ANTIGEN – ANTIBODY REACTIONS

Antibodies produced against antigens reacts specifically with that antigen in an observable
manner to form Ag-Ab complex.
 Ag- Ab reaction serve several purposes.
{-They form the basis of Ab- mediated immunity in infectious diseases.
Invivo{
-{Tissue injury in some type of Hypersensitivity and autoimmune disorders.

{-In lab they help in the diagnosis of infections.

Invitro{-In epidemiology , they assist to in the identification of infectious and non-infectious
agents.
{-In detection and quantification of either antigen or antibodies.
 Antigen-Antibody reaction in vitro are known as serological reactions.
 General features/characteristics of Ag-Ab reaction
 Reaction is specific, an antigen combines only with its homologous antibody and
vice-versa. However cross reaction may occur sometimes due to antigenic
similarity.
 The epitope makes contact specifically with paratope and are held together in
lock and key arrangement by complementarity.
 The reaction is firm, but reversible. The firmness of combination depends on the
affinity and avidity of the reaction.
 Affinity refers to the intensity of attraction b/w the antigen and antibody
molecule. Avidity refers to the strength of the bond after formation of antigen-
antibody complexes.
 Antigen and antibodies can combine in varying portions, because both antigen
and antibodies are multivalent, unlike chemicals with fixed valencies.
The reaction b/w antigens and antibodies occur in three stages.
 The primary stage: It is the initial interaction b/w the two, without any visible effects. This
reaction is rapid and obeys laws of physical chemistry and thermodynamics. The reaction is
reversible.
 The secondary stage :In most cases the primary stage is followed by secondary stage leading to
demonstrable events such as precipitation , agglutination, lysis of cells, killing of live antigens,
neutralization of toxin etc.
 The tertiary stage : some Ag-Ab reaction occurring Invivo, initiate chain reaction that lead to
neutralization or destruction of injurious antigens or tissue damage.
 The imp. Parameters of serological tests
 Sensitivity refers to the ability of the test to detect even very minute quantities of antigen or
antibody.
 Specificity refers to the ability of the test to detect reactions b/w homologous antigens and
antibodies only, with no other.
 Types of antigen antibody reaction

I. Precipitation reaction
 when soluble antigen combines with its specific antibody in the presence of
electrolyte (NaCl) at an optimal pH(7.4) and temp.(37 C), the Ag-Ab complex
forms an insoluble precipitate.
 The precipitate formed usually sediments at the bottom of the tube.
 Flocculation : when precipitate remains suspended as floccules instead of
sedimenting, the reaction is called flocculation.(Eg. VDRL test)
 The precipitation reaction can take place in liquid media or in gels such as agar,
agarose or polyacrylamide.
 Zone or prozone phenomenon
 The amount of ppt. formed is greatly influenced by the relative proportions of antigens
and antibodies.
 When same amount of antiserum (Ab) is mixed with increasing quantities of antigen
in different tubes, the ppt. found to occur most rapidly and abundantly in one of the
middle tubes in which the antigen and antibody are present in optimal/ equivalent
proportions (zone of equivalence).
 In the preceding tubes in which the antibody is in excess and in the later tubes in
which the antigen is in excess, the ppt. will be weak or even absent.

1. zone of Ab excess / prozone: Ab in excess quantity

2. Zone of equivalence: Both Ag and Ab are completely combined to form precipitate
and no Ag or Ab left.
3. Zone of Ag excess/post-zone: Some free Ags are found in excess.
Mechanism of precipitation (Lattice hypothesis)

 Marrack proposed the lattice hypothesis for precipitation.

