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In immunology, an antigen (Ag) is a molecule, moiety, foreign particulate matter, or an allergen, such as pollen, that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response.
The major histocompatibility complex (MHC) is a large locus on vertebrate DNA containing a set of closely linked polymorphic genes that code for cell surface proteins essential for the adaptive immune system. These cell surface proteins are called MHC molecules.
An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that binds to the epitope is called a paratope. Although epitopes are usually non-self proteins, sequences derived from the host that can be recognized are also epitopes.
In biochemistry, biotinylation is the process of covalently attaching biotin to a protein, nucleic acid or other molecule. Biotinylation is rapid, specific and is unlikely to disturb the natural function of the molecule due to the small size of biotin. Biotin binds to streptavidin and avidin with an extremely high affinity, fast on-rate, and high specificity, and these interactions are exploited in many areas of biotechnology to isolate biotinylated molecules of interest. Biotin-binding to streptavidin and avidin is resistant to extremes of heat, pH and proteolysis, making capture of biotinylated molecules possible in a wide variety of environments. Also, multiple biotin molecules can be conjugated to a protein of interest, which allows binding of multiple streptavidin, avidin or neutravidin protein molecules and increases the sensitivity of detection of the protein of interest. There is a large number of biotinylation reagents available that exploit the wide range of possible labelling methods. Due to the strong affinity between biotin and streptavidin, the purification of biotinylated proteins has been a widely used approach to identify protein-protein interactions and post-translational events such as ubiquitylation in molecular biology.
Superantigens (SAgs) are a class of antigens that result in excessive activation of the immune system. Specifically they cause non-specific activation of T-cells resulting in polyclonal T cell activation and massive cytokine release. Superantigens act by binding to the MHC proteins on antigen-presenting cells (APCs) and to the TCRs on their adjacent helper T-cells, bringing the signaling molecules together, and thus leading to the activation of the T-cells, regardless of the peptide displayed on the MHC molecule. SAgs are produced by some pathogenic viruses and bacteria most likely as a defense mechanism against the immune system. Compared to a normal antigen-induced T-cell response where 0.0001-0.001% of the body's T-cells are activated, these SAgs are capable of activating up to 20% of the body's T-cells. Furthermore, Anti-CD3 and Anti-CD28 antibodies (CD28-SuperMAB) have also shown to be highly potent superantigens.
Streptavidin is a 52 kDa protein (tetramer) purified from the bacterium Streptomyces avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin. With a dissociation constant (Kd) on the order of ≈10−14 mol/L, the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature. Streptavidin is used extensively in molecular biology and bionanotechnology due to the streptavidin-biotin complex's resistance to organic solvents, denaturants, detergents, proteolytic enzymes, and extremes of temperature and pH.
Antigen processing, or the cytosolic pathway, is an immunological process that prepares antigens for presentation to special cells of the immune system called T lymphocytes. It is considered to be a stage of antigen presentation pathways. This process involves two distinct pathways for processing of antigens from an organism's own (self) proteins or intracellular pathogens, or from phagocytosed pathogens ; subsequent presentation of these antigens on class I or class II major histocompatibility complex (MHC) molecules is dependent on which pathway is used. Both MHC class I and II are required to bind antigens before they are stably expressed on a cell surface. MHC I antigen presentation typically involves the endogenous pathway of antigen processing, and MHC II antigen presentation involves the exogenous pathway of antigen processing. Cross-presentation involves parts of the exogenous and the endogenous pathways but ultimately involves the latter portion of the endogenous pathway.
MHC class I molecules are one of two primary classes of major histocompatibility complex (MHC) molecules and are found on the cell surface of all nucleated cells in the bodies of vertebrates. They also occur on platelets, but not on red blood cells. Their function is to display peptide fragments of proteins from within the cell to cytotoxic T cells; this will trigger an immediate response from the immune system against a particular non-self antigen displayed with the help of an MHC class I protein. Because MHC class I molecules present peptides derived from cytosolic proteins, the pathway of MHC class I presentation is often called cytosolic or endogenous pathway.
The T-cell receptor (TCR) is a protein complex found on the surface of T cells, or T lymphocytes, that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. The binding between TCR and antigen peptides is of relatively low affinity and is degenerate: that is, many TCRs recognize the same antigen peptide and many antigen peptides are recognized by the same TCR.
A tetrameric protein is a protein with a quaternary structure of four subunits (tetrameric). Homotetramers have four identical subunits, and heterotetramers are complexes of different subunits. A tetramer can be assembled as dimer of dimers with two homodimer subunits, or two heterodimer subunits.
Protein tags are peptide sequences genetically grafted onto a recombinant protein. Tags are attached to proteins for various purposes. They can be added to either end of the target protein, so they are either C-terminus or N-terminus specific or are both C-terminus and N-terminus specific. Some tags are also inserted at sites within the protein of interest; they are known as internal tags.
A thymocyte is an immune cell present in the thymus, before it undergoes transformation into a T cell. Thymocytes are produced as stem cells in the bone marrow and reach the thymus via the blood.
