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IMMUNODIFFUSION:

Immunodiffusionis a diagnostic test which involves diffusion through a substance such as agar.

Immunodiffusion techniques det ect ant ige n-a nti bod y precipitation reacti ons in a semi sol idmedium. The formation of antigen a n d antibody comp lexes c a n be influenced by a number of

factors: relat ive concentrat ion of antigens a n d antibodies, ionic strength of t h e buffer , pH ,a n d temperature. Two techniques most often used in a clinical laboratory are single a n d double

diffusion.

RADIAL IMMUNO DIFFUSION:

Radial immunodiffusionor !ancini method, !ancini immunodiffusion, single radial immunodiffusion

assay" is an immunodiffusiontechnique used in immunologyto determine the quantity of anantigenby

measuring the diameters of circles ofprecipitin complexessurrounding samples of the antigen that mar# theboundary between the antigen and an antibodysuspended in a medium, such as an agargel. The diameters of

the circles increase with time as the antigen diffuses into the medium, reacts with the antibody, and forms

insoluble precipitin complexes.Aim: -$adial immunodiffusion technique for the quantitative analysis of the given antigen.

Requirements:

Glassware:%onical flas#, measuring cylinder.

Reaents:&lcohol, 'istilled water.

Ot!er Requirements:!icropipette, Tips, !oist chamber box with wet cotton".

"it des#ri$tion : (tandard antigen at different concentration ).*+ mgml, ).+ mgml, .)mgml, *.)mgml"

%rin#i$le:

(ingle radial immunodiffusion $I'" is used extensively for the quantitative estimation of antigens. The

antigen-antibody precipitation is made more sensitive by the incorporation of antiserum in the agarose. &g isthen allowed to diffuse from wells cut in the gel in which the antiserum is uniformly distributed. Initially, as the

antigen diffuses out of the well, its concentration is relatively high and soluble &g-&b adducts are formed.However, as &g diffuses farther from the well, the &g-&b complex reacts with more amount of antibody

resulting in a lattice that precipitates to form a precipitin ring. Thus, by running a range of #nown antigen

concentrations on the gel and by measuring the diameters of their precipitin rings, a calibration graph is plotted.

&ntigen concentrations of un#nown samples, run on the same gel can be found by measuring the diameter ofprecipitin rings and extrapolating this value on the calibration graph.

%ro#edure:

. repare ) ml of .)/ agarose ). g)ml" in 0 assay buffer by heating slowly till agarose dissolvescompletely. Ta#e care not to scorch or froth the solution.

*. &llow the molten agarose to cool to ++1%.2. &dd *) ml of antiserum to 3 ml of agarose solution. !ix by gentle swirling for uniform distribution ofantibody.

4. our agarose solution containing the antiserum onto a grease free glass plate set on a hori5ontal surface.

6eave it undisturbed to form a gel.

+. %ut wells using a gel puncher using the template provided.3. &dd *) ml of the given standard antigens and test antigens to the wells.

7. 8eep the gel plate in a moist chamber box containing wet cotton" and incubate overnight at room

http://en.wikipedia.org/wiki/Agarhttp://biotech-anaj.blogspot.com/2007/09/radial-immuno-diffusion.htmlhttp://en.wikipedia.org/wiki/Immunodiffusionhttp://en.wikipedia.org/wiki/Immunologyhttp://en.wikipedia.org/wiki/Antigenhttp://en.wikipedia.org/wiki/Antigenhttp://en.wikipedia.org/wiki/Precipitinhttp://en.wikipedia.org/wiki/Precipitinhttp://en.wikipedia.org/wiki/Antibodyhttp://en.wikipedia.org/wiki/Agarhttp://en.wikipedia.org/wiki/Gelhttp://en.wikipedia.org/wiki/Agarhttp://biotech-anaj.blogspot.com/2007/09/radial-immuno-diffusion.htmlhttp://en.wikipedia.org/wiki/Immunodiffusionhttp://en.wikipedia.org/wiki/Immunologyhttp://en.wikipedia.org/wiki/Antigenhttp://en.wikipedia.org/wiki/Precipitinhttp://en.wikipedia.org/wiki/Antibodyhttp://en.wikipedia.org/wiki/Agarhttp://en.wikipedia.org/wiki/Gel

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temperature.

9. !ar# the edges of the circle and measure the diameter of the ring. ote down your observations.

;. lot a graph of diameter of ring on here the two diffusion fronts meet, if any of the

antibodies recogni5e any of the antigens, they will bind to the antigens and form what is #nown as an immunecomplex. This immune complex precipitates in the gel to give a thin white line, which is a visual signature of

antigen recognition.

http://en.wikipedia.org/wiki/Agar_gelhttp://en.wikipedia.org/wiki/Immunodiffusionhttp://en.wikipedia.org/wiki/Immunodiffusionhttp://en.wikipedia.org/wiki/Passivehttp://en.wikipedia.org/wiki/Passivehttp://en.wikipedia.org/wiki/Doublehttp://en.wikipedia.org/wiki/Immunodiffusionhttp://en.wikipedia.org/wiki/Extractable_nuclear_antigenshttp://en.wikipedia.org/wiki/Agar_gelhttp://en.wikipedia.org/wiki/Immunodiffusionhttp://en.wikipedia.org/wiki/Passivehttp://en.wikipedia.org/wiki/Doublehttp://en.wikipedia.org/wiki/Immunodiffusionhttp://en.wikipedia.org/wiki/Extractable_nuclear_antigens

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The method can be conducted in parallel with multiple wells filled with different antigen mixtures and multiple

wells with different antibodies or mixtures of antibodies, and antigen-antibody reactivity can be seen by

observing between which wells the precipitate is observed. >hen more than one well is used there are manypossible outcomes based on the reactivity of the antigen and antibody selected. The 5one of equivalence lines

may give a full identity i.e. a continuous line", partial identity i.e. a continuous line with a spur at one end", or

a non-identity i.e. the two lines cross completely".

+!eor(

recipitation occurs with most antigens because the antigen is multivalent i.e. has several antigenic

determinants per molecule to which antibodies can bind". &ntibodies have at least two antigen binding sites

and in the case of Ig! there is a multimeric complex with up to ) antigen binding sites", thus large aggregates

or gel-li#e lattices of antigen and antibody are formed. Bxperimentally, an increasing amount of antigen isadded to a constant amount of antibody in solution, initially at low antigen concentration, all of the antigen is

contained in the precipitate. This is called the antibody-excess 5one i.e.pro5one phenomenon". &s more

antigen is added, the amount protein precipitated increases until the antigenantibody molecules are at an

optimal ratio. This is #nown as the 5one of equivalence or equivalence point. >hen the amount of antigen insolution exceeds the amount of antibody, the amount of precipitation will decrease. This is #nown as the antigen

excess 5one.

http://en.wikipedia.org/wiki/Prozone_phenomenonhttp://en.wikipedia.org/wiki/Prozone_phenomenonhttp://en.wikipedia.org/wiki/Prozone_phenomenon