IMMUNOPRECIPITATION & IMMUNODIFFUSION

 IMMUNOPRECIPITATION & IMMUNODIFFUSION

·      Immunoprecipitation is the reaction between antibody and soluble antigen (if we had used particulate Ag , we would have said this to be an agglutination reaction) to form a soluble Ag-Ab complex lattice that eventually turns into an insoluble precipitate when the size of the lattice is greater than the critical value

·      The paratope present in Fab(Fragment Antigen Binding) of Antibody(Ab) only binds to a particular epitope on Antigen(Ag).

FIGURE 1,2





·      For a lattice to be formed an Ag is required to be bound by two Fab parts of two different Abs. [The 2 Abs can have the same epitope specificity in that case the Ag must have two same epitopes (if similar not identical, chances of cross-reactivity – game of probability), or the 2 Abs can be specific against two different epitopes of the same Ag]

FIGURE 3, 4





·      Hence Polyclonal antibodies are a better choice in cases of carrying out immunoprecipitation reactions as polyclonal antibodies are secreted by multiple clones of B cells activated by a particular Ag and thus against a complex Ag it is specific to various epitopes on the surface of Ag.

·      Thus, Ab needs to be bivalent and Ag needs to be atleast bivalent or better yet polyvalent.

·      To know the concentration of Ag required to establish a “zone of equivalence i.e., for every Ag, there are two Abs” thus, the lattice formation is maximum, a graph is plotted from the data obtained by measuring the amount of precipitate formed versus the increasing amount of soluble Ag keeping the amount of Ab constant (known) FIGURE 5



·      Immunoprecipitation/Precipitation can take in liquid or gel( agar/agarose/polyacrylamide) medium. [Also note, Agarose is a polysaccharide derived from red seaweed Gracilaria/Gellidium, it is a homopolymer of agarobiose (D- galactose and 3,6- Anhydro L- galactose dimer)]

·      When precipitation reactions occur in a gel medium where Ag/Ab/both diffuses through the medium, such reactions are called immunodiffusion – Single Radial Immunodiffusion (SRID) or Mancini’s Technique and Ouchterlony’s Double Diffusion (ODD) or Ouchterlony’s Technique.

 

SINGLE RADIAL IMMUNODIFFUSION (SRID)

·      It is used to determine the relative concentration of Antigen.

·      On a grease-free slide, we evenly spread around 6ml of agarose gel mixed homogeneously as possible with Antibody.

·      After the agarose solidifies, holes are dug out using a puncher and filled with Ag solution having different concentrations using a micropipette.

·      The slide is then incubated at room temperature in a moist box for 24 hours.

·      The following day, we observe Precipitin Ring due to precipitation at the zone of equivalence preceded by lattice formation.

·      The diameter of the precipitin ring is directly proportional to the concentration of Ag in the well, more concentration – more diameter.

·      Direct proportionality is the virtue of the fact that for a particular concentration of Ab, the higher concentration Ag will form a precipitin ring farther apart from the well than a lesser concentration one. This is because, from the well, the concentration of Ag gradually decreases as it diffuses through the medium and forms a lattice and thereby precipitation at the zone of equivalence.

·      How to quantify? – For example, we dug out 6 wells, we add 4 known concentration Ag solutions and 2 Test samples.

§  With the 4 known concentration values, we measure the diameter of the precipitin ring and thereby plot a graph.

§  For the 2 unknown concentration value, we measure the diameter, and see the corresponding value of antigen concentration from the already calibrated graph.

FIGURE 6, 7





 

OUCHTERLONY’S DOUBLE DIFFUSION (ODD)

·      It is used to compare 2 Ags.

·      The agarose is spread on a plate by itself and 3 holes are dug out for each set. 2 are filled with 2 different Ag samples and 1 is filled with Ab solution.

·      If the precipitin line formed after incubation –

§  Arc shaped: then the two Ags are identical

§  Y shaped: then the two Ags are partially identical

§  X shaped: then the two Ags are non-identical

FIGURE 8



 

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