What is Antigen – antibody reactions :uses and characterstics ?

 

Antigen – antibody reactions :

Antigen combines with its specific antibody in an observable manner and the reaction between antigen antibody is specific.

A.     Uses :

1.      In the body or in vivo

v  It forms the basis of immunity against infectious diseases.

v  It may lead to tissue injury in some hypersensitivity reactions and autoimmune diseases.

2.      In the laboratory or in vitro :

v  For diagnosis of infections

v  Detection and quantitation of either antigens or antibodies.

B.     Characterstics

a.       Reaction is specific, an antigen combines only with its homologous antibody and vice-versa. However , cross reactions may occur due to antigenic similarity .

b.      Entire molecules of antigen and antibody reaction and not the fragments.

c.       The reaction is firm, but reversible.

Characters of antigen & antibody reaction :

1.      Specificity :

Specifity refers to the property of an antigen antibody reacts with only its specific antigenic determinant .

The specificity is depending upon chemical composition and physical force of the antibody.

2.      Affinity :

Affinity is the strength of interaction 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 of the antibody.

3.      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.

 

4.      Cross reaction :

An antibody produced due to specific antigenic stimuli may be sometimes reacts with another closely related antigen is called cross reaction .

Antiserum raised against the albumen of hen’s egg cross react with albumen of duck’s egg.

 

Mnemonic of Ag-Ab reactions

 In this context various antigen antibody reactions are described below with its mnemonic form,

“ preaggconeeliwife”

1.      Pre :  precipitation

2.      Agg : agglutination

3.      Co  : complement fixation

4.      Ne : neutralization

5.      Eli : ELISA

6.      W :  western blot

7.      If : immunofluorescence assay

8.      Ic :  immunochromatograph

 

AgAb I

1.      Precipitation

·         It is the specific antigen antibody reactions in which soluble antigens are reacted with its specific antibody.

·         In this reaction , two soluble reactants that come together to make one insoluble product called precipitate. The amount of precipitate is generally formed according to the proportion of antigen and antibody.

·         When the proportion of antigen antibody in optimum, the formation precipitate will be accounted as more.

·         The amount of formation of precipitate  will be reportedly less when the amount of antibody or antigen is in excess.

The precipitation reaction is following types.

1.      Precipitation in liquid medium

2.      Precipitation in Gel (immune diffusion)

3.      Precipitation in Gel using electric current

1.      Precipitation in liquid medium :

Eg. Ring test

  Flocculation test

 

Ring test :

In this reaction precipitate ring appears at the junction of two liquids (antigen and antibody)

Eg . Ascolic Thermo precipitation test for anthrax.

 

Flocculation test :

When a drop of an antigen is mixed with patient serum the precipitate is resulted. As the precipitate found to be appeared as floccules it is called flocculation. It is further classified into , slide flocculation test eg. VDRL and RPR for diagnosis of syphilis tube flocculation test e.g. Khan test.  

 

2.      Precipitation in gel (immune diffusion)

This reaction is performed in an Agarose Gel based on the principles of diffusion of antigen and antibody . it includes 

1.      Single diffusion in one dimension

2.      Double diffusion in one dimension

3.      Single diffusion in two dimensions

4.      Double diffusion in two dimensions

1.      Single diffusion in one dimension

In this, antigen can be difuses in one direction towards antibody when it is poured over the layer of gel containing antibody.

2.      Double diffusion in one dimension :

Initially agar gel is prepared in a test tube and incorporated with antibody, above that column of plain agar is added and then antigenic solution is poured in it. 

Both antigen and antibody move towards each other in opposite direction, thus line of precipitation appearing with band where they meet in plain agar.

3.      Single diffusion in two dimensions or radial immuno diffusion (Mancini).

This test is commonly used in the clinical laboratory for the determination of immunoglobulin levels in patient samples.

In this test, prepare agar gel in a sterile glass slide, to this add suitable dilution of an antiserum and then wells were made, places the antigen into well, the antigen diffusion and reacts with antibody radially to form precipitate.

Precipitate form a ring is called precipitatin ring. This ring is formed around well, by which concentration of antigen can be determined. 

4.      Double diffusion in two dimensions or ouchterlony double diffusion method :

Ouchterlony method is an effective qualitative tool for the determination of relationship between antigens and the number of different antigen antibody systems present.

Procedure :

a.       In the ouchterlony method, agar gel is prepared. Then make two wells on agar gel. Add antigen in one well and antibody in another well incubate the wells for some time.

b.      After that both antigen and antibody diffuse symmetrically or eaually from wells towards each other.

c.       Then form a concentration gradient. Finally equivalence of antigen antibody gradient reached there by invisible line of precipitation is formed.

i.                    Identity if the precipitin line is completely fused in reaction called identity.

ii.                  Non identity if the precipitate line gives rise to spur that is known as non identity.

iii.                Lines of partial identity in this, one antigen antibody pair crosses another forming double spur.

