Antigen – Antibody reactions

Dr. Pendru Raghunath Reddy

Assistant Professor of Microbiology
Dr. VRK Women’s Medical College
 Antigens and antibodies combine with each other
specifically and in an observable manner

 In the body, they form the basis of antibody mediated

immunity in infectious diseases, or hypersensitivity and
autoimmune diseases

 Antigen – antibody reactions in vitro are known as

serological reactions

 In laboratory, they help in diagnosis of infections, in

epidemiological surveys, in the identification of infectious
agents, enzymes
 Stages of Ag – Ab reactions

Primary stage

 Initial interaction between Ag & Ab – invisible

 Rapid, occurs at low temperatures & obeys the general

laws of physical chemistry & thermodynamics

 Reaction is reversible

 Ag & Ab is bound to each other by weak Van der Waal’s

forces, Ionic bonds & Hydrogen bonding
Ag-Ab interactions
Bonds:
 Hydrogen
 Ionic
 Hydrophobic interactions
 Van der Waals forces

Each bond is weak; many

are
strong
To “hold” they must be close
 requiring high amts of
complementarity!
 Secondary stage

 Demonstrable events – Precipitation, agglutination, lysis of

cells, killing of live antigens, neutralization of toxins,
complement fixation, immobilization of motile organisms &
enhancement of phagocytosis.

Precipitin – Ab participate in precipitation

Agglutinin - Ab participate in agglutination

Precipitinogen – Ag participate in precipitation

Agglutinogen - Ag participate in agglutination

 Tertiary stage

 Includes neutralization or destruction of injurious agents

or tissue damage

 Also includes humoral immunity against infectious

diseases as well as clinical allergy & other
immunological diseases
 GENERAL FEATURES OF Ag – Ab
REACTIONS
1. The reaction is specific

2. Entire molecules react and not the fragments

3. There is no denaturation of the antigen or antibody during

the reaction

4. The combination occurs at the surface. So surface antigens

are immunologically relevant

5. The combination is firm but reversible. The firmness is

influenced by the affinity & avidity of the reaction

6. Antigens & antibodies can combine in varying proportions.

Both Ags & Abs are multivalent
 Affinity
• Refers to the intensity of attraction between the
antigen & antibody molecules. It is the function of
closeness of fit between the epitope & antigen
binding region of its Ab

High Affinity Low Affinity

Ab Ab

Ag Ag

Affinity = ∑ attractive and repulsive forces

 Avidity
• Strength of the bond after the formation Ag-Ab complexes

• The overall strength of binding between an Ag with many

determinants and multivalent Abs

Keq = 104
Affinity 106 1010
Avidity Avidity
 Specificity
 The ability of an individual antibody combining site to
react with only one antigenic determinant

 The ability of a population of antibody molecules to react

with only one antigen
 Cross Reactivity
• The ability of an individual Ab combining site to
react with more than one antigenic determinant.
• The ability of a population of Ab molecules to
react with more than one Ag

Cross reactions

Anti-A Anti-A Anti-A

Ab Ab Ab

Ag A Ag B Ag C

Shared epitope Similar epitope

 Factors Affecting Measurement of
Ag/Ab Reactions

• Affinity
• Avidity Ab excess Ag excess

• Ag:Ab ratio
• Physical form of Ag

Equivalence – Lattice formation

Types of Antigen – Antibody Reactions
1. Precipitation reaction
2. Agglutination reaction
3. Neutralization reaction
4. Opsonisation

Serological tests based on Ag – Ab reactions

1. Complement fixation test
2. Immunofluorescence
3. Radioimmunoassay
4. Enzyme immunoassay
 PRECIPITATION REACTION

PRINCIPLE
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 – Flocculation
reaction

 Precipitation can take place in liquid media or in

gels such as agar, agarose or polyacrylamide.
 ZONE PHENOMENON

 The amount of precipitate formed is greatly influenced by

the relative proportions of Ags & Abs

 If increasing quantities of Ags are added to the same

amount of antiserum in different tubes, precipitation is
found to occur most rapidly & abundantly in the middle
tubes

Preceding tubes – Ab excess (Prozone)
 Middle tubes – Ag & Ab in equivalent proportions

(Zone of equivalence)
 Later tubes – Ag excess ( Post zone)
 Mechanism of precipitation

