Introduction to Antibodies and Immunoglobulins

Antibodies are multipart proteins the immune system produces in response to a foreign substance, such as a virus, bacteria, or toxin. They are also known as immunoglobulins. The primary function of antibodies is to bind to antigens and mark them for destruction by other cells of the immune system. Antibodies can also activate the complement system, which leads to the destruction of bacteria. There are five main types of antibodies: Immunoglobulin A (IgA), Immunoglobulin D (IgD), Immunoglobulin E (IgE), Immunoglobulin G (IgG), and Immunoglobulin M (IgM).

Antibody Structure

The structure of an antibody is key to its function. Antibodies are composed of four main polypeptide chains:

• Two heavy chains
• Two light chains

The heavy chains are the larger of the two types of polypeptide chains, and they determine the class of the antibody. There are five classes of antibodies, each with a different heavy chain: IgA, IgD, IgE, IgG, and IgM.

The light chains are smaller than the heavy chains and provide specificity to the antibody for a particular antigen. Each antibody has two light chains: kappa (κ) and lambda (λ). The variable region of each light chain contains the antigen-binding site. The variable regions of the heavy chains also contain antigen-binding sites. The constant regions of both the heavy and light chains determine the effector function of the antibody.

Light Chain

The light chain of an antibody is a polypeptide chain typically composed of two domains. Each domain has a unique sequence of amino acids that confers a specific three-dimensional structure. Interdomain disulfide bridges maintain the overall structure of the light chain.

The light chain is responsible for binding to antigens. Each light chain domain has a distinct binding site for an antigen. The domains are connected by a flexible hinge region that allows the antibody to bind to different epitopes on an antigen.

The variable domain of the light chain is responsible for the antibody's specificity. This domain undergoes somatic mutation during B cell development, resulting in a vast repertoire of antibodies with different specificities. The constant domain of the light chain contains the sites for interchain disulfide bridges and carbohydrate attachment.

Heavy Chain

The heavy chain of an antibody is a large protein responsible for most of the antibody's mass. It is composed of two domains, each with a different function. The first domain binds to the antigen, while the second domain binds to the light chain. The heavy chain also contains the variable region, which is responsible for the specificity of the antibody.

Immunoglobulins Types, Function, and Immunoglobulin Diversity

Immunoglobulin A (IgA)

It is secreted by plasma cells in mucosal tissues and protects against infections at sites where entry of pathogens is likely, such as the respiratory tract and gastrointestinal tract.

Immunoglobulin D (IgD)

It is found on the surface of B cells and helps regulate the immune response by activating B cells when they come into contact with an antigen.

Immunoglobulin E (IgE)

It is produced in response to allergens and helps protect against parasitic infections. It is also involved in allergic reactions.

Immunoglobulin G (IgG)

It is the most abundant type of antibody in the blood and tissue fluids. It protects against bacterial and viral infections and is the only type of antibody that can cross the placenta to protect a developing fetus.

Immunoglobulin M (IgM)

It is produced early in an infection and is the first line of defense against bacteria and viruses. It is also present in small amounts in blood and tissue fluids.

Effector Functions and Antibody Applications

Various Ig isotopes, including IgG, IgM and IgA, include Fc regions that mediate effector functions in mammalian cells. Therapeutic mAbs consist of IgG type antibodies that elicit well-known effector cell responses.

Commercialization of therapeutic mAbs requires a detailed characterization of important mAb structural and functional properties that includes analyzing and determining effector function.

Effector function analyses include characterizations of Antibody-dependent Cellular Cytotoxicity (ADCC), Complement-dependent Cytotoxicity (CDC), Antibody-dependent Cellular Phagocytosis (ADCP), T-cell Dependent Cellular Cytotoxicity (TDCC) and apoptosis.

Antibody-dependent Cellular Cytotoxicity (ADCC)

It is a cell-mediated defense response that involves an effector cell, such as natural killer (NK) cells, which interact with the Fc region of IgG antibodies, and results in the lysis and elimination of targeted cells. Other immune effector cells include eosinophils, neutrophils and macrophages.

mAbs cancer treatments take advantage of ADCC by targeting specific antigens on the outer membranes of cancerous cells (Fab region binding) and activate destruction (Fc region binding) by effector cells. Clinical studies of multiple myeloma treatments with daratumumab (Darzalex) indicate that ADCC is actively involved in successfully identifying and targeting cancer cells.

Complement-dependent Cytotoxicity (CDC)

It similarly starts with mAbs binding to cancer-specific antigens on the surface of malignant cells. A complement cascade activated by a variety of mechanisms leads to complement factors binding to mAb-cancer antigen moieties, resulting in the induction of cytolysis and the elimination of cancerous cells.

Antibody-dependent Cellular Phagocytosis (ADCP)

It involves mAb cancer antigen specificity, along activation and induction of phagocytosis with macrophages, that result in the destruction of cancerous cells targeted by mAbs.

T-cell Dependent Cellular Cytotoxicity (TDCC)

It includes mAb cancer antigen targeting properties that additionally engage with T-cells to initiate T-cell mediated cytolysis.

Apoptosis

It is programmed cell death, and it plays important role in modulating the immune response, performing cytotoxicity, and removing aberrant immune cells. mAbs can be designed to induce apoptosis in cancer cells that leads to their removal.

Antibody and Antigen Interactions

The structure of the antibody’s binding site matches the corresponding shape on the surface of the antigen, and binding occurs due to hydrophobic, electrostatic, and other interactions. This lock-and-key interaction between antibodies and antigens allows the immune system to specifically target and destroy pathogens.

The binding of an antibody to an antigen is a reversible process governed by hypervariable loops that determine specificity. It means the antibody-antigen complex can dissociate or break apart under certain conditions. For example, if the concentration of either the antibody or antigen becomes too dilute, they may no longer interact with each other.

