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I'd like to clear something up about antibodies that I'm not sure I've understood in the articles I've read. Looking at concepts such as "affinity maturation", "monovalent antigens" and "polyvalent antigens" it seems as if there are multiple antibodies which can bind to the same antigen. (Although I think I'm right in saying that individual antibodies can only bind to one antigen?) However, when I read sentences like "It has been estimated that humans generate about 10 billion different antibodies, each capable of binding a distinct epitope of an antigen" on the Wikipedia page (for antibody) it does make it sound like it's one antibody per antigen? Is this the case? Or is it one antibody per epitope. Either way, to put this question in a nutshell:
Is it true to say that the same substance could bind with (and, therefore, be recognised by) more than one antibody, or not?
Also, if the answer is no, does that mean that antibodies with different binding affinities are classed as the same antibody or not?
Let's clarify the terms. An antigen is a molecule that can be associated with a particular substance (virus, pollen, dander.) When an immunoglobulin or antibody recognizes an antigen it binds to a specific epitope. An antibody recognizes an epitope using its paratope.
Some antigens have multiple epitopes; this means that different antibodies can simultaneously bind to them (if the epitopes are far-enough apart from each other so that binding to one doesn't preclude binding to the others.)
An example from my work is influenza (flu) hemagglutinin, which is a major viral coat protein. Here's a low-resolution structure showing a "head-binding" antibody fragment (actually three of them), and here's a similar structure showing a "stem-binding" antibody fragment. Our immune repertoire (and physiology) greatly predisposes us towards producing head-binding antibodies over stem-binders. Note that each structure has three antibody fragments bound in the same state. Because the head-binder and stem-binder antibodies are well-separated in space they can both be accommodated in a single complex (as is experimentally confirmed.) In this case, up to six antibodies, using two distinct paratopes, could simultaneously bind to a single antigen.
For matured (high affinity and high specificity) antibodies, each binds a specific epitope. The typical state of affairs is then explained "one antibody per epitope" which is overly simplistic because some epitopes can be recognized by multiple paratopes (which would be found on different antibodies.) So a given epitope can be recognized by no antibodies (a new antigen, or one that is poorly immunogenic) or by multiple ones.
Is it true to say that the same substance could bind with (and, therefore, be recognised by) more than one antibody, or not?"
Yes, that's true.
Its all binding kinetics - even a monoclonal antibody will bind many different antigens. The affinity/avidity will be different depending on the interactions. The target antigen may have the lowest known K_d, but antibodies will still bind to many many other antigens. This is why when doing research using antibodies you often have to "block" your specimen with something like BSA or milk to try and reduce the "non-specific" binding.
Polyclonal vs. Monoclonal Antibodies
Antibodies, also known as immunoglobulins, are secreted by B cells (plasma cells) to neutralize antigens such as bacteria and viruses. The classical representation of an antibody is a Y-shaped molecule composed of four polypeptides-two heavy chains and two light chains. Each tip of the "Y" 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. The ability of binding to an antigen has led to their ubiquitous use in a variety of life science and medical science. These antibodies can be classified into two primary types (monoclonal and polyclonal) by the means in which they are created from lymphocytes. Each of them has important role in the immune system, diagnostic exams, and treatments.
This overview will describe the synthesis of monoclonal and polyclonal antibodies, their differentiating properties, and their role in clinical diagnostics and therapeutics.
Fig 1. The structure of the antibody
Polyclonal Antibodies vs. Monoclonal Antibodies: Production.
Polyclonal antibodies (pAbs) are mixture of heterogeneous which are usually produced by different B cell clones in the body. They can recognize and bind to many different epitopes of a single antigen.
Polyclonal antibodies are produced by injecting an immunogen into an animal. After being injected with a specific antigen to elicit a primary immune response, the animal is given a secondary even tertiary immunization to produce higher titers of antibodies against the particular antigen. After immunization, polyclonal antibodies can be obtained straight from the serum (blood which has had clotting proteins and red blood cells removed) or purified to obtain a solution which is free from other serum proteins.
Fig 2. The process to generate the polyclonal antibody
Monoclonal antibodies (mAbs) are generated by identical B cells which are clones from a single parent cell. This means that the monoclonal antibodies have monovalent affinity and only recognize the same epitope of an antigen.
Unlike polyclonal antibodies, which are produced in live animals, monoclonal antibodies are produced ex vivo using tissue-culture techniques. The process begins with an injection of the desired antigen into an animal, often a mouse, multiple times. Once the animal develops an immune response, the B-lymphocytes are isolated from the animal’s spleen and fused with a myeloma cell line, creating immortalized B cell-myeloma hybridomas. The hybridomas, which are able to grow continuously in culture while producing antibodies, are then screened for desired mAb.
Fig 3. The process to generate the monoclonal antibody
Polyclonal Antibodies vs. Monoclonal Antibodies: Advantages and Disadvantages
Both polyclonal and monoclonal antibodies have their own advantages and disadvantages which make them useful for different applications.
Fig 4. The specificity of polyclonal antibodies and monoclonal antibodies
The advantages and disadvantages of polyclonal antibodies were mainly determined by their multi-epitope specificity. The key advantages and disadvantages are listed below:
- Short production time and low cost.
- Highly stable and tolerant of pH or buffer changes.
- High affinity. Since the antibodies bind to more than one epitope, they can help amplify the signal from target protein even with low expression level. This makes these antibodies ideal for immunoprecipitation and chromatin immunoprecipitation.
- Tolerant of minor changes of antigen. Polyclonal antibodies are less sensitive to antigen changes (slight denaturation, polymorphism, heterogeneity of glycosylation) than monoclonal antibodies.
- Prone to batch to batch variability.
- Multiple epitopes make it important to check immunogen sequence for any cross-reactivity.
The advantages and disadvantages of monoclonal antibodies were mainly based on their high specificity to the same epitope of an antigen. The key advantages and disadvantages are listed below:
- Highly specific recognition of only one epitope of an antigen
- Immortal hybridoma cell lines have the ability to produce unlimited quantities of antibodies
- High consistency among experiments
- Minimal background noise and cross-reactivity
- Excellent for affinity purification
- Developing a monoclonal takes time and requires high technical skills.
- They can produce large amounts of specific antibodies but may be too specific to detect in across a range of species.
- Vulnerable to the change of epitope. Even a slight change in conformation may lead to dramatically reduced binding capacity.
Polyclonal Antibodies vs. Monoclonal Antibodies: Diagnostic Studies
Which is better, a monoclonal or a polyclonal antibody? It depends on the different characteristics of monoclonal and polyclonal antibodies.
Polyclonal antibodies are ideal reagents in diagnostic assays and hemagglutination reactions due to their ability to recognize different epitopes of a target molecule. The best use of polyclonal antibodies is to detect unknown antigens. Polyclonal antibodies are used as a secondary antibody in immunoassays (e.g. ELISA, western blotting, microarray assays, immunohistochemistry, flow cytometry). Their role is to bind to different epitopes and amplify the signal, leading to better detection.
Monoclonal antibodies, in contrast, provide an unlimited source of antibody that is homogeneous and, once characterized, predictable in its behavior. Monoclonal antibodies are often used as primary antibodies in immunoassays due to their ability of specifically binding to a single epitope of an antigen. Through the use of clinical application, some of the disadvantages of using each type of antibody has been nullified. Companies can purify polyclonal antibodies to limit the degree of cross-reactivity in their assays. The combination of monoclonal antibodies leads to the capture of multiple epitopes and expanding its’ specificity.