 According to this concept, multivalent antigens combine with bivalent antibodies in varying
proportions, depending on the Ag-Ab ratio in the mixture.
 Precipitation results only when a large lattice is formed consisting of alternating Ag and Ab
molecules. This occurs in the zone of equivalence.
 In the zone of Ag or Ab excess, the lattice does not enlarge, as the valencies of the Ab and Ag
are fully satisfied.
Application of precipitation reaction
 Precipitation reactions are very sensitive and can detect as little as 1µg protein
Ag. The test may be done either as qualitative or quantitative test.
 Forensic application in the identification of blood and seminal stains.
 Grouping of Streptococci by the Lancefield technique.
 The detection of syphilis antibody in VDRL test.
 Standardisation of toxins and antitoxins
 To test toxigenecity in diphtheria bacilli (Elek’s gel ppt test)
 Food adulteration tests.
Types of precipitation reaction
1. Ring test: This is the simplest type of ppt. test consists of layering
the antigen solution over an antiserum in a narrow tube.
A ppt. ring forms at the junction of two liquids.
Eg: Streptococcal grouping by Lancefield technique.

2.Slide flocculation test: when a each drop of Ag and antiserum (Ab) are placed on a slide and
mixed by shaking, floccules appear.
Eg. VDRL slide test for syphilis.

3.Tube flocculation test: Kahn test for syphilis is an Eg. Of tube flocculation test that was once
used earlier in diagnosis of syphilis.
A quantitative tube flocculation test used for standardisation of toxins and toxoids.
4.Immunodiffusion tests
 These are the precipitation reactions performed in 1% agar or agarose gel.
 The reaction is visible as a distinct line /band of precipitation, which is stable and can be
stained and preserved.
 Immunodiffusion also indicates identity, non-identity and cross reaction b/w antigens.
 Immunodiffusion results are more sensitive and specific.
a) Single diffusion in one dimension (Oudin) The Ab is incorporated in agar gel in a
Test tube and Ag solution is layered over it. The Ag diffuses downward through
the agar gel, forming line of precipitation that appears to move downwards.
b) Double diffusion in one dimension (Oakley-Fulthrope procedure) : The Ab is incorporated
in agar gel, above which is placed a column of plain agar with top layer of Ag. The Ag and Ab
move towards each other through the intervening column of plain agar and form a line of ppt
where they meet at optimum proportion.
c) Single diffusion in two dimensions (Radial immunodiffusion):

 Here the Ab is incorporated in agar gel poured on a flat slide or petridish. The Ag is added to
the wells cut on the surface of gel. It diffuses radially from the well and forms ring-shaped
bands of precipitation (halo) concentrically around the well.
 The diameter of the ring gives an estimate of the concentration of the Antigen.
USES: To estimate the concentration of Ag or Ab.
Widely used for estimation of immunoglobulin classes in sera.
d) Double diffusion in two dimensions ( Ouchterlony method)

 This method is widely employed and used to compare different Ags and Abs.
 Agar gel is poured on a slide and wells are cut. The antiserum (Ab) is placed in the central
well, and different antigens in the surrounding wells.
 If two adjacent Ags are identical, the lines of precipitate formed by them will fuse.
 If they are unrelated, the lines will cross each other.
 Partial identity(cross reaction) is indicated by spur formation.
e) Immunoelectrophoresis
 This method combines electrophoresis and immunodiffusion.
 Used for testing normal and abnormal proteins in the urine and
blood.
 The technique is performed on agar or agarose gel on a slide,
with an Ag well and an Ab trough cut on it.
 The serum containing Ag mixture is added to the well and
electrophoresed for about half an hour.
 Depending on charge and size they are pulled by electric field to
different locations on the gel.
 Then electric field is turned off and antiserum is added to trough
and allowed to diffuse for 18-24 hrs.
 Ags and Abs diffuse each other forming a series of bands of pptn.
f) Electroimmunodiffusion
 Immunodiffusion can be speeded up if Ags and Abs are driven by electricity.
a) Counterimmunoelectrophoresis (CIE)

 This involves simultaneous electrophoresis of the Ag and Ab in agar gel in opposite direction
resulting in a precipitation line at the point b/w them.
 This method produced visible pptn lines within 30 mns. and is more sensitive than std.
immunodiffusion technique.
 Used for detection of specific Ags of Cryptococcus and Meningococcus in CSF.
 Detection of various microbial Ags from urine, blood and body fluids.
ii) Rocket electrophoresis
( one dimentional single
Electroimmunodiffusion)

 Mainly used for quantitative estimation of Ags.

