1. Immunological Reactions and Techniques W. Robert Fleischmann, Ph.D. Department of Urologic Surgery University of Minnesota Medical School rfleisch@umn.edu (612) 626-5034

2. Objectives Become familiar with the various types of immunological techniques that are used for clinical diagnosis. Understand how each of these immunological techniques work.

3. The purposes of immunological tests are to aid in the determination or monitoring of diseases or conditions that may be present in a patients.

4. Immunologic Techniques Agglutination reactions Direct antiglobulin test Indirect antiglobulin test Electrophoresis Immunofixation Immunofluorescence Enzyme-linked immunosorbent assay Western blot Flow cytometry

5. Agglutination Reactions Purpose: –To detect antibodies in a patient’s serum –To detect antigens in a patient’s serum Examples: –Detect whether or not a patient has previously been exposed to an agent such as German measles (rubella) either by vaccination or by contracting the disease –Testing for the presence of Hemophilus influenzae type B capsular antigens in cerebral spinal fluid

6. Agglutination Reactions (Cont.) Agent detected: –Specific antibody –Specific antigen Indicator: –Agglutinated particles. Use particles coated with antigen or with antibody. These particles could be colored or white latex beads The particles could even be RBCs (hemagglutination).

7. Agglutination Reaction: Method Harvest a patient specimen. Clarify the specimen by centrifugation. Mix specimen with white or colored latex beads that have Bound antibodies on their surface. Allow binding, if any, to occur. Read the test as agglutination or no agglutination of the beads.

8. Results of an Agglutination Test Wikipedia

9. Direct Antiglobulin Test Aka: Coombs Test Purpose: –Detect antibody on a patient’s RBCs Examples: –Performed in patients with hemolytic anemia caused by autoimmune disease –Diagnosis of a transfusion reaction –Diagnosis of hemolytic disease of the newborn (blue baby syndrome, Rh factor incompatibility)

10. Direct Antiglobulin Test (Cont.) Agent detected: –Antibody –Complement Indicator: –Agglutinated (clumped) RBCs

11. Direct Antiglobulin Test: Method Wikipedia

12. Indirect Antiglobulin Test Aka: Indirect Coombs Test Purpose: –Detect antibodies that are directed against RBC antigens Examples: –Performed in patients about to undergo a blood transfusion to detect the presence of antibodies to the RBCs about to be transfused (mismatch) –During pregnancy or at delivery for a woman who is Rh negative

13. Indirect Antiglobulin Test (Cont.) Agent detected: –Antibody to RBCs in a recipient’s blood Indicator: –Agglutinated (clumped) RBCs in a donor’s blood

14. Indirect Antiglobulin Test: Method Wikipedia

15. Sample Results for Indirect or Direct Antiglobulin Kuby

16. Electrophoresis Purpose: –To detect the levels of various proteins Examples: –Detect monoclonal IgG produced by myeloma patients –Measuring amount of albumin and other blood constituents

17. Electrophoresis (Cont.) Agent detected: –Antibody –Antigen –Any protein Indicator: –Visible band of agent on a gel –Stained band of an agent on a gel Coomassie blue or silver stain for protein Ethidium bromide for nucleic acid

18. Electrophoresis: Method Prepare a polyacrylamide or agarose gel Load sample on the gel Apply an electric current across the gel for a period of time Visualize the band on the gel –With the naked eye –By exposure of the band to UV-light –By staining the band with a dye. Quantify the amount of material in the band by densitometer reading

19. From: Wikipedia

20. Multiple Myeloma Antibodies are produced by B cells and plasma cells. When a single plasma cell becomes transformed into a cancerous cell, it causes myeloma. –Myeloma patients over-produce a homogeneous Ig produced by a single plasma cell. This can be observed as a heightened peak of Ig by electrophoresis of blood proteins. –Myeloma patients also have some immunoglobulin proteins that spill over into their urine. These Bence-Jones proteins are dimers of kappa or lambda light chains. Albumin Globulins  Migration distance Absorbance IgX from Myeloma patients Densitometer Scan

21. From: Parslow, Medical Immunology Use of Electrophoresis to Diagnosis Immunodeficiencies

22. Immunofixation Purpose: –Identification of composition of monoclonal antibody Examples: –Detection of monoclonal antibody type in myeloma patients (plasma cell lymphoma giving an overproduction of IgG, IgA, IgE) –Detection of monoclonal antibody in Waldenstom’s macroglobulinemia (B cell lymphoma giving an overproduction of IgM)

