Immunologists Toolbox

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Immunologists Toolbox

• Tuesday, January 29, 2008

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Haptens

• Small, simple organic molecules that do not
  provoke antibodies when injected on their own.
• Examples phenyl arsonates and nitrophenyls.
• Can elicit an immune response if it is attached
  to a larger Carrier molecule.
• Commonly proteins
• Examples of protein carrier molecules - BSA,
  ovalbumin.

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Medical importance of anti-hapten antibodies

• Penicillin allergies
• Penicillin is too small to elicit an immune
  response on its own.
• Poison ivy
• Toxin is oxidized in the skin cell to create a
  quinone molecule.
• Quinone reacts with proteins in skin cells to
  create hapten-carrier conjugates.

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Three types of antibodies produced against
hapten-carrier conjugates.
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Purification of specific antibody

• Antigen is bound covalently to small beads, which
  are loaded into a column.
• Antiserum is passed over beads.
• Specific antibodies bind to the immobilized
  antigen
• Everything else passes through the column.
• Purified antibodies are eluted from the beads by
  raising or lowering pH.

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Affinity Chromatography

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Enzyme-Linked Immunosorbant Assay (ELISA)

• Labeled antibody used for detection scheme.
• Commonly used for viral diagnostics such as HIV
  detection.
• Detects specific viral proteins.
• Detects specific anti-viral antibodies.
• Does NOT allow one to measure amount of antigen
  present.
• Two common methods.
• Direct binding.
• Sandwich method.

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Direct labeling of antigen

• Coat plate with specific antigen.
• Add specific, labeled antibody .
• Rinse out extra antibody.
• Add enzyme, commonly horseradish peroxidase.
• Add substrate to cause color change.
• Measure color.

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Sandwich Method

• Coat plate with specific antibody.
• Add antigen of interest, serum, etc.
• Add labeled antibody specific for a different
  epitope of the antigen.
• Add enzyme linked 2 antibody
• Add substrate to cause color change.
• Measure color

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Quantifying antigen or antibody concentration
using competitive inhibition assay.

• Fixed amount of antibody attached to well.
• Standard concentration of labeled standard added
  to wells.
• Unlabeled standard or test samples are then added
  in various amounts to multiple wells.
• Displacement of labeled antigen is then measured
  and compared to standard curve.

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Precipitation reaction

• Addition of sufficient amounts of antibody to
  soluble antigen causes a precipitate to form.
• The first method used to quantify antibody
  concentration.
• Now seldomly used in immunology.
• Good example of the mechanisms for
  antigenantibody complex formation.
• Antigen must have multiple binding sites for
  precipitation to occur.

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Precipitation Curve

• At equivalence, the largest immune complexes
  form.
• Excessive antibody will form small amounts of
  complexes.
• In antigen excess some of the complexes are too
  small to precipitate out.
• Can cause damage to small blood vessels in vivo.

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Antigen Valence

• Valence is the maximum number of antibodies that
  can bind to a single antigen at one time
• Valence of an antigen does not have to equal the
  number of epitopes the antigen has.
• Steric hinderance prevents all different
  antibodies from binding.

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Hemagglutination and Blood typing

• Three primary blood types A, B, and O.
• Common gut bacteria bear antigens on their
  surfaces that are similar or identical to
  blood-group antigens.
• Therefore we produce antibodies against these
  antigens if we do not bear them on our own red
  blood cells.
• We can use these antibodies to test for blood
  type.
• When serum from an individual is added to blood
  of another individual, if blood type does not
  match antibodies will cause the red blood cells
  to clump up.

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The use of antibodies as research and diagnostic
tools.

• Detection of bound antibodies.
• Monoclonal and polyclonal antibodies.
• Immuno fluorescence.
• Flow Cytometry
• Immunoelectron microscopy.
• Immunoprecipitation.
• Immunoblotting.

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Detection of bound antibodies.

• Protein A.
• Produced in S. aureus.
• Binds Fc portion of some immunoglobulins)
• Can be labeled for detection of specific
  antibodies or used for purification.
• Protein G
• isolated from Group C Strep.
• Works the same is protein A
• Binds some of the same Igs as Protein A and some
  others as well.
• Protein A/G
• Combination of Fc binding domains of Protein A
  and Protein G.

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• Anti-immunoglobulin antibodies
• Can be specific for
• Ig heavy chains
• Ig light chains.
• Unique features within the antigen-binding site
  (idiotype).
• Individual species.
• Antibodies raised in goats to mouse antigen.
• Different alleles within the same species
  (allotypes).
• Due to genetic polymorphisim.
• Individual (isotypes) such as
• IgG, IgA, IgD.

