Single radial immunodiffusion (SRID)

3.4 Single radial immunodiffusion (SRID)

Oudin originally used analytical techniques involving the diffusion of antigens into an antibody- containing gel (single diffusion in one direction). Feinberg and later Mancini, Carbonara and Heremans extended this technique by incorporating the antiserum into a thin layer of agar and placing the antigen in wells cut into the antibody-containing agar.

As the antigen diffuses radially a ring of precipitation forms around the well and moves out- wards, eventually becoming stationary at equivalence. At equivalence, the diameter and area of

3.4SINGLE RADIAL IMMUNODIFFUSION (SRID) 3.4SINGLE RADIAL IMMUNODIFFUSION (SRID)

The optimal dilution for the antiserum will, of course, depend upon the strength of the antiserum and antigen as the diameter of the precipitation ring is inversely proportional to the antiserum concentration. In practice, with rabbit antisera to human IgG, a final dilution of approximately 1 : 40 in the agar is suitable for measuring IgG concentrations in the range of 50–200 µg/ml. However, this is only a guide; a standard curve should be determined for each antiserum.

MATERIALS AND EQUIPMENT 2% agar in barbitone buffer Precoated slides Antiserum, e.g. anti-human IgG Standard antigen solution, e.g. human IgG Phosphate-buffered saline (PBS) Flat, level surface (use a spirit level) Gel punch Humid chamber (plastic box with damp filter paper) Pasteur pipettes Water vacuum pump

METHOD

1 Melt the agar in a microwave oven and transfer to a 56°C water bath. This temperature will keep the agar molten but is low enough to avoid denaturation of the antibody.

2 Dilute the antiserum with PBS. Typically, add 75 µl of an antiserum to 1.9 ml of PBS and warm to 56°C.

3 Add the diluted antiserum (∼ 2 ml) from step 2 to 1 ml of agar at 56°C and mix well.

4 Layer the agar onto a precoated slide standing on a levelled surface and allow to set.

5 After the agar has set, use a gel punch to cut about eight wells per slide. The wells should be 2–3 mm in diameter, and must have vertical sides.

6 Remove the agar plug with a Pasteur pipette attached to a water vacuum pump.

7 Fill each of four wells with standard solutions of 50, 100, 150 and 200 µg/ml IgG. Use the other wells for the IgG solutions of unknown concentrations. Maintain a standard volume by filling the wells quickly until the meniscus just disappears. Alternatively a measured volume, such as 10 µl, may be accurately pipetted into each well.

8 Leave the slide in a humid box to equilibrate. (Although a satisfactory standard curve may

be obtained by overnight equilibration, the points will better approximate to a straight line if the slide is allowed to equilibrate longer: IgG and IgA 48 h, IgM 72 h. IgG concentrations may also be determined by incubation at 37°C for 4 h.)

3.4.1 Measurement of precipitation rings

The diameter of each ring may be measured either directly using a magnifying glass with a µm scale or, after staining, with a plastic ruler.

90 C H A P T E R 3: Antibody interactions with antigens

Direct measurement Hold the slide over a black background and illuminate it from the side. Measure the rings from

the reverse side through the glass plate, do not rest the magnifying glass on the gel. If the rings are not distinct, soak the slides in 4% tannic acid for 1 min to increase resolution. (This is not a permanent preparation.)

Stained preparations

1 Wash the slide for 24 h in several changes of PBS to remove free protein from the agar.

2 Cover the slide with good-quality, lint-free, filter paper and dry overnight.

3 Remove the filter paper after dampening it slightly. The slide may then be stained with any protein dye, but we suggest Coomassie blue R-250.

MIXTURE FOR STAINING SOLUTION Coomassie brilliant blue R-250 (1.25 g) Glacial acetic acid (50 ml) Distilled water (185 ml)

METHOD

1 Dissolve the Coomassie dye in the glacial acetic acid and distilled water.

2 Stain the slide for 5 min and differentiate in the same solution without the dye. Staining with this dye is reversible so do not leave the slide too long in the destaining solution.

3 Place the dry, stained slides in a photographic enlarger and measure the diameter of the precipitation rings with a ruler.

The staining solution may be stored for several weeks in a stoppered bottle. The destaining solu- tion can be regenerated by passing through powdered charcoal.

Calculation of results Figure 3.5 shows a typical determination. The diameters of the rings were measured and plotted

on a linear scale against the log of the antigen concentration. With a semilog transformation, the points should approximate to a straight line. If the assay has been allowed to reach equilibrium the areas of the rings should be calculated and plotted against the antigen concentration on a linear scale.

TECHNICAL NOTES • The assay can be made more sensitive by incorporating from 2 to 4% polyethylene glycol in the

agar to enhance precipitation. • If chicken antisera are used, 7–8% NaCl should be added to the agar to improve precipitation. • Although single radial immunodiffusion is now used as a quantitative technique, its first use was

to compare the identities of different antigen solutions. If two antigens, placed in neighbouring wells close together, are identical in terms of their antigenic determinants then the two rings of precipitation fuse completely. If the antigens share no determinants recognized by the anti- serum then each ring forms independently. Since the work of Ouchterlony, simpler procedures have been introduced to test the relationships between antigens or antibodies.

3.4SINGLE RADIAL IMMUNODIFFUSION (SRID)

4.3 8.6 17.2 IgG standards (mg/ml)

Fig. 3.5 Measurement of IgG concentration by single radial

immunodiffusion. A calibration curve Concentration of IgG (mg/ml)

2 is constructed from the diameter of the precipitation rings formed at equilibrium by IgG standards of known

1 concentration. The concentration 4 5 6 7 of unknown samples can then be

Diameter of ring (mm) determined with reference to the standard curve.