 Marrack (1934) proposed the lattice hypothesis –

mechanism of precipitation

 The multivalent antigens combine with bivalent Abs in

varying proportions, depending on the Ag – Ab ratio on
the reacting mixture

 Precipitation results when a large lattice is formed

consisting of alternating Ag & Ab
Marrack’s hypothesis
 Applications of Precipitation reaction

 It can be carried out as either a quantitative or qualitative test

 Sensitive for the detection of Ags

1. Identification of bacteria eg: Lancefield’s grouping of

Streptococcus

2. Detection of antibody for diagnostic purposes

eg: VDRL in syphilis
Types of precipitation reactions

1.Ring test

2.Flocculation test

3.Immunodiffusion

4.Electroimmunodiffusion
 RING TEST
 Consists of layering Ag solution over a column of
antisera in a narrow tube

Eg: Ascolis thermoprecipitin test, Grouping of

Streptococci by Lancefield technique
 Flocculation test
Slide test

 When a drop of Ag & antiserum is placed on a slide &

mixed by shaking, floccules will appear

 Eg: VDRL test & RPR test for syphilis

 Tube test

 The Kahn test (tube flocculation) for syphilis

 This is also employed for the standardization of toxins &

toxoids

 Serial dilutions of toxin/toxoid are added to the tubes

containing a fixed quantity of antitoxin

 The amount of toxin that flocculates optimally with one

unit of the antitoxin – Lf dose
IMMUNODIFFUSION (precipitation in gel)

Advantages of immunodiffusion:

 Reaction is visible as a distinct band of precipitation

 Stable, can be stained for preservation

 Indicates identity, cross reactions, non identity between

different Ags
 Various immunodiffusion tests
1. Single diffusion in one dimension (Oudin
procedure)
 Ab is incorporated in agar gel in a test tube & Ag
solution is layered over it

 Ag diffuses downward through the agar gel – forming a

line of precipitation.
2. Double diffusion in one dimension (Oakley
Fulthorpe procedure)

 Ab is incorporated in agar gel

 Above which is placed a column of plain agar

 The Ag is layered over it

 The Ag & Ab move towards each other through the

intervening column of plain agar & form the precipitate
3. Single diffusion in two dimensions (Radial
immunodiffusion)

 Here the antisera is incorporated in a gel & poured on a

flat surface

 Wells are cut on the surface to which Ag is added

 It diffuses radially from the well & forms ring shaped

bands of precipitation concentrically around the well
 Radial Immunodiffusion (Mancini)

• Method Ab in gel

– Ab in gel Ag Ag Ag Ag

– Ag in a well
 Interpretation
 Diameter of ring is
proportional to the
concentration Diameter2

 Quantitative
 Ig levels
Ag Concentration
 Uses

1. It has been widely employed for estimation of immunoglobulin

classes i.e. IgG, IgM, IgA in sera

2. It has also been used for screening sera for antibodies to

influenza viruses
4. Double diffusion in two dimensions (Ouchterlony
procedure)

 Helps to compare different antisera & antigens directly

 Agar gel is poured on a slide & wells are cut

 Antiserum – central well

 Different Ags in the surrounding wells

Reaction of identity

Lack of relatedness

Partial identity
Elek’s gel precipitation
5. Immunoelectrophoresis

 This involves the electrophoretic separation of composite Ag

into its constituent proteins, followed by immunodiffusion
against its antiserum – separate precipitin lines

 It is performed on an agarose gel with an Ag well & Ab trough

cut on it

 The test serum is placed in the antigen well & electrophoresed

for about 1 hour

 Ab against human serum is placed in the trough & diffusion

allowed for 18 – 24 hrs
Immunoelectrophoresis
 Uses

1. By this technique, a number of antigens can be identified in

human serum

2. It is particularly useful for detection of normal and abnormal

serum proteins like myeloma proteins
 ELECTROIMMUNODIFFUSION

 The development of precipitin lines can be speeded up

by electrically driving the Ag & Ab

 Two types

1. Counterimmunoelectrophoresis (One dimensional

double electroimmunodiffusion)

2. Rocket electrophoresis (One dimensional single

electroimmunodiffusion)
1. Counterimmunoelectrophoresis (CIE)

