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:

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•

•

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.