Antigen-Antibody reaction, Precipitation, Agglutination, CFT, Neutralization, Complement activation pathway

1. Antigen
Antibody
Reactions

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OUTLINE
ANTIGEN-ANTIBODY REACTION
PRIMARY ANTIGEN-ANTIBODY REACTIONS
• Precipitation
• Agglutination
• Complement fixation
• Neutralization
COMPLEMENT SYSTEM
• Biological role
• Pathways
• Stages of action

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ANTIBODY –
• Antibodies also known as Immunoglobulin are antigen- binding
proteins present on the B-cell membrane and secreted by plasma
cells.
• The antibodies are the gamma globulins
• Antibodies are one of the major plasma proteins, and against
infection often referred to as “first line of defense”
• The most important function of antibodies is to confer protection
against microbial pathogens
ANTIGEN –
• Molecules that can be recognized by the immunoglobulin receptor
of B cells or by the T-cell receptor when complexed with major
histocompatibility complex (MHC) are called Ag
• The word antigen is a shortened form of the words “antibody
generator”
• The antigens that are not immunogenic but can take part in
immune reactions are haptens

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•The antigens and the antibodies combine specifically with each other
•This interaction between them is called Antigen-Antibody reaction
(Ag –Ab reaction)
•These form the basis for humoral immunity or antibody mediated
immunity
•These reactions form the basis for detection of infectious disease
causing agents
•When Ag – Ab reactions occur invitro, they are known as serological
reactions
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The reactions between Ag and Ab occur in three stages
• In first stage the reaction involves formation of Ag-Ab complex
• The second stage leads to visible events like precipitation,
agglutination etc.
• The third stage includes destruction of Ag or its neutralization
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8. 1. Specificity of antigen –antibody reaction
2. Immune complex
3. Binding site of antigen –antibody reaction
4. Binding force of antigen –antibody reaction.
FEATURES OF ANTIGEN ANTIBODY REACTION
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9. • Specificity refers to the ability of an individual antibody
combining site to react with only one antigenic determinant
or the ability of a population of antibody molecules to react
with only one antigen
1. Specificity of antigen –antibody reaction
• Lock and key phenomenon
A standard lock can be opened by its own key only as one
antibody can react with its own antigen
e.g., the antibody produced against lens antigen will react
only with lens-antigen
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11. • An immune complex is formed from the integral binding of an antibody
to a antigen
• Antigen-antibody immune complex formation results in complement
activation, opsonization of target cells, assembly of membrane attack
complexes and release of complement activators for chemotaxis
2. Immune complex
3. Binding site of antigen –antibody reaction
• An epitope, also known as antigenic determinant, is the part of an
antigen that is recognized by the immune system, specifically by
antibodies, B cells, or T cells
• The part of an antibody that recognizes the epitope is called a paratope
• Ag may contain10-50 epitopes may go up to 200
• Ab – are may be Monovalent to multivalent (5- 10 binding sites)
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13. 4. Binding force of antigen –antibody reaction
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• Closeness between antigen and antibody: A strong antigen – antibody
interaction depends on a very close fit between the antigen and
antibody which requires high degree of specificity
• Non – Covalent Bonds: The bonds that hold the antigen to the antibody
combining site are all non-covalent in nature
These include hydrogen bonds, electrostatic bonds, Van der Waals
forces and hydrophobic bonds
• Affinity of antibody: Antibody affinity is the strength of the reaction
between a single antigenic determinant and a single combining site on
the antibody
The binding between antigen and antibody in Ag – Ab reaction is due to

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PROPERTIES OF ANTIGEN- ANTIBODY
REACTIONS
• Antibody Affinity
• Antibody Avidity
• Cross reactions
• Sensitivity
• Specificity

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AFFINITY OF ANTIBODY
• Interactions between Ag & Ab involve noncovalent binding of epitope
to the variable region (CDR-complementarity determining region) of
both the heavy and light Ig chains
• To describe the strength of the antigen-antibody interaction, one can
define the affinity constant (K) as shown:

