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2.

Basic Immunologic
Procedures
Terry Kotrla, MS, MT(ASCP)BB

This section of Unit 2 Contains


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

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.

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.

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.

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.

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.

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.

Nephelometry

Measures SCATTERED
light bouncing off antigenantibody complexes.
The more light that is
scattered the higher the
concentration.

Turbidimetry versus Nephelometry


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

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.

Factors Affecting Rate of Diffusion


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

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

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, antigenantibody 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.

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

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.

Radial Immunodiffusion
Precipitin Rings

Standards

Standard Curve

Samples

Standard Curve

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.

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.

Ouchterlony Immunodiffusion

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.

Ouchterlony Partial 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

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

Ouchterlony-Interpret

Determine which interpretation fits for samples 1, 2 and


3.

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

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.

Rocket Electrophoresis

Immunoelectrophoresis

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.

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.

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.

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.

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

Production of large amount of specific protein.


Will be excreted in urine

Electrophoresis Sources of Error


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

End of this lecture.