This document discusses the analysis of cellular signaling using activation-state specific antibodies. It begins by describing different types of activation-state specific antibodies, such as phospho-specific, cleavage-specific, and motif-specific antibodies. It then discusses techniques for validating and applying these antibodies for imaging cytometry and high content analysis, including immunofluorescence, antibody titration, and conjugation to fluorescent dyes. Finally, it covers protocols and applications for using activation-state specific antibodies in flow cytometry, including clinical assays for diseases like chronic myelogenous leukemia and chronic lymphocytic leukemia.
This document discusses the use of activation-state specific antibodies for analyzing cellular signaling using imaging cytometry techniques like immunofluorescence and high content analysis. It provides examples of how these antibodies can be used to study phosphorylation events, apoptosis, and validate optimal antibody concentrations. The document also discusses the potential for using these antibodies in flow cytometry applications like clinical assays to study disease signaling pathways.
1) Ab-XTEN fusions enable genetic fusion of antibody fragments to XTEN polymers for easy, specific drug conjugation and tunable pharmacokinetics. This allows for high drug loads and monovalent or multivalent formats.
2) Ab-XTEN-Drug conjugates specifically link cytotoxic drugs to Ab-XTEN fusions via cleavable linkers, offering many drug options for tumor-targeted delivery.
3) Preliminary studies show Ab-XTEN fusions maintain antigen binding, resist aggregation, and facilitate increased tumor uptake compared to antibody fragments alone.
This study characterized a modified release multiparticulate tablet formulation consisting of acetaminophen-loaded beads and placebo beads. Acetaminophen beads containing 40-60% ethylcellulose and placebo beads containing 30% calcium silicate were developed using extrusion-spheronization. Tablets were compressed containing a 50:50 mixture of the drug and placebo beads. The drug beads released acetaminophen over 2 hours while the tablet formulation provided modified release up to 5 hours, though some coating damage occurred during compression.
This document discusses XTEN, a protein polymer that can be fused to human proteins or peptides to extend their half-life and allow for less frequent dosing. Key points:
- XTEN fusion enables once-monthly or longer dosing by providing a long pharmacokinetic profile with maximal efficacy and minimal toxicity.
- XTEN has been clinically validated in two products and shows non-immunogenicity, stability, and biodegradability advantages over PEGylation.
- Several XTEN-fused product candidates are in preclinical development for indications like short bowel syndrome, AAT deficiency, diabetes. Two products have completed Phase I trials.
XTEN conjugates provide rapid access to many high-value product formats including antibody-XTEN-drug conjugates, peptide-XTEN-drug conjugates, and multifunctional conjugates. XTEN can be customized based on polymer length, conjugation site type and number, and coupling chemistry. Conjugation to XTEN is highly efficient (>90%) and homogeneous based on HPLC and mass spectrometry analysis. XTEN conjugates demonstrate similar pharmacokinetics to non-conjugated versions in animal studies. XTEN is expected to be manufactured at commercial scale at lower cost than PEG, with advantages including biodegradability and a defined single molecular species.
This document discusses techniques for exploring protein-protein interactions within cells. It begins by defining the proteome and interactome. The proteome is the entire complement of proteins in a cell, while the interactome refers to the network of protein-protein interactions. The document then describes several techniques used to study the interactome, including affinity purification, immunoprecipitation, epitope tagging, and tandem affinity purification. These techniques allow researchers to purify a target protein along with any interacting protein partners in order to map out the social network of proteins within the cell.
XTEN is a protein polymer composed of natural amino acids that mimics the properties of PEG. It has a precisely controlled sequence encoded in DNA that can be produced at large scale through fermentation. XTEN is biodegradable, has no risk of kidney vacuolation, and produces no toxic metabolites. It has versatility in genetic fusions and chemical conjugations. XTEN meets goals of long serum half-life, stability in plasma, intracellular degradation, high expression level, genetic stability, and lack of non-specific binding or aggregation.
This document discusses the use of activation-state specific antibodies for analyzing cellular signaling using imaging cytometry techniques like immunofluorescence and high content analysis. It provides examples of how these antibodies can be used to study phosphorylation events, apoptosis, and validate optimal antibody concentrations. The document also discusses the potential for using these antibodies in flow cytometry applications like clinical assays to study disease signaling pathways.
1) Ab-XTEN fusions enable genetic fusion of antibody fragments to XTEN polymers for easy, specific drug conjugation and tunable pharmacokinetics. This allows for high drug loads and monovalent or multivalent formats.
2) Ab-XTEN-Drug conjugates specifically link cytotoxic drugs to Ab-XTEN fusions via cleavable linkers, offering many drug options for tumor-targeted delivery.
3) Preliminary studies show Ab-XTEN fusions maintain antigen binding, resist aggregation, and facilitate increased tumor uptake compared to antibody fragments alone.
This study characterized a modified release multiparticulate tablet formulation consisting of acetaminophen-loaded beads and placebo beads. Acetaminophen beads containing 40-60% ethylcellulose and placebo beads containing 30% calcium silicate were developed using extrusion-spheronization. Tablets were compressed containing a 50:50 mixture of the drug and placebo beads. The drug beads released acetaminophen over 2 hours while the tablet formulation provided modified release up to 5 hours, though some coating damage occurred during compression.
This document discusses XTEN, a protein polymer that can be fused to human proteins or peptides to extend their half-life and allow for less frequent dosing. Key points:
- XTEN fusion enables once-monthly or longer dosing by providing a long pharmacokinetic profile with maximal efficacy and minimal toxicity.
- XTEN has been clinically validated in two products and shows non-immunogenicity, stability, and biodegradability advantages over PEGylation.
- Several XTEN-fused product candidates are in preclinical development for indications like short bowel syndrome, AAT deficiency, diabetes. Two products have completed Phase I trials.
XTEN conjugates provide rapid access to many high-value product formats including antibody-XTEN-drug conjugates, peptide-XTEN-drug conjugates, and multifunctional conjugates. XTEN can be customized based on polymer length, conjugation site type and number, and coupling chemistry. Conjugation to XTEN is highly efficient (>90%) and homogeneous based on HPLC and mass spectrometry analysis. XTEN conjugates demonstrate similar pharmacokinetics to non-conjugated versions in animal studies. XTEN is expected to be manufactured at commercial scale at lower cost than PEG, with advantages including biodegradability and a defined single molecular species.
This document discusses techniques for exploring protein-protein interactions within cells. It begins by defining the proteome and interactome. The proteome is the entire complement of proteins in a cell, while the interactome refers to the network of protein-protein interactions. The document then describes several techniques used to study the interactome, including affinity purification, immunoprecipitation, epitope tagging, and tandem affinity purification. These techniques allow researchers to purify a target protein along with any interacting protein partners in order to map out the social network of proteins within the cell.
XTEN is a protein polymer composed of natural amino acids that mimics the properties of PEG. It has a precisely controlled sequence encoded in DNA that can be produced at large scale through fermentation. XTEN is biodegradable, has no risk of kidney vacuolation, and produces no toxic metabolites. It has versatility in genetic fusions and chemical conjugations. XTEN meets goals of long serum half-life, stability in plasma, intracellular degradation, high expression level, genetic stability, and lack of non-specific binding or aggregation.
This document discusses immunofluorescence, a technique used to detect antibodies in serum or body fluids. It involves using a primary antibody that binds to the target antigen, then a fluorescent secondary antibody that binds to the primary. This allows visualization under a microscope. Two types are described: direct uses a fluorescent primary antibody, indirect uses a non-fluorescent primary and fluorescent secondary for signal amplification. Applications include detecting autoantibodies associated with various diseases by looking for fluorescence patterns on tissue sections like Hep-2 cells or mouse organs. Indirect immunofluorescence is considered the standard technique as it has high sensitivity and specificity.
Presentation of Frank Hills in 1st International Antibody Validation Forum 2014St John's Laboratory Ltd
After graduating with an honours degree in Biochemistry Dr Hills worked for several years as a Clinical Scientist at St Bartholomew's hospital in London. He was awarded his PhD in 2002 from the faculty of Medicine at Queen Mary University of London. He continued his interest in reproductive science at Imperial College London where he worked as a postdoctoral researcher before joining Middlesex in 2004 as a lecturer. Dr Hills has published many high profile original research articles on various aspects of obstetric pathology including pre-eclampsia, recurrent miscarriage, preterm labour and fetal distress as well as several articles in the area of assisted reproduction. Currently, he is research interests include investigating the role of glycosaminoglycans and proteoglycans on the development of placental pathology and breast cancer. Dr Hills teaches a range of topics in biomedical science including clinical biochemistry, cellular and developmental biology as well as statistical analysis. He is author of around 30 peer-reviewed scientific articles and has refereed manuscripts for a variety of journals in the area of reproduction and endocrinology.
For more details about 1st international antibody validation forum please check on http://www.stjohnslabs.com/ac_cms/blog
The document provides an overview of the enzyme-linked immunosorbent assay (ELISA) technique. It describes three main types of ELISA - the sandwich ELISA, indirect ELISA, and competitive ELISA. For each type, it provides a brief example of clinical applications, such as detecting neural proteins in urine to diagnose Alzheimer's disease (sandwich ELISA) or detecting HIV antibodies in serum (indirect ELISA). It emphasizes that ELISA is a sensitive, cost-effective, and widely used assay for diagnosing infections and diseases.
Immunoassays are biochemical methods that use the specificity of antigen-antibody reactions to detect and quantify target molecules in biological samples. The document defines immunoassay and provides details on various types including enzyme immunoassays (ELISA, EMIT), radioimmunoassay (RIA), counting immunoassay (CIA), fluoroimmunoassay (FIA), and chemiluminescence immunoassay (CLIA). It describes the principles, components, procedures, and applications of each type of immunoassay for detecting molecules like antibodies, hormones, and proteins.