 This involves simultaneous electrophoresis of Ag & Ab in

gel in opposite directions resulting in precipitation at a point
between them

 Used only when Ag and Ab have opposite charges

 Produce precipitation lines within 30 mins

 Clinical application: detecting Ags like alphafetoprotein in

serum, Ags of Cryptococcus & Meningococcus in the CSF

 It is also applied for detecting hepatitis B antigens and

antibodies
- +
Ag Ab
2. Rocket electrophoresis
 Used for quantitative estimation of Ags

 The antiserum to the Ag to be quantitated is incorporated in

agarose gel on a slide

 Ag in increasing concentrations, is placed in wells punched

in the set gel

 The Ag is electrophoresed into the Ab containing agarose

 The pattern of immunoprecipitation resembles a ROCKET

 The length of these rocket like structures corresponds to

the concentration of the antigen
Rocket electrophoresis
Laurell’s two dimensional electrophoresis

 Variant of rocket electrophoresis

 Used to quantitate each of the several Ags in a mixture

 In the first stage, the Ag mixture is electrophoretically

separated

 In second stage, electrophoresis is done perpendicular to

that of first stage to get rocket like precipitation
Agglutination

When particulate antigen combines with its antibody in the

presence of electrolytes at an optimal temperature and pH,
resulting in visible clumping of particles

 More sensitive than precipitation for the detection of antibodies

 The agglutination reaction takes place better with IgM antibody

 Lattice formation hypothesis holds good for aggltination too

 Blocking antibodies inhibit the agglutination by the complete

antibody added subsequently
 Types of agglutination reactions

1.Side agglutination test

2.Tube agglutination test

3.The antiglobulin (Coombs) test

4.Heterophile agglutination test

5.Passive agglutination test

 Slide agglutination test
 A uniform suspension of antigen is made in a drop of saline
on a slide and a drop of the appropriate antiserum is added

 Reaction is facilitated by mixing the antigen and the antiserum

with a wire loop or by gently rocking the slide

 Clumping occurs instantly or within seconds when agglutination

test is positive

 A control consisting of antigen suspension in saline, without

adding antiserum must be included on the same slide
Uses

1. It is a routine procedure to identify the bacterial strains isolated

from clinical specimens

(eg: Identification of Salmonella species)

2. It is also used for blood grouping and cross matching

Tube agglutination test
What is the titer of Ab?

The titer is customarily reported as the reciprocal of the

highest dilution of Ab that causes an obvious agglutination
 Tube Agglutination Test

Agglutination No agglutination

1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl

In this case, the titre is 40

Uses

Used for serological diagnosis of

1.Enteric fever (Widal test)

2.Typhus fever (Weil-Felix reaction)

3.Infectious mononucleosis (Paul-Bunnel test)

4.Brucellosis (SAT)

5.Primary atypical pneumonia

(Streptococcus MG agglutination test)
 Problems related to tube agglutination

1.Prozone phenomenon

2.Blocking antibodies

Blocking or incomplete antibodies may be detected by

performing the test in hypertonic (5%) saline or albumin saline

Antiglobulin (Coombs) test is more reliable for detecting these

antibodies
The antiglobulin (Coombs test)

Originally devised by Coombs, Mourant and Race (1945) for the

detection of incomplete anti-Rh antibodies

There are two types of Coombs test

1.Direct Coombs test

2.Indirect Coombs test

 Direct Coombs test

+ ↔

Patient’s RBCs Coombs Reagent

(Antiglobulin)
 Indirect Coombs test

Step 1
+ ↔

Patient’s Target
Serum RBCs

Step 2

+ ↔

Coombs Reagent
(Antiglobulin)

The only difference between the two is that the sensitisation of

the erythrocytes with incomplete antibodies takes place in vivo
in direct type whereas it occurs in vitro in indirect type
Uses of Coombs test

1.For detection of anti-Rh antibodies

2.For demonstration of any type of incomplete antibody

(eg: Brucellosis)
 Heterophile agglutination test

 Heterophile antibodies have a property to react with

microorganisms or cells of unrelated species due to common
antigenic sharing

i) Weil-Felix reaction

Some proteus (OX19, OX2, and OXK) strains are agglutinated

by sera of patients with rickettsial infections

This is due to antigenic sharing between these Proteus strains

and Rickettsial species
ii) Paul-Bunnel test
Sheep erythrocytes are agglutinated by sera of infectious
-mononucleosis’

iii) Streptococcus MG agglutination test

It is positive in primary atypical pneumonia
 Passive agglutination test

 A precipitation reaction can be converted into agglutination

test by attaching soluble antigens to the surface of carrier
particles such as latex particles, bentonite and red blood cells

