Antigen and Antibody

Reactions

basics

Dr.T.V.Rao MD

Dr.T.V.Rao MD 1

Classification of antigen-antibody

interactions 1. Primary serological tests: (Marker techniques) e.g.

– Enzyme linked immuono sorbent assay (ELISA)

– Immuno florescent antibody technique (IFAT)

– Radio immuno assay (RIA)

2. Secondary serological tests: e.g. – Agglutination tests

– Complement fixation tests (CFT)

– Precipitation tests

– Serum neutralization tests (SNT)

– Toxin-antitoxin test

3. Tertiary serological test: e.g. – Determination of the protective value of an anti serum in an

animal.

Dr.T.V.Rao MD 2

Types of Antigen and Antibody

reactions

1. Agglutination tests

2. Double diffusion precipitation tests

3. Immunoelectrophoresis

4. Western blot tests

5. Complement fixation tests

6. Immunofluorescence testing

7. Immunoassays

Dr.T.V.Rao MD 3

Serological Tests

• Antigen and antibody reactions in vitro are known as serological tests

What Happens can be studied in 3 stages

1st Antigen and antibody react with visible effects, obeys the laws of physical dynamics. But reversible

The reaction is effected by weaker intermolecular forces – Vander Waal-s forces

Ionic bonds, Hydrogen bonding not by covalent bonding

Can be detected by Radioactive isotopes, fluorescent dyes

Dr.T.V.Rao MD 4

What happens in Antigen and

Antibody reactions

• React with each other in a observable manner.

• Uses

1 Helps antibody mediated immunity in infection, and tissue injury

2 Helps diagnosis of Infections.

3 In epidemiological surveys

4 Detections and quantization of antigens and antibodies

Dr.T.V.Rao MD 5

Detectable reactions 2nd Stage

• Reaction can occur as

1 Precipitation

2 Agglutination

3 Lysis and killing of live antigens

4Neutralizatiobn of toxins

5 Fixation of complement

6 Immobilization of motile microbes

7 Enhancement of Phagocytosis.

Dr.T.V.Rao MD 6

General features of Antigen and

antibody reactions. • Specific reaction – combines with specific

antigen

• Entire molecule reacts not fragments

• No denaturation of antigen or antibody

• Combination occurs as surface antigens to surface of antibodies

• Commination is firm but reversible depends on affinity and avidity

• Both antigens and antibodies participate

• Combine in varying proportions Bivalent and multivalent

Dr.T.V.Rao MD 7

Terms used in evaluating test

Methodology

• Sensitivity – Analytical Sensitivity – ability of a test to

detect very small amounts of a substance

– Clinical Sensitivity – ability of test to give

positive result if patient has the disease (no

false negative results)

Dr.T.V.Rao MD 8

Specificity

• Analytical Specificity – ability of test

to detect substance without

interference from cross-reacting

substances

• Clinical Specificity – ability of test to

give negative result if patient does not

have disease (no false positive

results)

Dr.T.V.Rao MD 9

Affinity

• Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody.

• It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site .

Dr.T.V.Rao MD 10

Avidity • Avidity is a measure of

the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies

• Avidity is influenced by both the valence of the antibody and the valence of the antigen.

• Avidity is more than the sum of the individual affinities.

Dr.T.V.Rao MD 11

Dr.T.V.Rao MD 12

Dilution

• Estimating the

antibody by

determining the

greatest degree to

which the serum may

be diluted without

losing the power to

given an observable

effect in a mixture

with specific antigen

Dr.T.V.Rao MD 13

Measurement of Antigen and

Antibody reactions

• Measured as

Mass

Nitrogen

(microgram)

• As Units

• As titer

Dr.T.V.Rao MD 14

Titer

• Different dilutions

of serum are tested

in mixture with a

constant amount of

antigen and

greatest reacting

dilution is taken as

the measure or

Titer

Dr.T.V.Rao MD 15

Common methods in creating

dilutions

Dr.T.V.Rao MD 16

Expression of Titers

• Expressed in term of the

was in which they are

made

• Dilution 1 in 8 is a

dilution made by mixing

one volume of serum

with seven volumes of

diluents (Normal Saline )

• Incorrect to express

dilution as 1/8

Dr.T.V.Rao MD 17

Majority Diagnostic tests are

Serological tests

• There are several

serology techniques

that can be used

depending on the

antibodies being

studied. These

include: ELISA,

agglutination,

precipitation,

complement-fixation,

and fluorescent

antibodies. Dr.T.V.Rao MD

Antigens x Antibody

Dr.T.V.Rao MD 19

How Antigen – Antibody reaction

occurs

• Primary stage – Interaction between antigen and antibody occurs without any visible effects

• The reaction is rapid even at low temperature but the reaction is reversible

• Weaker _ intermolecular forces Van der wall’s force and ionic bonds

• Can be measured with radioactive isotopes and Florescent dyes.

