Antigen-antibody reaction
Antigen= immunogen: any foreign substance which, when introduced will evoke a specific immune response.
In terms of infectious diseases, the following may act as antigens:
Microbial structures (cell walls, capsules, flagella, pili, viral capsids, envelope-associated glycoproteins, etc.).
Microbial exotoxins.Certain noninfectious materials may also act
as antigens if they are recognized as "nonself" by the body. These include:
1. Allergens.2. Foreign tissues and cells.3. The body's own cells that the body fails to
recognize as "normal self" as cancer cells.
Antibodies or
immunoglobulins are specific
protein configurations
produced by B-lymphocytes
and plasma cells in response to
a specific antigen and capable
of reacting with that antigen.
Antibody structure (IgG)
Antigen-antibody reactions can be used in the laboratory to identify
unknown microorganisms
Antigen antibodies reaction
1. agglutination2. precipitation3. complement fixation4. Toxin antitoxin neutralization5. virus neutralization6. Immunefluorescence7. Flow cytometry8. Enzyme linked immunesorbant assay
(ELISA)9. Radioimmunoassay
Serologic testing may be used in the clinical laboratory in two distinct ways:
To identify unknown antigens (such as
microorganisms).
To detect antibodies being made against a
specific antigen in the patient's serum.
AgglutinationAntigen: suspension of
microorganisms, cells or latex
particles coated with antigens.
Antibodies: specific anti serum
Reaction ……………………
Agglutination or clumping.
If one (antigen or antibody) is
known, the reaction can be used
for identification of the other.
I. Direct agglutination1. Slide agglutination:Antigen + Antibody Clumping occurs
if the serum is specific to the organism.
Blood grouping
2. Tube agglutination
It is a quantitative test.
Used to determine the amount of antibodies
in the patient’s serum.
Serial dilution of the patient serum showing agglutination
titre is
Widal test
Serological method used for diagnosis of enteric fever.
The idea is to detect the antibodies to salmonella in patient’s serum.
This test demonstrates the presence of somatic (O) and flagellar (H)
agglutinins to Salmonella typhi in the patient's serum using suspensions
of O and H antigens.
Antigens of S. paratyphi A, S. paratyphi B, S. paratyphi C are included in
most commercial kits.
We can do by using :
1. Slide agglutination method.
2. Tube agglutination method.
Slide method:Slide Widal test is more popular among
diagnostic laboratories as it gives rapid results.
I. Qualitative test: 1. Dispense 0.08 of undiluted patient serum
sample onto a raw of circles.2. Add one drop of each O or H antigen
suspension to the serum (shake the reagent bottle before use) .
3. Mix well using a stirring stick gently rotate the slide for one minute.
Appearance of agglutinationgives qualitative positive results.
II. Quantitative test (slide titration method) :
1. 80 μl, 40 μl, 20 μl, 10 μl and 5 μl of patient’s
serum for each antigens are placed on the slides.
2. To each series of serum specimen, one drop of
specific antigen is added.
3. Mixed well and rotate the slide for one minute.
4. Agglutination in each of these is noted.
80 μl corresponds to 1in 20 dilution, 40 μl to 1 in 40,
20 μl to 1 in 80, 10 μl to 1 in 160 and 5 μl
corresponds to 1 in 320 titre.
Tube agglutination methodPrepare 8 sets each is prepared as follows:1. 8 plastic tubes in a rack.2. 1.9 ml of saline in the 1st tube and 1 ml into the
remaining seven.3. Dispense 0.1 ml of the of the serum in 1 st tube
and mix well.4. Dispense 1 ml from 1st tube into 2nd tube and mix
well.5. Continue this doubling of dilutions to the other
tubes.6. Discard 1 ml from the last tube.7. Shake the reagent bottle well.8. Add one drop of the antigen suspension into each
tube and mix.9. Incubate .
10. Examine for agglutination.
11. The titre to be taken is the highest dilution tube
to show agglutination.
Titres below 1/80 are of no significance (indicates
previous sub clinical infection) due to the endemicity of
the disease in the area.
Agglutination of O suspension indicates recent infection.
