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Multiplex Detection of Functional Tyrosine Phosphorylated

Multiplex Detection of Functional Tyrosine Phosphorylated Signaling Proteins by a Microbead Assay Imran H. Khan, Sara Mendoza, *Hsing-Jien Kung, and Paul A. Luciw Center for Comparative Medicine, *Cancer Center, University of California, Davis. Salient Features of Multiplexing by Luminex.

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Multiplex Detection of Functional Tyrosine Phosphorylated

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  1. Multiplex Detection of FunctionalTyrosine Phosphorylated Signaling Proteins by a Microbead Assay Imran H. Khan, Sara Mendoza, *Hsing-Jien Kung, and Paul A. Luciw Center for Comparative Medicine, *Cancer Center, University of California, Davis

  2. Salient Features of Multiplexing by Luminex • Multi-analyte detection in each sample (up to 100 targets) • Small sample size required (30 l) • Large number of replicates per sample (>100) • Sensitivity at least as good as ELISA • Dynamic range of up to four logs • Short assay time of 30 to 90 minutes • Easily amenable to high-throughput analysis (robotics)

  3. Technology Technology Hardware Software Assay Development

  4. 2 Color Suspension Array

  5. IL-2 IL-4 TNFa GmCSF Cytokine Capture/Sandwich

  6. 1 0 0 0 0 1 0 0 0 1 0 0 Mean Fluorescence Intensity G M - C S F I L - 2 1 0 I L - 4 T N F - a 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 C y t o k i n e C o n c e n t r a t i o n ( p g / m l )

  7. Near-TermGoals for Multiplex Detection of Phosphotyrosine Proteins in Signaling Pathways • Multiplex detection of tyrosine phosphorylated signaling proteins in TCR pathway in T-cell lines and peripheral blood mononuclear cells (PBMC): 1. Non-specific T-cell activation by sodium pervanadate 2. TCR specific activation by anti-CD3 antibody • Multiplex detection of tyrosine phosphorylated signaling proteins in EGFR pathway in cancer cell lines (e.g. A431 cervical, LnCap prostate cell lines)

  8. Immediate Focus • Phospho-tyrosine signaling proteins in T-Cell Receptor Pathway: 1. Initiated with Lck, Zap70 and LAT 2. Other components currently under development are PI3K, PLC; SLP76; Fyn; Lyn; Vav; Wasp etc. • Phospho-tyrosine signaling proteins in EGF Receptor Pathway: • initiated with EGF receptor

  9. K2 K2 K2 K2 Multiplexed Detection of Tyrosine Phosphorylated Proteins -kinase 3 -kinase 2 -kinase 1 Cell lysate Biotin BSA K1 K3 K3 Y Y Y Y Y Y K1 Phosphotyrosine (PY) Biotinylated -PY K1 K3 K1 K3 Y Y Y Y Y Y Phycoerythrin-labeled streptavidin Bead set PE signal Kinase 1 + Kinase 2 - Kinase 3 + BSA - B iotin+ Analyse mixture for bead identification and PE signal

  10. Immunoprecipitation/W. Blotting of Tyrosine Phosphorylated Proteins From Sodium Pervanadate Treated Jurkat T-Cells LAT IP-Antibody Sodium Pervanadate Lck Zap70 + - + - + - Zap70 Lck LAT

  11. Multiplex Analysis of Tyrosine Phosphorylated Proteins in Jurkat T-Cell Line Treated with Sodium Pervanadate 3000 PBS 2500 PBS + 4G10 2000 Jurkat-untreated Luminex Units 1500 Jurkat-sod. pervanadate 1000 500 0 BSA Lck Zap70 LAT Antibodies Coated on Luminex Beads

  12. T Cell Receptor Signaling Precedes Immunological Synapse Formation Lee et al., (2002) Science 295: 1539-1542 TCR LFA-1 Overlay DIC 2 min 15 min 30 min 60 min 15 min 30 min TCR LFA-1 Overlay

  13. Immunoprecipitation/W. Blotting of Tyrosine Phosphorylated Proteins From Anti-CD3 Activated Jurkat T-Cells LAT Zap70 IP-Antibody Sod. Pervan. Anti-CD3 Anti-CD3 Untreated Untreated Treatment Zap70 LAT

  14. Multiplex Analysis of Tyrosine Phosphorylated Proteins in Jurkat T-Cell Line Activated with anti-CD3 Antibody 1200 1000 BSA Lck 800 Zap70 LAT 600 Luminex Units 400 200 0 0 60 120 180 240 300 360 420 480 540 600 Time Post-Treatment (Sec)

  15. Dynamics of Activated Zap70 in Immunological Synapse Lee et al., (2002) Science 295: 1539-1542 2 min 5 min 15 min 30 min 60 min TCR Zap70 pY319 Zap70 DIC

  16. Dynamics of Activated Lck in Immunological Synapse Lee et al., (2002) Science 295: 1539-1542 2 min 5 min 15 min 30 min 60 min TCR Lck pY394 Lck DIC

  17. Dynamics of Total Phosphotyrosine in Immunological Synapse Lee et al., (2002) Science 295: 1539-1542 2 min 5 min 15 min 30 min 60 min TCR pY DIC

  18. Immunoprecipitation/Western Blotting of Tyrosine Phosphorylated Proteins from EGF Activated A431 Cervical Cancer Cell Line EGF - + 206 kDa EGFR 119 kDa 91 kDa 51 kDa ELISA AND LUMINEX RESULTS A431 Cells ELISA MULTIPLEX Untreated 0 .39 180(EGFR coated beads 100ug/ml) EGF(100ng/ml) 2.670 878(EGFR coated beads100ug/ml) BSA ___ 14 (BSA couted beads)

