Application of immunological tests in diagnosis.

1. Application of immunological tests in diagnosis.

2. Antigen -Antibody Reactions . • Antigen – antibody reactions are performed to determine the presence of either the antigen or antibody. ( serological tests ). • One of the two components has to be known. • e.g. with a known antigen, such as influenza virus , a test can determine whether antibody to the virus is present or not .

3. Types of serological Tests • Agglutination: • In this test the antigen is particulate(e.g. bacteria and red blood cells) or an inert particle (latex beads) coated with antigen. • Antibody is divalent and cross links the multivalent antigen to form a lattice network or clumps (agglutination). • This reaction can be performed in a tube or on a glass slide e.g. ABO blood grouping.

4. Agglutination Test positive. negative. Antibody. antigen

5. Antigen Antibody Reactions • Haemaggultination Tests: • Viruses can clump red blood cells from one species or another (active hemagglutination) . • This can be inhibited by specific anti-viral antibodies. • Red cells can also absorb many antigens and when mixed with specific antibodies will form clumps (passive hemagglutination) i.e. red cells are passive carriers .

6. Haemaggultination Tests

7. Precipitation test : • In this test antigen is in soluble form (solution). • Antibody cross -links antigen molecules to form aggregates (precipitates) in the zone of equivalence: optimal proportion of antigen and antibody. • Precipitation test can be performed in solution or in semi- solid medium (agar).

8. Zone of Equivalence

9. Precipitation in Agar. • Single radial immunodiffusion: • Antibody is incorporated into agar and antigen introduced into the well. • As antigen diffuses into agar precipitation rings form depending on the concentration of the antigen. • Radial Immunodiffusion is used to measure IgG, IgM and complement components.

10. Single Radial Immunodiffusion.

11. Double immunodiffusion. • Antigen and antibody are placed in different wells in agar and allowed to diffuse and form precipitation lines at the points of optimal concentrations. • This method is used to determine whether antigens are related, identical or non –identical.

12. Precipitation Test:

13. Double immunodiffusion

14. RAST Radioimmunoassay ( RAST) – measure specific IgE.

15. Enzyme Linked Immunosorbent Assay (ELISA) • This method is used for measuring either antigen or antibody in patient serum .. • For measurement of antibody, known antigen is fixed to a surface i.e. bottom of small wells on a plastic plate. • Incubated with dilutions of the patient’s serum. • Washed and then re-incubated with anti-human antibody labeled with an enzyme i.e. horseradish peroxidase.

16. ELISA . antigen Antibody. Enzyme Labelled antibody Enzyme substrate.

17. ELISA . • Enzyme activity is measured by adding the substrate for the enzyme that leads to development of a color. • Color reaction is estimated in a spectrophotometer. • The amount of antibody bound is proportional to the enzyme activity. • The titer of antibody in patient’s serum is the highest dilution of the serum that gives a positive color reaction .

18. ELISA Intensity of color correspond to concentration of antibody.

19. Immunofluoresence: • Fluorescent dyes e.g. fluorescein and rhodamine can be covalently attached to antibody molecules and made visible by ultraviolet (UV) light in a fluorescent microscope. • Such labeled antibody can be used to identify antigens on surface of microorganisms ( e.g. treponemes), in histological section or in other specimens.

20. Immunofluoresence: • Immunofluoresence reaction is called direct when a known labeled antibody interacts directly with unknown antigen . • Indirect Immunofluoresence involves a two stage process: • Patient’s serum is added, incubated and the preparation is washed. • Antigen is attached to a slide. • Antibody of interest if present will remain attached and can be detected by addition of fluorescent dye labeled antibody under UV light.

21. Immunofluoresence . Antigen fixed on slide e.g. nuclear antigen . Biopsy specimen from patient.

22. Antigen Antibody Reactions Immunofluoresence .

23. Antigen Antibody Reactions • Complement Fixation: • Based on the principle that antigen and antibody reaction activates complement . • Antigen and antibody, one known and the other unknown are mixed. • A measured amount of complement is added . • If antigen-antibody reaction has occurred it will combine “fix” complement.

24. Complement Fixation: • An indicator system consisting of “sensitized” red blood cells (red blood cells plus anti-red blood cell antibody) is added. • If the complement was fixed because of antigen antibody reaction red cells will not be hemolyzed i.e. the test is positive. • If the antigen antibody reaction did not occur in the first step complement will not be fixed and will be available to lyse RBCs – a negative test.

25. Complement Fixation Test

26. Diagnosis of cell-mediated responses: • 1. Delayed hypersensitivity reactions . - delayed skin test. - patch test. • 2. Lymphocyte transformation test . lymphocyte activation test. ( detect markers by flow cytometry .)

27. contact dermatitis diagnosed by patch test .

28. Patch test for contact dermatitis .

29. Type 1 allergy diagnosed by skin prick test .