23. Immunofixation (Cont.) Agent detected: –Specific heavy and light chain of monoclonal antibody Indicator: –Antibody against heavy and light chains

24. From: Kristine Krafts

25. IgG monoclonal antibody composed of a specific gamma and kappa chains Monoclonal antibody fragment Composed of specific kappa chain IgA antibody composed of a specific alpha and lambda chains Note absence of smear Note presence of smear

26. Immunofluorescence Purpose: –Detection of an antigen in a specimen Examples: –Detection of specific proteins in cells, such as a tumor antigen or a viral antigen –Detection of bacterial organisms –Detection of antigen-antibody complexes that have been deposited on cell membrane or basement membrane surfaces

27. Immunofluorescence (Cont.) Agent detected: –Antigen that is precipitated on a cell –Antigen that is part of the cell membrane –Agent that is within the cell (must permeablize the cell to detect it) Indicator: –Antibody that has a fluorescent tag

28. Immunofluorescence: Method Direct Fix cells to a slide Add antibody (IgG) specific to the target antigen that is tagged with fluorescent compound Visualize fluorescence by looking through a fluorescence microscope Indirect Fix cells to a slide Add primary antibody (IgG) specific to the target antigen Add secondary anti-antibody (anti- IgG) that is tagged with fluorescent compound Visualize fluorescence Kuby

29. Immunofluorescence Negative for the presence of antigen in the cells Positive for the presence of antigen in the cells Cytoplasmic Staining bright green Nuclear Staining pale blue Nuclear Staining: dark blue (DAPI)

30. Enzyme-Linked Immunosorbent Assay (ELISA) Purpose: –Detection of antibodies or antigens in a patient specimen Examples: –Home pregnancy test –Detection of antibody to a virus, bacterium or other microorganism HIV test –Detection of antibody to a foreign antigen –Detection of a viral antigen or bacterial antigen

31. Kinds of ELISA Indirect ELISA Sandwich ELISA Radioimmunoassay

32. ELISA (Cont.) Agent detected: –Antibody –Antigen Indicator: –Antibody with a bound enzyme that can catalyze conversion of a colorless molecule to a colored one Alkaline phosphatase Horseradish peroxidase –Antibody with a bound radioisotope

33. Y Y Y Y Y Y Y Y Y YY YY Y Y YY YY Y YYYY Y YY YY Y YYYY Y YY YY Y YYYY Add Antigen Add 2° Ab Add Reagent Incubate Y YY YY Y YYYY YYYYYYYYYYYY Y Y Y Y YYYYYY YYYYY YYYYYY YYYYY Add 1° Ab Add 2° Ab Add Reagent Incubate Indirect ELISASandwich ELISA

34. Elispot This is a variation of the ELISA test. A petri plate is coated with specific capture antibody. Cells are added to the plate and allowed to settle for a period of time. The cells produce a specific cytokine that binds to the specific antibody in the area where the cell settled. The cells are washed away. Detection antibody bearing an enzyme is added and unbound excess antibody is washed away. An appropriate substrate is added. An area of the petri plate where the cytokine was produced turns color.

35. Western Blot Purpose: –Detection of antibodies or proteins in a patient specimen Examples: –Detection of antibodies to HIV in a patient’s blood –Detection of HIV proteins in a patient’s blood

36. Western Blot (Cont.) Agent detected: –Antibody –Antigen Indicator: –Antibody tagged with fluorescent molecule –Antibody tagged with enzyme to convert non- colored compound to a colored compound –Antibody tagged with chemiluminescence enzyme (luciferase) –Antibody tagged with radiolabel

37. Detection of HIV Infection- Western Blotting Western blot –is the confirming test for HIV infection. –detects the presence of antibodies to the various protein components of HIV (e.g., anti-p18, anti-p24, anti-gp41). –consists of four steps: 1. Electrophoresis of HIV proteins on cellulose acetate 2. Reacting putative serum antibodies of patient with HIV proteins 3. Reacting conjugated (enzyme or radiolabeled) anti-antibody with the serum antibodies and the HIV proteins 4. Reading color change or radioactivity for positive result Pt 1 Pt 2 Stnd

38. Flow Cytometry Purpose: –Determine the number or percentage of cells that express a given antigen Examples: –Monitoring CD4+ T cell levels in HIV- infected patients –Diagnosis of leukemia and lymphoma

39. Flow Cytometry (Cont.) Agent detected –Cells bearing a specific antigen Indicator –Antibody tagged with fluorescent molecule

40. Flow Cytometry a.Separation of CD8+ and CD3+ cells from total WBCs. b.Rerun of CD8+/CD3+ T cells.