 Such tests are called passive agglutination tests

 When instead of antigen, the antibody is adsorbed on the

carrier particles for estimation of antigens, it is known as
reversed passive agglutination
Latex agglutination test

Polystyrene latex particles (0.8 – 1 µm in diameter) are widely

employed to adsorb several types of antigens
 This test is convenient, rapid and specific

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

HCG and many other antigens

 Latex agglutination tile is used to perform this test

 Haemagglutination test

 Erythrocytes sensitised with antigen are used for detection of

antibodies

 Rose-Waaler test for detection of RA factor in patient serum

 The antigen used for the test is sheep red blood cells sensitised
with rabbit antisheep erythrocyte antibody (amboceptor)
 Coagglutination

 Some strains of Staphylococcus aureus (especially Cowan 1

strain) possess protein A on their surface

 When specific IgG molecule is coated on these strains, Fc

portion of IgG molecule binds to protein A whereas antigen
combining Fab terminal reamains free

 When the corresponding antigen is mixed with these coated

cells, Fab terminal binds to antigen resulting in agglutination

 This test is used for detection of bacterial antigens in blood,

urine and CSF
(eg: Gonocooci, Streptococcus pyogenes and
Haemophilus influenzae)
 Complement fixation test

 Complement is a protein (globulin) present in normal serum

 Whole complement system is made up of nine components:

C1 to C9

 Complement proteins are heat labile and are destroyed by

heating at 56°C for 20 – 30 minutes
 Principle

 The antigen-antibody complexes have ability to fix complement

 This reaction has no visible effect

 To detect the fixation of complement, an indicator system

consisting of sheep erythrocytes coated with amboceptor
is used
 Components of CFT

Test System
 Antigen: It may be soluble or particulate.
 Antibody: Human serum (May or may not contain Antibody
towards specific Antigen)
 Complement: It is pooled serum obtained from 4 to 5
guinea pigs. It should be fresh or specially preserved as the
complement activity is heat labile (stored at -30 °C in small
fractions). The complement activity should be initially
standardized before using in the test

Indicator System (Haemolytic system)

 Erythrocytes: Sheep RBC
 Amboceptor (Hemolysin): Rabbit antibody to sheep red
cells prepared by inoculating sheep erythrocytes into rabbit
under standard immunization protocol.
 Controls

 Antigen and serum controls are included in the test

 Complement control is used to ensure that the desired amount

has been added

 Cell control to make sure that sensitised erythrocytes do not

undergo lysis in the absence of complement
 Positive Test

 Step 1:
At 37°C
Antigen + Antibody + Complement Complement gets fixed
(from serum) 1 Hour

 Step 2:
At 37°C
Fixed Complement complex + Haemolytic system No Haemolysis
1 Hour (Test Positive)
 Negative Test
 Step 1:
At 37°C
Antigen + Antibody absent + Complement Complement not fixed
1 Hour

 Step 2:
At 37°C
Free Complement + Haemolytic system Haemolysis
1 Hour (Test Negative)
 Results and Interpretations:

 No haemolysis is considered as a positive test

 Haemolysis of erythrocytes indicative of a negative test

1 2 3 4

 Microtiter plate showing Haemolysis (Well A3, A4 and B4)

and No Haemolysis (Well A1, B1, B2, and B3)
 Indirect complement fixation test

 Certain avian (eg: duck, parrot) and mammalian (eg: horse, cat)
sera cannot fix guinea pig complement

 Test is done in duplicate and after the first step, the standard
antiserum known to fix complement is added in one set
 Positive test

 First step: Antigen + test serum (positive for antibody) +

guinea pig complement

 Second step: Standard antiserum cannot react with antigen

because has been used up by antibody in the first step, hence,
complement is free

 Indicator system: Haemolysis occurs because complement is

free
 Negative test

 First step: Antigen + test serum (negative for antibody) +

guinea pig complement

 Second step: Standard antiserum will react with antigen and

fix the free complement

 Indicator system: No haemolysis because complement is not

free
 Conglutination
 This is an alternative method for systems which do not fix
guinea pig complement

