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

Methodological aspects of the analytical study of proteins
Study of native proteins
Study of native proteins by immunochemical methods

At the end of the last century, The Study of specific precipitation occurring during the in vitro interaction of Antibodies and Antigens led to The Emergence of a new scientific discipline—immunochemistry, which encompasses the Study of the Chemical aspects of Immunity, primarily the chemistry of antigens, antibodies, and their interaction. The high sensitivity and Specificity of immunological reactions have made them highly useful for protein research. Immunochemistry has not only expanded the methodological options for studying Proteins but has also established a new approach to their analysis.

TERMINOLOGY

If foreign substances, such as proteins of a different species or Bacteria, are introduced parenterally (i.e., bypassing the digestive tract) into an animal, soon after one or several such injections, specific substances capable of reacting with the introduced proteins will appear in the animal's serum. These substances are called antibodies, while the factors that trigger their appearance are termed antigens. The antigenicity of a protein molecule is determined by those regions of its constituent polypeptide chains that are not found in the molecular Structure OF THE recipient (i.e., the immunized animal). These regions are called antigenic determinants.

If an antigen solution (for example, a protein solution) is mixed in appropriate proportions with a serum containing specific antibodies (immune serum), the reaction results in The formation of a precipitate composed of antigen and antibody molecules. The highest dilution of the immune serum at which a precipitation reaction still occurs with a constant concentration of the antigen is called the titer of that serum. The titer reflects its activity; for example, a serum with a titer of 1:25 000 is considered five times more active than a serum with a titer of 1 : 5000.

General Information on PRECIPITATION REACTIONS

The precipitation reaction occurs in two stages, during which the reacting antigen and antibody molecules bind to each other without any noticeable changes in their original chemical structure. The binding of the antigen to the antibodies is specific, and this reaction is partially or completely reversible.

In The First stage of the precipitation reaction, antigen molecules bind to antibodies, but no visible precipitates are formed. In the second stage of the reaction, the previously formed antigen-antibody complexes aggregate to form large, insoluble particles visible to the naked eye. The first stage of the reaction is faster, more reversible, and more specific than the second.

The main feature of immunochemical reactions is their high specificity; however, it must be emphasized that the structural similarity of certain proteins, as well as the antibody heterogeneity sometimes characteristic of sera, can under certain conditions cause so-called cross-reactions. This means that the precipitation reaction may, to some extent, depend on non-specific factors. However, this latter circumstance only slightly limits The Use of immunochemical reactions in protein research.

In A number of areas, such as determining relationships between proteins, comparing fractions of pathological and normal proteins, etc., cross-reactions are of significant benefit. Precipitation reactions are highly sensitive. For example, using an antiserum of the appropriate titer, Ovalbumin can be detected at a concentration of 1 mcg/ml. Depending on the nitrogen determination method used, between 75 and 125 mcg of antigenic nitrogen can be detected in a quantitative precipitation reaction.

In some precipitation reactions, quantitative Analysis of the resulting precipitate yields absolute values of antigen or antibody content (the quantitative precipitation method). Other reactions provide only relative values (titer determination).

Precipitate formation depends on several factors. The most significant of these are the titer of the immune serum, The ratio of antigen and antibody concentrations, and the species Water/144.html">Origin of the immune serum.

An optimal ratio of antigen and antibody concentrations is required for the precipitation reaction. In antigen excess, the precipitate partially or completely dissolves; As a result, it may be impossible to evaluate the reaction, or the evaluation may be erroneous. While precipitates formed by rabbit antibodies dissolve only in antigen excess, precipitates formed by horse antibodies can often dissolve in an excess of both antigen and antibodies.

The optimal Temperature, pH, and Ionic strength of the medium for the precipitation reaction are those that exist in vivo. The course of the precipitation reaction depends on the ionic strength of the solution: as the salt concentration increases above 0,15 M, precipitate formation gradually slows down. At the same time, the pH of the medium, within a fairly wide range from 6,5 to 8,6, does not affect the precipitation reaction.

