Immunodiffusion Single Radial

George Feinberg, formerly of The Rayne Institute, The United Medical Dental School, London

As the term implies, immunodiffusion refers to an immunological reaction in which the reactants diffuse into a supporting medium, i.e. a gei. The reaction involved is that of precipitation, the objective being to visualize and immobilize an in vitro reaction between a precipitating antibody and a soluble antigen. Thus, immunodiffusion is a modification of the precipitin reaction first observed in fluid media by Kraus in 1897. As one of the reactants is placed in wells cut in the gel, the diffusion is radial.

As commonly practised, immunodiffusion is carried out in either of two forms: double diffusion and 'single radial immunodiffusion' (SRI; also RID, i.e. radial immunodiffusion). In double diffusion, both antigen and antiserum diffuse from wells in the gel, whereas in SRI one reactant - usually the antibody -is included in the gel and only the other diffuses in.

Thus, in double diffusion precipitation lines are formed in the gel where antigen and antibody from juxtaposed wells overlap; while in SRI the precipitate takes the form of a ring round the well.

SRI may be traced back to the observation of Petrie in 1932 that a ring of precipitate formed round a colony of an exotoxin-producing bacterium growing on agar which contained the specific antitoxin. In 1949, Ouchterlony and Elek independently-adapted this observation to produce a double immunodiffusion technique. Wells were produced in an agar plate, using a template or cutter. Individual wells received either antigen or antiserum. As the two reactants diffused into the gel, a line of precipitate formed within the zone of overlap. This has become a most useful qualitative technique for demonstrating and comparing antigens and

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Figure 1 Assay of an antigen from the straight line plot of D2

versus concentration on arithmetic graph paper.

antibodies, but proved inadequate for quantitative determinations.

Simultaneously, Oudin was transferring Petrie's observations to a tube, showing that rings of precipitate would form in a gel containing antiserum when it was overlaid with a solution of the corresponding antigen. This was single linear diffusion, as for the purpose of the method only the antigen diffusion was of consequence. It was a dynamic system, the precipitate rings migrating linearly down the gel with time; thus, single linear immunodiffusion. As the rate of the ring migration was determined by antigen concentration, it could be used (albeit not very conveniently) for quantification of antigens against known standards.

In 1957, to achieve a more convenient immunodiffusion assay system, Feinberg combined Oudin's tube single diffusion with the Ouchterlony-Elek plate to produce a single radial immunodiffusion system.

As in Oudin's technique, serum was incorporated in a molten gel, but as with Ouchterlony-Elek the gel was poured into a plate and allowed to set. With a specially designed gel cutter, uniform wells were cut in the antiserum-gel and filled with antigen solution, using standard concentrations of the antigen for comparison with test antigens. Diffusion of antigen and formation of the precipitate rings were allowed to go to completion, assay being based on a limiting dilution end-point, i.e. the lowest concentration of antigen showing a ring of precipitate.

In 1963, Tomasi and Zigelbaum modified the Feinberg technique, eliminating the need to achieve an end-point. More conveniently, they measured the diameters of the precipitate rings by placing a transparent ruler beneath the plate and plotting the diameters against antigen concentrations. With suitable standards they were able to assay secretory immunoglobulin A (IgA) in saliva, colostrum and urine. In 1965, Fahey and McKelvey confirmed the validity of the system and used it to measure levels of immunoglobulins in serum.

In 1965, Mancini and coworkers extended observations to include a diversity of antigens, simultaneously observing the reliability of SRI assay and noting that it was not influenced by variations in temperature. However, in their estimation, merely recording ring diameters could give erroneous results. They advocated projection of the rings on to strong paper, outlining the projected rings with pencil, cutting the paper discs outlined and weighing them. This so-called 'Mancini technique' proved to be cumbersome, as subsequently acknowledged by Heremans. Because of this, in practice it has largely been abandoned, whereas the Tomasi and Zigelbaum modification of the Feinberg technique has generally been adopted as the method of choice and now forms the basis of commercially supplied immunodiffusion plates for the immunoassay of proteins in plasma and body fluids.

Though SRI is most widely used for the assay of antigens, Feinberg observed it could also be used for the assay of antibodies, for which purpose the placing of the reactants is reversed: the antigen is incorporated in the gel, while dilutions of antibody are placed in the wells, i.e. reversed single radial immunodiffusion (RSRI). This was confirmed by Vaerman and colleagues in 1969.

