Precipitation test Agglutination test Immunofluorescence RIA ELISA

1. SALIPUR AUTONOMOUS COLLEGE
ZOOLOGY DEPARTMENT
TOPIC- ANTIGEN ANTIBODY
INTERACTION
NAME-SONALI GHOSAL
+3 2RD YEAR SCIENCE

2. INTERODUCTION :-
• Immune system of the body rejects or degrades pathogenic microorganisms their toxins
and other kind of foreign substances(non self organisms )or substances through various
defence mechanism .
• The resistant to immunity of body is called as immunity , which means freedom from
diseases .
• The branch of biology which deals with immunity or resistance of the body against non-
self organisms is called immunology .
• Immunology got recognition in biological science by the work of Louis Pasteur of
France(1879) .
• Thus he is called as founder father of the science of immunology .
• Antigen antibody interaction is a biomolecular association similar to enzyme substrate
interaction .
• The antigens and the antibodies combine specifically with each other the interaction
between them is called antigen -antibody reaction .
• It may be abbreviated Ag –Ab reaction .

3. Antigen ---
a substance that enters the body and starts a process that can cause disease.
the body then usually produce antibodies to fight the antigens .
Antibody ----
it is also known as immunoglobulins .these are Y shaped gamma globulins protein
found in the blood ,lymph and produce by B cells as an immune defence against foreign
agents(antigen) .

4. Itinvolves two process such as
-In vivo
-In vitro
A. IN VIVO:-
 As we know that interaction between the
antigen and antibody are the central dogma
of antibody mediated (humoral) immunity
inside the host body.
 As a result of these interactions the antigen
is altered .
 The different antigen antibody interactions
that takes place in the body are –
(1)Neutralization
(2)Immune complex formation

5. (3)Opsonization
(4)Antibody dependent cell mediated cytotoxicity (ADCC)
(5)Complement system
(1) NEUTRALIZATION:-
• Certain antibodies called neutralizing antibodies react with antigen and neutralize them
so,that they fail to attach on host cell surface .
A . Toxin neutralization:-
• It is the process during which antibodies capable of neutralizing a toxins produced by
the different pathogens like bacteria and these neutraling antibodies are called
antitoxins . However bacterial pathogens are causing tetanus ,diphtheria etc. are some
toxin producing bacteria .
B . viral neutralization:-
• viral neutralization is the process during which specific antibodies (IgG ,IgA & IgM)bind
to virus and inactivate them .
• viral neutralization prevents viral infection due to inability of virus to bind to and enters
it’s target cell .

6. (2) IMMUNE COMPLEX FORMATION:-
• Antibody possess at least 2 antigen binding sites and most antigens have atleast two
epitopes(antigenic determinants) .
• The antibodies cross link antigen forming large aggregates of antibody and antigen
referred to as immune complexes which are more readily phagocytized than are free
antigens.
• Depending upon their physical properties ,immune complex forming antibodies are of 2
types –precipitines and agglutins .

7. • The precipitins react with antigens that are soluble molecule and form immune complex
large enough to precipitate this process is called precipitation (L.,-praecipitare - to cast
down )
• The agglutins , however react with surface bound antigens of bacterial or other cells
and form immune complex .
• The agglutins however react with surface bound antigens of bacterial or other
cells and form immune complex .

8. • This process is called agglutination or agglutin reaction .
• Agglutination specifically involving red blood cells is called haemagglutination and is
caused by antibodies called hemagglutin .
(3) OPSONIZATION :-
• It is also called as opsonin dependent recognition or opsonization (Gr.,-opson-to prepare
victim for ) is a process in which the phagocytic cells readily recognize the microbial
pathogens that are coated by serum components called opsonins .

9. (4) COMPLEMENT SYSTEM:-
• Complement system is a part of the immune system called the innate immune system
i.e.,not adaptable and does not change over the course of an individuals life time .
• The serum of the blood contains a group of over a dozen of glycoproteins
(C1,C2,C3,C4,C5,C6,C7,C8,C9 and their components ) referred to collectively as
complement system .
• Complement play a very important role in 1st 2nd &3rd line of defence mechanism and
involve
• classical ,alternative and lectin pathway against macro and micro parasites
CLASSICAL PATHWAY:-
• It is triggered by the binding of antibody (IgM or IgG)to antigen on the surface of the
host cell to be attacked ,
• This antigen - antibody complex attaches with C1 forming complex Ag AbC1 which
follows a series of interactions resulting in the formation of complex C5bC6C7C8C9 that
creates a pore in the outer membrane .

10. • If the cell is eukaryotic ,there is rapid leakage of cytoplasmic content through the pore
causing the death of the cell .
• If the cell is gral –ve bacteria the enzyme lysozymes enters the pore and digests the
peptidoglycan thus causing lysis of the cell .
• If the cell is gram +ve bacteria however pore reistance to the lytic action and they donot
possess on outer membrane .
 ALTERNATIVE PATHWAY:-
• This pathway is not stimulated by antigen –antibody complex rather by other factors .
• It begins with cleavage of C3 into C3a and C3b enzymatically
• C3b is unstable components and is stabilized by binding to lipo polysaccharides of outer
membrane of gram –ve bacteria or to a IgA and IgG antibodies in immune complexes .
 LECTIN PATHWAY:-
• Lectins are proteins that recognize and bind to specific (CHO)targets .
• The lectin pathway, like alternative pathway does not depend on antibody for it’s
activation .

