IMMUNOLOGY AND SEROLOGY - LECTURE
Topic: Precipitation Reaction (week 6)
OVERVIEW
Precipitation
 Involves combining soluble antigen with soluble antibody to
produce insoluble complexes that are visible.
 Precipitation reactions are based on the interaction of antibodies
and antigens. They are based on two soluble reactants that come
together to make one insoluble product, the precipitate.
 These reactions depend on the formation of lattices (cross-links)
when antigen and antibody exist in optimal proportion.
Agglutination
 It is the process by which particulate antigens such as cells
aggregate to form larger complexes when a specific antibody is
present.
ANTIGEN-ANTIBODY BINDING
The primary union of binding sites on antibody with specific epitopes
on an antigen depends on two characteristics of antibody known as
affinity and avidity
Affinity
 Is the initial force of attraction that exists between a single Fab site
on an antibody molecule and a single epitope or determinant site
on the corresponding antigen.
 As epitope and binding site come into close proximity to each
other, several type of non-covalent bonds hold them together
 Ionic bonds - oppositely charged particles
 Hydrogen bonds - positive charge resides on H atom
 Hydrophobic bonds - non polar molecules that associate
and exclude water as they do
 Van der Waals forces - interaction between electron
clouds
 The strength of attraction depends on the specificity of antibody
for a particular antigen.
 Antibodies are capable of reacting with antigens that are
structurally similar to the original antigen that induced antibody
production. This is known as Cross-reactivity
The more the cross-reacting antigen resembles the original
antigen, the stronger the bond will be between the antigen and
the binding site.
 Perfect fit > Cross-reactivity
 The epitope and the binding site have a perfect lock-and-key fit, as
is the case with the original antigen, the affinity will be maximal.
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 Question: Which is more fit, is it the original or the one that has
cross-reactive? Answer: The original, since it has more points of
binding which makes it more fit. It has stronger affinity.
Avidity
 It represents the sum of all the attractive forces between an
antigen and an antibody.
 Once binding has occurred, it is the force that keeps the molecules
together.
 Avidity also refers to the strength with which a multivalent
antibody binds a multivalent antigen and is a measure of the
overall stability of an antigen-antibody complex.
 A high avidity can actually compensate for a low affinity.
 Both affinity and avidity contribute to the stability of the antigen-
antibody complex, which is essential to detecting the presence of
an unknown, whether it is antigen or antibody.
Law of Mass Action
 All antigen-antibody binding is reversible and is governed by the
law of mass action.
 This law states that free reactants are in equilibrium with bound
reactants.
 This constant can be seen as a measure of a goodness of fit.
 The higher the value of K, the larger the amount of antigen-
antibody complex and the more visible or easily detectable the
reaction is.
 K = K1/K2 = [AgAb] / [Ab][Ag] (K = equilibrium constant)
When the value of K
is higher, the
amount of antigen-
antibody complex is
larger and the assay
reaction is more
visible or easily
detectable
 The ideal conditions in the clinical laboratory would be to have an
antibody with a high affinity, or initial force of attraction, and a
high avidity, or strength of binding. The higher the values are for
both of these and the more antigen-antibody complexes that
are formed, the more sensitive the test will be
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PRECIPITATION CURVE
Precipitation also depends on the relative proportions of antigen and
antibody present. Optimum precipitation occurs in the zone of
equivalence.
Zone of Equivalence
 Point of optimum precipitation
 The number of multivalent sites of antigen and antibody are
approximately equal.
 In this zone, precipitation is the result of random, reversible
reactions whereby each antibody binds to more than one antigen
and vice versa, forming a stable network or lattice.
 Lattice hypothesis, as formulated by Marrack, is based on the
assumptions that each antibody molecule must have atleast two
binding sites and the antigen must be multivalent. > As they
combine, this arrangement results in a multimolecular lattices that
increases in size until it precipitates out of solution.
 As illustrated by the precipitin curve, when the same amount of
soluble antigen is added to increasing dilutions of antibody, the
amount of precipitation increases up to the zone of equivalence.
 When the amount of antigen overwhelms the number of antibody-
combining sites present, precipitation begins to decline because
fewer lattice networks are formed.
Prozone and Postzone
 Prozone phenomenon - antibody excess antigen combines with
only one or two antibody molecules and no cross-linkages are
formed.
 In the prozone, usually only one site on an antibody molecule is
used and many free antibody molecules remain in solution.
 A false-negative reaction = high antibody concentration
 Remedy: Dilution
 Postzone phenomenon - antigen excess ; small aggregates are
surrounded by excess antigen. No lattice networks are formed.
 In this case, every available antibody site is bound to single antigen
and no cross-links are formed.
 In the postzone, excess antigen = obscure the presence of a small
amount of antibody.
 Remedy: Additional patient specimen taken a week later
 This would give time for further production of
antibody. If the test is negative on this
occasion, it is unlikely that the patient has
that particular antibody.
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MEASUREMENT OF PRECIPIATION BY LIGHT SCATTERING
 Precipitation is one of the simplest methods of detecting antigen-
antibody reactions, because most antigens are multivalent and thus
capable of forming aggregates in the presence of corresponding
antibody.
 Precipitates in fluid can be measured by means of tubidimetry and
nephelometry.
Turbidimetry
 It is a measure of turbidity or cloudiness of a solution.
 A detection device is placed in direct line with an incident light,
collecting the light after it has passed through the solution.
 This device measures the reduction in light intensity due to
reflection, absorption, or scatter.
 Scattering - occurs when beam of light passes through
solution and encounters molecules in its path. Light then
bounces off the molecules and travels in all direction.
 The amount of scatter is proportional to the size, shape,
