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

Methods of Immunochemical Analysis
Protein Analysis by Precipitation Reaction
Heidelberger's Quantitative Precipitation Reaction

Applications. Determination of antibody nitrogen in test immune sera using an antigen solution of known concentration. Determination of protein antigen concentration using antisera with a known concentration of antibody nitrogen.

PROCEDURE

1. Determination of the optimal antigen-antibody ratio. The antigen-antibody equivalence zone is determined in preliminary experiments (see p. 123).

2. Reaction setup and incubation. Once the equivalence zone is determined, 1 ml of the test immune serum is added to each of 5 centrifuge tubes, followed by The addition of antigen solution in increasing concentrations. The concentration of the added antigen must be known from preliminary experiments. The antigen and immune serum are gently mixed by rolling each tube between the palms, incubated for 1 h at 37°C, and then left in a refrigerator for 24 h.

3. Centrifugation and washing of the precipitate. After the precipitation reaction is complete, the resulting precipitate is sedimented by centrifugation at 2000 rpm for 60 min.

The supernatant (I) is carefully decanted, ensuring that no precipitate fragments are lost. If there is any doubt that the precipitate has settled sufficiently, the supernatant should be aspirated rather than decanted. After removing the supernatant, the centrifuge tubes are inverted and placed vertically on filter paper.

To the pellet, 0.5 ml of chilled 0.9% NaCl solution is added, and the tube is vigorously shaken to suspend the precipitate. Then, the same solution (2.5 ml) is used to wash down precipitate particles from the tube walls (the total sample volume will be 3 ml). The suspended precipitate is centrifuged again at 2000 rpm, the supernatant is decanted, and the washing step is repeated once more.

4. Dissolution of the precipitate. To the twice-washed precipitate, a few drops of distilled Water and 1 drop of 0.5 M NaOH solution are added. After the precipitate dissolves, the nitrogen content in the resulting solution is determined using an appropriate method.

5. Determination of the equivalence zone. To determine in which of the five tubes the precipitation reaction actually occurred in the equivalence zone, the excess of antigen or antibody in the supernatant of each sample (I) is determined by adding immune serum or antigen solution, respectively (see p. 120).

NOTES

1. Precipitation is usually maximal in those tubes where the supernatant (I) contains a slight excess of antigen. The amount of antibody nitrogen in the test antiserum can be determined by subtracting the known amount of antigen nitrogen from the nitrogen content of the precipitate in such a sample. The results are typically expressed as the amount of antibody nitrogen per 1 ml of immune serum.

2. The results of the quantitative precipitation reaction can be expressed graphically by plotting the amount of precipitate nitrogen against the amount of antigen added. The resulting curve can be divided into three regions. The first part of the curve is the antibody excess zone. In the corresponding tubes, antigen-antibody complexes form a precipitate that is sedimented by centrifugation, while free antibody molecules can be detected in the supernatant. The second part of the curve (its peak) is the equivalence zone. The supernatant in the corresponding tubes contains neither free antigen nor free Antibodies. The third part of the curve is the antigen excess zone; the supernatant in the corresponding tubes contains unbound antigen molecules. As the amount of added antigen increases, the amount of precipitate nitrogen begins to decrease. A further increase in antigen concentration can lead to greater dissolution of the precipitate, up to its complete solubilization.

3. The amounts of antigen and antiserum required for this reaction depend on the sensitivity of the chosen protein determination method. For example, when using the Markham micro-Kjeldahl method, it is sufficient for the immune serum to contain 75–125 µg of antibody nitrogen. The biuret reaction requires twice as much antibody nitrogen. At the same time, if protein is determined spectrophotometrically, half of the specified amount of antibody can be used in the reaction, whereas with the Folin–Ciocalteu method or the ninhydrin reaction, one-third of this amount is sufficient. Accordingly, the volume of immune serum in the centrifuge tube can vary from 0.5 to 4.0 ml.

4. To avoid Protein Denaturation, The Use of a refrigerated centrifuge is recommended.