Antibodies

Antibody Proteins and Antigen Binding

A region at the tip of the antibody protein is very variable, allowing millions of antibodies with different antigen-binding sites to exist.

An antibody (Ab), also known as an immunoglobulin (Ig), is a large Y-shaped protein produced by B-cells that is used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, called an antigen. Each tip of the "Y" of an antibody contains a paratope (a structure analogous to a lock) that is specific for one particular epitope (similarly analogous to a key) on an antigen, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody can tag a microbe, or an infected cell, for attack by other parts of the immune system, or can neutralize its target directly; for example, by blocking a part of a microbe that is essential for its invasion and survival. The production of antibodies is the main function of the humoral immune system.

Antibody Functions

Antibody functions include the following:

• Combine with viruses/toxins to prevent them from invading cells
• Attach to flagella of bacterium, restricting their movement
• Multi-bind to many bacteria at once, causing them to accumulate and prevent movement around the body
• Burst bacteria cell walls
• Attach to bacteria, making it easier for phagocytes to ingest them

Antibody Structure

Antibodies are heavy (~150 kDa) globular plasma proteins. They have sugar chains added to some of their amino acid residues; in other words, they are glycoproteins. Antibodies are typically made of the same basic structural units, each with two large heavy chains and two small light chains.

Heavy and light chains, variable and constant regions of an antibody

There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals, which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter.

The general structure of all antibodies is very similar. The Ig monomer is a Y-shaped molecule that consists of four polypeptide chains: two identical heavy chains, and two identical light chains connected by disulphide bonds. Each chain is composed of structural domains called immunoglobulin domains. These domains contain about 70-110 amino acids and are classified into different categories according to their size and function; for example, variable or IgV, and constant or IgC. The constant region determines the class of an immunoglobulin. All chains have a characteristic immunoglobulin fold in which two beta sheets create a "sandwich" shape, held together by interactions between conserved cysteines and other charged amino acids.

However, a small region at the tip of the protein is extremely variable, allowing millions of antibodies with slightly different tip structures, or antigen binding sites, to exist. This region is known as the hypervariable region. Each of these variants can bind to a different antigen. This enormous diversity of antibodies allows the immune system to recognize an equally wide variety of antigens. The large and diverse population of antibodies is generated by random combinations of a set of gene segments that encode different or paratopes, followed by random mutations in this area of the antibody gene, which create further diversity. The paratope is shaped at the amino terminal end of the antibody monomer by the variable domains from the heavy and light chains. The variable domain is also referred to as the FV region, and is the most important region for binding to antigens. More specifically, variable loops of β-strands, three each on the light (VL) and heavy (VH) chains are responsible for binding to the antigen.

Antibodies can occur in two physical forms, a soluble form that is secreted from the cell, and a membrane-bound form that is attached to the surface of a B cell and is referred to as the B cell receptor (BCR). The BCR is only found on the surface of B cells and facilitates the activation of these cells and their subsequent differentiation into either antibody factories called plasma cells, or memory B cells that will survive in the body and remember that same antigen so the B cells can respond faster upon future exposure. In most cases, interaction of the B cell with a T helper cell is necessary to produce full activation of the B cell and, therefore, antibody generation following antigen binding.

Antibody Genes and Diversity

Complex genetic mechanisms evolved which allow vertebrate B cells to generate a diverse pool of antibodies from relatively few antibody genes.

Virtually all microbes can trigger an antibody response. Successful recognition and eradication of many different types of microbes requires diversity among antibodies (glycoproteins belonging to the immunoglobulin superfamily). It is the variety in their amino acid composition that allows them to interact with many different antigens. It has been estimated that humans generate about 10 billion different antibodies, each capable of binding a distinct epitope of an antigen. Although a huge repertoire of different antibodies is generated in a single individual, the number of genes available to make these proteins is limited by the size of the human genome. Several complex genetic mechanisms have evolved that allow vertebrate B cells to generate a diverse pool of antibodies from a relatively small number of antibody genes.

Antibody Structure

Antibodies are typically made of basic structural units—each with two large heavy chains and two small light chains. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals, which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter. Though the general structure of all antibodies is very similar, a small region at the tip of the protein is extremely variable, allowing millions of antibodies with slightly different antigen binding sites to exist. This region is known as the hypervariable region. Each of these variants can bind to a different antigen. This enormous diversity of antibodies allows the immune system to recognize an equally wide variety of antigens.

