Antibody structure and isotypes

Antibody structure

Antibodies, also known as immunoglobulins (Ig), are large, Y-shaped glycoproteins produced by B-cells as a primary immune defense. Antibodies specifically bind unique molecules of a pathogen, called antigens.

Antibodies exist as one or more copies of a Y-shaped unit composed of four polypeptide chains (Fig. 1). Each Y unit contains two identical copies of a heavy chain (H) and two identical copies of a light chain (L); heavy and light chains differ in their sequence and length. The top of the Y shape contains the variable region (V), also known as the fragment antigen-binding (F(ab)) region. This region binds tightly to a specific part of an antigen called an epitope.

The antibody base consists of constant domains (C) and forms the fragment crystallizable region (Fc). This region is essential for the function of the antibody during an immune response.

Figure 1. Antibody structure. The Y-shaped antibody is joined in the middle by a flexible hinge  region. Antigen binding occurs at the variable domain (V), consisting of immunoglobulin heavy (H) and light chains (L). The base of the antibody includes constant domains (C). V_H – heavy chain variable domain, V_L- light chain variable domain, C_H – heavy chain constant domain, C_L – light chain constant domain.

F(ab) and Fc Regions

The Y-shape of an antibody can be cleaved into three fragments by the proteolytic enzyme pepsin: two F(ab) regions and an Fc region. The F(ab) regions contain the variable domain that binds to cognate (specific) antigens. The Fc fragment provides a binding site for endogenous Fc receptors on the surface of lymphocytes and secondary antibodies. Also, dye and enzymes can be covalently linked to antibodies on the Fc portion of the antibody for experimental visualization.

Antibody fragments have distinct advantages in specific immunochemical techniques. Fragmenting IgG antibodies is sometimes useful because F(ab) fragments (1) will not precipitate the antigen, and (2) will not be bound by immune cells in live studies because of the lack of an Fc region. Often, because of their smaller size and lack of cross-linking (due to the Fc region's loss), F(ab) fragments are radiolabeled in functional studies. Fc fragments are often used as Fc receptor-blocking agents in immunohistochemical staining.

Learn more about the advantages of F(ab) and F(ab')2 fragments.

Heavy chains

The type of heavy chain defines the overall class or isotype of an antibody. There are five types of mammalian Ig heavy chains denoted by Greek letters: α, δ, ε, γ and μ. These chains are found in IgA, IgD, IgE, IgG, and  IgMantibodies, respectively. Heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, while μ and ε have about 550 amino acids.

Each heavy chain has two regions: constant (C_H) and variable (V_H). The constant region is identical in all the same isotype antibodies but differs in antibodies of different isotypes. Heavy chains γ, α, and δ have a constant region composed of three tandem Ig domains – CH^1, CH², CH³ – and a hinge region for added flexibility. Heavy chains μ and ε have a constant region composed of four immunoglobulin domains. The heavy chain's variable region (V_H) differs depending on the B cell that produced it but is the same for all antibodies produced by a single B cell or B cell clone. Each heavy chain's variable region is approximately 110 amino acids long and composed of a single Ig domain.

Light chains

Mammals have only two types of light chains, lambda (λ) and kappa (κ), which have minor differences in the polypeptide sequence. A light chain has two successive domains: constant (C_L) and variable (V_L). The approximate length of a light chain is 211–217 amino acids. Each antibody contains two light chains that are always identical. Other types of light chains, such as the iota (ι) chain, are found in lower vertebrates like Chondrichthyes and Teleostei.

Antibody isotypes

In mammals, antibodies are divided into five isotypes: IgG, IgM, IgA, IgD, and IgE. Each isotype has a unique structure, as depicted in Figure 2. The isotypes vary based on the number of Y units and the type of heavy chain. They will also differ in their biological properties, functional locations, and ability to deal with different antigens (Table 1).

Table 1. Structure and functions of different antibody isotypes.

Figure 2. Antibody structure and isotypes.

Antigen-antibody interactions: how and where antibodies bind

The F(ab) antibody region contains the antigen-binding site called paratope. The paratope binds to a specific part of an antigen called the epitope, which is a small part of the antigen – sometimes just a few amino acids long (Fig. 3).

The paratope and epitope are held together by complementary shapes and intermolecular interactions such as Van der Waals, hydrogen bonds, electrostatic and hydrophobic interactions; the strength of these forces determines the antibody's affinity.

Figure 3. A schematic representation of antigen-antibody interactions.

Antigens: overview and considerations for your experiment

The basic principle of any immunoassay is that a specific antibody binds with its specific antigen, forming an exclusive antibody-antigen complex. This chapter defines what an antigen is and how to choose one to make an antibody.

What are antigens?

An antigen is any foreign substance that can elicit an immune response in the body (eg, antibody production) and is bound by the specific antibodies produced against it by the immune system.

Antigens generally have high molecular weight and are commonly proteins or polysaccharides. Polypeptides, lipids, nuclear acids, and many other materials can also function as antigens.

Haptens

Haptens are smaller substances that can also generate immune responses if chemically coupled to a larger carrier protein. Common carrier proteins include bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or other synthetic matrices.