Dr.T.V.Rao MD 20

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!

Dr.T.V.Rao MD 21

- Four types of non-covalent forces operates over a very short distance

( generally 1 angstrom )

Nature of Ag/Ab Reactions

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What happen futher ?

• 1 Precipitation

• 2 Lysis of cells.

• 3 Killing of live antigen

• 4 Neutralization of toxins

• 5 Complement fixation

• 6 Immobilization of motile microbes

• 7 Enhancement of Phagocytosis

• 8 Entire molecule react no fragmented

• 9 No denaturation

10 Combination occurs on surface

11 Firm but reversible

Dr.T.V.Rao MD 23

Karl Landsteinar (1868-1943)

• An Austrian physician

by training,

Landsteiner played an

integral part in the

identification of blood

groups. He

demonstrated the

catastrophic effect of

transfusing with the

wrong type of blood,

Dr.T.V.Rao MD 24

Serology • The branch of

laboratory medicine

that studies blood

serum for evidence of

infection and other

parameters by

evaluating antigen-

antibody reactions in

vitro

Dr.T.V.Rao MD 25

Serology

• Serology is the scientific study of blood serum. In practice, the term usually refers to the diagnostic identification of antibodies in the serum

We can detect antigens too

Dr.T.V.Rao MD 26

Types of serological tests

1. Agglutination tests

2. Double diffusion precipitation tests

3. Immunoelectrophoresis

4. Western blot tests

5. Complement fixation tests

6. Immunofluorescence testing

7. Immunoassays

Dr.T.V.Rao MD 27

Immunology/ Serology?

Precipitation Reactions • Capillary tube precipitation (Ring Test)

• Ouchterlony Double Diffusion

(Immunodiffusion)

• Radialimmunodiffusion (RID)

• Immunoelectrophoresis (IEP)

• Rocket Electroimmunodiffusion (EID)

• Counterimmunoelectrophoresis (CIEP)

Dr.T.V.Rao MD

Antigen – Antibody reactions

presenting with precipitation

Dr.T.V.Rao MD 29

Precipitation - Reaction

• In precipitation antigen combines with its

antibody in the presence of electrolytes

( Nacl ) at a suitable temperature and Ph

the antigen and antibody complexes form

a insoluble precipitate suspended as

floccules.

• Reaction can take place in liquid medium,

gels, agar, agarose, polyacrylamide

Dr.T.V.Rao MD 30

Precipitation

• Principle – Soluble antigen + antibody (in proper proportions) –>

visible precipitate

– Lattice formation (antigen binds with Fab sites of 2 antibodies)

• Examples – Double diffusion (Ouchterlony)

– Single diffusion (radial Immunodiffusion)

– Imunoelectrphoresis

– Immunofixation

Dr.T.V.Rao MD 31

How precipitation occurs

• Precipitation occurs with most antigens because the antigen is multivalent (i.e. has several antigenic determinants per molecule to which antibodies can bind). Antibodies have at least two antigen binding sites (and in the case of IgM there is a multimeric complex with up to 10 antigen binding sites), thus large aggregates or gel-like lattices of antigen and antibody are formed

• Specific reaction Antigen combines with homologusantibody - vice versa

Dr.T.V.Rao MD 32

Mechanism of Precepitation

• Lattice hypothesis –

Multivalent antigens

combine with bivalent

antibodies in varying

proportions

depending on the

antigen and antibody

ratio in the reacting

mixture

Dr.T.V.Rao MD 33

Precipitation reactions in fluids yield a precipitin curve.