 The antiserum to the Ag to be quantitated is incorporated in agarose gel on a glass slide.
 The wells are punched and filled with increasing con.of Ag.
 The Ag is then electrophoresed into the Ab containing agarose.
 Pptn is formed in the shape of cone (rocket).
 II Agglutination
Def: It is an Ag-Ab reaction, in which an Ab combines with a particulate Ag in presence of
electrolytes at opt. pH and temperature resulting in visible clump or agglutination of the particles.
 The agglutination is more sensitive than ppt for detection of antibodies.
 The same Principle governs agglutination and precipitation.
 The Lattice hypothesis holds good for agglutination too.
Types;
1. Slide agglutination:
 This is widely used method to detect unknown Ag or Ab.
 A uniform suspension of Ag is made in a drop of saline on a clean glass slide or on a tile.
 A drop of appropriate antiserum is added to it, and mixed with the help of applicator stick or by
gently rocking the slide.
 Clumping occurs with in few secs when the agglutination test is positive, which is visible to the
unaided eye.
Uses: 1. Identification of bacterial isolates from clinical specimens.
2. Blood grouping, WIDAL slide test(qualitative)
2. Tube Agglutination

 This is a standard quantitative method for measurement of Antibodies.

 When a fixed volume of a particular Ag suspension is added to an equal volume of serial
dilutions of an antiserum in test tubes, the agglutination titre of the serum can be estimated.
 The highest dilution at which agglutination occurs is called “antibody titre”.
Uses:
 Routinely employed method for serological diagnosis of typhoid (WIDAL tube test), typhus
fever (Weil-Felix reaction), Brucellosis and infectious mononucleosis (Paul-Bunnel test).
3. Coomb’s test (Antiglobulin test)
 This was devised by Coombs for detection of Rh Abs.
 Certain Ab such as Rh antibodies fail to agglutinate with corresponding Rh Ag
unlike Abs to ABO grp. These are called blocking/incomplete Ab.
Thus detection of these Abs was difficult until coombs serum was developed.
 Antiglobulin or Coombs test is used to detect these incomplete Rh Abs that
causes haemolytic disease of newborn called erythroblastosis foetalis by using
antiglobulin Abs OR anti-immunoglobulin.
 Coomb’s test (Antiglobulin test) Principle and procedure
 when sera containing incomplete anti-Rh Abs are mixed with
Rh-positive RBCs, these incomplete Abs coats the surface of
RBCs though they are not agglutinated.
 When such coated RBCs are mixed with antiglobulin or
Coombs sera (anti-immunoglobulin raised in rabbit against
human Ab) the cells are agglutinated.
Types: a) Direct Coombs test: (Invivo)
 used to detect Rh Abs on the RBCs of the ERF suffering infant.
 When RBCs of infants are washed to remove unbound Ab
found in fetal serum and then mixed with a drop of Coombs
serum, agglutination occurs.
b) Indirect Coombs test (Invitro)
 Used to detect Rh Abs in mother’s serum.
Uses:
 To detect any type of Incomplete Ab or non-agglutinating Ab
4. Passive Agglutination :
 As Agglutination test are more convenient and more sensitive than precipitation reaction, it is
possible to convert precipitation tests into agglutination tests by attaching soluble Ag to the
surface of carrier particles such as RBCs and latex particles. Such tests are called passive
agglutination test.
a) Haemagglutination test :
 Human/Sheep RBCs adsorb a variety of Ags.
Eg. Rose-waller test for detection of autoantibody (RA) in rheumatoid arthritis
b) Latex agglutination test :
 Polystyrene latex of 0.8-1,0 µm in dia are used, that adsorb several types of Ags.
widely employed in the clinical laboratory for the detection of ASO, CRP, RA factor, HCG and
many other Ags.
c) Co-agglutination test :
 Passive agglutination tests are highly sensitive and yield high titres, but may give false +ve
results. When instead of Ag, the Ab is adsorbed to carrier particles in test for estimation of Ags,
the technique is known as reversed passive agglutination.
 III –Compliment fixation test
 Complement refers to a system of some non –specific proteins found normally in human and
animals which are activated/induced only by Ag-Ab complex formation.
 In presence of appropriate Ab, complement lyses bacteria, RBCs or cells containing Ags. In
some cases they immobilise motile organisms, promotes phagocytosis, inflammatory
response, hypersensitivity reaction and immune adherence.
 Complement do not bind to free Ag or Ab but only to antibody which is complexed with Ag.
 The ability of Ag-Ab complex to fix complement is made use in the CFT .
 CFT is versatile and sensitive capable of detecting little quantity of Ags and Abs.

