Trends in Biotechnology 150415 TB 12 Antibodies 1 Acquired or Adaptive Immunity. A. Recognizes foreign invaders and responds to the invader, called an “antigen”. B. Can also recognize the body as self and the tissues of others as non-self. C. Antigens can be protein, glycoprotein, polysaccharides, and nucleic acids, and small parts of the antigen can trigger a response (called the “antigenic determinant”). D. Based on the complex interactions of different types of cells and other parts. 2 3 4 • T cells and B cells are activated, some become “memory” cells. • The next time that an individual encounters that same antigen, the immune system is primed to destroy it quickly. • This is active immunity because the body’s immune system prepares itself for future challenges. 5 • Long-term active immunity can be naturally acquired by infection or artificially acquired by vaccines made from infectious agents that have been inactivated or, more commonly, from minute portions of the microbe. • Short-term passive immunity can be transferred artificially from one individual to another via antibody-rich serum. 6 7 • Any non-self substance capable of triggering an immune response is known as an antigen. • The distinctive markers on antigens that trigger an immune response are called epitopes. 8 9 • Very important to the immune response is the ability to distinguish between “self” and “nonself.” • Every cell in your body carries the same set of distinctive surface proteins that distinguish you as “self.” • Normally your immune cells do not attack your own body tissues, which all carry the same pattern of self-markers. 10 • This set of unique markers on human cells is called the major histocompatibility complex (MHC) proteins. • There are two classes: MHC Class I proteins, which are on all cells, and MHC Class II proteins, which are only on certain specialized cells. 11 12 • Cells work chiefly by secreting soluble substances known as antibodies. • They wait around a lymph node, waiting for a macrophage to bring an antigen or for an invader such as a bacteria to arrive. 13 When an antigen-specific antibody on a B cell matches up with an antigen: • The antigen binds to the antibody receptor, the B cell eats it, and, after a special helper T cell joins the action, the B cell becomes a large plasma cell factory that produces identical copies of specific antibody molecules very quickly -up to 10 million copies an hour. 14 An antigen may have one or more of the same antigenic determinants or different antigenic determinants. 15 An antibody molecular binds to an antigenic determinant (epitope) of a cell that has different antigens on its surface 16 • Antigenic Determinants (Epitopes) • http://highered.mcgrawhill.com/sites/0072556781/student_view0/ch apter32/animation_quiz_5.html 17 Structure of an antibody 18 The generation of a functional antibody L-chain by recombination of DNA regions 19 • Antibody Diversity • http://highered.mcgrawhill.com/sites/0072556781/student_view0/ch apter32/animation_quiz_2.html 20 Monoclonal antibodies A general representation of the method used to produce monoclonal antibodies. http://en.wikipedia.org/wiki/Monoclonal_antibody#/media/File:Monoclonals.png Adenosine CC BY-SA 3.0 21 • Monoclonal antibodies (mAb or moAb) are monospecific antibodies that are made by identical immune cells that are all clones of a unique parent cell, in contrast to polyclonal antibodies which are made from several different immune cells. 22 • Monoclonal antibodies have monovalent affinity, in that they bind to the same epitope. • Given almost any substance, it is possible to produce monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. • This has become an important tool in biochemistry, molecular biology and medicine. • When used as medications, the nonproprietary drug name ends in –mab 23 Monoclonal Antibody Production http://highered.mcgrawhill.com/sites/0072556781/student_view0/chap ter32/animation_quiz_3.html 24 • Hybridoma technology is a technology of forming hybrid cell lines (called hybridomas) by fusing an antibody-producing B cell with a myeloma (B cell cancer) cell that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis. • The antibodies produced by the hybridoma are all of a single specificity and are therefore monoclonal antibodies (in contrast to polyclonal antibodies). 25 (1) Immunisation of a mouse (2) Isolation of B cells from the spleen (3) Cultivation of myeloma cells (4) Fusion of myeloma and B cells (5) Separation of cell lines (6) Screening of suitable cell lines (7) in vitro (a) or in vivo (b) multiplication (8) Harvesting 26 • Laboratory animals (mammals, e.g. mice) are first exposed to the antigen that an antibody is to be generated against. • Usually this is done by a series of injections of the antigen. • Splenocytes are removed from the mammal's spleen, the B cells are fused with immortalized myeloma cells. 27 • The myeloma cells are selected to ensure they are not secreting antibody themselves and that they lack the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene, making them sensitive to HAT medium. • Electrofusion causes the B cells and Myeloma cells to align and fuse with the application of an electric field. 28 • Fused cells are incubated in HAT medium (hypoxanthine-aminopterin-thymidine medium) for roughly 10 to 14 days. • Aminopterin blocks the pathway that allows for nucleotide synthesis. • Unfused myeloma cells die, as they cannot produce nucleotides by alternate pathways because they lack HGPRT. 