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

Methods of Immunochemical Analysis
Protein Analysis by Gel Diffusion Methods
Quantitative Immunodiffusion Methods

Numerous attempts have been made to develop immunodiffusion Methods for the quantitative estimation of antigen and antibody levels. One group of such methods is based on Ouchterlony Double Gel diffusion (see p. 131), while another relies on measuring the diffusion rate and the turbidity of precipitation bands in Immunoelectrophoresis (p. 137). As yet, these methods have not found widespread application.

FEINBERG'S ANTIBODY CONCENTRATION GRADIENT METHOD [6]

Principle of the method. Diffusion of the antigen into an Agar gel plate containing immune serum results in The formation of a precipitate. The highest dilution of the antigen at which precipitation still occurs is considered its titer.

Scope of application. Titration of antigen solutions.

PROCEDURE

1. Preparation of the agar gel plate. Molten 1% agar is poured onto the bottom of a Petri dish (see p. 131). In the solidified agar gel, wells for the test antigen and antiserum are cut using a cork borer or another suitable tool (Fig. 34), as described on p. 131. The central well should be approximately 4 times larger in diameter than the equidistant peripheral wells (12-16 and 3-4 mm, respectively). Two adjacent peripheral wells should be spaced further apart from each other than from the central well.

2. Antigen titration. 0.2 ml of immune serum is introduced into the central well, and the agar plate is placed in a humid chamber and left at 37°C for 24—72 h. As the immune serum diffuses from the central well toward the peripheral ones, an antibody concentration gradient is established.

Once sufficient time has elapsed for the antibody concentration gradient to form, the pre-prepared dilutions of the test antigen are added to the peripheral wells. Exactly the same volume of solution is introduced into each well using a micropipette or a graduated capillary pipette.

After 24 h of re-incubation in a humid chamber at 37°C, precipitation bands (or rings) form in the agar gel around the peripheral wells. By recording these, the highest dilution of the antigen at which the precipitation reaction is still visible is determined and taken as the titer of the test antigen.

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Fig. 34. Punches for cutting wells in agar gel.

NOTES

There is another, less sensitive method for titrating Antigens or Antibodies, also developed by Feinberg, which is performed as follows.

For antigen titration, the specific immune serum (or a solution of a known antigen for antiserum titration) is mixed with molten agar and poured onto the bottom of a Petri dish.

After the agar solidifies, wells are cut in the gel and filled with the antigen or antiserum solution. As they diffuse through the gel, the antigen or antibodies meet their specific counterpart, resulting in the rapid appearance of precipitation rings around the wells. The reaction can be read as early as 30 min, but the final evaluation should be made after 24 h of incubation at 37°C in a humid chamber. The margin of error for this quantitative method of antigen and antibody determination is ±25%. The accuracy of the method increases when multiple parallel samples are run.

Quantitative determination of PROTEIN ANTIGEN IN AN AGAR GEL LAYER CONTAINING SPECIFIC ANTIBODIES [5]

Principle of the method. The protein antigen under study, diffusing from the well, forms a ring-shaped precipitate in an agar gel containing monospecific antibodies, the diameter of which is proportional to the concentration of this antigen.

Scope of application. Quantitative determination of Proteins, Determination of the concentration of individual components of a protein mixture (e.g., Blood serum).

PROCEDURE

1. Preparation of the antibody-containing agar plate. A 3% agar solution is prepared in 0.03 M potassium phosphate buffer, pH 8.0, containing 0.1 M NaCl, and placed in a Water bath at 56°C. The appropriate monospecific antiserum is diluted with phosphate buffer depending on its titer (see Note 2) and also heated to 56°C. 8 ml of the heated 3% agar and 8 ml of the warmed antiserum are mixed at 56°C, and 15 ml of the mixture is poured onto an 8 x 10 cm Glass plate kept on a horizontal surface. After the agar solidifies, the plate is placed in a humid chamber and stored in a refrigerator until use. (It is recommended to add merthiolate at a 1 : 10 000 dilution to the agar-antiserum mixture.)

2. Preparation of wells for the test antigen. Using a suitable tool (see p. 131), wells for the antigen with a diameter of 2.4 mm are cut in the agar gel, spaced 12 mm apart. Usually, the agar surface at the corners of the plate is not perfectly even, and the resulting precipitation rings do not have regular shapes; therefore, the four corner wells are left empty.

3. Filling the wells. Some of the wells in the agar gel are filled using a capillary pipette with pre-prepared protein solutions of known concentration at appropriate dilutions. The remaining wells are filled with the test protein mixture samples, such as blood serum. Care must be taken to ensure that the same volume of solution is added to all wells, filling them exactly to the brim. The agar plate is then placed in a humid chamber and left in a refrigerator for 24 h.

4. Evaluation of results. Precipitation rings form in the agar gel containing monospecific antibodies and the diffusing antigen. The diameter of such a ring is easiest to measure by viewing it against a dark Background with oblique illumination. First, the diameters of the precipitation rings around the wells with diluted standard protein solutions are measured, and a plot of these values (in mm) against the logarithm of the solution concentration is constructed. By measuring the diameters of the precipitation rings around the wells of the test samples, the concentration of the analyzed protein is determined using the resulting calibration curve.

NOTES

1. Two factors are of paramount importance for the successful execution of this method. First, the availability of an immunochemically pure (i.e., free of other protein fractions) preparation of the protein whose concentration in the mixture is to be determined. This protein is used in the reaction as a standard solution of known concentration. Second, it is necessary to have a monospecific (i.e., reacting only with the given protein) immune serum.

2. Antibody titers in immune sera can vary widely. Therefore, preliminary experiments should determine the antiserum dilution that yields the sharpest and most easily recorded precipitation rings. Depending on the titer of the available antiserum, it can be used at an 8-, 16-, or 32-fold dilution.

3. When evaluating the reaction results after 24 hours, a logarithmic relationship is observed between the antigen concentration and the diameter of the precipitation ring. However, the reaction can also be read over a longer period by continuing to monitor antigen diffusion in the gel and measuring the diameters of the precipitation rings daily. After a few days, equilibrium is reached in the system, i.e., the excess antigen in the well disappears and the increase in the diameter of the precipitation ring ceases. At this stage, There is a linear relationship between the antigen concentration and the diameter of the precipitation ring.

4. This method has proven highly effective for determining the levels of blood serum fractions that can be isolated in a sufficiently pure form (e.g., IgG, IgA, IgM, transferrin, and albumin). In general, this method can be used to determine the concentration of any homogeneous protein, provided that a specific antiserum is available.

5. Reaction results can be recorded by photographing the native preparation or by staining it with protein-detecting Dyes (see p. 135). In some cases, staining facilitates the recording of results, as stained precipitation rings are easier to measure. However, it can also complicate measurement if, for example, the shape of the ring is altered in the process.

QUANTITATIVE IMMUNOELECTROPHORESIS ACCORDING TO BACKHAUS? [1]

First, the antigen under study is subjected to Electrophoresis using the macromethod described on p. 137. Then, in the immediate vicinity of the antigen Separation zone in the agar gel, a wide trough is cut and filled with a mixture of molten agar and immune serum. The proteins separated in the electric field diffuse into the agar gel containing the antibodies. The distance traveled by the proteins is determined by the laws of linear diffusion in the gel (see p. 128). Therefore, by measuring the Displacement of the peaks of the precipitation bands from the edge of the trough, one can determine The rate of diffusion, i.e., the value of k, and thereby the concentration of the protein fractions under study.