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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200137	1/4	Homo sapiens	Blood plasma	Streptavidin bead precipitation of biotinylated cholera toxin B chain-bound EV	Tan, Kok Hian	2017	25%
Study summary Full title Extracellular vesicles yield predictive pre-eclampsia biomarkers All authors Kok Hian Tan, Soon Sim Tan, Mor Jack Ng, Wan Shi Tey, Wei Kian Sim, John Carson Allen, Sai Kiang Lim Journal J Extracell Vesicles Abstract Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-bind (show more...)Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-binding EVs were previously shown to be rich sources of biomarkers. Here we test if previously identified pre-eclampsia (PE) candidate biomarkers, TIMP-1 in CTB-EVs (CTB-TIMP) and PAI-1 in AV-EVs (AV-PAI) complement plasma PlGF in predicting PE in a low-risk obstetric population. Eight hundred and forty-three prospectively banked plasma samples collected at 28 + 0 to 32 + 0 gestation weeks in the Neonatal and Obstetrics Risk Assessment (NORA) cohort study were assayed by sandwich ELISAs for plasma PlGF, CTB-TIMP1 and AV-PAI1. Nineteen patients subsequently developed PE 7.3 (±2.9) weeks later at a mean gestational age of 36.1 ± 3.5 weeks. The biomarkers were assessed for their predictive accuracy for PE using stepwise multivariate logistic regression analysis with Firth correction and Areas under the curve (AUC). To achieve 100% sensitivity in predicting PE, the cut-off for plasma PlGF, CTB-TIMP1 & AV-PAI1 were set at <1235, ≤300 or >1300 and <10,550 pg/mL plasma, respectively. The corresponding AUCs, specificity and PPV at a 95% confidence interval were 0.92, 52.1% and 4.7%; 0.72, 44.5% and 4.0%; and 0.69, 21.5% and 2.9%, respectively. At 100% sensitivity, the three biomarkers had a combined AUC of 0.96, specificity of 78.6%, and PPV of 9.9%. This is the first large cohort validation of the utility of EV-associated analytes as disease biomarkers. Specifically, EV biomarkers enhanced the predictive robustness of an existing PE biomarker sufficiently to justify PE screening in a low-risk general obstetric population. (hide) EV-METRIC 25% (56th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Healthy pregnant Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Streptavidin bead precipitation of biotinylated cholera toxin B chain-bound EV Protein markers EV: GH/ PCT/ PAI1/ PlGF/ TIMP1 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Other Name other separation method Streptavidin bead precipitation of biotinylated cholera toxin B chain-bound EV Characterization: Protein analysis Protein Concentration Method Not determined ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins GH/ PCT/ PAI1/ PlGF/ TIMP1 Characterization: Lipid analysis No

	EV200137	2/4	Homo sapiens	Blood plasma	Streptavidin bead precipitation of biotinylated annexin AV-bound EV	Tan, Kok Hian	2017	25%
Study summary Full title Extracellular vesicles yield predictive pre-eclampsia biomarkers All authors Kok Hian Tan, Soon Sim Tan, Mor Jack Ng, Wan Shi Tey, Wei Kian Sim, John Carson Allen, Sai Kiang Lim Journal J Extracell Vesicles Abstract Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-bind (show more...)Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-binding EVs were previously shown to be rich sources of biomarkers. Here we test if previously identified pre-eclampsia (PE) candidate biomarkers, TIMP-1 in CTB-EVs (CTB-TIMP) and PAI-1 in AV-EVs (AV-PAI) complement plasma PlGF in predicting PE in a low-risk obstetric population. Eight hundred and forty-three prospectively banked plasma samples collected at 28 + 0 to 32 + 0 gestation weeks in the Neonatal and Obstetrics Risk Assessment (NORA) cohort study were assayed by sandwich ELISAs for plasma PlGF, CTB-TIMP1 and AV-PAI1. Nineteen patients subsequently developed PE 7.3 (±2.9) weeks later at a mean gestational age of 36.1 ± 3.5 weeks. The biomarkers were assessed for their predictive accuracy for PE using stepwise multivariate logistic regression analysis with Firth correction and Areas under the curve (AUC). To achieve 100% sensitivity in predicting PE, the cut-off for plasma PlGF, CTB-TIMP1 & AV-PAI1 were set at <1235, ≤300 or >1300 and <10,550 pg/mL plasma, respectively. The corresponding AUCs, specificity and PPV at a 95% confidence interval were 0.92, 52.1% and 4.7%; 0.72, 44.5% and 4.0%; and 0.69, 21.5% and 2.9%, respectively. At 100% sensitivity, the three biomarkers had a combined AUC of 0.96, specificity of 78.6%, and PPV of 9.9%. This is the first large cohort validation of the utility of EV-associated analytes as disease biomarkers. Specifically, EV biomarkers enhanced the predictive robustness of an existing PE biomarker sufficiently to justify PE screening in a low-risk general obstetric population. (hide) EV-METRIC 25% (56th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Healthy pregnant Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Streptavidin bead precipitation of biotinylated annexin AV-bound EV Protein markers EV: GH/ PCT/ PAI1/ PlGF/ TIMP1 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Other Name other separation method Streptavidin bead precipitation of biotinylated annexin AV-bound EV Characterization: Protein analysis Protein Concentration Method Not determined ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins GH/ PCT/ PAI1/ PlGF/ TIMP1 Characterization: Lipid analysis No