3.      Precipitation in Gel using electric current :

It includes immune electrophoresis , countercurrent electrophoresis and rocket immune electrophoresis . 

3.1.IMMUNOELECTROPHORESIS:

It is done by using electric current. It is used for the separation of protein from the complex protein mixture.

In immunoelectrophoresis , a complex mixture of antigens is placed in a well punched out of an agar gel and the the antigen allowed being electrophoresed using electric current. Antigens 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 where the antigen and antibody meet.

It is used for analyzing the components from the patient serum.

3.2.Counter current electrophoresis :

·         In this test , the antigen and antibody are placed in wells punched out of an agar gel and antibody are simultaneiously electrophoresedin gel.

·         Line of precipitation is formed where the antigen and antibody reacts.

·         The reaction takes place if antigen and antibody found to have opposite charges.

·         This test is primarily qualitative and is used for detection of capsular antigens of Cryptococcus from the CSF.

3.3.Rocket immunoelectrophoresis:

·         In this test , antigen is placed in wells punched out of an agar gel to which antibody has been incorporated and the antigen is electrophoresed into the gel.

·         Precipitin arcs are produced. As the arcs appeared like rocket is called “ immunoelectrophoresis”.

·         The height of the arc (rocket) is proportional to the concentration of the antigen, when the concentration of antigen is low it will be immobilized by the antibody before it moves.

·         In contrast when the amount of antigen is high, it will move farther before it is all tied up with antibody.

Agglutination :

    It is an antigen-antibody reaction, in which a particulate antigen contains with its antibody in the presence of electrolytes at an optimal temperature and pH, resulting in visible clumping of particles.

1.      It differs from precipitation in which soluble antigen is present in contrast to particulate antigen of agglutination.

2.      Principles governing agglutination are the same as that of precipitation.

3.      Agglutination occurs when antigen and antibody are present in optimal proportions.

4.      Lattice formation hypothesis holds good for agglutination too.

5.      The zone phenomenon may occur when either an antigen or an antibody is in excess.

Types of agglutination reaction :

1.      Slide agglutination test :

A uniform suspension of antigen is made in a drop of saline on a slide or tile and a drop of the appropriate antiserum is added.

Clumping occurs instantly or within seconds when agglutination test is positive.

Uses :

i.                    It is a routine procedure to identify the bacterial strains isolated from clinical specimens.  One example is to identify salmonella species.

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

2.      Tube agglutination test :

·         This is a standard quantitative method for determination of antibodies.

·         Serum is diluted serially by doubling dilution in test tubes.

·         An equal volume of a particulate antigen is added to all tubes.

·         The highest dilution of serum at which agglutination occurs is antibody titre.

·         Tube agglutination is routinely employed for antibody detection in diagnosis of typhoid (widal test), brucellosis and typhus fever ( Weli-felix reaction).

3.      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.

·         This conversion is done because agglutination tests are more sensitive for detection of antibodies.

·         Passive agglutination tests are very sensitive.

·         When instead of antigen, the antibody is absorbed on the carrier particles for estimation of antigens, it is known as reversed passive agglutination.

i.                    Latex agglutination test :

·         Polystyrene latex particles are employed to adsorb several types of antigens.

·         These tests are very convenient , rapid and specific.

·         These are used for detection of hepatitis B antigen, ASO,CRP,RA factor and many other antigens.

ii.                  Haemagglutination test :

Instead of latex particles,erythrocytes sensitized with antigen are used for detection of antibodies.

 

iii.                Coagglutination

Principle

·         It is based on the presence of protein A on the surface of some strains of staph. Aureus (especially cowan 1 strain).

·         Specific IgG immunoglobulin is coated on these Cowan 1 strains of Staphylococcus aureus. Fc portion  of IgG molecule binds to protein A where as antigen combining Fab terminal remains 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.Nisseria  gonorrhea and Human influenza antigens can be detected by this method.  

Complement fixation test : (CFT)

  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 (rabbit antibody to sheep erythrocytes) is used.

·         Complement can lyse these erythrocytes coated with antibodies.

·         If complement is fixed and utilized in the antigen-antibody reaction, there is no free complement to act on the indicator system and hence no lysis of erythrocytes, which indicates the positive complement fixation test.

·         Lysis of erythrocytes indicates that complement was not fixed in the first step and therefore, the serum is negative for antibodies(negative CFT).

Immunofluorescence :

·         Fluorescence is the property of certain dyes which absorb rays of one particular wave length (ultraviolet) and emit rays with a different  wave length (visible light).