Mechanisms

When antigen diffuses radially into a gel containing antiserum, immune complexes are formed and a precipitate begins to deposit as a ring at the interface between the two reactants. The higher the concentration of antigen in the well, the more rapidly precipitation takes place, i.e. rings appear sequentially in direct relationship to antigen concentration. Ongoing diffusion of antigen from the well builds up the precipitate at the outer edge of the ring, where the antigen encounters additional antibody. Therefore, the system is initially in a dynamic state in which the rings increase in size with time. A static-stage is reached when all the antigen has diffused into the gel and precipitation is complete. At this stage, the rings are most distinct and remain constant in size, so readings can conveniently be taken at any time thereafter.

The time to reach the static state and the ultimate size of the rings are dependent on the molecular size of the antigen, as well as the antigen concentration. The larger the molecule, the longer the time and the smaller the ring. As these are constants for any one antigen and apply equally to standard and test anti-

Figure 2 Measuring ring diameter with the convenient wedge-shaped rule.

gen, they do not ordinarily interfere with the validity and accuracy of SRI. However, they do become significant factors when assaying antigens which exist in different polymeric forms, or which tend to complex with other proteins, so one must remain aware of such possibilities.

At the static stage, the area circumscribed by the ring is proportional to the concentration of the antigen in the well. Since area is proportional to the square of the radius (and, hence, to the square of the diameter, D), then the square of the diameter (D2) of the ring is proportional to the concentration of antigen. Such being the case, plotting D2 against concentration on arithmetic graph paper will produce a straight line (Figure 1). Alternatively, to avoid the requirement of calculating D2, semilog graph paper may be used, plotting D itself on the linear scale against antigen concentration on the log scale.

Methodology

SRI is generally carried out in an agarose gel. The molten gel is cooled to ~50°C to avoid heat denatur-ation of the antiserum, which is warmed to a similar temperature and added to the molten gel in desired concentration. The two are mixed gently, but thoroughly, and poured into a dish, or on to a glass plate (e.g. a microscope slide), supported on a perfectly horizontal surface to ensure uniform gel thickness. When the gel has set, wells are cut using a sharp cutting instrument designed for the purpose. Alternatively, a plastic mask with a pattern of holes can be floated on the molten gel, which is then allowed to set to form a firm seal between mask and gel. In this case, wells need not be cut, the antigen solution being applied to the exposed areas of gel.

Three known standards, spanning the anticipated range of concentrations of the test samples, are used along with the test samples. Uniform measured volumes must be used throughout. Alternatively, when wells are used, comparable volumes can be achieved by filling each well until the meniscus just disappears;

this is preferable if the gel may not be of uniform thickness.

The plates are incubated in a humid atmosphere, preferably until ring formation is complete. Measurement of the ring diameters may be done with a transparent ruler or, more conveniently, with the wedge-shaped scale introduced by McSwiney (Figure 2). Mechanical aids for reading ring diameters are also available commercially. These include a modified jeweller's eye-piece with an in-built graduated scale. Most sophisticated and best suited for making large numbers of readings routinely is an electronic plate-reader.

For the purpose of assay, graphs are constructed, as described above, from three standard dilutions. The ring measurement of each test sample is then positioned on the graph and the corresponding concentration read off.

Investigating the sensitivity and accuracy of SRI in 1965, Mancini and colleagues reported that as little as 1.25 pg of antigen could be measured when using 2 pi samples of antigen solution. They found the standard deviation of SRI antigen assays to be less than 2% of the mean.

See also: Antibodies, detection of; Antibody-antigen intermolecular forces; Immune complexes; Precipitation reaction.

Further reading

Berne BH (1974) Differing methodology and equations used in quantitating immunoglobulins by radial immunodiffusion. Clinical Chemistry 20: 61-69. Feinberg JG (1957) Identification, discrimination and quantification in Ouchterlony gel plates. International Archives of Allergy 11: 129-152. Feinberg JG (1964) A new device for immunoprecipitation in agar gels. Nature 201: 631-632. Herzenberg LA, Weir DM, Herzenberg LA and Blackwell C (eds) (1997) Handbook of Experimental Immunology, 5th edn. Oxford: Blackwell Science. Hudson L and Hay FC (1989) Practical Immunology, 3rd edn. Oxford: Blackwell Science. Ingild A (1983) Single radial immunodiffusion. Scandinavian Journal of Immunology 17 (suppl 10): 41-56. Mancini G, Carbonara AO and Heremans JF (1965) Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2: 235-254. Voller A, Bartlett G and Bidwell D eds) (1981) Immunoassays for the 80s. Lancaster: MTP Press.

[This article is reproduced from the 1st edn (1992).]

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