Many molecules may function as haptens, including drugs, simple sugars, amino acids, small peptides, phospholipids, or triglycerides. Thus, given enough time, the immune system will identify just about any foreign substance and evoke specific antibody production. However, this specific immune response is highly variable and depends on the antigen's size, structure, and composition. Antigens eliciting strong immune responses are called strongly immunogenic.

Antigen-antibody Interaction

An epitope is a part of an antigen that the specific antibody recognizes and binds to. For efficient interaction between the antigen and the antibody, the epitope must be readily available for binding. If the target molecule is denatured (eg, through fixation, reduction, pH changes, or during preparation for gel electrophoresis), the epitope may be altered, which may affect its ability to interact with an antibody.

For example, some antibodies might be ineffective in western blot but very good in immunohistochemistry (IHC) because a complex antigenic site might be maintained in the tissue in IHC. In contrast, in western blot, the sample preparation process alters the protein conformation sufficiently to destroy the antigenic site and eliminate antibody binding.

Thus, the epitope may be present in the antigen's native cellular environment or only exposed when denatured. In their natural form, epitopes may be cytoplasmic (soluble), membrane-associated, or secreted. The epitopes' number, location, and size depend on the antigen amount available during the antibody-making process.

Characteristics of a good antigen include:

• Areas of structural stability and chemical complexity within the molecule
• Significant stretches lacking extensive repeating units
• A minimal molecular weight of 8,000–10,000 Da, although haptens with molecular weights as low as 200 Da have been used in the presence of a carrier protein.
• The ability to be processed by the immune system
• Immunogenic regions accessible to the antibody-forming mechanism
• Structural elements sufficiently different from the host
• For peptide antigens, regions containing at least 30% of immunogenic amino acids: lysine, arginine, glutamic acid, aspartic acid, glutamine, and asparagine.
• For peptide antigens, significant hydrophilic or charged residues

The immune system and the antibody response

This chapter provides a general overview of the immune system and its role in generating specific antibodies.

Overview of the immune system

The function of the immune system is to protect animals from foreign agents and infectious organisms. It responds to pathogens in a specific way and can display a long-term memory of infectious agents' exposure. The immune system consists of two functional components:

1. The innate or non-specific immune system
2. The adaptive or specific immune system

The innate immune system

The innate immune system components provide the first line of defense against infection. Physical barriers to infection include skin, which prevents pathogen penetration, and bodily fluids, like mucus, which collect and clear pathogens.

Many cellular and biochemical components, including complement proteins, innate leukocytes, and phagocytic cells, identify and eliminate pathogens from the body.

The innate immune system's function and efficiency do not change with repeated exposure to foreign pathogens.

The adaptive immune system

The adaptive immune system is activated when the innate system fails to clear pathogens from the body. It consists of various cells and molecules, with lymphocytes and antibodies being the key elements.

Lymphocytes arise continuously from progenitor cells in the bone marrow. Lymphocytes synthesize cell surface receptors or secrete proteins that specifically bind to foreign molecules. These secreted proteins are known as antibodies. Any molecule that can bind to an antibody is called an antigen. The term antibody is used interchangeably with immunoglobulin.

Pathogens bound to antibodies are marked for clearance or destruction.

Most functions of the adaptive immune system can be described by grouping lymphocytes into three basic types:

1. B cells
2. Cytotoxic T cells (T _C cells)
3. Helper T cells (T _h cells)

The adaptive immune response can be either humoral or cell-mediated. B lymphocytes mediate the humoral response by releasing antibodies specific to the infectious agent. The cell-mediated response involves binding T_C cells to foreign or infected cells, followed by the lysis of these cells.

T_h cells are involved in both responses through the release of cytokine proteins. All three types of lymphocytes carry cell surface receptors that can bind antigens. All antigen receptors are glycoproteins, and only one kind of receptor is synthesized within any one cell. The specificity of the immune system is impacted by the fact that one cell recognizes only one antigen.

View our poster on human T cell development

Antibody response

The antibody-antigen interaction forms the basis of all immunoassays but is also the basis for the immune response.

The region of the antibody that reacts with the antigen is called the paratope. The region of an antigen that interacts with an antibody is defined as an epitope. Affinity measures the strength of the epitope's binding to an antibody and is often represented by the dissociation constant K_D. Avidity measures the overall stability of the complex between antibodies and antigens.

An antibody response is the culmination of a series of interactions between macrophages, T lymphocytes, and B lymphocytes. Infectious agent antigens are engulfed and partially degraded by antigen-presenting cells (APCs), such as macrophages, Langerhans cells, dendritic cells, lymph nodes, and monocytes.

The antigen's fragments will appear on the APC's surface attached to a cell surface glycoprotein known as MHC II (major histocompatibility complex). There are two types of MHC molecules: MHC class I, expressed on the surfaces of most cells, and class II, expressed exclusively on APCs' surfaces. The antigen-MHC II complex allows T_h cells to bind to the APC, leading to a proliferation of T_h cells and cytokine release. T cells then bind to the MHC complex on B cells, leading to B cells' proliferation and differentiation. B cells change into plasma cells, secreting large quantities of finely tuned antibodies specific to the foreign agent. Some B cells are transformed into memory cells, allowing for a faster antibody-mediated immune response upon future infection.