Lecture presented by: Dr.D.W.Deshkar

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Antigen – Antibody ReactionsAntigen – Antibody Reactions

Antigen combines with its specific Antibody in observable manner.

Reaction between Ag & Ab specific.

Ag – Ab reactions in – vitro Serological

tests.

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Uses of Antigen – Antibody Uses of Antigen – Antibody ReactionsReactions

In the Body or in Vivo :

Forms the basis of antibody mediated (humoral) immunity against infectious diseases.

May lead to tissue injury in some hypersensitivity reactions & autoimmune diseases.

In the Lab.or in Vitro :

For diagnosis of infection.

Helpful in epidemiological studies.

For identification of non infectious agents such as enzymes.

Detection & quantitation of either Ag or Ab.

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Nature of Ag/Ab ReactionsNature of Ag/Ab Reactions

Lock & Key concept.

Non covalent bonds. - Hydrogen bonds - Electrostatic bonds - Van der Waal forces - Hydrophobic bonds

Multiple Bonds

Reversible

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Stages of Antigen – Antibody ReactionsStages of Antigen – Antibody Reactions Ag – Ab Reaction occurs in two stages

1. PRIMARY 2. SECONDARY

Primary Stage:

Initial, rapid, reversible, reaction between Ag & Ab without any visible effect, occurring at low temp.

Binding between Ag & Ab by weaker intermolecular forces.

No covalent bonding

Detected by estimating free & bound Ag or Ab separately by radioisotopes, fluorescent dyes or ferritin.

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Secondary StageSecondary Stage

In most but not all primary stage followed by secondary one.

Leads to demonstrable (visible) effects -------* Precipitation.* Agglutination.* Lysis of cell.* Killing of live antigens.* Neutralisation of toxins.* Complement fixation.* Immobilisation of motile organisms.* Enhancement of phagocytosis.

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Secondary Stage contdSecondary Stage contd. ---------. --------- Previous belief : different antibodies responsible for each

type of reaction.

Antibodies named according to type of reaction they produce

Antibody causing Agglutination Agglutinin & its corresponding Antigen Agglutinogen.

Antibody causing precipitation Precipitin & its corresponding antigen Precipitinogen.

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SecondarySecondary Stage contd Stage contd. ---------. --------- The previous belief replaced by Zinsser’s

unitaranian hypothesis (1920).

According to this hypothesis Ag give rise to only one class of Ab, capable of producing all different reactions depending on the nature of the Ag & conditions of reaction.

Both views fallacious.

True that single Ab can cause precipitation & agglutination & other serological reactions, but an Ag can stimulate different classes of immunoglobulins which differ in their reaction capabilities,

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Role of Immunoglobulin Classes Role of Immunoglobulin Classes in different Serological Reactions.in different Serological Reactions.

Serological Reactions

Immunoglobulin Class

IgG IgM IgA

Precipitation Strong Weak Variable

Agglutination Weak Strong Moderate

Complement fixation Strong Weak Negative

Lysis Weak Strong Negative

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Antigen – Antibody reactionsAntigen – Antibody reactions

Some Ag - Ab reactions initiate chain reactions in vivo.

Lead to neutralisation, destruction of injurious Ag or to tissue damage.

Tertiary Reactions

-Humoral immunity against infectious diseases , Clinical allergy , other immunological diseases.

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Characteristics of Ag – Ab ReactionCharacteristics of Ag – Ab Reaction Specific: Ag combines only with its homologous Ab &

vice versa.

Specificity not absolute. Cross reactions occur due to antigenic similarity or relatedness.

Entire molecules of Ag & Ab & not fragments react.

No denaturation of Ag or Ab during reaction.

Only surface Ag participate in reaction.

Ag – Ab combination firm but reversible.

Firmness of combination depends on - Affinity & Avidity

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AffinityAffinity Strength of the reaction between a single antigenic

determinant and a single Ab combining site.

Intensity of attraction between Ag & Ab molecules.

Function of closeness of fit between an epitope & Ag combining region of Ab.

Affinity = ∑ attractive and repulsive forces

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AvidityAvidity

The overall strength of binding between an Ag with many determinants and multivalent Abs.

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Affinity & AvidityAffinity & Avidity

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Measurement of Ag & AbMeasurement of Ag & Ab Measurement in terms of mass ( eg mg nitrogen) or

commonly units or titre.

Antibody titre of serum = Highest dilution of serum showing an observable reaction with antigen in the test.

Two parameters of serological tests :-

Sensitivity - Ability of test to detect even minute quantities of antigen or antibody.

Specificity – Ability of the test to detect reactions between homologous antigens & with no other.

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Cross ReactivityCross 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

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Factors Affecting Measurement of Factors Affecting Measurement of Ag/Ab ReactionsAg/Ab Reactions

Affinity.

Avidity.

Ag : Ab ratio.

Physical form of Ag.

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Tests Based on Ag/Ab ReactionsTests Based on Ag/Ab Reactions

All tests based on Ag/Ab reactions will have to depend on lattice formation or they will have to utilize ways to detect small immune complexes.

All tests based on Ag/Ab reactions can be used to detect either Ag or Ab

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Precipitation TestsPrecipitation Tests

Lattice Formation

Equivalence – Lattice formation

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Precipitation ReactionPrecipitation Reaction

When a soluble antigen combines with its antibody in presence of electrolytes ( NaCl) at suitable temperature & pH, the Ag – Ab complex forms an insoluble precipitate.