Polyclonal Antibodies vs. Monoclonal Antibodies: Treatment
Where monoclonal antibodies have stood out in a clinical setting is their ability to find and target specific molecules. Monoclonal antibodies’ Fc regions are initially tagged with markers and are used to discover cellular surface components. This research promotes the monoclonal antibodies used as medicine. OKT3 (also called Muromonab) was first approved by FDA in 1985 as a specific transplant rejection drug for organ transplant patients preventing graft disease. Since then, forty-one other antibodies have been approved by FDA to fight cancers, rheumatoid arthritis, and asthma and other illnesses. Monoclonal antibodies are also being used as vectors to bring drug to the target cell (e.g. a cancerous cell). When the Antibody-drug conjugates meet the target cell, the drug is released and exerts its effect.
Polyclonal antibodies, in contrast, are not as adept as monoclonal antibodies at treating cancer cells due to their lack of specificity and a high degree of cross reactivity. Research is showing that polyclonal antibody therapy can be useful in the treatment of some diseases and as an immunosuppressant for transplant patients.
Lesson Explainer: Specific Immune Response: Antibodies Biology
In this explainer, we will learn how to describe the process of clonal expansion and antibody formation in response to an antigen.
Adaptive or specific immunity can be thought of as a “third line of defense” when it comes to fighting pathogens and illnesses. The first line of defense is the barriers present in the body, which are specialized to keep pathogens and foreign materials out. The second line of defense is the nonspecific immune response involving inflammation, phagocytic cells, and certain aspects of the complement system.
The first and second lines of defense are both part of the innate immune system. Innate immunity reacts to every infection or potential threat in the same way and is the immunity you were born with.
If an infection or pathogen makes it past these two lines of defense and persists in the body for a period of time, the adaptive immune system responds. Adaptive immunity is also referred to as “specific immunity” or “acquired immunity” because it is the part of the immune system that creates a response specific to the pathogen that needs to be eliminated. Adaptive immunity is the immunity you develop, or acquire, over time. A diagram showing the combined response of innate and adaptive immunity is shown in Figure 1.
Key Term: Specific Immunity (Adaptive or Acquired Immunity)
Specific immunity describes the antigen-specific immune response. Specific immunity is immunity that develops over time as a result of exposure to different pathogens.
The adaptive immune system is often divided into two categories: cell-mediated immunity and humoral immunity. Cell-mediated immunity is sometimes called T cell immunity because it mostly relies on the action of cytotoxic T cells.
Humoral immunity is also sometimes called B cell immunity because it primarily relies on the action of B lymphocytes, a type of cell that makes proteins called antibodies. Humoral immunity is also sometimes referred to as antibody immunity. However, the B cells do not act on their own and often need the assistance of several other types of lymphocytes to mount an effective immune response.
Key Term: B Lymphocytes (B Cells)
B lymphocytes are lymphocytes that mature in the bone marrow and are involved in the production of antibodies.
Key Term: Antibody (Immunoglobulin)
An antibody is a globular protein produced by B lymphocytes that is adapted to bind with a specific antigen.
Let’s begin by getting acquainted with B cells. Generally, B cells, also called B lymphocytes, are lymphocytes that reach maturity in the bone marrow. B cells have a special genetic makeup that allows them to each make a different kind of B cell receptor. The genes that code for B cell receptors are mixed up by genetic recombination so that many random varieties are produced, as you can see in Figure 2 below.
Figure 2 shows us how a single stem cell can differentiate into several different types of B cells with different antigens on their cell surface membrane through genetic recombination.
Since different B cell receptors are randomly produced, it is important to make sure that they will not react with the normal cells of the body. So, before the B cells leave the bone marrow, they are tested to make sure that they do not carry receptors against “self.” B cells that fail this test are eliminated.
Every B cell has only one kind of receptor on its surface. The B cell receptor is essentially an antibody that is embedded in the B cell’s membrane. Antibodies are globular proteins that bind to substances we call antigens.
An antigen is basically anything that can bind to an antibody, and most antigens trigger an immune response. Antigens can be molecules on the surface of cells, pathogens like bacteria or viruses, or other substances like toxins or parasites.
When antibodies are attached to the surface of a naïve, not yet activated, B cell, they are called B cell receptors or antibody receptors.
Key Term: Antigen
Antigens are substances that can trigger an immune response.
Key Term: B Cell Receptor (BCR)
A B cell receptor is an antibody attached to the cell membrane of a B lymphocyte. When a B cell receptor binds with a complementary antigen, it stimulates the B cell to begin activation.
Example 1: Recalling the Characteristics of B Lymphocytes
The figure shows a B lymphocyte with antibody (immunoglobulin) receptors on its surface membrane.
Which statement is not true?
- Each B cell will have a different type of antibody receptor on its surface.
- Each B cell has only one type of antibody receptor that binds to one specific antigen.
- The B cell antigen receptor has a similar structure to a soluble antibody.
- The B cell can bind to extracellular and intracellular antigens.
- The B cell antibody receptor is complementary to an antigen.
B cells, also called B lymphocytes, are cells involved in the specific, also called adaptive or acquired, immune response. This immune response is often divided into humoral and cell-mediated immunity. Humoral immunity relies on the action of B cells and is most effective against extracellular pathogens, whereas cell-mediated immunity relies on the action of cytotoxic T cells and is effective against intracellular pathogens. Both types of adaptive immunity work simultaneously to fight infections.
B cells have a special genetic makeup. The genes for antibodies are mixed up by some type of genetic recombination so that many random varieties of antibodies are produced. This means that each B cell has a different antibody on its cell surface, called a B cell receptor, and also means that each B cell has only one kind of antibody on its surface.
Antibodies are globular proteins that bind to substances we call antigens. Each antibody only binds to one kind of antigen. An antigen is basically anything that can bind to an antibody, and most antigens trigger an immune response. Antigens can be molecules on the surface of pathogens like bacteria or viruses, or other substances like toxins or parasites. Antibodies and B cell receptors are only able to bind with extracellular antigens, antigens found outside of the cells.
This tells us that the statement that is not true about B lymphocytes is that the B cell can bind to extracellular and intracellular antigens.
When a pathogen, such as a bacterium or a virus, enters the body and begins to multiply, the first step in humoral immunity is to find a B cell with antibodies that can bind to the antigens on the pathogen. Once a B cell comes into contact with an antigen complementary to its B cell receptor, it matures and becomes activated and is no longer referred to as naïve.
The immune system facilitates this process in several ways. Some phagocytic cells carry bits of the pathogen with them on their cell surface. These cells, such as neutrophils and macrophages, engulf the pathogen through a process called phagocytosis, a type of endocytosis. They then break down the pathogen and present recognizable parts, called antigens, on their cell surface attached to special proteins called the major histocompatibility complex, or MHC.
Key Term: Major Histocompatibility Complex (MHC)
Major histocompatibility complex is a protein that functions to bind processed antigens and present them on the cell surface for recognition by immune cells.
Cells that are able to present antigens to other cells in the immune system are called antigen-presenting cells, or APCs for short. An illustration of a phagocytic APC is shown in Figure 3.
Key Term: Antigen-Presenting Cell (APC)
An antigen-presenting cell is a type of immune cell that facilitates an immune response by showing antigens bound to MHC proteins on its surface to other cells of the immune system.
These cells travel to the lymph nodes of the immune system, where they interact with the lymphocytes housed there, including B cells. Alternatively, the pathogen itself may be present in the lymph fluid or in the blood that is filtered through the lymph node and may come into contact with immune cells that way.
When the antibody on the surface of a B cell binds with an antigen it recognizes, the B cell becomes activated. This activation occurs in one of two ways, one of which is shown in Figure 4 below.
Some B cells are simply activated due to the presence of a complementary antigen, which you can see occurring in Figure 4 above. Once the B cells bind with an antigen that matches their antibody, they are activated and differentiate into what we call plasma cells. These cells multiply rapidly and produce large amounts of antibodies, which are secreted from the cell into the body fluids.
