Antigenic determinant. What are antigens and antibodies
Antigenic properties of immunoglobulins served as those phenotypic characteristics, the study of which made it possible to establish the patterns of genetic regulation of the biosynthesis of immunoglobulins. Any immunoglobulin molecule apparently has one or another antibody specificity, that is, it is capable of interacting with substances foreign to a given organism - antigens. However, the immunoglobulin molecule itself can act as an antigen in cases where immunoglobulins of one species (for example, humans) are administered to individuals of another species (for example, rabbits).
There are three types antigenic determinants immunoglobulin molecules: isotypes, allotypes, idiotypes. Isotypic antigenic determinants are those sections of immunoglobulin molecules whose antigenic properties are identical in all individuals of a given species.
Every class immunoglobulins has its own isotypic antigens, characteristic only for this class, which are localized on the constant region of the heavy chains. Isotypic determinants characteristic of kappa- and lambda-type light chains are also localized in the constant region of the chain. Different classes of immunoglobulins and different types light chains do not have common antigenic determinants, despite the presence of homologous sequences.
However, subclasses immunoglobulins have both antigenic determinants common to different subclasses and determinants specific only to a given subclass.
TO allotypic antigenic determinants(allotypes) include those antigenic determinants of immunoglobulin molecules that are present in some individuals of a given species and absent in others, and these differences are determined by allelic genes. The presence of allotypes is a reflection of intraspecific polymorphism in the antigenic structure of immunoglobulin molecules.
And finally third type of antigenic determinants- these are idiotypic determinants (idiotypes). Idiotypes include those individual antigenic properties that are inherent only to antibody molecules of a given specificity or individual myeloma immunoglobulins. The antigenic specificity of idiotypes depends on the structure of the variable region of the antibody molecule, and in some cases there is some evidence that idiotypes are a reflection of the antigenic properties of the active center of the antibody molecule.
Antibodies to isotypic determinants are used to identify different classes and subclasses of immunoglobulins and light chain types. Antibodies to allotypes serve to detect genetic variants of immunoglobulins, and allotype markers are localized, as a rule, on the constant part of the polypeptide chains of immunoglobulins. As for idiotypic determinants, their localization on the variable part of the immunoglobulin molecule allows them to be used as genetic markers of the variable part.
Story detection of genetic markers of polypeptide chains of immunoglobulins is briefly as follows. It has long been known that the serum of patients with rheumatoid arthritis often contains so-called agglutinators, which can specifically interact with autologous IgG. To detect agglutinators, erythrocytes of Rh+ people are used, coated with incomplete aHTH-Rh antibodies, i.e. antibodies that are unable to agglutinate erythrocytes. Agglutination occurs only after the addition of an agglutinator capable of interacting with anti-Rh antibodies on the surface of red blood cells.
Specificity - this is the ability of an antigen to interact with strictly defined antibodies or antigen receptors of lymphocytes.
In this case, the interaction does not occur with the entire surface of the antigen, but only with its small section, which is called the “antigenic determinant” or “epitope”. One antigen molecule can have from several units to several hundred epitopes of varying specificity. The number of epitopes determines the valency of the antigen. For example: egg albumin (M 42,000) has 5 epitopes, i.e. 5-valentene, thyroglobulin protein (M 680,000) - 40-valentene.
In protein molecules, the epitope (antigenic determinant) is formed by a set of amino acid residues. The size of the antigenic determinant of proteins can include from 5 - 7 to 20 amino acid residues. Epitopes that are recognized by antigen receptors of B and T lymphocytes have their own characteristics.
B-cell epitopes of the conformational type (formed by amino acid residues from different parts of the protein molecule, but close in the spatial configuration of the protein globule) are located on the outer surface of the antigen, forming loops and protrusions. Typically, the number of amino acids or sugars in an epitope is from 6 to 8. Antigen recognition receptors of B cells recognize the native conformation of the epitope, rather than a linear sequence of amino acid residues.
T-cell epitopes are a linear sequence of amino acid residues that make up part of an antigen and include a larger number of amino acid residues compared to B-cell epitopes. Their recognition does not require saving the spatial configuration.
Immunogenicity - the ability of an antigen to induce immune defense of the macroorganism. The degree of immunogenicity is determined by the following factors:- Foreignness . In order for a substance to act as an immunogen, it must be recognized as “not its own.” The more foreign the antigen is, that is, the less similar it is to the body’s own structures, the stronger the immune response it causes. For example, the synthesis of antibodies to bovine serum albumin is easier to induce in a rabbit than in a goat. Rabbits belong to the order of lagomorphs and are further away in phylogenetic development from the goat and bull, which belong to the artiodactyls.
- Nature of the antigen . The most powerful immunogens are proteins. Pure polysaccharides, nucleic acids and lipids have weak immunogenic properties. At the same time, lipopolysaccharides, glycoproteins, and lipoproteins are capable of sufficiently activating the immune system.
- Molecular mass . All other things being equal, the larger molecular weight of the antigen provides greater immunogenicity. Antigens are considered good immunogens if their molecular weight is more than 10 kDa. The higher the molecular weight, the more binding sites (epitopes), which leads to an increase in the intensity of the immune response.
- Solubility. Corpuscular antigens associated with cells (erythrocytes, bacteria) are usually more immunogenic. Soluble antigens (serum albumin) may also be highly immunogenic, but are cleared more quickly. To increase the time they remain in the body, necessary for the development of an effective immune response, adjuvants (depositing substances) are used. Adjuvants are substances that are used to enhance the immune response, for example, liquid paraffin, lanolin, aluminum hydroxide and phosphate, potassium alum, calcium chloride, etc.
- Chemical structure of antigen . Increasing the number of aromatic amino acids in synthetic polypeptides increases their immunogenicity. With equal molecular weight (about 70,000), albumin is a stronger antigen than hemoglobin. At the same time, the collagen protein, whose molecular weight is 5 times greater than that of albumin and amounts to 330,000, has significantly less immunogenicity compared to albumin, which is undoubtedly due to the structural features of these proteins.
Immunity to various infectious diseases develops as a response to exposure to antigens. The term "antigens" refers to molecules that are recognized by the immune system and induce an immune response. An antigen stimulates the formation of antibodies and/or cellular immune responses that will specifically interact with this antigen. The reaction between antigen and antibody can be compared to the interaction between a key and a lock. This reaction is specific, so antibodies to a particular antigen do not react at all or react only slightly with other antigens.
An antigen can be a soluble substance produced by microorganisms - for example, a toxin or its non-toxic form - toxoid (see figure), as well as a substance located on the surface of bacteria, viruses or other cells or localized in the cell wall. Most antigens are proteins, but some antigens are bacterial capsule polysaccharides, or glycolipids.
The part of the antigen to which antibodies attach is called the antigenic determinant, antigenic locus, or epitope. Typically, antigens contain multiple determinants, which may differ from each other or may be repeating molecular structures.
Each microorganism contains many different antigens. Protozoa, fungi and bacteria have from several hundred to several thousand antigens. Viruses have fewer antigens - from three (for example, the polyoma virus) to a hundred or more (herpes viruses and poxviruses). During an infectious process, an immune response develops to many of these antigens. However, resistance to infection depends mainly on the immune response to a small number of antigens located on the surface of microorganisms.
Relevant surface antigens have been identified and characterized in several viruses. Much less is currently known about antigens that induce resistance to bacteria, fungi and protozoa. It is all the more obvious that the currently used vaccines, which consist of killed bacteria, induce a much more irrelevant immune response. For example, the pertussis vaccine, which includes whole cells, contains several components - polysaccharides, a heat-labile toxin and a cytotoxin. Although these components have antigenic activity, they are not important in inducing immunity to pertussis.
note
When reacting with an antigen, the antibody binding centers do not always cover the entire antigen. The part of the latter that directly interacts with the antibody is called the antigenic determinant. One molecule may contain one or more antigenic determinants. To study antibody specificity, it is necessary to have antibodies directed against individual antigenic determinants. A small functional group that represents a single antigenic determinant is called a hapten. Haptens can be various organic compounds, such as TNP (trinitrophenyl group), phenylarsonate, mono- and oligosaccharides such as glucose and lactose, and oligopeptides such as pentalysine. Although these haptens are capable of binding to antibodies, they are usually not immunogens, i.e., immunization with them does not lead to the formation of antibodies.
At the same time, an immune response can often be generated using a hapten covalently attached to a large molecule called a carrier. This carrier is immunogenic in itself, and immunization with hapten-carrier conjugates produces antibodies against both the vehicle and the hapten. The hapten-specific antibodies thus obtained can be studied by equilibrium dialysis in the presence of a free (not bound to a carrier) hapten, by immunoprecipitation using a hapten linked to another carrier, or by using the method of inhibiting precipitation with a free hapten.
The described method for obtaining and studying antihapten antibodies, first used by Landsteiner, helped to clarify which elements of the fine structure of the antigenic determinant determine its specificity.
A comparative study of the binding of antibodies to various haptens showed that recognition of “their” hapten by antibodies turns out to be specific, even despite the heterogeneity of the resulting antibody population. Unlike antibodies directed against multideterminate antigens, the population of antibodies specific to one determinant (hapten) is relatively limited. This is due to the fact that the binding of the hapten to the antigen-binding site of the antibody requires certain structural restrictions for their exact correspondence to each other. At the same time, the specificity of the antiserum depends on the specificities of all antibodies included in it, which in turn is determined by the structures of their antigen-binding centers.
