Antibodies and antitoxins perform the following function. Antitoxins. Biological properties of antibodies

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BELARUSIAN STATE UNIVERSITY

Faculty of Biology

ANTIBODIES, CLASSIFICATION AND FUNCTIONS

Abstract

4th year student, 6th group

KOVALCHUK K.V.

Minsk 2004

Discovery of antibodies

Antibody structure

Antibody classification

Antibody functions

Literature

Discovery of antibodies

The term "antibody" was coined at the end of the 19th century. In 1890, Behring and Kitasato conducted experiments in which they studied the effects of diphtheria and tetanus toxins on guinea pigs. They injected the animals with a sublethal dose of the toxin, after some time they took the serum from them and injected it along with the lethal dose of the toxin into other animals, as a result of which the animals did not die. It was concluded that after immunization with a toxin, a substance appears in the blood of animals that can neutralize it and thereby prevent the disease. This substance was called antitoxin, and then a more general term was introduced - antibody; Substances that cause the formation of antibodies are called antigens.

It was not until 1939 that Tiselius and Kabat showed that antibodies were contained in a specific fraction of serum proteins. They immunized the animal with ovalbumin and took two samples from the resulting serum; ovalbumin was added to one of them and the resulting precipitate (antibody-ovalbumin complex) was removed. Electrophoresis revealed that in the sample to which ovalbumin was added, the content of g-globulins was significantly lower than in the other sample. This indicated that the antibodies were g-globulins. To distinguish them from other proteins contained in this fraction of globulins, antibodies were called immunoglobulins. It is now known that antibodies are also found in significant quantities in the β- and β-globulin fractions.

The structure of antibodies was established through a variety of 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, the model of the antibody molecule described below was constructed.

Antibody structure

The antibody molecule consists of four polypeptide chains (Fig. 1): two heavy (H; molecular weight 50-70 kDa) and two light (L; molecular weight 23 kDa). The chains are connected by non-covalent bonds (hydrogen, hydrophobic bonds) and disulfide bridges and consist of two (light chain) or four (heavy chain) domains with a length of about 110 amino acid residues. The VH and VL variable domains, which are the N-terminal portions of the chains, form the antigen-binding site. In addition to them, light chains contain one (CL), and heavy chains three or four (CH1-4) constant domains.

When antibodies are enzymatically digested by the proteolytic enzyme papain, three fragments are formed: two identical antigen-binding fragments (Fab) and one crystallizable fragment (Fc). The Fab fragment consists of an intact L chain linked by a disulfide bond to the CH1 and VH domains; its N-terminal part (Fv fragment) has antigen-binding activity. The Fc fragment consists of two pairs of CH2 and CH3 domains connected by a disulfide bond. This fragment is not involved in the binding of antigens, but performs effector functions - reacting with cells and complement factors.

The ability of an antibody to bind to a particular antigen is determined by the amino acid composition of the variable domains, or rather their hypervariable regions. These regions are characterized by very high amino acid sequence variability. Each VH and VL domain contains three hypervariable regions, which actually form antigen-binding sites. The sequences between them are called frame sequences; they are characterized by lower structural variability.

Rice. 1. The structure of the antibody molecule. H and L, heavy and light chains; CDRs, hypervariable regions.

The amino acid sequence of the constant region varies slightly. Light chain sequencing revealed the existence of two main variants of the amino acid sequences of the CL domains, which led to the identification of two types of light chains - kappa (k) and lambda (l). An antibody molecule can simultaneously contain either two k-chains or two l-chains (k-chains are more common in human antibodies).

Also, the determination of amino acid sequences made it possible to identify five types of CH regions and, accordingly, heavy chains (b, e, f, d, l). Chains m and e contain four constant domains, the remaining chains contain three constant domains, as well as a hinge region between the CH1 and CH2 domains. Depending on what type of heavy chain the antibody contains, five classes of immunoglobulins are distinguished: IgA (heavy chain type b), IgD (e), IgE (e), IgG (g), IgM (m). Due to some differences in amino acid sequences, several types of l-chains are distinguished, as well as several types of b- and g-chains (and, accordingly, several subclasses of IgG and IgA). Associated with the heavy chains (primarily the CH2 domains) are several oligosaccharide chains that likely increase antibody solubility and are involved in binding to complement components and cellular receptors.

In domains, polypeptide chains are stacked to form B-folded layers in which antiparallel chains are connected by loops (Fig. 2). These loops can have different lengths and amino acid sequences, which is very important because they form the antigen-binding site. Within each domain, two β-sheets are connected by a disulfide bond and stabilized by hydrophobic interactions. The Y-shaped quaternary structure (Fig. 3) is formed due to non-covalent interactions between domains. Between the CH2 domains are carbohydrate molecules, which causes these domains to protrude and make them more accessible to interact with a variety of molecules, such as components of the complement system.

Fig.2. Two-dimensional diagram of the folding of a polypeptide chain within the VL domain: two β-pleated layers connected by a disulfide bond (black stripe).

Fig.3. Diagram showing the interaction between the light and heavy chain domains. Between the CH2 domains are carbohydrate molecules. Hypervariable regions (CDRs) are shown.

Antibody classification

As mentioned above, depending on the type of heavy chain, five classes of immunoglobulins are distinguished.