Some B cells require what is called T cell-dependent activation. In this case, the antibody, or B cell receptor, on the surface of the B cell recognizes a compatible antigen, which triggers the B cell to engulf the antigen by phagocytosis. The antigen is then broken apart, and pieces are attached to a molecule called MHC, which stands for major histocompatibility complex. The MHC attached to the antigen is then presented on the surface of the B cell. A diagram illustrating a helper T cell interacting with a B cell is shown in Figure 5.
T lymphocytes called helper T cells have receptors that recognize and attach to this MHC-antigen complex. Helper T cells are characterized by a cell surface marker called CD4. They also have receptors called T cell receptors. These T cell receptors along with the CD4 cell surface molecules together recognize and attach to the MHC-antigen complex on the surface of B cells, which activates the helper T cells.
The activated helper T cells in turn release cytokines called interleukins that complete the activation of the B cell. This fully activates the B cell, which then begins to divide. Some of the activated B cells differentiate to become plasma cells. These plasma cells multiply rapidly and secrete large amounts of antibodies. The activated T cell also begins to multiply in order to activate more B cells.
Helper T cells can also be activated by antigens presented in an MHC-antigen complex on the surface of other phagocytic cells, such as macrophages. These T cells then release interleukins that can activate B cells that have antibodies bound to an antigen. The interleukins also activate other immune cells, such as cytotoxic T cells. In this way, helper T cells play a role in both humoral and cell-mediated immunity.
In this process of B cell activation, only B cells that possess antibodies that are able to bind to the antigens of the pathogen actively infecting the body are activated by cytokines. We call this clonal selection, which is illustrated in the first stage of Figure 6, where the B cell is activated.
The activated B cells differentiate into effector cells called plasma cells and multiply rapidly, resulting in the same antibody present on the original activated B cell. We call this process clonal expansion. You can see clonal expansion occurring as the activated B cell at the top of Figure 6 proliferates via mitosis.
We get these two terms from the fact that one version of a B cell is chosen or selected. Then, it is reproduced many times, or its population is expanded. All of these subsequent cells are clones of the first cell and produce the same antibody.
Key Term: Clonal Selection
Clonal selection is the process by which T cells and B cells with receptors that bind with specific antigens are selected for clonal expansion.
Key Term: Clonal Expansion
Clonal expansion is the production of daughter cells all arising from a single parent cell. In the clonal expansion of lymphocytes, all of the daughter cells are specific to the same antigen.
Each pathogen often possesses several surface molecules that function as antigens. In this case, more than one type of B cell may become activated and may go through clonal expansion so that more than one type of antibody is generated against the pathogen.
Example 2: Determining the Effect of HIV on Antibody Production
HIV infects and destroys T-helper cells. How would an HIV infection affect the body’s antibody response to a new bacterial infection?
- The antibody concentrations would rise higher and faster.
- Only one type of antibody would be produced.
- The same level of antibodies would be produced but it would take longer.
- Fewer antibodies would be produced by B cells.
- There would be no effect on the antibody response.
HIV stands for human immunodeficiency virus, which is a bloodborne pathogen that, over time, can lead to acquired immunodeficiency syndrome (AIDS). The term immunodeficiency describes a situation in which the immune system is not fully functioning. The question explains that HIV infects and destroys T-helper cells.
T-helper cells are a type of T lymphocytes, a type of white blood cell that plays a role in the immune system. T-helper cells play a role in both the humoral and the cell-mediated aspects of the specific, also called adaptive or acquired, immune response. They become activated when the T cell receptors on their surface recognize and bind with a specific antigen on a phagocytic antigen-presenting cell.
Once activated, T-helper cells go through the process of clonal expansion, increasing the population of activated T-helper cells that recognize a particular antigen. These cells, in turn, activate B cells and cytotoxic T cells. These cytotoxic T cells recognize host cells infected with the virus and destroy them.
B cells are cells that make antibodies. Antibodies are globular proteins that are specific to a particular antigen. This means that each type of antibody binds with only one type of antigen. B cells are often activated by cytokines released by activated T-helper cells before they differentiate into plasma cells that make and secrete large quantities of antibodies into the bloodstream.
Since HIV infects T-helper cells and destroys them, there are usually fewer T-helper cells available during an infection to become activated. Since there are fewer activated T-helper cells, there will also be fewer activated B cells. This means that there will be fewer antibodies produced since there will be fewer activated B cells and, therefore, fewer plasma cells that can produce them.
This means that an HIV infection will affect the body’s antibody response to a new bacterial infection because fewer antibodies would be produced by B cells.
The process of clonal selection and expansion of B cells explains where antibodies come from. Antibodies are proteins present on the surface of B lymphocytes that, once activated, are produced in large quantities and released into the blood and lymph. Let’s now answer the question of how antibodies allow the immune system to fight infections.
Once the right B cell is activated, it differentiates into what we call a plasma cell. This plasma cell is a type of effector B cell that makes and secretes the same antibodies that it had on its cell surface as a B cell. This secreted antibody is sometimes called a soluble antibody. The plasma cell multiplies rapidly, creating more cells that make the same antibody. Effector cells are cells that actively respond to a stimulus and initiate a change. These plasma cells produce this soluble antibody in large quantities and release it into the bloodstream.
Key Term: Plasma Cell (Plasmacyte, Plasma B Cell)
A plasma cell is a type of immune cell that makes large amounts of a specific antibody. Plasma cells are effector cells that develop from B cells that have been activated.
Example 3: Recalling the Immune Cells that Secrete Antibodies
What type of immune cells secrete antibodies?
- Memory cells
- T-helper cells
- Plasma cells
Antibodies, also called immunoglobulins, are globular proteins that bind to one specific antigen. Antibodies are made by B cells. Immature B cells have antibody receptors, also called B cell receptors. These B cell receptors are antibodies attached to the cell membrane of B cells. When the B cell receptor binds with a complementary antibody, the B cell becomes activated. B cells may also require additional signaling from helper T cells to become activated. Once the B cell is activated, it begins to proliferate. This creates a population of B cells that all possess B cell receptors that recognize a particular antigen. The mature, activated B cells then differentiate into one of two cell types. Some become memory B cells, which live for a long time in the immune system, ready to rapidly activate if stimulated by the same antigen in the future. The majority differentiate into plasma cells. Plasma cells are B lymphocytes that, instead of surface-bound antibody receptors, generate large quantities of soluble antibodies that they secrete into the bloodstream and other body fluids.
This means that the type of immune cells that secrete antibodies is plasma cells.
The soluble antibodies bind to extracellular antigens wherever they find them. This can be on the surface of the pathogen or on the surface of infected host cells. Antibodies cannot enter cells because they are relatively large proteins. Antibodies are only effective against extracellular antigens, or antigens found outside of host cells.
Generally, antibodies are very specific. Each antibody binds with only one type of antigen. However, antibodies are extremely well adapted to destroy and remove anything bearing the antigens they attach to. This is why antibodies targeting “self” antigens are very dangerous and is also why immature B cells must be screened and carefully selected.
Let’s have a look at some of the many ways that antibodies can fight infections.
The antibodies attached to the surface of a pathogen cause macrophages to engulf the pathogen. If the pathogen is soluble, for example, a type of toxin, the antibodies can make it insoluble. This process is called precipitation. This makes it easier for macrophages to identify and phagocytose the pathogen.
Antibodies can also cause the pathogens in the blood to clump together, a process called agglutination, which makes them easier to locate and destroy. Antibodies can trigger the complement system to create a chemical complex that can lyse the cell membrane of the pathogen. This causes the pathogen to break apart, which kills it, and the parts are removed by phagocytes.
