Course: Immunology
Lecturer: Dr. Weam Saad
Practical Lecture: Precipitation Reactions
Precipitation Reactions
The precipitation reaction occurs when a soluble antigen reacts with
a specific antibody to form the Immunocomplex, this reaction
depend on :
1. The nature of Ag is soluble.
2. The amounts of reacting antigens and antibodies.
3. The numbers of combining sites on each. Antigens may have
one or many antibody-binding sites, also antibodies have at
least two antigen binding site e.g. IgG antibodies are bivalent
having 2 antigen-binding sites.
Repeated antigen-antibody linkages can result in large insoluble
complexes at antibody optimal ratio or concentration (AOR). The
AOR is the ideal antibody/antigen ratio for formation of insoluble
immune complexes (equivalence zone). When the number of antigen
particles is much higher than the number of antibody molecules,
many antibody binding sites on the antigen will remain empty. The
complexes that are formed are small and not visible with eye. When
number of antibodies is much higher than the number of antigen
molecules, there is not enough antigen to form cross-linkages. The
complexes that are formed are small and not visible with eye.
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Types of Precipitation Reactions Techniques:
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Capillary tube precipitation (Ring Test)
Ouchterlony Double Diffusion (Immunodiffusion)
Radial Immunodiffusion (RID)
Immunoelectrophoresis (IEP)
Rocket Electroimmunodiffusion (EID)
CounterImmunoelectrophoresis (CIEP)
1-Tube precipitation (Ring Test) Qualitative test
It is the simplest test among precipitation tests. Clinical
example is Milk ring test (MRT), this test is a fast investigation
used for cows Brucellosis identification by mixing the cow milk
(suspected with Brucella sp. Infection), the blue ring formed
after incubation time indicates for Ab present means infection
with Brucella sp. present.
Principles:
1. Occurs after adding a layer Ag over Ab in a test tube
2. Qualitative: means investigation of the specificity of Ab to the
Ag used.
3. Precipitation occurs at the interface between the two reagents
(forming a ring).
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2-Double Immunodiffusion (Ouchterlony Qualitative test)
The scientist Ouchterlony (1966) developed this test; the double
diffusion technique has many applications e.g. determining whether
a given antigen shares structural characteristics (cross-reacts) with
other molecules, also; this technique has the advantage that several
antigens can be compared around a single well of antibody.
Principles:
1. Cutting wells in a gel (agar or agarose); one well contains the
antibody and the other well contains the antigens.
2. Antigens and antibodies diffuse towards each other at rates that
increase and decrease according to:
a) Ag and Ab concentrations in the wells.
b) The sizes and molecular weight of both Ag and Ab
c) The shape of both Ag and Ab
d) The solubility of the Ag.
3. Ag and Ab form a line of precipitation (precipitin line) where
they meet at equivalence zoon after incubation time.
4. The gel does not interfere with the diffusion of the Ag and Ab.
5. The formation of the precipitin line in the equilibrium between
the antibody and antigen acts as a barrier, which components
cannot pass.
6. There are three basic patterns of precipitation as shown in the
figure below. The figures show an Agarose gel plates. The
antiserum well contains antibodies (blue color) used against the
determinants in two different antigens A and B in two different
wells (red color), results after diffusion can be one of the
followings:
A
B
Ab
Figure 1
A
B
Ab
Figure 2
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A
B
Ab
Figure 3
 Figure1: The precipitation lines have fused completely
showing the presence of identical determinants between the
two Ags.
 Figure2: Ag A is different from Ag B and both react with the
Abs, the precipitin lines cross and a double spur is formed; this
is a line of nonidentity determinants.
 Figure3: Ag A and Ag B share a common determinants but are
not exactly the same, a single spur is formed. This is the line
of partial identity.
3-Radial Immunodiffusion (RID) (QuantitativeTest)
Principles:
1. The technique based on the reaction between an Ag, and a
specific Ab.
2. The Ag placed in a well diffuses into a gel ( usually agarose)
containing the Ab (e.g. anti-IgG looking for serum IgG).
3. A ring of precipitation around the Ag well will be formed after
the Ag-Ab reaction. This ring represents the immunocomplex
that formed at equivalence zoon.
4. The gel is prepared by mixing the antibodies with the agarose
gel then poured into plate. After the gel become solid, different
dilutions of the antigen placed in wells cut into the agarose.
5. A after incubation time, calculating rings diameters, then a
standard curve is plotted to determine the concentration of
unknown sample Ag.
6. There are two methods of RID:
1. Fahey method
2. Mancini method
Mancini method (endpoint): This test is, commonly used in the
clinical laboratories for the determination of immunoglobulin levels
and complement components in patient samples. The technique of
Single Radial Immunodiffusion (SRID) is one of these method
techniques.
Single Radial Immunodiffusion (SRID)
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The scientists; Mancini and Oudin developed this technique. This
technique used to measure the concentration of Ag in a solution
mixed with other Ags when specific antiserum used. The
equivalence zoon is called the endpoint because it represents the
point where the diameter of the well stops to increase. This endpoint
diameter represents the actual concentration of Ag in sample used.
Notes:
1. Temperature is a very effective factor in this test. Some
reactions need room temperature rather than 37 o C.
2. Single radial immune diffusion need mixing of antiserum
with the gel after cooling before pouring in plates. Hence
do not need for antiserum well.
Experimental design for Double Diffusion Method:
In this experiment, human serum will be used as antigen source for
albumin protein (Ag) and prepared bovine albumin antiserum
(commercial) is used as antibodies source.
Procedure:
1. Dissolve agarose gel powder (20 g/l) in boiling phosphate
buffered saline PBS, pH 7.4, after cool (50 o C) then pour into
special plates, thickness of less than 1mm.
2. After the gel has set, cut out wells about 2mm in diameter.
3. Extract the core by pipette tip with a negative pressure pump.
4. Cover plates with fitted lids, and store in sealed packets at
refrigerator (4 o C) until used.
5. Application of serum and commercial prepared bovine
albumin antibodies using micropipette 10 µper well for both.
6. Incubation for 24-48 hr. at 37 o C. then read results.
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