IgG make up the majority of serum antibodies. Most antibodies of the secondary immune response and antitoxins are represented by class G immunoglobulins. Maternal IgG provides passive immunity to the child in the first few months of life, entering the fetal blood through the placenta. IgG activates the complement system and binds to cell surface antigens, thereby making these cells more accessible to phagocytosis (opsonization). Capable of binding to tissues causing anaphylactic reactions.

IgM molecules consist of five identical four-chain subunits connected by disulfide bonds. They also contain an additional polypeptide chain (J-chain), which forms an immunoglobulin-type domain and is linked by disulfide bonds to the C-terminal peptides (18 amino acid residues) of the heavy chains of individual monomers. Presumably, it is involved in the polymerization of monomers. Class M immunoglobulins are found predominantly in the blood. They dominate as “early” antibodies (the first to appear during the development of an immune response). Due to the multiple binding sites, they cause cell agglutination. More effective than IgG in activating complement.

IgA predominate among the antibodies of serous-mucosal secretions (saliva, colostrum, milk, respiratory tract secretions), where they are represented mainly in the dimeric form. Like IgM, they contain a C-terminal peptide to which a J chain can attach, linking two monomers into a dimer. A protein called the secretory component additionally binds to this complex, which promotes the delivery of antibodies to secretions and protects them from proteolysis. In human serum they are presented mainly in the monomeric form, and in the serum of other mammals they are mainly in the dimeric form. Prevents the penetration of viruses and microorganisms through mucous membranes.

IgD And IgE present in serum in very low concentrations. IgDs are often found on the cytoplasmic membranes of B cells and are thought to be involved in antigen-dependent lymphocyte differentiation. IgE is found on the membranes of basophils and mast cells. They participate in allergic reactions, causing the IgE carrier cell to secrete histamine and other vasoactive substances in response to the binding of the IgE molecule to the antigen. Perhaps they play a significant role in anthelmintic immunity.

Antibody functions

Antibodies are synthesized by B lymphocytes and plasma cells formed from them. Their molecules are embedded in the cytoplasmic membrane of B lymphocytes, where they function as antigen-specific receptors. Most human blood B lymphocytes express two classes of immunoglobulins on their surface - IgM and IgD. But in certain areas of the body, B cells carrying other classes of antibodies (for example, IgA in the intestinal mucosa) may occur at high frequencies. Plasma cells secrete antibodies into the blood plasma and tissue fluid. All antibodies produced by a single B cell (or plasma cell) have an identical antigen-binding site and can bind to only one antigen.

The primary function of antibodies is binding to foreign (normally) antigens with their subsequent inactivation. Antibodies are capable of inactivating toxins by binding to areas of the toxin molecule responsible either for adsorption on cellular receptors or directly for the toxic effect. Similarly, the binding of antibodies to proteins necessary for the adsorption of the virus onto cell receptors leads to inactivation of virions.

In addition, antibodies are capable of involving other elements of the immune system in the immune response: the complement system and host cells. The complement component C1q is capable of binding to the constant domains of the heavy chain of antibodies of classes G and M (with the CH2 and CH3 domains, respectively). This causes a cascade of reactions (the process of complement activation along the classical pathway), ultimately leading to the lysis of the cell to whose antigens the antibodies were bound. Some body cells carry Fc receptors on their surface, to which antibody molecules can bind via the Fc fragment. These receptors are present in macrophages, which allows them to recognize antigen-antibody complexes with their subsequent phagocytosis (antibodies are opsonins, i.e. molecules that, when bound to antigens, facilitate their phagocytosis). The Fc fragment is also responsible for the fixation of antibodies on cells of certain tissues and the development of anaphyloxic reactions.

Antibodies initially exist for any antigen in an animal’s body. This suggests that each organism produces millions of different immunoglobulins, differing in their antigen binding sites. This diversity is ensured by several mechanisms. The light and heavy chains of antibody molecules are encoded by several types of gene segments: the light chain - by three types of segments (V, J, C), the heavy chain - by four (V, D, J, C). The genome usually contains from several to several hundred segments of each type, slightly different in nucleotide sequence. To synthesize a whole polypeptide (light or heavy chain), it is necessary to combine the nucleotide sequences of segments of each type. This association occurs first at the DNA level (somatic recombination) and then at the messenger RNA level (splicing). As a result, a huge number of variants of mRNA and, accordingly, polypeptide chains are formed. During somatic recombination and splicing, insertions and deletions of nucleotides can occur, which, together with an increased frequency of mutations in antibody genes, further increases the diversity of these unique proteins.

Literature

1. Immunology / Royt A., Brostoff J., Mail D.-M.: Mir, 2000.-592 p.

2. Immunology: In 3 volumes; v.1 / Ed. U. Pola.-M.: Mir, 1987-88.-476 p.

History of the study

The very first antibody was discovered by Behring and Kitazato in 1890, but at that time nothing definite could be said about the nature of the discovered tetanus antitoxin, other than its specificity and its presence in the serum of an immune animal. Only in 1937, with the research of Tiselius and Kabat, did the study of the molecular nature of antibodies begin. The authors used the method of protein electrophoresis and demonstrated an increase in the gamma globulin fraction of the blood serum of immunized animals. Adsorption of serum by the antigen that was taken for immunization reduced the amount of protein in this fraction to the level of intact animals.