	EV200137	3/4	Homo sapiens	Blood plasma	Streptavidin bead precipitation of biotinylated cholera toxin B chain-bound EV	Tan, Kok Hian	2017	25%
Study summary Full title Extracellular vesicles yield predictive pre-eclampsia biomarkers All authors Kok Hian Tan, Soon Sim Tan, Mor Jack Ng, Wan Shi Tey, Wei Kian Sim, John Carson Allen, Sai Kiang Lim Journal J Extracell Vesicles Abstract Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-bind (show more...)Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-binding EVs were previously shown to be rich sources of biomarkers. Here we test if previously identified pre-eclampsia (PE) candidate biomarkers, TIMP-1 in CTB-EVs (CTB-TIMP) and PAI-1 in AV-EVs (AV-PAI) complement plasma PlGF in predicting PE in a low-risk obstetric population. Eight hundred and forty-three prospectively banked plasma samples collected at 28 + 0 to 32 + 0 gestation weeks in the Neonatal and Obstetrics Risk Assessment (NORA) cohort study were assayed by sandwich ELISAs for plasma PlGF, CTB-TIMP1 and AV-PAI1. Nineteen patients subsequently developed PE 7.3 (±2.9) weeks later at a mean gestational age of 36.1 ± 3.5 weeks. The biomarkers were assessed for their predictive accuracy for PE using stepwise multivariate logistic regression analysis with Firth correction and Areas under the curve (AUC). To achieve 100% sensitivity in predicting PE, the cut-off for plasma PlGF, CTB-TIMP1 & AV-PAI1 were set at <1235, ≤300 or >1300 and <10,550 pg/mL plasma, respectively. The corresponding AUCs, specificity and PPV at a 95% confidence interval were 0.92, 52.1% and 4.7%; 0.72, 44.5% and 4.0%; and 0.69, 21.5% and 2.9%, respectively. At 100% sensitivity, the three biomarkers had a combined AUC of 0.96, specificity of 78.6%, and PPV of 9.9%. This is the first large cohort validation of the utility of EV-associated analytes as disease biomarkers. Specifically, EV biomarkers enhanced the predictive robustness of an existing PE biomarker sufficiently to justify PE screening in a low-risk general obstetric population. (hide) EV-METRIC 25% (56th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Pre-eclampsia Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Streptavidin bead precipitation of biotinylated cholera toxin B chain-bound EV Protein markers EV: GH/ PCT/ PAI1/ PlGF/ TIMP1 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Other Name other separation method Streptavidin bead precipitation of biotinylated cholera toxin B chain-bound EV Characterization: Protein analysis Protein Concentration Method Not determined ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins GH/ PCT/ PAI1/ PlGF/ TIMP1 Characterization: Lipid analysis No

	EV200137	4/4	Homo sapiens	Blood plasma	Streptavidin bead precipitation of biotinylated annexin AV-bound EV	Tan, Kok Hian	2017	25%
Study summary Full title Extracellular vesicles yield predictive pre-eclampsia biomarkers All authors Kok Hian Tan, Soon Sim Tan, Mor Jack Ng, Wan Shi Tey, Wei Kian Sim, John Carson Allen, Sai Kiang Lim Journal J Extracell Vesicles Abstract Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-bind (show more...)Circulating extracellular vesicles (EVs) such as cholera toxin B chain (CTB)- or annexin V (AV)-binding EVs were previously shown to be rich sources of biomarkers. Here we test if previously identified pre-eclampsia (PE) candidate biomarkers, TIMP-1 in CTB-EVs (CTB-TIMP) and PAI-1 in AV-EVs (AV-PAI) complement plasma PlGF in predicting PE in a low-risk obstetric population. Eight hundred and forty-three prospectively banked plasma samples collected at 28 + 0 to 32 + 0 gestation weeks in the Neonatal and Obstetrics Risk Assessment (NORA) cohort study were assayed by sandwich ELISAs for plasma PlGF, CTB-TIMP1 and AV-PAI1. Nineteen patients subsequently developed PE 7.3 (±2.9) weeks later at a mean gestational age of 36.1 ± 3.5 weeks. The biomarkers were assessed for their predictive accuracy for PE using stepwise multivariate logistic regression analysis with Firth correction and Areas under the curve (AUC). To achieve 100% sensitivity in predicting PE, the cut-off for plasma PlGF, CTB-TIMP1 & AV-PAI1 were set at <1235, ≤300 or >1300 and <10,550 pg/mL plasma, respectively. The corresponding AUCs, specificity and PPV at a 95% confidence interval were 0.92, 52.1% and 4.7%; 0.72, 44.5% and 4.0%; and 0.69, 21.5% and 2.9%, respectively. At 100% sensitivity, the three biomarkers had a combined AUC of 0.96, specificity of 78.6%, and PPV of 9.9%. This is the first large cohort validation of the utility of EV-associated analytes as disease biomarkers. Specifically, EV biomarkers enhanced the predictive robustness of an existing PE biomarker sufficiently to justify PE screening in a low-risk general obstetric population. (hide) EV-METRIC 25% (56th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Pre-eclampsia Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Streptavidin bead precipitation of biotinylated annexin AV-bound EV Protein markers EV: GH/ PCT/ PAI1/ PlGF/ TIMP1 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Other Name other separation method Streptavidin bead precipitation of biotinylated annexin AV-bound EV Characterization: Protein analysis Protein Concentration Method Not determined ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins GH/ PCT/ PAI1/ PlGF/ TIMP1 Characterization: Lipid analysis No

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EV-TRACK ID	EV200137
species	Homo sapiens
sample type	Blood plasma
condition	Healthy pregnant	Healthy pregnant	Pre-eclampsia	Pre-eclampsia
separation protocol	Streptavidin bead precipitation of biotinylated cholera toxin B chain-bound EV	Streptavidin bead precipitation of biotinylated annexin AV-bound EV	Streptavidin bead precipitation of biotinylated cholera toxin B chain-bound EV	Streptavidin bead precipitation of biotinylated annexin AV-bound EV
Exp. nr.	1	2	3	4
EV-METRIC %	25	25	25	25