The strength of the bond between an antibody and antigen also plays a role in determining how long the complex will stay together. If the bond is weak, the complex will quickly dissociate. However, if the bond is strong, the complex may remain intact for a longer period of time.

Antibody and Antigen Binding Sites

The antigen binding site is the part of the antibody that recognizes and binds to antigens. Antibodies are proteins the immune system produces in response to foreign substances called antigens. Each antibody has two parts: a variable region and a constant region. The variable region is responsible for binding to the specific antigen, while the constant region is responsible for mediating the antibody's effector function.

The structure of an antibody's variable region is highly flexible, allowing it to bind to various antigens. The variable region comprises several smaller units called domains, each contributing to the binding site. The number and arrangement of these domains vary from one antibody to another.

The antigen binding site is formed at the interface between the variable domains of the heavy and light chains. The amino acid residues that make up the binding site are located in both chains and are held together by non-covalent interactions, such as hydrogen bonds and van der Waals forces. These interactions are relatively weak, allowing the binding site to readily change shape so it can bind to different antigens.

The specificity of an antibody's binding site arises from its three-dimensional shape, determined by the sequence of amino acids in the variable domains. Because there are a limited number of possible amino acid sequences, each with its own three-dimensional shape, antibodies exhibit a high degree of specificity for their target antigens.

Monoclonal Antibodies (mAbs)

A monoclonal antibody (mAb), originating from a single B lymphocyte, is produced in cell culture and binds to a specific epitope on antigens in the body, including viruses, bacteria, and cancer cells. mAbs are used in the diagnosis and treatment of many diseases. For example, they can be used to identify a specific cancer cell to target it for destruction. mAbs can also boost the immune system’s response to infection.

There are different types of antibodies that include Fragment antigen-binding (Fab) regions, which consists of a variable and a constant region from a light and a heavy chain. Monovalent Fab antibodies are Y-shaped and bind to a single, identical antigen, while bivalent antibodies can contain different antigen and epitope binding regions.

Polyclonal Antibodies

Polyclonal antibodies are antibodies that are produced by multiple B-cells, each of which creates a unique antibody. These antibodies can recognize and bind to various epitopes on a given antigen. Polyclonal antibodies are often used in research and diagnosis, as they can provide a broader range of protection than monoclonal antibodies.

Research Detection Methods and Applications for Antibodies

There are a number of different research applications for antibodies, including:

ELISA (Enzyme-linked Immunosorbent Assay)

The most common method used to identify and characterize antibodies in a sample is ELISA, which is a test that uses immobilized antigens to capture specific antibodies from a sample of blood or other bodily fluid.

Western Blotting

This technique is used to identify specific proteins in a sample by their size and/or charge.

Immunohistochemistry

This technique is used to study the distribution and function of specific proteins in tissue samples.

Flow Cytometry

This technique measures the number of specific antibodies present in cells.

Immunofluorescence

Antibodies and antigens labeled with fluorescent compounds can be used to track specific protein locations in cells and monitor cellular processes.

Medical Applications for Monoclonal Antibodies

Monoclonal antibodies (mAbs) are now commonly used as therapeutic agents to treat a variety of diseases. mAbs that target a specific epitope on an antigen are usually delivered via intravenous (IV) infusions, although subcutaneous injections are also used. This can lead to the destruction of the antigen, the prevention of its function, or an effect on other targeted, intracellular processes.

There are many different medical applications for mAbs, including the treatment of cancer, autoimmune disorders, and infectious diseases. mAbs can be used alone or in combination with other therapies, such as chemotherapy or radiation.

Cancer Treatment

mAbs can be used to treat many different types of cancer, including breast cancer, ovarian cancer, and leukemia. In some cases, mAbs are used as part of a targeted therapy approach that specifically targets cancer cells while sparing healthy cells. This allows for more effective treatment with fewer side effects.

Herceptin (trastuzumab) is an example of a mAb used to treat breast cancer by binding to cancer cell specific receptors and inhibiting their growth. Other mAb cancer treatment examples include rituximab and Campath.

Treating Autoimmune Disorders

Autoimmune disorders occur when the body’s immune system attacks healthy tissues and organs. mAbs can be used to modulate the immune response and reduce inflammation in these disorders. Common autoimmune disorders treated with mAbs include rheumatoid arthritis, Crohn’s disease, and psoriasis.

Examples include Humira (adalimumab) for the treatment of moderate to severe rheumatoid arthritis, psoriatic arthritis and Crohn’s disease. Humira inhibits disease progression, reduces visible signs and symptoms, and can improve physical function. Rituximab and Cimzia are mAbs that also treat rheumatoid arthritis. Amevive (alefacept) is a mAb used as an immunosuppressant for treating chronic plaque psoriasis.

Treating Infectious Diseases

mAbs can also be used to treat infections caused by bacteria, viruses (e.g., SARS-CoV-2), and potentially for fungi. They can be used to prevent infection or to treat an active infection.

Covid-19 Veklury (Remdesivir) is an example of a therapeutic mAb specific for SARS-CoV-2 that treats Covid-19 patients in an outpatient setting. Zinplava (bezlotoxumab) is a therapeutic mAb used to prevent C. difficile bacterial reinfection. Clinical research is underway for therapeutic applications including the use of mAbs to treat various fungal infections including Candida, dimorphic fungi and molds.

Treating Respiratory Diseases

mAbs are effective for treating asthma by blocking respiratory-related responses. Examples include Xolair (omalizymab), a mAb used to help reduce severe asthma by reducing reactions caused by antigens like pollen and bacteria. Dupixent (dupilumab) is a mAb that blocks specific interleukins and is used for treating allergenic diseases including asthma.

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