General principle of immunoassay Theoretical basis and optimization of immun...Ashish Gadage
Unlock the mysteries of immunoassays with this comprehensive PowerPoint presentation. Delve into the fundamental principles that underpin immunoassay techniques, exploring the theoretical foundations and key concepts. From antigen-antibody interactions to signal amplification strategies, this presentation provides valuable insights into the world of immunoassay science.
Key Topics:
Basics of Immunoassay: Antigen-Antibody Interactions
Types of Immunoassays: ELISA, Western Blot, and More
Signal Detection and Amplification Techniques
Factors Affecting Assay Sensitivity and Specificity
Optimization Strategies for Enhanced Performance
Emerging Trends in Immunoassay Technology
Who Should View:
Designed for scientists, researchers, and students in the fields of immunology, biochemistry, and medical diagnostics. Whether you're new to immunoassays or seeking advanced insights, this presentation caters to a broad audience.
Presenter: Mr. Gadage Ashish Rambhau
(M Pharm Pharmacology)
Pravara Rural Education Society pravaranagar,Loni .
Immunofluorescence is a technique that uses fluorescent-labeled antibodies to detect antigens in tissue sections. It has two main types: direct immunofluorescence uses antibodies directly labeled with fluorophores, while indirect immunofluorescence uses a secondary antibody labeled with a fluorophore to detect the unlabeled primary antibody. Indirect immunofluorescence provides signal amplification and allows detection of multiple antigens using the same labeled secondary antibody. It is considered the standard technique for detecting autoantibodies associated with various diseases. Hep-2 cells are commonly used as the substrate for detecting antinuclear antibodies due to advantages like high antigen expression and visibility of nuclear details. Different staining patterns on substrates can indicate specific autoantibodies present.
This document discusses principles of immunodetection and antigen-antibody interactions. It describes how antibodies recognize antigens through epitopes and bind through various forces. Techniques like ELISA, RIA, western blotting use this interaction to detect antigens. The generation of polyclonal and monoclonal antibodies is also summarized. The document outlines clinical applications in diagnostics and therapeutics using antibodies and discusses research problems requiring immunoanalysis techniques.
Since antigen and antibody reactions are specific, they can be used to identify each other.
These diagnostic tests are particularly useful in diagnosing for examples: infectious diseases, autoimmune diseases, and in typing of blood and tissues prior to transplantation.
An Over view on Bioassay, structure & principles, types & methods of bioassay. Also mention of other assay's like biotechnology, microbio assay, immunoassay etc.
Bioassay techniques involve estimating the concentration or potency of a substance using biological responses. There are three main types of bioassays: in vitro assays using cell cultures, in vivo assays using live animals, and ex vivo assays using isolated tissues. Bioassays work by comparing the biological effects of test substances to a reference standard under controlled conditions. The results are used to standardize drugs, diagnose conditions, and more. Common bioassay methods include graded response assays, end point assays, and multi-point assays using interpolation. Related techniques include ELISA assays, microbioassays, radioimmunoassays, and applications of biotechnology.
The document discusses the state of coagulation testing and summarizes key findings from surveys of clinical coagulation laboratories. It finds that (1) screening tests like APTT and PT have limitations in detecting low titer lupus anticoagulants, (2) there is significant variability between laboratories in factor assays due to differences in reagents and methods, and (3) further standardization could help improve the performance and interpretation of coagulation tests.
Antigen-Antibody Interactions, Immune Assays and Experimental SystemsMd Azizul Haque
1. The document discusses various techniques used in serology and immunology including antigen-antibody interactions, precipitation reactions, agglutination reactions, immunoassays like ELISA and western blot, and fluorescent labeling.
2. It explains key concepts like affinity, avidity, titer, direct and indirect Coombs tests.
3. The production of monoclonal antibodies is described involving fusing B cells from immunized animals with myeloma cells to form immortalized hybridomas that each secrete a single antibody specificity.
OriGene Technologies Capabilities Overview Feb 2011mwatson26
Opportunity overview with OriGene Technologies. OriGene is looking to identify strategic collaboration partners for its full-length human proteins, validated monoclonal antibodies and "gene centric" tool box.
The document describes the enzyme-linked immunosorbent assay (ELISA) technique. ELISA uses the coupling of antigens and antibodies and relies on their specificity and affinity. It can detect proteins, hormones, antibodies, bacteria, or viruses in a sample. The ELISA technique involves coating a microtiter plate with an antigen or antibody, adding the sample and secondary antibody linked to an enzyme, washing unbound components, and detecting the bound complex using a colorimetric or fluorescent substrate. There are different types of ELISA including indirect, sandwich, direct, competitive, and multiplex.
This document summarizes the key steps involved in translational research for systemic sclerosis (SSc), from target identification to preclinical testing. It discusses expression profiling of SSc patient samples to identify potential drug targets. Functional characterization of targets involves in vitro testing using patient cells and in vivo testing in mouse models. Promising targets then undergo therapeutic modification and efficacy/toxicity testing. The document provides examples of STAT3 and SOCS3 as potential targets, showing how their expression and role is characterized in SSc and mouse models. Epigenetic changes like DNA methylation are also explored, with DNMT3A and SOCS3 methylation implicated in the SSc fibrotic process.
