ANTIGEN

ANTIBODY
REACTION
Presented by,
July Mary Johnson
Definition

Antigen combines with antibody in an observable manner and the reaction

is specific and irreversible.
Characters:

1. Reaction is specific; an antigen combines only with homologous antibody

and vice versa. Cross reaction occurs due to antigenic similarity

2. Entire molecule of antigen and antibody will react not the fragments

3. Reaction is firm and irreversible due to affinity and avidity

• Affinity : defined as intensity of attraction between antigen and
antibody molecules

• Avidity: defined as the binding strength after formation of antigen-

antibody complex
 Uses
1. In the body or In Vivo

• It form the basis of immunity against infectious disease

• It may lead to tissue injury in some hypersensitivity reaction or autoimmune disease

2. In Laboratory or In Vitro or immunoassay

• For diagnosis of infections

• Epidemiological studies

• Detection and quantitation of either antigens or antibodies

 Marrack’s Lattice hypothesis
• When same amount of antiserum (antibody) is mixed with
increasing quantities of antigen in different tubes following
reaction occurs

1. Prozone: In the preceding tubes due to excess antibody no

reactions occurs

2. Zone of equivalence: both antigen and antibody present in

optimal or equal amount, so the reaction occurs

3. Post zone: the later tubes antigen is excess, and no

reaction occurs
Types of Ag-Ab Reactions

1. Precipitation

2. Agglutination

3. Complement fixation test

4. Immunofluorescence

5. Radioimmunoassay (RIA)

6. Enzyme linked immunosorbent assay (ELISA)

7. Immunochromatography
Precipitation reaction

• When a soluble antigen reacts with its antibody in presence of electrolyte

(NaCL), at an optimum temperature and pH, the Ag-Ab complex forms an
insoluble precipitate, and it is called as precipitation

• The precipitate usually sediment at bottom of the tube

Flocculation: when instead of sedimenting, the precipitate is suspended as

floccules, this is called flocculation. Modified form of precipitation

E.g., VDRL (Venereal disease research laboratory) test for syphilis

Precipitation reaction
Application:
May be used for either qualitative or quantitative test
Very sensitive can detect as little as 1μg of protein antigen
Used in
• Identification of bacteria e.g., Lancefield's grouping of Streptococcus
• Detection of antibody for diagnostic purpose e.g., VDRL in syphilis
• Forensic application in identification of human blood and seminal stains
• Testing for food adulterants
 Types of precipitation reaction
1. Ring test
2. Flocculation test
3. Immunodiffusion test
• Single diffusion in one dimension ( Oudin procedure)
• Double diffusion in one dimension (Oakley- Fultrope procedure)
• Single diffusion in two dimension (radial immunodiffusion)
• Double diffusion in two dimension (Ouchterlony procedure)
• Electroimmunodiffusion
Precipitation reaction
1.Ring test

• Antigen solution is layered over an antiserum in a

narrow tube

• A precipitation ring appears at the junction of two

liquids

• E.g., C-reactive protein test (CRP) and streptococcal

grouping
Precipitation reaction

2. Flocculation test

Slide test: A drop of antigen solution is added to a

drop of patient's serum on a slide & mixed by
shaking, floccules appear. VDRL test for syphilis

Tube test: Kahn test and standardization of toxins &

toxoids is done by tube flocculation test
Precipitation reaction

3. Immunodiffusion test

These are precipitation tests in gel (1% agar gel)

Advantages:

• Reaction band is visible ,stable and can be stained for preservation

• The number of different antigens can observe because each Ag-Ab reaction rises a
line of precipitate

• Cross reacting or non reacting Antigens can also be identified

Various immunodiffusion tests

1. Single diffusion in one dimension ( Oudin

procedure)

• The antiserum (antibody) is incorporated in

melted agar and mixture is poured into a tube
and allowed to solidify.

• Antigen solution is placed above the agar.

• The precipitation band appears in the agar.

Various immunodiffusion tests
2. Double diffusion in one dimension (Oakley- Fultrope
procedure)

• Antibody (antiserum) is incorporated in agar, poured into a

tube and allowed to harden

• A second layer of agar without antibody is placed above

and allowed to solidify

• Antigen solution is placed above the agar

• The precipitin band appears in the plain agar column.

Various immunodiffusion tests
3. Single diffusion in two dimension (Radial
immunodiffusion)
• An agar containing an appropriate antiserum (antibody) is
poured in plates
• Carefully circular wells are cut and removed from the plates
• Known antigen is added to the well
• As the antigen diffuses radially, a ring of precipitate will form
in the area of optimal antigen – antibody concentration
• The ring diameters are measured, diameter of the ring is
directly proportional to the concentration of antigen
 Various immunodiffusion tests
4. Double diffusion in two dimension (Ouchterlony
procedure)
• Agar gel is pored on a slide
• Wells are cut, to the center well antibody (antiserum)
is placed, and surrounding wells are filled with
different antigen
• If two adjacent antigens are similar, the line of
precipitate will fuse
• If they are unrelated lined cross each other
• Spur formation shows cross reaction or partial identity
Various immunodiffusion tests
5. Electro immunodiffusion
Immunodiffusion can be speeded up if antigen and antibody are driven by
electricity
Two types
a) Counterimmunoelectrophoresis (counter current immuno
electrophoresis, CIE/ CIEP)
b) Rocket electrophoresis
 Electro immunodiffusion
a) Counterimmunoelectrophoresis (counter current immuno
electrophoresis, CIE/ CIEP)

