immunodiffusion presentation

8/6/2019 Immunodiffusion Presentation

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Submitted by:Swati singh

Neha verma

Monika pandey


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Introductiony Antibody and soluble antigen interacting in aq sol

form a lattice that eventually develops into a visibleprecpitate.

y Antibody that aggregate soluble antigens are calledprecipitins.

y Immnune precipitates can form not only in solutionbut also in agar matrix

yWhen antigen and antibody diffuse towards oneanother in agar or when antibody is incorporated intoagar and antigen diffuses into the antibody containing matrix ,a visible precipitate will form.


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y Immunodiffusion is a technique for the identificationand quantification of any of the immunoglobulins.

y It is based on the presence of a visible precipitate thatresults from an Ag-Ab combination under certaincircumstances.

y Gel diffusion is a technique that involves evaluation ofthe precipitin reaction in a clear gel.

y Two commonly known immunodiffusion techniquesare: double immunodiffusion(ouchterlony method)and radial immunodiffusion(mancini method).


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Double immunodiffusiony In this method both antigen and antibody diffuse

radially from wells towards each other,thereby

establishing a concentration gradient.y Precipitation occurs with most antigens because the

antigen is multivalent (i.e. has several antigenicdeterminants per molecule to which antibodies can

bind).y Antibodies have at least two antigen binding sites,

thus large aggregates or gel-like lattices of antigen andantibody are formed.


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y Experimentally, an increasing amount of antigen isadded to a constant amount of antibody in solution,

initially at low antigen concentration, all of the antigenis contained in the precipitate.

y This is called the antibody-excess zone i.e. prozonephenomenon.

yAs more antigen is added, the amount proteinprecipitated increases until the antigen/antibodymolecules are at an optimal ratio.


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y This is known as the zone of equivalence or equivalencepoint.

y The zone of equivalence lines may give a full identity (i.e.

a continuous line), partial identity (i.e. a continuous linewith a spur at one end), or a non-identity (i.e. the twolines cross completely).

y Where the two diffusion fronts meet, if any of theantibodies recognize any of the antigens, they will bind

to the antigens and form what is known as an immunecomplex.y This immune complex precipitates in the gel to give a

thin white line, which is a visual signature of antigenrecognition.


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Radial immunodiffusiony Radial immunodiffusion (or Mancini method)is a

technique used in immunology to determine thequantity of an antigen by measuring the diameters of

circles of precipitin complexes,surrounding samples ofthe antigen that mark the boundary between the antigenand an antibody suspended in a medium, such as an agargel.

y In radial immunodiffusion ,an antigen sample is placed

in a well and allowed to diffuse into agar containing asuitable dilution of an anti-serum.y As the antigen diffuses into the agar the region of

equivalence is establised and a ring of preciptation ,aprecipitin ring,forms around the well.


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y The area of precipitin ring is proportion to theconcentration of antigen.

y

By comparing the area of precipitin ring with a standardcurve,the concentration of the antigen sample can bedetermined.

y The diameters of the circles increasewith time as theantigen diffuses into the medium, reacts with the antibody,

and forms insoluble precipitin complexes.

y It can be applied when sensitivity is not required butspecificity is, and antibody is plentiful. It is commonly usedin clinically detecting levels of blood proteins in a patient.


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Thank you

immunodiffusion presentation

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