This document discusses antigen-antibody reactions and their clinical applications. It describes the principles of primary, secondary, and tertiary antigen-antibody reactions. Various serological tests are discussed, including precipitation reactions, agglutination reactions, complement fixation tests, ELISA, immunofluorescence, and radioimmunoassay. These tests can help diagnose infections, identify infectious agents, and detect non-infectious substances. The document also provides examples of clinical applications for many common serological tests.
Bioassay techniques involve measuring the biological response of a test system to determine the potency or concentration of a physical, chemical, or biological substance. There are three main types of bioassay techniques: in vitro, in vivo, and ex vivo. Bioassays can be qualitative, to assess effects, or quantitative to estimate concentration/potency by measuring biological responses. Common bioassay methods include graded response assays, endpoint assays, and multi-point assays using interpolation. ELISA, microbioassays, and radioimmunoassays are also important specialized bioassay techniques.
This document discusses immunofluorescence, a technique used to detect antibodies in serum or body fluids. It involves using a primary antibody that binds to the target antigen, then a fluorescent secondary antibody that binds to the primary. This allows visualization under a microscope. Two types are described: direct uses a fluorescent primary antibody, indirect uses a non-fluorescent primary and fluorescent secondary for signal amplification. Applications include detecting autoantibodies associated with various diseases by looking for fluorescence patterns on tissue sections like Hep-2 cells or mouse organs. Indirect immunofluorescence is considered the standard technique as it has high sensitivity and specificity.
Presentation of Frank Hills in 1st International Antibody Validation Forum 2014St John's Laboratory Ltd
After graduating with an honours degree in Biochemistry Dr Hills worked for several years as a Clinical Scientist at St Bartholomew's hospital in London. He was awarded his PhD in 2002 from the faculty of Medicine at Queen Mary University of London. He continued his interest in reproductive science at Imperial College London where he worked as a postdoctoral researcher before joining Middlesex in 2004 as a lecturer. Dr Hills has published many high profile original research articles on various aspects of obstetric pathology including pre-eclampsia, recurrent miscarriage, preterm labour and fetal distress as well as several articles in the area of assisted reproduction. Currently, he is research interests include investigating the role of glycosaminoglycans and proteoglycans on the development of placental pathology and breast cancer. Dr Hills teaches a range of topics in biomedical science including clinical biochemistry, cellular and developmental biology as well as statistical analysis. He is author of around 30 peer-reviewed scientific articles and has refereed manuscripts for a variety of journals in the area of reproduction and endocrinology.
For more details about 1st international antibody validation forum please check on http://www.stjohnslabs.com/ac_cms/blog
The document provides an overview of the enzyme-linked immunosorbent assay (ELISA) technique. It describes three main types of ELISA - the sandwich ELISA, indirect ELISA, and competitive ELISA. For each type, it provides a brief example of clinical applications, such as detecting neural proteins in urine to diagnose Alzheimer's disease (sandwich ELISA) or detecting HIV antibodies in serum (indirect ELISA). It emphasizes that ELISA is a sensitive, cost-effective, and widely used assay for diagnosing infections and diseases.
Immunoassays are biochemical methods that use the specificity of antigen-antibody reactions to detect and quantify target molecules in biological samples. The document defines immunoassay and provides details on various types including enzyme immunoassays (ELISA, EMIT), radioimmunoassay (RIA), counting immunoassay (CIA), fluoroimmunoassay (FIA), and chemiluminescence immunoassay (CLIA). It describes the principles, components, procedures, and applications of each type of immunoassay for detecting molecules like antibodies, hormones, and proteins.
General principle of immunoassay Theoretical basis and optimization of immun...Ashish Gadage
Unlock the mysteries of immunoassays with this comprehensive PowerPoint presentation. Delve into the fundamental principles that underpin immunoassay techniques, exploring the theoretical foundations and key concepts. From antigen-antibody interactions to signal amplification strategies, this presentation provides valuable insights into the world of immunoassay science.
Key Topics:
Basics of Immunoassay: Antigen-Antibody Interactions
Types of Immunoassays: ELISA, Western Blot, and More
Signal Detection and Amplification Techniques
Factors Affecting Assay Sensitivity and Specificity
Optimization Strategies for Enhanced Performance
Emerging Trends in Immunoassay Technology
Who Should View:
Designed for scientists, researchers, and students in the fields of immunology, biochemistry, and medical diagnostics. Whether you're new to immunoassays or seeking advanced insights, this presentation caters to a broad audience.
Presenter: Mr. Gadage Ashish Rambhau
(M Pharm Pharmacology)
Pravara Rural Education Society pravaranagar,Loni .