Antigen presentation is a vital immune process that is essential for T cell immune response triggering. Because T cells recognize only fragmented antigens displayed on cell surfaces, antigen processing must occur before the antigen fragment can be recognized by a T-cell receptor. Specifically, the fragment, bound to the major histocompatibility complex (MHC), is transported to the surface of the cell, a process known as presentation. If there has been an infection with viruses or bacteria, the cell will present an endogenous or exogenous peptide fragment derived from the antigen by MHC molecules. There are two types of MHC molecules which differ in the behaviour of the antigens: MHC class I molecules (MHC-I) bind peptides from the cell cytosol, while peptides generated in the endocytic vesicles after internalisation are bound to MHC class II (MHC-II). Cellular membranes separate these two cellular environments - intracellular and extracellular. Each T cell can only recognize tens to hundreds of copies of a unique sequence of a single peptide among thousands of other peptides presented on the same cell, because an MHC molecule in one cell can bind to quite a large range of peptides. Predicting which antigens will be presented to the immune system by a certain MHC/HLA type is difficult, but the technology involved is improving.
MHC-restricted antigen recognition, or MHC restriction, refers to the fact that a T cell can interact with a self-major histocompatibility complex molecule and a foreign peptide bound to it, but will only respond to the antigen when it is bound to a particular MHC molecule.
In biochemistry, avidity refers to the accumulated strength of multiple affinities of individual non-covalent binding interactions, such as between a protein receptor and its ligand, and is commonly referred to as functional affinity. Avidity differs from affinity, which describes the strength of a single interaction. However, because individual binding events increase the likelihood of occurrence of other interactions, avidity should not be thought of as the mere sum of its constituent affinities but as the combined effect of all affinities participating in the biomolecular interaction. A particular important aspect relates to the phenomenon of 'avidity entropy'. Biomolecules often form heterogenous complexes or homogeneous oligomers and multimers or polymers. If clustered proteins form an organized matrix, such as the clathrin-coat, the interaction is described as a matricity.
A tetramer assay is a procedure that uses tetrameric proteins to detect and quantify T cells that are specific for a given antigen within a blood sample. The tetramers used in the assay are made up of four major histocompatibility complex (MHC) molecules, which are found on the surface of most cells in the body. MHC molecules present peptides to T-cells as a way to communicate the presence of viruses, bacteria, cancerous mutations, or other antigens in a cell. If a T-cell's receptor matches the peptide being presented by an MHC molecule, an immune response is triggered. Thus, MHC tetramers that are bioengineered to present a specific peptide can be used to find T-cells with receptors that match that peptide. The tetramers are labeled with a fluorophore, allowing tetramer-bound T-cells to be analyzed with flow cytometry. Quantification and sorting of T-cells by flow cytometry enables researchers to investigate immune response to viral infection and vaccine administration as well as functionality of antigen-specific T-cells. Generally, if a person's immune system has encountered a pathogen, the individual will possess T cells with specificity toward some peptide on that pathogen. Hence, if a tetramer stain specific for a pathogenic peptide results in a positive signal, this may indicate that the person's immune system has encountered and built a response to that pathogen.
The Strep-tag system is a method which allows the purification and detection of proteins by affinity chromatography. The Strep-tag II is a synthetic peptide consisting of eight amino acids (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys). This peptide sequence exhibits intrinsic affinity towards Strep-Tactin, a specifically engineered streptavidin, and can be N- or C- terminally fused to recombinant proteins. By exploiting the highly specific interaction, Strep-tagged proteins can be isolated in one step from crude cell lysates. Because the Strep-tag elutes under gentle, physiological conditions, it is especially suited for generation of functional proteins.
MHC multimers are oligomeric forms of MHC molecules, designed to identify and isolate T-cells with high affinity to specific antigens amid a large group of unrelated T-cells. Multimers generally range in size from dimers to octamers; however, some companies use even higher quantities of MHC per multimer. Multimers may be used to display class 1 MHC, class 2 MHC, or nonclassical molecules from species such as monkeys, mice, and humans.
Immunodominance is the immunological phenomenon in which immune responses are mounted against only a few of the antigenic peptides out of the many produced. That is, despite multiple allelic variations of MHC molecules and multiple peptides presented on antigen presenting cells, the immune response is skewed to only specific combinations of the two. Immunodominance is evident for both antibody-mediated immunity and cell-mediated immunity. Epitopes that are not targeted or targeted to a lower degree during an immune response are known as subdominant epitopes. The impact of immunodominance is immunodomination, where immunodominant epitopes will curtail immune responses against non-dominant epitopes. Antigen-presenting cells such as dendritic cells, can have up to six different types of MHC molecules for antigen presentation. There is a potential for generation of hundreds to thousands of different peptides from the proteins of pathogens. Yet, the effector cell population that is reactive against the pathogen is dominated by cells that recognize only a certain class of MHC bound to only certain pathogen-derived peptides presented by that MHC class. Antigens from a particular pathogen can be of variable immunogenicity, with the antigen that stimulates the strongest response being the immunodominant one. The different levels of immunogenicity amongst antigens forms what is known as dominance hierarchy.
IBA Lifesciences is a biotechnology company providing products and custom specific services for life science applications in academia and industry worldwide. IBA focusses on two business segments: cell selection and protein purification.
References
- ↑ Knabel, M., Franz, T.J., Schiemann, M., Wulf, A., Villmow, B., Schmidt., B., Bernhard, H., Wagner, H. and Busch, D. (2002) Reversible MHC multimer staining for functional isolation of T-cell populations and effective adoptive transfer. Nature Medicine 8 (6), 631-637.
- ↑ Schmidt TGM and Skerra A, 2007. The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. NATURE PROTOCOLS 2, 1528-1535.
- ↑ "Home". strep-tag.com. Archived from the original on 2004-09-18. Retrieved 2023-08-13.