Antibody structure

Antibodies are relatively large (~150 kDa - IgG) glycoproteins with a complex structure. Consist of two identical heavy chains (H-chains, in turn consisting of V H, C H 1, hinge, CH 2- and C H 3-domains) and two identical light chains (L-chains, consisting of V L - and C L - domains). Oligosaccharides are covalently attached to the heavy chains. Using papain protease, antibodies can be cleaved into two Fabs. fragment antigen binding- antigen-binding fragment) and one (eng. fragment crystallizable- fragment capable of crystallization). Depending on the class and functions performed, antibodies can exist both in monomeric form (IgG, IgD, IgE, serum IgA) and in oligomeric form (dimer-secretory IgA, pentamer - IgM). In total, there are five types of heavy chains (α-, γ-, δ-, ε- and μ-chains) and two types of light chains (κ-chain and λ-chain).

Heavy chain classification

There are five classes ( isotypes) immunoglobulins, differing:

amino acid sequence
molecular weight
charge

The IgG class is classified into four subclasses (IgG1, IgG2, IgG3, IgG4), the IgA class into two subclasses (IgA1, IgA2). All classes and subclasses make up nine isotypes that are normally present in all individuals. Each isotype is determined by the amino acid sequence of the heavy chain constant region.

Antibody functions

Immunoglobulins of all isotypes are bifunctional. This means that immunoglobulin of any type

recognizes and binds antigen, and then
enhances the destruction and/or removal of immune complexes formed as a result of activation of effector mechanisms.

One region of the antibody molecule (Fab) determines its antigen specificity, and the other (Fc) performs effector functions: binding to receptors that are expressed on body cells (for example, phagocytes); binding to the first component (C1q) of the complement system to initiate the classical pathway of the complement cascade.

This means that each lymphocyte synthesizes antibodies of only one specific specificity. And these antibodies are located on the surface of this lymphocyte as receptors.

As experiments show, all cell surface immunoglobulins have the same idiotype: when a soluble antigen, similar to polymerized flagellin, binds to a specific cell, then all cell surface immunoglobulins bind to this antigen and they have the same specificity, that is, the same idiotype.

The antigen binds to receptors, then selectively activates the cell to produce large amounts of antibodies. And since the cell synthesizes antibodies of only one specificity, this specificity must coincide with the specificity of the initial surface receptor.

The specificity of the interaction of antibodies with antigens is not absolute; they can cross-react with other antigens to varying degrees. An antiserum raised against one antigen can react with a related antigen that carries one or more of the same or similar determinants. Therefore, each antibody can react not only with the antigen that caused its formation, but also with other, sometimes completely unrelated molecules. The specificity of antibodies is determined by the amino acid sequence of their variable regions.

Clonal selection theory:

Antibodies and lymphocytes with the required specificity already exist in the body before the first contact with the antigen.
Lymphocytes that participate in the immune response have antigen-specific receptors on the surface of their membrane. B lymphocytes have receptor molecules of the same specificity as the antibodies that the lymphocytes subsequently produce and secrete.
Any lymphocyte carries receptors of only one specificity on its surface.
Lymphocytes that have the antigen undergo a proliferation stage and form a large clone of plasma cells. Plasma cells synthesize antibodies only of the specificity for which the precursor lymphocyte was programmed. Signals for proliferation are cytokines, which are released by other cells. Lymphocytes can themselves secrete cytokines.

Antibody variability

Antibodies are extremely variable (up to 10 8 antibody variants can exist in the body of one person). All the diversity of antibodies stems from the variability of both heavy chains and light chains. Antibodies produced by one or another organism in response to certain antigens are distinguished:

Isotypic variability - manifested in the presence of classes of antibodies (isotypes), differing in the structure of heavy chains and oligomerity, produced by all organisms of a given species;
Allotypic variability - manifests itself at the individual level within a given species in the form of variability of immunoglobulin alleles - is a genetically determined difference between a given organism and another;
Idiotypic variability - manifests itself in differences in the amino acid composition of the antigen-binding site. This applies to the variable and hypervariable domains of the heavy and light chains that are in direct contact with the antigen.

Control of proliferation

The most effective control mechanism is that the reaction product simultaneously serves as its inhibitor. This type of negative feedback occurs during the formation of antibodies. The effect of antibodies cannot be explained simply by neutralization of the antigen, because whole IgG molecules suppress antibody synthesis much more effectively than F(ab")2 fragments. It is assumed that the blockade of the productive phase of the T-dependent B-cell response occurs as a result of the formation of cross-links between the antigen , IgG and Fc receptors on the surface of B cells. Injection of IgM enhances the immune response. Since antibodies of this particular isotype appear first after the introduction of an antigen, they are credited with an enhancing role at the early stage of the immune response.