Immunofluorescence is a technique that uses fluorescent-labeled antibodies to detect antigens in tissue sections. It has two main types: direct immunofluorescence uses antibodies directly labeled with fluorophores, while indirect immunofluorescence uses a secondary antibody labeled with a fluorophore to detect the unlabeled primary antibody. Indirect immunofluorescence provides signal amplification and allows detection of multiple antigens using the same labeled secondary antibody. It is considered the standard technique for detecting autoantibodies associated with various diseases. Hep-2 cells are commonly used as the substrate for detecting antinuclear antibodies due to advantages like high antigen expression and visibility of nuclear details. Different staining patterns on substrates can indicate specific autoantibodies present.
This document discusses principles of immunodetection and antigen-antibody interactions. It describes how antibodies recognize antigens through epitopes and bind through various forces. Techniques like ELISA, RIA, western blotting use this interaction to detect antigens. The generation of polyclonal and monoclonal antibodies is also summarized. The document outlines clinical applications in diagnostics and therapeutics using antibodies and discusses research problems requiring immunoanalysis techniques.
Since antigen and antibody reactions are specific, they can be used to identify each other.
These diagnostic tests are particularly useful in diagnosing for examples: infectious diseases, autoimmune diseases, and in typing of blood and tissues prior to transplantation.
An Over view on Bioassay, structure & principles, types & methods of bioassay. Also mention of other assay's like biotechnology, microbio assay, immunoassay etc.
Bioassay techniques involve estimating the concentration or potency of a substance using biological responses. There are three main types of bioassays: in vitro assays using cell cultures, in vivo assays using live animals, and ex vivo assays using isolated tissues. Bioassays work by comparing the biological effects of test substances to a reference standard under controlled conditions. The results are used to standardize drugs, diagnose conditions, and more. Common bioassay methods include graded response assays, end point assays, and multi-point assays using interpolation. Related techniques include ELISA assays, microbioassays, radioimmunoassays, and applications of biotechnology.
The document discusses the state of coagulation testing and summarizes key findings from surveys of clinical coagulation laboratories. It finds that (1) screening tests like APTT and PT have limitations in detecting low titer lupus anticoagulants, (2) there is significant variability between laboratories in factor assays due to differences in reagents and methods, and (3) further standardization could help improve the performance and interpretation of coagulation tests.
Antigen-Antibody Interactions, Immune Assays and Experimental SystemsMd Azizul Haque
1. The document discusses various techniques used in serology and immunology including antigen-antibody interactions, precipitation reactions, agglutination reactions, immunoassays like ELISA and western blot, and fluorescent labeling.
2. It explains key concepts like affinity, avidity, titer, direct and indirect Coombs tests.
3. The production of monoclonal antibodies is described involving fusing B cells from immunized animals with myeloma cells to form immortalized hybridomas that each secrete a single antibody specificity.
OriGene Technologies Capabilities Overview Feb 2011mwatson26
Opportunity overview with OriGene Technologies. OriGene is looking to identify strategic collaboration partners for its full-length human proteins, validated monoclonal antibodies and "gene centric" tool box.
The document describes the enzyme-linked immunosorbent assay (ELISA) technique. ELISA uses the coupling of antigens and antibodies and relies on their specificity and affinity. It can detect proteins, hormones, antibodies, bacteria, or viruses in a sample. The ELISA technique involves coating a microtiter plate with an antigen or antibody, adding the sample and secondary antibody linked to an enzyme, washing unbound components, and detecting the bound complex using a colorimetric or fluorescent substrate. There are different types of ELISA including indirect, sandwich, direct, competitive, and multiplex.
This document summarizes the key steps involved in translational research for systemic sclerosis (SSc), from target identification to preclinical testing. It discusses expression profiling of SSc patient samples to identify potential drug targets. Functional characterization of targets involves in vitro testing using patient cells and in vivo testing in mouse models. Promising targets then undergo therapeutic modification and efficacy/toxicity testing. The document provides examples of STAT3 and SOCS3 as potential targets, showing how their expression and role is characterized in SSc and mouse models. Epigenetic changes like DNA methylation are also explored, with DNMT3A and SOCS3 methylation implicated in the SSc fibrotic process.
This document discusses antigen-antibody reactions and their clinical applications. It describes the principles of primary, secondary, and tertiary antigen-antibody reactions. Various serological tests are discussed, including precipitation reactions, agglutination reactions, complement fixation tests, ELISA, immunofluorescence, and radioimmunoassay. These tests can help diagnose infections, identify infectious agents, and detect non-infectious substances. The document also provides examples of clinical applications for many common serological tests.
Bioassay techniques involve measuring the biological response of a test system to determine the potency or concentration of a physical, chemical, or biological substance. There are three main types of bioassay techniques: in vitro, in vivo, and ex vivo. Bioassays can be qualitative, to assess effects, or quantitative to estimate concentration/potency by measuring biological responses. Common bioassay methods include graded response assays, endpoint assays, and multi-point assays using interpolation. ELISA, microbioassays, and radioimmunoassays are also important specialized bioassay techniques.