When a colloidal antigen combines with its antibody in presence of electrolytes ( NaCl) at a suitable temperature & pH, the Ag – Ab complex forms an insoluble precipitate which remains suspended instead of suspended

‘ Flocculation’.

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ApplicationsApplications Qualitative & Quantitative test.

Very sensitive in detecting antigens & as little as 1g of protein.

Less sensitive to detect antibody.

1) Ring test - Ascoli’s thermoprecipitin test , Grouping of streptococci by Lancefield technique.

2) Slide test – VDRL test for syphilis = eg of flocculation test.

3) Tube test – Kahn test eg of tube flocculation test.

4) Quantitative tube flocculation test – used for standardisation of toxins & toxoides.

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Immunodiffusion ( Precipitation in Immunodiffusion ( Precipitation in Gel)Gel)

Several advantages in allowing precipitation to occur in gel than in liquid.

Reaction visible as distinct & stable band of precipitation . Can be stained for preservation.

Each Ag – Ab reaction forms a line of ppt. & the number of different Ag in the mixture readily observed.

Immunodiffusion indicates identity, cross reaction & nonidentity betn different Ag.

Performed in soft (1%) agar or agarose gel.

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ImmunodiffusionImmunodiffusion

‘Oudin procedure’‘Oakley – Fulthorpe

procedure

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Radial Immunodiffusion (Mancini)Radial Immunodiffusion (Mancini) Method

• Ab in gel

• Ag in a well Interpretation

• Diameter of ring is proportional to the concentration

Quantitative

• Ig levels

• Screening sera for antibodies to influenza virus

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Oucheterlony procedureOucheterlony procedure

e.g. : Elek’s test for toxigenicity in C. diphtheriae

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ImmunoelectrophoresisImmunoelectrophoresis

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Countercurrent electrophoresisCountercurrent electrophoresis

Method• Ag and Ab migrate toward each other by

electrophoresis• Used only when Ag and Ab have opposite charges• Qualitative• Rapid• For Hepatitis B Ag & Ab , Antigens of Cryptococcus in

C.S.F.

Ag Ab

- +

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Rocket ElectrophoresisRocket Electrophoresis One dimensional single immunodiffusion :

Mainly applied for quantitation of antigens.

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Laurell’s two dimensional Laurell’s two dimensional electrophoresiselectrophoresis

Variant of Rocket electrophoresis Several Ag in mixture quantitated. Antigen mixture electrophoretically separated first. Secondly electrophoresis done perpendicular to that of first

stage to get rocket like precipitation.

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Agglutination ReactionsAgglutination Reactions

Lattice Formation

Equivalence – Lattice formation

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Agglutination ReactionsAgglutination Reactions

Particulate antigen combining with its antibody in presence of electrolyte at optimal temp. & pH, resulting in visible clumping of particles.

Lattice formation. Zonal phenomenon. Incomplete or monovalent Ab do not cause

agglutination, though combine with Ag. They act as blocking Ab inhibiting agglutination.

More sensitive than precipitation for detection of Ab.

Better with IgM than IgG.

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ApplicationsApplications

Slide Agglutination : -

1. Routine procedure to identify bacterial strains

from clinical specimens e.g. Salmonella sp.

2. Blood grouping & cross matching.

+ ↔

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ApplicationsApplications

Tube Agglutination test : -

1. Enteric fever ( Widal test).

2. Typhus fever (Weil Felix test).

3. Infectious mononucleosis ( Paul Bunnel test).

4. Brucellosis.

5. Primary atypical pneumonia .

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Coombs (Antiglobulin)TestsCoombs (Antiglobulin)Tests

Incomplete Ab Direct Coombs Test

• Detects antibodies on erythrocytes

+ ↔

Patient’s RBCs Coombs Reagent(Antiglobulin)

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Coombs (Antiglobulin)TestsCoombs (Antiglobulin)Tests

Indirect Coombs Test• Detects anti-erythrocyte antibodies in serum

Patient’s Serum

TargetRBCs

+ ↔Step 1

+ ↔Coombs Reagent

(Antiglobulin)

Step 2

Detection of Anti Rh Ab & Autoimmune hemolytic anemia.

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Passive Agglutination TestPassive Agglutination Test A precipitation reaction can be converted to agglutination

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

1. Latex Agglutination test : # For detection of Hepatitis B Ag.

# ASO, CRP,

# RA factor ( Rose Waller test)

# HCG

# N. meningitidis typing

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Passive Agglutination TestPassive Agglutination Test

2. Hemagglutination Test :

Erythrocytes sensitised with antigen used for detection of antibodies. In rheumatoid arthritis, an auto Ab ( RA factor)appears in the serum which acts as Ab to gammaglobulin.RA factor can agglutinate red cells coated with gammaglobulins. Ag used is sheep red blood cells sensitised with rabbit antisheep erythrocyte Ab (amboreceptor). = Principle of Rose Waller test for RA factor detection.

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Passive Agglutination TestPassive Agglutination Test Based on the presence of protein A on the surface of some

strains of Staph. Aureus ( Cowan I strain) . Specific IgG coated on Cowan I strains of Staph. Aureus . Fc portion of IgG binds to protein A while antigen combining Fab portion remains free. When the corresponding Ag is mixed with these coated cells , Fab terminal binds to Ag resulting in agglutination. = coagglutination.

Used for detection of bacterial antigens in blood, urine, CSF.

N.gonorrhoeae, Strepto. Pyogenes, H. infuenzae Ag can be detected.

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That’s all for Now!

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