There was no engagement and Bolkonsky’s engagement to Natasha was not announced to anyone; Prince Andrei insisted on this. He said that since he was the cause of the delay, he must bear the entire burden of it. He said that he was forever bound by his word, but that he did not want to bind Natasha and gave her complete freedom. If after six months she feels that she does not love him, she will be within her right if she refuses him. It goes without saying that neither the parents nor Natasha wanted to hear about it; but Prince Andrei insisted on his own. Prince Andrei visited the Rostovs every day, but did not treat Natasha like a groom: he told her you and only kissed her hand. After the day of the proposal, a completely different, close, simple relationship was established between Prince Andrei and Natasha. It was as if they didn't know each other until now. Both he and she loved to remember how they looked at each other when they were still nothing; now both of them felt like completely different creatures: then feigned, now simple and sincere. At first, the family felt awkward in dealing with Prince Andrei; he seemed like a man from an alien world, and Natasha spent a long time accustoming her family to Prince Andrei and proudly assured everyone that he only seemed so special, and that he was the same as everyone else, and that she was not afraid of him and that no one should be afraid his. After several days, the family got used to him and without hesitation led with him the same way of life in which he took part. He knew how to talk about the household with the Count, and about outfits with the Countess and Natasha, and about albums and canvas with Sonya. Sometimes the Rostov family, among themselves and under Prince Andrei, were surprised at how all this happened and how obvious the omens of this were: the arrival of Prince Andrei in Otradnoye, and their arrival in St. Petersburg, and the similarity between Natasha and Prince Andrei, which the nanny noticed on their first visit Prince Andrei, and the clash in 1805 between Andrei and Nikolai, and many other omens of what happened were noticed by those at home.
The house was filled with that poetic boredom and silence that always accompanies the presence of the bride and groom. Often sitting together, everyone was silent. Sometimes they got up and left, and the bride and groom, remaining alone, were still silent. Rarely did they talk about their future lives. Prince Andrei was scared and ashamed to talk about it. Natasha shared this feeling, like all his feelings, which she constantly guessed. One time Natasha started asking about his son. Prince Andrei blushed, which often happened to him now and which Natasha especially loved, and said that his son would not live with them.
- Why? – Natasha said in fear.
- I can’t take him away from my grandfather and then...
- How I would love him! - Natasha said, immediately guessing his thought; but I know you want there to be no excuses to blame you and me.
The old count sometimes approached Prince Andrei, kissed him, and asked him for advice on the upbringing of Petya or the service of Nicholas. The old countess sighed as she looked at them. Sonya was afraid at every moment of being superfluous and tried to find excuses to leave them alone when they didn’t need it. When Prince Andrei spoke (he spoke very well), Natasha listened to him with pride; when she spoke, she noticed with fear and joy that he was looking at her carefully and searchingly. She asked herself in bewilderment: “What is he looking for in me? He's trying to achieve something with his gaze! What if I don’t have what he’s looking for with that look?” Sometimes she entered into her characteristic insanely cheerful mood, and then she especially loved to listen and watch how Prince Andrei laughed. He rarely laughed, but when he laughed, he gave himself entirely to his laughter, and every time after this laugh she felt closer to him. Natasha would have been completely happy if the thought of the upcoming and approaching separation did not frighten her, since he too turned pale and cold at the mere thought of it.
On the eve of his departure from St. Petersburg, Prince Andrei brought with him Pierre, who had never been to the Rostovs since the ball. Pierre seemed confused and embarrassed. He was talking to his mother. Natasha sat down with Sonya at the chess table, thereby inviting Prince Andrey to her. He approached them.
– You’ve known Bezukhoy for a long time, haven’t you? – he asked. - Do you love him?
- Yes, he is nice, but very funny.
And she, as always speaking about Pierre, began to tell jokes about his absent-mindedness, jokes that people even made up about him.
“You know, I trusted him with our secret,” said Prince Andrei. – I have known him since childhood. This is a heart of gold. “I beg you, Natalie,” he said suddenly seriously; – I’ll leave, God knows what might happen. You might spill... Well, I know I shouldn't talk about it. One thing - no matter what happens to you when I’m gone...
- What will happen?...
“Whatever the grief,” continued Prince Andrei, “I ask you, m lle Sophie, no matter what happens, turn to him alone for advice and help.” This is the most absent-minded and funny person, but the most golden heart.
Neither father and mother, nor Sonya, nor Prince Andrei himself could foresee how parting with her fiancé would affect Natasha. Red and excited, with dry eyes, she walked around the house that day, doing the most insignificant things, as if not understanding what awaited her. She did not cry even at that moment when, saying goodbye, he kissed her hand for the last time. - Don't leave! - she just said to him in a voice that made him think about whether he really needed to stay and which he remembered for a long time after that. When he left, she didn't cry either; but for several days she sat in her room without crying, was not interested in anything and only sometimes said: “Oh, why did he leave!”

Antibodies: these are proteins produced by cells of lymphoid organs (B lymphocytes) under the influence of an antigen and capable of entering into a specific relationship with them. In this case, antibodies can neutralize the toxins of bacteria and viruses; they are called antitoxins and virus-neutralizing antibodies.

They can precipitate soluble antigens - precipitins, and glue corpuscular antigens - agglutinins.

Nature of antibodies: antibodies belong to gammaglobulins. In the body, gammaglobulins are produced by plasma cells and make up 30% of all proteins in the blood serum.

Gammaglobulins that carry the function of antibodies are called immunoglobulins and are designated Ig. Ig proteins are chemically classified as glycoproteins, that is, they consist of proteins, sugars, and 17 amino acids.

Ig molecule:

Under electron microscopy, the Ig molecule is shaped like a game with a varying angle.

The structural unit of Ig is a monomer.