This document describes a doctoral thesis that investigates the regulation of apoptosis induced by inhibition of phosphatidylcholine synthesis. The thesis was presented by Michiel Henrik Marie van der Sanden to obtain a doctoral degree from Utrecht University. It includes chapters that examine how inhibition of phosphatidylcholine synthesis affects cellular morphology, lipid composition, and generation of lipid droplets. It also analyzes the induction of endoplasmic reticulum stress and apoptosis-related proteins, such as C/EBP-Homologous Protein, during inhibition of phosphatidylcholine synthesis.
This certificate certifies that Michiel van der Sanden of Eurofins Medinet successfully passed a web-based examination on the International Conference on Harmonisation - Good Clinical Practice Guideline Course. The certificate is endorsed by the Course Director and Professor and Head of the School of Biological Sciences at Royal Holloway, University of London.
Technical support training cell signalingmichielvds
The document discusses technical training on signal transduction and CST products. It describes kinases and their role in phosphorylating effectors, and the use of phosphospecific antibodies, siRNA kits, and kinases/kinase assays. Application of phosphospecific and total antibodies are summarized for techniques like western blotting, immunohistochemistry, immunofluorescence, flow cytometry, immunoprecipitation, in cell western, and ELISA. Protocols are provided for some of these techniques.
This document provides an overview of Eurofins Medinet, a central laboratory services provider. It details Eurofins' global network of laboratories and key services, including central laboratory services, bioanalysis, biomarkers, and specialized testing. Eurofins offers a full range of analytical testing services to support pharmaceutical and biotech drug development.
The document provides advice for career options after completing a PhD, including positions in academia such as postdoctoral research, or industry roles in pharmaceutical/biotech companies, government institutes, hospitals, medical writing, sales, and life science companies. It then outlines the author's specific career path, beginning with a postdoc position before becoming a product specialist and later product manager at a life science company, highlighting the rewarding and developmental aspects of those roles.
6. Total Antibodies
Untreated Wnt 5’ Whole Blood
HeLa Cells
CD5
β-Catenin (C-term)
CD13
LY294002 Insulin
Side Scatter
C2C12 Cells
Akt (pan) (11E7)
CD4
7. Phospho-specific Antibodies
Untreated EGF-Treated
Phospho-EGFR (PE)
EGF Receptor (FITC)
Green = Phospho-EGF Receptor (Tyr1068)
Blue = DRAQ5
8. Caspase-3 Cleavage in Apoptosis
Untreated Staurosporine
TUNEL
Cleaved-Caspase 3
Green = Cleaved Caspase-3 (Asp175) (5A1) RmAb
Red = F-Actin (Phalloidin)
Blue = DRAQ5
9. Antibody Validation & Applications
CST’s activation-specific antibodies are purified to distinguish
between only one or two posttranslationally modified amino acid
residues
Most antibodies raised against such modified sites have a lower
affinity when compared with antibodies recognizing whole
proteins
Thus, all product development follows rigorous in-house testing on
a wide range of assay applications by our clinical applications
specialists
Stringent validation requirements
Protocol optimization
Cross platform functionality
10. Outline
What are activation state-specific antibodies?
Imaging Cytometry
• immunofluorescence & antibody validation
• antibody conjugation
• high content analysis (HCA)
Flow Cytometry
• protocols
• clinical assays
11. Antibody Validation for Immunofluorescence/HCA
Part I - Titration to determine Part II - Specificity Testing
optimal dilution/concentration • treat cells with specific
ligands, drugs, inhibitors, etc.
• test antibody on cells that do
and do not express target
• verify expression or treatment
efficacy with another antibody
(same target, or different
target in same pathway)
Confocal Imaging on Chamber Slides
Titration Curve
45000.00 6.00
40000.00
5.00
35000.00
30000.00 4.00
2966
Mean Channel Fluorescence
Signal to Noise Ratio
25000.00
3.00 Control
20000.00
S/N Ratio
15000.00 2.00
10000.00
1.00
5000.00
0.00 0.00
0 5 10 15 20 25 30 35 40 45
Antibody Dilution (ug/ml)
12. Antibody Validation for Immunofluorescence/HCA
[Ab]
Antibody
Isotype
Antibody
1:25
1:50
1:100
1:200
1:400
1:800
16000.00 4.50
14000.00 4.00
12000.00 3.50
3.00
10000.00
Isotype
4694
Mean Channel Fluorescence
2.50
Signal to Noise Ratio
8000.00 Control
2.00
6000.00 S/N Ratio
1.50
4000.00
1.00
2000.00 0.50
0.00 0.00
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
Antibody Dilution (ug/ml)
MEK1/2 (red)
actin (green)
DNA (blue)
HeLa cells
