In vitro Antigen-

Antibody-Reactions
 I- Agglutination Reactions:
Requirements:
1-The antigen(Ag) has to be in particulate
form.
2-The presence of electrolytes (saline).
Agglutination can be performed on slides,
in tubes, or even in plastic plates with
wells (micro-titration plates).
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Agglutinins
Agglutination
 Types:
1-Slide agglutination:
i- Identification and typing of isolated bacteria
(Serotyping):It is a quick reliable test, if the
proper antibody (Ab ) is available.
Two drops of saline are placed on a clean slide.
A suspension of the isolated organism is
prepared in both drops. To, one drop, a drop of
known serum (Ab) is added and mixed, the other
is left as a control.
Clumping occurs if the serum is specific to the
organism.
Slide agglutination
ii) Blood grouping: Two drops of blood are
placed on a clean slide. To one drop, anti-
A is added, and to the other drop, anti-B is
added and mixed. Group A red cells will
agglutinate with anti-A serum. Group B will
agglutinate with anti-B serum. Group AB
will agglutinate with anti-A and anti-B sera.
Group O will not agglutinate with any of
the antisera. This is an example of active
haemagglutination.
2-Tube agglutination : It is a semiquantitative
test, used mainly to detect Abs in patients' sera.
To serial dilutions of patient's serum, a constant
amount of known bacterial suspension is added,
then incubated for the required time at the
desired temperature. The tubes are examined
for the presence of agglutination. The last tube
showing agglutination is the end point.
• The reciprocal of the dilution of this end
point is the titre, for example, if the dilution
is 1/400 then the titre is 400. e.g.Widal
test for the diagnosis of enteric fever
(typhoid and paratyphoid) and Weil - Felix
test for the diagnosis of typhus fever are
examples of such tube agglutination tests .
Tube agglutination
• 3-Haemagglutination(Passive&Active):
i) Passive haemagglutination: Red cells
sensitized with the Ag can be used to
detect antibodies directed to these
antigens that are prepared in a special
way and used to coat the RBCs. If
antibodies are present, haemagglutination
will occur. Example TPHA for detection of
treponemal antibodies.
Hemagglutination
Hemagglutination(tube method)
• ii) Active haemagglutination: Some viruses
for example influenza virus have
haemagglutinin spikes on their surface
that are capable of haemagglutinating
RBCs. This character can be used in a
haemagglutination inhibition test to detect
viral antibodies.
• 4-Latex agglutination:
Particles such as latex beads could be
conjugated to antibodies and used for
agglutination of antigens in solutions. These
latex agglutination tests are the basis of a
number of kits for the rapid identification of
pathogenic bacteria and fungi such as
N.meningitidis and Cryptococcus neoformans.
It is also possible to conjugate latex particles
with the Ags and used for detecting antibodies in
patients' sera or other body fluids.
Latex agglutination
Latex agglutination
Antigen & Antibodies
• 5-Coagglutination test:
This assay uses Staphylococcus aureus
as an antibody – carrying particle. It takes
the advantage of the ability of protein A on
the surface of this bacterium to bind the Fc
portion of IgG antibodies. In
coagglutination assays, the binding of the
antigen to the antibody immobilized via
protein A to the bacterial surface causes
the S.aureus cells to agglutinate.
Coagglutination
• 6-Coomb' s test:
i) Direct Coomb' s test: It detects Abs
already present on red cells. In
erythroblastosis foetalis (E.F.), baby' s red
cells are coated with anti- Rh Abs. The
cells are washed, then anti-human globulin
is added resulting in agglutination.
Coomb’s test
 Incomplete or blocking Abs
Coombs (Antiglobulin)Tests
• Direct Coombs Test
– Detects antibodies on erythrocytes

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Patient’s RBCs Coombs Reagent

