ELISA Enzyme-linked Immunosorbent Assays (ELISA) - Activity 3 Slides PDF

ELISA Enzyme-linked Immunosorbent Assays (ELISA) → Method for detecting and quantifying a specific protein in a protein solution “Immuno” in Immunosorbent Assay → Antibody-antigen binding “Sorbent” in Immunosorbent Assay → Non-specific adsorption of the antigen or the antibody (depending on the type...

ELISA Enzyme-linked Immunosorbent Assays (ELISA) → Method for detecting and quantifying a specific protein in a protein solution “Immuno” in Immunosorbent Assay → Antibody-antigen binding “Sorbent” in Immunosorbent Assay → Non-specific adsorption of the antigen or the antibody (depending on the type of ELISA) to the well surface Benzene → Microplates used for ELISA are made of polystyrene → Polystyrene is a very hydrophobic compound. It is a long carbon chain with pendant benzene rings on every other carbon. Proteins and other biomolecules passively adsorb to the well surface through hydrophobic interactions → Polystyrene structure can also be modified by incorporating negatively charged carboxyl groups to some of the benzene rings. In this case, proteins are immobilized on the well surface through hydrophobic and ionic interactions Direct ELISA Indirect ELISA → Note that the secondary antibody binds to the primary antibody, NOT to the antigen (our protein of interest) → The primary antibody binds to the antigen Direct and Indirect ELISA Sandwich ELISA → 2 primary antibodies are used, one of which is attached to the well surface and is called the capture antibody Sandwich ELISA can either be direct or indirect General Steps for Direct ELISA → Add sample solution containing the antigen Discard solution and wash the wells → Add blocking buffer/solution. In our experiment, the wash buffer/solution already contains the blocking agent (An inert protein or a detergent) Discard solution and wash the wells → Add enzyme-conjugated antibody (For direct ELISA, only a primary antibody is used, and it is conjugated to the enzyme) Discard solution and wash the wells → Add the substrate that will react with the enzyme attached to the antibody. The reaction results in a change of color of the solution Wash buffer/solution & the blocking agent → In our experiment, the wash buffer/solution, contains Tween 20, a detergent Washing step after discarding antigen solution and antibody solutions ensures removal of excess, unbound antigens or antibodies in the well → decreases background signal Tween 20, the blocking agent added to the wash buffer, blocks unoccupied binding sites on the well surface to prevent direct binding of antibodies → decreases background signal → Commonly used blocking agents are proteins that do not bind to the antibody and detergents Different types of labels/tags for antibodies that allow detection of bound antigens in the well → In our experiment, Indirect ELISA, the secondary antibody is labeled with an enzyme Enzyme-linked antibody → In our experiment, Indirect ELISA, the secondary antibody is labeled with an enzyme Enzyme – Horse Radish Peroxidase (HRP) Chromogenic substrate – 3,3',5,5'-Tetramethylbenzidine (TMB) → HRP oxidizes TMB in the presence of hydrogen peroxide; reaction results in the formation of a blue-colored product → The color intensity of the solution varies directly with antigen (target protein) concentration → Another commonly used reporter enzyme for ELISA is alkaline phosphatase Chromogenic substrate – para-nitrophenyl phosphate (pNPP) Closer look at the antibody → Antibodies are immunoglobulins (Ig) → They are glycoproteins Paratope The 4 polypeptides of an antibody are held together by disulfide bonds between cysteines Closer look at the antibody → Antibodies are immunoglobulins (Ig) → They are glycoproteins Oligosaccharide (carbohydrate), colored black, bound to the constant tail region of an antibody B cell activation and production of antibody (Secreted B cell receptor) → Mature naive (not yet exposed to antigen) B cells that recognize different epitopes and antigens of pathogens that the body has never encountered already exists in the body. → During initial encounter with a pathogen, an antigen (of the pathogen) binds to a mature naive B cell through the B cell receptor (BCR). → The B cell acts as an antigen-presenting cell by internalizing and digesting the antigen and then presenting it on the cell surface through the MHC II molecule. → A helper T cell specific for the same antigen recognizes the MHC II–antigen complex and activates the B cell. → The B cell differentiates into memory B cells and plasma cells. → Plasma B cells secrete antibodies to fight against the pathogen. → Memory B cells do not secrete antibodies. They remain in the body after the initial infection, allowing the body to generate a faster, stronger response against the same pathogen during subsequent exposures. → Tremendous diversity in antigen-binding sites results from random gene recombination and mRNA splicing Gene sequence for antibody heavy chain has multiple V, D, and J segments Gene sequence for antibody light chain has multiple V and J segments Note: No need to memorize left figure on this slide for the exam Supplementary: https://www.youtube.com/watch?v=Na-Zc-xWCLE&list=PL1dwMQTGwkB3nctd-8QGt2QXNuZtCZhTM&index=7 → 5 different classes or isotypes of immunoglobulins (Ig)/antibodies in humans Functions of the antibody inside the body → The complement system refers to an array of different types of soluble proteins in the blood → Complement proteins bind to the surfaces of pathogens and are particularly attracted to pathogens that are already bound by antibodies → Certain complement proteins can form attack complexes that destroy pathogens by opening pores in their membranes Functions of the antibody inside the body Agglutination → Binding of antibodies to different pathogens can result in the clumping of these pathogens → The clumping of pathogens increases the efficacy of their removal by phagocytosis, the process by which certain white blood cells engulf and digest the pathogen Neutralization Opsonization Antibodies are part of the adaptive immune system Antibody production Antigen from a different organism Antigen from a different organism The antibody produced by mouse (the 1˚ antibody) is introduced to another organism Antibody production and nomenclature 2˚ antibodies bind to the tail (Fc) region of the 1˚ antibody → 2˚ antibody is produced by goat, and the antigen it detects is a 1˚ antibody from mouse → 1˚ antibody is produced by mouse, and it binds to an antigen from humans → An advantage of using enzyme-labeled 2˚ antibodies is that they can bind to different 1˚ antibodies (for different antigens) produced from the same organism Ex. If you need 5 different mouse 1˚ antibodies to detect 5 different antigens, instead of labeling each of these 1˚ antibodies with an enzyme, you can just use one enzyme-linked anti-mouse 2˚ antibody that can bind to the Fc region of diff. mouse 1˚ antibodies of the same class, since the Fc or tail region is constant for all 1˚ antibodies of the same class produced by one organism → Antibodies (primary or secondary) used for research can be monoclonal or polyclonal → Antibodies are utilized in other common experimental techniques LATERAL FLOW IMMUNOASSAY → When sample solution flows from L to R, antigen in the sample will bind to tagged (e.g., gold-labeled) antibodies along the way → As the solution keeps flowing, antibody-bound antigens reach the test line and bind to another antigen-specific antibody that is bound to the surface on the test line → Excess tagged antibodies (bound or not bound to antigen) keep flowing and reach the control line, where there are surfacebound antibodies specific for the tagged antibodies (They bind the tagged antibodies, not the antigen) LATERAL FLOW IMMUNOASSAY → In this version of the lateral flow immunoassay, there is a second antibody (Chicken IgY) present in the conjugation pad that does not bind the antigen (COVID virus in this example) → As sample solution flows, the chicken IgY antibodies will not stop on the test line and will continue flowing up to the control line where there are surface-bound antibodies specific for the chicken IgY → The appearance of colored line on the control line ensures that if the result is negative (No colored line on the test line), it is because there is really no antigen in the sample and NOT because insufficient volume of sample solution was used for the assay LATERAL FLOW IMMUNOASSAY LATERAL FLOW IMMUNOASSAY