Amino Acids, Peptides and Proteins - Dévényi T., Gergely J. 1976

Methods of Immunochemical Analysis
Protein Analysis via Precipitation Reactions
Ring precipitin test (precipitation reaction at the interface of layers containing antigen and antibodies

Principle of the method. When an antigen solution is layered over a specific antiserum, a precipitation reaction occurs at the interface of the two layers.

Applications. Determination of antibody titers in immune sera.

Materials REQUIRED FOR THE REACTION

1. Glass (precipitation) tubes, 35 mm in length and 2.5 mm in diameter.

2. Capillary (Pasteur) pipettes.

PROCEDURE

1. Preparation of serial antigen dilutions (see page 121).

2. Performing the assay. Dispense 0.04 ml of the test immune serum into the bottom of each precipitation tube using a capillary pipette. Then, holding the tubes at an angle, carefully layer approximately 0.04 ml of antigen from the corresponding serial dilutions onto the serum in each tube using a capillary pipette.

3. Incubation and reading of results. Keep the tubes in a refrigerator and read the results the following day.

NOTES

1. In a positive reaction, a whitish, opalescent ring visible to the naked eye appears at the interface between the antigen and immune serum layers. In a strong precipitation reaction, this ring appears within a few minutes, and precipitate fragments may settle to the bottom of the tube.

2. The precipitin ring remains strictly horizontal in the tube, even when the tube is gently tilted. This feature distinguishes the specific precipitate from a non-specific sediment, which shifts when the tube is tilted.

3. The dilution factor of the antigen depends on the required accuracy of the antibody titer determination; ten-fold, five-fold, three-fold, or two-fold antigen dilutions can be prepared.

4. When reading the results, it is helpful to examine the tubes against a dark Background, as this makes the precipitin rings more clearly visible.

5. As with other precipitin titration Methods, the titer of the ring precipitation reaction is defined as the highest antigen dilution that still produces a visible precipitin ring.

6. The main advantage of this antibody titration method is the small volume of Reagents required. Since the diffusion of antigen and antibody solutions occurs relatively slowly, the solubilizing effect of antigen excess is less pronounced in the ring precipitation reaction than in other assays.

7. The disadvantages of the ring precipitation reaction include its lower reproducibility compared to the methods described above, as well as the inability to determine the quantitative ratio of antigen to antibody.