Pankaj Oberoi

Global standardization measurement of cerebral spinal fluid for Alzheimer's disease: An update from the Alzheimer's Association Global Biomarkers Consortium

Recognizing that international collaboration is critical for the acceleration of biomarker standardization efforts and the efficient development of improved diagnosis and therapy, the Alzheimers Association created the Global Biomarkers Standardization Consortium (GBSC) in 2010. The consortium brings together representatives of academic centers, industry, and the regulatory community with the common goal of developing internationally accepted common reference standards and reference methods for the assessment of cerebrospinal fluid (CSF) amyloid β42 (Aβ42) and tau biomarkers. Such standards are essential to ensure that analytical measurements are reproducible and consistent across multiple laboratories and across multiple kit manufacturers. Analytical harmonization for CSF Aβ42 and tau will help reduce confusion in the AD community regarding the absolute values associated with the clinical interpretation of CSF biomarker results and enable worldwide comparison of CSF biomarker results across AD clinical studies.

Abstract 3346: Development of a multiplex screening panel for Akt signaling pathway biomarkers in cell and tissue lysate models

Major challenges face the large-scale implementation of intracellular tumor biomarkers for clinical diagnosis and therapeutic development. These challenges include non-quantitative results, insufficient assay sensitivity, and lack of multiplexing capability. For many studies, sample volumes are limited (e.g., tumor lysates); however, it is essential to extract as much biomarker information as possible from a given sample while maintaining the quality and consistency necessary to sustain ongoing studies. To address these challenges, MSD® has developed multiplex panels for assaying cell signaling biomarkers using fit-for-purpose methods that emphasize optimal multiplex combinations and rigorous development of critical reagents. Here we report the development and verification of a multiplex screening panel of immunoassays for simultaneous measurement of total and phosphorylated analytes of the Akt signalling pathway using MSD technology. Markers in the panel include phosphorylated and total forms of GSK3B, p70S6K, FOXO3a, PTEN, Akt, S6RP, PRAS40, and ERK1. We demonstrate the ability of these assays to measure analyte levels in multiple tumor-derived cell lines and human tissue samples (normal and tumor) with excellent sensitivity and performance. Most analytes can be quantified using no more than 10 µg of sample. By using recombinant proteins to calibrate some of the assays, we were able to quantify analytes in cultured cell lysates, including MCF-7 and Jurkat T-cells. Lack of analyte specificity is a well-known issue when multiplexing intracellular signaling analytes, especially those in a common signaling pathway such as the Akt pathway. Through rigorous optimization, we achieved non-specific binding less than or equal to 1% for all analytes in this panel. Spike recovery and dilution linearity were characterized to demonstrate matrix tolerance in tumor cell line and tissue lysates as well as dynamic ranges between 3 and 4 logs, allowing quantification of analytes at different abundance levels without using multiple dilutions. In conclusion, MSD MULTI-SPOT® Akt signaling assay panels have been developed and verified for measurement of intracellular tumor biomarkers of relevance to clinical diagnosis, therapeutic development, and treatment of various cancers. The accuracy, reliability, ease of use, and high-throughput features of these multiplex assays make them ideally suited for use in large-scale clinical studies. Citation Format: Thomas W. Miller, Karen Tressler, Jill Dunty, Paula Eason, Leonid Dzantiev, Sripriya Ranganathan, Laura Schaefer, David Stewart, Pankaj Oberoi, Jacob Wohlstadter. Development of a multiplex screening panel for Akt signaling pathway biomarkers in cell and tissue lysate models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3346. doi:10.1158/1538-7445.AM2014-3346

Abstract 4731: Discovering cancer biomarkers using a novel cytokine screening panel.