Finally, antibodies can neutralize a pathogen, such as a virus, by coating its surface and preventing it from entering cells to infect them, or by preventing it from releasing its genetic material once inside.
Once our immune system responds to an initial infection, how do we retain this immunity to the same pathogens if they infect our bodies again?
Activated B cells differentiate into plasma cells, which can produce and secrete the large quantities of antibody needed to fight an infection. Activated B cells also differentiate into what we call memory B cells. Figure 7 illustrates how B lymphocytes differentiate into plasma cells and memory B cells.
Memory B cells do not secrete antibodies. They have antibodies on their cell surface, like naïve B cells. Memory B cells are dormant cells that live in the immune system. These cells are specialized to have an especially long lifespan. They are adapted to allow the immune system to more rapidly respond to a second infection by the same pathogen.
Key Term: Memory B Cell
A memory B cell is a cell formed following a primary infection. Memory B cells have a long lifespan and are able to mount a rapid response upon secondary infection.
Example 4: Describing the Difference between Memory Cells and Plasma Cells
How are memory cells different from plasma cells?
- Memory cells are no longer able to multiply and differentiate.
- Memory cells have only one type of antibody receptor on their cell surface.
- Memory cells have a higher rate of protein synthesis.
- Memory cells remain in the circulation for a longer time period.
- Memory cells are able to respond to a wider range of antigens.
The adaptive immune system is often divided into two categories: cell-mediated immunity and humoral immunity. Cell-mediated immunity is sometimes called T cell immunity because it mostly relies on the action of cytotoxic T cells. Humoral immunity is also sometimes called B cell immunity because it primarily relies on the action of B lymphocytes, a type of cell that makes proteins called antibodies.
Antibodies are globular proteins that bind with one specific antigen. Immature B cells have antibody receptors, also called B cell receptors. These B cell receptors are antibodies attached to the cell membrane of B cells. When the B cell receptor binds with a complementary antibody, the B cell becomes activated. B cells may also require additional signaling from helper T cells to become activated.
Once the B cell is activated, it begins to reproduce. This creates a population of B cells that all possess B cell receptors that recognize a particular antigen. This process is called clonal selection. The mature, activated B cells then differentiate into one of two cell types. Some become memory B cells, which live for a long time in the immune system, ready to rapidly activate if stimulated by the same antigen in the future. The majority differentiate into plasma cells. Plasma cells are B lymphocytes that, instead of surface-bound antibody receptors, generate large quantities of soluble antibodies that they secrete into the bloodstream and other body fluids.
Using this information, we can conclude that memory cells are different from plasma cells because memory cells remain in the circulation for a longer time period.
Our body makes memory cells because if an organism is exposed to a pathogen once, it is likely to encounter it again. The presence of memory cells means that, upon second or subsequent exposure, the immune system can react more rapidly, and we may not even notice or become ill in the process.
Humoral and cell-mediated immune responses are often described separately, but they occur simultaneously within the body. Many aspects of the humoral immune response impact the cell-mediated response, and vice versa. These two types of immune response are both facets of the specific, or acquired, immune system.
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Can more than one antibody bind the same antigen - Biology
Enzyme-linked Immunosorbent Assay (ELISA)
Enzyme-linked immunosorbent assay (ELISA) is a plate-based assay technique designed for detecting and quantifying peptides, various proteins and hormones. As its name indicates, ELISA involves the specific antigen-antibody binding and the use of enzymes. In an ELISA, an antigen (or antibody) must be immobilized to a solid surface. After incubating with specific antibody (or antigen), enzyme-conjugated antibody will be added. Detection is finally accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measurable product.
ELISA is typically performed in 96-well polystyrene plates, which will passively bind antibodies and proteins. Binding and immobilization of reagents make ELISA easier to design and perform. Having the reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound from non-bound material by repeated washing steps. This ability to wash away nonspecifically bound agents makes the ELISA a powerful tool for measuring specific analytes within a crude preparation.
According to how it works, ELISA can be divided into four major types, namely direct, indirect, sandwich, and competitive ELISA. Advantages and disadvantages of four major ELISA types are illustrated as follows.
- Simple protocol.
- Time-saving, and reagents-saving.
- No cross-reactivity from secondary antibody.
- High background.
- No signal amplification, since only a primary antibody is used.
- Low flexibility, since the primary antibody must be labeled.
- Signal amplification, since one or more secondary antibodies can be used to bind to the primary antibody.
- High flexibility, since the same secondary antibody can be used for various primary antibodies.
- Complex protocol compared with direct ELISA.
- Cross-reactivity from secondary antibody.
- High flexibility.
- High sensitivity.
- Best for the detection of small antigens, even when they are present in low concentrations.
- Relatively complex protocol.
- Needs the use of inhibitor antigen.
- High flexibility.
- High sensitivity.
- High specificity, since different antibodies bind to the same antigen for detection.
- The antigen of interest must be large enough so that two different antibodies can bind to it at different epitopes.
- It's sometimes difficult to find two different antibodies that recognize different epitopes on the antigen of interest and cooperate well in a sandwich format.
Due to the specific needs of the experiment, the availability of reagent materials as well as limitations and defects of the method itself, many other detailed and specific ELISA modes are derived on the basis of these four models. Here is the summary about types of ELISA.
In addition to the ELISA types above, there are other ELISA types that help meet the various demands of experiments. For example:
1. ELISPOT, enzyme-linked immunospot, is used to measure the frequency of protein-secreting cells at the single-cell level. The technique that ELISPOT uses is very similar to that of sandwich ELISA.
2. In-cell ELISA is used to measure the levels of the target protein within cells that are fixed on the plate. It also involves the use of the technique used by sandwich ELISA. Cells are fixed to the plate and are permeabilized. Next, a primary antibody is added to react with the target protein within the cells. Lastly, a labeled secondary antibody is added to react with the primary antibody. In this way, the target protein within cells is detected.
Difference Between B Cell Receptor and Antibody
B cell receptor refers to an immunoglobulin molecule which serves as a type of transmembrane protein on the surface of B cells while an antibody refers to a blood protein that the B cells produce in response to and counteracting a specific antigen. Thus, this is the main difference between B cell receptor and antibody.
While a B cell receptor is a type of membrane-bound immunoglobulin, an antibody is a type of a secreted immunoglobulin.
The only structural difference between B cell receptor and antibody is the presence of C-terminal, hydrophobic region in the heavy chains to serve as transmembrane domains and the presence of another transmembrane domain for the signal transduction in the B cell receptors. Antibodies do not contain such transmembrane domains.
The two types of B cell receptors expressed by a mature B cell are IgD and IgM while the five classes of antibodies are IgA, IgD, IgE, IgG, and IgM.
Another difference between B cell receptor and antibody is that the B cell receptors bind with a specific antigen to activate the B cell while antibodies can bind to the antigen and elicit immune responses through the complement pathway and recruit other immune cells to destroy the pathogen.
B cell receptor is the type of immunoglobulin that a particular clone of B cells produce in response to a particular pathogen. These immunoglobulins are not secreted into the circulation but, they are inserted into the cell membrane. They bind to their specific antigen and the antigen-bound B cell receptors are processed and presented again to the T cells. On the other hand, antibodies are the immunoglobulins that secrete into the circulation. Their main function is to neutralize the antigens in order to destroy them by recruiting other immune cells or elicit an immune response through the complement system. In conclusion, the main difference between B cell receptor and antibody is their significance and role in the immune system.