When studying cross-reactions with hapten analogs, it turned out that some analogs form complexes with all antibodies present in the serum, while the binding of other analogs quickly reaches saturation, since their structure matches well the structure of the antigen-binding center of only some antibodies. Antibodies obtained from different animals may exhibit different abilities to undergo immunological crossover when interacting with the same set of related haptens. Even in the same animal, as is known, under certain conditions, the affinity and specificity of antibodies can increase with increasing time since the start of immunization.
Thus, the presence or absence of immunological crossover of any two haptens reflects both the structural differences between them, which determine the ability or impossibility of the antigen and antibody to contact each other, and the diversity of antigen-binding centers present in a given antiserum.
Chapter 3. Works of Karl Landsteiner
Antigen as the root cause of the development of the immune process has been of interest to immunologists since ancient times, when immunology was born. However, only thanks to the research of K. Landsteiner in the 20s - 30s of the XX century. conditions have arisen for studying the subtle nature of antigen specificity. The scientist took simple organic compounds - haptens - as the object of his research. As already noted, these compounds themselves are not capable of causing an immunological reaction. The presence of foreignness at a low molecular weight deprives them of immunogenicity. In this case, the hapten complex with the carrier protein is immunogenic. Landsteiner's research revealed at least two significant points: extremely high level specificity (sometimes only one radical takes part in determining specificity - a carboxyl or amino group); the specificity of a high-molecular-weight antigen is represented by individual sites (epitopes) - binding sites for antibodies or antigen-recognition receptors, and the greater the molecular weight of the antigen, the more binding sites.
The experimental design developed by Landsteiner included immunization of rabbits with a hapten-protein complex and subsequent analysis of antisera from immunized animals with the same or a different hapten, but conjugated to another unrelated protein. This technique made it possible to work only with antibodies to the hapten taken for immunization, and excluded those antibodies that were formed by protein capitopes. As a result, it was possible to demonstrate the decisive role of the fine configuration of the hapten in determining specificity.
What are antigens
These are any substances contained in (or secreted by) microorganisms and other cells that carry signs of genetically foreign information and that can potentially be recognized by the body's immune system. When introduced into the internal environment of the body, these genetically foreign substances are capable of causing an immune response of various types.
Each microorganism, no matter how primitive it is, contains several antigens. The more complex its structure, the more antigens can be found in its composition.
Various elements of a microorganism have antigenic properties - flagella, capsule, cell wall, cytoplasmic membrane, ribosomes and other components of the cytoplasm, as well as various protein products secreted by bacteria during external environment, including toxins and enzymes.
There are exogenous antigens (entering the body from the outside) and endogenous antigens (autoantigens - products of the body's own cells), as well as antigens that cause allergic reactions - allergens.
What are antibodies
The body continually encounters a variety of antigens. It is attacked both from the outside - from viruses and bacteria, and from the inside - from body cells that acquire antigenic properties. - serum proteins that are produced by plasma cells in response to the penetration of an antigen into the body. Antibodies are produced by cells of lymphoid organs and circulate in blood plasma, lymph and other body fluids.
The main important role of antibodies is to recognize and bind foreign material (antigen), as well as trigger the mechanism for destroying this foreign material. Essential and unique property Antibodies serve as their ability to bind antigen directly in the form in which it enters the body.
Antibodies have the ability to distinguish one antigen from another. They are capable of specific interaction with an antigen, but they interact only with the antigen (with rare exceptions) that induced their formation and fits them in spatial structure. This antibody ability is called complementarity.
Full understanding molecular mechanism antibody formation does not yet exist. The molecular and genetic mechanisms underlying the recognition of millions of different antigens found in the environment have not been studied.
Antibodies and immunoglobulins
At the end of the 30s of the 20th century, the study of the molecular nature of antibodies began. One of the methods for studying molecules was electrophoresis, which was introduced into practice in the same years. Electrophoresis allows proteins to be separated based on their electrical charge and molecular weight. Serum protein electrophoresis usually produces 5 main bands, which correspond (from + to -) to the albumin, alpha1, alpha2, beta and gamma globulin fractions.
In 1939, Swedish chemist Arne Tiselius and American immunochemist Alvin Kabat used electrophoresis to fractionate the blood serum of immunized animals. Scientists have shown that antibodies are contained in a certain fraction of serum proteins. Namely, antibodies relate mainly to gamma globulins. Since some also fell into the area of beta globulins, a better term was proposed for antibodies - immunoglobulins.
In accordance with the international classification, the totality of serum proteins that have the properties of antibodies is called immunoglobulins and are designated by the symbol Ig (from the word “Immunoglobulin”).
Term "immunoglobulins" reflects the chemical structure of the molecules of these proteins. Term "antibody" determines the functional properties of the molecule and takes into account the ability of the antibody to react only with a specific antigen.
Previously, it was assumed that immunoglobulins and antibodies were synonyms. Currently, there is an opinion that all antibodies are immunoglobulins, but not all immunoglobulin molecules have the function of antibodies.
We talk about antibodies only in relation to the antigen, i.e. if the antigen is known. If we do not know the antigen complementary to a certain immunoglobulin that we have in our hands, then we only have an immunoglobulin. In any antiserum, in addition to antibodies against a given antigen, there is a large number of immunoglobulins, the antibody activity of which could not be detected, but this does not mean that these immunoglobulins are not antibodies to any other antigens. The question of the existence of immunoglobulin molecules that initially do not have the properties of antibodies remains open.
Antibodies (AT, immunoglobulins, IG, Ig) are the central figure of humoral immunity. The main role in the body's immune defense is played by lymphocytes, which are divided into two main categories - T-lymphocytes and B-lymphocytes.
Antibodies or immunoglobulins (Ig) are synthesized by B lymphocytes, or more precisely by antibody-forming cells (AFC). Antibody synthesis begins in response to antigens entering the internal environment of the body. To synthesize antibodies, B cells require contact with an antigen and the resulting maturation of B cells into antibody-forming cells. A significant number of antibodies are produced by so-called plasma cells formed from B-lymphocytes - AOC, which are detected in the blood and tissues. Immunoglobulins are contained in large quantities in serum, intercellular fluid and other secretions, providing a humoral response.
Immunoglobulin classes
Immunoglobulins (Ig) differ in structure and function. There are 5 different classes of immunoglobulins found in humans: IgG,IgA,IgM,IgE,IgD, some of which are further divided into subclasses. There are subclasses for immunoglobulins of classes G (Gl, G2, G3, G4), A (A1, A2) and M (M1, M2).
Classes and subclasses taken together are called isotypes immunoglobulins.
Antibodies of different classes differ in molecular size, charge of the protein molecule, amino acid composition and content of the carbohydrate component. The most studied class of antibodies is IgG.
In human blood serum, immunoglobulins of the IgG class normally predominate. They constitute approximately 70–80% of the total serum antibodies. IgA content - 10-15%, IgM - 5-10%. The content of immunoglobulins of the IgE and IgD classes is very small - about 0.1% for each of these classes.
One should not think that antibodies against a particular antigen belong only to one of the five classes of immunoglobulins. On the contrary, antibodies against the same antigen can be represented by different classes of Ig.
The most important diagnostic role is played by the determination of antibodies of classes M and G, since after a person is infected, class M antibodies appear first, then class G, and immunoglobulins A and E appear last.
Immunogenicity and antigenicity of antigens
In response to the entry of antigens into the body, a whole complex of reactions begins, aimed at freeing the internal environment of the body from the products of foreign genetic information. This set of protective reactions of the immune system is called immune response.
Immunogenicity is called the ability of an antigen to cause an immune response, that is, to induce a specific protective reaction of the immune system. Immunogenicity can also be described as the ability to create immunity.
Immunogenicity largely depends on the nature of the antigen, its properties (molecular weight, mobility of antigen molecules, shape, structure, ability to change), on the route and mode of entry of the antigen into the body, as well as additional influences and the genotype of the recipient.
As mentioned above, one of the forms of response of the immune system in response to the introduction of an antigen into the body is the biosynthesis of antibodies. Antibodies are able to bind the antigen that caused their formation, and thereby protect the body from the possible harmful effects of foreign antigens. In this regard, the concept of antigenicity is introduced.
Antigenicity- this is the ability of an antigen to specifically interact with immune factors, namely, to interact with the products of the immune response caused by this particular substance (antibodies and T- and B-antigen-recognizing receptors).
Some terms of molecular biology
Lipids(from ancient Greek λίπος - fat) - a large group of fairly diverse natural organic compounds, including fats and fat-like substances. Lipids are found in all living cells and are one of the main components of biological membranes. They are insoluble in water and highly soluble in organic solvents. Phospholipids- complex lipids containing higher fatty acids and a phosphoric acid residue.
Conformation molecules (from Latin conformatio - shape, structure, arrangement) - geometric forms that molecules of organic compounds can take when rotating atoms or groups of atoms (substituents) around simple bonds while maintaining the order of the chemical bond of the atoms (chemical structure), the length of the bonds and bond angles.
Organic compounds (acids) of a special structure. Their molecules simultaneously contain amino groups (NH 2) and carboxyl groups (COOH). All amino acids consist of only 5 chemical elements: C, H, O, N, S.
Peptides(Greek πεπτος - nutritious) - a family of substances whose molecules are built from two or more amino acid residues connected into a chain by peptide (amide) bonds. Peptides whose sequence is longer than about 10-20 amino acid residues are called polypeptides.
In the polypeptide chain there are N-terminus, formed by a free α-amino group and C-end, having a free α-carboxyl group. Peptides are written and read from N-terminal to C-terminal - from N-terminal amino acid to C-terminal amino acid.
Amino acid residues- These are monomers of amino acids that make up peptides. An amino acid residue that has a free amino group is called N-terminal and is written on the left, and one that has a free α-carboxyl group is called C-terminal and is written on the right.
