ELISA Techniques PDF

Enzyme Linked Immunosorbent Assay (ELISA) FDSC 624 1  Definitions Antibodies (also known as immunoglobulins, Ig) are globulin proteins found in blood and are used by the immune system to identify and neutralize foreign objects, s...

Enzyme Linked Immunosorbent Assay (ELISA) FDSC 624 1  Definitions Antibodies (also known as immunoglobulins, Ig) are globulin proteins found in blood and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.  Definitions Antigens A substance stimulates the production of an antibody when introduced into the body. Immunoassay A technique that makes use of the binding between an antigen and its homologous antibody to identify and quantify the specific antigen or antibody in a sample. Analyte Samples being analyzed; in immunoassays the analyte is either antibody or antigen.  Definitions similar to lock and key specificity, sensitivity, linear range. specificity is the most important Antibody Production (Polyclonal antibody) Specific antibodies are produced by injecting an antigen into a mammal, such as a mouse, rat or rabbit for small quantities of antibody, or goat, sheep, or horse for large quantities of antibody. Text Blood isolated from these animals contains polyclonal antibodies — multiple antibodies that bind to the same antigen. Antibody Production (Polyclonal antibody) Antigen injected Polyclonal antibodies containing serum is taken out from the animal into animal Antigen activated B cell. Animal selection: Based on the need of the amount of antibodies, select animal with different sizes  Antibody Production (monoclonal antibody) To obtain antibody that is specific for a single antigen, antibody-secreting lymphocytes are isolated from the animal and immortalized by fusing them with a cancer cell line. The fused cells are called hybridomas, and will continuously grow and secrete antibody in culture. Single hybridoma cells are isolated by dilution cloning to generate cell clones that all produce the same antibody; these antibodies are called monoclonal antibodies. Antibody Mouse immunized with X Mouse myeloma cells Production (monoclonal Lymphocyte antibody) Fusion Cell death Cell death what you need to “Immortal” know: Antibody, hybrid cells antigen, mono and poly difference Screen for Anti-X and clone cells producing Anti-X Clone 1 Clone 2 Clone 3 Monoclonal antibodies to X  ELISA technique A biochemical technique used in immunology to detect the presence of an antibody or an antigen in a sample. The technique is divided into: 1. Competitive ELISA; 2. Sandwich ELISA (also called direct ELISA); 3. Indirect ELISA.  Competitive ELISA The labelled antigen competes for primary antibody binding sites with the sample antigen (un-labelled). The more antigen in the sample, the less labelled antigen is retained in the well and the weaker the signal. It is mainly used for the detection of “small molecules”.  Competitive ELISA Solid phase coated with antibody Add free and the higher the signal labelled antigen intensity, the higher of the real analyte Free and labelled antigen are captured substrate HRP enzyme Color generation by oxidation of substrate into a colored compound  Sandwich ELISA (Direct ELISA) The ELISA plate is coated with antibody to detect specific antigen. It is mainly used for the detection of “large molecules”.  Sandwich ELISA (Direct ELISA) 1. Prepare a surface to which a known quantity of capture antibody is bound; 2. Block any non-specific binding sites on the surface; 3. Apply the antigen-containing sample to the plate; if my primary bacteria is salmonella then salmonella will be the antigen  Sandwich ELISA (Direct ELISA) 4. Wash the plate, so that unbound antigen is removed; 5. Apply enzyme-linked primary Secondary antibodies as detection antibodies which also bind specifically to the antigen; 6. Wash the plate, so that the unbound antibody- enzyme conjugates are removed;  Sandwich ELISA (Direct ELISA) 7. Apply a chemical (e.g., TMO substrate) which is converted by the enzyme (e.g., HRP) into a coloured product; 8. Measure the absorbency of the plate wells to determine the presence and quantity of antigen. Sandwich ELISA (Direct ELISA)  Indirect ELISA 1. The protein antigen to be tested is added to each well of ELISA plate, where it is given time to adhere to the plastic through charge interactions; 2. A solution of non-reacting protein is added to block any plastic surface in the well that remains uncoated by the protein antigen;  Indirect ELISA 3. Then the serum is added, which contains a mixture of the serum antibodies, some of which may bind specifically to the test antigen that is coating the well; 4. Afterwards, a secondary antibody is added, which will bind to the antibody bound to the test antigen in the well. This secondary antibody often has an enzyme attached to it;  Indirect ELISA 5. A substrate for this enzyme is then added. This substrate changes colour upon reaction with the enzyme. The colour change shows that secondary antibody has bound to primary antibody, which strongly implies that the donor had an immune reaction to the test antigen; 6. The higher the concentration of the primary antibody that was present in the serum, the stronger the colour change. A spectrometer is often used to give quantitative values for colour intensity. Indirect ELISA Indirect ELISA & Sandwich ELISA (direct ELISA) 21  Example: an ELISA experiment Before starting the work read kit instruction carefully 1. The 96 well plate is labeled carefully and the first wells are used to draw standard curve;  Example: an ELISA experiment 2. The sample is added to plate in duplicate or triplicate and then the mean result is calculated; 3. The quality control sample which is provided with the kit is treated as the test samples;  Example: an ELISA experiment 4. Standard concentration is specified on the x-axis and the reading of each standard is specified on the y-axis and the standard curve is drawn;  Example: an ELISA experiment 5. The standard curve is used to determine the unknown concentration of each sample by fitting the concentration to the absorbance Absorption Concentration Colloidal Gold-based Immunoassay test strip Colloidal Gold-based Immunoassay test strip Reaction format of the competitive format immunochromatographic assay (left) and sandwich format immunochromatographic assay (right). 27