Cell mol lab

• Enzyme-linked Immunosorbent Assays (ELISA) are a method for detecting and quantifying a specific protein in a protein solution.
• Antibody-antigen binding is a crucial aspect of Immuno sorbent Assay.
• Microplates used for ELISA are made of polystyrene, a very hydrophobic compound.
• Proteins and other biomolecules passively adsorb to the well surface through hydrophobic interactions.
• The polystyrene structure can also be modified by incorporating negatively charged carboxyl groups to some of the benzene rings, immobilizing proteins on the well surface through hydrophobic and ionic interactions.
• In InDirect ELISA, the secondary antibody binds to the primary antibody, not to the antigen, the protein of interest.
• The primary antibody binds to the antigen in Direct and Indirect ELISAs, and in Sandwich ELISA, two 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.
• The general steps for Direct ELISA are: discard solution and wash the wells, add sample solution containing the antigen, add blocking buffer/solution, add enzyme-conjugated antibody, add the substrate that will react with the enzyme attached to the antibody.
• Antibodies are utilized in other common experimental techniques.
• When sample solution flows from L to R, antigen in the sample will bind to tagged (e.g., gold-labeled) antibodies along the way.
• The appearance of a 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.
• 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.
• The Fc or tail region is constant for all 1˚ antibodies of the same class produced by one organism.
• 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).
• Antibodies (primary or secondary) used for research can be monoclonal or polyclonal.
• 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.
• Excess tagged antibodies (bound or not bound to antigen) keep flowing and reach the control line, where there are surface-bound antibodies specific for the tagged antibodies.
• The gene sequence for antibody light chain has multiple V and J segments.
• Binding of antibodies to different pathogens can result in the clumping of these pathogens, increasing the efficacy of their removal by phagocytosis.
• Certain complement proteins can form attack complexes that destroy pathogens by opening pores in their membranes.
• Antibodies are part of the adaptive immune system.
• If you need 5 different mouse 1st antibodies to detect 5 different antigens, instead of labeling each of these 1st antibodies with an enzyme, you can just use one enzyme-linked anti-mouse 2nd antibody that can bind to the Fc region of different 1st antibodies.
• The 1st antibody is produced by mouse, and it binds to an antigen from humans.
• The functions of the antibody inside the body include the complement system, which refers to an array of different types of soluble proteins in the blood.
• Memory cells remain in the body after the initial infection, allowing the body to generate a faster, stronger response against the same pathogen during subsequent exposures.
• 2nd antibodies bind to the tail (Fc) region of the 1st antibody.
• Tremendous diversity in antigen-binding sites results from random gene recombination and mRNA splicing.
• There are 5 different classes or isotypes of immunoglobulins (Ig)/antibodies in humans.
• The antibody produced by mouse (the 1st antibody) is introduced to another organism.
• The gene sequence for antibody heavy chain has multiple V, D, and J segments.
• Complement proteins bind to the surfaces of pathogens and are particularly attracted to pathogens that are already bound by antibodies.
• An advantage of using enzyme-labeled 2nd antibodies is that they can bind to different 1st antibodies (for different antigens) produced from the same organism.
• Wash buffer/solution and the blocking agent are discarded after the washing step to remove excess, unbound antigens or antibodies in the well, decreasing background signal.
• 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.
• The 4 polypeptides of an antibody are held together by disulfide bonds between cysteines.
• Mature naive B cells recognize different epitopes and antigens of pathogens that the body has never encountered.
• HRP oxidizes TMB in the presence of hydrogen peroxide; reaction results in the formation of a blue-colored product.
• Tween 20, the blocking agent added to the wash buffer, blocks unoccupied binding sites on the well surface to prevent direct binding of antibodies, decreasing background signal.
• During initial encounter with a pathogen, an antigen (of the pathogen) binds to a mature naive B cell through the B cell receptor (BCR).