Gels are used in immunoprecipitation techniques to stabilize the precipitate, enabling both the position and the area of the precipitate to be measured. The point has already been made that maximum precipitation occurs when the equivalent proportions of both antigen and antibody are available. Hence, if a high concentration of antigen is permitted to diffuse into a gel that contains a uniform concentration of antibody, at some point in the concentration gradient of antigen that is
> Single radial immunodiffusion (SRID) is a quantitative immunoprecipitation technique.
formed there will be optimum concentrations of both reactants and a precipitate will form. The dimensions of the gradient will depend on the original concentration of the antigen and hence the distance between the precipitate and the original starting point of the antigen will be proportional to its initial concentration.
This principle forms the basis of single radial immunodiffusion (SRID). a technique first developed in 1965 by Mancini. SRID involves pipetting a measured volume of antigen into holes cut into a buffered agar gel containing the antibody. The loaded gel is placed in a moist chamber at room temperature for at least 18 hours to permit diffusion. Rings of precipitate form around each well (Procedure 7.1), the precipitate being maximal at the periphery and less
Procedure 7.1: Quantitation of albumin by single radial immunodiffusion
Agar gel containing 10% (v/v) of a polyclonal antibody to human albumin.
Standard solutions of human albumin with concentrations ranging from 10 to 100 mg 1 '.
Small wells were cut into the gel.
10 ¡jA volumes of albumin solutions were pipetted into the wells. Allowed to stand at 4°C for 24 h.
The zones of precipitation were measured in two directions at right angles. Results
Well -\lhumin roncrnlration King diaim-U-r
6 Sample A 6.5
A calibration graph was prepared from iho ivmiIi- of the standard solution* h\ plnlliiiL' the square of the diameter ayainM the alhuinin cniii.emruik'n. The conccntiaiion ul" the test sample can «lien Iv determined Irorii the graph and L'iu's ,i \,ilue of 54 niu I '.
intense towards the well. The time taken for samples containing a high antigen concentration to arrive within the zone of equivalence will be longer than for lower concentrations of antigen and so the diameter of the formed rings will be larger. The radial nature of the diffusion is such that the diameter of the precipitation ring will be related to the concentration of the antigen. In practice, the diameter of the ring is measured and a plot of the square of the diameter against concentration generally gives a straight line relationship (Procedure 7.1). Two measurements of the diameter are taken at right angles in order to allow for any slight irregularities in the shape of the rings. SRID techniques provide useful and specific methods for the quantitation of individual proteins. The limit of sensitivity is about 5 mg 1_1 and it is dependent on the ability to detect and measure very small precipitation rings. The concentration of
Immunoelectrophoresis is a quantitative immunoprecipitation technique.
antibody in the gel has to be carefully selected; reducing the amount of antibody in the gel will increase the average ring size but will result in less precipitate and a compromise has to be made between ring diameter and precipitate intensity. The visualization of the precipitate may be improved by staining the protein with a suitable dye.
One of the drawbacks to SRID is the time for the reaction to take place. Laurell, in 1966, sought to overcome this difficulty by using electrophoresis rather than diffusion to produce the concentration gradient. In electro-immunoassay the antibody is incorporated into the gel in a similar way to SRID. The electrophoresis is usually performed at pH 8.6, conditions under which the antibodies do not migrate significantly but the test proteins do. Because the samples are driven towards the equivalence zone by electrophoresis rather than by diffusion the precipitation lines characteristically appear as peaks or 'rockets' in the gel (Figure 7.6). A calibration curve for the quantitation of unknown samples is constructed by plotting peak height against the concentration of antigen.
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