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AVIDITY OF ANTIBODY
• Avidity is the strength of multiple interaction between the
multivalent antigen and antibody
• A multivalent Ag has many types of epitopes
• The various antibodies produced by a single Ag combine
with the different antigenic determinants of the Ag
• High avidity can compensate for low affinity

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CROSS REACTIONS
• When an antibody elicited by one antigen can react with an related
antigen, the phenomenon is called cross-reactivity
• Such cross-reactivity occurs if two different antigens share an
identical or very similar epitope
• The antibody’s affinity for the cross-reacting epitope is usually less
than that for the original epitope
• The Ag which produces the cross reaction is called Cross reactive Ag
Example:
• The serum raised against albumin of hen’s egg can cross react with
albumin obtained from duck’s egg
• The antiserum raised against human insulin will react with the insulin of
Pig, Sheep, whale etc.
• ABO blood group is a good example of cross-reactivity

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SENSITIVITY
• The ability of test to detect even minute quantity of antigen
and antibody
• When the test is highly sensitive false negative result may be
absent or minimal
SPECIFICITY
• ability of the test to detect homologus antigen and antibody
• In highly specific test false positive reactions are absent or
minimal

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METHODS OF DETECTION ANTIGEN ANTIBODY
REACTIONS
I-Primary reactions
Ag-Ab complexes visible
by naked eye or by
microscope
1. Precipitation
2. Agglutination
3. Complement fixation
4. Neutralization
II-Secondary reactions
Ag-Ab complexes visible
by using labels
e.g. enzyme, radioisotopes,
Fluorescent substance
1. IF
2. ELISA
3. RIA
4. Immunoblotting
5. Flow cytometry

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PRECIPITATION REACTIONS
A soluble Ag combines with its Ab in the presence of an electrolyte (NaCl) at a
particular temperature and pH, it forms an insoluble precipitate of Ag-Ab complex
• The Ab causing precipitation is called Precipitin
• Formation of an Ag-Ab lattice depends on the valency of both the antibody and
antigen
• The antibody must be bivalent; a precipitate will not form with monovalent Fab
fragments
• The Ag must be either bivalent or polyvalent; that is, it must have at least two
copies of the same epitope, or have different epitopes that react with different
antibodies present in polyclonal antisera
• Precipitation occurs in two media: Liquid, Gel
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PRECIPITATION – MECHANISM OF ACTION
As each Ab is a bivalent molecule, it can bridge two multivalent Ag molecule
This bridging leads to the formation of a lattice which forms the precipitation
The Ag is multivalent and the Ab is bivalent

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Precipitation in Liquid:
• Antigen – Antibody reaction perform by placing a constant
amount of antibody in a series of tubes and adding increased
amount of antigen
• Antigen –Antibody reacts together resulting in precipitation
• The amount of precipitate formed is greatly influenced by the
relative proportions of Ags and Abs

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Plotting the amount of precipitate against increasing antigen concentration yields a precipitation curve
Precipitation in Liquid:
Plotting the amount of precipitate against increasing antigen concentration yields a
precipitation curve

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• Zone of antibody excess: Precipitation is inhibited and antibody doesn't bind to
antigen and can be detected in supernatant This is also called as Prozone
• Zone of equivalence: maximal precipitation in which Ab and Ag form large
insoluble complexes and neither antibody nor Ag can be detected in the supernatant
• Zone of antigen excess: Precipitation is inhibited and antigen not bound to
antibody can be detected in suprernatent. This is also known as Post zone

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There are several advantages in allowing precipitation to occur in
a gel rather than in a liquid medium
• The reaction is visible as a distinct band of precipitation, which
is stable and can be stained for preservation, if necessary
• As each antigen antibody reaction gives rise to a line of
precipitation, the number of different antigens in the reacting
mixture can be readily observed

