Antigen -Antibody Reactions .
• Antigen – antibody reactions are performed to determine the presence of either the antigen or antibody. ( serological tests ).
• One of the two components has to be known.
• e.g. with a known antigen, such as influenza virus , a test can determine whether antibody to the virus is present or not .

Types of serological Tests
• Agglutination:
• In this test the antigen is particulate (e.g. bacteria and red blood cells) or an inert particle (latex beads) coated with antigen.
• Antibody is divalent and cross links the multivalent antigen to form a lattice network or clumps
(agglutination).
• This reaction can be performed in a tube or on a glass slide e.g. ABO blood grouping.

Agglutination Test
Antibod y.
antigen

Antigen Antibody Reactions
• Haemaggultination Tests:
• Viruses can clump red blood cells from one species or another (active hemagglutination) .
• This can be inhibited by specific anti-viral antibodies.
• Red cells can also absorb many antigens and when mixed with specific antibodies will form clumps (passive hemagglutination) i.e. red cells are passive carriers .

Haemaggultination Tests

Precipitation test :
• In this test antigen is in soluble form (solution).
• Antibody cross -links antigen molecules to form aggregates (precipitates) in the zone of equivalence: optimal proportion of antigen and antibody.
• Precipitation test can be performed in solution or in semi- solid medium (agar).

Zone of Equivalence

Precipitation in Agar .
• Single radial immunodiffusion:
• Antibody is incorporated into agar and antigen introduced into the well.
• As antigen diffuses into agar precipitation rings form depending on the concentration of the antigen.
• Radial Immunodiffusion is used to measure IgG, IgM and complement components.

Single Radial Immunodiffusion.

Double immunodiffusion.
• Antigen and antibody are placed in different wells in agar and allowed to diffuse and form precipitation lines at the points of optimal concentrations.
• This method is used to determine whether antigens are related, identical or non – identical.

Double immunodiffusion

RAST
Radioimmunoassay ( RAST) – measure specific IgE.

Enzyme Linked Immunosorbent Assay (ELISA)
• This method is used for measuring either antigen or antibody in patient serum ..
• For measurement of antibody, known antigen is fixed to a surface i.e. bottom of small wells on a plastic plate.
• Incubated with dilutions of the patient’s serum.
• Washed and then re-incubated with anti-human antibody labeled with an enzyme i.e. horseradish peroxidase.

ELISA .
antigen
Antibody.
Enzyme Labelled antibody
Enzyme substrate.

ELISA .
• Enzyme activity is measured by adding the substrate for the enzyme that leads to development of a color.
• Color reaction is estimated in a spectrophotometer.
• The amount of antibody bound is proportional to the enzyme activity.
• The titer of antibody in patient’s serum is the highest dilution of the serum that gives a positive color reaction .

Intensity of color correspond to concentration of antibody.

Immunofluoresence:
• Fluorescent dyes e.g. fluorescein and rhodamine can be covalently attached to antibody molecules and made visible by ultraviolet (UV) light in a fluorescent microscope.
• Such labeled antibody can be used to identify antigens on surface of microorganisms ( e.g. treponemes), in histological section or in other specimens.

Immunofluoresence:
• Immunofluoresence reaction is called direct when a known labeled antibody interacts directly with unknown antigen .
• Indirect Immunofluoresence involves a two stage process:
– Patient’s serum is added, incubated and the preparation is washed.
– Antigen is attached to a slide.
– Antibody of interest if present will remain attached and can be detected by addition of fluorescent dye labeled antibody under UV light.

Biopsy specimen from patient.
Immunofluoresence .
Antigen fixed on slide e.g.
nuclear antigen .

Antigen Antibody Reactions
Immunofluoresence .

Antigen Antibody Reactions
• Complement Fixation:
• Based on the principle that antigen and antibody reaction activates complement .
• Antigen and antibody, one known and the other unknown are mixed.
• A measured amount of complement is added .
• If antigen-antibody reaction has occurred it will combine “fix” complement.

Complement Fixation:
• An indicator system consisting of “sensitized” red blood cells (red blood cells plus anti-red blood cell antibody) is added.
• If the complement was fixed because of antigen antibody reaction red cells will not be hemolyzed i.e. the test is positive.
• If the antigen antibody reaction did not occur in the first step complement will not be fixed and will be available to lyse RBCs – a negative test.

Complement Fixation Test

Diagnosis of cell-mediated responses:
• 1. Delayed hypersensitivity reactions .
- delayed skin test.
- patch test.
• 2. Lymphocyte transformation test .
lymphocyte activation test.
( detect markers by flow cytometry .)

contact dermatitis diagnosed by patch test .

Patch test for contact dermatitis .

Type 1 allergy diagnosed by skin prick test .