Antigenic determinants/Epitopes on Immunoglobulins (Part 13- Antibody Basics)
Welcome to the 13th part of the 13-part series on Antibody basics.
Previous parts: Part 1, Part 2, Part 3, Part 4, Part 5, Part 6, Part 7, Part 8, Part 9, Part 10, Part 11, and Part 12
As already discussed in previous parts, antigens are proteins that can induce an immune response. In other words, antibodies are generated against them. And since antibodies are glycoproteins, they can also induce an immune response. There are specific regions on antibodies that can induce the immune system to produce antibodies against them. These regions on antibodies are called antigenic determinants or epitopes.
The epitopes or antigenic determinants of the antibody molecule fall into three major categories: Isotypic determinants, Allotypic determinants, and Idiotypic determinants
Isotypic determinants
The first type of antigenic determinant on Ig is the isotypic determinants. The prefix “iso” means the same in all members of the same species. Isotypic determinants are present in the constant region and define the class and subclass of each heavy chain and similarly define the type and subtype of each light chain of an immunoglobulin within a species. There are five different classes of antibodies; IgM, IgA, IgG, IgE, and IgD; based on the type of heavy chain, they have µ, α, γ, ε, and δ, respectively. Each of these five different heavy chains is called an isotype. And each isotype is encoded by a separate constant region gene.
Within a species, say in humans, each individual will carry the same constant region genes and express the same isotypes in a serum. In contrast, different species inherit different constant region genes and therefore express different isotypes. In other words, all individuals of the same species have the same isotype.
But the isotypic determinants between different species are different.
This is the reason when an antibody, e.g., IgM from one species, say human, is injected into another species, say mouse, the isotypic determinants of human antibody are recognized as foreign by mouse. Thus, the mouse generates an antibody response or anti-antibodies to the isotypic determinants on the foreign human IgM antibody. And these anti-antibodies bind to the isotypic determinants in the constant region of injected human antibodies. The anti-antibodies generated are also called anti-isotype antibodies.
These anti-antibodies are used in many immunological tests like immunofluorescence, enzyme-linked immunosorbent assays (ELISA), and western blots to quantify Ig classes and subclasses produced during an immune response in humans, characterization of B cell leukemia, and in the diagnosis of various immunodeficiency diseases.
Role of anti-isotype antibodies in the detection of viral diseases
Suppose if the person is infected with HIV, then his immune system starts producing antibodies against HIV. Since HIV binds to CD4 cells via its envelope proteins, if the person is infected with HIV, his immune system makes antibodies to the HIV proteins, including envelope proteins.
To detect if the person is infected with HIV, ELISA can be done. For this, the HIV envelope protein is coated on the solid support, such as a microtiter plate. Then the blood of the patient is added to the microtiter plate. If antibodies against HIV are present in the blood sample, they bind to the viral antigen coated on the solid support. The bound anti-HIV antibodies are then detected using secondary antibodies.
The secondary antibodies are the antibodies that can bind to anti-HIV antibodies. These are raised in other species such as rabbits, mice, or goats by injecting them with anti-HIV antibodies. The animals of other species recognize anti-HIV antibodies as foreign and induce an immune response by producing antibodies against anti-HIV antibodies. Now, these anti-anti HIV antibodies act as secondary antibodies that are used for diagnostic purposes.
Suppose the patient’s blood contains anti-HIV antibodies. In that case, the secondary antibody binds to the isotypic determinants in the constant region of the anti-HIV antibody present in the blood sample. The secondary antibody is attached to an enzyme that catalyzes a color change when the substrate is added. If the substrate changes color, it indicates that the person is suffering from HIV. In this way, anti-antibodies can be used in the diagnosis of diseases.
Allotypic determinants
The second type of antigenic determinant on immunoglobulins is the allotypic determinants. The prefix “allo” means the difference in individuals of the same species. Individuals of the same species inherit the same set of genes for the constant region, hence having the same isotypic determinants. Yet within a species, multiple alleles may exist for certain isotypic genes. And these alleles encode for subtle differences in the amino acid sequences called allotypic determinants that occur in some but not all members of the species.
Consequently, some members within the same species have different antigenic determinants or allotypic determinants. The sum of the individual allotypic determinants displayed by an antibody determines its allotype. In humans, 25 different allotypes have been characterized for all four IgG subclasses, 2 allotypes for IgA2 subclass, and 3 allotypes for the kappa light chain. Each of these allotypic determinants represents differences in 1 to 4 amino acids of the constant region encoded by multiple alleles. Therefore, when antibodies from one member of a species, say from person 1 are injected into another member of the same species, say person 2, allotypic determinants of person 1 are recognized as foreign in person 2. Thus person 2 generates antibodies against the allotypic determinants of the injected antibodies of person 1. The antibodies generated against the allotypic determinants are called anti-allotypic antibodies. These anti-allotypic antibodies then bind to the allotypic determinants in the constant region of injected Ab.
For instance, antibodies to allotypic determinants or anti-allotypic antibodies are sometimes produced by the mother during pregnancy in response to paternal allotypic determinants on the fetal immunoglobulins. Antibodies to allotypic determinants in a person can also arise from a blood transfusion.
Idiotypic determinants
The third type of antigenic determinant on immunoglobulins is idiotypic determinants. The unique amino acids of the domains of variable regions of heavy chain and light chain can function not only as antigen-binding site but also as a set of antigenic determinants or epitopes. Each individual antigenic determinant of a variable region of Ab is called an idiotope. In some cases, the idiotope may be the actual binding site of the antibody, also called paratope and in some cases, idiotope may comprise variable sequences outside of the antigen-binding site. Each antibody contains multiple idiotopes, and the combination of all idiotopes for each antibody is called idiotype of the antibody.