(Antiglobulin)
ii) Indirect Coomb' s test: It detects anti-
Rh Abs in mother's serum.
Mother 's serum suspected to have anti-
Rh Abs is incubated with Rh positive red
cells. Abs will bind to the RBCs but
produce no agglutination (similar to E.F.).
The cells are then washed , and anti-
human globulin prepared in rabbit is
added. If the serum contains Abs,
clumping of the cells will occur.
 Coombs (Antiglobulin)Tests
• Indirect Coombs Test
– Detects anti-erythrocyte antibodies in serum

Step 1
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Patient’s Target
Serum RBCs

Step 2

+ 
Coombs Reagent
(Antiglobulin)
 II -Precipitation Reactions:

• Requirements:
1-The Ag has to be soluble(in
solution).
2-The Ag and the Ab have to be in
optimal concentration.
• Types:
• i) Slide precipitation: as RPR and VDRL
for diagnosis of syphilis.
• ii)Tube precipitation :as Lancefield test
for grouping streptococci ( Ring test).
iii)Agar ( Gel) Diffusion tests:
A) Double immunodiffusion :
a) Agar gels are poured in plates or on
slides. Wells are punched in the gel. The
Ag is placed in one well, and the Ab in
another well. Both the Ag and Ab will
diffuse, and at the site of optimum
proportion a precipitation band will occur.
Double Immunodiffusion
• b) Toxin-antitoxin reaction as in Elek's
test to diagnose toxigenic strains of
C.diphtheria. Strains isolated from carriers
are not considered pathogenic unless
proved to produce the toxin.
Elek’s test
• Elek's test: Strip of filter paper is immersed in
diphtheria antitoxin, then placed on the surface
of serum agar plate. A heavy inoculum of
diphtheria bacilli isolated from case or carrier is
inoculated at right angles to the strip. The plate
is examined after two days incubation at 37ºC. If
the organism is toxigenic, the toxin will diffuse
and will precipitate with the antitoxin diffusing
from the filter paper and a line of precipitation
will be formed at the optimum concentration.
Positive and negative strains should also be
included in the test as controls.
 Double immunodiffusion could be
electrically accelerated to give a rapid
result in half an hour.
This is termed
counter current immunoelectrophoresis.
B) Single (Radial) immunodiffusion:
The Ab is incorporated in the agarose gel
plate. The Ag is placed in a well punched in the
agarose. The Ag diffuses in all directions and a
precipitation ring will form around the well. The
diameter of the ring is proportional to the Ag
concentration. This is a quantitative method to
measure the concentration of the unknown Ag.
Single radial immunodiffusion
Single radial immunodiffusion
• This technique is used for estimating the amount of
different Ig classes in the serum for example IgG. An
agarose plate containing anti-IgG is used.

• IgG preparations of known concentration, e.g.(2.5, 5.0

and 10.0 µg/ml) are placed in the first three wells, the
unknown sera are placed in the other wells. The plate
is left for two days, then the squares of the diameter
of the rings containing the known concentrations are
plotted against their concentration .

