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

Methods of Immunochemical Analysis
Protein Analysis by Gel Diffusion Methods
Qualitative and quantitative analysis of proteins using electrophoresis in antibody-containing agarose gel

Principle of the method. The analysis consists of two stages: a) Electrophoresis of the protein of interest in an agarose gel, and b) second-dimension electrophoresis of the obtained fractions in an antibody-containing gel, perpendicular to the direction of the first Separation. The gel strip containing the protein fractions obtained in The First stage is placed on an agarose gel plate containing antiserum, and electrophoretic separation is performed again at a right angle to the original direction. The concentration of the various fractions of the protein under study can be estimated from the height of the precipitin peaks formed As a result of antigen migration in the antibody-containing gel.

Applications. Identification and quantitative Analysis of Protein fractions.

PROCEDURE

1. Preparation of the agarose gel plate. A 1% agarose solution containing 1 mmol of calcium lactate is prepared in a veronal buffer solution with an Ionic strength of 0.05 and pH 8.6.

The warmed agarose is poured onto a Microscope slide to form a gel layer 1.5 mm thick; the slides are placed in a humid chamber and left in a refrigerator until solidified.

Afterwards, a rectangular trough is cut in the cathodic third of the agarose plate for the antigen sample (Fig. 33).

2. Electrophoresis. The antigen solution (the protein or protein mixture under study) is introduced into the trough, and electrophoresis is carried out for 3 h at a voltage gradient of 10 V/cm.

It is recommended to use a cooled electrophoresis apparatus. Upon completion of electrophoresis, the separation area of the sample in the agarose gel is cut out as a narrow longitudinal strip containing the protein fractions (Fig. 33) and placed on an agarose gel plate containing antiserum.

3. Preparation of the antibody-containing agarose gel and the Second Stage of the analysis (electrophoresis of the fractions obtained in the first stage). The specific antiserum, at an appropriate dilution, is mixed with 1% agarose and poured onto a 10 x 10 cm Glass plate to form a layer 1.5 mm thick.

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Fig. 33. Protein analysis by electrophoresis in an antibody-containing agarose gel (see text for details).

The gel strip containing the protein fractions under study is placed on this plate, and electrophoresis is performed. The plate is mounted in the apparatus so that the migration of the protein fractions occurs at a right angle to the longitudinal axis of the applied agarose gel strip (Fig. 33). Electrophoresis is continued for 30–90 min at a voltage gradient of 10 V/cm. During electrophoresis, the protein fractions diffuse from the strip into the antibody-containing gel. As they migrate through it under the Influence of the electric field, they form precipitin "peaks" as a result of a specific reaction with the corresponding Antibodies. The contours of the precipitin peaks are formed due to the electrophoretic migration of the antigen, as well as the gradual decrease of its excess as a result of this migration in the agarose gel. (It is known that antigen excess solubilizes immune precipitates; therefore, precipitation bands form only where the antigen excess is depleted due to electrophoretic migration.)

4. The height of the precipitin peaks, i.e., the distance between the gel strip originally containing the protein fractions and the apex of the peak, is proportional to the antigen concentration.

NOTES

1. In preliminary experiments, it is recommended to optimize The ratio of agarose solution to antiserum under specific conditions, i.e., for the given antigen under study and the available antiserum. If the antiserum is sufficiently active, its concentration in the agarose gel should not exceed 1–5%.

2. This method allows the Determination of the concentration of a given protein in a protein mixture, provided that the researcher has the appropriate monospecific antiserum. As an example, the procedure for determining the concentration of IgG in Blood serum is described below.

A 1% agarose gel containing 2.5% anti-IgG antiserum is cast onto a glass plate in the usual manner. In the solidified agarose gel, the required number of circular wells is cut for the test serum and IgG, and a few drops of agarose solution are poured into them. IgG preparations of known concentration at appropriate dilutions are added to several wells; the remaining wells are filled with the test serum samples, and electrophoresis is carried out as described above. Depending on the ratio of antigen to antibody concentration, the duration of electrophoresis can vary from 2 to 10 h. Electrophoresis should be stopped when the growth of the precipitin peaks ceases, i.e., when the antigen excess has been resolved by electrophoretic migration. Upon completion of electrophoresis, the distance from the apex of the peak to the center of the well from which the migration of the test sample began is measured. The value obtained is directly proportional to the concentration of the antigen, in this case IgG, in the well. Based on the data obtained from appropriate dilutions of the IgG preparation of known concentration, a calibration curve is plotted on graph paper to determine the IgG concentration in the test serum samples.

3. Quantitative evaluation of the results and photographic recording of the precipitin peaks can be performed on both unstained and stained preparations.