Precipitation Reactions in Gels Yield Visible Precipitin Lines

Immune precipitates can form not only in solution but also in an agar matrix.When antigen and antibody diffuse toward one another in agar, or when antibody is incorporated into the agar and antigen diffuses into the antibody-containing matrix, a visible line of precipitation will form. As in a precipitation reaction in fluid, visible precipitation occurs in the region of equivalence, whereas no visible precipitate forms in regions of antibody or antigen excess. Two types of immunodiffusion reactions can be used to determine relative concentrations of antibodies or antigens, to compare antigens, or to determine the relative purity of an antigen preparation. They are radial immunodiffusion (the Mancini method) and double immunodiffusion (the Ouchterlony method); both are carried out in a semisolid medium such as agar. In radial immunodiffusion, an antigen sample is placed in a well and allowed to diffuse into

Assay

Sensitivity of various immunoassays

Sensitivity* (lg antibody/ml)

RADIAL IMMUNODIFFUSION

Precipitation reaction in fluids 20-200 Precipitation reactions in gels

Mancini radial immunodiffusion 10-50

Ouchterlony double immunodiffusion 20-200

Immunoelectrophoresis 20-200

Rocket electrophoresis 2 Agglutination reactions

Direct 0.3

Passive agglutination 0.006-0.06

Agglutination inhibition 0.006-0.06

Radioimmunoassay 0.0006-0.006 Enzyme-linked immunosorbent assay (ELISA) <0.0001-0.01

ELISA using chemiluminescence <0.0001 -0.01

Immunofluorescence 1.0

Flow cytometry 0.06-0.006

*The sensitivity depends upon the affinity of the antibody as well as the epitope density and distribution.

'Note that the sensitivity of chemiluminescence-based ELISA assays can be made to match that of RIA.

SOURCE: Adapted from N. R. Rose et al., eds., 1997, Manual of Clinical Laboratory Immunology, 5th ed., American Society for Microbiology, Washington, D.C.

Antibody incorporated in agar

RADIAL IMMUNODIFFUSION

Precipitation Curve Antibody Antigen

Antigen diffusion

Antigen

Precipitate forms ring

Antigen diffusion

Antigen

Precipitate forms ring

DOUBLE IMMUNODIFFUSION Antibody

Antigen

DOUBLE IMMUNODIFFUSION Antibody

Antigen

Double Immunodiffusion

Agar matrix

FIGURE 6-5

Precipitate

Diagrammatic representation of radial immunodiffusion (Mancini method) and double immunodiffusion (Ouchterlony method) in a gel. In both cases, large insoluble complexes form in the agar in the zone of equivalence, visible as lines of precipitation (purple regions). Only the antigen (red) diffuses in radial immunodiffusion, whereas both the antibody (blue) and antigen (red) diffuse in double immunodiffusion.

Agar matrix

FIGURE 6-5

Precipitate

Diagrammatic representation of radial immunodiffusion (Mancini method) and double immunodiffusion (Ouchterlony method) in a gel. In both cases, large insoluble complexes form in the agar in the zone of equivalence, visible as lines of precipitation (purple regions). Only the antigen (red) diffuses in radial immunodiffusion, whereas both the antibody (blue) and antigen (red) diffuse in double immunodiffusion.

agar containing a suitable dilution of an antiserum. As the antigen diffuses into the agar, the region of equivalence is established and a ring of precipitation, a precipitin ring, forms around the well (Figure 6-5, upper panel). The area of the pre-cipitin ring is proportional to the concentration of antigen. By comparing the area of the precipitin ring with a standard curve (obtained by measuring the precipitin areas of known concentrations of the antigen), the concentration of the antigen sample can be determined. In the Ouchterlony method, both antigen and antibody diffuse radially from wells toward each other, thereby establishing a concentration gradient. As equivalence is reached, a visible line of precipitation, a precipitin line, forms (Figure 6-5, lower panel).

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  • arttu
    What is precipitation reaction in microbiology?
    8 years ago

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