LBYBI37 ELISA

• ELISA: Enzyme-linked Immunosorbent Assays

  • 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 absorption of antigen/antibody to the well surface
• • microplates used for ELISA are made of polystyrene
  • polystyrene is very hydrophobic, proteins and other biomolecules passively absorb to the well surface through hydrophobic interactions
• Direct ELISA

  • only one antibody is used
  • single antibody is conjugated directly to detection enzyme
• Indirect ELISA

  • uses two antibodies (primary antibody and enzyme-linked secondary antibody)
• Sandwich ELISA

  • can be direct or indirect
  • uses two primary antibodies, one of which is attached to the well surface and is called the capture antibody
• General Steps for Direct ELISA

  1. Coating: antigen is absorbed onto well in ELISA plate in coating buffer
  2. Blocking: buffer containing unrelated protein is used to block free sites in the wells
  3. Detection: enzyme conjugated detection antibody binds antigen
  4. Readout: substrate is catalyzed by enzyme to generate colored readout
• Wash Buffer/Solution and The Blocking Agent

  • wash buffer/solution contains detergent Tween 20 (blocking agent)
  • washing step ensure removal of excess, unbound antigens or antibodies in the well; decreases background signal
  • Tween 20 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
• Enzyme-linked antibody

  • in the experiment, indirect ELISA, is labeled with an enzyme
  • enzyme - horse radish peroxidase (HRP)
  • chromogenic substrate - 3,3',5,5'-Tetramethylbenzidine (TMB)
  • HRP oxidizes TMB in presence of hydrogen peroxide; reaction results in blue-colored product
  • color intensity of solution varies directly with antigen (target protein) concentration
  • another commonly used reporter enzyme for ELISA is alkaline phosphatase
  • chromogenic substrate - para-nitrophenyl phosphate (pNPP)
• • Antibodies are immunoglobulins (lg)
  • Antibodies are glycoproteins
• Mature naive B cells that recognize different epitopes and antigens of pathogens the body has never encountered already exist in the body.
• [1] During initial encounter with pathogen, an antigen binds to a mature naive B cell through the B cell receptor (BCR).
• [2] 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.
• [3] Helper T cell specific for the same antigen recognizes the MHC II-antigen complex and activates the B cell.
• [4] B cell differentiates into memory B cells and plasma cells.
• [5] Plasma B cells secrete antibodies to fight against pathogen.
• [6] Memory B cells do not secrete antibodies. They remain in the body after initial infection, allowing for a stronger response against the same pathogen.
• Memory B cells remain in the body after initial infection, allowing for a stronger response against the same pathogen.
• 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
  • 5 different classes or isotypes of antibodies in humans
  • monomer: IgD, IgE, IgG
  • dimer: IgA
  • pentamer: IgM
• Functions of the antibody inside the body

  • neutralization: prevents virus or toxic proteins from binding to their target
  • opsonization: pathogen tagged by antibodies is consumed by macrophage or neutrophil
  • complement activation: antibodies attached to the surface of a pathogen cell activate the complement system
  • complement system: array of different types of soluble proteins in the blood
  • agglutination: binding of antibodies to different pathogens can result in clumping; increases efficacy of removal by phagocytosis
• Antibodies are part of the adaptive immune system.
• Adaptive Immune System

  1. specific response
  2. lag time between antigen exposure and response
  3. retains immunological memory
  4. found in only jawed vertebrates
• Innate Immune System

  1. nonspecific responce
  2. immediate response
  3. retains no immunological memory
  4. found in nearly all forms of life
• Primary or secondary antibodies used for research can be monoclonal or polyclonal.