Compliment fixation test Procedure :(Wasserman test)

 The classical example of CFT is Wasserman test, formerly used method for the diagnosis of
Syphilis by detecting syphilis antibodies.
 CFT consists of 2 steps
 I step: The patient serum is inactivated by heating
at 56 C for half an hour to destroy any complement
activity and also to remove inhibitors of complement
present in some sera.
The inactivated serum of the patient is incubated at
37 C for 1 hr. with Wasserman Ag and fixed amt of
Complements freshly obtained from guinea pig.
If the serum contains syphilitic Ab the complement Will
be utilised drg. Ag-Ab complex formation. If the serum
does not contain Ab, no Ag-Ab reaction Occurs and
therefore complement will be left intact.
II Step: (Indicator step)
Constitute testing of free Complements in the mixture.
Free/unused complement in the mixture is detected by
adding sheep RBCs that are coated with anti-erythrocyte
Ab or Amboceptor (rabbit Ab to sheep RBCs) to the
Interpretation:
 Absence of erythrocyte lysis indicates that the
complement were used up in the first step and
therefore the serum contained the Ab
( Positive CFT) .
 Lysis of RBCs indicates that the complement was
not fixed in the first step and therefore, the serum
did not have the Ab (Negative CFT)

Other CFT tests:

1.Indirect CFT-
2.Conglutinating complement absorption test:
Used for certain sera do not fix guinea pig complement.
Use horse complement which is non-haemolytic.
 IV Immunofluorescence
 Fluorescence is the property of certain dyes to absorb light rays of one particular
wavelength(shorter) and emitting rays with different wavelength (higher).
 Fluorescent dyes like Fluroescein isothiocyanate (green) and Lissamine rhodamine (red)
glow brightly UV light.
 Such fluorescent dyes can be conjugated to antibodies, and such “labelled Abs” can be used to
locate and identify Ags in tissues or clinical samples.
Types of Immunofluorescence techniques
1. Direct Immunoflurescence
2. Indirect Immunoflurescence
1. Direct Immunoflurescence ( for detection of Ag)
 This can be used for identification of bacteria, viruses or
other Ags, in blood, CSF, urine, faeces, tissues and other
clinical specimens using specific primary labelled antibody.
 Labelled Primary Abs are added to the sample that are fixed
on the slide. If Ag is present in the sample, it react with
labelled Abs to form Ag-Ab complex which fluoresce under
UV light of Fluorescent microscope.
 Used to diagnose rabies virus Ags in brain smear.
 Disadvantage- the separate fluorescent conjugates have to be
prepared for each Ag.
2. Indirect Immunofluroscence ( for detection of Ab)
 This method is better than DIF for detection of Abs, as it overcomes the difficulty
of preparing individual conjugate.
 A drop of test serum is placed on a smear of Tr. Pallidum (Ag) on a slide and
washed after incubation. If test serum contain Ab it attaches to Ag. For detection
of this Ag-Ab complex Fluorescein conjugated Secondary Ab(antiglobulin)
is added in the 2nd step. The fluorescent Secondary Ab reacts with Ab bound to Ag.
 After washing the slide is observed, if the test sample is +ve they fluoresce under
UV of Fluroscent Microscope. If test serum is -ve no fluorescence seen.