FIGURE 6-4

( Lattices or

large aggregates )

( no precipitate is formed

if an Ag contains only a

single copy of each epitope )

Precipitation Reactions

Dr.T.V.Rao MD 34

Applications of precipitation

reactions

• Qualitative and

quantitative

• More useful for

antigen detection to

as least as 1

microgram

• Blood, seminal stains,

Food adulteration

Dr.T.V.Rao MD 35

Slide test -Flocculation test –

VDRL test

• VDRL Test – a drop of

VDRL antigen to a drop

of patients serum,

• Shake

• The reaction observed

under microscope

• Observe for flocculation

reaction

• A Khan test is done in a

test tube

Dr.T.V.Rao MD 36

Non reactive and Reactive VDRL

Tests

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Agglutination+: ve RPR Agglutination : -

Dr.T.V.Rao MD 38

Tube test - Precipitation

• Kahn test for Syphilis – a tube flocculation test.

• Quantitative tube flocculation test used in

standardarisation of toxin/toxoid

• Serum dilution of toxin or toxoid is added to

tubes containing a fixed quantity of antitoxin

• The amount of toxin that flocculates optimally

with one unit of antitoxin is defined as Lf dose

Dr.T.V.Rao MD 39

Single diffusion in one

Direction

O

Oudin Procedure

Dr.T.V.Rao MD 40

Ring Test

• The reaction is demonstrated by layering antigen solution over the column of antigen in a narrow tube, The precipitate forms at the junction of two liquids.

• Eg Ascoli’s thermo precipitate test

• Grouping of streptococci

• C-reactive protein

Dr.T.V.Rao MD 41

Oudin Procedure

• Antibodies in agar gel

• Above antigen is layered

• Single diffusion in one direction

• Called Qudin procedure

• Antibody is incorporated in agar

• Antigen diffuses down

• Precipitation concentration of antigen at the site increases due to diffusion

Dr.T.V.Rao MD 42

Dr.T.V.Rao MD 43

Double diffusion in one dimension

Oakley Fulthrope procedure

• Antibodies in gel

• Agar layer

• Antigen layered

• Antigens and antibodies moves towards

each other

• Forms precipitate

Dr.T.V.Rao MD 44

Single Diffusion In two Dimensions

Radial Immunodiffusion

• Radial Immunodiffusion

is an Immunodiffusion

technique used in

immunology to detect

quantity of antigen by

measuring the radius

surrounding samples of

the antigen, marking the

boundary between it and

antibody

Dr.T.V.Rao MD 45

Single Diffusion In two Dimensions

Radial Immunodiffusion

• Antigen is added to the wells cut on the surface of the gel

• It diffuses radially from well and forms a ring shaped band of precipitation

• The halo of precipitation diameter gives the estimate of concentration of antigen

• Used in estimation of Immunoglobulin's

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Precipitation in Agar

• These tests are done

in agar

• Tested for passive

immuno diffusion in

Agarose

• There are several

methods in testing

this procedure

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Gel electrophoresis allows to

separate pieces of DNA.

Dr.T.V.Rao MD 48

Elek’s Immunodiffusion Test

• Elek Immunodiffusion test. Sterile filter paper impregnated with diphtheria antitoxin is imbedded in agar culture medium. Isolates of C

diphtheria are then streaked across the plate at an angle of 90° to the antitoxin strip. Toxigenic C diphtheria is detected because secreted toxin diffuses from the area of growth and reacts with antitoxin to form lines of precipitin Dr.T.V.Rao MD 49

Immuno Diffusion Test

• Can demonstrate

the

immunological

identity (or not)

of two antigen

samples; radial

Immunodiffusion

Dr.T.V.Rao MD 50

Radial Immunodiffusion

• Radial Immunodiffusion, a variation of the agar precipitation technique, is used in clinical immunology for the detection and quantitation of all classes of Immunoglobulin's, complement, ceroplastic, transferring, and other serum components

Dr.T.V.Rao MD 51

Double Diffusion in one Dimension

Oakley – Fulthrope procedure

• In Double diffusion in one direction antibody in agar gel moves through the layer of plain agar to the antigen above

• They react to each other

• Form a band of precipitation

• Virus antigen is placed in the central well and diffuses outwards. Wells A and C contain positive sera, well B contains a negative sample. The black areas show where antibody in the positive sera have bound to virus antigen and formed a precipitate

Dr.T.V.Rao MD 52

Double diffusion in two dimensions

Ouchterlony procedure

• Both antigen and antibody diffuses independently through agar gel in two dimensions horizontally and vertically

• Done in a slide or petridish

• In 12 – 48 hours line of precipitates are formed

• Useful in serological identification

Dr.T.V.Rao MD 53

Immunoelectrophoresis:

In Immunoelectrophoresis, a complex mixture of

antigens is placed in a well punched out of an

agar gel and the antigens are electrophoresed

so that the antigen are separated according to

their charge. After electrophoresis, a trough is

cut in the gel and antibodies are added. As the

antibodies diffuse into the agar, precipitin lines

are produced in the equivalence zone when an

antigen/antibody reaction occurs.