1. Treanor, Bebhinn. “B-Cell Receptor: From Resting State to Activate.” Immunology 136.1 (2012): 21–27. PMC. Web. 9 Oct. 2018. Available Here
2. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science 2002. B Cells and Antibodies. Available Here
1. “Figure 42 02 06” By CNX OpenStax – (CC BY 4.0) via Commons Wikimedia
2. “Antibody” By Fvasconcellos 19:03, 6 May 2007 (UTC) – Color version of Image:Antibody.png, originally a Work of the United States Government (Public Domain) via Commons Wikimedia
3. “Antibody action” By Becky Boone (CC BY-SA 2.0) via Flickr
About the Author: Lakna
Lakna, a graduate in Molecular Biology & Biochemistry, is a Molecular Biologist and has a broad and keen interest in the discovery of nature related things
Affinity, Avidity, and Cross Reactivity
Not all antibodies bind with the same strength, specificity, and stability. In fact, antibodies exhibit different affinities (attraction) depending on the molecular complementarity between antigen and antibody molecules, as illustrated in Figure 23.25. An antibody with a higher affinity for a particular antigen would bind more strongly and stably, and thus would be expected to present a more challenging defense against the pathogen corresponding to the specific antigen.
Figure 23.25. (a) Affinity refers to the strength of single interaction between antigen and antibody, while avidity refers to the strength of all interactions combined. (b) An antibody may cross react with different epitopes.
The term avidity describes binding by antibody classes that are secreted as joined, multivalent structures (such as IgM and IgA). Although avidity measures the strength of binding, just as affinity does, the avidity is not simply the sum of the affinities of the antibodies in a multimeric structure. The avidity depends on the number of identical binding sites on the antigen being detected, as well as other physical and chemical factors. Typically, multimeric antibodies, such as pentameric IgM, are classified as having lower affinity than monomeric antibodies, but high avidity. Essentially, the fact that multimeric antibodies can bind many antigens simultaneously balances their slightly lower binding strength for each antibody/antigen interaction.
Antibodies secreted after binding to one epitope on an antigen may exhibit cross reactivity for the same or similar epitopes on different antigens. Because an epitope corresponds to such a small region (the surface area of about four to six amino acids), it is possible for different macromolecules to exhibit the same molecular identities and orientations over short regions. Cross reactivity describes when an antibody binds not to the antigen that elicited its synthesis and secretion, but to a different antigen.
Cross reactivity can be beneficial if an individual develops immunity to several related pathogens despite having only been exposed to or vaccinated against one of them. For instance, antibody cross reactivity may occur against the similar surface structures of various Gram-negative bacteria. Conversely, antibodies raised against pathogenic molecular components that resemble self molecules may incorrectly mark host cells for destruction and cause autoimmune damage. Patients who develop systemic lupus erythematosus (SLE) commonly exhibit antibodies that react with their own DNA. These antibodies may have been initially raised against the nucleic acid of microorganisms but later cross-reacted with self-antigens. This phenomenon is also called molecular mimicry.
A person’s blood becomes more potent over time as that person prevails and overcomes pathogens and parasites with its newly formed antibodies in high numbers. Antibodies have the secret locked away to defeat the germ that has invaded. The second infection usually has minimal harm hence, the wise blood has defeated the enemy. When a microbe pathogen or parasite invades the blood, the immune system goes to work. The immune system acts like a Star Wars anti-missile action (Behe 1996). The first action is to recognize the invader. Bacteria have to be distinguished from blood cells, viruses from connective tissue, malaria parasites from liver and spleen cells, etc. Unlike microbiologists, they cannot look at them under a microscope rather, they have to rely on a chemical sense of “taste/smell/touch.” They have “fingers” of antibody arms. This is part of the fearfully and wonderfully made design of the human body.
The body must solve the problem of binding to a specific invader with a match of an estimated 1 in 100,000. There may be billions or trillions of antibody types possibly needed in a lifetime.
There may be billions of different kinds of antibodies. Each antibody is made in a separate B lymphocyte cell. Once made, a factory of antibodies is generated in many plasma cells, and memory cells recall the information for long-term storage of remembering what was encountered. Immunization derives from the brilliant pioneering work of Edward Jenner and Louis Pasteur: they solved the time problem of a quick and efficient immune response for the body. Exposing the body to a weakened or “killed” pathogen shrinks the time for the secondary response of IgG, allowing the body to flood the battle scene with prepared antibodies and overwhelm intruders. The immune response, crafted wisely, was engineered with a mitigating mercy in light of a fallen world. It remembers “germs” and responds swiftly with a counter defense that prolongs life.
Clonal Selection of Antibody-Producing Cells
The clonal selection hypothesis is a widely accepted model for the immune system’s response to infection.
Describe the clonal selection hypothesis in regards to the production of B cells
- In 1954, immunologist Niels Jerne put forth the hypothesis that there is already a vast array of lymphocytes in the body before 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 it.
- B cells exist as clones derived from a particular cell. Thus the antibodies and their differentiated progenies can recognize and/or bind the same specific surface components composed of biological macromolecules of a given antigen. Clonality has important consequences for immunogenic memory.
- The clonal selection hypothesis states that an individual B cell expresses receptors specific to the distinct antigen, determined before the antibody ever encounters the antigen.
- clonal selection: An hypothesis which states that an individual lymphocyte (specifically, a B cell) expresses receptors specific to the distinct antigen, determined before the antibody ever encounters the antigen. Binding of Ag to a cell activates the cell, causing a proliferation of clone daughter cells.
- clone: A group of identical cells derived from a single cell.
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.
A schematic view of clonal selection: Clonal selection of lymphocytes: 1) A hematopoietic stem cell undergoes differentiation and genetic rearrangement to produce 2) immature lymphocytes with many different antigen receptors. Those that bind to 3) antigens from the body’s own tissues are destroyed, while the rest mature into 4) inactive lymphocytes. Most of these will never encounter a matching 5) foreign antigen, but those that do are activated and produce 6) many clones of themselves.
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.
Hematopoiesis in Humans: This diagram shows hematopoiesis as it occurs in humans.
Role of Antibodies and Antigens | Human Immunology
Humoral immunity is antibody-mediated. It protects body from extracellular pathogenic agents by combining with them to form antigen-antibody complex, leading eventually to their elimination. The antibodies play at least in three different ways in this process of elimination of an antigenic agent.
Firstly, the antibody can bind to the surface epitopes of the antigen making it more susceptible to phagocytosis. This is known as opsonization.
Secondly, the antibody molecule can bind to the antigen forming an antigen-antibody complex which then combines with the complement in a step-wise manner to initiate and facilitate phagocytosis of the antigen.
Thirdly, the antibody can bind to toxin molecules elaborated by microbes making them non-toxic. In a similar manner, antibodies can inactivate free virus particles by combining with the epitopes on viral particles to make them incapable of attachment to host cell membranes.
In opsonization, the Fc-domain of the antibody molecule binds to the receptors on phagocytes and the antigen-binding sites to the epitopes of antigen. Thereby the antigen such as a microbial cell is brought close enough to the phagocytic cell, so that it can be ingested and destroyed by the contents of the phagolysosomes.
Opsonization by antibodies is more effective than by complement (C3b) in case of encapsulated bacterial cells. The capsular material, mostly polysaccharides, prevents interaction with the complement C3b. In contrast, these polysaccharides provoke immune response to produce antibodies that can bind specifically to the capsule, so that the bacterial cells can be effectively opsonized and phagocytosed. Opsonization by antibodies is schematically represented in Fig. 10.25A.
There is another mode of destroying antigens by antibodies which is known as antibody dependent cell-mediated cytotoxicity (ADCC) (Fig. 10.25B). By this method, virus-infected body cells and large parasites which cannot be ingested by phagocytes are eliminated.