The monomer consists of 4 polypeptide chains linked to each other by disulfide bonds. Of the 4 chains, two chains are long and curved in the middle. Molecular weight from 50-70 kDa are the so-called heavy H chains, and two short chains are adjacent to the upper sections of the H chains, molecular weight 24 kDa are light L chains.

Variable light and heavy chains together form a site that specifically binds to the antigen - the antigen-binding center Fab fragment, Fc fragment responsible for complement activation.

Fab (English fragment antigen binding - antigen-binding fragment) and one Fc (English fragment crystallizable - fragment capable of crystallization).

Immunoglobulin classes:

Ig M - makes up 5-10% of serum immunoglobulins. It is the largest molecule of all five classes of immunoglobulins. Molecular weight 900 thousand kDa. The first to appear in the blood serum when the antigen is introduced. The presence of Ig M indicates an acute process. Ig M agglutinates and lyses antigen, and also activates complement. Attached to the bloodstream.

Ig G - makes up 70-80% of serum immunoglobulins. Molecular weight 160 thousand kDa. It is synthesized during the secondary immune response, is able to overcome the placental barrier and provide immune protection to newborns for the first 3-4 months, then is destroyed. At the beginning of the disease, the amount of Ig G is insignificant, but as the disease progresses, their amount increases. It plays a major role in protecting against infections. High titers of Ig G indicate that the body is at the stage of recovery or has recently suffered an infection. Found in blood serum and distributed through the intestinal mucosa into tissue fluid.

Ig A - ranges from 10-15%, molecular weight 160 thousand kDa. Plays an important role in protecting the mucous membranes of the respiratory and digestive tracts and the genitourinary system. There are serum and secretory Ig A. Serum neutralizes microorganisms and their toxins, does not bind complement and does not pass through the placental barrier.

Secretory Ig A activate complement and stimulate phagocytic activity in the mucous membranes, found mainly in secretions of the mucous membranes, saliva, tear fluid, sweat, nasal discharge, where it provides protection of surfaces communicating with the external environment from microorganisms. Synthesized by plasma cells. In human serum, it is presented in a monomeric form. Provides local immunity.

Ig E - its amount in serum is small and only a small part of plasma cells synthesize Ig E. They are formed in response to allergens and interacting with them cause an HNT reaction. Synthesized by B lymphocytes and plasma cells. Does not pass through the placental barrier.

Ig D - its participation has not been sufficiently studied. Almost all of it is located on the surface of lymphocytes. Produced by cells of the tonsils and adenoids. IgD does not bind complement and does not cross the placental barrier. Ig D and Ig A are interconnected and activate lymphocytes. The concentration of Ig D increases during pregnancy, with bronchial asthma, and with systemic lupus erythematosus.

Normal antibodies (natural)

The body contains a certain level of them, they are formed without the phenomena of antigenic stimulation. These include antibodies against erythrocyte antigens, blood groups, and against intestinal groups of bacteria.

The process of antibody production, their accumulation and disappearance have certain characteristics that are different in the primary immune response (this is the response to the initial encounter with the antigen) and the secondary immune response (this is the response to repeated contact with the same antigen after 2-4 weeks).

The synthesis of antibodies in any immune response occurs in several stages - these are the latent stage, the logarithmic stage, the stationary stage and the antibody decline phase.

Primary immune response:

Latent phase: during this period, the process of recognition of the antigen and the formation of cells that are capable of synthesizing antibodies to it occur. The duration of this period is 3-5 days.

Logarithmic phase: The rate of antibody synthesis is low. (duration 15-20 days).

Stationary phase: titers of synthesized antibodies reach maximum values. Antibodies belonging to class M immunoglobulins are synthesized first, then G. Later, Ig A and Ig E may appear.

Declining phase: Antibody levels decrease. Duration from 1-6 months.

Secondary immune response.

Antibodies (immunoglobulins, IG, Ig) are a special class of glycoproteins present on the surface of B cells in the form of membrane-bound receptors and in blood serum and tissue fluid in the form of soluble molecules. They are the most important factor in specific humoral immunity. Antibodies are used by the immune system to identify and neutralize foreign objects - such as bacteria and viruses. Antibodies perform two functions: antigen-binding and effector (cause one or another immune response, for example, trigger the classical complement activation scheme).

Antibodies are synthesized by plasma cells, which become B lymphocytes in response to the presence of antigens. For each antigen, specialized plasma cells corresponding to it are formed, producing antibodies specific to this antigen. Antibodies recognize antigens by binding to a specific epitope - a characteristic fragment of the surface or linear amino acid chain of the antigen.

Antibodies consist of two light chains and two heavy chains. In mammals, there are five classes of antibodies (immunoglobulins) - IgG, IgA, IgM, IgD, IgE, which differ in the structure and amino acid composition of the heavy chains and in the effector functions performed.

History of the study

The very first antibody was discovered by Behring and Kitazato in 1890, however, at this time about the nature of what was discovered tetanus antitoxin, besides its specificity and its presence in serum immune animal, nothing definite could be said. Only with 1937- research by Tiselius and Kabat, the study of the molecular nature of antibodies begins. The authors used the method electrophoresis proteins and demonstrated an increase in the gamma globulin fraction of the blood serum of immunized animals. Adsorption serum antigen, which was taken for immunization, reduced the amount of protein in this fraction to the level of intact animals.

