II MBBS

Antigen – Antibody Reactions

 Ag – Ab reactions in vitro are known as
Serological reactions.

 Help in - the diagnosis of infections

- epidemiological surveys
- identification of infectious and
noninfectious antigens
Stages of Ag-Ab reactions
 3 stages - Primary
- Secondary
- Tertiary

Primary Stage
 Initial interaction between Ag & Ab
 No visible effect
 Rapid & reversible
 Held by weak forces
 Secondary stage
 Follows 10 stage.
 Visible effects - Precipitation
- Agglutination
- Cell lysis
- Killing of live Ags
- Neutralisation of toxins
- Complement fixation
- Phagocytosis
 Tertiary Stage Reactions
 Seen in vivo
 Ag- Ab reactions initiate chain reactions that
lead to
 Neutralisation or destruction of injurious Ags.
 Tissue damage.
 Include humoral immunity against infectious
diseases as well as clinical allergy & other
immunological diseases.
General features of Ag-Ab reactions
 Specific reaction, but cross reactions may occur due to
antigenic relatedness.

 Entire molecule of Ag & Ab reacts and participates in

the formation of agglutinates or precipitates

 No denaturation of Ag or Ab occurs during the reaction

 Combination occurs at the surface.

 Combination is firm but reversible.

Serological Reactions

 Precipitation
 Agglutination
 Complement fixation test (CFT)
 Neutralisation
 Immunofluorescence (IF)
 Enzyme immunoassay (EIA/ ELISA)
Other Ag-Ab reactions
 Opsonisation
 Radio immunoassay (RIA)
 Immunochromatographic tests.
 Immunoelectroblot techniques
 Immunoelectronmicroscopic tests
 Measurement of Ag & Ab
 Titre or units - Ab titre of a serum is the reciprocal
of the highest dilution of serum which shows an
observable reaction with the Ag in the particular
test.

 2 important parameters of serological tests :

1. Sensitivity – ability of the test to detect even very minute
quantities of Ag or Ab.
False –ve results are absent or minimal.

2. Specificity – ability of the test to detect reactions between

Ags & its homologous Abs.
False +ve reactions are absent or minimal.
 PRECIPITATION
 When a soluble Ag combines with its
Ab in the presence of electrolytes
(NaCl) at a suitable temperature &
pH, the Ag-Ab complex forms an
insoluble precipitate.
 When instead of sedimenting, the
precipitate remains suspended as
floccules, the reaction is called
Flocculation.
 It can take place in liquid media or in
gels such as agar, agarose or
polyacrylamide.
Mechanism of precipitation
 Lattice formation - lattice hypothesis by
Marrack
Zone phenomenon
 Precipitation and some other serological reactions are
best seen when the antigen and antibody are mixed in
optimal (equivalent) proportion.

 Since in these test antigen used is in fixed quantity, the

only variable is the antibody. Serial dilutions of antibody
when treated with antigen show that there is no
precipitation where the antibody is in excess. This is
called as PROZONE PHENOMENON and is caused due to
failure to form lattice.
 Optimal
Antibody excess proportions Antigen excess

Serial Dilution of Serum containing Abs-

decreasing concentration of Ab
Graphical Representation
 If amount of precipitate in the different tubes are
plotted on a graph, the resulting curve will have
three phases.
 Prozone or Zone of Ab excess.
 Zone of equivalence (peak).
 Post zone or Zone of Ag excess.
Prozone – imp. in clinical serology.
- Several dilutions are tested.
Applications
 Very sensitive in Ag detection (1µg of protein)
 Forensic – identification of blood & seminal stains
 Food adulteration

Types of Precipitation Test

 Ring test
 Slide / Tube flocculation test
 Immunodiffusion (precipitation in gel)
 Electroimmunodiffusion
 Types of Precipitation & Flocculation
1. RING TEST
- Simplest
- Layering Ag solution over a column of
antiserum in a narrow tube
- Ppt forms at the junction.
e.g. Ascoli’s thermopreciptin test
Lancefield grouping of streptococci

2. SLIDE TEST – A drop each of Ag &

antiserum are placed on a slide & mixed –
floccules appear
e.g. VDRL test for syphilis
Types
3. TUBE TEST - Tube flocculation
e.g. Kahn test for syphilis
Standardisation of toxins & toxoids

4. IMMUNODIFFUSION (Precipitaiton in gel)

Advantage – Distinct band of ppt form which is
stable & can be stained for preservation.
- Number of different Ags in the
reacting mixture can be identified.
Modifications of Immunodiffusion
 Single diffusion in one dimension (Oudin
procedure)