Proteins usually called polypeptides containing approximately 50 amino acid residues. The term “proteins” is also used as a synonym for the term “proteins” (from the Greek protos - first, most important). The molecule of any protein has a clearly defined, fairly complex, three-dimensional structure.
Amino acid residues in proteins are usually designated using a three-letter or one-letter code. The three-letter code is an abbreviation of the English names of amino acids and is often used in scientific literature. Single-letter codes, for the most part, do not have an intuitive connection to amino acid names and are used in bioinformatics to represent amino acid sequences in text for easy computer analysis.
Peptide backbone. In the polypeptide chain, the sequence of atoms -NH-CH-CO- is repeated many times. This sequence forms the peptide backbone. The polypeptide chain consists of a polypeptide backbone (skeleton), which has a regular, repeating structure, and individual side groups (R-groups).
Peptide bonds combine amino acids into peptides. Peptide bonds are formed by the interaction of the α-carboxyl group of one amino acid and the α-amino group of a subsequent amino acid. Peptide bonds are very strong and do not spontaneously break under normal conditions existing in cells.
Groups of atoms -CO-NH- that are repeated many times in peptide molecules are called peptide groups. The peptide group has a rigid planar (flat) structure.
Protein conformation- location of the polypeptide chain in space. The spatial structure characteristic of a protein molecule is formed due to intramolecular interactions. Due to the interaction of functional groups of amino acids, linear polypeptide chains of individual proteins acquire a certain three-dimensional structure, which is called “protein conformation.”
The process of formation of a functionally active protein conformation is called folding. The rigidity of the peptide bond reduces the number of degrees of freedom of the polypeptide chain, which plays an important role in the folding process.
Globular and fibrillar proteins. The proteins studied to date can be divided into two large classes according to their ability to take on a certain geometric shape in solution: fibrillar(stretched into a thread) and globular(rolled into a ball). The polypeptide chains of fibrillar proteins are elongated, located parallel to each other and form long threads or layers. In globular proteins, polypeptide chains are tightly folded into globules - compact spherical structures.
It should be noted that the division of proteins into fibrillar and globular is conventional, since there are a large number of proteins with an intermediate structure.
Primary protein structure(primary structure of protein) is a linear sequence of amino acids that make up a protein in a polypeptide chain. Amino acids are connected to each other by peptide bonds. The amino acid sequence is written starting from the C-terminus of the molecule, towards the N-terminus of the polypeptide chain.
P.s.b is the simplest level of structural organization of a protein molecule. First P.s.b. was established by F. Sanger for insulin (Nobel Prize for 1958).
(secondary structure of protein) - the folding of the polypeptide chain of a protein as a result of the interaction between closely spaced amino acids within the same peptide chain - between amino acids located a few residues apart from each other.
The secondary structure of proteins is a spatial structure that is formed as a result of interactions between the functional groups that make up the peptide backbone.
The secondary structure of proteins is determined by the ability of peptide bond groups to undergo hydrogen interactions between the -C=O and -NH- functional groups of the peptide backbone. In this case, the peptide tends to adopt a conformation with the formation of the maximum number of hydrogen bonds. However, the possibility of their formation is limited by the nature of the peptide bond. Therefore, the peptide chain does not acquire an arbitrary, but a strictly defined conformation.
The secondary structure is formed from segments of the polypeptide chain that participate in the formation of a regular network of hydrogen bonds.
In other words, the secondary structure of a polypeptide refers to the conformation of its main chain (backbone) without taking into account the conformation of side groups.
The polypeptide chain of a protein, folding under the influence of hydrogen bonds into a compact form, can form a number of regular structures. Several such structures are known: α (alpha)-helix, β (beta)-structure (another name is β-pleated layer or β-pleated sheet), random coil and turn. A rare type of protein secondary structure is π-helices. Initially, researchers believed that this type of helix did not occur in nature, but later these helices were discovered in proteins.
The α-helix and β-structure are the energetically most favorable conformations, since they are both stabilized by hydrogen bonds. In addition, both the α-helix and β-structure are further stabilized by the close packing of the backbone atoms, which fit together like pieces of a picture puzzle.
These fragments and their combination in a certain protein, if present, are also called the secondary structure of this protein.
In the structure of globular proteins, fragments of a regular structure of all types can be found in any combination, but there may not be any. In fibrillar proteins, all residues belong to one type: for example, wool contains α-helices, and silk contains β-structures.
Thus, most often the secondary structure of a protein is the folding of the protein polypeptide chain into α-helical regions and β-structural formations (layers) involving hydrogen bonds. If hydrogen bonds are formed between the bending areas of one chain, then they are called intrachain; if between chains, they are called interchain. Hydrogen bonds are located perpendicular to the polypeptide chain.
α-helix-formed by intrachain hydrogen bonds between the NH group of one amino acid residue and the CO group of the fourth residue from it. The average length of α-helices in proteins is 10 amino acid residues
In an α-helix, hydrogen bonds are formed between the oxygen atom of the carbonyl group and the hydrogen of the amide nitrogen of the 4th amino acid from it. All C=O and N-H groups of the main polypeptide chain are involved in the formation of these hydrogen bonds. The side chains of amino acid residues are located along the periphery of the helix and do not participate in the formation of the secondary structure.
β-structures are formed between the linear regions of the peptide backbone of one polypeptide chain, thereby forming folded structures (several zigzag polypeptide chains).
The β-structure is formed due to the formation of many hydrogen bonds between the atoms of the peptide groups of linear chains. In β-structures, hydrogen bonds are formed between amino acids or different protein chains that are relatively distant from each other in the primary structure, and not closely located, as is the case in an α-helix.
In some proteins, β-structures can be formed due to the formation of hydrogen bonds between atoms of the peptide backbone of different polypeptide chains.
Polypeptide chains or parts thereof can form parallel or antiparallel β-structures. If several chains of a polypeptide are connected in opposite directions, and the N- and C-termini do not coincide, then antiparallelβ-structure, if they coincide – parallelβ-structure.
Another name for β-structures is β-sheets(β-folded layers, β-sheets). A β-sheet is formed from two or more β-structural regions of a polypeptide chain called β-strands. Typically, β-sheets are found in globular proteins and contain no more than 6 β-strands.
β-strands(β-strands) are regions of a protein molecule in which the bonds of the peptide backbone of several consecutive polypeptides are organized in a planar conformation. In illustrations, the β-strands of proteins are sometimes depicted as flat "arrowhead bands" to emphasize the direction of the polypeptide chain.
The main part of the β-strands is located adjacent to other strands and forms with them an extensive system of hydrogen bonds between the C=O and N-H groups of the main protein chain (peptide backbone). β-strands can be packaged
A messy tangle- this is a section of the peptide chain that does not have any regular, periodic spatial organization. Such regions in each protein have their own fixed conformation, which is determined by the amino acid composition of this region, as well as the secondary and tertiary structures of adjacent regions surrounding the “chaotic coil”. In regions of a random coil, the peptide chain can bend relatively easily and change conformation, while the α-helices and β-sheet layer are fairly rigid structures
Another form of secondary structure is denoted as β-turn. This structure is formed by 4 or more amino acid residues with a hydrogen bond between the first and last, and in such a way that the peptide chain changes direction by 180°. The loop structure of such a turn is stabilized by a hydrogen bond between the carbonyl oxygen of the amino acid residue at the beginning of the turn and the N-H group of the third residue along the chain at the end of the turn.
If antiparallel β-strands approach the β-turn from both ends, then a secondary structure is formed, called β-hairpin(β-hairpin)
Protein tertiary structure(tertiary structure of protein) - In solution under physiological conditions, the polypeptide chain folds into a compact formation that has a certain spatial structure, which is called the tertiary structure of the protein. It is formed as a result of self-folding due to interactions between radicals (covalent and hydrogen bonds, ionic and hydrophobic interactions). For the first time T.s.b. was established for the myoglobin protein by J. Kendrew and M. Perutz in 1959 (Nobel Prize for 1962). T.s.b. almost completely determined by the primary structure of the protein. Currently, using the methods of X-ray diffraction analysis and nuclear magnetic spectroscopy (NMR spectroscopy), the spatial (tertiary) structures of a large number of proteins have been determined.
Quaternary structure of protein. Proteins consisting of one polypeptide chain have only tertiary structure. However, some proteins are built from several polypeptide chains, each of which has a tertiary structure. For such proteins, the concept of quaternary structure has been introduced, which is the organization of several polypeptide chains with a tertiary structure into a single functional protein molecule. Such a protein with a quaternary structure is called an oligomer, and its polypeptide chains with a tertiary structure are called protomers or subunits.
Conjugate(conjugate, lat. conjugatio - connection) - an artificially synthesized (chemically or by recombination in vitro) hybrid molecule in which two molecules with different properties are connected (combined); widely used in medicine and experimental biology.
Haptens
Haptens- these are “defective antigens” (the term was proposed by the immunologist K. Landsteiner). When introduced into the body under normal conditions, haptens are not capable of inducing an immune response in the body, since they have extremely low immunogenicity.
Most often, haptens are low molecular weight compounds (molecular weight less than 10 kDa). They are recognized by the recipient's body as genetically foreign (i.e., they have specificity), but due to their low molecular weight, they do not themselves cause immune reactions. However, they have not lost their antigenic property, which allows them to specifically interact with ready-made immune factors (antibodies, lymphocytes).
Under certain conditions, it is possible to force the immune system of the macroorganism to specifically respond to the hapten as a full-fledged antigen. To do this, it is necessary to artificially enlarge the hapten molecule - to connect it with a strong bond to a sufficiently large protein molecule or other carrier polymer. The conjugate synthesized in this way will have all the properties of a full-fledged antigen and cause an immune response when introduced into the body.