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i) Single diffusion in one dimension
The Ab is incorporated in agar gel in a test tube and the Ag
solution is layer over it.
The Ag diffuses downward through the agar gel, forming a line of
precipitation that appears to move downwards.
This is due to the precipitation formed at the advancing front of
the Ag and is dissolved as the concentration of Ag at the site
increases due to diffusion.
The no of bands indicate the no of different Ag present.zzzzzzzzzzzzzzzzzzzz
i) Single diffusion in one dimension (Oudin
Procedure)
• Ab is incorporated in agar gel in a test tube & Ag
solution is layered over it
• Ag diffuses downward through the agar gel – forming
a line of precipitation
• The no of bands indicate the no of different Ag present
ii) Double diffusion in One dimension (Oakley-
fulthorpe procedure)
• Ab is incorporated in a gel above which is placed a
column of plain agar. The Ag is layered on top of this
• The Ag and Ab move towards each other through the
intervening column of plain agar and form a band of
precipitate where they meet at optimum proportion

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iii) Single diffusion in two dimension (Radial immunodiffusion)
The antiserum is incorporated in agar gel poured on a flat surface
The Ag is added to the wells cut on the surface of the gel. It diffuses radially from
the well and forms ring- shaped bands of precipitation concentrically around the well
This method has been employed for the estimation of the immunoglobulin classes in sera

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iv) Double diffusion in two dimensions (Ouchterlony double diffusion)
Agar gel is poured on a slide and wells are cut using a template
The antiserum is placed in the central well and different Ags in the surrounding well
If two adjacent Ags are identical, The lines of precipitate form by them will fuse. If
they are unrelated, the lines will cross each other

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v) Immunoelectrophoresis:
This technique involves the electrophoretic separation of a composite Ag (such as serum)
into its constituent proteins and then tested by double immunodiffusion
resulting in separate precipitin lines, indicating reaction between each individual protein
with its antibody
It is performed on agar or agarose gel on a slide with an Ag well and Ab trough cut on it
The test serum is placed in the antigen well and electrophoresed for about an hour
Ab against human serum is then placed in the trough and diffusion allowed to proceed for
18-24 hrs
The resulting precipitin lines can be photographed and the slides dried, stained and
preserved for record
This is used for testing normal and abnormal proteins in serum and urine

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E) Electroimmunodiffusion
The development of precipitin can be speeded up by electrically driving the Ag and Ab
Two most frequently technique used are:
i) Counter immunoelectrophoresis
ii) Rocket electrophoresis
i. Counter immunoelectrophoresis
It involves simultaneous electrophoresis of the Ab & Ag in gel in opposite directions
resulting in precipitation at a point between them
Produces visible precipitation line within 30 mins and is ten times more sensitive than the
standard double diffusion techniques
Used for the detection of alpha- fetoprotein in serum and specific Ags of Cryptococcus
in the CSF

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ii) Rocket- electrophoresis
The Ag in increasing concentration, is placed in wells in the set gel. The Ag is
then electrophoresed into the Ab containing agarose
The pattern of immunoprecipitation resembles a rocket and hence the name
Application- it is used for quantitative estimation of Ags

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APPLICATIONS OF PRECIPITATION RECATION
• Diagnosis of disease by demonstration of antibodies
• Identification of pathogen and classification of microorganisms
• Titration of Antibody
• To compare two unknown antigens
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AGGLUTINATION REACTIONS
A particular Ag is mixed with its Ab’s in the presence of electrolytes
at a suitable temperature and pH, the particles are clumped or
agglutinated
• The Ab of the serum causes the cellular Ag’s to form clumps and
these are called Agglutinins
• The particulate antigens that are aggregated are termed
Agglutinogens
• More sensitive than precipitation for detection of Abs
• Types: Slide agglutination, Tube agglutination, Antiglobulin test
Passive agglutination
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SLIDE AGGLUTINATION
rapid method to determine the presence of agglutinating antibodies
when a drop of the appropriate anti serum is added to a smooth uniform suspension
of a particulate Ag in a drop of saline on a slide, agglutination take place
Uses:
◦ Blood grouping and cross matching
◦ Confirmation of cultural isolates

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TUBE AGGLUTINATION
• Standard quantitative method for measuring Abs
• The serum containing Ab is diluted serially with saline in several small
test tubes, to which a constant volume of particulate Ag suspension is
added, the agglutination titre of the serum can be estimated
• A control tube is kept which has no antiserum
• The tubes are incubated until visible agglutination is observed
• Tube agglutination is employed for the serological diagnosis of
typhoid (Widal test), brucellosis