• Slide slides are layered with agar gel and wells are cut on the
surface

• One well is filled with Ag and other with Ab

• When electric current is passed, antigen & Ab moves towards each

other and raises a line of precipitation

• Very sensitive and result within 30 minutes

 Electro immunodiffusion
b. Rocket immunoelectrophoresis
• This technique mainly applied for quantitation of antigens
• The antiserum to the antigen is mixed with agar gel and layered
on the slide
• The wells are punched and filled with increasing concentration
of antigen
• It is then electrophorized
• The precipitation is in the form of cone (rocket appearance)
• The length of rocket like structure represent the concentration
of antigen
AGGLUTINATION
Agglutination

• It is an antigen –antibody reaction, in which particulate antigen

combines with antibody in presence of electrolyte at an optimum
temperature and pH, resulting in visible clumping of particle

• Agglutination is more sensitive than precipitation for detection of

antibodies

• Agglutination reaction takes place better with IgM than IgG

Agglutination
Types

I. Slide agglutination

II. Tube agglutination

III. Antiglobulin (Coombs) test

IV. Passive agglutination test: latex agglutination & hemagglutination

 Agglutination
1. Slide Agglutination

• A drop of antigen is mixed with appropriate antibody on a

slide or tile, mixed well by shaking or rocking, clumping
occurs instantly or within seconds, then its positive test

• If clumping occurs after a minute, it may be due to drying

& should be discarded

• Controls should be maintained

• Uses: blood grouping, bacterial identification

 Agglutination

2. Tube agglutination test

• For quantitative determination of antibodies

• Serum is diluted serially by doubling the dilution in test tube

• An equal volume of particulate antigen is added to all tubes

• The highest dilution in which agglutination occurs is known

as the titre

• Uses: Widal test, Weil felix reaction

 Agglutination

3. Antiglobulin (Coombs) test

• For detection of incomplete anti-Rh antibodies

• When sera containing incomplete anti-Rh antibodies are mixed

with corresponding Rh-positive erythrocyte, incomplete
antibody coats the RBC without causing agglutination

• When this coated RBCs are treated with antiglobulin or

Coombs serum (rabbit antiserum against human gamma
globulin), cells are agglutinated
Agglutination

• Types of coombs test

i. Direct Coombs test: in vivo sensitization

ii. Indirect Coombs test: invitro sensitization

• Uses

To detect anti Rh antibodies

For demonstrate incomplete antibodies e.g. brucellosis

 Agglutination

5. Passive Agglutination test

• A precipitation reaction can be converted into agglutination test by attaching

soluble antigen to the surface of carrier particles such as latex, bentonite or RBC

• Such tests are called as passive agglutination test

• This conversion is done because agglutination test is more sensitive

• When antibody is attached to carrier molecule the test is known as reverse

passive agglutination
Agglutination

i. Latex agglutination test

• Polystyrene latex particles are widely employed for adsorb

several types of antigens

• Latex particle can also be coated with antibody for detection

of antigen

• Test is convenient, rapid and specific

• Used for detection of hepatitis B antigen, ASO, CRP, RA factor

COMPLEMENT FIXATION TEST
Complement Fixation Test (CFT)

• The antigen- antibody complexes can “fix” complement but this

reaction has no visible effect

• To detect the fixation of complement, an indicator system consisting

of sheep erythrocytes coated with amboceptor (rabbit antibody to
sheep erythrocytes) is used
 Complement Fixation Test (CFT)
Principle

• CFT depends upon two different systems, namely test and indicator system

• A test system includes antigen, antibody and a complement

• The indicator system involves the addition of sheep erythrocytes with amboceptors to test
whether a free complement is present or not

• If there will be specific binding between an antigen and antibody, a complement will fix with
the Ag-Ab complex, no lysis

• In case of antibodies absence, there is no fixation, a complement will remain free and lyse
the sheep RBCs
 Complement Fixation Test (CFT)

Procedure

1st step (Test system)

Antigen (soluble/ particulate) + inactivated test
serum (56ᵒC for 30 mins) + complement (from fresh
guinea pig serum) → add all together

2nd step (indicator system)

To this add sheep receptor coated with amboceptor
Complement Fixation
Test (CFT)
Interpretation

• If the test serum is positive for antibody there is no

free complement in serum to bind to amboceptors
bound on sheep RBCs there is no hemolysis.

