This article will be devoted to the phenomenon of the precipitation reaction. Here we will consider the features of the formulation of this phenomenon, the phenomenon of diffusion, general characteristics, role in human life and much more.
Introduction to the phenomenon
Precipitation is a phenomenon of a serological type, during which soluble antigens interact with antibodies and, as a result, precipitation is observed - a precipitate.
The general characteristic of the precipitation reaction is a form of coordinated influence of antigen and antibody. These types of interactions make it possible to determine the presence of unknown antigens in the test substance by adding known antibodies and antigens. The precipitation process without the presence of s alts will proceed worse, and the best optimum lies within the range of 7.0-7.4 pH.
Constituents of a reaction
There are three main elements among the components of the precipitation reaction:
- An antigen having a molecular nature. It is in the fine type state, in other words, itsoluble. Also, such an antigen is called a precipitogen, which is a lysate or tissue extract, etc. A precipitogen has a characteristic difference from an agglutinogen, which lies in the size of the particles of which it consists. Agglutinogen has an inherent cell size, and precipitogens are commensurate with the size of the molecule. The antigen solution is characterized by transparency.
- An antibody found in human blood serum, as well as in immune diagnostic serum, which contains the studied antibodies.
- Electrolytes are sodium chloride solutions that are isotonic.
Precipitogen production
Setting up a precipitation reaction is impossible without a precipitogen, which is obtained by grinding materials and extracting protein antigens from them. Extraction occurs by boiling or other methods.
Lysates, as well as tissue and organ extracts, blood serum, various types of filtrates based on microbial broth cultures, as well as s alt extract of microorganisms and autolysate substances, are a striking example of precipitogens.
Setting up in precipitation
Now let's consider the method of setting up the precipitation reaction.
A ring-precipitation reaction is performed, which takes place in specially prepared test tubes. Serum is introduced into the cavity of the dishes, pouring it over the wall with the help of a pipette spout. Next, the appropriate amount of precipitater is carefully layered on top, and then the tube is brought to a vertical position from a horizontal one. Setting up and taking into account the precipitation reaction is a very meticulous operation. The result is recorded after the appearance of a white ring on the border between the antigen and antibody. If the reacting elements of the reaction correspond to each other, then they bind, but this becomes noticeable after a long period of time of their interaction.
The precipitation reaction is also carried out in a Petri dish or on a glass slide, where agar gel is transferred, applying it in a small layer. After it hardens in the gel, a small number of wells are cut out into which antigens and antibodies will be placed. There are two ways to perform this action: the method of radial immunodiffusion and double immunodiffusion.
General information
The mechanics of the work of precipitation is similar to the agglutination device. Under the influence of immune-type serum, the antigen, which has already entered into the reaction, reduces its degree of dispersion. An important condition is the transparency of both the serum and the antigen.
You can improve the registration of the reaction if you superimpose antigens on antibodies. As a result, the appearance of precipitates in the form of a ring can be observed. This phenomenon is called ring precipitation and is carried out in special tubes having a diameter of 2.5 to 3.5 mm. One of the most common examples of a precipitation reaction is the diagnosis of anthrax.
Precipitation makes it possible to determine the level of toxigenicity of a diphtheria culture in agar.
During the reaction under considerationprecipitation of antigenic complexes and antibodies occurs. Precipitation is an immunological phenomenon that allows you to determine the amount of antibodies in the blood serum of a sick or vaccinated person and animals.
Titration Consequence
It is important to know that the data obtained by titrating the above method is not quantifiable. To create and analyze a quantitative assessment of the contained number of antibodies, a special reaction technique was developed by M. Heidelberger and E. Kabat, which is based on the search and identification of the equivalence zone. Mixing the age number of antigens with a constant volume of antiserum leads to an increase in the initially formed precipitate, and then again it decreases due to an increase in the ability to dissolve the antigen complexes. By determining the amount of antibodies in the supernatants contained in each tube, you can find that a certain number of dishes with antibodies will be missing liquid. Here, in comparison with other test tubes, the largest precipitate will be formed. Due to this and subtraction of the antigenic protein precipitate from the total value of proteins, it is possible to obtain the exact value of the antibodies contained in the volume of the specific serum being studied. Further, the amount of protein molecules of the precipitate is determined by the amount of nitrogen or using colorimetric methods.
Evaluation of values
Estimation of precipitation values in diagnostic methodology should take into account the possibility of the presence in the immune serum of an antibody that does not have the property of precipitin, from which it follows that the precipitate itself may not be formed after reacting with antigens. The list of such molecules includes incomplete antibodies and some species from the group of gamma-A globulins.
The precipitation reaction in laboratory conditions finds its application in various types of modifications. For example, the thermoprecipitation reaction is used to detect bacterial antigens of botulism, anthrax, etc., which do not undergo thermal denaturation. Unlike ring precipitation, this type of reaction uses filtrates of the material in question in a boiled state.
Analysis of the precipitation reaction in a complex mixture does not allow one to characterize the properties of individual elements of the mixture. In such cases, a person resorts to the method of precipitation in agar, and also use immunoelectropheresis.
Diffuse precipitation
In this area of research, there is the concept of a diffuse precipitation reaction (RPD). It is based on the ability to diffuse in the gel of antibodies and soluble antigens. Diffusion is the ability of a molecule of a certain substance to penetrate into the molecules of another, which is caused by thermal movement.
Gel is a dispersed type system in which the liquid phase is distributed evenly in the solid phase. Most often, agar gel is used for such a reaction.
After giving the parameters, inconditions under which the molecules can diffuse with respect to each other, their meeting will be accompanied by the formation of an antigen + antibody complex. Such a neoplasm is able to diffuse, being in the gel itself, and it will precipitate, taking the form of a strip that can be detected with the naked eye. If the antigen and antibody are homologous, no band will form.
Creating the conditions under which diffusion will take place while in the agar layer involves pouring the components, but the total number of wells and their relative position is determined by the type of task that is required decide. RPD gives a person the ability to detect and identify unknown isolated viruses by testing using known antibody sera.
Application
Precipitation is widely used not only in the diagnosis of diseases, but also finds its application in forensic medical examination. It is difficult to imagine an analysis in which it is possible to determine the species of blood, part of an organ or tissue found on a crime weapon that does not use the precipitation reaction. During this process, precipitating sera are used, which are obtained by immunizing various animals and birds. It is important that the serum titer level is not less than 1:10000, and it must also have sufficient specificity. From the detected spot of blood or its crust, an extract is made for physical. solution, which will be further exposed toprecipitating serum. According to this reaction, it is possible to establish the types of tissue and organ proteins of both humans and animals. Obtaining cloudy extracts forces one to resort to precipitation on agar.
Conclusions
Analyzing the read information, we can conclude that precipitation reactions are extremely important for a person, as they allow diagnosing various antigens using antibodies, this phenomenon is also widely used in forensic medical examination and allows you to identify the type of blood, tissue or organ by relation to a particular subject. There are several types and methods of precipitation, which are used in accordance with the emerging needs of the problem being solved.
