done by bilal m marwa abdullah al harby from the slides of dr jad alrab n.
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Application of immunological tests in diagnosis.

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Done by: Bilal M. Marwa , Abdullah Al- Harby . From the slides of: Dr. Jad AlRab. Application of immunological tests in diagnosis. LECTURE outline. Serological Tests: types of tests where serum is used to measure the amount of antibodies present in it. Serological tests.

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antigen antibody reactions

Antigen – antibody reactions are performed to determine the presence of either the antigen or antibody. (serological tests ).

Either the antigen or the antibody have 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 .

Antigen -Antibody Reactions .
1 agglutination

In this test the antigen is particulate (visible, big and insoluble) (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.
1. Agglutination
agglutination test positive negative
Agglutination Test positive. negative.



2 haemaggultination tests

It is a type of agglutination test performed on RBCs.

  • It has two types:
    • Active: the antigen is the RBC itself.
      • Viruses can clump red blood cells from one species or another (active hemagglutination)
      • This can be inhibited by specific anti-viral antibodies.
      • Another example is the test used in ABO grouping.
    • Passive: the antigen here is not the RBC. The RBC absorbs it and expresses it on the surface.
      • It will form clumps when mixed with antibodies.
      • i.e. red cells are passive carriers .
2. Haemaggultination Tests:
precipitation test

In this test, the 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).

Precipitation test :
zone of equivalence
Zone of Equivalence

For antibody-antigen reaction to form a precipitate that we can see, the amount of antigen and antibody should be the same (optimum concentration).

This happens in the zone of equivalnce.

3 4 precipitation in agar

In this test, we want to measure the amount of antibodies in a serum sample.

  • We use the fact that a precipitation will happen from an anitobdy-antigen reaction ONLY if their amount is the same.
  • This can be made by 2 methods:
    • Single Radial Immunodiffusion
    • Double Immunodiffusion.
3,4 Precipitation in Agar.
3 single radial immunodiffusion


    • We use a plate with a known amount of antigen (let’s say 1 mg).
      • The plate is special in that it has a hole for insertion of serum (see next slide)
    • Then we add serum (containing antibodies) into the hole.
    • If the amount of antibody is equal to the amount of antigen (1 mg), it will precipitate immediately, forming a small precipitation ring.
    • If the amount of antibody is higher (let’s say 10 mg) than the amount of antigen in the plate, the antibodies will radiate out away from the center, to be diluted, and will stop only if it is diluted enough to be equal to the amount of antigen.

10 mg antiobdy

3 mg antibody

1 mg antibody

The solution contains

1 mg antigen

4 double immunodiffusion

Here, we have a plate with 2 holes. We add the antigen in one of them, and the serum in the other, and we allow them 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.

(see the next slides)

4. Double immunodiffusion.
5 radioimmunoassay rast measure specific i g e
5. Radioimmunoassay (RAST): measure specific IGE

Labeled anti-IgE helps us to measure the amount of reaction

6 enzyme linked immunosorbent assay elisa

This method is used for measuring either antigen or antibody in patient serum.

  • For measurement of antibody, a 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.

(see next slide)

6. Enzyme Linked Immunosorbent Assay (ELISA)



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 .

Intensity of color correspond to concentration of antibody.

7 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.
7. Immunofluoresence:
7 immunofluoresence1

It can be:

    • Direct: a known labeled antibody interacts directly with an 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.
7. Immunofluoresence:
Immunofluoresence .

Antigen fixed on slide e.g.

nuclear antigen .

Biopsy specimen

from patient.

antigen antibody reactions1
Antigen Antibody Reactions

Immunofluoresence .

8 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.

8. Complement fixation
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:
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 .)

Diagnosis of cell-mediated responses: