2. Basic Immunologic Procedures

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2. Basic Immunologic Procedures

• Terry Kotrla, MS, MT(ASCP)BB

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This section of Unit 2 Contains

• Part 2 Measurement of Light Scattering
• Part 3 Passive Immunodiffusion Techniques
• Part 4 Electrophoretic Techniques

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Introduction

• How are antigen antibody reactions detected?
• Multitude of test methodologies have been
  developed.
• Go from simple to complex.
• Can be used either as a screening test or a
  confirmatory test.

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Measurement of Precipitation by Light Scattering

• Antigen-antibody complexes, when formed at a high
  rate, will precipitate out of a solution
  resulting in a turbid or cloudy appearance.
• Turbidimetry measures the turbidity or cloudiness
  of a solution by measuring amount of light
  directly passing through a solution.
• Nephelometry indirect measurement, measures
  amount of light scattered by the antigen-antibody
  complexes.

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Precipitation/Flocculation

• When soluble antibody binds to soluble antigen
  (sensitization) there will come a point where
  lattice formation will occur resulting in
  precipitation occurring resulting in a visible
  reaction
• These immune complexes have fallen out of
  solution. The Ab at the bottom in the
  illustration at right is still in the soluble
  phase.

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Turbidimetry

• Measures turbidity or cloudiness of a solution by
  measuring the amount of light PASSING THROUGH the
  solution.
• Soluble antigen and antibody join and once they
  join in sufficient amounts precipitate, results
  in cloudiness.
• The more cloudy the solution, the less light can
  pass through.

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Turbidimetry

• The amount of substance being quantitated is
  calculated based on the results obtained on
  standards (aka calibrators) and controls.
• Calibrator is a substance of an EXACT amount,
  i.e. 50 mg/dL, used to create standard curve.
• Controls are substances similar to patient
  samples and have a range, i.e., 43-56 mg/dL
• Turbidimetry is very simple but not very
  sensitive.

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Standard Curve
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Nephelometry

• Widely used in clinical laboratories because it
  is relatively easily automated.
• Based on the principle that a dilute suspension
  of small particles will scatter light passed
  through it rather than simply absorbing it.
• The amount of scatter is determined by collecting
  the light at an angle (usually about 70 or 75
  degrees).

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Nephelometry

• Can detect either antigen or antibody.
• Endpoint tests all Ag/Ab reaction to go to
  completion.
• If complexes get to large will fall out of
  solution.
• Causes falsely decreased results
• Kinetic tests add antigen and antibody then
  measure at a specific time.
• Rate of formation must be known.
• Calculate concentration based on standards.

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Nephelometry

• Measures SCATTERED light bouncing off
  antigen-antibody complexes.
• The more light that is scattered the higher the
  concentration.

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Turbidimetry versus Nephelometry

• Turbidimetry measures light which PASSES
  through.
• Nephelometry measures light which is SCATTERED.

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Passive Immunodiffusion

• Reactions of antigens and antibodies in agar gel.
• Migrate towards each other and where they meet in
  optimal proportions form a precipitate.
• Passive because they are allowed to react to
  completion with no enhancements such as an
  electrical charge applied.

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Factors Affecting Rate of Diffusion

• Size of the particles.
• Temperature
• Gel viscosity and hydration
• Interaction of reactants with gel

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Four Methodologies

• Single diffusion, single dimension
• Single diffusion, double dimension
• Double diffusion, single dimension
• Double diffusion, double dimension

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Ouidin Double Diffusion, Single Dimension
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Oudin Precipitation

• Solution of antibody is carefully layered on top
  of a solution of antigen, such that there is no
  mixing between the two. 
• With time at the interface where the two layers
  meet, antigen-antibody complexes form a visible
  precipitate.  The other two tubes are negative
  controls, containing only antibody or only
  antigen plus an irrelevant protein in the second
  layer. 

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Radial Immunodiffusion

• Antibody mixed with agar poured into plate.
• Holes punched.
• Add standards, controls and patients to wells.
• Antigen will diffuse out and form precipitin
  ring.
• The diameter of the ring directly proportional to
  concentration.
• Create standard curve and read results.

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Radial Immunodiffusion
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Radial Immunodiffusion

• Two methods
• Endpoint allows reaction to go to completion.
• Kinetic measurements taken at a specific time
  before zone of equivalence is reached.
• This is a QUANTITATIVE technique which will give
  the actual concentration.

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Radial Immunodiffusion
Precipitin Rings
A B C
a b c
Standards
Samples
Standard Curve
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Standard Curve
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RID Sources of Error

• Over or under filling the well.
• Spilling sample onto the outside of the well.
• Nicking the well with the pipette tip.
• Improper incubation time or temperature.
• Incorrect measuring of the wells.