Dr.T.V.Rao MD 54

Immuno-electrophoresis

• Mixtures containing multiple antigen

species which cross react with the same

antiserum may be analysed by running

them first on an analytical gel, then cutting

a strip from that gel and laying it in a slit

cut into the immuno-electrophoresis gel to

form a large well. The result is a pattern

which shows the positions of strongly

reacting antigen species.

Dr.T.V.Rao MD 55

Immunoelectrophoresis

• Immunoelectrophores

is--migration of

molecules due to

electric

charge Positive

particles travel to

cathode Negative

particles travel anode

Precipitin specificity

provides a critical

indicator of identity Dr.T.V.Rao MD 56

Immuno Electrophoresis

• In this procedure the

electrophoretic separation

of compatible antigen into

constituent protein

followed by

Immunodiffusion against

its antiserum resulting in

separate precipitation

line, indicating relation

between each individual

protein in the antibody

Dr.T.V.Rao MD 57

Electrical gel

Immunoelectrophoresis

• The reaction is

electrically driven,

different antigens are

separated according

to their charges under

electrical fields

• Number of antigens

can be identified

Dr.T.V.Rao MD 58

Countercurrent electrophoresis

• Method

– Ag and Ab migrate toward each other by electrophoresis

– Used only when Ag and Ab have opposite charges

• Qualitative –Rapid

Ag Ab

- +

Dr.T.V.Rao MD 59

Counter current

Immunoelectrophoresis

• In this procedure

movement of antigens

towards the anode and

antibody towards the

cathode through agar

under electric field

• Useful studies on CSF

• Hepatitis B surface

antigen detection

• Detection of fetoproteins

Dr.T.V.Rao MD 60

Imunoelectrphoresis (IEP)

Qualitative • A serum sample is

electrophoresed through an agar medium.

• A trough is cut in the agar and filled with Ab.

• A precipitin arc is then formed.

• Because Ag diffuses radially and Ab from a trough diffuses, the reactants meet in optimal proportions for precipitation.

Dr.T.V.Rao MD 61

Rocket electrophoresis

Laurel

• The reactions appear

as rocket

• Driven by electric

current

• Rocket” Immuno-

Electrophoresis is

used as a rapid way

to quantitate antigen

in complex samples.

Dr.T.V.Rao MD 62

Immuno-electrophoresis gel

• Mixtures containing multiple antigen

species which cross react with the same

antiserum may be analyzed by running

them first on an analytical gel, then cutting

a strip from that gel and laying it in a slit

cut into the immuno-electrophoresis gel to

form a large well. The result is a pattern

which shows the positions of strongly

reacting antigen species.

Dr.T.V.Rao MD 63

Counterimmunoelectrophoresis

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Electro Immunodiffusion

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Rocket electrophoresis

• Crossed

Immunoelectrophoresis of

antigens and antiserum.

In the first dimension,

proteins are separated by

standard electrophoresis.

The separated proteins

are then run into the

second dimension gel at

an angle of 90° from the

first dimension.

Dr.T.V.Rao MD 67

Serology can be done on various

specimens

• Some serological tests are not limited to blood serum, but can also be performed on other bodily fluids such as semen and saliva, which have (roughly) similar properties to serum.

• Serological tests may also be used forensically, generally to link a perpetrator to a piece of evidence (e.g., linking a rapist to a semen sample).

Dr.T.V.Rao MD 68

Measurement of Precipitation by

Light

• Antigen-antibody complexes, when formed

at a high rate, will precipitate out of a

solution resulting in a turbid or cloudy

appearance.

• Turbidimetry measures the turbidity or

cloudiness of a solution by measuring

amount of light directly passing through a

solution. • Nephelometry indirect measurement, measures amount of light

scattered by the antigen-antibody complexes. Dr.T.V.Rao MD 69

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

Anti-A

Ab

Ag A

Anti-A

Ab

Ag B

Shared epitope

Anti-A

Ab

Ag C

Similar epitope

Cross reactions

Dr.T.V.Rao MD 70

Dr.T.V.Rao MD 71

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