Recently vaccinated individuals will possess agglutinins
to S. typhi and para typhi. The agglutination will occur
with more than one suspension.
For proper interpretations of the widal test, two serum
samples separated by ten days should be tested and the
rising titre in the second serum sample is indicative of
active enteric fever.
II.Passive (indirect) agglutination
What is a difference between passive and
active agglutination?
In active agglutination you have a particulate Ag
+ Ab, since the Ag is particulate, large, when a
complex is formed it is visible.
In passive agglutination the Ag is soluble so it
must first be attached to something like latex
beads, red blood cells so when agglutination
occurs it can be seen with the naked eye.
Passive agglutiniation
include:
1. Pregnancy test.
2. ASO testing.
3. C- reactive protein testing.
4. Rapid plasma reagen test.
5. Infectious mononucleosis latex
agglutination test.
6. H. pylori testing.
1. Latex method pregnancy
testIt depends on the appearance of HCG in blood
& urine of pregnant females.
Method: Drop of latex particles coated with
anti HCG + drop of urine
Agglutination
2. ASO ( anti streptolysin O)
testingSlide agglutination test performed for patients with
streptococcal ß hemolytic infections.
Latex particles coated with SO antigens + serum
containing Abs
Agglutination
3. CRP (C- reactive protien)Slide agglutination test.
Latex particles coated with specific antihuman
C- reactive protein antibodies + serum
containing CRP.
Agglutination
4. RPR (Rapid plasma reagen)Slide test used for diagnosis of sypillis.
Syphillis is a venereal disease caused by Treponema
pallidum.
The test measures antibodies against the lipoidal
material released from the damaged host cell, so it is
not very specific.
Specific Ag containing micro particulate carbon +
Serum containig the Abs
Agglutination
5. Infectious mononucleosis latex agglutination (IM Latex Test)
Latex agglutination slide test used for
determination of infectious mononucleosis
in non-diluted serum or plasma.
Infectious mononucleosis is a disease caused
by the Epstein Barr virus.
The IM latex test provides: Infections with IM is associated with
production of heterophile antibodies.These heterophile antibodies can cross react
with glycoprotien on the surface of the sheep or bovine RBCS .
Latex particles coated with partially purified glycoprotein (from bovine red blood cells). + Serum which contains heterophile antibodies associated with IM
Agglutination.
Agglutination identifies the presence of
the heterophile antibodies in the sample
(It is a non specific test that can be used
for screening of patients).
6. One step H. pylori Test
H. pylori is a small, spiral-shaped bacterium
that lives in the surface of the stomach and
duodenum.
This test is a rapid one step test for the
qualitative detection of antibodies to
Helicobacter pylori (H. pylori) in human
serum or plasma.
The one step H.pylori Test device is a simple
test that utilize a combination of H.pylori
antigen coated particles and anti-human IgG
to detect H.pylori antibodies in serum or
plasma.
In this test procedure:
Anti human IgG is immobilized in the line region of
the test.
Specimen contain H.pylori antibodies will be added to
the “specimen well” of the device, it will react with
H.pylori antigen coated particles in the test.
This mixture migrates chromatographically along the
length of the test device and interacts with the
immobilized anti-human IgG.
If the specimen contains H.pylori antibodies, a colored
line will appear in the test line region indicating a
positive result.
PrecipitationThis is an Ag-Ab reaction in which the Ag is
soluble (bacterial toxin).
When antigens and antibody mixed in the
proper proportion, they form large
macromolecular complexes called precipitates.Precipitation
Tube precipitation Agar gel diffusion
Double diffusion Single diffusion
Tube precipitationUsed in estimation of C3. CRP,
immunoglobulins.
Agar gel diffusionDiffusion of Ag & Ab is allowed to occur in agar gel.
1. Double diffusion
Ag & Abs are put in wells in the agar.
Used in grouping of Streptococci
( groups: A, B, C, D).
Eleck’s test: for diphtheria
Strain toxin production.2. Single diffusion
Ab is mixed with the agar
before pouring in plates and
the Ag is
placed in agar wells
Precipitation lines