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