·         The commonly used fluorescent dyes are fluresin isothiocyanate and lissamine rhodamine, exhibiting blue green and orange red fluorescence respectively.

Immunofluorescence test  is of two types:

1.      Direct immunofluorescence test

2.      Indirect immunofluorescence test .

Direct immunofluorescences test :

Principle

The specific antibodies tagged with fluorescent dye are used for detection of unknown antigen in a specimen .

If antigen is present it reacts with labeled antibodies and fluorescent microscope.

Uses :

1.      It is commonly employed for detection of bacteria viruses or other antigens in blood, CSF, urine and other specimens.

2.      It is a sensitive method to diagnose rabies, by detection of the rabies virus antigens in brain smears.

2.Indirect immunofluorescence test :

  The indirect method is employed for detection of antibodies in serum or other body fluids.

Principle :

1.      A known antigen is fixed on a slide.

2.      The unknown antibody (serum) is applied to the slide.

3.      If antibody is present in the serum, it attaches to known antigen on the slide.

4.      For detection of this antigen-antibody reaction, fluorescein-tagged antibody to human globulin is added.

5.      In positive test, fluorescence occurs under ultraviolet light.

Enzyme linked immuno sorbent assay (ELISA)

1.      Elisa has been applied widely for detection of a variety of antibodies and antigens.

2.      The principle of ELISA is almost the same as that of immunofluorescence, the only difference being, an enzyme is used instead of fluorescent dye.

3.      The enzyme acts on substrate to produce a colour in a positive test.

4.      The test can be done in polystyrene tubes or polyvinyl microtitre plates (micro-ELISA).

SANDWICH ELISA:

1.      For antigen detection in a specimen, the wells of microtitre plate are coated with specific antibody against the antigen to be detected.

2.      Specimens to be tested are added in coated wells.

3.      If antigen is present in specimen, it binds to coated antibody.

4.      To detect this antigen-antibody reaction , antiserum conjugated with an enzyme is added.

5.      This conjugated antiserum binds to an antigen already attached to coated antibody.

6.      A substrate is added to know the binding of conjugated antiserum to antigen-antibody complex.

7.      In case of binding an enzyme acts on substrate to produce colour, intensity of which can be read by spectrophotometer or ELISA reader.

8.      Colour detection can also be seen by naked eye.

9.      This type of ELISA test is known as sandwich ELISA.

INDIRECT ELISA :

1.      For antibody detection, the wells of micro titre plate are coated with antigen.

2.      Sera to be tested are added in these coated wells.

3.      If antibody is present in specimen, it binds to coated antigen.

4.      To detect this antigen-antibody reaction, a goat antihuman immunoglobulin antibody conjugated with an enzyme is added.

5.      Enzyme conjugated antihuman immunoglobulin binds to antibody.

6.      To detect this binding, a substrate is added and enzyme acts on substrate to produce colour in a positive reaction.

7.      This procedure is also named as indirect ELISA.

8.      Reading of the test is the same as described in sandwich ELISA.

COMPETITIVE ELISA :

1.      It has been used for detection of HIV antibodies.

2.      Positive result shows no colour whereas appearance of colour indicates a negative test.

3.      The microtitre plate wells are coated with HIV antigen.

4.      Sera to be tested is added to these wells.

5.      If antibody are present,antigen-antibody reaction occurs.

6.      To detect this reaction, enzyme labeled specific  HIV antibodies are added.

7.      There is no antigen left for these antibodies to act.

8.      These antibodies remain free and washed off during washing.

9.      Substrate is added but there is no enzyme to act on it.

10.  Therefore , positive results show no colour.

11.  If serum to be tested is negative for antibodies, antigen is there to combine with enzyme conjugated antibodies and enzyme acts on substrate to produce colour.

Uses of ELISA :

It has been used for detection of antigen and antibodies of various microorganisms. Some example are :

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.

Immuno chromatography :

1.      Immuno chromatography or lateral flow immunoassay is one of the most popular form of rapid immunoassays.

2.      It can detect both antigens and antibodies.

3.      It has advantage to being a one step test.

4.      it can be completed within 30 minute. It is a strip based test.

5.      The strip contains a chromatographic pad with three zones: sample pad, conjugate pad capture line.

6.      The conjugate pad may be having colloidal gold, dye, or latex beads as conjugate which produces signal.

7.      The specimen is applied to the sample pad and flows laterally by capillary action.

8.      Upon reaching the conjugate pad, it may bind to conjugate if antigen or antibody is present in the specimen and forms antigen antibody complex.

9.      This complex then flow laterally to capture line.

10.  Here it is captured by antigen or second antibody present in the capture line.

11.  The presence of colour line is a positive test.

12.  There is a positive control line also to check that the test was properly performed.