• If the serum is negative for antibody the free

complements are fixed to amboceptors bound on
sheep RBCs --- Results in haemolysis.
Complement Fixation Test (CFT)
Uses
For diagnosis of Syphilis, Rickettsia, Chlamydia, Brucella
IMMUNOFLUORESCENCE
Immunofluorescence
• Fluorescence is the property of certain dyes which absorb rays of
particular wavelength (UV light) & emit rays with different wavelength
(visible light)
• Fluorescent dyes can be conjugated to antibodies and these
“labelled” antibodies can be used for detection of antigen
• Commonly used fluorescein dyes are fluorescein isothiocyanate &
lissamine rhodamine, exhibiting blue green and red orange
fluorescence respectively
• Two types: direct and indirect
Immunofluorescence
1. Direct immunofluorescence test

• Used for detection of unknown

antigen
Specimen(unknown antigen)
+
Labelled specific antibody
↓
Fluorescence observed
(antigen is present)
Immunofluorescence
Direct immunofluorescence test
Uses
• Employed to detect bacteria, virus, fungus or other antigens in blood,
urine, feces, tissue & tissue & another specimen
• Sensitive method for diagnosis of rabies
Disadvantage
• Separate specific fluorescent labelled antibody has to be prepared
against each antigen to be tested
 Immunofluorescence
Used to detect the antibodies in serum or
body fluids
Principle
• A known Ag is fixed on the slide and
unknown antibody (serum) is applied to the
slide
• If antibody is present, it will form complex
• For detecting this Ag-Ab complex a
fluorescein tagged antiglobulin is added
• Positive test produce fluorescence
 ENZYME LINKED
IMMUNOSORBENT ASSAY (ELISA)
Enzyme Linked Immunosorbent
Assay (ELISA)
• Applied for detection of wide variety of antibodies
and antigens

• The principle is similar to immunofluorescence here

instead of dye we use enzymes

• This enzymes act on substrate to produce colour

• It is done in solid phase, the ELISA plate (96 wells)

 Enzyme Linked Immunosorbent
Assay (ELISA)
1. Direct ELISA

• Used for detection of antigen in test serum

• Step 1: Wells of microtitre plate are empty, not precoated
• with Ag or Ab
• Step 2: Test serum (containing antigen) is added in to
• the wells. Antigen becomes attached to the plate
• Step 3: The enzyme-labeled primary
• antibodies are added.
• Step 4: a substrate-chromogen system is
• added and color is measured.
 Enzyme Linked Immunosorbent
Assay (ELISA)
2. Indirect ELISA
• Used to detect antibodies
• The wells of microtiter plate is pre-coated
with Ag on addition of test serum, the Ag-Ab
complex will be formed
• A secondary anti human immunoglobin with
enzyme is added
• To detect this binding substrate chromogen
complex is added, colour produced in positive
test
Enzyme Linked Immunosorbent
Assay (ELISA)
3. Sandwich ELISA

• For antigen detection in a specimen, the wells of mitrotitre plate is coated with specific antibody
against antigen to be detected

• Specimen to be tested is added to the wells, if antigen present in specimen it will bind with coated
antibody

• To detect this Ag-Ab complex enzyme conjugated antibody is added, this will bind with the antigen
already attached to coated antibody

• A substrate is added to know the binding of conjugated antiserum to Ag-Ab complex

• If positive colour will be produced

• Negative no colour produced

Enzyme Linked Immunosorbent
Assay (ELISA)
4. Competitive ELISA

• It is used for detection of HIV antibodies

• Positive result shows no colour whereas appearance of colour indicates a

negative test

• In this test there are 2 specific antibodies, one conjugated with enzyme &
other present in serum

• Competition occurs between 2 antibodies for same antigen

Enzyme Linked Immunosorbent
Assay (ELISA)
Procedure
• The microtiter plate wells are coated with HIV antigen
• Sera to be tested is added to these wells & incubated at 37ᵒC & then washed
• If antibodies are present, Ag-Ab reaction occurs
• To detect this reaction, enzyme labelled specific HIV antibodies are added
• There is no antigen left for these antibodies to act on it
• These antibodies remain free & washed off during washing
• Substrate is added but there is no enzyme to act on it
• Therefore, positive result is colorless
• If serum to be tested is negative for antibodies, antigen is there to combine with
enzyme conjugated antibodies & enzyme acts on substrate to produce colour
Competitive ELISA
IMMUNOCHROMATOGRAPHY
Immunochromatography

• Also known as lateral flow immunoassay, it’s a rapid immunoassay

• Both antigen & antibody can be detected

• It can be completed within 30 mins

• It is a strip-based test

• The strip contains a chromatographic pad with three zone: sample

pad, conjugate pad & capture line
 Immunochromatography
• The specimen (e.g., serum, urine) containing the antigen to be detected is placed on the sample
pad, which soaks up the specimen fluid.

• The fluid then migrates to the conjugate pad, which contains conjugated antibodies (conjugated
with gold, colored latex, or a chromophore) directed against the antigen.

• Here, the antigen-antibody-conjugate complex is formed. Ag-Ab complex continues to migrate

across the membrane until it reaches the capture zone where the complex will bind to
immobilized antibodies.

• As more and more Ag-Ab complexes are captured at the “test” line, the line becomes visible on
the membrane.