Ryan Hamnett, PhD | 30th September 2024
Antibodies are a critical component of the immune system’s ability to protect the body from foreign invaders.
The immune system has two main branches: the innate immune system, and the adaptive immune system. Where the innate immune system offers general protection in the form of barriers and some non-specific immune cell types such as phagocytes, the adaptive immune system is highly specialized and specifically targets the pathogen causing the infection. The adaptive immune system also has a “memory”, meaning it can quickly recognize and destroy pathogens that it has encountered before.
Antibodies are a key part of the adaptive immune system, which takes over if the innate immune system fails to rid the body of pathogens. They recognize particular pathogens with high affinity and high specificity to neutralize them or recruit other components of the immune system.
There are two broad classes of lymphocytes (white blood cells) that mediate the actions of the adaptive immune system: B-cells and T-cells. Both B- and T-cells are derived from lymphoid progenitors (Figure 1).
Figure 1: Immune cell development. Created using BioRender.
B-cells are made in the bone marrow, and are ultimately responsible for generating antibodies. Each B-cell recognizes a specific pathogen and will produce antibodies only against that pathogen. B-cells must first be activated by a specific type of T-cell called a T helper cell. Following activation, B-cells become plasma cells, which can produce and release large amounts of antibodies, or memory B cells, which can rapidly reactivate if a repeat infection occurs months or years later.
T-cells are made in the thymus. There are several subtypes of T-cells that carry out different functions, including T helper cells, cytotoxic T cells, memory T cells, and T regulatory cells. Many of these can be further subdivided based on their activity and their expression of specific cytokines.
The recruitment of B-cells and antibodies in fighting pathogens generally involves the following steps:
Genetic Mechanisms of Antibody Diversity
Each individual antibody is specific to one antigen and is produced by a single type of B-cell. However, there are countless potential pathogens, antigens, and harmful factors that the body might have to defend against, requiring a large amount of diversity among antibodies.
Differences between antibodies is mainly found in the amino acid composition of their variable region (see Antibody Structure). Instead of having a separate gene for each antibody, which would result in an enormous genome, some complex genetic mechanisms manage to create the necessary diversity from comparatively few genes.
The variable regions of both the light and heavy chains is encoded by multiple gene segments, which are recombined to form a functional gene (Figure 2). These segments are referred to as the variable (V), diversity (D) and joining (J) segments. While there are many potential gene segments to choose from for each of V, D and J, only one of each type is selected to create a given antibody. This method of recombination along with the number of different combinations of light- and heavy-chain pairings can create millions of unique antibodies. Coupled with junctional diversity, in which minor amino acid changes are introduced by the recombination process, billions of unique antibodies can be produced.
Figure 2: VDJ recombination. Antibody variable regions are constructed from multiple gene segments (V, D and J for heavy chain, V and J for light chain) that recombine to produce the final mRNA. Only one segment of each type is selected, creating a large potential for antibody diversity. The number of each segment to choose from is illustrated for each type of segment in humans. The CH gene segments are arranged in the order Cµ, Cδ, C𝛾3, C𝛾2, C𝛾1, C𝛾4, Cε, Cα1, and Cα2, resulting in different antibody isotypes. Unlike kappa, the lambda locus comprises tandem cassettes of J and C segments. Created using BioRender.
Somatic Hypermutation
A final mechanism, termed somatic hypermutation, introduces even more diversity. Somatic hypermutation introduces point mutations at a high rate into the rearranged variable region genes of activated B-cells – B-cells that have successfully recognized an invading pathogen.
These slight mutations can create B-cells that have lesser or greater affinity for the pathogen. B-cells that express higher affinity antibodies will outcompete others and be maintained by the body in a process called affinity maturation, which is dependent on T helper cells.
Immune Tolerance
Given the large number of unique antibodies that are made by the body, it is inevitable that some will recognize the host’s own proteins, called self-antigens. B-cells that recognize self-antigens are eliminated in the bone marrow as part of a feature of the immune system called immune tolerance. Other immune cells, such as T regulatory cells, are also capable of recognizing and inactivating these anti-self B-cells.
There are instances where a host does want its immune system to attack its own cells, such as in cancer, or if the cell has been infected by a bacteria or virus and is presenting antigens on its surface. In most other cases, however, aberrant targeting of host cells creates autoimmune disorders, including rheumatoid arthritis, multiple sclerosis, lupus, and Type 1 diabetes.
Once antibodies have been produced against an antigen on a specific pathogen or invading substance, they can affect the activity of that pathogen in several ways:
An antigen is a foreign substance that induces the immune system to generate antibodies against it. The antigen can be a protein, polysaccharide, lipid or nucleic acid, and includes types of foreign substance such as a molecule on the surface of a pathogen, a toxin, or an allergen.
Antigen presentation is a key part of the immune response, in which infected cells transport antigens or antigen fragments to their cell surface for immune cells to bind. This stimulates a number of different immune processes, including immune cell activation, proliferation, lysis or apoptosis of the infected cell, and cytokine release.
Antigen Properties
Though many types of molecule can be antigenic, there are some characteristics of the molecule that make an immune response more likely. These include:
Haptens
An antigen does not require all properties listed above, and there are ways to generate antibodies against molecules that are not particularly antigenic in their native form. For example, haptens are low molecular weight molecules that lack antigenicity on their own, and must be attached to a carrier protein to evoke an immune response. Molecules such as small molecule drugs and small peptides can be haptens, while common carrier proteins include bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH).
Antibody-Antigen Interactions
The part of an antigen that an antibody recognizes and binds to is called an epitope. Antibodies typically do not recognize the entire antigen. Epitopes can be just a few amino acids in length, and many epitopes can exist on an antigen. An epitope must be available for binding, so they are often on the external surface of a pathogen where antibodies can access them.
The part of the antibody that binds to the epitope is known as the paratope. The paratope-epitope interaction is facilitated by Van der Waals, hydrogen bonds, electrostatic and hydrophobic interactions. The stronger these interactions are, the higher the affinity of the antibody for that specific antigen.
Paratope-epitope interactions are extremely specific, which allows antibodies to distinguish between even closely related antigens. It also makes generating antibodies against self-proteins, for example if they resemble pathogenic proteins, less likely. This antibody specificity makes antibodies useful to researchers, allowing them to single out a protein of interest from millions of others in the cell.
Antigens for Research
When generating antibodies for research use, the antigen does not necessarily need to be easily accessible in its native, endogenous location. This is because an isolated or recombinant peptide or protein fragment is typically used as the antigen for such antibodies. Permeabilization reagents such as detergents are then used during experiments to make intracellular antigens accessible for visualization or isolation.
Nonetheless, the epitope in target cells must still be recognizable to the antibody. Aspects of biochemical processing, such as fixation or denaturation, can alter the epitope such that an antibody no longer recognizes it. This is why antibodies should be validated for a specific application.