Epitopes (antigenic determinants)
The body can form antibodies to almost any part of the antigen molecule, but this usually does not happen during a normal immune response. Complex antigens (proteins, polysaccharides) have special areas to which a specific immune response is actually formed. Such areas are called epitopes(epitope), from Greek. epi - on, above, over and topos - place, area. Synonym - antigenic determinant.
These sections consist of a few amino acids or carbohydrates, each section is a group of amino acid residues of a protein antigen or a section of a polysaccharide chain. Epitopes are able to interact with both specific receptors lymphocytes, thereby inducing an immune response, and with antigen-binding centers of specific antibodies.
Epitopes are diverse in their structure. An antigenic determinant (epitope) can be a region of the protein surface formed by amino acid radicals, a hapten or a prosthetic group of a protein (a non-protein component associated with a protein), especially often polysaccharide groups of glycoproteins.
Antigenic determinants or epitopes are specific regions of the three-dimensional structure of antigens. There are different types of epitopes - linear And conformational.
Linear epitopes are formed by a linear sequence of amino acid residues.
As a result of studying the structure of proteins, it was found that protein molecules have a complex spatial structure. When coiled (into a ball), protein macromolecules can bring together residues that are distant from each other in a linear sequence, forming a conformational antigenic determinant.
In addition, there are terminal epitopes (located at the ends of the antigen molecule) and central ones. “Deep,” or hidden, antigenic determinants, which appear when the antigen is destroyed, are also determined.
The molecules of most antigens are quite large. One protein macromolecule (antigen), consisting of several hundred amino acids, can contain many different epitopes. Some proteins may have the same antigenic determinant in multiple copies (repeated antigenic determinants).
A wide range of different antibodies are formed against one epitope. Each of the epitopes is capable of stimulating the production of different specific antibodies. Specific antibodies can be produced for each of the epitopes.
There is a phenomenon immunodominance, which manifests itself in the fact that epitopes differ in their ability to induce an immune response.
Not all epitopes in a protein are characterized by equal antigenicity. As a rule, some epitopes of an antigen have special antigenicity, which is manifested in the preferential formation of antibodies against these epitopes. A hierarchy is established in the spectrum of epitopes of the protein molecule - some of the epitopes are dominant and most antibodies are formed specifically to them. These epitopes are named immunodominant epitopes. They are almost always located on prominent parts of the antigen molecule.
Structure of antibodies (immunoglobulins)
IgG immunoglobulins based on experimental data. Each amino acid residue of a protein molecule is depicted as a small ball. Visualization was built using the RasMol program.
During the 20th century, biochemists sought to find out what variants of immunoglobulins exist and what is the structure of the molecules of these proteins. The structure of antibodies was established through various experiments. Basically, they consisted in the fact that the antibodies were treated with proteolytic enzymes (papain, pepsin), and were subjected to alkylation and reduction with mercaptoethanol.
Then the properties of the resulting fragments were studied: their molecular weight (by chromatography), quaternary structure (by X-ray diffraction analysis), ability to bind to antigen, etc. was determined. Antibodies to these fragments were also used to determine whether antibodies to one type of fragment could bind to fragments of another type. Based on the data obtained, a model of the antibody molecule was built.
More than 100 years of research into the structure and function of immunoglobulins has only emphasized the complex nature of these proteins. Currently, the structure of human immunoglobulin molecules has not been fully described. Most researchers have concentrated their efforts not on describing the structure of these proteins, but on elucidating the mechanisms by which antibodies interact with antigens. In addition, antibody molecules
Despite the supposed diversity of immunoglobulins, their molecules have been classified according to the structures included in these molecules. This classification is based on the fact that immunoglobulins of all classes are built according to a general plan and have a certain universal structure.
Immunoglobulin molecules are complex spatial formations. All antibodies, without exception, belong to the same type of protein molecules that have a globular secondary structure, which corresponds to their name - “immunoglobulins” (the secondary structure of a protein is the way its polypeptide chain is laid out in space). They can be monomers or polymers built from several subunits.
Heavy and light polypeptide chains in the structure of immunoglobulins
Peptide chains of immunoglobulins. Schematic illustration. Variable regions are highlighted with dotted lines.
The structural unit of immunoglobulin is a monomer, a molecule consisting of polypeptide chains connected to each other by disulfide bonds (S-S bridges).
If an Ig molecule is treated with 2-mercaptoethanol (a reagent that destroys disulfide bonds), it will disintegrate into pairs of polypeptide chains. The resulting polypeptide chains are classified by molecular weight: light and heavy. Light chains have a low molecular weight (about 23 kDa) and are designated by the letter L, from the English. Light - light. Heavy chains H (from the English Heavy - heavy) have a high molecular weight (varies between 50 - 73 kDa).
The so-called monomeric immunoglobulin contains two L chains and two H chains. The light and heavy chains are held together by disulfide bridges. Disulfide bonds connect light chains to heavy chains and heavy chains to each other.
The main structural subunit of all classes of immunoglobulins is the light chain-heavy chain (L-H) pair. The structure of immunoglobulins of different classes and subclasses differs in the number and location of disulfide bonds between heavy chains, as well as in the number of (L-H) subunits in the molecule. The H-chains are held together by varying numbers of disulfide bonds. The types of heavy and light chains that make up different classes of immunoglobulins also differ.
The figure shows a diagram of the organization of IgG as a typical immunoglobulin. Like all immunoglobulins, IgG contains two identical heavy (H) chains and two identical light (L) chains, which are linked into a four-chain molecule through interchain disulfide bonds (-S-S-). The only disulfide bond connecting the H and L chains is located near the C-terminus of the light chain. There is also a disulfide bond between the two heavy chains.
Domains within an antibody molecule
The light and heavy polypeptide chains in the Ig molecule have a specific structure. Each chain is conventionally divided into specific sections called domains.
Both light and heavy chains do not form a straight thread. Within each chain, at regular and approximately equal intervals of 100-110 amino acids, there are disulfide bridges that form loops in the structure of each chain. The presence of disulfide bridges means that each loop in the peptide chains must form a compactly folded globular domain. Thus, each polypeptide chain in the immunoglobulin forms several globular domains in the form of loops, including approximately 110 amino acid residues.
We can say that immunoglobulin molecules are assembled from separate domains, each of which is located around a disulfide bridge and is homologous to the others.
In each of the light chains of antibody molecules, there are two intrachain disulfide bonds; accordingly, each light chain has two domains. The number of such bonds in heavy chains varies; heavy chains contain four or five domains. Domains are separated by easily organized segments. The presence of such configurations was confirmed by direct observations and genetic analysis.
Primary, secondary, tertiary and quaternary structure of immunoglobulins
The structure of the immunoglobulin molecule (as well as other proteins) is determined by the primary, secondary, tertiary and quaternary structure. The primary structure is the sequence of amino acids that make up the light and heavy chains of immunoglobulins. X-ray diffraction analysis showed that the light and heavy chains of immunoglobulins consist of compact globular domains (the so-called immunoglobulin domains). The domains are arranged in a characteristic tertiary structure called the immunoglobulin fold.
Immunoglobulin domains are regions in the tertiary structure of the Ig molecule that are characterized by a certain autonomy of structural organization. Domains are formed by different segments of the same polypeptide chain, folded into “balls” (globules). The globule contains approximately 110 amino acid residues.
Domains have similar general structure and specific functions to each other. Within the domains, the peptide fragments that make up the domain form a compactly folded antiparallel β-sheet structure stabilized by hydrogen bonds (protein secondary structure). There are practically no regions with an α-helical conformation in the structure of the domains.
The secondary structure of each domain is formed by folding an extended polypeptide chain back and forth upon itself into two antiparallel β-sheets (β-sheets) containing several β-sheets. Each β-sheet has a flat shape - the polypeptide chains in the β-sheets are almost completely elongated.
The two β-sheets that make up the immunoglobulin domain are arranged in a structure called a β-sandwich (“like two pieces of bread on top of each other”). The structure of each immunoglobulin domain is stabilized by an intradomain disulfide bond—the β-sheets are covalently linked by a disulfide bond between the cysteine residues of each β-sheet. Each β-sheet consists of antiparallel β-strands connected by loops of varying lengths.
The domains, in turn, are interconnected by a continuation of the polypeptide chain, which extends beyond the β-sheets. The open sections of the polypeptide chain present between the globules are especially sensitive to proteolytic enzymes.
The globular domains of a light and heavy chain pair interact with each other to form a quaternary structure. Due to this, functional fragments are formed that allow the antibody molecule to specifically bind the antigen and, at the same time, perform a number of biological effector functions.
Variable and constant domains
Domains in peptide chains differ in the consistency of their amino acid composition. There are variable and constant domains (regions). Variable domains are designated by the letter V, from the English. variable - “changeable” and are called V-domains. Permanent (constant) domains are designated by the letter C, from the English constant - “permanent” and are called C-domains.
Immunoglobulins produced by different clones of plasma cells have variable domains of different amino acid sequences. The constant domains are similar or very similar for each immunoglobulin isotype.
Each domain is designated by a letter indicating whether it belongs to the light or heavy chain and a number indicating its position.
The first domain on the light and heavy chains of all antibodies is extremely variable in amino acid sequence; it is denoted as V L and V H respectively.
The second and subsequent domains on both heavy chains are much more constant in amino acid sequence. They are designated CH or C H 1, C H 2 and C H 3. Immunoglobulins IgM and IgE have an additional C H 4 domain on the heavy chain, located behind the C H 3 domain.