Antibody structure

General plan of the structure of immunoglobulins: 1) Fab; 2) Fc; 3) heavy chain; 4) light chain; 5) antigen-binding site; 6) hinge section

Antibodies are relatively large (~150 k Yes- IgG) glycoproteins, having a complex structure. Consist of two identical heavy chains(H-chains, in turn consisting of V H, C H1, hinge, C H2 and C H3 domains) and two identical light chains(L-chains consisting of V L and C L domains). Oligosaccharides are covalently attached to the heavy chains. Using protease papaina antibodies can be split into two Fab (English fragment antigen binding- antigen-binding fragment) and one Fc (English fragment crystallizable- fragment capable of crystallization). Depending on the class and functions performed, antibodies can exist in both monomeric form (IgG, IgD, IgE, serum IgA) and in oligomeric form (dimer-secretory IgA, pentamer - IgM). In total, there are five types of heavy chains (α-, γ-, δ-, ε- and μ-chains) and two types of light chains (κ-chain and λ-chain).

Heavy chain classification

There are five classes ( isotypes) immunoglobulins, differing:

size

amino acid sequence

Antibody functions

Immunoglobulins of all isotypes are bifunctional. This means that immunoglobulin of any type

recognizes and binds antigen, and then

enhances killing and/or removal of immune complexes formed as a result of activation of effector mechanisms.

IgG is the main immunoglobulin serum healthy person (accounts for 70-75% of the total fraction of immunoglobulins), most active in secondary immune response and antitoxic immunity. Thanks to its small size ( sedimentation coefficient 7S, molecular weight 146 kDa) is the only fraction of immunoglobulins capable of transport across the placental barrier and thereby providing immunity to the fetus and newborn. Contains IgG 2-3% carbohydrates; two antigen-binding F ab fragments and one F C fragment. F ab fragment (50-52 kDa) consists of the whole L-chain and the N-terminal half of the H-chain, connected to each other disulfide bond, while the F C fragment (48 kDa) is formed by the C-terminal halves of the H chains. There are a total of 12 domains in the IgG molecule (regions formed from β-structures And α-helices Ig polypeptide chains in the form of disordered formations interconnected by disulfide bridges of amino acid residues within each chain): 4 on heavy and 2 on light chains.

IgM are a pentamer of a four-chain basic unit containing two μ chains. In this case, each pentamer contains one copy of a polypeptide with a J-chain (20 kDa), which is synthesized by an antibody-producing cell and covalently binds between two adjacent F C fragments of immunoglobulin. They appear during the primary immune response of B-lymphocytes to an unknown antigen and constitute up to 10% of the immunoglobulin fraction. They are the largest immunoglobulins (970 kDa). Contains 10-12% carbohydrates. The formation of IgM also occurs in pre-B-lymphocytes, in which they are primarily synthesized from the μ-chain; the synthesis of light chains in pre-B cells ensures their binding to μ-chains, resulting in the formation of functionally active IgM, which are integrated into the surface structures of the plasma membrane, acting as an antigen recognition receptor; from this point on, pre-B lymphocyte cells become mature and are able to participate in the immune response.

IgA Serum IgA makes up 15-20% of the total immunoglobulin fraction, with 80% of IgA molecules present in monomeric form in humans. Secretory IgA is presented in dimeric form in a complex secretory component, contained in serous-mucosal secretions (for example, in saliva, tears, colostrum, milk, separated from the mucous membrane of the genitourinary and respiratory systems). Contains 10-12% carbohydrates, molecular weight 500 kDa.

IgD makes up less than one percent of the plasma immunoglobulin fraction and is found mainly on the membrane of some B lymphocytes. Functions not fully understood, presumably an antigen receptor with a high content of protein-bound carbohydrates for B lymphocytes, not yet presented to the antigen. Molecular weight 175 kDa.

Classification by antigens

so-called “antibodies that are evidence of disease”, the presence of which in the body signals the familiarity of the immune system with this pathogen in the past or current infection with this pathogen, but which do not play a significant role in the body’s fight against the pathogen (they do not neutralize either the pathogen itself or its toxins, but bind to minor proteins of the pathogen ).

auto-aggressive antibodies, or autologous antibodies, autoantibodies- antibodies that cause destruction or damage to normal, healthy tissues of the body host and triggering the development mechanism autoimmune diseases.

alloreactive antibodies, or homologous antibodies, alloantibodies- antibodies against antigens of tissues or cells of other organisms of the same biological species. Alloantibodies play an important role in the processes of allograft rejection, for example, during transplantation kidneys, liver, bone marrow, and in reactions to transfusion of incompatible blood.

heterologous antibodies, or isoantibodies- antibodies against antigens of tissues or cells of organisms of other biological species. Isoantibodies are the reason for the impossibility of xenotransplantation even between evolutionarily close species (for example, a chimpanzee liver transplant to a human is impossible) or species that have similar immunological and antigenic characteristics (a pig's organ transplant to a human is impossible).

anti-idiotypic antibodies - antibodies against antibodies produced by the body itself. Moreover, these antibodies are not “in general” against the molecule of a given antibody, but specifically against the working, “recognizing” region of the antibody, the so-called idiotype. Anti-idiotypic antibodies play an important role in binding and neutralizing excess antibodies and in the immune regulation of antibody production. In addition, the anti-idiotypic “antibody against antibody” mirrors the spatial configuration of the original antigen against which the original antibody was developed. And thus the anti-idiotypic antibody serves as an immunological memory factor for the body, an analogue of the original antigen, which remains in the body even after the destruction of the original antigens. In turn, anti-idiotypic antibodies can be produced anti-anti-idiotypic antibodies, etc.