Uses: widely used to detect Ab in Syphilis, ameobiasis, toxoplasmosis and in some viral diseases like EB virus infn,
and Herpes
ADVANTAGE: A single antihuman globulin fluorescent conjugate can be employed for detection of Ab to various
Ag.
V - Radioimmuno Assay (RIA)
 Besides flurorescent dyes, radioisotopes can also be conjugated
to Ags or Abs.
 RIA is extremely sensitive and measure up to Picogram(10-12gm)
 RIA is a competitive assay b/w fixed amount of known radio-
labelled Ag and unknown unlabelled test Ag for a fixed amount
of antibody.
 If the test sample has more Ag, the chances are less for labelled
Ag to combine with Ab and viceversa.
 After Ag-Ab rean. The Ags are separated into free and bound
fraction and their radioactivity is measured.
 The concentration of unlabelled Ag is calculated from the ratio
of bound and total Ag labels, using Standard dose response
curve.
 VI- Enzyme Linked Immunosorbent Assay (ELISA)
 In 1971, enzyme labelled Ags and Abs were developed as serological reagents
for assay of antibodies and antigens.
 Their versatility, sensitivity, simplicity, economy, absence of radiation hazard,
availability of test kits have made ELISA the most widely used procedure in
clinical serology.
 ELISA is so named because it involves the use of enzymes and
immunosorbent, an adsorbing material specific for one of the components of
the reaction either Ag or Ab.
 This immunosorbent is a solid phase made up of plastic like polystyrene
(Macro-ELISA) or polyvinyl micro titre plates (Micro-ELISA) consisting of
96 wells suitable for automation.
 Enzymes Substrate
Horse radish peroxidase---------O-phenyl diamine dihydrochloride
Alkaline phosphatase ---------P- nitrophenyl phosphate
Types of ELISA
1. Sandwich ELISA (Detection of Ag or Ab)
2 Indirect ELISA (Detection of Ab)
3. Competitive ELISA (Detection of Ab)
4.Cylinder or Cassette ELISA (modified ELISA)
 Here each specimen is tested in a separate disposable cassette. The test is rapid(10-15 mns).
 There is no need of microtitre plate or readers. The result is read visually.
 In built +ve and –ve controls are provided for validation of the test procedure.
 Eg. Cassette ELISA for detection of Ab in HIV 1 and HIV 2.
Uses of ELISA
 HIV detection
 Detection of infectious deseases like Hepatitis, EBV, Cytomegalovirus IgM/IgG,
dengue IgG, influenza.
 Rotavirus detection in faecal specimen and enterotoxin of E.coli in feces.
 Syphilis Ab and Ag detection
 Food toxin and food adulterants detection.
 Mycobacterial Ab detection in TB.
 Human allergen-specific IgE and IgA ELISA
Immunoelectroblot or
Western Blot technique – Highly specific
 Used for identification of specific Abs(proteins)
in a sample containing mixture of Abs each targeted
against different Ags of same pathogen. (HIV Ags)
Confirmatory test for serodiagnosis of HIV
I step –SDS PAGE Serparation: HIV Ags are
separated by Gel electrophoresis.
II step- Blotting : The separated Ags are then
Transferred(blotted) to Nitrocellulose membrane(NCM)
Filter.
III Step – Enzyme immunoassay: NCM strips
containing various separated Ags are treated with
patient’s sample containing Abs. Individual Ab bind to
respective Ag fragments. Ag-Ab complex is then
detected by addition of Enzyme labelled antiglobulin
followed by addition of substrate.
ASSIGNMENT
1. Neutralization
2. Opsonisation
3. Chemiluminescence Immunoassay (CLIA)
4. Immunochromatographic tests
5. Immunoelectromicroscopic tests

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