Immunoassays PDF Fall 2023-2024

Biotechniques (BMS 34010A) Fall semester 2023 -2024 Dr. Tania Tahtouh [email protected] Immunoassays  Immunoassay  An immunoassay is a biochemical test that measures the presence or the concentration of a molecule through the use of an antibody or an antigen.  The b...

Biotechniques (BMS 34010A) Fall semester 2023 -2024 Dr. Tania Tahtouh [email protected] Immunoassays  Immunoassay  An immunoassay is a biochemical test that measures the presence or the concentration of a molecule through the use of an antibody or an antigen.  The basic principle of these assays is the specificity of the antibody-antigen reaction. There are several different formats of immunoassays, but all of them involve a specific antibody binding to its antigen, and a label that is used to detect the antigen-antibody complex.  Definitions  Antigen (Ag): is a substance capable of causing an immune response leading to the production of antibodies and they are also the targets to which antibodies will bind.  Antibodies (Ab) (= immunoglobulins, Ig): are antigen specific proteins and will only bind to the antigen that initiated their production. There are five different types: IgA, IgD, IgE, IgG, and IgM.  Haptens: are small molecules which could never induce an immune response unless coupled to a carrier molecule (foreign protein).  Adjuvant: are substances which increase the immunogenicity of the antigen and are used to reduce the amount of antigen required as well as stimulate specific immunity to it. Immunoglobulin classes Due to its small size (monomeric) and high diffusibility, IgG is the prevalent type in the extracellular fluid IgG accounts for 70–75% of all human immunoglobulins found in the blood.  Antibody structure  An antibody is a Y-shaped protein molecule consisting of four polypeptide chains held together by disulfide bonds. ▪ 2 types of chains: heavy & light Fab ▪ 2 types of domains: variable & constant ▪ 2 parts of the molecule: Fab (fragment antigen-binding) & Fc (fragment crystallizable) Fc  Antibody properties  Specificity: Each individual antibody protein is capable of binding specifically with one unique epitope thanks to the unique antigen binding site located at the tip of the variable region on the antibody.  Affinity: It measures the strength of interaction between an epitope and an antibody’s antigen binding site.  Avidity: It gives a measure of the overall strength of an antibody- antigen complex. Polyclonal antibody  Polyclonal antibodies (pAbs) are a complex mixture of several antibodies that recognize and bind to many different epitopes of a single antigen. ▪ Raised in appropriate donor animals, generally rabbits for smaller amounts and sheep or goats for larger quantities.  Monoclonal antibodies (mAbs) are laboratory produced antibodies that recognize and bind to specific receptors and act like human antibodies in the immune system. Monoclonal Antibodies vs Polyclonal Antibodies  Agglutination  The first immunoassay formats described were methods based on the agglutination reaction.  Agglutination is the visible expression of the aggregation of antigens and antibodies. Agglutination only occurs when there is the right amount of antibody and antigen present ! ! ! Agglutination  Immunoassays by signal  A useful way to classify immunoassays is by the type of signal that is measured, as this has an impact on its sensitivity and the instrumentation needed to measure them.  ELISA (Enzyme-Linked Immunosorbent Assay) if the signal is provided by an enzyme that produces a colored substrate that is measured using absorbance.  FIA (Fluorescent ImmunoAssay) if the signal is provided by fluorescent dyes (e.g. FITC) and is measured by a microplate fluorometer. FITC (Fluorescein isothiocyanate) is the form of fluorescein used for conjugation to ABs  RIAs (Radio ImmunoAssay) if the signal is provided by radiation coming from a radioisotope, such as 125I (Iodine). As they are often gamma-emitting isotopes, they are usually measured using a gamma counter. ELISA types  Direct Sandwich ELISA 1. Coat the ELISA plate with capture antibody 2. Add sample (antigen) 3. Add enzyme labelled detection antibody that will bind to any target antigen already bound to the plate 4. Add substrate that will be converted into a colored product following the standard curve. Sandwich ELISA ELISA (Enzyme-linked Immunosorbent Assay): https://youtu.be/JFXVPOym3fs Principle of ELISA: https://youtu.be/lUWpWKVcmc4 The Enzyme Linked Immunosorbent Assay (ELISA): https://youtu.be/zR_xlV5v_f4  Immunohistochemistry (IHC)  Immunohistochemistry is a powerful microscopy based technique for visualizing and localizing cellular components, proteins or other macromolecules in tissue samples.  Immunofluorescence (IF) microscopy uses antibodies conjugated to fluorescent markers to locate specific structures on specimens and allows them to be visualized by illuminating them with ultraviolet light. Common fluorophores for Ab conjugation  Immunoblotting  Western blotting is used to identify proteins from samples after electrophoresis. 1. Electrophoresis 2. The separated proteins are transferred onto a nitrocellulose or polyvinyl membrane 3. The membrane is treated with a protein-blocking solution to prevent non-specific binding of antibody to the membrane itself. 4. Either direct or indirect antibody systems can be used but often indirect methods are used for reasons of cost.  Flow cytometry  Flow cytometry is a laser-based technology used to study cells as they move in fluid suspension. ▪ It allows simultaneous multi-parameter analysis of single cells. ▪ Physical and chemical characteristics of a population of cells or particles can be detected and measured.  It is widely used for: ▪ analyzing the expression of cell surface and intracellular molecules. ▪ characterizing and defining different cell types in a heterogeneous cell population. ▪ assessing the purity of isolated subpopulations. ▪ analyzing cell size and volume.  Fluorescent Activated Cell Sorting (FACS)  Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry.  FACS machines are capable of sorting a heterogeneous mixture of biological cells into several containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. ▪ The technique is used on live cells and allows recovery and subsequent culture of the cells after separation.  FACS steps 1. The cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid. 3. A vibrating mechanism causes the stream of cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell per droplet. 4. Just before the stream breaks into droplets, the flow passes through a fluorescence measuring station where the fluorescent character of interest of each cell is measured. 5. An electrical charging ring is placed just at the point where the stream breaks into droplets. Droplets get a charge. 6. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge.  Autofluorescence  Autofluorescence (primary fluorescence) is the fluorescence of naturally occurring substances, such as chlorophyll, collagen and fluorite.  Most plant and animal tissues show some autofluorescence when excited with ultraviolet light ~365 nm. Visualization of chlorophyll autofluorescence in the cotyledons of Ipomoea nil growing under continuous light. DOI: 10.1007/s11738-016-2244-1. References  https://www.sinobiological.com/category/fcm-facs-facs