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Immunology Assays for Clinical Research

Immunology Assays for Clinical Research. Yan Ge, Ph.D. University of Virginia Email: yg9z@virginia.edu. Common Immunology Assays. ELISA ELISPOT Flow cytometry Immunofluorescent staining. ELISA. Enzyme-linked immunosorbent assay

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Immunology Assays for Clinical Research

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  1. Immunology Assays for Clinical Research Yan Ge, Ph.D. University of Virginia Email: yg9z@virginia.edu

  2. Common Immunology Assays • ELISA • ELISPOT • Flow cytometry • Immunofluorescent staining

  3. ELISA • Enzyme-linked immunosorbent assay • To detect the level of an antibody or an antigen in a sample

  4. Types of ELISA • Indirect ELISA • Sandwich ELISA • Competitive ELISA

  5. Indirect ELISA – Antibody Detection

  6. Sandwich ELISA– Antigen Detection

  7. Enzymes and Substrates • horse radish peroxidase (HRP) • OPD o-phenylenediamine dihydrochloride, maximum absorbance: 492 nm • TMB 3,3',5,5'-tetramethylbenzidine, maximum absorbance: 450 nm • ABTS 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt, maximum absorbance: 410 nm

  8. Enzymes and Substrates • alkaline phosphatase (AP) • PNPP p-Nitrophenyl Phosphate, Disodium Salt, maxium absorbance: 405 nm

  9. Measurement • Optical density (OD) at a certain wavelength • Standard curve  absolute concentration or arbitrary units

  10. ELISPOT • Enzyme Linked Immunospot • Principle: sandwich ELISA • To detect secreted proteins (e.g. cytokines, growth factors)

  11. Flow Cytometry (FACS, Flow) • A technique used to measure the physical and chemical properties of cells or cellular components • Cells are measured individually, but in large numbers.

  12. Applications • Immunophenotyping • Identification of cells using cell-surface markers • Intra-cellular proteins (e.g. cytokines, chemokines) • Kinetics • E.g. Ca2+ mobilization using Indo-1 • DNA and cell cycle • DAPI, propidium iodide, 7-AAD • Cell sorting

  13. Cell Staining with Fluorochromes

  14. Flow Cytometer • Components: • Fluidics • Optics • Detectors • Electronics

  15. Fluidics

  16. FSC and SSC • Side-scattered light (SSC): cell granularity or internal complexity • Forward-scattered light (FSC): cell-surface area or size

  17. Compensation

  18. An example • Stain cells with: • FL1: CD4-FITC • FL2: CD19-PE • FL3: 7-AAD (live/dead cells) • FL4: CD3-Alexa Fluor 647 • FL5: CD8-Alexa Fluor 750

  19. Data Analysis Live cells (7-AAD negative) lymphocytes

  20. Data Analysis FL5: CD8-Alexa 750 CD4+ T cells CD8+ T cells FL1: CD4-FITC FL4: CD3-Alexa 750 FL4: CD3-Alexa 750

  21. Data Analysis Counts B cells FL2: CD19-PE

  22. References • Introduction to Flow Cytometry: A learning Guide. Manual Part Number: 11-11032-01, April 2000, BD Sciences. • Introduction to Flow Cytometry. Flow cytometry core facility. Biomedical Instrumentation Center, Unifored Services University. • Introduction to Flow Cytometry. By Misha Rahman, AbD Serotec.

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