• By measuring the diameters of the unknown sera,

one can get out the concentration of the unknown
Ag.
• Single immunodiffusion could be
electrically accelerated to get rapid results
in half an hour. This is termed rocket
electrophoresis.
C) Immunoelectrophoresis:
It is electrophoretic separation of the serum into its
components followed by immunoprecipitation by specific
antisera.
It is done by separating different Ags in an agarose gel
poured on a glass slide by placing an electric charge
across it. The pH is chosen so that positively charged
proteins move to the negative electrode, and the
negatively charged proteins to the positive electrode.
Then an antiserum(Ab) is allowed to diffuses from a
trough cut into the agar along the line of protein
migration . As each Ag meets its specific Ab, a curve of
precipitin forms in the agarose ( the Ags and Abs form
precipitin arcs). This method allows the study of
complicated mixtures of Ags as found in serum.
Immunoelectrophoresis
 III-Complement Fixation Test
(C.F.T):
• This is an antigen antibody reaction that
occurs in the presence of a third
component known as the complement.
The antigen reacts with its specific
antibody and the resulting complex fixes
the complement.
• The complement is a complex protein
component of normal serum and body
fluids (except urine and C.S.F.) of man
and other animals. It is heat labile and is
destroyed by heating at 56ºC for 30
minutes. The guinea pig serum is a
common laboratory source of complement.
• The complement can be fixed by Ab after
it binds with its specific Ag giving Ag-Ab
complex. If the Ag-Ab complex is red cells
coated with their antibodies , the fixed
complement causes haemolysis of red
cells.
The C.F.T. is used in the diagnosis of
many diseases by detecting
C.F.antibodies in the serum of patients, as
in syphilis, whooping cough, chronic
gonorrhoea, typhus and other diseases as
well as many viral infections.
• Principle of C.F.T:
The heated serum of a patient (unknown
antibody) is added to the known antigen,
then the standardized complement is
added. If the serum contains antibodies
specific to the added antigen, they will
unite together and fix the complement. If
the serum is negative for antibodies, no
Ag-Ab complex is formed and the
complement is not fixedi.e.left free in
solution.
• Up to this stage of the test there is no visible
phenomenon to show if the complement is fixed
or not(no visible reaction).
• A second step should be done in which an
“indicator system” is added made of sheep red
cells coated with their antibody. If the
complement is free (as with negative sera), it will
cause haemolysis of red cells.
• Thus, haemolysis means negative serum. If the
complement is fixed (as with positive sera); no
haemolysis occurs. Thus no haemolysis means
positive serum.
 IV- Antibody (Ab) –labeled
assays:

1-Fluorescent Antibody techniques:

• A)Direct immunofluorescence(D I F):
The Ag to be detected is present in a smear or
frozen tissue section.
The fluorescein labeled specific Ab is added, and
then the slide is washed to remove the unbound
Ab. The slide is then examined by an ultraviolet
microscope(U.V.). The background is dark and
areas with bound fluoresceinated Ab fluoresce
green.
• B) Indirect immunofluorescence(I I F):
i-For detection of antibodies:
This test can be used for detecting
specific antibody in patient's sera. It is
done by placing the Ag on a clean slide,
the patient' serum is added, then the slide
is washed. Antihuman gamma globulin
labeled with fluorescein is added, then the
slide is washed and examined by the
ultraviolet microscope.If green
fluorescence is seen, it means that the
patient has specific Ab to the Ag.
ii-For detection of antigens:
Specimen suspected to contain the
antigen is fixed on a slide, specific
antibody is added, then the slide is
washed. Antispecies antibody conjugated
with fluorescent dye is added, then
washed and examined by U.V microscope.
The indirect IF is more sensitive than the
direct method, and is more economic and
it gives more magnification to the reaction.
Indirect Immunoflourescence
• C-Flow cytometer for the detection of
leukocyte antigens ( markers or cell
surface molecules ).
-It is an apparatus capable of analyzing
single cells using monoclonal antibodies
that are conjugated with different
fluorochromes (e.g. fluorescein
isothiocyanate for green light emission,
rhodamine for red /orange light emission)
as they pass through an orifice at high
velocity.
• -This method is termed leukocyte
phenotyping based on their size and
intracellular granules.The data are
analysed by computer.
• -It allows us quantify B-cells, T-cells,
monocytes and granulocytes within
different sites in the body ( blood, bone
marrow, spleen, lymph nodes ) in tissue
sections or in fresh cell suspensions.
• -In addition , we can enumerate subsets of
these cell types .For example, T-
lymphocyte subsets that differ in their
functional properties can be distinguished
phenotypically.
• -The surface phenotype can also provide
clues as to the differentiation state of the
cell.
• Precise quantitation of T and B cells in
human peripheral blood has made
important contributions to our
understanding of immunodeficiency
disorders, autoimmune diseases, tumour
immunity, and immunity to infections.
 2- Enzyme Linked
Immunosorbent Assay(ELISA):
• Principle of the test:
It is based on two assumptions that
antigen or antibody can be attached to a
solid phase (plastic surface), but still it
retains its immunological activity and that
either antigen or antibody can be linked to
an enzyme such as alkaline phosphatase
or peroxidases and the complex retain
both immunological and enzymatic activity.
• Types:
A-Sandwich method ELISA( Double antibody)
for detection and measurement of unknown
Ag.
1.The wells in polystyrene plastic plates are coated
with specific antibody to the antigen.Plates are
washed.
2. The test solutions thought to contain antigen are
incubated in the sensitized wells. Washing
removes unreacted materials, and any antigen
remains attached to the immobilized antibody on
the plastic surface.
3.The conjugate consisting of enzyme- labelled
specific antibody is then incubated in each well.
This will react with any antigen already
“captured” by the antibody on the well surface. A
further washing removes excess conjugate.
4.Finally, the enzyme substrate is added. Its rate
of degradation depends on the amount of
enzyme- labelled antibody present and that, in
turn, depends on the amount of antigen in the
test sample. The enzyme substrate is chosen to
give a colour change upon degradation, and this
can be assessed visually or measured in a
spectrophotometer (ELISA reader).
 B-Indirect ELISA for detection and
measurement of unknown antibody.
1.Wells of polystyrene microplates are
sensitized by passive adsorption with the
relevant antigen; the plates are then
washed.
2.The test samples are incubated in the
sensitized wells and the plates
are again washed; antibody present
reacts with the immobilized antigen on
the well surfaces.
 3.Enzyme-labelled anti-human Ig conjugate is
incubated in the wells, this reacts with any
“captured” antibody in step 2. Enzyme substrate