Cytokines and chemokines that are produced in the tumor micro-environment play major roles in tumorigenesis, tumor progression, and apoptosis. They are involved in the modulation of angiogenesis and consequently, in tumor growth and metastases. Cytokine/chemokine expression by tumor cells leads to tumor proliferation and enhances tumor cell survival. Profiling these biomarkers may be useful for cancer diagnosis. For example, biomarkers such as IFNγ, TNFα, IL-1β, MCP-1, and IL-8 are overexpressed in breast carcinoma; IL-2, IL-4, IL-6, IL-8, IL-10, IL-16, and TARC are associated with prostate cancer; and increased expression levels of TNFα, IL-6, and IL-1β are implicated in colon cancer. We have developed and validated multiplexed panels of 30 assays that allow users to simultaneously detect cytokines and chemokines with high precision and accuracy in different human matrices. These panels detect IFNγ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNFα, IL-5, IL-7, GM-CSF, IL-1α, IL-17A, IL-15, IL-16, IL-12/IL-23 p40, TNFβ, and VEGF MIP-1α, MIP-1β, Eotaxin, Eotaxin-3, IP-10, TARC, IL-8 (high abundance), MDC, MCP-1, and MCP-4. The MSD ® platform has the ability to quantitate these biomarkers in multiple sample types with superior sensitivity and performance, quantifying many low abundance analytes at levels below 0.5 pg/mL. A challenge with current multiplex assays is a lack of analytical validation and lot-to-lot reproducibility. These new multiplex panels were developed with highly characterized and qualified reagents, and the assays were developed in accordance with industry guidelines. The panels demonstrated suitable spike recovery and dilution linearity performance in different biological matrices. Diluents, other kit components, and an optimized assay protocol are provided to achieve optimum performance. Each assay in the panel is well characterized and shows low non-specific binding. Each has been evaluated for potential assay and analytical interference. Controls are available for each panel. These panels are suitable tools for accurately quantitating cytokines and chemokines in various tumor samples. Data are presented for samples from normal subjects as well as samples from benign prostatic hyperplasia and prostate cancer patients (serum), cirrhosis and liver cancer patients (serum), kidney cancer patients (plasma), and colon cancer patients (serum). Citation Format: Joseph Manimala, Prachi Gupte, Ashok Swaminathan, Jorge P. Tituana, Caitlin Hansen, Anu Mathew, David Stewart, Pankaj Oberoi, Jacob Wohlstadter. Discovering cancer biomarkers using a novel cytokine screening panel. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4731. doi:10.1158/1538-7445.AM2013-4731

Abstract 5159: Quantitative immunoassays to measure total Akt-1 and phospho-Akt(Ser473) in cell and tissue lysate models.

Akt1, Akt2, and Akt3 kinases are critical effectors of the activated tyrosine receptor kinase/phosphoinositol 3-kinases, influencing cell growth, proliferation, and survival. Akt1-specific substrates have been implicated in cell cycle progression and cell motility, processes that underlie increased proliferation and metastatic spread of cancer cells. In this study, we report development and analytical validation of singleplex immunoassays for quantification of total Akt1 and phospho-Akt(Ser473) using fit-for-purpose and CLSI principles. We also demonstrate the ability of these assays to quantify total Akt1 and phospho-Akt(Ser473) in tumor-derived cell lines and mouse xenograft tumor lysates. These assays allow cancer researchers to correlate characteristics of cancer cells with absolute protein levels. The Akt1 singleplex assays were developed using MSD9s MULTI-ARRAY® technology. A monoclonal mouse antibody specific for total Akt1 was used to capture Akt1 in the solid phase, and MSD SULFO-TAGTM-labeled antibodies specific for an alternate epitope of total Akt1 or phospho-Akt(Ser473) were used as assay-specific reporters. Both assays were calibrated using full length recombinant human Akt1 protein expressed in baculovirus and phosphorylated in vitro by sequential incubation with phosphoinositide-dependent protein kinase-1 (PDK1) and MAP kinase-activated protein kinase 2 (MAPKAPK2). Akt calibrator concentrations were assigned following multi-day testing. Akt1 concentrations were confirmed by amino acid analyses, and units of phosphorylation (UP473) were assigned. The functional performance of the Akt1 calibrator was verified to be equivalent across three lots. Both assays demonstrated excellent sensitivity (lower limits of detection were 0.028 pg total Akt/well for the total Akt1 assay and 0.005 UP473/well for the phospho-Akt(Ser473) assay) and inter-plate reproducibility (coefficients of variation were below 12.1% for both assays). Calibration with recombinant protein enabled the absolute quantification of both total and phospho-Akt(Ser473) levels in cultured cell lysates, including MCF-7, Jurkat, NIH3T3, rat L6, and COS7, as well as epithelial, renal cell, and kidney carcinomas. Dilutional linearity and spike recovery testing demonstrated minimal matrix effects and accurate quantitation in these samples. (Recovery values in the range of 80-120% of the expected values were observed.) The assays showed no significant cross-reactivity with Akt2 or Akt3 or with the active forms phospho-Akt(Ser472) and phospho-Akt(Ser474). These assays can accurately quantify total Akt1 and phospho-Akt(Ser473) in multiple cell models. Citation Format: Allison Ellington, Seamus Webb, Thomas Miller, Paula Eason, Leonid Dzantiev, Sripriya Ranganathan, Laura Schaefer, Pankaj Oberoi, Jacob N. Wohlstadter. Quantitative immunoassays to measure total Akt-1 and phospho-Akt(Ser473) in cell and tissue lysate models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5159. doi:10.1158/1538-7445.AM2013-5159

