Practical

Microbiology

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Antigen Antibody interactions:
reversible binding of antigen to antibody by
formation of weak bonds between the epitopes of
the antigen and the antigen binding sites of the
antibody.

Ag-Ab reactions in vitro are known as serological

reactions.

Ag-Ab assays are used for:

1. Identification of microorganisms
2. Diagnosis of disease

These assays can be either:

1. Qualitative to detect the presence of antigen
or antibody.
2. Quantitative to measure the level
concentration or of antigen or antibody.

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Specificity
the ability of an individual antibody to react with only
one epitope or the ability of a population of antibody
molecules to react with only one antigen.

Cross reactivity
occurs when epitopes on different molecules of
different sources are identical → antibody directed
against one antigen also reacts with another. These are
known as heterophile Ab and heterophile Ag

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Factors and forces involved in Ag-Ab reactions
1. Environmental factors:
a. The presence of electrolytes and their concentration (NaCI).
b. pH: the reactions are weaker in acidic (pH4) or alkaline (pH10).
c. The temperature: the rate of antigen-antibody reactions
increases with rising of the temperature up to a maximum
temperature of 55°C

2- The physical state of the antigen: soluble or particulate.

This will affect the visible result of the reaction
antibody + soluble antigen = precipitation
the same antibody + particulate antigen = agglutination.

3. The presence of a third factor → complement → affects the end

result of reaction.
Antigen + antibody + complement → complement fixation and lysis
of cell Antigen.

4. Concentration of antigen and antibody "Zone phenomenon"

The amount of precipitate is determined by proportion of Ag and Ab.
The maximum amount of precipitates forms when optimal
proportions of Ag and Ab combine → Zone of equivalence. Page | 4
Excess of either Ab or Ag interferes with precipitation → Prozone.
Ab Titer: Highest dilution or lowest
concentration of serum that gives
visible reaction with the antigen.

Rising titer: when the level of

antibody in the patient serum is
measured twice with one week
interval, an increase in the antibody
titer of 2 folds or more indicates active
infection.
It is more diagnostic

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 Precipitation Reactions
It is the interaction between an Ab and a
soluble Ag.
The Ab causing precipitation is called
precipitin

A. Precipitation in tubes:
rarely used now
a) Test tubes
b)Capillary tubes

B. Precipitation in agar:
a) Single radial immunodiffusion
b)Double diffusion (Ouchterlony)
c) Immunoelectrophoresis

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1-Single radial immunodiffusion:
 Ab is incorporated into the agar gel before pouring it in
the plates and different dilutions of the Ag are placed in
holes punched into the agar.
 As the Ag diffuses into the gel, it reacts with the Ab → a
ring of precipitation is formed

Interpretation:
diameter of ring is proportional to the concentration of Ag.

Application: determination of:

1. immunoglobulin levels
2. C-reactive proteins
3. complements components

2- Immunoelectrophoresis:
Technique for the separation and identification of
mixtures of proteins, consisting of electrophoresis
followed by immunodiffusion Page | 8
3- Double diffusion (Ouchterlony):
❖ Compare between different antigens.
❖ Agar gel is poured on plates and wells are cut.
❖ The antiserum is placed in the central well and different antigens are poured in
surrounding wells.
❖ Both will diffuse in the agar → precipitations bands.

Applications:
1. Identification of toxigenic strains of bacteria → Elek’s test
2. Medicolegal applications to identify whether blood spots of human or animal origin.

Interpretation:
1. Identity occurs when two antigens share identical epitope.
a single curved line of identity
2. Nonidentity occurs when two antigens are unrelated
independent precipitin lines that cross
3. Partial identity occurs when two antigens share some epitopes but one or the
other has a unique epitope(s).
a line of identity with the common epitope(s) and a curved spur with the unique
epitope(s)

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 No
Identity Identity Partial
identity

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Elek’s test

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Elek’s test

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 Agglutination Reactions
When the Ag is particulate, the reaction of an
Ab with the Ag can be detected by agglutination
(clumping) of the Ag.
antibodies are called → agglutinins.