The antibodies bind with their antigen-binding sites to the virus infected cells or large parasites by recognizing viral epitopes expressed on the body cells, or the natural epitopes of large parasites, like a helminth. Then the Fc-domains of the antibodies bind to the receptors on phagocytic cells, like neutrophils. Release of toxic chemical mediators from lysosomes of the phagocytes kills the infected body cell or the parasite.
Antibodies can neutralize the toxins liberated by bacteria, like Clostridium tetani, Corynebacterium diphtheriae, Vibrio cholerae etc. by combining with them, so that the toxins are no longer able to bind to their target cells. The antibodies produced in response to toxins are called antitoxins. Exotoxins are generally proteins and endotoxins mostly lipolysaccharides, both of which are strongly antigenic (Fig. 10.25C).
Another major role of antibodies of the IgG and IgM classes is activation of complement by the classical pathway. The role of the secretory form of IgA antibodies in the mucous membranes is to trap any microbial or viral antigen that come in contact with mucin.
The IgA antibodies are anchored in mucin with their Fc domains while their antigen-binding sites remain free to trap any incoming microbe or viral entity, thereby preventing its entry into the deeper layers of the membrane (Fig. 10.25D).
An important feature of all classes of immunoglobulin’s is that any one antibody molecule, whether it is monomeric, dimeric or pentameric, can recognize a single antigenic determinant. It follows, therefore, that for each of the thousands of such determinants which can possibly exist in thousands and thousands of antigens, there is a cognate antibody in the human body.
Also, such an antibody or the antibody producing cell must be present before the body encounters the antigen. In other words, the human body is endowed with the potentiality to produce thousands of antibodies, each having specificity for a different antigenic determinant, to meet the challenge of any antigen that may come across during the lifetime.
As antibodies are proteins, the sequences of their amino acids are determined by DNA. The basis of the great diversity of antibodies is genetical.
While discussing the origin of blood cells, it has been seen that majority of B-lymphocytes produced from stem cells in bone marrow which possess receptors capable of interacting with self- antigens are eliminated by apoptosis and the surviving B-cells which have receptors that do not bind to self-antigens migrate to the secondary lymphoid organs. It is in these organs that the B-cells come to meet the non-self or foreign antigens for the first time.
The question that confronts immunologists at this point is: How — from a large and diverse population of B-lymphocytes each carrying a specific receptor — only those are selected by the antigen which carry specific receptors appropriate to their antigenic determinants?
The outline of the underlying principle of this process was first proposed by Burnet and Medawar in the 1950s in their clonal selection theory (Burnet and Medawar were awarded Nobel Prize in 1960). According to this theory, an immunologically responsive cell can respond to only one kind of antigen (precisely one antigenic determinant or epitope) and this property is acquired before the particular cell encounters the antigen.
Clonal Selection Theory:
It has been estimated that the human body can develop immune response against as many as 100 million or more different antigenic determinants which means that the body can produce as many different clones of B-lymphocytes each carrying a specific receptor. The receptors of B-cells are antibodies which bind to the antigenic determinants.
The clonal selection theory explains how from the multitude of different B-cells, the antigenic determinants select only those which carry appropriate receptors. The basis of this theory is diagrammatically explained in Fig. 10.26.
The diagrammatic representation of clonal selection in Fig. 10.26 is over-simplified. A more precise and modern version of clonal selection is discussed below. In the figure (Fig. 10.26), after capturing an antigenic determinant, the appropriate (cognate) B-cell is activated.
Activation of B-Cells:
For activation of B-cells to proliferate and differentiate into plasma cells, binding with cognate antigenic determinants carried, by antigens is not enough. For majority of antigens, participation of T-cells of the T-helper class (TH-cells) is also essential. Such antigens are, therefore, known as T-dependent. Certain antigens, however, can stimulate B-cells directly without the help of these T-cells and they are known as T-independent antigens.
(a) T-dependent pathway:
The mature B-cells, when they first meet the antigens in the secondary lymphoid organs like lymph nodes and spleen, have receptors in the form of IgM and IgD on their surfaces. The antigen selects the B-cells with cognate receptors.
The selected B-cell then acts like an antigen-presenting cell in that it internalizes the antigen together with the receptor molecules into its cytoplasm and displays the epitopes complexed with MHC class II proteins on its surface. This behaviour of B-cells is unique and is essential for binding to the TH-cells for further activation, because the T-cells are unable to recognize antigens unless they are presented along with MHC proteins.
In the T-dependent B-cell activation, binding of the antigen to the B-cell receptor constitutes the initial step which provides the first signal for activation, but is not adequate. The second signal is provided by its binding to an appropriate TH-cell with its T-cell receptor and the MHC Class II proteins with the epitope embedded in it. The binding between the B-cell and TH-cell is further stabilized by other ligands of both partners.
The bound TH-cell then secretes cytokines, like interleukin-2 which stimulate the B-cell to proliferate and differentiate into antibody-secreting plasma cells (Fig. 10.27). But the picture is not yet complete, because the TH-cell that can bind to the appropriate B-cell needs also to be activated.
The activation of TH-cells is effected by its interaction with an antigen-presenting cell, like a macrophage. Such a macrophage, in the meantime, has engulfed the same antigen which had selected the B-cell and displayed the epitopes complexed with its MHC Class II proteins on its surface. A TH-cell now binds to the macrophage with its T-cell receptor.
This binding is also helped by other proteins ligands, specially the CD4 protein of the TH-cell and the MHC protein of the macrophage. The macrophage secretes cytokines like interleukin-1 which stimulates the TH-cell to become activated and to proliferate producing a clone of activated TH-cells (Fig. 10.28).
One of these activated TH-cells then binds to the activated B-cell and the B-cell is stimulated to become larger in size and to divide producing a clone of antibody-producing plasma cells. A tight binding not only stimulates the B-cell, but also co-stimulates the TH-cell to secrete cytokines, like interleukins 2, 4 and 5 which play vital roles in activation of B-cells.
The plasma cells are short-lived, having a life-span of 3 to 4 days, but they are extremely efficient in antibody synthesis, producing some 2,000 Ig molecules per second. The antibody molecules are secreted into circulatory system. Not all of the activated B-cells are transformed into plasma cells, but a small proportion (about 5%) circulates as memory cells.
An overall picture of T-dependent pathway of B-cell activation leading to antibody production is shown in Fig. 10.29:
(b) T-independent pathway:
There are some antigens which can bind directly to B-cells to induce them to produce antibodies. They do not require participation of TH-cells and, hence, are known as T-independent antigens. These antigens are mostly bacterial surface polysaccharides, like those forming the capsule. The antibodies produced in response to these polysaccharides bind with the capsule to opsonize capsulated bacteria, thereby facilitating ingestion by the phagocytic cells of the body.
T-independent activation of B-cells proceeds via a pathway which is different from that of T-dependent pathway. At high concentration, the polysaccharide antigens can directly interact with the B-cells and activate them to produce antibodies.
The polysaccharide molecules consist of repetitive units. With the help of these units, they can bind to B-cells irrespective of their specific antigenic receptors. The antigen-bound B-cells are induced to proliferate and produce antibody (Fig. 10.30). The T-independent antigens generally induce a weaker response than T-dependent ones. Also no memory cells are the produced in the T-independent pathway.
Interactions Between Antigens and Antibodies:
In humoral immunity, antibodies interact with antigens by their antigen-binding sites to form antigen-antibody complexes. Such complex is formed by actual combination of the two components. An antigen, whether it is a protein or polysaccharide or any other antigenic molecule, or a particulate body, like a microbial cell, always contains a number of antigenic determinants each of which can bind to the antigen-binding site of a cognate antibody.