Antibodies obtain their diversity through two processes:

V(D)J Recombination

The first stage is called somatic, or V(D)J, which stands for variable, diverse, and joining regions recombination. Several sets of genes are located within each of the three regions. During cell maturation, the B cell will splice out the DNA of all but one of the genes from each region and combine the three remaining genes together to form one VDJ segment. This segment, along with a constant region gene, forms the basis for subsequent antibody production.

It is estimated that given the number of variants in each of the three regions, approximately 10,000-20,000 unique antibodies are producible. V(D)J recombination takes place in the primary lymphoid tissue (bone marrow for B cells, and thymus for T cells ) and nearly randomly combines variable, diverse, and joining gene segments. It is due to this randomness in choosing different genes that it is able to diversely encode proteins to match antigens.

Somatic Hypermutation

The second stage of recombination occurs after the B cell is activated by an antigen. In these rapidly dividing cells, the genes encoding the variable domains of the heavy and light chains undergo a high rate of point mutation, by a process called somatic hypermutation (SHM). SHM is a cellular mechanism by which the immune system adapts to the new foreign elements that confront it and is a major component of the process of affinity maturation. SHM diversifies B cell receptors used to recognize antigens and allows the immune system to adapt its response to new threats during the lifetime of an organism. Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. SHM results in approximately one nucleotide change per variable gene, per cell division. As a consequence, any daughter B cells will acquire slight amino acid differences in the variable domains of their antibody chains. This serves to increase the diversity of the antibody pool and impacts the antibody's antigen-binding affinity. Some point mutations will result in the production of antibodies that have a lower affinity with their antigen than the original antibody, and some mutations will generate antibodies with a higher affinity. B cells that express higher affinity antibodies on their surface will receive a strong survival signal during interactions with other cells, whereas those with lower affinity antibodies will not, and will die by apoptosis. Thus, B cells expressing antibodies with a higher affinity for the antigen will outcompete those with weaker affinities for function and survival. The process of generating antibodies with increased binding affinities is called affinity maturation. Affinity maturation occurs after V(D)J recombination, and is dependent on help from helper T cells.

Antibody genes also re-organize in a process called class switching, which changes the base of the heavy chain to another. This creates a different isotype of the antibody while retaining the antigen specific variable region, thus allowing a single antibody to be used by several different parts of the immune system.

Clonal Selection of Antibody-Producing Cells

The clonal selection hypothesis is a widely accepted model for the immune system's response to infection.

The clonal selection hypothesis has become a widely accepted model for how the immune system responds to infection and how certain types of B and T lymphocytes are selected for destruction of specific antigens invading the body.

Four predictions of the clonal selection hypothesis

• Each lymphocyte bears a single type of receptor with a unique specificity (by V(D)J recombination).
• Receptor occupation is required for cell activation.
• The differentiated effector cells derived from an activated lymphocyte will bear receptors of identical specificity as the parental cell.
• Those lymphocytes bearing receptors for self molecules will be deleted at an early stage.

In 1954, Danish immunologist Niels Jerne put forward a hypothesis which stated that there is already a vast array of lymphocytes in the body prior to any infection. The entrance of an antigen into the body results in the selection of only one type of lymphocyte to match it and produce a corresponding antibody to destroy the antigen. This selection of only one type of lymphocyte results in it being cloned or reproduced by the body extensively to ensure there are enough antibodies produced to inhibit and prevent infection. Australian immunologist Frank Macfarlane Burnet, with input from David W. Talmage, worked on this model and was the first to name it "clonal selection theory. " Burnet explained immunological memory as the cloning of two types of lymphocyte. One clone acts immediately to combat infection whilst the other is longer lasting, remaining in the immune system for a long time, which results in immunity to that antigen. In 1958, Sir Gustav Nossal and Joshua Lederberg showed that one B cell always produces only one antibody, which was the first evidence for clonal selection theory.

B cells exist as clones. All B cells derive from a particular cell, and as such, the antibodies and their differentiated progenies can recognize and/or bind the same specific surface components composed of biological macromolecules ( epitope ) of a given antigen. Such clonality has important consequences, as immunogenic memory relies on it. The great diversity in immune response comes about due to the up to 109 clones with specificities for recognizing different antigens. Upon encountering its specific antigen, a single B cell, or a clone of cells with shared specificity, divides to produce many B cells. Most of such B cells differentiate into plasma cells that secrete antibodies into blood that bind the same epitope that elicited proliferation in the first place. A small minority survives as memory cells that can recognize only the same epitope. However, with each cycle, the number of surviving memory cells increases. The increase is accompanied by affinity maturation which induces the survival of B cells that bind to the particular antigen with high affinity. This subsequent amplification with improved specificity of immune response is known as secondary immune response. B cells that have not been activated by antigen are known as naive lymphocytes; those that have met their antigen, become activated, and have differentiated further into fully functional lymphocytes are known as effector B lymphocytes.

