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PRECIPITATION REACTIONS IN IMMUNOLOGY

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ejpmr, 2020,7(2), 214-216 SJIF Impact Factor 6.222
Review Article
EUROPEAN JOURNAL OF PHARMACEUTICAL
Deshmukh et al. European Journal of Pharmaceutical and Medical Research
ISSN 2394-3211
AND MEDICAL RESEARCH
www.ejpmr.com EJPMR

PRECIPITATION REACTIONS IN IMMUNOLOGY

Amol S. Deshmukh1*, Pravin R. Dighe1, Kundan J. Tiwari2 and Supriya T. Garud2

1
S.M.B.T College of Pharmacy, Dhamangaon, Nashik, Maharashtra, India.
2
S.M.B.T Institute of D. Pharmacy, Dhamangaon, Nashik, Maharashtra, India.

*Corresponding Author: Mr. Amol S. Deshmukh

Assistant Professor, Department of Pharmaceutics, S.M.B.T College of Pharmacy, Dhamangaon, Nashik, Maharashtra, India.

Article Received on 23/11/2019 Article Revised on 14/12/2019 Article Accepted on 04/01/2020

ABSTRACT
Precipitation reactions in immunology are based on the interaction between antigens and antibodies. These are
based on two reactants which are soluble that combine to make one product which is insoluble, and that product is
called precipitate. The interaction of the antigen and antibody is a chemical reaction and is specific. When antigen
and antibody exist in optimal proportions there is a formation of lattices (cross-links). The molecules are held
together by intermolecular forces which are effective only when the antibody combining site and the antigenic
determinant group are able to make close contact. Present review gives detail study about the precipitation
reactions in immunology.

KEYWORDS: Antigen, Antibody, Precipitation, Immunology, Serology.

INTRODUCTION Precipitation reactions involve the reaction of soluble

An antigen is defined as any substance when introduced antigen with IgG or IgM antibodies to form large
into the body, stimulates the production of an antibody interlocking molecular aggregates called lattices.
with which it reacts specifically and in a manner that Precipitation reactions occur in two distinct stages. First,
reaction is observable. Antibodies are specific substances the antigens and antibodies rapidly form small antigen-
in the bodies of the vertebrate secreted in the tissue fluids antibody complexes. This interaction occurs within
from the lymphoid cells that have been stimulated by seconds and is followed by a slower reaction, which may
foreign substances (antigens) with which they react take minutes to hours, in which the antigen-antibody
specifically. The interaction of the antigen and antibody complexes form lattices that precipitate from solution.
is a chemical reaction and is specific.[1,2] The reaction Precipitation reactions occur only when the ratio of
between an Ab and an Ag involves numerous non- antigen to antibody is optimal.[7]
covalent interactions between the epitope and paratope.
Epitope is the binding site on the Ag, and the paratopes
is the binding site on the Ab.[3] The study of reaction
between the antigen and antibody is known as Serology.
Serology is classified as precipitation, agglutination,
complement fixation, neutralization, immobilization and
intra-dermal reaction. Serology is also defined as a
branch of science which deals with the study of
serum.[1,4] A basic immunological reaction involves in
Antigen-antibody reactions and it describes a specific
response among antigen and antibody.[5]

PRECIPITAION REACTIONS
Antigens and antibodies are both complexes of amino
acids and also have positive and negative polar groups
distributed over their surfaces in specific but reciprocal
patterns. When the antigen and corresponding antibody
molecules are mixed, electrical attraction and repulsion
takes place and results in orientation of the Figure 1: Precipitation curve.
corresponding antigen and antibody molecules with
respect to their molecular forms and electrical charges, Above figure 1 shows that no visible precipitate forms
so that an absolute ‘fit’ (mold and cast) is obtained.[6] when either component is in excess. The best possible

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Deshmukh et al. European Journal of Pharmaceutical and Medical Research