The precipitation reaction occurs soon after mixing the antigen and immune serum solutions. Its preliminary result can usually be read after 30—60 min of incubation of the resulting mixture at 37° C. Precipitation is typically complete after 24 ч of incubation in a refrigerator. In immunodiffusion assays, where the Reagents combine only after diffusing through a supporting medium (such as Agar), the time required for precipitate formation depends, among other factors, on The rate of diffusion.

Immunochemical Methods based on the precipitation reaction are highly convenient for the Qualitative and quantitative Analysis of proteins, for determining the homogeneity of protein preparations and the presence of impurities in them, as well as for identifying components of protein mixtures. As an auxiliary method, the precipitation reaction is used to study Cell/13.html">Protein Structure. The advantage of this method compared to others is that it can be used when quantitative chemical analysis is not feasible, for example, when determining a specific component in a protein mixture. It is in these cases that results obtained using immunochemical reactions can be extremely useful.

IMMUNODIFFUSION METHODS

The simplest way to study soluble antigens in a precipitation reaction is to mix the test solution with a specific immune serum and then observe the formation of a precipitate. This reaction is mainly used for titer determination. Only when the reaction is performed with an immune serum specific to a given protein can the titer value serve as a characteristic of the protein preparation. Truly widespread application of the immunochemical approach to protein analysis began only after the advent of immunodiffusion methods.

In 1905, Bechhold, by layering goat serum onto rabbit antiserum to goat proteins mixed with gelatin, observed the formation of two precipitation bands in the gel. The immunological nature of this “colloid-chemical” phenomenon was uncovered only several decades later. Based on the precipitation reaction in gel, a number of new Research Methods were developed, which not only allowed the characterization of the immunological Properties of Proteins but also opened up new Perspectives for their in-depth analysis.

In essence, all immunodiffusion methods are based on the phenomenon observed by Bechhold: the antigen-antibody interaction in gel. The soluble protein antigen and the immune serum diffuse toward each other in the gel. At the meeting point of both reagents, a precipitation band forms, which can be easily observed and recorded.

A number of substances are used to prepare the semi-liquid gel. Their most important property must be inertness toward the proteins under study and the immune serum. These substances must not enter into any reactions with them. Such substances include agar, starch, gelatin, pectin, etc. Currently, agar is the most widely used, as it has proven to be the best medium for routine immunodiffusion assays. Upon heating, agar easily dissolves in water and salt solutions, forming a firm gel at room temperature. The transparency of agar allows the precipitation lines arising in the gel to be clearly distinguished and stained with appropriate Dyes.

The particular significance of immunodiffusion methods in protein research lies in the fact that they allow proteins to be characterized based on properties different from those previously known. Electrophoresis enables the fractionation of proteins according to their physicochemical properties, whereas classical immunological methods allow further differentiation of protein components based on their antigenic properties.

Therefore, immunodiffusion methods offer additional opportunities for researchers:

1. They allow the simultaneous analysis of several antigen-antibody systems (in a protein mixture).

2. Different protein antigens can be compared with one another.

3. Both the immunological and electrophoretic properties of Proteins can be studied simultaneously.

4. Quantitative determination of proteins can be performed.

Each component of the protein mixture and the corresponding antibodies

of the immune serum diffuse toward each other in the gel, and each pair of reactants forms a single precipitation line upon merging. The number of precipitation lines that appear corresponds to the minimum number of different antigens and corresponding antibodies present in the system. Immunodiffusion methods are highly sensitive: they can detect as little as 2—18 µg of protein nitrogen per 1 ml [3], making them suitable for studying low-concentration protein solutions that are otherwise unfit for electrophoretic analysis.

Electrophoresis and classical immunological reactions are also used to compare protein mixtures and individual native proteins. However, the resolving power of these methods is limited, as they provide either a physicochemical or an immunochemical characterization. In comparison, the immunodiffusion method is more informative, because it allows for the detection of not only identical components in two or more compared protein mixtures, but also potential shared antigenic structures present in different components [11—13].