Antibody specificity

Means that everyone lymphocyte synthesizes antibodies of only one specific specificity. And these antibodies are located on the surface of this lymphocyte as receptors.

As experiments show, all surface immunoglobulins of cells have the same idiotype: when soluble antigen similar to polymerized flagellin binds to a specific cell, then all cell surface immunoglobulins bind to this antigen and they have the same specificity, that is, the same idiotype.

The antigen binds to receptors, then selectively activates the cell to produce large amounts of antibodies. And since cell synthesizes antibodies of only one specificity, then this specificity must match the specificity of the initial surface receptor.

The specificity of the interaction of antibodies with antigens is not absolute; they can cross-react with other antigens to varying degrees. Antiserum received for one antigen can react with a related antigen carrying one or more identical or similar determinant. Therefore, each antibody can react not only with the antigen that caused its formation, but also with other, sometimes completely unrelated molecules. The specificity of antibodies is determined by the amino acid sequence of their variable regions.

Clonal selection theory:

Antibodies and lymphocytes with the required specificity already exist in the body before the first contact with the antigen.

Lymphocytes that participate in the immune response have antigen-specific receptors on the surface of their membrane. U B lymphocytes receptors are molecules of the same specificity as antibodies that lymphocytes subsequently produce and secrete.

Any lymphocyte carries receptors of only one specificity on its surface.

Lymphocytes having antigen, are going through a stage proliferation and form a large clone of plasma cells. Plasma cells synthesize antibodies only of the specificity for which the precursor lymphocyte was programmed. Signals for proliferation are cytokines that are secreted by other cells. Lymphocytes can themselves secrete cytokines.

Antibody variability

Isotypic variability - manifested in the presence of classes of antibodies (isotypes), differing in the structure of heavy chains and oligomerity, produced by all organisms of a given species;

Allotypic variability - manifests itself at the individual level within a given species in the form of variability of immunoglobulin alleles - is a genetically determined difference between a given organism and another;

Idiotypic variability - manifests itself in differences in the amino acid composition of the antigen-binding site. This applies to the variable and hypervariable domains of the heavy and light chains that are in direct contact with the antigen.

Control of proliferation

The most effective control mechanism is that the product of the reaction simultaneously serves as its inhibitor. This type of negative feedback occurs during the formation of antibodies. The effect of antibodies cannot be explained simply by neutralization of the antigen, because whole IgG molecules suppress antibody synthesis much more effectively than F(ab")2 fragments. It is assumed that the blockade of the productive phase of the T-dependent B-cell response occurs as a result of the formation of cross-links between the antigen , IgG and Fc receptors on the surface of B cells. Injection IgM, enhances immune response. Since antibodies of this particular isotype appear first after the introduction of an antigen, they are assigned an enhancing role at the early stage of the immune response.

1. Opsonization (immune phagocytosis).

2. Antitoxic effect.

3. Activation of complement.

4. Neutralization.

5. Circulating complexes (bound soluble Ags form complexes with Abs, which are excreted from the body with bile and urine).

6. Antibody-dependent cytotoxicity.

Dynamics of antibody formation.

Serological reactions in laboratory diagnosis of infectious diseases.

In protecting the body from foreign antigens, a decisive role is played by immunological mechanisms carried out by antibodies and immunocompetent cells. The basis of immunological mechanisms is a specific reaction between antibodies or lymphocytes (formed under the influence of an antigen that has entered the body) and the antigen. The main function of antibodies is to bind the antigen and its further removal from the body.

However, such reactions between antibodies and antigens can also occur outside the body (in vitro) in the presence of an electrolyte and are possible only if there is complementarity (structural similarity, affinity) of the antigen and antibody.

Having specific antibodies against a certain antigen, one can recognize and identify it among other antigens, and in blood serum there are antibodies against a known antigen.

The antigen-antibody reaction in vitro is accompanied by the occurrence of a certain phenomenon - agglutination, precipitation, lysis.

Thus all serological reactions are used for two purposes:

detection of antibodies in the patient’s serum using standard diagnostic antigens ( for serological diagnosis of infectious diseases);

to identify unknown antigens using known standard sera containing antibodies of a certain specificity ( for serological identification of pathogens).

For example, if the patient’s serum reacts with a specific microbial antigen, it means that the patient’s serum contains antibodies against this microorganism.

Serological diagnosis– take a standard antigen (diagnosticum), which is inactivated or live bacteria, viruses or their antigens (components) in an isotonic solution.

Serological identification– use standard immune sera, which are obtained from immunized animals (a large number of antibodies appear in the blood of animals as a result of repeated immunization with the pathogen).

Agglutination.

Agglutination– a serological reaction between antibodies (agglutinins) and antigens (agglutinogens) located on the surface of a bacterial cell, resulting in the formation of an antigen-antibody complex (agglutinate).