  19. Other Applications of Luminex Multiplex System • Detection and titration of antibodies to mouse infectious agents (MHV, Reo, GD7, Sendai, Ectro, MPV, MVM, Helicobacter, etc.) (Khan, Luciw, Kendall, Griffey) • Detection and titration of antibodies to rhesus monkey infectious agents (Herpes B virus, SRV, SIV, STLV, SFV, RRV, rhCMV) (Khan, Luciw, Lerche) • Antibody repsonses in vaccinated animals (Khan, Lerche, Barry) • Tyrosine kinase activity profiling in cancer cells and tissues (Luciw, Khan, Kung) • Cytokine profiling: quantitation of up to 15 cytokines has been published (Bassiri,Greenhalgh, Khan)

  20. Multiplex Viral (MHV, EDIM, Sendai, Ectromelia, Reo3) Antibody Detection in Mouse Sera Antigen Coated Beads Serum Dilution Reo3 - cell lysate Infected Sham Sample MHV EDIM Sendai Ectromelia MFI Ratio of antigen beads to BSA beads Normal serum 1:250 4.27 1.54 4.52 0.52 0.84 0.94 Standard Mouse Sera (Charles River) MHV Recom. Diln. 92.99 1.86 2.31 0.29 0.60 0.57 EDIM Recom. Diln. 2.48 71.6781.91 0.44 1.13 0.84 Sendai Recom. Diln. 2.97 0.96 461.0 2.90 83.77 0.75 Ectro Recom. Diln. 2.44 2.36 2.88 698.0 0.56 0.59 Reo 3 Recom. Diln. 2.97 1.47 4.48 0.38 161.0 0.83 Unknown Mouse sera 4029 1:250 48.6257.69 1.49 0.25 0.54 0.47 4030 1:250 96.2255.02 1.78 0.22 0.62 0.59 4031 1:250 19.3811.67 3.29 0.24 0.55 0.59 4032 1:250 43.5749.77 3.98 0.29 1.19 1.26 4033 1:250 60.1865.71 2.45 0.39 0.76 0.76 4034 1:250 35.9543.75 1.68 0.29 0.42 0.54 4035 1:250 56.5219.24 1.26 0.30 0.87 0.73

  21. Multiplex Viral (SIV, SRV, Herpes B) Antibody Detection in Mixed Rhesus Macaque Sera Serum Dilution SRV SIV HVP2 (Herpes-B) Luminex Units Animal # Uninfected controls 47 86 23 1:100 #30365 11 10 15 1:100 #25872 Mixed serum 1:1 #30365+#25872 1:100 75 82 32 Herpes-B Infected (#29683) 78 138 4375 1:100 SRV vaccinated (#26082) 1:100 2665 25 24 SIV vaccinated (#23577) 1:100 294 1084 367 1:100 1835 962 3804 Mixed sera 1:1:1 SRV+SIV+Herpes-B 1221 904 3913 1:300 1:900 460 554 2291 Mixed sera 1:1 SRV+SIV 2830 1478 41 1:100 1:300 1290 991 26 1:900 657 790 26 Mixed sera 1:1 SIV+Herpes-B 1:100 59 1170 4000 1:300 26 864 3780 1:900 13 593 2315 Mixed sera 1:1 SRV+Herpes-B 1:100 2387 91 4049 1290 79 4982 1:300 1:900 477 39 2396

  22. Advantageous Features • Statistical advantage of >100 replicates per analyte • Proteins maintained in solution environment throughout the assay 1. Recovery of signaling complexes for further analysis 2. Use of recovered proteins in functional assays, e.g. kinase activity • Opportunities for development of quantitative assays • Flexibility: mix and match bead sets according to need - not possible for microarray • Data analysis is in real time - takes less time • Easily amenable to high throughput format • Combination with other multiplex methodologies, e.g.Quantum Dots (Center for Biophotonics, UCD)

  23. Comparison of Luminex to ELISA Parameter ELISA Luminex Analytes per assay 1 Up to 100 Sample Size 50-100 l/analyte 30-100 l for multi-analytes Replicate Measurements 1-3 100 Sensitivity 10-20 pg/ml Equal Dynamic Range 2-2.5 log 2.5-3.5 log Assay Time 3-24 Hr 30-90 min High-throughput Difficult Simple

  24. Cost Per Sample for a 10-Plex (10 Analytes) Assay Materials Cost ($) Microbeads 1.00 Antigens 2.00 Antibodies 1.00 Buffers 0.10 Tubes,pipete tips, plates etc. 0.10 Labor Bead preparation 0.15 Sample preparation 0.30 Analysis/data processing 0.30 Total 4.85 Current estimate for ELISA, per serum sample, for 10 analytes is $50.00

  25. Long-TermGoals for Multiplex Detection of Phosphotyrosine Proteins in Signaling Pathways • Molecular dissection of multiple signaling pathways e.g. TCR, BCR, EGFR etc. • for a more comprehensive understanding of the signal dynamics • than that obtained by conventional biochemistry. • Development of phosphotyrosine profile of signaling proteins in cancer (e.g. chronic mylogenous leukemia, breast cancer, prostate cancer, etc.). • Multiplex molecular profiling in well defined mouse models of cancer. • Multiplex detection of cancer markers in serum e.g., osetopontin, p53, IL-6, CRP etc. • Applications for monitoring anti-cancer therapy, e.g. Gleevac, Herceptin. • Development of phosphotyrosine profile of signaling proteins in inflammatory, autoimmune, and infectious diseases.

  26. 104 105 106 107 103 Titration of Capture Ab

  27. 104 105 106 107 103 Titration of Capture Ab

  28. Titration of Reporter Ab

  29. IL-8 Standard Curve

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