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Ouchterlony Gel Diffusion

• Holes punched in agar.
• Known antibody or antigen added to center well.
• Known sample added to outer well.
• Unknown sample added to outer well next to
  unknown sample.
• Wait for bands to form.
• This is a QUALITATIVE technique, simply
  determines the presence NOT the concentration.

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Ouchterlony Immunodiffusion
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Ouchterlony - Identity

• Precipitation appears as a continuous line in the
  form of an arc between the two outer wells and
  the center well. There are no spurs at the angle
  and this type of reaction is termed a band of
  identity.

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Ouchterlony Partial Identity

• FIGURE 2If a solution with antigens X and Y is
  placed in well 1, a solution with antigen X only
  is placed in well 2, and antiserum containing
  antibodies specific for both X and Y is placed in
  well 3, a reaction similar to that appearing in
  Fig. 2 will occur. Notice that there is a spur
  reaction towards the XY well. This indicates that
  the two antigenic materials in wells 1 and 2 are
  related, but that the material in well 1
  possesses an antigenic specificity not possessed
  by the material in well 2. Such a reaction with
  spur formation indicates partial identity

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Ouchterlony Non-Identity

• If the material in wells 1 and 2 do not possess
  common antigens and the antiserum in well 3
  possesses specificities for both materials, the
  reaction will appear as two crossed lines as in
  Fig. 3

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Ouchterlony-Interpret

• Determine which interpretation fits for samples
  1, 2 and 3.

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Electrophoretic Techniques

• Immunodiffusion can be combined with electrical
  current to speed things up.
• Electrophoresis is a technique which separates
  molecules using electrical current.
• Small molecules move faster than large.
• For immunolectrophoresis antigen and antibody
  migrate through gel faster.
• Can be single or double diffusion.

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Rocket Immunoelectrophoresis

• Adaptation of radial immunodiffusion (RID).
• Antibody incorporated (mixed) into the gel.
• Antigen added to wells.
• Apply electrical current and antigen will move
  forward and will bind to antigen.
• Dissolution and reformation occurs.
• Height of precipitin band related to
  concentration of antigen.
• Much faster than RID.

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Rocket Immunoelectrophoresis

• Antigen is electrophoresed into gel containing
  antibody. The distance from the starting well to
  the front of the rocket shaped arc is related to
  antigen concentration.

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Rocket Electrophoresis
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Immunoelectrophoresis
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Immunoelectrophoresis

• Two step double diffusion technique.
• Electrophorese antigen.
• Antiserum added to trough parallel to line of
  separation.
• Incubate overnight.
• Diffusion occurs and bands of precipitate form.
• Most often used as a screening test.

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Immunoelectrophoresis

• Two-dimensional immunoelectrophoresis. Antigens
  are separated on the basis of electrophoretic
  mobility. The second separation is run at right
  angles to the first which drives the antigens
  into the antiserum-containing gel to form
  precipitin peaks the area under the peak is
  related to the concentration of antigen.

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Immunoelectrophoresis -Antivenom

• Each antibody molecule can bind two separate
  sites on an antigen molecule (venom toxin),
  consequently antibodies have the ability to cross
  link many antigen molecules simultaneously.   
  This cross-linking causes the antibody
  antigen-complex to become insoluble and
  precipitate out from the solution.
• The immunoelectrophoresis technique makes use of
  this capability of the antibodies to form giant
  insoluble complexes with their respective
  antigens.    The antigen-antibody precipitate
  which forms can be visualized by specific
  staining techniques, or quantified by various
  means.

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Immunofixation Electrophoresis

• Immunofixation Electrophoresis (IFE) combines
  electrophoresis with immunoprecipitation.
• This technique may be used to identify and
  characterize serum or urine proteins.
• In IFE, proteins of sample are first separated by
  electrophoresis on a support (agarose) according
  to their charge and after that the medium is
  overlaid with monospecific antisera reactive with
  specific protein - antigen.
• If the antigen is present a characteristic
  immunoprecipitin band will be formed.

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Immunofixation Electrophoresis
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Immunofixation Electrophoresis
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Immunofixation Electrophoresis

• Test is frequently ordered to identify moncolonal
  proteins.
• May be done on urine or serum depending upon what
  doctor suspects.
• Multiple myeloma
• Production of large amount of specific protein.
• Will be excreted in urine

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Electrophoresis Sources of Error

• Applying current in wrong direction.
• Incorrect buffer pH
• Incorrect timing
• Incorrect current applied.
• Concentration of reactants must be appropriate.

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End of this lecture.