Antibodies: Protein Detection and Quantitation (BIOC3570 F24 03)

# Protein detection and quantitation (2) ## Antibodies Antibodies are very useful biochemical reagents * Antibodies are specialized "recognition" proteins produced by the immune systems of vertebrates in an enormous variety. * We can immunize animals so that they produce large amounts of antibodi...

# Protein detection and quantitation (2) ## Antibodies Antibodies are very useful biochemical reagents * Antibodies are specialized "recognition" proteins produced by the immune systems of vertebrates in an enormous variety. * We can immunize animals so that they produce large amounts of antibodies that recognize any arbitrary specific protein. * Antibodies bind their targets very tightly ($KD 10^{-7} - 10^{-10} M$), and very specifically. * Antibodies are powerful enough to detect and/or label a single target protein, even within the full complexity of the cell's proteome. * They also are very important in medicine - both diagnostically and therapeutically. ## Terminology: Antigens and Epitopes * An antigen is the foreign substance which elicits antibody production. * Proteins and oligosaccharides are usually excellent antigens. * The antigen is effectively what you inject into your animal. * An epitope is the antigenic determinant recognized by a given antibody. * The epitope is a subset of the antigen. * A given antigen usually has many distinct epitopes. * Distinct antigens may have similar epitopes (e.g. a short peptide sequence). * This can lead to cross-reactivity of Ab. ## Antibodies are made by B-cells * Antibodies are synthesized by specialized B-cells in the immune systems of vertebrates. * One animal will have ~10⁸ B-cells, each of which encodes a different antibody gene, and which makes a unique antibody. * Any given protein (or oligosaccharide) as a small probability of tightly binding a random one of these antibodies. * But given that there are millions to choose from, there will be many B-cells in an organism whose antibody binds tightly to some specific macromolecule by chance. * B-cells with antibodies that recognize host molecules are usually eliminated *self cell recognition is bad*. * The rest serve as a sensory system to recognize foreign molecules. * B-cells that recognize foreign molecules being present divide, amplifying the amount of that antibody present. ## Antibodies genes are produced by recombination The image shows a diagram of the genes that encode antibodies in the germline genome. * Antibodies are not directly encoded in the germ-line genome of organisms. * Instead, the genome encodes two sets of multiple interchangeable modules. * The B-cell combines these modules at random (with inexact borders, point mutations and random insertions) as it matures. * This results in two genes unique to that B-cell, encoding two protein chains. * Each B-cell (and its descendants) produces a unique antibody *with the variable sequences concentrated within a small area of its surface*. ## Antibody Structure (IgG) The image shows a diagram of a typical IgG antibody. * Antibodies have two separate chains - a heavy and a light chain. * IgGs are considered to be "monomeric" (technically hetero-tetrameric). * Other classes are dimeric (IgA) pentameric (IgM. ## Antibody Fragments The image shows a diagram of how papain can be used to cleave an antibody into fragments. * Antibodies can be cleaved by partial digestion with papain. * This produces two types of fragment - Fab and Fc. * Fab will recognize antigens with the specificity of the original antibody, but is considerably smaller *as signal is 1/2 amplified*. * Fabs are often used in diagnostic and therapeutic applications. ## Antibody Recognition is by CDRs The image shows a diagram of how CDRs on an antibody recognize epitopes on an antigen. * B-cell recombination results in each heavy and each light chain having highly variable sequences in 3 specific regions. * These are termed complementarity determining regions (CDRs). * In the structure, the CDRs converge to form a set of loops at one tip of the Fab (orange/dark blue). * The CDRs contact and recognize the epitope in a sequence specific manner. ## Nanobodies The image shows a diagram of a nanobody binding to a green fluorescent protein antigen. * Antibodies from camelids (e.g. llamas) and sharks have only a heavy chain. * When cleaved these yield a single chain "nanobody". * Nanobodies are one quarter of the size of Fabs, with only one chain and one domain. * Nanobodies generally express well recombinantly (e.g., in *E. coli*), so are easy to scale up. * Nanobodies are used in biochemistry, diagnostics and are being explored for therapeutic purposes. ## Immunization The image shows a cartoon of an immunized rabbit. * (If the antigen g. a purified recombinant protein) is injected into an animal (e.g. a rabbit). > m imm ne serum can then later be ex!racted from this. > rabbit MVVIU--Yve yon '?l y h> ?C.., e * This immune serum is a complex mixture of Abs. * Some will (hopefully) recognize the antigen used in immunization. > We liglīt also col ect pre-immune serum - a control > serum, obtained before immunization. ## Making Polyclonal Antibodies The image shows a cartoon of a rabbit being immunized. * Typical