Measurement of Immune function:

Immunological tests rely upon: Ability of antibodies to aggregate particulate antigens (agglutination) Or to precipitate soluble antigens Antigens quantitation (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).

Epitope detection by antibodies: Detect the specific reaction between Ag & Ab  Particulate antigens: (agglutination) o Direct agglutination. o Indirect agglutination. o Agglutination inhibition.  Soluble antigens: (precipitation) o Radial immunodiffusion. o Double-diffusion

Epitopes detection: o Direct agglutination: Agglutination of particulate or cell- bounded antigen by antibodies (mainly IgM, multivalent Ab). Example: Blood grouping: group A RBCs +Anti-A antibodies = agglutination.

Agglutination of RBC is called haemagglutination Insert a picture

Epitope detection o Indirect (passive) agglutination : Adding of anti-immunoglobulins to detect low titers and non IgM antibodies. o Agglutination of RBCs: haemagglutination. Example: Latex agglutination : Rheumatoid factors test: Anti-human IgG Antibodies.

Epitope detection Agglutination inhibition Haemadsorption: is a direct agglutination of erythrocytes by certain viruses (Spontaneous agglutination). Inhibition of Erythrocytes agglutination is a quantitative method for calculation of anti-virus antibodies concentration in patient’s serum.

Haemadsorption and agglutination inhibition: N

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.

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.

Radial immunodiffusion to determine Ag concentration:

o Double diffusion: Ag serum

Other widely used techniques Immunofluorescent microscopy Flow cytometery Enzyme linked immunosorbent assay

N o Immunofluorescent Microscopy: Direct: 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. Indirect: Secondary anti-human globulin conjugated with fluorochrome.

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.

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 -Spectrophotometry.

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

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

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

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

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.