- Determination of Antibody’s Specificity Towards Phosphorylated Protein Targets with Automated In-capillary Enzyme Treatment and Immunoassay
- Profiling Immune Cell Populations in the Tumor Microenvironment with Complementary Capillary-based and Single-cell Western Assays
- Novel Approach for Automated Sequential Immunoassay for Quantitation and Characterization of PI3K/AKT Pathway Proteins
- Multiplexed Protein and RNA Quantification on a Single Instrument Harmonizes Multi-omic Analyses of Biomarkers for Immunotherapies and Targeted Therapies in Non-Small Cell Lung Cancer
Determination of Antibody’s Specificity Towards Phosphorylated Protein Targets with Automated In-capillary Enzyme Treatment and Immunoassay
Presented by Dr. Daryl Taketa
Abstract: Protein phosphorylation is a reversible reaction that is integral in numerous signaling cascades. Characterization of signaling cascades has been largely detected by immunoblotting with phospho-specific antibodies, which may or may not have enough specificity or affinity. Currently, a separate lysate without any phosphatase inhibitors or a separate blot is needed to determine an antibody’s specificity. Here we describe a simple assay that leverages automation and quantitation with capillary electrophoresis-based immunoassay (CEIA) to assess the specificity of these antibodies with a single lysate preparation. In this study, three lysate models are used: K562 ± TNFα treatment, 50 ng/mL phorbol myristate acetate (PMA) differentiated THP-1 ± 1 μg/mL lipopolysaccharide (LPS) treatment, and cytotoxic T lymphocytes (CTL) ± 10 ng/mL PMA and 500 ng/mL ionomycin treatment. K562 cell lysates are commercially purchased whereas THP-1 lysates are generated in-house. For CTL cells, whole blood cells from a single donor are isolated and expanded with commercially available kits. Expanded CTL cells are then stimulated with PMA and ionomycin for 15 minutes. Untreated and treated lysate samples are separated and captured to the inner lumen of the capillary wall with UV activated crosslink chemistry. Cross-linked proteins are treated with lambda phosphatase for 1 hour followed by the immunoassay to investigate the specificity of antibodies against phosphorylated protein targets respective to each activated pathway using either chemiluminescent or fluorescent detection. Preliminary data suggest phospho-specific signal decreased >90% with no significant changes to the non-specific noise. The method described here eliminates the need for multiple lysate preparations or an additional blot to assess an antibody’s specificity to a phosphorylated protein target.
Profiling Immune Cell Populations in the Tumor Microenvironment with Complementary Capillary-based and Single-cell Western Assays
Presented by Dr. Charles Haitjema
Abstract: The tumor microenvironment (TME) is a complex mixture of cancerous and non-cancerous cells, including immune cells like T-cells, macrophages, and neutrophils. The TME plays a key role in tumorigenesis and metastasis, and it has recently been recognized that it can dramatically shape a response to therapy. Thus, there is a pressing need to accurately identify and quantify the variety of cell types in any given TME. However, studying the TME presents major challenges. For example, the heterogeneity of the environment requires sensitive and high-resolution techniques to parse subpopulations of different cell types. This challenge is compounded by the severely limited sample size that can be obtained from donor tissues. To address these challenges, we use a capillary immunoassay (Wes from ProteinSimple) with small sample sizes (3 µL) to identify immune cells commonly found in the TME. We also leverage single-cell western (Milo from ProteinSimple) to uncover trends in population heterogeneity. Human peripheral blood mononuclear cells (PBMCs) were differentiated into dendritic cells (DCs) and regulatory T cells (Tregs), and natural killer (NK) cells were expanded from isolated NK cells. These samples were then analyzed by Wes and Milo. These analyses revealed the identification and characterization of cell types, at both the single-cell and population level, based on the differential expression of protein biomarkers. Specifically, Wes analysis identified mature populations by a CD56+/CD3- phenotype for NK cells, a CD209+/CD14- phenotype for DCs, and a CD25+/FoxP3+ phenotype for Tregs. Milo analysis provided further detail within these populations, for example, we observed FoxP3low and FoxP3high subpopulations in Tregs, and an unexpectedly large (81%) CD56-/CD3- subpopulation in undifferentiated PBMCs, suggesting the presence of other cell subtypes. We anticipate that the small sample size, automation, single-cell resolution, and multiplexing ability of these assays collectively will enable a more efficient and deeper characterization of the TME not possible with traditional immunoassays like western blot and flow cytometry.
Novel Approach for Automated Sequential Immunoassay for Quantitation and Characterization of PI3K/AKT Pathway Proteins
Presented by Dr. Jessica Dermody
Abstract: The P13K/Akt signaling pathway modulates growth, survival and apoptosis and this pathway is frequently modulated in human cancers contributing to resistance to radiation and chemotherapy treatments. Akt is a target for specific inhibition and recently, a number of small molecules have been developed the pharmacologic properties of known inhibitors like wortmannin and LY294002. However, Pan-Akt inhibitors can result in unanticipated side effects due to the lack of specificity for Akt isoforms 1, 2 and 3. Therefore, detection and quantitation of Akt isoforms and their downstream targets for both expression levels and phosphorylation states is crucial for therapeutic drug development. Here we demonstrate application of Replex™ to characterize the P13K/Akt signaling pathway. This approach uses sequential analysis of proteins separated and immobilized in a capillary, by performing either dual immune assays or immunoassay with total protein on the Simple Western platform chemiluminescense detection. Assays with control and LY294002 inhibitor-treated samples were developed. Proteins were first separated based on molecular weight based on capillary electrophoresis, followed by immobilization via UV-crosslinking. Next P13K/Akt pathway targets were sequentially probed in the same capillary with total and phospho-specific antibodies to determine the phosphorylated fraction relative to the total fraction. Primary antibodies from the first immunoprobe was removed with the detection probe with >95% efficiency, as confirmed by re-probing with the same secondary antibody. Target protein loss was negligible due to covalent immobilization to the capillary wall, which was confirmed with re-probing, thus validating the quantitative data generated using this sequential approach. In addition, total protein normalization was performed in tandem with the immunoassay in the same capillary. This approach enables normalization of phosphorylation levels and/or target abundance in cell line or tissue samples, correcting for change in protein content due to treatment, loading and/or other systemic errors. These results present the utility of the RePlex™ to quickly characterize and quantify proteins involved in signaling pathways targeted during development of cancer therapies.
Multiplexed Protein and RNA Quantification on a Single Instrument Harmonizes Multi-omic Analyses of Biomarkers for Immunotherapies and Targeted Therapies in Non-Small Cell Lung Cancer
Presented by Dr. Chris Heger
Abstract: Multi-omic approaches can combine protein, DNA, and RNA analyses to elucidate diagnostic biomarkers and pathways, advancing our understanding of complex diseases. These assays, however, require different technologies and platforms to resolve the distinct physico-chemistries of protein and DNA/RNA. In contrast, single-platform quantification of proteins and nucleic acid markers offers many potential benefits, including reduced sample requirements, decreased inter-assay variability, streamlined and less error-prone workflows, and integrated results reporting. Here we demonstrate expanded capabilities of an established protein analysis system (Simple Western, ProteinSimple®) to characterize nucleic acids, and show that this system can quantify oncogenic tyrosine kinases, immune checkpoint proteins, RNA translocations, and other mRNA transcripts associated with targeted or immune-based therapies for non-small cell lung cancer (NSCLC).
- View webinar: "Single-Platform Technology for Protein and Nucleic Acid Biomarker Analysis in Non-Small Cell Lung Cancer"