals Human Plasma Metabolomics for Biomarker Discovery: Targeting the Molecular Subtypes in Breast Cancer

Cancers ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 147
Author(s):  
Leticia Díaz-Beltrán ◽  
Carmen González-Olmedo ◽  
Natalia Luque-Caro ◽  
Caridad Díaz ◽  
Ariadna Martín-Blázquez ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Patients ◽  
Biomarker Discovery ◽  
Molecular Subtypes ◽  
Breast Cancer Subtype ◽  
P Value ◽  
Plasma Samples ◽  
Breast Cancer Patients ◽  
Clinical Value ◽  
Molecular Phenotypes

Purpose: The aim of this study is to identify differential metabolomic signatures in plasma samples of distinct subtypes of breast cancer patients that could be used in clinical practice as diagnostic biomarkers for these molecular phenotypes and to provide a more individualized and accurate therapeutic procedure. Methods: Untargeted LC-HRMS metabolomics approach in positive and negative electrospray ionization mode was used to analyze plasma samples from LA, LB, HER2+ and TN breast cancer patients and healthy controls in order to determine specific metabolomic profiles through univariate and multivariate statistical data analysis. Results: We tentatively identified altered metabolites displaying concentration variations among the four breast cancer molecular subtypes. We found a biomarker panel of 5 candidates in LA, 7 in LB, 5 in HER2 and 3 in TN that were able to discriminate each breast cancer subtype with a false discovery range corrected p-value < 0.05 and a fold-change cutoff value > 1.3. The model clinical value was evaluated with the AUROC, providing diagnostic capacities above 0.85. Conclusion: Our study identifies metabolic profiling differences in molecular phenotypes of breast cancer. This may represent a key step towards therapy improvement in personalized medicine and prioritization of tailored therapeutic intervention strategies.

scholarly journals Radioimmunoassay of plasma ouabain in healthy and pregnant individuals

2000 ◽  
Vol 165 (3) ◽  
pp. 669-677 ◽  
Author(s):  
O Vakkuri ◽  
SS Arnason ◽  
A Pouta ◽  
O Vuolteenaho ◽  
J Leppaluoto
Keyword(s):  
Pregnant Women ◽  
Human Plasma ◽  
High Performance ◽  
Chemical Nature ◽  
Solid Phase ◽  
Plasma Concentrations ◽  
First Trimester ◽  
Plasma Samples ◽  
Third Trimester ◽  
Endogenous Substances

Ouabain was recently isolated from human plasma, bovine hypothalamus and bovine adrenal in attempts to identify endogenous substances inhibiting the cell membrane sodium pump. A number of radioimmunoassays have been developed in order to study the clinical significance of ouabain. The results have been controversial with regard to the presence and chemical nature of plasma ouabain-like immunoreactivity. We have now measured ouabain in healthy and pregnant individuals using solid-phase extraction of plasma samples followed by a new radioimmunoassay with the extraordinary sensitivity of at least 2 fmol/tube (5 pmol/l). Plasma extracts, a previously isolated human plasma ouabain-like compound and bovine hypothalamic inhibitory factor displaced the tracer in parallel and eluted identically with ouabain in high-performance liquid chromatography. Plasma ouabain immunoreactivity was found to be much lower than reported previously: 12.6+/-1.3 pmol/l in healthy men (mean+/-s.e., n=20) and 9.4+/-0.7 pmol/l in women (n=14). In pregnant women (n=28) plasma ouabain concentration was 16.3+/-4.0 pmol/l during the first trimester, 18.8+/-4.3 pmol/l during the second trimester and 24.3+/-4.0 pmol/l during the third trimester (all P<0.01 compared with non-pregnant women). Plasma ouabain 3-5 days after the delivery was 13.6+/-1.1 pmol/l (n=10, P<0.05-0.01 compared with second and third trimesters). The pregnancy-related changes in the plasma concentrations of ouabain resembled those of cortisol. Therefore cortisol was measured from the same plasma samples and a significant positive correlation was found (r=0.512, P=0.006). The similar profiles of plasma ouabain and cortisol during pregnancy and their rapid decreases postpartum are consistent with the adrenal cortical origin of ouabain and also show that the secretions of these hormones are possibly under the control of same factors.

scholarly journals Protocol to analyze circulating small non-coding RNAs by high-throughput RNA sequencing from human plasma samples

2021 ◽  
Vol 2 (3) ◽  
pp. 100606
Author(s):  
Giuseppina E. Grieco ◽  
Guido Sebastiani ◽  
Daniela Fignani ◽  
Noemi Brusco ◽  
Laura Nigi ◽  
...  
Keyword(s):  
Human Plasma ◽  
Rna Sequencing ◽  
High Throughput ◽  
Plasma Samples ◽  
Human Plasma Samples ◽  
Non Coding Rnas

Optimizationvia experimental design of an SPE-HPLC-UV-fluorescence method for the determination of valsartan and its metabolite in human plasma samples

2006 ◽  
Vol 29 (15) ◽  
pp. 2265-2283 ◽  
Author(s):  
Gorka Iriarte ◽  
Nerea Ferreirós ◽  
Izaskun Ibarrondo ◽  
Rosa Maria Alonso ◽  
Miren Itxaso Maguregi ◽  
...  
Keyword(s):  
Experimental Design ◽  
Human Plasma ◽  
Fluorescence Method ◽  
Plasma Samples ◽  
Uv Fluorescence ◽  
Human Plasma Samples
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