is added and the plates are incubated, the rate

of degradation of the substrate is indicated by a
colour change, which is proportional to the
antibody concentration in the test samples in
step 2.
4.The reaction is stopped, and the colour
changes is assessed visually or in a
spectrophotometer.
C-Competitive ELISA; both labelled
(known) and unlabelled (unknown) Ag
compete for the active site of the
enzyme.
3-Radioimmuno assay (RIA):
*Principle:It is based on labelling either the
Ag or the Ab with radioactive substance
such as iodine or tritiated thymidine. One
of them should be known
(radioactivelabelled) and the other is
unknown.
The amount of radioactivity is measured
by gamma or beta counter.
RIA
 Types:
i) Liquid phase RIA:The test is done in
solution.
ii) Solid phase RIA:Ag is coated onto a
plastic plate (or tube), then the plate is
washed, and the test Ab is added. The
plate is washed , then radioactive labeled
anti-species Ab is added, then the
radioactivity is measured.
iii) Competitive RIA.
4-Chemiluminescence:
Principle: It depends on labelling the Ab
with acetylaminofluorene ;a luminescent
substance detected by luminometer.
 Intradermal skin tests
I-Neutralization tests:

These are in- vivo manifestations of

Ag-Ab reactions.
A-Schick test: To test the susceptibility of
a person to diphtheria. A small amount of
the diluted diphtheria toxin, is injected
intradermally in one forearm and in the
other a heated toxin. The development of
a localized erythema indicates that, there
is no antitoxin to neutralize the toxin, so
the person is susceptible to suffer from
diphtheria.
B-Dick test:To test the susceptibility of a
person to get scarlet fever.The test is
done exactly as the above, but using the
erythrogenic toxin of Streptococcus
pyogenes.
C-Schultz-Charlton reaction:Used for
the diagnosis of scarlet fever. A small
amount of the antitoxin (anti-erythrogenic
toxin) is injected in the red rash. If
blanching occurs, it means that the rash
is caused by the erythrogenic toxin, and
that the person has scarlet fever.
 II-Hypersensitivity tests:
a) Immediate type skin test:
As testing for the sensitivity to penicillin.
A small amount of penicillin is injected in
the forearm. An immediate wheal and flare
indicates that the person is hypersensitive
to penicillin.
b)Delayed type skin test:
The Ag is injected intradermally, and the
test is read after 48-72 hours.
Development of induration and redness at
the site of injection indicates a positive
test. Delayed type hypersensitivity tests
are induced by sensitized lymphoid
cells(cellular reaction). Examples:
tuberculin, lepromin, brucellin and Ducrey
tests.