Of particular significance is the combination of immunodiffusion and electrophoresis, known as Immunoelectrophoresis [4]. This technique allows for the simultaneous electrophoretic Separation of protein mixture components and their immunological characterization. Its simplicity, high resolving power, and the extremely small amount of material required for analysis make immunoelectrophoresis one of the most valuable Methods for the analytical study of proteins. Combining immunoelectrophoresis with double immunodiffusion in gel enables the identification of identical components in two different protein mixtures [10].

Applications OF IMMUNODIFFUSION METHODS

Serum protein analysis is performed using various immunochemical methods, but here we will focus on only two: double immunodiffusion and immunoelectrophoresis. Both methods have found wide and versatile applications. They are used to study unfractionated serum as well as to analyze individual protein fractions. The advent of these methods has enabled the discovery and characterization of several new Serum proteins.

In an agar gel or on a Cellulose acetate membrane, the protein antigen and specific antibodies diffuse toward each other, forming precipitation lines at their point of contact, which can be seen with the naked eye or visualized using special staining. When analyzing two protein mixtures, such as two different sera or isolated native proteins, this method not only reveals the number of individual proteins present in the system but also provides data on their immunochemical identity, relatedness, or differences. All these Conclusions can be drawn from the number and relative positions of the precipitation lines.

Immunoelectrophoresis has played a significant role in the advancement of serum protein research. Historically, free-boundary and zone electrophoresis allowed for the analysis of a relatively small number of individual serum proteins. Except for the 5 classic proteins detected by free-boundary and zone electrophoresis, other serum fractions are not always easily identified. Consequently, specialized types of zone electrophoresis with higher resolving power than simple paper electrophoresis never became routine in clinical laboratories, remaining important primarily for scientific research. Determining the percentage of albumin, α-, β-, and γ-globulins often helps in making an accurate clinical Diagnosis, but we must remember that protein fractions that appear homogeneous in zone electrophoresis may contain different proteins that form a single fraction solely due to their similar electrophoretic mobility. This is also evidenced by the staining of Lipoproteins and Glycoproteins.

According to modern concepts, circulating Blood Plasma contains over a hundred proteins, including Hormones and Enzymes. The capabilities of electrophoretic Separation Methods are quite limited, yielding only about 20 protein fractions. However, immunochemical methods, particularly immunoelectrophoresis, offer greater possibilities, allowing for the identification of up to 30 serum protein fractions. The Significance of immunoelectrophoresis becomes even more apparent considering that Scheidegger's micromethod [16] can analyze proteins in extremely small quantities—down to 5—10 µg.

If, upon completion of the electrophoretic Separation of proteins in agar gel, a specific immune serum is allowed to diffuse toward them, arc-shaped precipitation bands form at the meeting points of antigen and antibodies, each corresponding to a single type of serum protein. The precipitation bands are clearly visible in the native preparation, but they can be stained for better visualization.

The number of precipitation lines on the electrophoregram depends on the immune serum used. For example, the well-proven rabbit antiserum to human serum proteins from Behringwerke (FRG) and the similar horse antiserum from the Human Institute (Hungarian People's Republic) allow for the identification of approximately 20 serum protein fractions. The fundamental possibility of preparing an antiserum to virtually any protein greatly expands the applicability of immunoelectrophoresis. Furthermore, by preparing antisera specific to a single protein (or a small number of proteins), individual components within a protein mixture can be studied. Immune sera designed for the immunochemical analysis of individual human Plasma Proteins are already commercially available.

These include antisera to fractions of albumin, α-1-, α-2-lipoproteins, α-2-macroglobulin-transferrin, IgG, IgM, IgA, etc. Obviously, by having the appropriate fractions of native proteins and mastering the methods of antiserum production, the range of commercially available preparations can be significantly expanded to meet the demands of experimental research.