On the other hand, an antibody molecule has two or more antigen-binding sites. Thus both antigen and antibody are mutually multivalent. When they meet each other — either in vitro or in vivo — each antigen is linked to more than one antibody and, in turn, one antibody is linked to more than one antigen. Through a series of bimolecular interactions, antigens and antibodies produce a lattice or framework with alternating antigens and antibodies leading to formation of an antigen-antibody complex.
The characteristic features of an antigen-antibody complex are discussed below:
(a) Antigen-antibody complex:
The basic principles governing antigen-antibody interaction can be best understood in a simple in vitro system when a soluble antigen is treated with its antiserum (serum containing the specific antibody). The antigen-antibody reaction results in a complex that builds up a lattice which grows in size and is eventually precipitated. When such precipitate is analysed, the proportion of antigen and antibody present in it can be quantitatively estimated.
If, in such an antigen-antibody reaction mixture, the quantity of antigen is gradually increased, keeping the quantity of antibody fixed, and the quantity of precipitate and that of the antibody in the precipitate are determined, it is observed that both the quantities increases gradually until a maximum is reached. With further increase of antigen, the quantity of antibody in the precipitate decreases.
A graphical representation of the antibody present in the precipitate as a function of antigen concentration produces a more or less normal curve as shown in Fig. 10.31:
It is seen from the figure that the precipitate of antigen-antibody complex does not contain a fixed proportion of the two components, as it is expected by the law of fixed proportions applicable to the normal chemical reactions. Rather, the relative proportions of antigen and antibody in the precipitated complex depend on the concentrations of the reactants in the reaction mixture.
Thus, when the antigen concentration in the mixture is low, all the antibody-binding sites of the antigen molecules are engaged, but the antigen-binding sites of all the antibodies are not engaged. This is the antibody excess zone.
As the antigen concentration is gradually increased, more and more of antigen-binding sites of antibodies become engaged. Ultimately, all such sites of the antibody molecules become bound to antigens. This is represented by the equivalence zone.
With further increase of antigen, the binding-sites of all antibodies are engaged but some antigen molecules do not find sites to bind. This is represented by the antigen excess zone of the curve. The supernatant fractions of the reaction mixtures do not contain any free antigen in the first phase (antibody excess zone), neither antigen nor antibody in the second phase (equivalence zone) and no antibody in the third phase (antigen excess zone).
Antigen-antibody complex forming a lattice can be diagrammatically represented as shown in Fig. 10.32:
Antigen-antibody reaction in vivo also results in the formation of complexes, similar to those formed in vitro. At the outset of an infection, only the antigen is present. Antibodies are then produced by plasma cells as a result of immune response. Gradually, antibody concentration increases due to continued production. Interaction of antibody and antigen leads to formation of large complexes as in the equivalence of the in vitro system.
These complexes are generally removed effectively by the phagocytic activity of the polymorphonuclear cells, like neutrophils. Thus, the antigen is destroyed. With the elimination of the antigen, antibody formation slows down due to the absence of antigenic stimulations of the B-cells and finally stops, because the plasma-cells die within 4 to 5 days. Only the memory cells produced in the immune response continue to circulate in the body and can initiate another immune response promptly if the same antigen happens to attack the body.
(b) Antibody-mediated complement activation:
A very important aspect of antigen-antibody interaction is the complement activation by the classical pathway. The complement is a set of constitutive serum proteins which, on activation, performs several important functions including destruction of cellular antigens. Complement activation by the alternative pathway does not involve antibodies, but the classical pathway is dependent on antigen-antibody interaction.
Not all classes of antibodies, but only IgG and IgM can bind complement to their Fc-domain. Such binding can only occur when the antibodies have formed complex with the antigen. Binding of complement to the antigen-antibody complex leads to complement activation by the classical pathway. Such activation eventually leads to lysis of the target cell carrying the antigen. The binding of complement to antigen-antibody complex is known as complement fixation.
In complement activation by the alternative pathway, the components of complement are activated sequentially. This happens also in case of activation by the classical pathway. Complement activation by this pathway begins with binding of an IgM molecule or two adjacent IgG molecules to the antigenic determinants on the surface of the target cell. The target cell is thereby sensitized.
The first complement component, Clq, which is a subcomponent of CI, then binds to the Fc-domain of the heavy chain(s) of the antigen-bound antibody. Clq does not require to be activated, because it has a stable binding site with which it can bind directly to the antibody molecule (Fig. 10.33).
The bound Clq then activates the other subcomponents of CI complex which become catalytically active to cause proteolytic cleavage of the components C2 and C4 to produce C2a and C2b, and C4a and C4b. The fragments C4b and C2a then bind to the target cell forming an enzyme, C3-convertase, which catalytically cleaves C3 component into C3a and C3b.
Next, C3b binds to the target cell surface to form a cell bound complex containing Cl-C4b-C2a-C3b. This complex then activates the binding of the complement components C5, C6 and C7 of which the C5 component is cleaved into C5a and C5b. The fragment C5b is incorporated into the complex which is now Cl-C4b-C2a-C3b-C5b-C6-C7.
Finally, the components C8 and C9 are also added to form the membrane attack complex. This complex is responsible for formation of pores in the membrane of the target cell through which cell contents come out and the cell undergoes lysis. The sequential activation of the complement cascade is shown in Table 10.6 and diagrammatically in Fig. 10.34:
Besides causing cell lysis, the activated complement exert other effects. One important effect is opsonization by the complement fraction C3b which binds both to specific receptors on phagocytic cells and the microbial cell facilitating phagocytosis.
It has been estimated that human body can produce approximately one billion (10 9 ) different antibodies, each having the ability to interact with a specific antigenic determinant. It is also known that antibodies are, proteins of the class immunoglobulin’s and that like other proteins they are synthesized from m-RNA coded by DNA.
The variable domains of H and L polypeptide chains of Ig molecules possess amino acid sequences which are different in each antibody and determine its antigen-binding specificity. The problem that baffled immunologists for a long time is how the human organism having some 100,000 (10 5 ) genes manages to produce 10 9 different proteins.
Dreyer and Bennett (1965) suggested for the first time that the genes encoding the variable and constant domains of Ig molecules are separate in DNA. This meant that the two parts (V arid C) of the same polypeptide molecule are coded by DNA segments which were not continuous. This was not in conformity with the accepted postulate.
The mystery of antibody diversity was solved by Tonegawa and Hozumi when they worked out the mechanism of somatic recombination in 1976 (Tonegawa was awarded the Nobel Prize in 1987). It was shown that the DNA’ segments of H and L chains of Ig molecules were located in different chromosomes and that for each H and L chains were are a number of genes encoding the different domains.
These genes are shuffled to produce a unique set of genes for different H and L chains. The H and L chains are synthesized separately and joined later to produce a monomeric Ig molecule consisting of two identical H chains and two identical L-chains.
The process by which different DNA segments are joined to produce a gene encoding an H or L chain is somatic recombination. This occurs in the embryonic cells when B-cells are differentiated from stem cells. The salient features of antibody-diversity according to the currently accepted views are briefly discussed below.
The genes encoding immunoglobulin’s are distributed in three human chromosomes, viz. genes for H-chains are located in human chromosome 14, those for k-chain (L-chain) in chromosome 2 and those of λ-chain (L-chain) in chromosome 22.
In the DNA of embryonic cells (germ-line DNA) there are many copies of genes encoding the V-domains of H and L chains. The coding segments of these genes are not continuous, but interrupted by non-coding segments (introns).
Besides the genes controlling V and C domains of Ig molecules, other genes code for the joining segments (J) and diversity segments (D).
Thus, an H-chain and an L-chain consist of die following segments:
H-chain: V segment – D segment – J segment – C segment
L-chain: V segment – J segment – C segment.