Isotype Class Switching

Isotype class switching is a biological mechanism that changes a B cell's production of antibody from one class to another.

Isotype Class Switching

Antibodies can come in different varieties, known as isotypes or classes. In placental mammals there are five antibody isotypes: IgA, IgD, IgE, IgG and IgM. They are each named with an "Ig" prefix that stands for immunoglobulin (another name for antibody) and differ in their biological properties, functional locations, and ability to deal with different antigens.

The antibody isotype of a B cell changes during cell development and activation. Immature B cells, which have never been exposed to an antigen, are known as naïve B cells and express only the IgM isotype in a cell surface bound form. B cells begin to express both IgM and IgD when they reach maturity; the co-expression of both of these immunoglobulin isotypes renders the B cell 'mature' and ready to respond to an antigen. B cell activation follows engagement of the cell-bound antibody molecule with an antigen, causing the cell to divide and differentiate into an antibody-producing cell, called a plasma cell. In this activated form, the B cell starts to produce antibody in a secreted form rather than a membrane-bound form. If these activated B cells encounter specific signaling molecules via their CD40 and cytokine receptors (both modulated by T helper cells), they undergo antibody class switching to produce IgG, IgA or IgE antibodies (from IgM or IgD) that have defined roles in the immune system.

Immunoglobulin class switching (or isotype switching, or isotypic commutation, or class switch recombination (CSR)) is a biological mechanism that changes a B cell's production of antibody from one class to another; for example, from an isotype called IgM to an isotype called IgG. During this process, the constant region portion of the antibody-heavy chain is changed, but the variable region of the heavy chain stays the same (the terms "constant" and "variable" refer to changes or lack thereof between antibodies that target different epitopes). Since the variable region does not change, class switching does not affect antigen specificity. Instead, the antibody retains affinity for the same antigens, but can interact with different effector molecules. This allows different daughter cells from the same activated B cell to produce antibodies of different isotypes or subtypes (e.g. IgG1, IgG2 etc.).

Class switching occurs by a mechanism called class switch recombination (CSR) binding. Class switch recombination is a biological mechanism that allows the class of antibody produced by an activated B cell to change during a process known as isotype or class switching. During CSR, portions of the antibody-heavy chain locus are removed from the chromosome, and the gene segments surrounding the deleted portion are rejoined to retain a functional antibody gene that produces antibody of a different isotype. Double-stranded breaks are generated in DNA at conserved nucleotide motifs, called switch (S) regions, which are upstream from gene segments that encode the constant regions of antibody-heavy chains; these occur adjacent to all heavy chain constant region genes with the exception of the δ-chain. DNA is nicked and broken at two selected S-regions by the activity of a series of enzymes, including Activation-Induced (Cytidine) Deaminase (AID), uracil DNA glycosylase and apyrimidic/apurinic (AP)-endonucleases. The intervening DNA between the S-regions is subsequently deleted from the chromosome, removing unwanted μ or δ heavy chain constant region exons and allowing substitution of a γ, α or ε constant region gene segment. The free ends of the DNA are rejoined by a process called non-homologous end joining (NHEJ) to link the variable domain exon to the desired downstream constant domain exon of the antibody-heavy chain. In the absence of non-homologous end joining, free ends of DNA may be rejoined by an alternative pathway biased toward microhomology joins. With the exception of the μ and δ genes, only one antibody class is expressed by a B cell at any point in time.

Making Memory B Cells

Memory B cells are a B cell sub-type that are formed following primary infection.

Making Memory B Cells

Memory B cells are a B cell sub-type that are formed following a primary infection. In the wake of the first (primary response) infection involving a particular antigen, the responding naïve cells (ones which have never been exposed to the antigen) proliferate to produce a colony of cells. Most of them differentiate into the plasma cells, also called effector B cells (which produce the antibodies) and clear away with the resolution of infection. The rest persist as the memory cells that can survive for years, or even a lifetime.

To understand the events taking place, it is important to appreciate that the antibody molecules present on a clone (a group of genetically identical cells) of B cells have a unique paratope (the sequence of amino acids that binds to the epitope on an antigen).

