MEASURING RECEPTOR LIGAND INTERACTION

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MEASURING RECEPTOR LIGAND INTERACTION

• UMESH SHARMA
• CELL MOL ENDO

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RECEPTOR- ASSAY

• Receptor- molecule in cell membrane/
  cytoplasm/nucleus,
• capable of binding a distinct chemical entity, so
  that information is transduced to the cell,
  regulating a variety of biochemical processes
  necessary for the cell (and the organism) to
  function and survive.
• Ligands- drugs, neurotransmitters, hormones
• Types of assays
• 1) Immuno assays
• a) Enzyme immuno assay- Enzyme linked immuno
  sorbent assay (ELIZA)
• b) Radio Immuno assay
• c) Radio immunometric assay
• d) Fluorescent Immuno assay
• 2) Receptor assay
• a) Competitive, non- competitive
• b) Association, Dissociation assay
• 3) Others
• a) Western Blotting
• b) Electronic method
• c) Recombinant Proteins/Fusion Tag Antibodies

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EIAs for Antigen Detection
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EIAs for Antibody DetectionNon Competitive
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Competitive EIA
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Capture EIAto detect a specific type of
antibody, such as IgG or IgM
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Radio immuno assay (RIA)
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Radio immuno metric assay
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Receptor Assays

• General criteria
• (a) They should bind to the same receptor site as
  the unlabeled ligands.
• (b) They should be chemically stable and
  resistant to enzymatic and hydrolytic
• degradation. The latter might be of particular
  concern when peptides are
• used as labeled ligands.
• (c) Non-specific binding to non-receptor sites
  and materials used must be
• minimal.
• (d) They should preferably be eutomers, i.e. the
  pharmacologically most potent
• enantiomers.
• Radioligands have to meet some additional
  criteria
• (e) They should be of high specific activity.
  This is necessary because the
• receptor density in most tissues and organs is
  limited.
• (f) They have to be radiochemically pure.

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Receptor Assays

• R L L RL RL
• Competitive binding experiments measure
  equilibrium binding of a single concentration of
  radioligand at various concentrations of an
  unlabeled competitor.
• Saturation binding experiments measure
  equilibrium binding of various concentrations of
  the radioligand. Analyze the relationship between
  binding and ligand concentration to determine the
  number of sites, Bmax, and the ligand affinity,
  Kd.
• Kinetics experiments measure binding at various
  times to determine the rate constants for
  radioligand association and dissociation.
• Dissociation ("off rate") experiments
• A dissociation binding experiment measures the
  "off rate" for radioligand dissociating from the
  receptor. Initially ligand and receptor are
  allowed to bind, perhaps to equilibrium. At that
  point, you need to block further binding of
  radioligand to receptor so you can measure the
  rate of dissociation. There are several ways to
  do this

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Dissociation Curve
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Western Blot

• SDS PAGE- separate proteins and transfer on to
  solid membrane
• Primary Ab specific to antigen added
• Second/Conjugate Ab added
• anti-immunoglobulin antibodies coupled to a
  reporter group such as the enzyme alkaline
  phosphatase are added (e.g. Goat anti-human IgG-
  alkaline phosphatase)
• Substrate added gives characteristic color to the
  band

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Typical Western Blot- HIV

• Band pattern Interpretation
• Lane 1, HIV serum (positive control)
• Lane 2, HIV- serum (negative control)
• Lane A, Patient A
• Lane B, Patient B
• Lane C, Patient C

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ELECTRONIC METHOD

• Uses the biotin-streptavidin interaction
• The biotin ligand is site-specifically anchored
  to a chemically modified silicon oxide surface
  and changes in conduction due to interaction are
  measured
• Atomic Force Microscopy is also used to
  characterize the morphology of the molecules on
  the surface. Finally, a control sample, i.e. a
  sample on which the biotin-streptavidin
  interaction is not allowed, exhibits no change in
  the measured conductance.

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Recombinant Proteins/Fusion Tag Antibodies

• Short pieces of well-defined peptides (Poly-His,
  Flag-epitope or c-myc epitope or HA-tag) or small
  proteins (bacterial GST, MBP, Thioredoxin,
  b-Galactosidase, VSV-Glycoprotein etc) are often
  cloned along with the target gene.
• Proteins are expressed as fusion proteins.
  Antibodies to these fusion-tags are already
  available to monitor fusion protein expression
  and purification. Therefore, fusion-tags serve as
  universal tags much like secondary antibodies.
  Ex- GST-fusion proteins can bind to
  glutathione-Sepharose. Therefore, a high degree
  of purification of fusion protein can be achieved
  in just one affinity purification