Immunological tests use antigen-antibody reactions to determine the presence of antigens or antibodies. Some common tests include agglutination tests which cause antigens to clump in the presence of antibodies, hemagglutination tests using red blood cells, precipitation tests forming aggregates, ELISA measuring antibodies or antigens bound to an enzyme, immunofluorescence using fluorescent dyes to identify antigens, and complement fixation testing antigen-antibody activation of complement. These various serological tests are important in the diagnosis of diseases.

1. Application of immunological tests
in diagnosis.

2. 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 .

3. 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.

4. Agglutination Test
positive. negative.
Antibod
y.
antigen

5. 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 .

6. Haemaggultination Tests

7. 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).

8. Zone of Equivalence

9. 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.

10. Single Radial Immunodiffusion.

11. 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.

12. Precipitation Test:

13. Double immunodiffusion

14. RAST
Radioimmunoassay ( RAST) – measure specific IgE.

15. 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.

16. ELISA .
antigen
Antibody.
Enzyme Labelled
antibody
Enzyme substrate.

17. • 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 .

18. ELISA
Intensity of color correspond to
concentration of antibody.

19. 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.

20. • 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:

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

22. Antigen Antibody Reactions
Immunofluoresence .

23. 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.

24. 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.

25. Complement Fixation Test

26. 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 .)

27. contact dermatitis diagnosed by patch test .

28. Patch test for contact dermatitis .

29. Type 1 allergy diagnosed by skin prick test .