protocol: antigen (up to 1 mg) administered subcutaneously four times at 2-week intervals. * Blood collected at day 60. * ~30 mL whole blood can be recovered from rabbit by ear-vein bleed. * Centrifugation removes cells, leaving half the volume as serum. > Contaminating proteins inactivated and removed by heat treatment and chromatography steps *antibodies are heat and acid stable*. * Yields ~150 mg total immunoglobulin after progressing. ## Monoclonal Antibodies * Polyclonal antibodies (directly from the animal) recognize a large number of epitopes (everything that animal's immune system considered a threat). * This can result in cross reaction with undesired proteins. * Alternatively, polyclonal antibodies can be affinity purified. * B-cells do not form stable cultures, and so cannot be grown alone. * Hybridomas are hybrid cells produced by fusing a B-cell (produce a specific antibody) and a B lymphocyte tumor cell (immortal) *keep dividing, but they don't die*. * This gives an immortal cell that produces a single antibody. * This cell can then be cultured, producing large amounts of just one antibody *don't dic*. * These monoclonal antibodies recognize a single epitope, and are much less likely to cross-react. ## Monoclonal Antibodies (Milstein & Köhler, 1975) The image shows a diagram of the process of producing hybridoma cells. * One mouse is immunized with a specific antigen and, a few days later, its spleen is removed. * The spleen cells are fused with myeloma cells. * These hybrid cells (hybridomas) are selected for their ability to produce antibodies against the specific antigen. * The hybridomas are then cloned and grown in culture, producing large amounts of monoclonal antibodies. ## Antibodies to hundreds of thousands of proteins (and other antigens) are commercially available. The image shows a screenshot of the Abcam website, which sells a wide variety of antibodies. * Almost 29,000 antibodies from a single company! * ~2.5 m antibodies are RRID Registered across all companies. * ...but if you work on something a little outside the mainstream, you might still need to make a new one. ## Key applications of antibodies * Detection and quantitation of the antigen (ELISA assays). * Detection of specific proteins on electrophoresis gels ("Western" immunoblotting; see electrophoresis lectures). * Detection of specific proteins in cells (immunostaining). * Purification of proteins by affinity chromatography. * Inhibiting the biological activity of a protein. * Cell sorting by surface antigen (e.g. FACS analysis). * Disease diagnostics (detecting bacteria, viruses, or protein markers of disease). * Serotyping bacteria (polysaccharide recognition). * Antibody therapeutics (e.g. antibodies against cancer markers train the immune system to kill cancer cells; COVID protective patient antibodies). ## Secondary antibodies The image shows a diagram of a primary antibody and a secondary antibody. * Antibodies per se are not directly detectable. * Labelling antibodies individually is inefficient. * Instead antibodies from a second species (e.g., goat) are raised to the antibodies the target species (è.g., rabbit). * This yields goat antibodies that recognize any antibody from rabbit. * These (normally commercial) secondary antibodies can be labelled with: * A fluorophore to gives a fluorescent signal. * An enzyme that generates a chromogenic product, to give a visible signal. ## ELISA (enzyme-linked immunosorbent assay) This is a common technique used to detect and quantify the presence of a specific antigen in a sample. * An ELISA assay uses antibodies to detect substrates adhered to a solid surface. * With the inclusion of suitable standards, the amount of the antigen can be quantified, even in complex samples (like blood). * In diagnostics, the plate may be coated with a capture antibody which specifically immobilizes the antigen of interest prior to detection. ## Immunohistochemistry The image shows a microscope image of breast carcinoma tissue stained for the estrogen receptor. The nuclei of the cells are stained, indicating this is ER+ breast cancer. * Antibodies can be used to label specific proteins in thin tissue slices. * A microtome is used to slice tissue into thin (~10 m) sections. * Cells are permeabilized by a detergent. * Antibody is added, along with any required coupling reagents (secondary antibodies, substrates). * This experiment shows which cells have the antigen, and where in the cell the antigen is located. ## Antibodies – buyer beware! * Antibodies are powerful reagents, but not foolproof. * Irreproducibility in cell biology is often an Ab problem. * Polyclonal antibodies will sometimes recognize a similar epitope in a very different protein. * Different antibodies “targeting” the same epitope from different vendors may differ greatly in sensitivity or specificity. * Commercial antibodies sometimes fail to recognize the target that they are supposed to bind; polyclonal Abs can show batch variability *Problem attu*. * If possible, validate the antibody against a known positive, and against a cell line that should be negative (e.g. gene knock-out). * If trying to build on previous work, make sure that you are using the exact same antibody (RRIDs are used to track Abs).