Measurement of Immune function: -Epitope detection by antibodies: A-Direct agglutination: -Agglutination of particulate antigen or cell-bounded antigen by antibodies (mainly multivalent antibodies). Example: Blood grouping : RBCs(group A)+Anti-A Antibodies= agglutination.

B-Passive agglutination : Refers to agglutination of antigen-coated cells or carbon particles by antibodies. -Agglutination of RBCs is called Haemagglutination. Examples: 1-Latex agglutination : -Rheumatoid factors test: Anti-human IgG Antibodies. clinical significance: diagnosis of Rheumatoid arthritis. 2-Direct Coomb’s test: Clinical significance: diagnosis of Alloimmune hemolytic anemia of newborn. 3-Treponema pallidium haemagglutination assay: (TPHA): Diagnosis of syphilis.

Passive agglutination: .

-Spontaneous agglutination is a direct agglutination of erythrocytes by certain viruses (haemadsorption). -Inhibition of Erythrocytes agglutination is a quantitative method for calculation of anti-virus antibodies concentration in patient’s serum. : The use of specific viral antigenic reagent with known concentration(0.1-10 μg/ml).

Haemadsorption and agglutination inhibition:

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

Quantitative precipitin curve: Prozone and Equivalence zone:

C-Radial immunodiffusion: (Mancini technique): -Soluble antigen reacts with soluble antibodies in buffer-semisolid medium. -Formation of immuno-precipitin lines. -Quantitative assay. Clinical applications: 1-Diagnosis of Complement deficiency (C1, C2, C3,..). 2-Calculation of Hb F for diagnosis of Beta-thalassemia. N

Immunodiffusion: Radial and double: Radial: (Mancini technique) Double: (Ouchterlony technique) :Detection of Toxigenic microbial strains.

D-Immunofluorescent Microscopy: -Direct: Cell surface bounded antigen (tissue section) is detected by monoclonal antibody conjugated with Fluorochrome (FITC). Clinical application: 1-Diagnosis of malignant tumor. 2-Diagnosis of intracellular infectious diseases (Chlamydia, Virus infections). -Indirect: -Secondary anti-human globulin conjugated with fluorochrome.

E-Enzyme Linked Immunosorbent Assay (ELISA): - High degree of safety and sensitivity. - Quantitative assay. - Soluble antigen (antibodies) fixation on micro-titer plate wells. -Patients sera antibodies (antigen) is added. - Secondary antibodies conjugated with enzyme (Peroxidase) reacts with the complex. -Substrate (colorless) converted into colored end product -Spectrophotometry. N

ELISA : Clinical application: -Diagnosis of infectious diseases: HIV infection and Hepatitis B and C infection.

Assessment of Cellular immunity: Cellular immunity could be measured by: 1-Determination of phagocyte function: -APCs incubated with microbes for 30 to 120 min. -The oxidative enzyme activity will be measured microscopically. 2-Determination of lymphocyte proliferation ability: -Lymphocyte culture (48-72 hrs). -Incubation with mitogen. -Incorporation of radioactive material H-thymidine. -Radioimmunoassay.

-A powerful modification of IF. 3-Flow Cytometry: -A powerful modification of IF. -Each clone of leukocytes can be stained by Monoclonal antibodies-dye conjugate. -The machine then counts each clone according to the scattered laser beam number. Clinical application: -Calculation of CD4/CD8 Ratio variation due to malignant tumor treatment.