International, multicenter training and validation of CSF Alzheimer's biomarker assays

pathogenesis. Despite accumulating evidence for the importance of Ab oligomers, currently there is no established method for allowing routine monitoring of Ab oligomers. We developed a platform technology called Multimer Detection System (MDS), which can detect oligomeric proteins in blood. In this study, we focused on examination of influence of anticoagulant type and sample preparationmethod for Ab oligomer detection in samples of patients with AD using MDS. Methods: Blood samples were collected in EDTA or heparin containing tubes and centrifuged at 850g for 30 min. In MDS, epitope-overlapping N-terminal Ab antibodies were used for capturing and detecting of Ab. Using this system, we tested Ab oligomers in samples from 20 patients with clinically characterized AD and 20 healthy normal.Results: For detection of Ab oligomers, different results came out depend on types of anticoagulant and changes in method of sample preparation. Conclusions:We confirmed influence of anticoagulant type and sample preparation method for Ab oligomers detection in AD sample. Especially, the difference in levels of Ab oligomers between AD and healthy normal was significantly increased by optimizing the sample preparation method.

Development of novel assay panels for the detection of Alzheimer's and Parksinson's disease biomarkers in human matrices

Background: Accurate and reproducible measurement of neurodegenerative disease biomarkers is required for biomarker validation and for the development of assays suitable for clinical studies. We describe the feasibility of a multiplexed immunoassay panel designed to measure both Alzheimer’s disease (AD) and Parkinson’s disease (PD) biomarkers, including Abeta 42, Abeta 40, Abeta 38, Tau, alpha-synuclein, and DJ-1, in clinically relevant humanmatrices.Methods:The assays were built usingMSD’sMULTI-ARRAY electrochemiluminescence technology. Antibody selection was based on sensitivity, specificity, analytical characteristics, and functional performance in human matrices. Protocol format was evaluated to achieve optimal signal and assay sensitivity. Matrix tolerance was assessed using dilution linearity and spike and recovery. Results: The multiplexed assays for AD and PD biomarkers demonstrated reproducible standard curves with quantitative ranges that support the accurate measurement of endogenous levels of each analyte. We present AD and PD biomarker levels measured in human cerebrospinal fluid samples from patients with AD and PD and from normal controls. Differences in biomarkers levels were observed between normal and disease populations for many of the assays developed. Conclusions: The development of multiplexed assays to measure several protein biomarkers associated with AD and PD is an important goal in the neurodegenerative biomarker field. Such assays would be particularly advantageous given that some biomarkers are relevant for multiple disease types; therefore, multiplexing allows measurement of biomarkers for multiple, related diseases while conserving sample. The panel described is anticipated to aid researchers in defining the relationship between disease progression and the relative abundance of specific neurodegenerative disease biomarkers.

The impact of assay format on sensitivity and matrix tolerance for plasma beta-amyloid peptide (Aβ-40 and Aβ-42) measurements

P1-323 THE IMPACT OFASSAY FORMAT ON SENSITIVITYAND MATRIX TOLERANCE FOR PLASMA BETA-AMYLOID PEPTIDE (Ab-40 AND Ab-42) MEASUREMENTS Allison Cicero, Pankaj Oberoi, Sid O’Bryant, Robert Barber, Jill Dunty, David Stewart, Robert Umek, Paula Eason, Meso Scale Diagnostics, Gaithersburg, Maryland, United States; 2 Meso Scale Discovery, Gaithersburg, Maryland, United States; 3 University of North Texas Health Science Center, Lubbock, Texas, United States; University of North Texas Health Science Center, Fort Worth, Texas, United States; Meso Scale Discovery, Gaithersburg, Maryland, United States.