The half of the light chain including the carboxyl terminus is called the constant region C L , and the N-terminal half of the light chain is called the variable region V L .
Carbohydrate chains are also associated with the CH2 domain. Immunoglobulins of different classes differ greatly in the number and location of carbohydrate groups. The carbohydrate components of immunoglobulins have a similar structure. They consist of a constant core and a variable outer part. Carbohydrate components affect the biological properties of antibodies.
Fab and Fc fragments of the immunoglobulin molecule
The variable domains of the light and heavy chains (V H and V L), together with the constant domains closest to them (C H 1 and C L 1), form Fab fragments of antibodies (fragment, antigen binding). The immunoglobulin region that binds to a specific antigen is formed by the N-terminal variable regions of the light and heavy chains, i.e. V H - and V L -domains.
The remaining part, represented by the C-terminal constant domains of the heavy chains, is designated as the Fc fragment (fragment, crystallizable). The Fc fragment includes the remaining CH domains held together by disulfide bonds. At the junction of the Fab and Fc fragments there is a hinge region that allows the antigen-binding fragments to unfold for closer contact with the antigen.
Hinge area
At the border of the Fab and Fc fragments there is the so-called. "hinge area" having a flexible structure. It provides mobility between the two Fab fragments of the Y-shaped antibody molecule. The mobility of antibody molecule fragments relative to each other is an important structural characteristic of immunoglobulins. This type of interpeptide connection makes the structure of the molecule dynamic - it allows you to easily change the conformation depending on the surrounding conditions and state.
The hinge region is a section of the heavy chain. The hinge region contains disulfide bonds that connect the heavy chains to each other. For each class of immunoglobulins, the hinge region has its own structure.
In immunoglobulins (with the possible exception of IgM and IgE), the hinge region consists of a short segment of amino acids and is found between the C H 1 and C H 2 regions of the heavy chains. This segment consists predominantly of cysteine and proline residues. Cysteines are involved in the formation of disulfide bridges between chains, and proline residues prevent folding into a globular structure.
Typical structure of an immunoglobulin molecule using IgG as an example
The schematic representation in the planar drawing does not accurately reflect the structure of Ig; in reality, the variable domains of the light and heavy chains are not arranged in parallel, but are closely intertwined with each other in a criss-cross pattern.
It is convenient to consider the typical structure of an immunoglobulin using the example of an IgG antibody molecule. There are a total of 12 domains in the IgG molecule - 4 on the heavy chains and 2 on the light chains.
Each light chain includes two domains - one variable (V L, variable domain of the light chain) and one constant (CL, constant domain of the light chain). Each heavy chain contains one variable domain (V H, variable domain of the heavy chain) and three constant domains (CH 1–3, constant domains of the heavy chain). About a quarter of the heavy chain, including the N-terminus, is classified as the variable region of the H chain (VH), the rest of it is the constant region (CH1, CH2, CH3).
Each pair of variable domains V H and V L located in adjacent heavy and light chains forms a variable fragment (Fv, variable fragment).
Types of heavy and light chains in antibody molecules
Based on differences in the primary structure of permanent regions, circuits are divided into types. The types are determined by the primary amino acid sequence of the chains and the degree of glycosylation. Light chains are divided into two types: κ and λ (kappa and lambda), heavy chains are divided into five types: α, γ, μ, ε and δ (alpha, gamma, mu, epsilon and delta). Among the variety of heavy chains of alpha, mu and gamma types, subtypes are distinguished.
Classification of immunoglobulins
Immunoglobulins are classified according to their H-chain (heavy chain) type. The constant regions of the heavy chains of immunoglobulins of different classes are not the same. Human immunoglobulins are divided into 5 classes and a number of subclasses, according to the types of heavy chains that are included in their composition. These classes are called IgA, IgG, IgM, IgD and IgE.
The H-chains themselves are designated by a Greek letter, corresponding to the capital Latin letter of the name of one of the immunoglobulins. IgA has heavy chains α (alpha), IgM – μ (mu), IgG – γ (gamma), IgE – ε (epsilon), IgD – δ (delta).
Immunoglobulins IgG, IgM and IgA have a number of subclasses. Division into subclasses (subtypes) also occurs depending on the characteristics of the H-chains. In humans, there are 4 subclasses of IgG: IgG1, IgG2, IgG3 and IgG4, containing heavy chains γ1, γ2, γ3 and γ4, respectively. These H chains differ in small Fc fragment details. For the μ-chain, 2 subtypes are known - μ1- and μ2-. IgA has 2 subclasses: IgA1 and IgA2 with α1 and α2 subtypes of α chains.
In each immunolobulin molecule, all heavy chains are of the same type, in accordance with the class or subclass.
All 5 classes of immunoglobulins consist of heavy and light chains.
The light chains (L-chains) of immunoglobulins of different classes are the same. All immunoglobulins can have either both κ (kappa) or both λ (lambda) light chains. Immunoglobulins of all classes are divided into K- and L-types, depending on the presence of κ- or λ-type light chains in their molecules, respectively. In humans, the ratio of K- and L-types is 3:2.
The classes and subclasses taken together are called immunoglobulin isotypes. The antibody isotype (class, subclass of immunoglobulins - IgM1, IgM2, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE) is determined by the C-domains of the heavy chains.
Each class includes a huge variety of individual immunoglobulins, differing in the primary structure of the variable regions; the total number of immunoglobulins of all classes is ≈ 10^7.
The structure of antibody molecules of various classes
Schemes of the structure of immunoglobulins. (A) - monomeric IgG, IgE, IgD, IgA; (B) - polymeric secretory Ig A (slgA) and IgM (B); (1) - secretory component; (2) - connecting J-chain.
1. Antibody classes IgG, IgD and IgE
Antibody molecules of the IgG, IgD and IgE classes are monomeric; they are Y-shaped.
IgG class immunoglobulins account for 75% of the total number of human immunoglobulins. They are found both in the blood and outside the blood vessels. An important property of IgG is its ability to pass through the placenta. Thus, maternal antibodies enter the body of the newborn child and protect him from infection in the first months of life (natural passive immunity).
IgD is mainly found on the membrane of B lymphocytes. They have a structure similar to IgG, 2 active centers. The heavy chain (δ chain) consists of a variable and 3 constant domains. The hinge region of the δ chain is the longest, and the location of carbohydrates in this chain is also unusual.
IgE - the concentration of this class of immunoglobulins in blood serum is extremely low. IgE molecules are mainly fixed on the surface of mast cells and basophils. IgE is similar in structure to IgG and has 2 active centers. The heavy chain (ε-chain) has one variable and 4 constant domains. It is assumed that IgE is essential in the development of anthelmintic immunity. IgE plays a major role in the pathogenesis of some allergic diseases (bronchial asthma, hay fever) and anaphylactic shock.
2. Antibody classes IgM and IgA
Immunoglobulins IgM and IgA form polymer structures. For polymerization, IgM and IgA include an additional polypeptide chain with a molecular weight of 15 kDa, called the J-chain (joint). This J-chain binds the terminal cysteines at the C-termini of the μ- and α-heavy chains of IgM and IgA, respectively.
On the surface of mature B lymphocytes, IgM molecules are located in the form of monomers. However, in serum they exist in the form of pentamers: the IgM molecule consists of five structural molecules arranged radially. The IgM pentamer is formed from five “slingshot” monomers, similar to IgG, linked together by disulfide bonds and a J chain. Their Fc fragments are directed to the center (where they are connected by a J-chain), and their Fab fragments are directed outward.
In IgM, the heavy (H) chains consist of 5 domains, since they contain 4 constant domains. IgM heavy chains do not have a hinge region; its role is played by the C H 2 domain, which has some conformational lability.
IgM is synthesized mainly during the primary immune response and is predominantly found in the intravascular bed. The amount of Ig M in the blood serum of healthy people is about 10% of the total amount of Ig.
IgA antibodies are built from varying numbers of monomers. Class A immunoglobulins are divided into two types: serum and secretory. Most (80%) of IgA present in blood serum has a monomeric structure. Less than 20% of IgA in serum is represented by dimeric molecules.
Secretory IgA is not found in the blood, but as part of exocretes on the mucous membranes and is designated sIgA. In the secretions of mucous membranes, IgA is presented in the form of dimers. Secretory IgA forms a dimer of two “slingshots” (Ig monomers). The C-termini of the heavy chains in the sIgA molecule are connected to each other by the J-chain and a protein molecule called the “secretory component”.
The secretory component is produced by epithelial cells of the mucous membranes. It attaches to the IgA molecule as it passes through epithelial cells. The secretory component protects sIgA from cleavage and inactivation by proteolytic enzymes, which are contained in large quantities in the secretions of the mucous membranes.
The main function of sIgA is to protect mucous membranes from infection. The role of sIgA in providing local immunity is very significant, because The total area of the mucous membranes in the adult human body is several hundred square meters and far exceeds the surface of the skin.
High concentrations of sIgA are found in human breast milk, especially in the first days of lactation. They protect the newborn's gastrointestinal tract from infection.
Children are born without IgA and receive it through their mother's milk. It has been reliably shown that children who are breastfed are significantly less likely to suffer from intestinal infections and respiratory tract diseases compared to children receiving artificial nutrition.
Antibodies of the IgA class make up 15-20% of the total content of immunoglobulins. IgA does not penetrate the placental barrier. Ig A is synthesized by plasma cells located mainly in submucosal tissues, on the mucous epithelial surface of the respiratory tract, urogenital and intestinal tract, and in almost all excretory glands. Part of Ig A enters the general circulation, but most of it is secreted locally on the mucous membranes in the form of sIgA and serves as a local protective immunological barrier for the mucous membranes. Serum IgA and sIgA are different immunoglobulins; sIgA is not found in blood serum.
