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Basic Immunologic
Procedures
Terry Kotrla, MS, MT(ASCP)BB
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.
Precipitation/Flocculation
Turbidimetry
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.
Standard Curve
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).
Nephelometry
Can detect either antigen or antibody.
Endpoint tests all Ag/Ab reaction to go to
completion.
Nephelometry
Measures SCATTERED
light bouncing off antigenantibody complexes.
The more light that is
scattered the higher the
concentration.
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.
Four Methodologies
Single diffusion, single dimension
Single diffusion, double dimension
Double diffusion, single dimension
Double diffusion, double dimension
Ouidin
Double Diffusion, Single Dimension
Oudin Precipitation
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.
Radial Immunodiffusion
Radial Immunodiffusion
Two methods
Radial Immunodiffusion
Precipitin Rings
Standards
Standard Curve
Samples
Standard Curve
Ouchterlony Immunodiffusion
Ouchterlony - Identity
FIGURE 2:
If 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
Ouchterlony Non-Identity
Ouchterlony-Interpret
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.
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.
Rocket Immunoelectrophoresis
Rocket Electrophoresis
Immunoelectrophoresis
Immunoelectrophoresis
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.
Immunoelectrophoresis
Immunoelectrophoresis -Antivenom
Immunofixation Electrophoresis
Immunofixation Electrophoresis
Immunofixation Electrophoresis
Immunofixation Electrophoresis
Test is frequently ordered to identify moncolonal
proteins.
May be done on urine or serum depending upon
what doctor suspects.
Multiple myeloma