Antigen
Precipitation band
Ab in agar gel
Modifications
 Double diffusion in one dimension (Oakley-
Fulthorpe procedure)
 Modifications
 Single diffusion in two dimensions (Radial
immunodiffusion)
 Uses : estimation of Ig classes in sera.
screening sera for Abs to Influenza viruses
 Modifications
 Double diffusion in two dimensions (Ouchterlony
procedure) e.g. Elek’s test for C.diphtheriae
- most widely used.
- helps to compare different Ags & antisera directly.
- reaction of identity
 Modifications
 Immunoelectrophoresis
– useful for testing
normal & abnormal
proteins in serum &
urine.
- identification &
quantitation of various
proteins in the serum.
5. Electroimmunodiffusion
 Development of precipitin lines can be speeded up
by electric current.
 2 methods :
a. Counterimmunoelectrophoresis (CIEP) – one
dimensional double electroimmunodiffusion
 Electroimmunodiffusion
b. Rocket electrophoresis –
one dimensional single
electroimmunodiffusion
- Ag in increasing
concentration
Uses : quantitative
estimation of Ags.
 Electroimmunodiffusion
c. Laurell’s two dimensional electrophoresis – variant
of Rocket electrophoresis.
- can quantitate each of several Ags.
AGGLUTINATION
 When a particulate Ag is mixed with its Ab in
the presence of electrolytes at a suitable
temperature & pH, the particles are clumped or
agglutinated.

 More sensitive than precipitation for the

detection of Abs.

 Incomplete or monovalent Abs do not cause

agglutination– “Blocking Abs”
Types of Agglutination
 SLIDE AGGLUTINATION
 drop of antiserum + a drop of uniform
suspension of Ag clump formation
 Control is must
 Visible to the naked eye.
 uses : blood grouping
identification of bacterial isolates
 TUBE AGGLUTINATION

 Standard quantitative method for the

measurement of Abs.
 Uses : WIDAL test for Typhoid fever.
 Heterophile agglutination test : Weil-Felix

 Haemagglutination – RBCs are used as antigens

e.g Paul Bunnell test, Cold agglutination test
PASSIVE AGGLUTINATION
 Soluble Ags are attached to the surface of
carrier particles to convert precipitation tests
into agglutination tests.
 More convenient & sensitive.
 Carrier particles : RBC’s, Latex, Bentonite
e.g. ASO test
 Reversed Passive Agglutination

 Instead of Ag, the Ab is adsorbed on the surface

of carrier particles.
 Very sensitive
 May give false positive results.
 ANTIGLOBULIN (Coomb’s) TEST

 Detection of
anti-Rh Abs
(incomplete
Abs)

 Can be direct
(in vivo) or
indirect (in
vitro) test
 Agglutination Inhibition

 Modification of
agglutination
e.g. pregnancy
test kit.
 COMPLEMENT FIXATION TEST
 Ability of Ag-Ab complexes to fix complement.
 Consists of two steps & five reagents
e.g. Wasserman reaction for Syphilis
1. ANTIGEN+ TEST SERUM
(contains Ab) COMPLEMENT FIXED
+ COMPLEMENT

+ HEMOLYTIC SYSTEM Result – No Hemolysis (Sheep

RBC + Amboceptor) POSITIVE CFT
2.ANTIGEN + TEST SERUM COMPLEMENT NOT FIXED
(contains no Ab)
+ COMPLEMENT

+ HEMOLYTIC SYSTEM Result – Hemolysis

NEGATIVE CFT
http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter31/animation_quiz_4.html
NEUTRALISATION TESTS
 Ability of the Ab to neutralize various effects
of micro organisms mediated through toxins,
enzymes or microbial Ags.

e.g. Nagler’s test for

Clostridium perfingens
(test to detect alpha toxin)
IMMUNOFLUORESCENCE
 FLUORESCENCE – property of certain compounds
to absorb light of shorter (UV) wavelength & emit
light of higher (visible) wavelength.
 Fluorescent dyes (Fluorochromes) are conjugated
to Abs – Labelled Abs.
 Fluoresce when binds to specific Ag in tissues.
 Can be direct or indirect.
 Detected by Fluorescent Microscope.
Fluorochromes
 FITC- Fluorescein Isothiocynate :– Green
 Rhodamine- Auramine :- Red
 Acridine orange :- Orange
 Calcofluor white :- fungal elements
Applications
 Direct IF – detection of bacteria, viruses or
other Ags using specific antiserum labelled
with fluorescent dye e.g. detection of Rabies
virus Ag in brain smears.

 Indirect IF – fluorescent treponemal Ab test.

 RADIO IMMUNOASSAY
 Radio isotopes are conjugated to Abs or Ags.

 A competitive binding assay in which fixed amounts of Ab &

radiolabelled Ag react in the presence of unlabelled Ag ( test
sample)

 High levels of unlabelled Ag– less radioactivity

 Applications – quantitation of hormones, drugs, tumor

markers, IgE & viral Ags.
ELISA
 Enzyme linked immunosorbent assay.
 Can be used for the detection of Ag or Ab.
 Corresponding Ag or Ab is conjugated with an enzyme.
 The enzyme is then detected by its ability to convert a
colorless substrate to a colored product.
 The color is then measured in a machine called ELISA
reader.
http://student.ccbcmd.edu/courses/bio141/labmanua/lab18/eia_flash.
html
 http://www.biology.arizona.edu/IMMUNOLOGY/activities/elisa/tech
nique.html
Immunochromatographic tests
 One step test.
 HBsAg detection
 Membrane impregnated with anti-HBsAg Ab
colloidal gold dye conjugate.
 Presence of a colored band is +ve.
Immunoelectron Microscopy
 Viral particles + specific antisera
EM

clumping of viruses