People with IgA immunodeficiency have a tendency to autoimmune diseases, infections of the respiratory tract, maxillary and frontal sinuses, and intestinal disorders.
Digestion of the immunoglobulin molecule by enzymes
Proteolytic enzymes (such as papain or pepsin) break down immunoglobulin molecules into fragments. At the same time, under the influence of different proteases, different products can be obtained. Immunoglobulin fragments obtained in this way can be used for research or medical purposes.
The globular structure of immunoglobulins and the ability of enzymes to break down these molecules into large components in strictly defined places, and not destroy them into oligopeptides and amino acids, indicates an extremely compact structure.
1. Cleavage of the immunoglobulin molecule by papain. Fab and Fc fragments of antibodies.
In the late 50s - early 60s, the English scientist R.R. Porter analyzed the structural characteristics of IgG antibodies by separating the molecule with papain (a purified enzyme from papaya juice). Papain destroys immunoglobulin in the hinge region, above the interchain disulfide bonds. This enzyme splits the immunoglobulin molecule into three fragments of approximately the same size.
Two of them were named Fab fragments(from the English fragment antigen-binding - antigen-binding fragment). Fab fragments are completely identical and, as studies have shown, are designed to bind to antigen. The heavy chain region of the Fab fragment is called Fd; it consists of V H and C H 1 domains.
The third fragment may crystallize out of solution and cannot bind antigen. This fragment is named Fc fragment(from the English fragment crystallizable - fragment of crystallization). It is responsible for the biological functions of the antibody molecule after binding the antigen and the Fab part of the intact antibody molecule.
The Fc fragment has the same structure for antibodies of each class and subclass and different for antibodies belonging to different subclasses and classes.
The Fc fragment of the molecule interacts with cells of the immune system: neutrophils, macrophages and other mononuclear phagocytes that carry receptors for the Fc fragment on their surface. If antibodies bind to pathogenic microorganisms, they can interact with phagocytes with their Fc fragment. Thanks to this, the pathogen cells will be destroyed by these phagocytes. In fact, antibodies act in this case as intermediary molecules.
Subsequently, it became known that the Fc fragments of immunoglobulins within one isotype in a given organism are strictly identical, regardless of the antigen specificity of the antibody. For this invariance, they began to be called constant regions (fragment constant - Fc, the abbreviation is the same).
2. Cleavage of the immunoglobulin molecule by pepsin.
Another proteolytic enzyme, pepsin, cleaves the molecule at a different location, closer to the C-terminus of the H chains than papain does. Cleavage occurs “downstream” of the disulfide bonds holding the H chains together. As a result, under the action of pepsin, a divalent antigen-binding F(ab")2 fragment and a truncated pFc" fragment are formed. The pFc" fragment is the C-terminal portion of the Fc region.
Pepsin cuts the pFc" fragment from a large fragment with a sedimentation constant of 5S. This large fragment is called F(ab")2 because, like the parent antibody, it is bivalent with respect to antigen binding. It consists of linked Fab fragments linked by a disulfide bridge at the hinge region. These Fab fragments are monovalent and homologous to papain Fab fragments I and II, but their Fd fragment is approximately ten amino acid residues larger.
Antigen-binding centers of antibodies (paratopes)
The Fab fragment of immunoglobulin includes V domains of both chains, C L and C H 1 domains. The antigen-binding region of the Fab fragment has received several names: the active or antigen-binding center of antibodies, antideterminant or paratope.
Variable segments of light and heavy chains participate in the formation of active centers. The active site is a cleft located between the variable domains of the light and heavy chains. Both of these domains participate in the formation of the active center.
Immunoglobulin molecule. L - light chains; H - heavy chains; V - variable region; C - constant region; The N-terminal regions of the L and H chains (V region) form two antigen-binding centers within the Fab fragments.
Each Fab fragment of IgG immunoglobulins has one antigen-binding site. The active centers of antibodies of other classes, capable of interacting with the antigen, are also located in Fab fragments. Antibodies IgG, IgA and IgE each have 2 active centers, IgM - 10 centers.
Immunoglobulins can bind antigens of different chemical natures: peptides, carbohydrates, sugars, polyphosphates, steroid molecules.
An essential and unique property of antibodies is their ability to bind to intact, native molecules of antigens, directly in the form in which the antigen has penetrated into the internal environment of the body. This does not require any pre-metabolic processing of antigens
Structure of domains in immunoglobulin molecules
The secondary structure of the polypeptide chains of the immunoglobulin molecule has a domain structure. Individual sections of heavy and light chains are folded into globules (domains), which are connected by linear fragments. Each domain is approximately cylindrical in shape and is a β-sheet structure formed from antiparallel β-sheets. Within the basic structure, there is a distinct difference between the C and V domains, which can be seen using the light chain as an example.
The figure schematically shows the folding of a single polypeptide chain of the Bence-Jones protein containing V L and C L domains. The scheme is based on X-ray diffraction data - a method that allows you to establish the three-dimensional structure of proteins. The diagram shows the similarities and differences between the V and C domains.
The upper part of the figure schematically shows the spatial arrangement of the constant (C) and variable (V) domains of the light chain of a protein molecule. Each domain is a cylindrical “barrel-shaped” structure in which sections of the polypeptide chain (β-strands) running in opposite directions (i.e., antiparelle) are packed to form two β-sheets held together by a disulfide communication
Each of the domains, V- and C-, consists of two β-sheets (layers with a β-sheet structure). Each β-sheet contains several antiparallel (running in opposite directions) β-strands: in the C-domain the β-sheets contain four and three β-strands, in the V-domain both layers consist of four β-strands. In the figure, the β-strands are shown in yellow and green for the C domain and red and blue for the V domain.
In the lower part of the figure, immunoglobulin domains are discussed in more detail. This half of the picture shows a diagram of the relative arrangement of β-strands for the V- and C-domains of the light chain. It is possible to more clearly examine the way in which their polypeptide chains are stacked when forming β-sheets, which creates the final structure. To show the folding, the β-strands are designated by letters of the Latin alphabet, according to the order of their appearance in the sequence of amino acids that make up the domain. The order of occurrence in each β-sheet is a characteristic of immunoglobulin domains.
The β-sheets (sheets) in the domains are linked by a disulfide bridge (bond) approximately in the middle of each domain. These bonds are shown in the figure: between the layers there is a disulfide bond connecting folds B and F and stabilizing the structure of the domain.
The main difference between the V and C domains is that the V domain is larger and contains additional β-strands, designated Cʹ and Cʹʹ. In the figure, the β-strands Cʹ and Cʹʹ, present in the V-domains but absent in the C-domains, are highlighted with a blue rectangle. It can be seen that each polypeptide chain forms flexible loops between successive β-strands when changing direction. In the V domain, flexible loops formed between some of the β-strands form part of the active site structure of the immunoglobulin molecule.
Hypervariable regions within V domains
The level of variability within variable domains is not evenly distributed. Not the entire variable domain is variable in its amino acid composition, but only a small part of it - hypervariable areas. They account for about 20% of the amino acid sequence of V-domains.
In the structure of the whole immunoglobulin molecule, the V H and V L domains are combined. Their hypervariable regions are adjacent to each other and create a single hypervariable region in the form of a pocket. This is the region that specifically binds to the antigen. Hypervariable regions determine the complementarity of the antibody to the antigen.
Since hypervariable regions play a key role in antigen recognition and binding, they are also called complementarity determining regions (CDRs). There are three CDRs in the variable domains of the heavy and light chains (V L CDR1–3, V H CDR1–3).
Between the hypervariable regions are relatively constant sections of the amino acid sequence, which are called frame regions (FR). They account for about 80% of the amino acid sequence of V-domains. The role of such regions is to maintain a relatively uniform three-dimensional structure of V-domains, which is necessary to ensure affinity interaction of hypervariable regions with the antigen.
In the variable domain sequence of region 3, hypervariant regions alternate with 4 relatively invariant “framework” regions FR1–FR4,
H1–3 – CDR loops included in the chains.
Of particular interest is the spatial arrangement of the hypervariable regions in three separate loops of the variable domain. These hypervariable regions, although located at a great distance from each other in the primary structure of the light chain, but, when the three-dimensional structure is formed, they are located in close proximity to each other.
In the spatial structure of V-domains, hypervariable sequences are located in the zone of bends of the polypeptide chain, directed towards the corresponding sections of the V-domain of the other chain (i.e., the CDRs of the light and heavy chains are directed towards each other). As a result of the interaction of the variable domain of the H- and L-chains, the antigen-binding site (active center) of the immunoglobulin is formed. According to electron microscopy, it is a cavity 6 nm long and 1.2–1.5 nm wide.
The spatial structure of this cavity, determined by the structure of hypervariable regions, determines the ability of antibodies to recognize and bind specific molecules based on spatial correspondence (antibody specificity). Spatially separated regions of the H- and L-chains also contribute to the formation of the active center. The hypervariable regions of the V domains are not completely included in the active center - the surface of the antigen-binding region covers only about 30% of the CDR.
The hypervariable regions of the heavy and light chain determine the individual structural features of the antigen-binding center for each Ig clone and the diversity of their specificities.
The ultra-high variability of CDRs and active centers ensures that immunoglobulin molecules synthesized by B lymphocytes of the same clone are unique, not only in structure, but also in their ability to bind various antigens. Despite the fact that the structure of immunoglobulins is quite well known and it is the CDRs that are responsible for their features, it is still not clear which domain is most responsible for antigen binding.
