Technical performance and biotemporal stability evaluation of Olink proximity extension assay for blood‐based biomarker discovery

There is a lack of reliable biomarkers for disorders of the central nervous system (CNS), and diagnostics still heavily rely on symptoms that are both subjective and difficult to quantify. The cerebrospinal fluid (CSF) is a promising source of biomarkers due to its close connection to the CNS. Extracellular vesicles are actively secreted by cells, and proteomic analysis of CSF extracellular vesicles (EVs) and their molecular composition likely reflects changes in the CNS to a higher extent compared with total CSF, especially in the case of neuroinflammation, which could increase blood–brain barrier permeability and cause an influx of plasma proteins into the CSF. We used proximity extension assay for proteomic analysis due to its high sensitivity. We believe that this methodology could be useful for de novo biomarker discovery for several CNS diseases. We compared four commercially available kits for EV isolation: MagCapture and ExoIntact (based on magnetic beads), EVSecond L70 (size-exclusion chromatography), and exoEasy (membrane affinity). The isolated EVs were characterized by nanoparticle tracking analysis, ELISA (CD63, CD81 and albumin), and proximity extension assay (PEA) using two different panels, each consisting of 92 markers. The exoEasy samples did not pass the built-in quality controls and were excluded from downstream analysis. The number of detectable proteins in the ExoIntact samples was considerably higher (~150% for the cardiovascular III panel and ~320% for the cell regulation panel) compared with other groups. ExoIntact also showed the highest intersample correlation with an average Pearson’s correlation coefficient of 0.991 compared with 0.985 and 0.927 for MagCapture and EVSecond, respectively. The median coefficient of variation was 5%, 8%, and 22% for ExoIntact, MagCapture, and EVSecond, respectively. Comparing total CSF and ExoIntact samples revealed 70 differentially expressed proteins in the cardiovascular III panel and 17 in the cell regulation panel. To our knowledge, this is the first time that CSF EVs were analyzed by PEA. In conclusion, analysis of CSF EVs by PEA is feasible, and different isolation kits give distinct results, with ExoIntact showing the highest number of identified proteins with the lowest variability.

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Inflammation biomarker discovery in Parkinson's disease and atypical parkinsonisms

10.21203/rs.2.14452/v3 ◽

2019 ◽

Author(s):

Anna Santaella ◽

H. Bea Kuiperij ◽

Anouke van Rumund ◽

Rianne A.J. Esselink ◽

Alain J. van Gool ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Growth Factor ◽

Biomarker Discovery ◽

Response To Treatment ◽

Csf Biomarkers ◽

Vascular Parkinsonism ◽

Epidermal Growth ◽

Atypical Parkinsonisms ◽

Proximity Extension Assay

Abstract Background: Parkinson’s disease (PD) and atypical parkinsonisms (APD) have overlapping symptoms challenging an early diagnosis. Diagnostic accuracy is important because PD and APD have different prognosis and response to treatment. We aimed to identify diagnostic inflammatory biomarkers of PD and APD in cerebrospinal fluid (CSF) using the multiplex proximity extension assay (PEA) technology and to study possible correlations of biomarkers with disease progression. Methods: CSF from a longitudinal cohort study consisting of PD and APD patients (PD, n = 44; multiple system atrophy (MSA), n = 14; vascular parkinsonism (VaP), n = 9; and PD with VaP, n = 7) and controls (n = 25) were analyzed. Results: Concentrations of CCL28 were elevated in PD compared to controls (p = 0.0001). Five other biomarkers differentiated both MSA and PD from controls (p < 0.05) and 10 biomarkers differentiated MSA from controls, of which two proteins, i.e. beta nerve growth factor (β-NGF) and Delta and Notch like epidermal growth factor-related receptor (DNER), were also present at lower levels in MSA compared to PD (both p = 0.032). Two biomarkers (MCP-1 and MMP-10) positively correlated with PD progression (rho > 0.650; p < 0.01). Conclusions: PEA technique identified potential new CSF biomarkers to help to predict the prognosis of PD. Also, we identified new candidate biomarkers to distinguish MSA from PD.

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Inflammation biomarker discovery in Parkinson's disease and atypical parkinsonisms

10.21203/rs.2.14452/v2 ◽

2019 ◽

Author(s):

Anna Santaella ◽

H. Bea Kuiperij ◽

Anouke van Rumund ◽

Rianne A.J. Esselink ◽

Alain J. van Gool ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Growth Factor ◽