Each time these cells are induced to proliferate due to an infection, the genetic region coding for the paratope undergoes spontaneous mutations with a frequency of about 1 in every 1600 cell divisions. This may not seem high, but because the cells divide so often, it ends up resulting in many mutations. The frequency of mutations in other cells is around 1 in 10⁶, which is much lower.

All these events occur in the highly "eventful" germinal centers of lymphoid follicles, within the lymph nodes.

Some of the resulting paratopes (and the cells elaborating them) have a better affinity for the antigen (actually, the epitope) and are more likely to proliferate than the others.

Moreover, the number of different clones responding to the same antigen increases (polyclonal response) with each such exposure to the antigen and a greater number of memory cells persist. Thus, a stronger (basically, larger number of antibody molecules) and more specific antibody production is the hallmark of secondary antibody response.

The fact that all the cells of a single clone elaborate one (and only one) paratope, and that the memory cells survive for long periods, is what imparts a memory to the immune response. This is the principle behind vaccination and administration of booster.

The paratope is the part of an antibody which recognizes an antigen, the antigen-binding site of an antibody. It is a small region (15–22 amino acids) of the antibody's Fv region and contains parts of the antibody's heavy and light chains. The part of the antigen to which the paratope binds is called an epitope.

Primary and Secondary Antibody Responses

The immune system protects organisms from infection first with the innate immune system, then with adaptive immunity.

The immune system is a system of biological structures and processes within an organism that protects against disease. To function properly, an immune system must detect a wide variety of agents, from viruses to parasitic worms, and distinguish them from the organism's own healthy tissue. Pathogens can rapidly evolve and adapt to avoid detection and neutralization by the immune system. As a result, multiple defense mechanisms have also evolved to recognize and neutralize pathogens. Even simple unicellular organisms such as bacteria possess a rudimentary immune system, in the form of enzymes that protect against bacteriophage infections. Other basic immune mechanisms evolved in ancient eukaryotes and remain in their modern descendants, such as plants and insects. These mechanisms include phagocytosis, antimicrobial peptides called defensins, and the complement system. Jawed vertebrates, including humans, have even more sophisticated defense mechanisms, including the ability to adapt over time to recognize specific pathogens more efficiently. Adaptive (or acquired) immunity creates immunological memory after an initial response to a specific pathogen, leading to an enhanced response to subsequent encounters with that same pathogen. This process of acquired immunity is the basis of vaccination.

Disorders of the immune system can result in autoimmune diseases, inflammatory diseases and cancer. Immunodeficiency occurs when the immune system is less active than normal, resulting in recurring and life-threatening infections. In humans, immunodeficiency can either be the result of a genetic disease such as severe combined immunodeficiency, acquired conditions such as HIV/AIDS, or the use of immunosuppressive medication. In contrast, autoimmunity results from a hyperactive immune system attacking normal tissues as if they were foreign organisms. Common autoimmune diseases include Hashimoto's thyroiditis, rheumatoid arthritis, diabetes mellitus type 1, and systemic lupus erythematosus. Immunology covers the study of all aspects of the immune system.

The immune system protects organisms from infection with layered defenses of increasing specificity. In simple terms, physical barriers prevent pathogens such as bacteria and viruses from entering the organism. If a pathogen breaches these barriers, the innate immune system provides an immediate, but non-specific response. Innate immune systems are found in all plants and animals. If pathogens successfully evade the innate response, vertebrates possess a second layer of protection, the adaptive immune system, which is activated by the innate response. Here, the immune system adapts its response during an infection to improve its recognition of the pathogen. This improved response is then retained after the pathogen has been eliminated, in the form of an immunological memory, and allows the adaptive immune system to mount faster and stronger attacks each time this pathogen is encountered. Both innate and adaptive immunity depend on the ability of the immune system to distinguish between self and non- self molecules. In immunology, self molecules are those components of an organism's body that can be distinguished from foreign substances by the immune system. Conversely, non-self molecules are those recognized as foreign molecules. One class of non-self molecules are called antigens (short for antibody generators) and are defined as substances that bind to specific immune receptors and elicit an immune response.

When B cells and T cells are first activated by a pathogen, memory B-cells and T- cells develop. Throughout the lifetime of an animal these memory cells will "remember" each specific pathogen encountered, and are able to mount a strong response if the pathogen is detected again. This type of immunity is both active and adaptive because the body's immune system prepares itself for future challenges. Active immunity often involves both the cell-mediated and humoral aspects of immunity as well as input from the innate immune system. The innate system is present from birth and protects an individual from pathogens regardless of experiences, whereas adaptive immunity arises only after an infection or immunization and hence is "acquired" during life.