and concentration of molecules in the solution.
 It is recorded in absorbance units (a measure of the incident light
to that of transmitted light)
 Measurements are made using a spectrophotometer or an
automated clinical chemistry analyzer.
Nephelometry
 Measures the light that is scattered at a particular angle from the
incident beam as it passes through a suspension.
 The amount of light scattered is an index of the solution's
concentration.
 The relationship between antigen concentration, as indicated by
antigen-antibody complex formation, and the amount of light
scattering will form a straight line if plotted Linearity
Beginning with a constant amount of antibody, increasing amount
of antigen results in an increase amount in antigen-antibody
complexes, and thus an increase in light scattering.
 Nephelometry is much more sensitive than turbidimetry
 It is preferred because it is simple and cheap
 It used in quantification of immunoglobulins such as IgG, IgA, IgM
and IgE, as well as kappa and lambda light chains.
Two method of Measurement:
1. End point nephelometry - the reaction is allowed to run
essentially to completion, but large particles tend to fall-out of
solution and decrease the amount of scatter.
2. Kinetic or Rate nephelometry - it was devised, in which the rate
of scattering increase is measured immediately after the reagent
is added.
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PASSIVE IMMUNODIFFUSION TECHNIQUES
 Use of gel derived from seaweed and agarose that acts as a
support medium for the precipitation of antigen-antibody
complexes.
 Agarose is preferred to agar, because agar has a strong negative
charge, while agarose has almost none, so interactions between
the gel and the reagents are minimized.
 Agarose, purified high molecular-weight complex
polysaccharide derived from seaweed
 It helps stabilize the diffusion process and allow visualization of
the precipitin bands.
 Antigen-antibody reaction occurs by diffusion.
 It is called Passive, because there is no electrical current used to
speed up the process
 Immunodiffusion reactions can be classified according to the
number of reactants diffusing and the direction of diffusion.
Radial Immunodiffusion
 James Oudin - was the first to use gels for precipitation reactions,
and he pioneered the technique known as single diffusion.
 A modificatin of single diffusion technique is called Radial
Immunodiffusion (RID).
 In this technique, antibody is uniformly distributed in the support
gel, and antigen is applied to a well cut into 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.
Two Methods of Measurement:
1. End point method (Macini) - antigen is allowed to diffuse to
completion. → Readings 24 to 72 hours
2. Kinetic method (Fahey and McKelvey) - measurement taken
before the point of equivalence is reached. → Readings 19 hours
The amount of
precipitate formed
is in proportion to
the antigen present
in the sample. In
the Mancini end-
point method,
concentration is in
proportion to the
diameter squared.
 Question: How does the reaction occur?
Answer: First it incorporates the antibodies on the medium, and
using the samples (which contains the patient unknown or antigen)
it is then allowed to diffuse on the exclusion. Given that it is radial
immunodiffusion, the contents of the solution diffuses radially.
You’ll know if precipitates are formed if the antibodies meets with
the antigen complex.
 Question: How does the measurements are obtained?
Answer: We compare them into different standards, and we
measure yung mga sukat nung mga nakuha natin sa standards.
Therefore identifying the amount of concentration of the antigen in
the solution.
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Sources of Error
1. Overfilling or underfilling the wells
2. Nicking the side of the wells when filling
3. Spilling sample outside the wells
4. Improper incubation time and temperature
5. Incorrect measurement
Ouchterlony Double Diffusion
 One of the older, classic immunochemical technique
 In this technique, both antigen and antibody diffuse independently
through a semisolid medium in two dimensions, horizontally and
vertically.
 Antibody that is multi-specific is placed in the central well, and
different antigens are placed in the surrounding wells to determine
if the antigens share identical epitopes.
 Diffusion takes place radially from the wells. After incubation
period between 12 and 48 hours in moist chamber, precipitin lines
forms.
 Precipitin lines form where the moving front of antigen meets that
of antibody.
 The density of the lines reflects the amount of immune complex
formed.
 This is technique is used in identifying whether the antigens share
an identical epitopes.
(1) Fusion of the lines at their junction to form an arc represents
serological identity or the presence of a common epitope,
(2) a pattern of crossed lines demonstrates two separate
reactions and indicates that the compared antigens share no
common epitopes, and
(3) fusion of two lines with a spur indicates partial identity.
In this last case, the two antigens share a common epitope, but
some antibody molecules are not captured by antigen and travel
through initial precipitin line to combine with additional epitopes
found in the more complex antigen. Therefore the spur always
points to the simpler antigen1
ELECTROPHORETIC TECHNIQUES
 Electrophoresis separates molecules according to differences in
their electric charge when they are placed in an electric field.
 A direct current is forced through the gel, causing antigen,
antibody, or both to migrate.
 As diffusion takes place, distinct precipitin bands are formed.
 Diffusion can be combined with electrophoresis to speed up or
sharpen the results.
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Rocket Immunoelectrophoresis
 Aka One-dimension electroimmunodiffusion
 Developed by Laurell
 An adaption of radial immunodiffusion with application of
electrophoresis
 The end result is a precipitin line that is conical in shape,
resembling a rocket.
 The height of the rocket, measured from the well to the apex, is
directly in proportion to the amount of antigen in the sample.
 Immunodiffusion takes place in a shorter time and results in a
higher resolution than when antibody diffuses from a trough.
Because diffusion is only across the thickness of the gel,
approximately 1mm, the reaction usually takes place in less than 1
hour.
 This technique is characteristically used to determine the presence
of antibodies to organisms of complex antigenic composition.
 Immunofixation technique is especially useful in demonstrating
those antigens present in serum, urine, or spinal fluid in low
concentrations.