Interaction of antibodies and antigens (interaction of epitope and paratope)
The antigen-antibody reaction is based on the interaction between the antigen epitope and the active center of the antibody, based on their spatial correspondence (complementarity). As a result of the binding of the pathogen to the active center of the antibody, the pathogen is neutralized and its penetration into the body's cells is difficult.
In the process of interaction with the antigen, not the entire immunoglobulin molecule takes part, but only a limited part of it - the antigen-binding center, or paratope, which is localized in the Fab fragment of the Ig molecule. In this case, the antibody does not interact with the entire antigen molecule at once, but only with its antigenic determinant (epitope).
The active center of antibodies is a structure that is spatially complementary (specific) to the determinant group of the antigen. The active center of antibodies has functional autonomy, i.e. capable of binding antigenic determinants in isolated form.
On the antigen side, epitopes that interact with specific antibodies are responsible for interaction with the active centers of antigen recognition molecules. The epitope directly enters into ionic, hydrogen, van der Waals and hydrophobic bonds with the active center of the antibody.
The specific interaction of antibodies with an antigen molecule is associated with a relatively small area of its surface, corresponding in size to the antigen-binding site of receptors and antibodies.
The binding of antigen to antibody occurs through weak interactions within the antigen-binding center. All these interactions appear only when the molecules are in close contact. Such a small distance between molecules can only be achieved due to the complementarity of the epitope and the active center of the antibody.
Sometimes the same antigen-binding site of an antibody molecule can bind to several different antigenic determinants (usually these antigenic determinants are very similar). Such antibodies are called cross-reactive, capable of polyspecific binding.
For example, if antigen A has common epitopes with antigen B, then some of the antibodies specific to A will also react with B. This phenomenon is called cross reactivity.
Complete and incomplete antibodies. Valence
Valence- this is the number of active centers of the antibody that are able to combine with antigenic determinants. Antibodies have a different number of active centers in the molecule, which determines their valency. In this regard, there is a distinction full And incomplete antibodies.
Full antibodies have at least two active centers. Full (divalent and pentavalent) antibodies, when interacting in vitro with the antigen in response to which they are produced, give visually visible reactions (agglutination, lysis, precipitation, complement fixation, etc.).
Incomplete or monovalent antibodies differ from regular (complete) antibodies in that they have only one active center; the second center does not work in such antibodies. This does not mean that the second active center of the molecule is absent. The second active center of such immunoglobulins is shielded by various structures or has low avidity. Such antibodies can interact with the antigen, block it, binding epitopes of the antigen and preventing the contact of full antibodies with it, but do not cause aggregation of the antigen. Therefore they are also called blocking.
The reaction between partial antibodies and antigen is not accompanied by macroscopic phenomena. Incomplete antibodies, when specifically interacting with a homologous antigen, do not give a visible manifestation of a serological reaction, because cannot aggregate particles into large conglomerates, but only block them.
Incomplete antibodies are formed independently of complete ones and perform the same functions. They are also represented by different classes of immunoglobulins.
Idiotypes and idiotopes
Antibodies are complex protein molecules that themselves can have antigenic properties and cause the formation of antibodies. In their composition, several types of antigenic determinants (epitypes) are distinguished: isotypes, allotypes and idiotypes.
Different antibodies differ from each other in their variable regions. The antigenic determinants of the variable regions (V regions) of antibodies are called idiotopes. Idiotopes can be constructed from characteristic sections of V-regions of only H-chains or L-chains. In most cases, both chains are involved in the formation of idiotope at once.
Idiotopes may be related to the antigen-binding site (site-associated idiotopes) or unrelated to it (non-associated idiotopes).
Site-associated idiotopes depend on the structure of the antigen-binding region of the antibody (belonging to the Fab fragment). If this site is occupied by an antigen, then the anti-idiotopic antibody can no longer react with an antibody that has this idiotope. Other idiotopes do not appear to have such close association with antigen-binding sites.
The set of idiotopes on the molecule of any antibody is designated as idiot. Thus, an idiotype consists of a set of idiotopes—antigenic determinants of the V region of an antibody.
Group constitutional variants of the antigenic structure of heavy chains are called allotypes. Allotypes are determinants encoded by alleles of a given immunoglobulin gene.
Isotypes are determinants that distinguish classes and subclasses of heavy chains and variants κ (kappa) and λ (lambda) of light chains.
Antibody affinity and avidity
The binding strength of antibodies can be characterized by immunochemical characteristics: avidity and affinity.
Under affinity understand the binding force between the active site of an antibody molecule and the corresponding antigen determinant. The strength of the chemical bond of one antigenic epitope with one of the active centers of the Ig molecule is called the binding affinity of the antibody to the antigen. Affinity is usually quantified by the dissociation constant (in mol-1) of one antigenic epitope with one active site.
Affinity is the accuracy of the coincidence of the spatial configuration of the active center (paratope) of the antibody and the antigenic determinant (epitope). The more connections are formed between the epitope and the paratope, the higher the stability and lifespan of the resulting immune complex will be. The immune complex formed by low-affinity antibodies is extremely unstable and has a short lifespan.
The affinity of antibodies for an antigen is called avidity antibodies. The avidity of the connection between an antibody and an antigen is the total strength and intensity of the connection between the entire antibody molecule and all the antigenic epitopes that it managed to bind.
Antibody avidity is characterized by the rate of formation of the antigen-antibody complex, the completeness of interaction and the strength of the resulting complex. Avidity, as well as the specificity of antibodies, is based on the primary structure of the determinant (active center) of the antibody and the associated degree of adaptation of the surface configuration of antibody polypeptides to the determinant (epitope) of the antigen.
Avidity is determined both by the affinity of the interaction between epitopes and paratopes, and by the valence of antibodies and antigen. Avidity depends on the number of antigen-binding centers in the antibody molecule and their ability to bind to numerous epitopes of a given antigen.
A typical IgG molecule, when both antigen-binding sites are involved, will bind to a multivalent antigen at least 10,000 times stronger than when only one site is involved.
Antibodies of class M have the greatest avidity, since they have 10 antigen-binding centers. If the affinities of the individual antigen-binding sites of IgG and IgM are the same, the IgM molecule (having 10 such sites) will exhibit incomparably greater avidity for the multivalent antigen than the IgG molecule (having 2 sites). Due to their high overall avidity, IgM antibodies, the main class of immunoglobulins produced early in the immune response, can function effectively even with low affinity of individual binding sites.
The difference in avidity is important because antibodies produced early in the immune response usually have much less affinity for the antigen than those produced later. The increase in the average affinity of antibodies produced over time after immunization is called affinity maturation.
Specificity of interaction between antigens and antibodies
In immunology, specificity refers to the selectivity of the interaction of inducers and products of immune processes, in particular, antigens and antibodies.
The specificity of interaction for antibodies is the ability of an immunoglobulin to react only with a specific antigen, namely, the ability to bind to a strictly defined antigenic determinant. The phenomenon of specificity is based on the presence of active centers in the antibody molecule that come into contact with the corresponding determinants of the antigen. The selectivity of the interaction is due to the complementarity between the structure of the active center of the antibody (paratope) and the structure of the antigenic determinant (epitope).
Antigen specificity is the ability of an antigen to induce an immune response to a strictly defined epitope. The specificity of an antigen is largely determined by the properties of its constituent epitopes.
One of the most important functions of immunoglobulins is antigen binding and the formation of immune complexes. Antibody proteins react specifically with antigens, forming immune complexes - complexes of antibodies associated with antigens. This connection is unstable: the resulting immune complex (IC) can easily disintegrate into its constituent components.
Each antigen molecule can be joined by several antibody molecules, since there are several antigenic determinants on the antigen and antibodies can be formed to each of them. As a result, complex molecular complexes arise.
The formation of immune complexes is an integral component of the normal immune response. The formation and biological activity of immune complexes depend, first of all, on the nature of the antibodies and antigen included in their composition, as well as on their ratio. The characteristics of immune complexes depend on the properties of antibodies (valence, affinity, rate of synthesis, ability to fix complement) and antigen (solubility, size, charge, valency, spatial distribution and epitope density).
Interaction of antigens and antibodies. Antigen-antibody reaction
The antigen-antibody reaction is the formation of a complex between an antigen and antibodies directed towards it. The study of such reactions is of great importance for understanding the mechanism of specific interaction of biological macromolecules and for elucidating the mechanism of serological reactions.
The effectiveness of the interaction of an antibody with an antigen significantly depends on the conditions under which the reaction occurs, primarily on the pH of the medium, osmotic density, salt composition and temperature of the medium. Optimal for the antigen-antibody reaction are the physiological conditions of the internal environment of the macroorganism: a close to neutral reaction of the environment, the presence of phosphate, carbonate, chloride and acetate ions, the osmolarity of the physiological solution (solution concentration 0.15 M), as well as a temperature of 36- 37 °C.
The interaction of an antigen molecule with an antibody or its active Fab fragment is accompanied by changes in the spatial structure of the antigen molecule.
Since no chemical bonds arise when an antigen is combined with an antibody, the strength of this connection is determined by the spatial accuracy (specificity) of the interacting sections of two molecules - the active center of the immunoglobulin and the antigenic determinant. The measure of bond strength is determined by the affinity of the antibody (the magnitude of the connection of one antigen-binding center with an individual epitope of the antigen) and its avidity (the total strength of interaction of the antibody with the antigen in the case of interaction of a polyvalent antibody with a polyvalent antigen).
All antigen-antibody reactions are reversible; the antigen-antibody complex can dissociate to release antibodies. In this case, the reverse antigen-antibody reaction proceeds much slower than the direct one.