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Monoclonal and polyclonal Antibodies

• Polyclonal antibodies are derived from multiple
  cell lines against one particular antigen each
  recognizing a different epitope.
• Most easily obtained by immunizing a suitable
  mammal, such as a mouse, rabbit or goat.
• Although all antibodies will bind to the specific
  antigen, some are better than others.
• Monoclonal antibodies are derived from a single
  cell line and recognize one single epitope.
• Obtained by fusing a B cell secreting the
  antibody with the desired specificity with
  myeloma cells to create an immortal cell line to
  produce mass quantities of antibody.
• Fused by adding polyethelyne glycol.
• Fused cells are called hybridomas
• In culture the cells will make many clones of
  itself all secreting identical antibodies.

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Creation of a hybridoma

• Monoclonal antibodies are derived from a single
  cell line and recognize one single epitope.
• Obtained by fusing a B cell secreting the
  antibody with the desired specificity with
  myeloma cells to create an immortal cell line to
  produce mass quantities of antibody.
• Fused by adding polyethelyne glycol.
• Fused cells are called hybridomas
• In culture the cells will make many clones of
  itself all secreting identical antibodies.

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Immunofluorescence

• Used to identify a particular molecule in cells,
  tissues or biological fluids.
• Can be used to locate target molecules accurately
  in single cells or tissues.
• Useful for a variety of methods

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How it works

• A fluorescent molecule is attached to
• An antibody against a molecule of interest
  (direct immunofluorescence).
• An anti-immunoglobulin antibody (indirect
  immunofluorescence).
• Add labeled antibody (for direct) or primary
  unlabeled antibody (for indirect) to single cell
  suspension. If indirect, add secondary (labeled
  antibody) to cell suspension.
• Expose the labeled cells to a specific wavelength
  of light.
• Fluorescent molecule will emit light at a greater
  wavelength.
• Multiple proteins can be identified in the same
  sample by using different antibody/fluorphor
  conjugates.

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Immunofluorescence microscopy

• Light microscope with filter sets that allow
  only certain wavelengths of light to pass
  through.
• Allows for the detection and location of specific
  proteins of interest.
• Usually limited to a maximum of 5 fluorophores.

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Laser confocal microscopy

• An improvement to fluorescence microscopy.
• Laser is tuned to a specific wavelength of light
  to excite the fluorescent molecules.
• Can excite and detect many fluorophores.
• Detects a variable range of fluorsecence set by
  user.
• Uses a pinhole to block out of focus light.
• Allows for better sectioning.
• Allows for better resolution.
• Can do live, real time, imaging.

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Confocal image of multi-colored image.
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Immunoelectron microscopy

• Gold particles are attached to antibodies used to
  identify intracellular molecules within cells.
• Can identify multiple molecules by varying the
  size of the gold particle attached to each
  antibody.
• Very time consuming and expensive.

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Flow Cytometry

• Counts and sorts thousands of individual cells
  per second.
• Newer machines can count and sort up to 21
  paramaters at a time.
• Identifys cells based upon
• Size
• Complexity
• Specific molecules tagged with fluorsecent
  probes.

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Immunoprecipitation

• Using an antigen-specific antibody to pull a
  protein out of solution.
• Can pull down a single protein or a complex of
  proteins (co-immunoprecipitation) if they are
  attached. If it is a DNA binding protein, the
  attached DNA can also be isolated (chromatin
  immunoprecipitation).
• Process will isolate the protein with a
  relatively high degree of purity.
• Proteins identified by
• SDS PAGE
• Western Blot
• Mass spectometry

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TUNEL assay to detect apoptosis.

• Apoptotic cells degrade their chromatin.
• Cells are exposed to TdT and biotin labeled UTPs.
• TdT adds the labeled UTPs to the 3 end of the
  fragmented DNA.
• Streptavadin tagged with fluorescent antibodies
  are then added to the mix.
• SA will bind to the biotin.
• Amount of labeled UTPs is measured with Flow
  Cytometery or fluorescence microscopy. Large
  signal indicates apoptosis.

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Transgenic mice

• Mice can be altered to contain altered or extra
  copies of a gene.
• Allows researchers to study the effects of
  specific mutations in the whole mouse such as
• Identification or regluatory gtenes.
• Effects of a genes over-expression
• Expression in inappropriate tissues
• Effects of mutations on gene function.

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Congenic Mice

• Transgenic mice need to be introduced into a
  well-categorized , stable genetic background.
• It is often difficult to successfully prepare
  transgenic embryos in inbred strains of mice.
• SOwe breed the transgene onto the well
  categorized, stable background.
• Usually a C57BL/6 mouse.
• Needs to be back crossed at least 10 generations.
• Creates 99 genetically identical mice.

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Knockout Mice

• Replacing a normal gene with a defective copy.
• Often the gene is disrupted by inserting another
  gene, commonly for antibiotic resistance, in the
  middle of it.
• Defective gene is cloned and inserted into
  embryonic stem cell.
• Defective gene then recombines with the
  homologous copy in the cells genome.
• These cells are then inserted into a mouse
  blastocyst which, in turn, are in placed into a
  pseudopregnant female.
• Some of the offspring will contain tissues
  derived from the injected cells (chimeric mice).

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