46. In this test Ab content of the patient’s serum, is
measured by adding a constant amount of antigen
(Salmonella typhi) to the serially diluted serum.
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• In this test Ab content of the patient’s serum, is measured by adding a
constant amount of antigen (Salmonella typhi) to the serially diluted serum
• The tube showing highest agglutination is referred to as the titre

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THE ANTIGLOBULIN TEST
It was devised by Coombs, Mourant and Race (1945)
Detection of anti- Rh antibodies that do not agglutinate Rh positive red cells in saline
Used for the detection of incomplete Abs
PRINCIPLE:
When sera containing incomplete anti-Rh Abs are mixed with Rh positive red cells ,
the Ab coats the surface of the RBCs , though they are not agglutinated
When such coated erythrocytes are washed off all unattached protein and treated
with a rabbit antiserum against human gamma globulin (antiglobulin or coombs
serum) the cells are agglutinated
The coombs test may be direct or indirect

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PASSIVE AGGLUTINATION TEST
• It is similar to haemagglutination test but physical nature of the reaction is altered
• The Ag is coated on the surface of a carrier particle and thereby helps to convert
a precipitation reaction into an agglutination reaction making the reaction more
sensitive for detection of antibodies
• The carrier particles used can be RBC, latex particles or bentonite. Some times
RBC coated with polystyrene (tanned RBC) can be used
• When patients serum is mixed with these, it leads to agglutination
• This test is used for the diagnosis of Rheumatoid arthritis

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LATEX AGGLUTINATION TEST

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Applications
•To check for the presence of Abs in
patient’s serum for influenza, measles,
mononucleosis, mumps viruses etc.
•Used for serotyping
•Used to measure Ab titer

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COAGGLUTINATION

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Lack of agglutination is indicator of a
positive reaction
Lack of agglutination is indicator of a positive reaction

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APPLICATIONS OF AGGLUTINATION REACTION
• Blood grouping and cross matching
• Demonstration of anti-blood group antibodies (haemolytic anaemia)
• Diagnosis of pregnancy (Haemagglutination Inhibition)
• Diagnosis of disease (Widal, Weil-Felix rxn, Paul-Bunnel test)
• Identification and classification of microorganisms by detection of Ag
LIMITATIONS :-
• - Time consuming (1 day)
• - Cannot distinguish IgG from IgM
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COMPLEMENT FIXATION TEST
• The ability of activated complement to lyse RBCs is made use of in
complement fixation test for detection of antibodies or antigens
• This reaction is not visible
• An indicator system is used to detect the fixation of complement
• This indicator system consists of sheep erythrocytes coated with amboceptor
(rabbit antibody to sheep RBCs)

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• Complement is a protein (globulin) present in normal serum
• Whole complement system is made up of nine
components: C1 to C9
• Complement proteins are heat labile and are destroyed
by heating at 56°C for 20 – 30 minutes
• Complement binds to Ag-Ab complex
• When the Ag is an RBC it causes lysis of RBC’s

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https://youtu.be/vboyla11Lyw

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It is a very versatile and sensitive test
Types of CFT-
◦ Wasserman reaction: serodiagnosis of syphilis
◦ Indirect complement fixation test
◦ Conglutinating complement adsorption
◦ Other complement- dependant serological tests

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NEUTRALIZATION REACTIONS
• In vivo Toxin antitoxin
neutralization
• In vitro toxin antitoxin
neutralization
Toxin
antitoxin
neutralization
• Viral neutralization
Viral
neutralization

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A)TOXIN ANTI TOXIN NEUTRALIZATION
In vivo Toxin antitoxin neutralization
e.g.
• Chick test: test susceptibility to diphtheria
• Dick test: test susceptibility to scarlet fever
• Schuiltz –Charlton test: diagnosis of scarlet fever
In vitro Toxin anti toxin neutralization
e.g: Anti streptolysin (O) test
• In neutralization reactions, the harmful effects of a bacterial
exotoxin or virus are eliminated by a specific antibody
• An antitoxin is an antibody produced in response to a bacterial
exotoxin or a toxoid that neutralizes the exotoxin

