PRECIPITATION REACTION

ZANDRA JOY P. COLUMNA, RMT

 PRECIPITATION
• One of the easiest of serological tests.
• It was first noted in 1987 by Kraus
• It involves combining SOLUBLE antigen with SOLUBLE antibody to
produce INSOLUBLE complexes that are visible.
• Occur best when antigen and antibody are present in optimal
proportions (Equivalence).

NOTE: All antigen and antibody bindings are reversible according to LAW OF
MASS ACTION.
 PRECIPITATION CURVE
• Plots the amount of Antigen-Antibody complexes precipitated when
increasing antigen concentrations are added to a constant antibody.

• Three zones:
1. ZONE OF ANTIBODY EXCESS- precipitation is inhibited and
antibody not bound to antigen can be detected in the supernatant.

2. ZONE OF EQUIVALENCE- Maximal precipitation in which antibody

and antigen form large insoluble complexes; Neither antigen or
antibody can be detected in the supernatant.

3. ZONE OF ANTIGEN EXCESS- precipitation is inhibited and antigen

not bound to antibody can be detected in the supernatant.
PRECIPITATION CURVE
 METHODS USED TO QUANTITATE PRECIPITATION

A. Measurement of Light Scattering

1. NEPHELOMETRY
2. TURBIDIMETRY
B. By Passive Immunodiffusion
1. SINGLE DIFFUSION
2. RADIAL IMMUNODIFFUSION
3. OUCHTERLONY DOUBLE DIFFUSION
C. By Electrophoretic Technique
1. ROCKET IMMUNOELECTROPHORESIS
2. IMMUNOELECTROPHORESIS
3. IMMUNOFIXATION ELECTROPHORESIS
 TURBIDIMETRY
• It measures the reduction in light intensity due to reflection, absorption,
or scatter.
• It is recorded in absorbance units, and is measured with a
spectrophotometer.

• PRINCIPLE: Particles in the solution cause a decrease in transmitted

light, that is incident light is blocked by particles in solution.

NOTE: As the number of particles INCREASES, the lesser transmittance of the

incident light.
 NEPHELOMETRY
• It measures the light that is scattered at a particular angle from the
incident beam.

• The amount of light scattered is an index of the solution’s concentration.

• END POINT NEPHELOMETRY- it allows total reaction to takes place

• KINETIC or RATE NEPHELOMETRY- measurement is done immediately

after the reagent is added.
 PASSIVE IMMUNODIFFUSION
• Makes use of a medium such as agar or agarose
• Agarose is preferred over agar because it has no charge of itself while
agar has a strong negative charge.
• Reactants are added to the gel and antigen-antibody combination
occurs by means of diffusion.

FACTORS AFFECTING THE RATE OF DIFFUSION:

1. Size of particle
2. Temperature
3. Gel viscosity
4. Amount of Hydration
 SINGLE DIFFUSION (OUDIN TEST)
PRINCIPLE:
An antibody is incorporated into agarose in a test tube. The antigen
is then layered on top and as the antigen moves into the gel, precipitation
will occur and moves down the tube in proportion to the amount of antigen
present.

SUMMARY:
• Antibody is incorporated into agarose in a test tube.
• Antigen is layered on top and precipitates as it moves down.
 RADIAL IMMUNODIFFUSION (RID)
PRINCIPLE:
An antibody is uniformly distributed in the support gel and an
antigen is applied to a well cut into the gel. As the antigen diffuses out from
the well, the antigen-antibody complex occurs in changing proportion until
the zone of equivalence is reached and a stable lattice network is formed in
the gel. The area of the ring obtained is a measure of ANTIGEN
concentration, and this can be compared to a standard curve obtained by
using antigens of known concentration.
 RADIAL IMMUNODIFFUSION (RID)
SUMMARY:
• Antibody is uniformly distributed in the support gel.
• Antigen is applied to a well cut into the gel.
• The area of the ring is a measure of antigen concentration (compared to
a standard curve).

The region of equivalence

 RADIAL IMMUNODIFFUSION (RID)
TWO TECHNIQUES:

1. MANCINI (END-POINT METHOD)

 Antigen is allowed to diffuse to completion.
 It takes 24-72 hours to complete.
 The square of diameter is DIRECTLY PROPORTIONAL to the
concentration of the antigen.

