1. Measurement of Immune function:

2. Detect antigens and / or antibodies. Immunological tests rely upon: ability of antibodies to aggregate particulate antigens (agglutination) Or to precipitate soluble antigens (precipitation) Antigens and antibodies quantitation tests e.g. ELISA: enzyme linked immunosorbent assay. Flourochrome labeled antibodies to detect intracellular antigens (e.g. immunofluorescence). Measurement of immune function (e.g., complement fixation and cytotoxic T lymphocyte assay) Clinical setting (assessment of hypersensitivity).

3. Antigen detection: Detect the specific reaction between the antigens & antibodies.  Agglutination: to detect particulate antigens: o Direct agglutination. o Indirect (passive) agglutination. o Agglutination inhibition.  precipitation: to detect soluble antigens: o Radial immunodiffusion. o Double-diffusion

4. Agglutination tests: o Direct agglutination: Agglutination of particulate or cell-bounded antigen by antibodies (mainly IgM, multivalent Ab). o Agglutination of RBCs: haemagglutination. Example: Blood grouping: group A RBCs + anti- A antibodies = agglutination. Coombs test*: diagnosis of alloimmune hemolytic anemia of newborn.

5. Agglutination of RBC is called haemagglutination Insert a picture

8. Agglutination o Indirect (passive) agglutination : Adding of anti-immunoglobulins to detect low titers and non IgM antibodies. o Example: Latex agglutination : rheumatoid factors test: Anti-human IgG Antibodies. Treponema pallidium haemagglutination assay: (TPHA): Diagnosis of syphilis.

11. RBCs + treponemal antigens TPHA

12. Agglutination o Agglutination inhibition Haemadsorption: is a direct agglutination of erythrocytes by certain viruses (spontaneous agglutination). Inhibition of erythrocytes agglutination by anti-virus antibodies in patient’s serum.

13. Haemadsorption and agglutination inhibition: N

14. N Antibody titer: the lowest concentration of antibody that causes agglutination in vitro. Serial doubling dilution of patient’s serum creates zone of equivalence; and so positive reaction. Prozone phenomena: False-negative agglutination due to excess antibodies or antigen concentration.

15. Negative Positive Negative

17. Precipitation: soluble antigens o Radial immunodiffusion: Mancini technique: Soluble antigen reacts with soluble antibodies in semisolid medium. Formation of immuno-precipitin lines. Quantitative assay. Clinical applications: Diagnosis of complement deficiency Calculation of Hb F for diagnosis of Beta- thalassemia.

20. Radial immunodiffusion to determine Ag concentration:

21. o Double diffusion: Ag serum

23. Other widely used techniques Immunofluorescent microscopy. Flow cytometery. Enzyme linked immunosorbent assay (ELISA).

24. N o Immunofluorescent Microscopy: Cell bounded antigen is detected by antibodies conjugated with fluorochrome. Clinical application: Diagnosis of malignant tumor. Diagnosis of intracellular infectious diseases e.g. Chlamydia, Virus infections. Direct: antibodies conjugated with fluorochrom. Indirect: Secondary anti-human globulin conjugated with fluorochrome.

26. N o Flow Cytometry: A powerful modification of IF. Each type of leukocytes can be stained by monoclonal antibodies fluorochrome conjugate. The computerized machine then counts each type using laser beam. Clinical application: Calculation of CD4/CD8 Ratio.

27. N o Enzyme Linked Immunosorbent Assay (ELISA): - Quantitative assay. - Soluble antigens or antibodies fixed on micro-titer plate wells. - Secondary antibodies linked with enzyme reacts with the complex. -Substrate (colorless) converted into colored end product which is measured by spectrophotometry.

28. Add the patient’s serum then wash add specific antibodies to the antigen wash add 2° antibody linked with the enzyme then wash add specific substrate. read the reaction. PN

29. Antigen coating 1° Antibody in the patient's serum 2° antibody (enzyme- linked) Chromogenic substrate Indirect ELISA

30. ELISA Clinical application: Diagnosis of infectious diseases: HIV, HBV, HCV …..

31. Assessment of Cellular immunity and function: Determination of phagocyte function: - APC incubated with microbes for 30-120 min. - Particle inclusion within the cell & oxidative enzyme activity is assessed by microscopy. Determination of lymphocyte proliferation: -Lymphocyte cultured for 48-72 hrs with added mitogen and radioactive material. - incorporation of radioactive material into the new formed DNA is measured by radioimmunoassay.

32. Assessment of hyper sensitivity Allergy skin testing (type I hypersensitivity): scratch or intradermal injection of a small amount of diluted allergen. Sensitive (atopic) individuals develop a wheal-and-flare (redness & swelling) reaction within 20 minutes. Complement fixation (types II and III): Contact dermatitis and delayed hypersensitivity (type IV): Application of antigen to the surface of the skin (contact dermatitis) or injected intradermally. Wheal-and-flare reactions are evident only 24 to 72 hours after challenge.

33. Type I hypersensitivity reaction

34. Type II (DTH) hypersensitivity reaction