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)

12. 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.

13. Applications Immunophenotyping Identification of cells using cell-surface markers Intra-cellular proteins (e.g. cytokines, chemokines) Kinetics E.g. Ca 2+ mobilization using Indo-1 DNA and cell cycle DAPI, propidium iodide, 7-AAD Cell sorting

14. Cell Staining with Fluorochromes

15. Flow Cytometer Components: Fluidics Optics Detectors Electronics

16. Fluidics

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

22. Compensation

23. 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

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

25. Data Analysis FL4: CD3-Alexa 750 FL1: CD4-FITCFL5: CD8-Alexa 750 FL4: CD3-Alexa 750 CD4+ T cells CD8+ T cells

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

27. 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.