The antibodies produced by B cells derived from the same clone have an identical variable sequence, thus having the same idiotype.
Suppose, if individual encounters two different antigens, i.e., Ag a and Ag b, and produce IgG1 antibody against both of these antigens, then, in this case, the idiotypic determinants for the IgG1 produced against Ag a will be different from the idiotypic determinants of IgG1 produced against Ag b.
Further, when antibodies from a genetically identical donor are injected into a recipient, the recipient may induce the production of anti-idiotypic antibodies against the idiotype of donor antibodies. This is because in genetically identical twins, the isotype and allotype are the same, but the idiotype may be different. The antibodies generated against the idiotypic determinants of the antibody are called anti-idiotypic antibodies or written as anti-ID Ab.
Types of anti-ID antibodies
There are three classifications of anti-ID antibodies.
1. The first type of anti-ID antibody is an antigen blocking anti-ID antibody, which competes with the antigen to bind to the target antibody. It is because the paratope and idiotope of the target idiotypic antibody overlap with one another (Fig 1a). Paratope is the antigen-binding site of Ab and idiotope is the antigenic determinant or epitope of the Ab. Because of this overlapping of paratope and epitope of the target idiotypic antibody, the antibody’s target antigen and the anti-ID antibody compete to bind to the target idiotypic antibody.
2. The second classification of anti-ID antibody is non-blocking anti-ID antibody, which can simultaneously bind to the target idiotypic antibody without affecting its antigen’s binding capability (Fig 1b). This is because the target antibody’s paratope and idiotope do not overlap. Because of this reason, the anti-ID antibody and the antigen can simultaneously bind to the target idiotypic antibody without affecting one another’s binding capability.
3. The third classification of anti-ID antibody is a complex specific anti-ID antibody. This anti-ID antibody cannot bind to the target idiotypic antibody unless the target Ab is already bound to its antigen (Fig 1c).
Applications of Anti-ID antibodies
Before studying the use of anti-ID antibodies, it is essential to get familiar with the term antibody-drug or antibody-drug conjugates. Antibody-drug is a highly potent biopharmaceutical drug composed of an antibody that is linked, via a chemical linker, to a biologically active drug or cytotoxic compound.
For example, a tumor-specific antibody drug, in which anticancer drug is coupled to an antibody that specifically targets a specific tumor marker, e.g., a protein that ideally is only to be found in or on cancer cells. In this case, the antibody-drug will only bind and kill the targeted cancerous cell.
But after administering, it is essential to do the pharmacokinetic (PK) analysis of the antibody-drug. The pharmacokinetic analysis is the study of drug metabolism throughout the body.
It is critical in defining the absorption rates, distribution, half-life, and excretion rate of the antibody-drug. This analysis is essential to evaluate the dosage, toxicity, and efficacy of the antibody drug. To accomplish this, it is required to track and measure the antibody drugs which are bound or unbound to their designated target at various time points post administering them into the body.
For this, different types of anti-ID antibodies against the antibody-drug are used to detect and quantify the various forms of antibody drugs in patient serum, blood, urine, or other body fluids. For instance,
- To examine if the antibody-drug is present in unbound form, i.e., not bound to its target antigen in the patient, then blocking anti-ID antibody is used, which is coated on the solid support. When antigen blocking anti-ID antibody is used, the antibody drug’s target antigen and the anti-ID antibody will compete with one another to bind to the antibody-drug. Therefore, when the patient’s sample like serum, blood, urine, or any other bodily fluid is added, the free or unbound antibody drugs present in an individual, i.e., antibody drugs that are not bound to their target antigens, will bind to the anti-ID antibodies coated on the substrate.
Then these free antibody drugs bound to the coated anti-ID antibodies are detected using enzyme-tagged secondary anti-ID antibodies. After this, the chromogenic substrate is added, and the subsequent formation of a colored product indicates the presence of free antibody drugs in the patient. Thus, it establishes that antibody drugs in the patient are not bound to their target antigens.
2. The other type of non-blocking anti-ID antibody is used to detect both the antigen-bound and unbound antibody drug in the patient. In this case, the antibody drug’s paratope and idiotope do not overlap. Therefore, the anti-ID antibody and the antigen can simultaneously bind to the antibody-drug without affecting one another’s binding capability. For detection in this case, non-blocking anti-ID antibodies are coated on the solid support followed by the addition of the patient’s sample.
Both unbound and bound antibody drugs present in the patient’s sample will bind to the non-blocking anti-ID antibody coated on the substrate. Subsequently, antibody drugs bound to coated anti-ID antibodies are detected using enzyme-tagged secondary anti-ID antibodies. Thus, indicating that antibody drugs in the patient are both bound and unbound to their target antigens.
Once established, the ratio of unbound and bound antibody drugs in the patient’s sample is evaluated in the subsequent steps.
3. To specifically detect if the antibody-drug is bound to its target antigen, the complex specific anti-ID antibodies coated on the solid support are used. The complex specific anti-ID antibody cannot bind to the antibody-drug unless the drug is already bound to its target antigen.
Therefore, complex specific anti-ID antibodies can specifically detect bound antibody drugs in a patient’s sample.
Using a combination of three different types of anti-ID antibodies, it is possible to get overall information about the availability of antibody drugs in a patient. These anti-ID antibodies help to detect and track the state of antibody drugs, whether they are bound or unbound to their designated target at various time points post administering. And once the state of antibody drugs is established, the quantity and ratio of bound and unbound antibody drugs can also be calculated in the patient’s serum, blood, urine, or any other body fluid.
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