Mechanism of agglutination– under the influence of electrolyte ions, the negative surface charge of the bacterial cell decreases and, therefore, they can come closer to such a distance that the bacteria stick together.

Macro- and microscopic view of agglutinate:

O-agglutination (somatic) – fine-grained, under microscopy – bacteria stick together at the poles of the cells, forming a network.

Vi-agglutination (capsular) – fine-grained; under microscopy, bacteria stick together over the entire surface of the cell.

H-agglutination (flagellar) - agglutinins interact with flagella immobilizing bacteria; microscopically - large cotton, gluing of bacterial cells in the area of flagella.

The agglutination reaction is used to determine antibodies in the blood serum of patients, for example, in brucellosis (Wright, Heddelson reaction), typhoid fever and paratyphoid fever (Vidal reaction) and other infectious diseases, as well as in determining the pathogen isolated from the patient. The same reaction is used to determine blood groups using monoclonal antibodies against red blood cell alloantigens.

Various options for the agglutination reaction are used: extensive, indicative, indirect, etc.

To determine the patient's antibodies, they put extensive agglutination reaction: a suspension of killed microbes (diagnosticum) is added to dilutions of the patient’s blood serum and after several hours of incubation at 37°C, the highest dilution (titer) of the serum at which agglutination occurred is noted, i.e. a precipitate formed.

The nature and speed of agglutination depend on the type of antigen and antibodies.

If it is necessary to determine the pathogen isolated from the patient, put indicative agglutination reaction, using diagnostic antibodies, i.e. carry out serotyping of the pathogen. An indicative reaction is carried out on a glass slide. A pure culture of the pathogen isolated from the patient is added to 1 drop of diagnostic immune serum at a dilution of 1:10 or 1:20. If a flocculent precipitate appears, then the reaction is carried out in test tubes with increasing dilutions of diagnostic serum; 2-3 drops of a pathogen suspension were added to each dose of serum. The reaction is considered positive if agglutination is observed in a dilution close to the titer of the diagnostic serum. In controls (serum diluted with isotonic sodium chloride solution, or a suspension of microbes in the same solution), there should be no precipitate in the form of flakes.

Different related bacteria can be agglutinated by the same diagnostic agglutinating serum, which makes their identification difficult. Therefore, they use adsorbed agglutinating sera, from which cross-reacting antibodies have been removed by adsorption to related bacteria. Such sera retain antibodies that are specific only to a given bacterium. The production of monoreceptor diagnostic agglutinating sera in this way was proposed by A. Castellani (1902). Indirect (passive) hemagglutination reaction(RNGA) is based on the use of erythrocytes (or latex) with antigens or antibodies adsorbed on their surface, the interaction of which with the corresponding antibodies or antigens of the patients' blood serum causes the erythrocytes to stick together and fall out to the bottom of the test tube or cell in the form of a scalloped sediment. RNGA is used to diagnose infectious diseases, determine gonadotropic hormone in urine when establishing pregnancy, to identify hypersensitivity to drugs and hormones, and in some other cases. Hemagglutination inhibition reaction(RTGA) is based on blockade, suppression of viruses by antibodies of immune serum, as a result of which viruses lose their ability to agglutinate red blood cells. RTGA is used to diagnose many viral diseases, the causative agents of which (influenza viruses, measles, rubella, tick-borne encephalitis, etc.) can agglutinate the red blood cells of various animals. Agglutination reaction for determining blood groups used to establish the ABO system using RA of erythrocytes, using antibodies to blood groups A (II), B (III). The control is serum that does not contain antibodies, i.e. AB(IV) blood groups, antigens contained in erythrocytes of groups A(II), B(III); negative control does not contain antigens, i.e. Group 0 (I) red blood cells are used. IN agglutination reactions to determine the Rh factor use anti-Rhesus serums (at least two different series). If there is a Rh antigen on the membrane of the erythrocytes under study, agglutination of these cells occurs. Standard Rh-positive and Rh-negative erythrocytes of all blood groups serve as control.

Agglutination reaction to determine anti-Rhesus antibodies(indirect Coombs reaction) is used in patients with intravascular hemolysis. Some of these patients have anti-Rhesus antibodies that are incomplete. They specifically interact with Rh-positive erythrocytes, but do not cause their agglutination. The presence of such incomplete antibodies is determined by the indirect Coombs test. To do this, antiglobulin serum (antibodies against human immunoglobulins) is added to the system of anti-Rh antibodies + Rh-positive erythrocytes, which causes agglutination of erythrocytes. Using the Coombs reaction, pathological conditions associated with intravascular lysis of erythrocytes of immune origin are diagnosed, for example, hemolytic disease of the newborn: erythrocytes of a Rh-positive fetus combine with incomplete antibodies to the Rh factor circulating in the blood, which have passed through the placenta from a Rh-negative mother.

Coagglutination reaction - type of RA: pathogen cells are determined using staphylococci pre-treated with immune diagnostic serum. Staphylococci containing protein A, having an affinity for immunoglobulins, nonspecifically adsorb antimicrobial antibodies, which then interact with active centers with the corresponding microbes isolated from patients. As a result of coagglutination, flakes are formed consisting of staphylococci, diagnostic serum antibodies and the detected microbe.