Biomarker Discovery ◽

Response To Treatment ◽

Csf Biomarkers ◽

Vascular Parkinsonism ◽

Epidermal Growth ◽

Atypical Parkinsonisms ◽

Proximity Extension Assay

Abstract Background: Parkinson’s disease (PD) and atypical parkinsonisms (APD) have overlapping symptoms challenging an early diagnosis. Diagnostic accuracy is important because PD and APD have different prognosis and response to treatment. We aimed to identify diagnostic inflammatory biomarkers of PD and APD in cerebrospinal fluid (CSF) using the multiplex proximity extension assay (PEA) technology and to study possible correlations of biomarkers with disease progression. Methods: CSF from a longitudinal cohort study consisting of PD and APD patients (PD, n = 44; multiple system atrophy (MSA), n = 14; vascular parkinsonism (VaP), n = 9; and PD with VaP, n = 7) and controls (n = 25) were analyzed. Results: Concentrations of CCL28 were elevated in PD compared to controls (p = 0.0001). Five other biomarkers differentiated both MSA and PD from controls (p < 0.05) and 10 biomarkers differentiated MSA from controls, of which two proteins, i.e. beta nerve growth factor (β-NGF) and Delta and Notch like epidermal growth factor-related receptor (DNER), were also present at lower levels in MSA compared to PD (both p = 0.032). Two biomarkers (MCP-1 and MMP-10) positively correlated with PD progression (rho > 0.650; p < 0.01). Conclusions: PEA technique identified potential new CSF biomarkers to help to predict the prognosis of PD. Also, we identified new candidate biomarkers to distinguish MSA from PD.

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Inflammation biomarker discovery in Parkinson's disease and atypical parkinsonisms

10.21203/rs.2.14452/v4 ◽

2020 ◽

Author(s):

Anna Santaella ◽

H. Bea Kuiperij ◽

Anouke van Rumund ◽

Rianne A.J. Esselink ◽

Alain J. van Gool ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Growth Factor ◽

Biomarker Discovery ◽

Response To Treatment ◽

Csf Biomarkers ◽

Vascular Parkinsonism ◽

Epidermal Growth ◽

Atypical Parkinsonisms ◽

Proximity Extension Assay

Abstract Background: Parkinson’s disease (PD) and atypical parkinsonisms (APD) have overlapping symptoms challenging an early diagnosis. Diagnostic accuracy is important because PD and APD have different prognosis and response to treatment. We aimed to identify diagnostic inflammatory biomarkers of PD and APD in cerebrospinal fluid (CSF) using the multiplex proximity extension assay (PEA) technology and to study possible correlations of biomarkers with disease progression. Methods: CSF from a longitudinal cohort study consisting of PD and APD patients (PD, n = 44; multiple system atrophy (MSA), n = 14; vascular parkinsonism (VaP), n = 9; and PD with VaP, n = 7) and controls (n = 25) were analyzed. Results: Concentrations of CCL28 were elevated in PD compared to controls (p = 0.0001). Five other biomarkers differentiated both MSA and PD from controls (p < 0.05) and 10 biomarkers differentiated MSA from controls, of which two proteins, i.e. beta nerve growth factor (β-NGF) and Delta and Notch like epidermal growth factor-related receptor (DNER), were also present at lower levels in MSA compared to PD (both p = 0.032). Two biomarkers (MCP-1 and MMP-10) positively correlated with PD progression (rho > 0.650; p < 0.01). Conclusions: PEA technique identified potential new CSF biomarkers to help to predict the prognosis of PD. Also, we identified new candidate biomarkers to distinguish MSA from PD.

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Inflammation biomarker discovery in Parkinson's disease and atypical parkinsonisms

10.21203/rs.2.14452/v1 ◽

2019 ◽

Author(s):

Anna Santaella ◽

H. Bea Kuiperij ◽

Anouke van Rumund ◽

Rianne A.J. Esselink ◽

Alain J. van Gool ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Growth Factor ◽

Biomarker Discovery ◽

Response To Treatment ◽

Csf Biomarkers ◽

Diagnostic Biomarkers ◽

Epidermal Growth ◽

Atypical Parkinsonisms ◽

Proximity Extension Assay

Abstract Background : Parkinson’s disease (PD) and atypical parkinsonisms (APD) have overlapping symptoms challenging an early diagnosis. Diagnostic accuracy is important because PD and APD have different prognosis and response to treatment. We aimed to identify inflammatory biomarkers in cerebrospinal fluid (CSF) using the multiplex proximity extension assay (PEA) technology to identify diagnostic biomarkers of PD and APD and to study possible correlations of biomarkers with disease progression. Methods : CSF from a longitudinal cohort study consisting of PD and APD patients (PD, n = 46; multiple system atrophy (MSA) , n = 15; vascular parkinsonism (VaP) , n = 9; and PD with VaP, n = 7) and controls (n = 25) were analyzed. Results : Concentrations of CCL28 were elevated in PD compared to controls (p < 0.05). Five other biomarkers differentiated both MSA and PD from controls (p < 0.05) and 10 biomarkers differentiated MSA from controls, of which two proteins, i.e. beta nerve growth factor (β-NGF) and Delta and Notch like epidermal growth factor-related receptor (DNER) , were also present at lower levels in MSA compared to PD (both p = 0.032). Two biomarkers (MCP-1 and MMP-10) positively correlated with PD progression (rho > 0.600; p < 0.01). Conclusions : PEA technique identified potential new CSF biomarkers to help to predict the prognosis of PD. Also, we identified new candidate biomarkers to distinguish MSA from PD.

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