 The indicator system is sheep erythrocytes sensitised with

bovine serum

 Bovine serum contains a a beta globulin component named

conglutinin

 Conglutinin can cause agglutination of sensitised sheep

erythrocytes, if these are combined with complement

 No agglutination – Positive result

 Agglutination – Negative result
 Complement dependent serological tests

1.Immobilisation test

2.Immune adherence

3.Cytolytic or cytocidal tests

Neutralisation test

Opsonisation
Immunofluorescence

 Fluorescence is the property of certain dyes which absorb rays

of one particular wavelength (UV light) and emit rays with a
different wavelength (visible light)

 Coons and his colleagues showed that fluorescent dyes can be

conjugated to antibodies and these labelled antibodies can be
used to detect antigens in tissues

 The commonly used fluorescent dyes are

i)Fluorescin isothiocyanate (Blue green fluorescence)

ii)Lissamine rhodamine (orange red fluorescence)

 Immunofluorescence test is of two types

1.Drect immunofluorescence test

2.Indirect immunofluorescence test

Direct immunofluorescence test
Uses
1. It is commonly employed for detection of bacteria, viruses or
other antigens in blood, CSF, urine, faeces, tissues and other
specimens

2. It is a sensitive method to diagnose rabies by detection of the

rabies virus antigens in brain smears

Disadvantage
Separate specific fluorescent labelled antibody has to be
prepared against each antigen to be tested
Indirect immunofluorescence test
Advantages

 A single antihuman globulin fluorescent conjugate can be

employed for detection of antibody to any antigen

 All antibodies are globulin in nature, therefore, antihuman

globulin attaches to all antibodies

 This has overcome the disadvantage of direct immuno-

-fluorescence test
 Radioimmunoassay (RIA)

 Berson and Yallow (1959) first described

 Since then it has been utilised for quantitation of hormones,

drugs, HBsAg, IgE and viral antigens

 Radioimmunoassay is widely-used because of its great

sensitivity

 Using antibodies of high affinity, it is possible to detect a few

picograms (10−12 g) of antigen

 The greater the specificity of the antiserum, the greater the

specificity of the assay
The principle involves competitive binding of radiolabeled Ag
and unlabeled Ag to the limited supply of a high affinity Ab

FIGURE 6-9
A solid-phase radioimmunoassay (RIA) to detect hepatitis B virus in blood
samples & A standard curve to determine the conc. of HBsAg in unknown serum.
 Disadvantages

 Radiation hazards: Uses radiolabelled reagents

 Requires specially trained persons

 Labs require special license to handle radioactive material

 Requires special arrangements for

 Requisition, storage of radioactive material

radioactive waste disposal
 Enzyme Linked Immunosorbent Assay (ELISA)

 Is a biochemical technique used mainly in immunology to

detect the presence of an antibody or an antigen in a sample

 Term was coined by Engvall and Pearlmann in 1971

 Similar to RIA, except no radiolabel

 Very sensitive, pg/mL

 Different types of ELISAs

1.Indirect ELISA

2.Sandwich ELISA

3.Competitive ELISA

4.Cassette or cylinder ELISA

Indirect ELISA

Two commonly used enzymes

1.Horseradish peroxidase (HRP)

2.Alkaline phosphatase (AP)

Sandwich ELISA

 Two antibodies required; must recognize different epitopes

 1st Antibody is referred to as capture Ab

 2nd antibody as detection Ab

Competitive ELISA
 The labelled antigen competes for primary antibody binding
sites with the sample antigen (unlabelled)

 The more antigen in the sample, the less labelled antigen is

retained in the well and the weaker the signal).
 Cassette or cylinder ELISA
 It is a simple modification of ELISA for testing one or few
samples of sera at a time

 The test is rapid (about ten minutes) as compared with the

2 - 4 hours taken for conventional ELISA
 Uses of ELISA
 Used for detection of antigens and antibodies for various
microorganisms

1)Detection of HIV antibodies in serum

2)Detection of mycobacterial antibodies in tuberculosis

3)Detection of rotavirus in faeces

4)Detection of hepatitis B markers in serum

5)Detection of enterotoxin of E. coli in faeces

Western blotting (Immunoblotting)
 Western Blot for detection of HIV antibody

HIV-1 Western Blot

 Lane1: Positive Control
 Lane 2: Negative Control
 Sample A: Negative
 Sample B: Indeterminate
 Sample C: Positive