ratio is formed when separate solutions of antigen and more advantageous than precipitation in liquid
antibody are sited adjacent to each other and permitted to medium. There are different types of
diffuse together. The equivalence zone represents the immunodiffusion tests are as follows:
concentration of antigen and antibody where complete a) Single diffusion in one dimension (Oudin
precipitation occurs.[4] Procedure): Agar gel having antibody is taken in a
tube and antigen solution is layered over it. The
The following types of precipitation and flocculation antigen diffuses downwards and wherever it reaches
tests are used commonly. in optimum concentration with antibody a line of
1. Ring test: The detection of antigens is very simple precipitation is formed. The number of bands
by using this test. In this test a layer of antigens is indicates the number of different antigens present.
formed over layer of serum in a narrow tube. The b) Double diffusion in one dimension (Oakley-
line of precipitate is observed at the junction of two Fulthorpe procedure): In a test tube antibody is
clear fluids as shown in figure 2. C-reactive protein incorporated in agar gel. Above this layer a column
test, Ascoli’s thermo precipitin test, diagnosis of of plain agar is placed which in turn is superimpose
anthrax and typing of streptococci are some of the with antigen, either as liquid or incorporated into
important applications of the ring test. Detection of agar. The antigen-antibody forms a band of
adulteration of food stuffs is also done by using ring precipitate where they meet at optimum proportion
test. by moving towards each other through the
intervening agar.
c) Single diffusion in two dimensions (Radial
immunodiffusion): A slide but generally petri plate
is used for this method. Agar gel is poured on a slide
or in a petriplate and antiserum is then incorporated
in agar gel. Well were prepared on the agar surface
and antigen is added to a well. Bands of precipitate
of ring shape are formed around wells. The
concentration of antigen is estimated by measuring
the diameter of ring.
d) Double diffusion in two dimensions (Ouchterlony
procedure): Agar gel is poured on a slide or petri
plate and different wells were prepared on
surrounding and one at center on agar surface. In the
Figure 2: Ring test. central well the antiserum is added and different
antigens are added in the surrounding well. If in
2. Slide test: In this test a drop of each of the antigen observation the two precipitin bands found totally
and the antiserum are added on a slide, then both combine, then the pattern is called as reaction of
antigen and antiserum are mixed by shaking. The identity. This indicates that the antigens in the
reaction observed in the form of floccules formation. adjacent wells are identical. If in observation the
The VDRL test for syphilis is one of the examples of precipitin bands form separately and cross each
slide flocculation. other, then the pattern is known as reaction of non-
3. Tube test: In this test a fixed quantity of antitoxin is identity. This indicates that the unrelated antigens
added in the tubes. Then serial dilutions of the are present. If in observation the precipitation bands
toxoid or toxin are added to those tubes. The amount fuse but form a spur like projection, in this case the
of toxoid or toxin which flocculates optimally with a antigens are cross-reacting, this is known as reaction
one unit of the antitoxin is known as Lf dose. One of of partial identity. For the diagnosis of small pox
the example of a tube test is the Kahn test for this method was a routine technique. For
syphilis. For the standardization of toxoids and toxigenicity of Diphtheria bacilli a special variety of
toxins a quantitative tube flocculation test is used. this test is used called as Elek test. Double diffusion
4. Immunodiffusion (Precipitation in Gel): The in two dimensions is used for comparison of
important characteristic property of this method is different antigens and antisera.[2,8]
that the precipitation reaction is observable in the 5. Immunoelectrophoresis: An electrochemical
form of distinct band which is more stable and if process is a process in which colloidal particles
necessary can be stained for preservation. The (suspended particles) or macromolecules which
number of different antigens in the reacting mixture having a net electric charge are travels in a solution
can be readily visualized, as each antigen-antibody or agar gel under the influence of an electric current
reaction gives rise to a line of precipitation. is known as electrophoresis. A characteristic of
Immunodiffusion also indicates identity, non- living cells in suspension and biological compounds
identity and cross-reaction between different (such as protein antigens) in solution or in gel is that
antigens. Immunodiffusion is usually performed in a in an electric field they travel to the positive or
soft agar (1%) or agarose gel. Precipitation in gel is negative electrode, depending on the charge on the

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substance. Positively charged substances travel to

the cathode while negatively charged ones go to the
anode, this movement is called as electrophoretic
mobility. When electrophoresis is applied to the
study of antigen-antibody reactions, it is called
immunoelectrophoresis. When a fluid containing
antigens in the form of proteins is placed in a well
which was prepared in a thin layer of buffered agar.
If an electric current is applied to this, the antigens
will be travels and distributed in separate spots along
a line passing throughout the well and that is parallel
to the current flow direction. When the current is
shut off, diffusion will begin from each of these
spots. By placing antiserum in a trench cut in the
agar parallel to the electroporetic distribution of the
antigens, the precipitin reaction can be used to
demonstrate the nature of the diffusing molecules. In
this case, a broad band of antibody diffuses toward
the antigens from the linear antibody trench, while
the antigens diffuse as expanding discs. This results
in a complex pattern of arc-shaped zones of
precipitate.[4]

CONCLUSION
Antigens and antibodies are playing an important role in
immunological reactions. Different reactions are
involved in immunology with special reference to
serology are precipitation, agglutination, complement
fixation, neutralization, immobilization and intra-dermal
reaction etc. among them precipitation reaction and its
subtypes were described in this review.

REFERENCES
1. Panjarathinam R. Medical Microbiology. 1st ed.,
New Age International Publishers, 2007.
2. Ananthnarayan R, Paniker C. Textbook of
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3. Inbal S, Vered K, Yanay O. The structural basis of
antibody-antigen recognition. Frontiers in
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4. Pelczar M, Chan E, Krieg N. Microbiology. 5th ed.,
McGraw Hill Education, 2017.
5. Shuang H, Guanyu W, Naijin X, Hui L. Quantitative
Assessment of the Effects of Oxidants on Antigen-
Antibody Binding In Vitro. Oxidative Medicine and
Cellular Longevity, 2016; 1-7.
6. Frobisher, Hinsdill, Carbtree, Goodheart.
Fundamentals of Microbiology. 9th ed., Toppan
Printings, 1974.
7. Tortora G, Funke B, Case C. Microbiology, an
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8. Kokare C. Pharmaceutical Microbiology, Principles
and Application. Nirali Prakashan, 2013.

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