Antigen and Antibody Reactions

basics
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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.

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Types of Antigen and Antibody reactions

1. 2. 3. 4. 5. 6. 7. Agglutination tests Double diffusion precipitation tests Immunoelectrophoresis Western blot tests Complement fixation tests Immunofluorescence testing Immunoassays
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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
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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
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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.
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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
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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)

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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)
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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 .
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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.
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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
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Measurement of Antigen and Antibody reactions

Measured as Mass Nitrogen (microgram) As Units As titer

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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
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Common methods in creating dilutions

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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

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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 Dr.T.V.Rao MD antibodies.

Antigens x Antibody

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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 walls force and ionic bonds Can be measured with radioactive isotopes and Florescent dyes.
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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!
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Nature of Ag/Ab Reactions

- Four types of non-covalent forces operates over a very short distance Dr.T.V.Rao MD 22 ( generally 1 angstrom )

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
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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,
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Serology
The branch of laboratory medicine that studies blood serum for evidence of infection and other parameters by evaluating antigenantibody reactions in vitro
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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
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Types of serological tests

1. 2. 3. 4. 5. 6. 7. Agglutination tests Double diffusion precipitation tests Immunoelectrophoresis Western blot tests Complement fixation tests Immunofluorescence testing Immunoassays
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Immunology/ Serology? Precipitation Reactions

Capillary tube precipitation (Ring Test) Ouchterlony Double Diffusion (Immunodiffusion) Radialimmunodiffusion (RID) Immunoelectrophoresis (IEP) Rocket Electroimmunodiffusion (EID) Counterimmunoelectrophoresis (CIEP)
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Antigen Antibody reactions presenting with precipitation

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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
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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
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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
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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
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Precipitation Reactions

( no precipitate is formed ( Lattices or if an Ag contains only a large aggregates ) single copy of each epitope ) FIGURE 6-4

Precipitation reactions inDr.T.V.Rao MD fluids yield a precipitin curve. 34

Applications of precipitation reactions

Qualitative and quantitative More useful for antigen detection to as least as 1 microgram Blood, seminal stains, Food adulteration

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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
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Non reactive and Reactive VDRL Tests

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Agglutination+: ve

RPR

Agglutination

:-

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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

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Single diffusion in one Direction O

Oudin Procedure

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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 Ascolis thermo precipitate test Grouping of streptococci C-reactive protein

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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
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Double diffusion in one dimension Oakley Fulthrope procedure

Antibodies in gel Agar layer Antigen layered Antigens and antibodies moves towards each other Forms precipitate

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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

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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 Dr.T.V.Rao MD Used in estimation of 46 Immunoglobulin's

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.

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Eleks 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

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Immuno Diffusion Test

Can demonstrate the immunological identity (or not) of two antigen samples; radial Immunodiffusion
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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
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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
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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
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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.
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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.
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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
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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
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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

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Countercurrent electrophoresis
Method
Ag and Ab migrate toward each other by electrophoresis Used only when Ag and Ab have opposite charges

Ag Qualitative
Rapid
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+ Ab

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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

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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.
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Rocket electrophoresis Laurel

The reactions appear as rocket Driven by electric current Rocket ImmunoElectrophoresis is used as a rapid way to quantitate antigen in complex samples.
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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.
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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.
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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).
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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
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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 Ab Anti-A Ab Anti-A Ab

Ag A

Ag B

Ag C

Shared epitope Dr.T.V.Rao MD

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