Application of immunological tests in diagnosis.

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 positive. negative. Antibody. 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.

Precipitation Test:

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 .

ELISA 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.

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

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 .