Application of immunological tests in diagnosis.

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.

• 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 .

ELISAIntensity 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 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:

Immunofluoresence .

Biopsy specimen from patient.

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 .