In the embryonic cells from which B-cells arise, the germ-line DNA contains multiple copies of the genes controlling the V, J, D and C segments. From these genes, one from each segment is selected at random and joined to each other by somatic recombination to yield the DNA of a B-cell. This is known as gene rearrangement or gene translocation. Gene rearrangement of the H-chain is accomplished first and then that of the L-chain is undertaken.
Again, of the two classes of L-chains, rearrangement of the k-chain is taken up first. In case rearrangement of the k-chain DNA fails, then only that of λ-chain is rearranged. As the genes of k-and λ-chains are located in different chromosomes, successful recombination of one chromosome prevents rearrangement of the other chromosome. Thus, it is seen that gene rearrangement in the differentiating B-cells proceeds in a specific order.
In the germ-line DNA, there are some 200 (or more) genes controlling the V segment of the H-chain of Ig molecule, located in chromosome 14. There are also 5 genes for D segment, 9 genes for J-segment and 9 genes for C segment, all located in chromosome 14. Similarly, for the kappa L-chain, there are 80 V genes, 5 J genes and one C gene, located in chromosome 2. The lambda L-chain is also controlled by multiple copies of V genes, J genes and one C gene.
These are shown in Fig. 10.35:
Random selection from some 300 immunoglobulin genes can produce an immensely large number of different antibodies. Thus, the main source of antibody diversity is somatic recombination, though two other factors contribute to it.
A simplified diagrammatic representation of somatic recombination of randomly selected gene segments is shown in Fig. 10.36:
(a) Somatic gene recombination:
In case of L-chains e.g. a kappa chain, recombination starts with joining of one of the VK genes with one of the JK genes, by a process called V-J joining. There are 80 VK genes and 5 JK genes for K-chain. Any one VK gene can be moved to any JK gene. This is controlled by highly conserved nucleotide sequences flanking the genes.
These sequences serve as recombinational signal. These sequences have been determined to be a heptamer and a nonamer separated by a 12 base-pair sequence (spacer) in case of the VK segments and a 23 base-pair spacer in case of JK segment. This unit i.e. a heptamer-spacer-nonamer serves as recognition signal and may precede or follow the coding sequences of the VK and JK genes. These recombinational signal sequences pair with each other and are cleaved to join VK and JK segments. Such pairing of the recombinational signal sequences is possible because the heptamer sequences and the nonamer sequences are exact inverted repeats.
This is diagrammatically shown in Fig. 10.37:
The actual joining of a VK and a JK gene is catalysed by the enzyme recombinase which is formed by two proteins, RAG 1 and RAG 2 (recombination activity gene products). As a result of recombinase activity the two genes are recombined and at the same time, the intervening DNA segments are eliminated in the form of a circle by DNA splicing.
The recombinase nicks each DNA strand at its signal sequence. The nicked ends are rejoined to produce a DNA segment containing a V gene adjacent to a J gene. The recognition signals assure that a VK gene is joined always to a JK gene and not to another VK gene.
Though a VK gene is always joined to a JK gene, the joining process may sometimes result in a loss of a small number of bases from the ends of both VK and J K genes during crossing over of the nicked strands. This imperfect crossing over gives rise to variations in the amino acid sequence of the resulting K-chain. This creates what is known as functional diversity which acts as another source of antibody diversity.
The basic mechanism of gene rearrangement in somatic recombination described with the example of kappa light chain is applicable to other Ig chains, like λ-light chain and heavy chains of different classes of Ig molecules. The DNA segments in all cases are characterized by similar recognition signals consisting of heptamer-spacer-nonamer sequences.
In case of H-chains, there is an extra D segment. During rearrangement, first of all a D segment is joined to one of the J segments producing a D-J combination. Then one of the V genes is trans-located to form a V-D-J combination and lastly, one of the nine C segment is added. Of these nine C segments, there is one for IgM (Cμ), one for IgD (Cδ), four for IgG (Cγ), two for IgA (Cα) and one for IgE (Cϵ).
After the genes of H-and L-chains have been successfully rearranged, the DNA segments are transcribed into a primary transcript (hn-RNA). It contains besides the essential coding segments, also extra segments including introns. These are removed by RNA-splicing to produce an m-RNA.
Translation of the m-RNA takes place on ribosomes attached to the endoplasmic reticulum of the B-cells. The resultant polypeptides of H-and L-chains are secreted into the lumen of E.R. where they combine to form a complete Ig molecule. It is then transported to the B-cell membrane where it is expressed as an antigenic receptor.
The process of gene rearrangement in one L-chain (kappa) and one H-chain (of IgM) is described below:
(b) Generation of kappa L-chain and its polypeptide:
The first step in gene rearrangement of the k-chain is the joining one of 80 V genes (say V 50) to one of the 5 J genes (say J4) in a B-cell. Then the V-J genes are joined to the Ck gene.
The process is shown in Fig. 10.38:
(c) Generation of H-chain of IgM:
Heavy chain generation starts with joining of a D-segment with a J-segment.
The process of H-chain formation of IgM is shown in Fig. 10.39:
The main factor which plays the key role in creating antibody diversity is doubtlessly the random combination of V, D, J and C segments, each of which has few too many representatives in the germ-line DNA. Additionally, the imprecise joining of V, D and J segments (junctional diversity) also is responsible for some diversity. Another factor that adds to antibody diversity is the high rate of somatic mutation of the V genes of germ-line DNA.
Affinity Maturation of Antibodies:
Antibodies produced as a result of secondary immune response generally show a higher affinity to antigens than those produced in the primary response. We know that affinity refers to the firmness of binding of an antibody molecule to its cognate antigen and that it is distinct from valence of an antibody which refers to the number of antigen-binding sites possessed by an antibody molecule. Affinity maturation is the increased affinity of the antibodies produced in the secondary response in comparison to that of primary response.
The higher affinity of antibodies for antigens has been thought to be due to the larger number of antibody-producing cells generated in secondary response which competes with each other for binding to the available antigens. As a result, those B-cells which produce antibodies with higher affinity are favoured by natural selection for proliferation and transformation into antibody-producing plasma cells.
The production of higher-affinity antibodies is due to somatic mutation in the V genes of germ-line DNA. Thus, as the immune response progresses, antibodies appear that bind more effectively due to affinity maturation.
Class switching refers to the ability of a B-cell to switch over production of one class of antibody to another class, both haying the same antigenic specificity. A B-cell initially expresses IgM and IgD on its surface. After being stimulated, the same B-cell expresses IgG or other antibodies. This is known as class switching. It occurs through interaction of the B-cell with a TH-cell.
TH-cells bind to B-cells with specific proteins ligands. Such binding induces the TH-cell to secrete specific cytokines. Class switching is effected through the cytokines as well as binding with specific ligands of both cells (TH and B).
TH-cells are divided into two main types, TH-1 and TH-2, both being CD4+ cells. While the main function of TH-1 cells is to activate macrophages through production of the cytokine — interferon y, they also induce class switching of B-cells to produce IgG instead of IgM.
The major function of Th-2 cells is in humoral immunity in which they participate in T-dependent antibody formation by B-cells. TH-2 cells secrete cytokines including interleukin-4 and interleukin-5. When a Th-2 cell binds to a B-cell, the latter produces IgE or IgA. Here also, the two cells interact by binding to each other with specific ligands. Binding of TH-1 and TH-2 cells with B-cells associated with class-switching is shown in Fig. 10.40.
The cytokines viz. interferon y, interleukins 4 and 5 are believed to induce differential cleavage and splicing of the primary transcript (hn-RN A) during formation of the H-chains of Ig molecules. As a result a V-D-J segment already formed by gene rearrangement is attached to a different C segment.