14. GPCR Activation
MEK PI3K
inhibitor inhibitor
LPA treatment 0 min 2 min 5 min 15 min 15 min 15 min
merge
phospho-Erk
phospho-Akt
DNA
C6 cells
15. Neuroscience - Development
Postnatal Day 1 Postnatal Day 14
Postnatal Rat Brain
p-Histone H3
Red=GFAP
Blue=Doublecortin
P1 P4 P14
16. Cell Cycle / Checkpoint
Untreated Untreated
UV Normal Colon Dexamethasone Colon Carcinoma
Metaphase
UV UV+PPT
Anaphase
#9309 Rb (4H1)
#3068 p-Aurora #2558 p-Kip2 (T310) p-Rb (S807/811) p-p53 (Ser37) p-Rad17 (S645)
#9309 Rb (4H1)
#9719 p-H2A.X (S139) Aurora A/AIK
A/B/C #9308
#4718 #9289 #3421
P Chk1/2 p53 Aurora
Cdc25A Kip1 P
cdc25B P
ATM
P
CDK4/6 CDK2 cdc2
CyclinD CyclinE CyclinB
G1 Phase R S Phase G2 Phase M Phase
P
P P Rad17
Abl
HDAC P
Rb
P X P P
Rb cdc2 H2A H3
E2F1 E2F1
OFF DP1 ON
DP1 DNA Damage
17. CST Conjugated Antibodies
Conjugated antibodies helpful for multiplex analyses
CST conjugates are optimized for cytometric applications
• high-quality pre-validated antibodies
• bright photostable Alexa dyes
• F/P trials to ensure bright signal
• antibody titration
• stability tested (accelerated and real-time)
• screened with flow cytometry, HCA, and IF
• lot-to-lot stability
18. Survivin (Alexa488-conjugate)
FAS, TNFa
• inhibits apoptosis and regulates mitosis
• over-expressed in most human cancers
FADD
• expression correlates with both accelerated Caspase-8/10
ER Stress Mitochondria
relapse and chemotherapy resistance
[Ca++] Smac/
P1 Rat Brain Diablo Cyto C
Caspase-12 Survivin
Caspase-9
Caspase-3
Caspase-6 Caspase-7
Lamin A a-Fodrin DFF PARP
CST’s conjugated Survivin antibody is currently being used to screen patient samples
19. The Bigger Picture
Single well immunofluorescence
+ ability to examine subcellular (co)localization in 4-dimension
(XYZt)
- difficult to quantify without specialized software
- imaging is time consuming and data files become massive
High Content Analysis
+ some systems able to analyze localization
+ rapid scanning (comparatively), sensitive, and quantifiable
+ ability to multiplex and dissect various pathways in tandem
20. High Content Analysis
• automated plate-based image analysis
• quantifiable signal intensity and subcellular
1:800
1:100
1:400
1:200
1:50
1:25
localization Untreated
• more predictive of drug activity in a cellular PDGF
p-Erk
environment compared to ELISAs U0126+PDGF
LY/Wort+PDGF
• can be used to determine: Untreated
efficacy and therapeutic dose
PDGF
p-Akt
U0126+PDGF
cell-permeability of drugs LY/Wort+PDGF
potential toxicity (DNA damage, apoptosis,
micronuclei)
downstream effects of drug/target interaction
off-target effects
21. Value of CST Antibodies in HCS
Current platforms have increased colors and resolution in an attempt to
quantify complex events
Example: nuclear translocation of Erk or NFkB
Requirements
• nuclear marker
• total antibody
• high resolution optics
• complex software
• lots of data storage
Using a phospho antibody will eliminate these costly requirements and
speed up the screen (on/off as opposed to determination of localization)
KEY: reliable simple affordable assay with clear robust results
22. Multiple, Parallel Analyses by Cellular Imaging (ICW)
Gleevec/PDGF
U0126/PDGF
LY294002
Untreated
Gleevec
PDGF
PDGF
PMA
blank
p-PDGFR
p-Erk
p-Akt
RTK signaling analysis using LI-COR Odyssey
Untreated Anisomycin UV
p-p38
p-Jnk
p-ATF2
p-H2A.X
DNA damage profiling analysis using LI-COR Odyssey
23. Future Plans: Broad Signal Profiling by HCA
• large antibody panel for profiling screens
• can be used on any cytometric platform
• detection = fluorescent, chromogenic, chemiluminescence
Starved
• up to 96 antibodies per plate
total and phosphorylation-specific antibodies
MAPK, Akt, NFkB, Jak/Stat, etc.
receptor tyrosine kinases (EGFR, VEGFR, FGFR, IGF-IR, cKit)
adaptor proteins and downstream targets EGF
transcription factors
motif antibodies (general serine or tyrosine phosphorylation)
• can be customized for analysis of different biological processes
toxicity
cell cycle/arrest Anisomycin
apoptosis
cell adhesion
96 prediluted cells treated with
CST antibodies compound X
UV
24. Antibody Development: FoxO1 Screening
PI3K inhibitor IGF-1 + serum
(Akt off) (AKT on)
How can we generate antibodies that
work well for cytometric applications?
Screen using cytometric applications
HTS & HCS Platforms
Nuclear Cytoplasmic
nuclear trigger
(half-width intensity)
25. Outline
What are activation state-specific antibodies?
Imaging Cytometry
• immunofluorescence & antibody validation
• antibody conjugation
• high content analysis (HCA)
Flow Cytometry
• protocols
• clinical assays
26. Optimization: Fixation/Permeabilization
CST 2-4% formaldehyde/90% methanol
Fix&Perm Kits 0.25-4% formaldehyde/detergent (triton or saponin)
Krutzik and Nolan (2004) Cytometry 55A:61-70.