29 • Unfused B cells die as they have a short life span. • Only the B cell-myeloma hybrids survive, since the HGPRT gene coming from the B cells is functional. • These cells produce antibodies (a property of B cells) and are immortal (a property of myeloma cells). 30 • The incubated medium is then diluted into multi-well plates to such an extent that each well contains only one cell. • Since the antibodies in a well are produced by the same B cell, they will be directed towards the same epitope, and are thus monoclonal antibodies. • The next stage is a rapid primary screening process, which identifies and selects only those hybridomas that produce antibodies of appropriate specificity. 31 The B cell that produces the desired antibodies can be cloned to produce many identical daughter clones. 32 Western Blotting • • • • Immunoblotting (Western Blot) proteins are separated by electrophoresis, blotted to nitrocellulose sheets, then treated with solution containing enzymetagged antibodies 33 Western blotting for antibodies to HIV proteins. 34 • The western blot (sometimes called the protein immunoblot). • Widely used analytical technique used to detect specific proteins in a sample of tissue homogenate or extract. • Gel electrophoresis seperates proteins by 3-D structure or denatured proteins by the length of the polypeptide. 35 • The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein. • The gel electrophoresis step is included in western blot analysis to resolve the issue of the cross-reactivity of antibodies. 36 • This method is used in the fields of molecular biology, immunogenetics and other molecular biology disciplines. • A number of search engines, such as CiteAb, Antibodypedia, and SeekProducts, are available that can help researchers find suitable antibodies for use in western blotting. 37 Other related techniques include dot blot analysis, immunohistochemistry and immunocytochemistry where antibodies are used to detect proteins in tissues and cells by immunostaining, and enzyme-linked immunosorbent assay (ELISA). 38 Immunofluorescence • Dyes coupled to antibody molecules will fluoresce (emit visible light) when irradiated with ultraviolet light • Direct-used to detect antigen-bearing organisms fixed on a microscope slide • Indirect-used to detect the presence of serum antibodies • Used for light microscopy with a fluorescence microscope. 39 • This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualisation of the distribution of the target molecule through the sample. • Immunofluorescence is a widely used example of immunostaining and is a specific example of immunohistochemistry that makes use of fluorophores to visualise the location of the antibodies. 40 Immunofluorescence can be used on tissue sections, cultured cell lines, or individual cells, and may be used to analyze the distribution of proteins, glycans, and small biological and nonbiological molecules. 41 • Immunofluorescence can be used in combination with other, non-antibody methods of fluorescent staining. • Several microscope designs can be used for analysis of immunofluorescence samples; the simplest is the epifluorescence microscope, and the confocal microscope is also widely used. • Various super-resolution microscope designs that are capable of much higher resolution can also be used. 42 The direct antibody assay where tagged antibody interacts directly with antigens on a cell surface. 43 Enzyme-linked immunosorbent assay The ELISA method. (a) The sandwich of substrate-bound antibody, specific antigen, and a second antibody with a bound enzyme, with the subsequent conversion of a substrate to a colored product. 44 • A test that uses antibodies and color change to identify a substance. • Uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample. • Used as a diagnostic tool in medicine and plant pathology, as well as a quality-control check. 45 • Antigens from the sample are attached to a surface. • A further specific antibody is applied over the surface so it can bind to the antigen. • This antibody is linked to an enzyme, and, in the final step, a substance containing the enzyme's substrate is added. • The subsequent reaction produces a detectable signal, most commonly a color change in the substrate. 46 Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either nonspecifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). 47 After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation. 48 Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are non-specifically bound. After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. 49 The steps in conducting an ELISA assay. 50 • ELISA Enzyme-Linked Immunosorbent Assay • http://highered.mcgrawhill.com/sites/0072556781/student_view0/ch apter33/animation_quiz_1.html 51 ELISA can perform other forms of ligand binding assays instead of strictly "immuno" assays. The technique essentially requires any ligating reagent that can be immobilized on the solid phase along with a detection reagent that will bind specifically and use an enzyme to generate a signal that can be properly quantified. 52 Looking back: Using antibodies means using molecules which can bind to other molecules. Look for similarities with other binding techniques. Eg. Nucleic acid hybridization, hydroxyapatite column, Also, think about different labeling techniques. 53