The document describes several immunology techniques: Flow cytometry analyzes individual cells or particles using laser light scattering and fluorescent dyes to label cellular molecules. ELISA uses antigen-coated plates and enzyme-linked antibodies to detect antibodies in serum samples. ELISPOT counts antibody-secreting cells by detecting individual antibody spots. Tissue culture methods grow cells in controlled conditions using culture medium, reagents, and equipment to maintain cells outside the body.

1. Flow cytometry, ELISA, ELISPOT & cell culture methods Sanjaya Adikari Department of Anatomy

4. Laser beam

5. Stain T cells with Anti –CD3-PE ab and Anti-CD4-FITC ab FITC PE Control FITC PE CD3 CD8 CD4 CD3 + CD3 + CD4 +

7. Enzyme-Linked Immunosorbent Assay (ELISA) 96-well plate

8. Main steps Patient serum which contains antibody Anti-human immunoglobulin coupled to an enzyme. This is the second antibody, and it binds to human antibodies. Chromogen or substrate which changes colour when cleaved by the enzyme attached to the second antibody Coat the plates with antigen

10. Positive ELISA Test Negative ELISA Test

11. The cut-off value indicating a positive result is 0.500 O D of 0.300 to 0.499 are indeterminate and need to be retested Values < 0.300 are considered to be negative (+) Control (-) Control Patient A Patient B Patient C Assay Control 1.689 0.153 O.055 0.412 1.999 0.123

12. OD Concentration (ng/pg) sample

13. Enzyme-Linked Immunosorbent Spot ELISPOT Coat the plates with antigens Cells Anti-human immunoglobulin coupled to an enzyme. This is the second antibody, and it binds to human antibodies. Chromogen or substrate which changes color when cleaved by the enzyme attached to the second antibody Cells secrete antibodies

14. Examples of images from the ELISpot technique: Production of Interferon-gamma by human Peripheral Blood Mononuclear Cells (PBMC): Interferon-gamma (IFN-  ) Human unstimulated (250.000 cells) PPD stimulation (250.000 cells) TT stimulation (250.000 cells)

15. Tissue Culture Methods Tissue culture organ culture cell culture p rimary cell cultures continuous cell lines

18. Procedure for MNC culture lymphoprep Peripheral blood MNCs RBC Neutrophils Density gradient centrifugation

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