2. FAHEY and MCKELVEY (KINETIC METHOD)

 Measurement is taken before the point of equivalence is reached.
 It takes 18 hours to complete.
 The diameter is proportional to the log of the concentration
 RADIAL IMMUNODIFFUSION (RID)
SOURCES OF ERROR IN RADIAL IMMUNODIFFUSION:

1. Overfilling or underfilling of the well.

2. Nicking the side of the well when filling.
3. Spilling the sample outside the well.
4. Improper incubation time.
5. Improper temperature.
6. Incorrect measurement.
 OUCHTERLONY DOUBLE DIFFUSION
PRINCIPLE:
Both antigen and antibody diffuse independently through a
semisolid medium in two dimensions (horizontally and vertically). Wells are
cut in the gel, and reactants are added to the wells. After an incubation
period of between 12-24 hours in a moist chamber, precipitin lines from the
moving front of antigen meets the antibody. The density of the line reflects
the amount of immune complex formed.

• Antibody that is placed MULTI-SPECIFIC is placed in the central well.

• Antigens are placed in the surrounding wells.
OUCHTERLONY DOUBLE DIFFUSION

SEROLOGICAL
IDENTITY
 The 2 antigens are the
same

NON-IDENTITY
 The 2 antigens are not
identical

PARTIAL IDENTITY
 They are not identical
but have certain
common antigenic
determinant.
 ELECTROPHORETIC TECHNIQUE
• Speeds up or sharpens results
• It is based on the migration of particles.

ELECTROPHORESIS
 Movement of macromolecule in an electric field.
 Migration of molecules depends on CHARGE and SIZE.

CHARGE:
• Molecules that positively charged move towards the negative
electrode.
• Molecules that are negatively charged move towards the positive
electrode.

SIZE:
• Large molecules migrate slowly and cover short distances
• Small molecules migrate faster and cover longer distances
 ROCKET IMMUNOELECTROPHORESIS
• Laurell Procedure
• One dimension electroimmunodiffusion

PRINCIPLE:
Antibody is distributed in the gel, and antigen is placed in wells cut
in the gel. However, instead of allowing diffusion to take place at its own
rate, electrophoresis is used to facilitate migration of antigen into the agar.
When the antigen diffuses out of well, precipitation begins. As the
concentration of antigen changes, there is dissolution and reformation of the
precipitate at ever-increasing distances from the well. The end result is a
precipitation line that is conical in shape.
 ROCKET IMMUNOELECTROPHORESIS

NOTE: The distance from the starting well to the front of the rocket shaped
arc is related to antigen concentration.
 IMMUNOELECTROPHORESIS
• Grabar and Williams
• Electrophoresis + Double diffusion Technique

PRINCIPLE:
1. Antigen in serum is electrophoresed to separate out the main protein
fractions then a trough is cut in the gel parallel to the line of separation.
Antiserum is placed in the trough, and the gel is incubated for 18-24
hours.
2. Double diffusion occurs at right angles to the electrophoretic separation,
and precipitin line develops where specific Antigen-Antibody
combination takes place. These lines or arcs can be compared in shape,
intensity and location to that of a normal serum to detect anibodies.
IMMUNOELECTROPHORESIS
 IMMUNOFIXATION ELECTROPHORESIS
• Similar with Immunoelectrophoresis except that after electrophoresis
takes place, the anti-serum is DIRECTLY applied rather than placing it in
a trough.
 COUNTER-CURRENT IMMUNOELECTROPHORESIS

• Reactions occur between migrating antigens and antibodies during

electrophoresis (Ag and Ab migrate toward each other by
electrophoresis).

• Used only when antigen and antibody have opposite charges.

• Pairs of wells are punched in agarose plates in which antigen is placed in

one well of each pair and antibody in the other.

• Following electrophoresis, precipitin lines will be visible between the wells

of a pair of wells of matching specificity.

- +
Ag Ab
COUNTER-CURRENT IMMUNOELECTROPHORESIS