11. • Lectin pathway is activated by the binding of mannose binding lectin(MBL) to manose
residues on glycoproteins .
• MBL is an acute phase produced in inflammatory responses .
(5) ANTIBODY DEPENDENT CELL MEDIATED CYTOTOXICITY (ADCC):-
• When an antibody specifically binds to a target cell the receptors of these cells can
bind to the Fc region .
• Thus when the cytotoxic cells bind to the target cells subsequently cause lysis of the
target cell .
• The cytotoxic cells are non-specific for the antigen ,the specificity of the antigen bound
to the target cells directs the cytotoxic cells to specific target cells .
• This process is called as antibody dependent cell mediated cytotoxicity .
• In the immune system the cells that mediate ADCC are NKcells , macrophages
monocytes ,neutrophils ,eosinophils,basophils .
• In the immune system the cells that mediate ADCC are NK cells,macrophages
monocytes ,neutrophils ,eosinophils,basophils .

12. • Ex-the ADCC to cells infected with measles virus can be observed invitro by adding
antimeasles antibody together with macrophages to a culture of measles infected cell .
• There are some other varieties of ADCC also but it does no involve a complement
mediated lysis .

13. B. IN-VITRO:- SEROLOGY (SEROLOGICAL REACTIONS )
a). Antigen antibody interaction in vitro (under laboratory conditions)are referred to as
serological reactions or serology (so name because they involve patients serum) .
b). Antigen antibody interactions were 1st adapted to laboratory test in 1880s .
c). At present, they have sophisticated ,and are widely used in clinical diagnosis .
d). The most important and often used serological reactions in laboratories are :-
(1) Precipitation test
(2)Agglutination test
(3)Fluorescent antibody technique (immunofluorescence)
(4)Radio Immune Assay (RIA
(5) Enzyme Linked Immuno Sorbent Assay(ELISA)
(1) PRECIPITATION TEST:-
• When soluble antigens and its homologous antibody molecules react ,they some times
form large polymeric macro-molecules terminating into visible precipitate .

14. • The precipitation occurs in two stages ---- first the antibodies bind to antigens forming
antigen antibody complex within a few seconds or minutes ,then the “constant
regions’’of the antibodies of the complexes bind to each other within some hours
resulting in the formation of visible precipitate .
• The formation of precipitate is dependent on the proper relative concentrations of the
antigen and antibody molecules in a specific region called the zone of equivalence or
zone of precipitation.

15. • These are performed either in fluid –fluid precipitation or gel-gel precipitation .
(2) AGGLUTINATION TEST:-
• Agglutination test is that in which visible clumping or aggregation of cells or particles
takes place due to the reaction of surface –bound antigens of such cells or particles
with homologous antibodies
• It is probably best known for its use in human blood typing(haemagglutination).

16. (3) FLUORESCENT –ANTIBODY TECHNIQUE:-
• The fluorescent –antibody technique (immunofluorescence) is often used to identify
unknown antigen .
• The technique is based on the behaviour of certain dyes which fluorescence (glow)
when exposed to certain wavelengths of light .
 Such dyes are :- fluor escenin isothiocyanate which emits an apple –green glow ,and
rhodamune isothiocyanate which emits orange-red light .
• It may be direct or indirect .
• This technique particularly helps identifying specific strains of microorganisms within a
microbial population ;it is also useful in identifying those pathogenic microbes that are
difficult or impossible to culture in vitro .
(4) RADIO IMMUNOASSAY (RIA):-
• All the assay principles are called binder ligand assay .

17. • RIA or radioimmunoassay is widely accepted and highly Sensitive serological test.
• It was first developed by Barson and Yallow in 1960 for which Yallow was awarded
Nobel Prize in 1977 .
• It was introduced to detect the human insulin using antinsulin antibodies .
• In this method either antigen or antibody or hapten is radiolabelled like 125I &3H and
detected by radioautography .
It has two method - * liquid phase RIA
* solid phase RIA
(5) ELISA(ENZYME LINKED IMMUNO SORBENT ASSAY):-
• This principle is similar to RIA but here an enzyme is used that reacts with a colourless
substrate to produce a chromogenic reaction product .
• It is a test that detects and measure antibodies in our blood .

18. • It is also called as enzyme immune assay(EIA) .
• The enzymes commonly used are alkaline phospatase , horse radish
peroxidase ,-galactosidase etc .
• This method is mostly useful in testing AIDS antibodies .
• However now -a -days a variety of ELISA kits are used and are studied for
different type of diagnosis .
CONCLUSION:-
Antigens are foreign particles which cause diseases in our bodies . But
antibodies protect from these antigen by destroy them .