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 & AbParticulate antigens: (agglutination)o Direct agglutination.o Indirect agglutination.o Agglutination inhibition.Soluble antigens: (precipitation)oRadial immunodiffusion. oDouble-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
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Epitope detectiono 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 inhibitionHaemadsorption: 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
oRadial 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
serum
serum
serum
serum
serum
Other widely used techniques
•Immunofluorescent microscopy•Flow cytometery•Enzyme linked immunosorbent assay
N
oImmunofluorescent 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.
ELISA for antigen detection• 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.
P N
ELISA for Antibody detection
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 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.
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.
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