Antibody is distributed in the gel, and the antigen is placed in wells

cut in the gel, just as in RID. However, instead of allowing diffusion
to take place as its own rate electrophoresis is used to facilitate
migration of the antigen into the agar. When the antigen diffuses
out of the well, precipitation begins. As concentrations of antigens
changes, there is dissolution and reformation of the precipitate at
ever-increasing distances from the well.
Immunoelectrophoresis
 Immunoelectrophoresis is a double-diffusion technique that
incorporates electrophoresis current to enhance results.
 Introduced by Grabar and Williams in 1953
 Has been used as screening tool for the differentiation of many
serum proteins, including the major classes of immunoglobulins
 It can be used as a qualitative and a semiquantitative technique.
 Typically the source of antigen is serum, which is electrophoresed
to separate out the main proteins. A trough is then cut in the gel
parallel to the line of separation. Antiserum is placed in the trough,
and the gel is incubated for 18 to 24 hours.
 Double diffusion occurs at the
right angles to the
electrophoretic separation and
precipitin lines develop where Sources of Error in Electrophoresis
specific antigen-antibody
combination takes place. These 1.
lines or arcs can be compared
in shape, intensity, and
location to that normal serum 2.
control to detect abnormalities. 3.
4.
Immunofixation Electrophoresis
 First described by Alper and Johnson
 Is similar to immunoelectrophoresis except that after
electrophoresis takes place, antiserum is applied directly to the
gel’s surface rather than placed in a trough.
 Uses agarose or cellulose acetate
5.
 The gel is washed to remove any non-precipitating proteins and
can then be stained for easier visibility.
6.
 One of the best known adaptations of this technique is the
Western blot.
7.
 Typically, patient serum is applied to six lanes of the
gel, and after electrophoresis, five lanes are overlaid
with one each of the following antibodies: Antibody to
gamma, alpha, or mu heavy chains to kappa or
lambda light chains.
 The sixth lane is overlaid with antibody to all serum
proteins and serves as the reference lane.
 Reaction in each of the five lanes are compared to the
reference lane.
 Hypogammaglobulinemias will exhibit faintly staining
bands
 Polyclonal hypergammaglobulinemias show darkly
staining bands in the gamma region.
 Monoclonal bands, such as as found in Waldenstrom’s
macroglobulinemia or multiple myeloma, have dark
and narrow bands in specific lanes.
Applying the current in the wrong direction
 If this occurs, samples may either run off the gel or
not be separated.
Incorrect pH of the buffer
Incorrect electrophoresis time
Wrong concentration of antigen and/or antibody
 If mali ang concentration it can lead to False-Negative
(prozone and post zone)
 Concentrations of antigen and antibody must be
carefully chosen so that lattice formation and
precipitation is possible.
Current is not strong enough
 There will be an incomplete separation of proteins
Current too strong
 Generation of heat leading to denaturation of proteins
Improper filling of wells

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 COMPARISON OF PRECIPITATION TECHNIQUES

Stevens 4th Ed.

TAKE NOTE:

SENSITIVITY (3rd Column)

→ Kapag mas mababa yung number sa
column na yan mas sensitive yung
test because the test can detect lower
amounts of analyte in the solution.
→ ↓ value = ↑ Sensitive

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