1. 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.

2. 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.

3. Passive agglutination:.

4. -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( μg/ml).

5. Haemadsorption and agglutination inhibition:

6. -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

7. Quantitative precipitin curve: Prozone and Equivalence zone:

8. 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

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

10. 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.

11. 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

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

13. 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.

14. -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.