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B) VIRUS NEUTRALIZATION :
• Used for: diagnosis of viral infection by detection of antibody in
the patient serum using known virus
• Neutralisation of animal viruses can be demonstrated in three
systems animals, eggs and tissue culture
• In a virus neutralization test, the presence of antibodies against a
virus can be detected by the antibodies’ ability to prevent
cytopathic effects of viruses in cell cultures
• Antibodies against certain viruses can be detected by their ability
to interfere with viral hemagglutination in viral hemagglutination
inhibition tests

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APPLICATIONS OF ANTIGEN-ANTIBODY REACTIONS
• Determination of blood groups for transfusion and cross matching
• Serological ascertainment of exposure to infectious agents
• Diagnosis of diseases by detecting antibody/ antigen
• Diagnosis of Immune disorders
• To detect the presence or absence of protein in serum
• Identification of microorganisms
• Diagnosis of pregnancy
• Detection of antibodies after vaccination
• Detection of HLA antigens e.g. for tissue transplantation.

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COMPLEMENT SYSTEM
• Complement – A series of serum proteins (normally inactive )
involved in the effector role of the immune response to pathogens
and are activated through sequential protease- based step
• Made up of approximately 20 circulating and membrane-bound
proteins
• This complement system comprises of 11 proteins and are
present in every individual
• Synthesized in the liver and by cells involved in the
inflammatory response
• Major effector mechanism of humoral immunity

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Smaller component Larger component

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CLASSICAL PATHWAY
C1 : large, multimeric, protein complex
Activated C1s cleaves C4, to generate C4b
C2, complexes with C4b
C3 convertase : C4b2b
C5 convertase : C4b2b3b
C5a : potent inflammatory activity
C5b initiate formation of MAC

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ALTERNATIVE PATHWAY
Activation : proteolysis of C3
C3b , binding site for factor B covalently
tethered to surface of microbial or host cell
Bound factor B cleaved by factor D (serine
protease)
Properdin bind to and stabilize C3bBb
complex
C3 convertase : C3bBb
C5 convertase : C3bBb3b

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LECTIN PATHWAY
Activation : D-mannose,GlcNAc
(microbial polysaccharides )
Binding to circulating lectins, eg. MBL, or to
N- acetylglucosamine resemble C1q
MASP-1 cleave C3
MASP-2 cleave C4 and C2
C3 convertase : MASP-1
C5 convertase : C4b2b3b

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MEMBRANE ATTACK COMPLEX
Cleavage of C5 into C5a and C5b
C5b initiates formation of MAC binds to C6, and C7 , recruits C8
and complex penetrates (complex of C5b, C6, C7, C8 and multiple
C9 molecules ) more deeply into the membrane
C9, a pore-forming molecule with homology to perforin
The complex of C5b678 is a nidus for C9 binding & polymerization
Penetrates membrane bilayers to form pores
Disrupt the osmotic barrier, leading to swelling and lysis of
susceptible cells

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LATE STEPS OF COMPLEMENT ACTIVATION AND FORMATION OF MAC

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COMPLEMENT FUNCTIONS
Host benefit:
● opsonization to enhance phagocytosis
● phagocyte attraction and activation
● lysis of bacteria and infected cells
● regulation of antibody responses
● clearance of immune complexes
● clearance of apoptotic cells
Host detriment:
Inflammation, anaphylaxis

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REGULATION OF COMPLEMENT ACTIVATION

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Regulation of Classical
Pathway
Regulation of Alternate
Pathway

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INHIBITION OF FORMATION OF C3 CONVERTASES REGULATION OF FORMATION OF MAC

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• Antigen-antibody interaction is vital process of our immune
system of the body
• This chemical interaction elicits an immune response in the
body against the foreign substances
• The specificity of the interaction has lead to the development
of a variety of immunological assay which can be used for
detection of presence of antigen or antibody and widely used
as immunodiagnostics
CONCLUSIONS

118. THANK
YOU
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