28. Surface and Signaling Markers
• Methanol diminishes or abolishes signal from some key surface markers
• Many signaling event are very transient so immediate fixation is critical, no
time to prelabel with surface markers, lyse RBCs, or perform FiColl
separations
• Staggered protocol: fix cells with aldehyde to stop all enzymes and preserve
phosphoepitopes, label with surface markers, permeabilize with methanol,
and then label with intracellular signaling antibodies (destroys some
fluorochromes)
• Need a better solution…
30. Protocol Comparison
4%PFA at RT for 10m
3% PFA at 37ºC for 10m 0.1% Triton X-100 at RT for 30m
90% ice cold MeOH at -20ºC for 10m 50% ice cold MeOH at 4ºC for 10m
CD19 (FITC) CD19 (FITC)
31. Flow Cytometry: Clinical Applications
• Staining of intracellular signaling molecules can be easily
incorporated with traditional cell surface marker labeling
4% Formaldehyde for 10m
0.1 Trinton X-100 ft RT for 30m
50% ice cold MeOH at 4oC for 10m
• Important implications in the Clinic
• Flow Cytometry and Disease-specific Signaling
• Chronic myelogenous leukemia (CML)
• Chronic lymphocytic leukemia (CLL)
• Acute myelogenous leukemia (AML)
33. Large Scale Bcr/Abl Pathway Profiling
K562 cells +/- Gleevec
bar height represents amount of Gleevec inhibition
34. Biomarkers for CLL
*
*
Staudt’s lab first identified Zap-70 as a potential biomarker of the aggressive form of CLL
CLL patients with Zap-70 (+) B-cells have poorer prognosis
Crespo et al. (2003) N Engl J Med 348:1746-75
35. CLL Assay using Zap-70 (136F12) RmAb
In House: In the Clinic:
B cells (Ramos)
T cells (Jurkat)
Primary CLL Cells: Source: Esoterix Center for Innovation
Difficulties persist in Zap-70 analysis
- Zap-70 levels decline significantly after blood is drawn
- Inconclusive results with weak expression
Zap-70 (low) Zap-70 (high) - Additional biomarkers to make assay more reliable?
(slow growing) (fast growing)
37. Flt3 Signaling
Flt3 • Mutation or overexpression of Flt3 kinase
is observed in AML, (~70%), B-ALL, T-
ALL, and some blast phase CML
• Most common mutations are internal
tandem duplications (ITD) and active loop
mutations (AL), both of which result in
constitutive activation
• Phospho-specific antibodies can be used
to profile downstream signaling and to
monitor the efficacy of specific Flt3
inhibitors
Scheijen and Griffin (2002) Oncogene 21:3314-33
38. Flt3 Inhibitor Pharmacodynamic Assay Design
Phospho-Flt3
Flt3 ITD Overexpressed Flt3 Alexa488 Conjugate
(MOLM-14(MOLM14)
Dose Response Cells) (SEM Cells)
Dose Response (SEM) (SEM cells)
1.4 1.4
Normalized mean fluorescence
Normalized mean fluorescence
1.2 1.2
1 1 p-S6
p-S6
p-Flt3 p-Flt3
0.8 0.8
p-Stat5 p-Stat5
0.6 p-Stat3 p-Stat3
0.6
PY p-Erk
0.4 p-AKT PY
0.4
p-AKT uninhibited Flt3 inhibitor
0.2
0.2
0
0
0 0.01 0.1 0.5 2
0 0.01 0.1 0.5 2
Concentration of Flt3 inhibitor (µM) Concentration of Flt3 inhibitor (µM)
Conclusion:
Phospho-S6 is a key indicator
of Flt3 inhibition, regardless of
mutation
This antibody is a reliable
Flt3 inhibitor decreases
downstream signaling,
Percentage of Cleaved Caspase-3 positive cells
robust biomarker for receptor
causes cell cycle
80
70 tyrosine kinase (RTK) activity
60
arrest, and induces
Percent (%)
50
apoptotic cell death 40
30
20
10
0
0 2 3 5 22
Time (hours)
39. Conclusion
• Activation state-specific antibodies are very useful in multiplex
_phosphoproteomics analyses on various fluorescence based platforms
• stringent validation assures highest quality antibody and lot-to-lot
reproducibility
• determine prognostic significance of signaling anomalies
• monitor patients for resistance or recurrence
• study effectiveness and optimal dosage of specific kinase inhibitors
• allow tumor “typing” to determine best treatment option
• improve clinical trial and basic research design (outcome and economics)
Gregory Innocenti
Clinical Applications/Image Cytometry
ginnocenti@cellsignal.com
978-867-2475
Mutations in EGFR have been identified in patients with nsclc. The L858R gain-of-function mutation is associated with a therapeutic response to the EGFR inhibitor Iressa. A secondary T790 mutation results in resistance to Iressa inhibition.