There are two main ways by which an already formed antigen-antibody complex can be partially or completely separated. The first is the displacement of antibodies by an excess of antigen, and the second is the impact on the immune complex of external factors, leading to the severing of bonds (decreased affinity) between the antigen and the antibody. Partial dissociation of the antigen-antibody complex can generally be achieved by increasing the temperature.
When using serological methods, the most universal way to dissociate immune complexes formed by a wide variety of antibodies is to treat them with dilute acids and alkalis, as well as concentrated solutions of amides (urea, guanidine hydrochloride).
Heterogeneity of antibodies
Antibodies formed during the body’s immune response are heterogeneous and differ from each other, i.e. They heterogeneous. Antibodies are heterogeneous in their physicochemical, biological properties and, above all, in their specificity. Main base heterogeneity (diversity of specificities) of antibodies - diversity of their active centers. The latter is associated with the variability of the amino acid composition in the V regions of the antibody molecule.
Antibodies are also heterogeneous in belonging to different classes and subclasses.
The heterogeneity of antibodies is also due to the fact that immunoglobulins contain 3 types of antigenic determinants: isotypic, characterizing the belonging of the immunoglobulin to a certain class; allotypic, corresponding to allelic variants of immunoglobulin; idiotypic, reflecting the individual characteristics of immunoglobulin. The idiotype-anti-idiotype system forms the basis of the so-called Jerne network theory.
Isotypes, allotypes, idiotypes of antibodies
Immunoglobulins contain three types of antigenic determinants: isotypic (the same for each representative of a given species), allotypic (determinants that are different among representatives of a given species) and idiotypic (determinants that determine the individuality of a given immunoglobulin and are different for antibodies of the same class or subclass).
In each biological species, the heavy and light chains of immunoglobulins have certain antigenic characteristics, according to which the heavy chains are divided into 5 classes (γ, μ, α, δ, ε), and the light chains into 2 types (κ and λ). These antigenic determinants are called isotypic (isotypes); for each chain they are the same in each representative of a given biological species.
At the same time, there are intraspecific differences in the named immunoglobulin chains - allotypes, determined by the genetic characteristics of the producing organism: their characteristics are genetically determined. For example, more than 20 allotypes have been described for heavy chains.
Even when antibodies to a particular antigen belong to the same class, subclass, or even allotype, they are characterized by specific differences from each other. These differences are called idiotypes. They characterize the “individuality” of a given immunoglobulin depending on the specificity of the inducer antigen. This depends on the structural features of the V-domains of the H- and L-chains and the many different variants of their amino acid sequences. All of these antigenic differences are determined using specific sera.
Classifications of antibodies according to the reactions in which they can participate
Initially, antibodies were conventionally classified according to their functional properties into neutralizing, lysing and coagulating. Neutralizing agents included antitoxins, antienzymes and virus-neutralizing lysines. Coagulating agents include agglutinins and precipitins; to lysing - hemolytic and complement-fixing antibodies. Taking into account the functional ability of antibodies, names were given to serological reactions: agglutination, hemolysis, lysis, precipitation, etc.
Antibody studies. Phage display.
Until recently, the study of antibodies was difficult due to technical reasons. Immunoglobulins in the body are a complex mixture of proteins. The immunoglobulin fraction of blood serum is a mixture of a huge number of different antibodies. Moreover, the relative content of each type of them is, as a rule, very small. Until recently, obtaining pure antibodies from the immunoglobulin fraction was difficult to obtain. The difficulty of isolating individual immunoglobulins has long been an obstacle both to their biochemical study and to the establishment of their primary structure.
In recent years, a new field of immunology has emerged - antibody engineering, which deals with the production of non-natural immunoglobulins with desired properties. For this, two main directions are usually used: the biosynthesis of full-length antibodies and the production of minimal fragments of the antibody molecule, which are necessary for effective and specific binding to the antigen.
Modern technologies for producing antibodies in vitro copy the selection strategies of the immune system. One of these technologies is phage display, which makes it possible to obtain fragments of human antibodies of different specificities. The genes from these fragments can be used to construct full-length antibodies.
In addition, very often therapeutic drugs created on the basis of antibodies do not require the involvement of their effector functions through the Fc domain, for example, in the inactivation of cytokines, blocking receptors or neutralizing viruses. Therefore, one of the trends in the design of recombinant antibodies is to reduce their size to a minimal fragment that retains both binding activity and specificity.
Such fragments in some cases may be more preferable due to their ability to penetrate tissue better and be eliminated from the body more quickly than full-length antibody molecules. At the same time, the desired fragment can be produced in E. coli or yeast, which significantly reduces its cost compared to antibodies obtained using mammalian cell cultures. In addition, this method of development allows one to avoid the biological hazard associated with the use of antibodies isolated from donor blood.
Myeloma immunoglobulins
Bence Jones protein. An example of a molecule of such an immunoglobulin, which is a dimer of kappa light chains
The term immunoglobulins refers not only to normal classes of antibodies, but also to a large number of abnormal proteins, commonly called myeloma proteins. These proteins are synthesized in large quantities in multiple myeloma, a malignant disease in which degenerated specific cells of the antibody-forming system produce large quantities of certain proteins, for example Bence-Jones proteins, myeloma globulins, fragments of immunoglobulins of various classes.
Bence Jones proteins are either single κ or λ chains or dimers of two identical chains linked by a single disulfide bond; they are excreted in the urine.
Myeloma globulins are found in high concentrations in the plasma of patients with multiple myeloma; their H and L chains have a unique sequence. At one time it was assumed that myeloma globulins are pathological immunoglobulins characteristic of the tumor in which they are formed, but now it is believed that each of them is one of the individual immunoglobulins, randomly “selected” from the many thousands of normal antibodies formed in the human body.
The complete amino acid sequence of several individual immunoglobulins has been determined, including myeloma globulins, Bence Jones proteins, and the light and heavy chains of the same myeloma immunoglobulin. Unlike the antibodies of a healthy person, all protein molecules of each named group have the same amino acid sequence and are one of many thousands of possible antibodies in an individual.
Hybridomas and monoclonal antibodies
Obtaining antibodies for human needs begins with immunizing animals. After several injections of the antigen (in the presence of immune response stimulants), specific antibodies accumulate in the blood serum of animals. Such sera are called immune sera. Of them special methods release antibodies.
However, the animal’s immune system produces special antibodies to a huge variety of antigens. This ability is based on the presence of a diversity of lymphocyte clones, each of which produces antibodies of the same type with narrow specificity. The total number of clones in mice, for example, reaches 10^7 –10^10 degree.
Therefore, immune sera contain many antibody molecules with different specificities, i.e., having affinity for many antigenic determinants. Antibodies obtained from immune sera are directed both against the antigen that was immunized and against other antigens that the donor animal encountered.
For modern immunochemical analysis and clinical use, the specificity and standardization of the antibodies used are very important. It is necessary to obtain absolutely identical antibodies, which cannot be done using immune sera.
In 1975, J. Köhler and S. Milstein solved this problem by proposing a method for producing homogeneous antibodies. They developed the so-called “hybridoma technology” - a technique for producing cell hybrids (hybridoma). Using this method, hybrid cells are obtained that can multiply indefinitely and synthesize antibodies of narrow specificity - monoclonal antibodies.
To obtain monoclonal antibodies, plasmacytic tumor cells (plasmocytoma or multiple myeloma) are fused with the spleen cells of an immunized animal, most often a mouse. Köhler and Milstein's technology includes several stages.
Mice are injected with a specific antigen, which causes the production of antibodies against that antigen. Mouse spleens are removed and homogenized to obtain a cell suspension. This suspension contains B cells that produce antibodies against the administered antigen.
The spleen cells are then mixed with myeloma cells. These are tumor cells that are capable of continuously growing in culture; they also lack a reserve pathway for nucleotide synthesis. Some antibody-producing spleen cells and myeloma cells fuse to form hybrid cells. These hybrid cells are now able to grow continuously in culture and produce antibodies.
The mixture of cells is placed in a selective medium that allows only hybrid cells to grow. Unfused myeloma cells and B-lymphocytes die.
Hybrid cells proliferate, forming a hybridoma clone. Hybridomas are tested for production of the desired antibodies. Selected hybridomas are then cultured to produce large quantities of monoclonal antibodies that are free of extraneous antibodies and so homogeneous that they can be treated as pure chemical reagents.
It should be noted that antibodies produced by one hybridoma culture bind only to one antigenic determinant (epitope). In this regard, it is possible to obtain as many monoclonal antibodies to an antigen with several epitopes as it has antigenic determinants. It is also possible to select clones that produce antibodies of only one desired specificity.
The development of technology for producing hybridomas was of revolutionary importance in immunology, molecular biology and medicine. It made it possible to create completely new scientific directions. Thanks to hybridomas, new ways have opened up for the study and treatment of malignant tumors and many other diseases.
Currently, hybridomas have become the main source of monoclonal antibodies used in basic research and in biotechnology to create test systems. Monoclonal antibodies are widely used in the diagnosis of infectious diseases of farm animals and humans.
Thanks to monoclonal antibodies, enzyme immunoassays, immunofluorescence reactions, flow cytometry methods, immunochromatography, and radioimmunoassays have become routine.
Many technologies have been developed to improve the synthesis of antibodies. These are DNA recombination technologies, cell cloning methods and other transgenic technologies. In the 90s, using genetic engineering methods, it was possible to minimize the percentage of mouse amino acid sequences in artificially synthesized antibodies. Thanks to this, in addition to mouse ones, chimeric, humanized and fully human antibodies were obtained.
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