Particulate antigen may be

1. Bacterial cell
2. Parasite
3. Red blood cells

Agglutination reactions include:

A. Direct Agglutination
1- Qualitative agglutination test
2- Quantitative agglutination test
B. Passive (latex) agglutination
C. Coomb's test (Antiglobulin test)
1- Direct Coomb's test
2- Indirect Coomb's test
D. Hemagglutination inhibition
E. Heterophile agglutination Page | 13
A. Direct Agglutination:
❖Ag- Ab reaction that forms agglutination which is
directly visible.
❖Antigen is directly present on surface of a cell.

N.B. When the antigen is an erythrocyte, the term

hemagglutination is used.

1- Qualitative agglutination test:

❖To assay for the presence of an antigen or an antibody.
❖The antibody is mixed with the particulate antigen and
a positive test is indicated by the agglutination of the
particulate antigen
Example → blood grouping

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2- Quantitative agglutination test:
❖used to quantitate the level of
antibodies to particulate antigens.
❖In this test one makes serial dilutions of
a sample to be tested for antibody and
then adds a fixed number of red blood
cells or bacteria and determines the
maximum dilution which gives
agglutination.
❖The maximum dilution that gives
agglutination is called the Ab titer

Applications:
To assess bacterial infections.
A rise (fourfold rise) in titer of an antibody
to a particular bacterium indicates a specific
infection with that bacterium

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B. Passive (latex) agglutination:
It is an agglutination reaction in which inert particles → latex or RBCs are
coated with a soluble antigen (viral antigen, a polysaccharide or a hapten)
and used in an agglutination test.

1. Detection of C-reactive protein (CRP) in patient’s serum: Used for

diagnosis of rheumatic fever.
Serum (containing CRP) agglutinate latex particles coated with anti-CRP

2. Pregnancy test:
The presence of human chorionic gonadotrophin (HCG) in the urine of pregnant
female indicates pregnancy.
It is detected by Latex particles coated by anti-HCG

3. Rheumatoid factor (Anti-IgG):

Used for the diagnosis of Rheumatoid arthritis.
In this disease, patient’s serum has abnormal IgM antibodies raised against his
own IgG and hence called (Anti-IgG).
the latex particles used in this test are coated with IgG

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CRP detection

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Rheumatoid factor

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C. Coomb's test (Antiglobulin test):
two clinical blood tests → done to find incomplete
antibodies that cause autoimmune hemolysis of
red blood cells (erythrocytes).

1- Direct Coomb’s test:

❖Antibodies that bind to RBCs but do not cause
RBCs agglutination are sometimes referred to as
incomplete antibodies.
❖In order to detect the presence of non-
agglutinating antibodies coating the surface of
red blood cells, add a second antibody directed
against the immunoglobulin (antibody) coating
the red cells.
❖This anti-immunoglobulin can now cross link the
red blood cells and result in agglutination.

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2- Indirect Coomb's test:
❖This test is done to detect the presence of
incomplete antibodies present in the sample not
covering the surface of RBCs.
❖done by adding the RBCs with to serum → then
adding a second anti-immunoglobulin to cross link
the cells.

Applications of Coomb’s test:

Detection of anti-rhesus factor (Rh) antibodies.
Antibodies to the Rh factor generally do not
agglutinate red blood cells.

Red cells from Rh+ children born to Rh- mothers, who

have anti-Rh antibodies, may be coated with these
antibodies.
To check for this, a direct Coomb’s test is performed.
To see if the mother has anti-Rh antibodies in her
serum, an indirect Coomb’s test is performed

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D. Hemagglutination inhibition:
❖This test is used for diagnosis of certain
viruses like influenza and mumps
viruses which cause hemagglutination.
❖If a person is suspected having
influenza, his serum is mixed with a
known influenza virus (Ag) and then
RBCs are added.
❖If Ab is present in the serum, it binds
to the virus and prevents agglutination
of RBCs.
❖If Ab is absent, hemagglutination
occurs

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E. Heterophile agglutination
for diagnosis of infections based on detection of
heterophile antibodies in patient’s serum,

Paul Bunnel test:

Used for diagnosis of infectious mononucleosis.
In the sera of these patients, there is a
heterophile Ab which agglutinates sheep RBCs.

Weil Felix test:

Heterophile Ab in patients’ sera with rickettsial
infections agglutinates bacterial suspension of
proteus OX-19, 2 or OX-K.

Cold agglutinin test:

Heterophile Ab in patients’ sera with Mycoplasma Cold agglutination
pneumoniae infections agglutinates human group
O red cells at 4°C
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