ANTIGEN,ANTIBODY AND

ANTIGEN-ANTIBODY REACTIONS
 ANTIGENS
• Definition : An antigen is a high molecular weight protein or
polysaccharide which when introduced into an immunocom -
petent host, generates an immune response stimulating T
cells or B cells or both .
• It should satisfy 2 properties –
 Immunogenicity : Ability of an antigen to induce immune
response in the body
 Antigenicity : Ability of an antigen to combine specifically with
antibodies or T cell surface receptors .
• Epitope / antigenic determinant : It is the smallest unit of
antigenicity that specifically binds to the antibody ( paratope).
• Hapten : These are low molecular weight molecules that lacks
immunogenicity but retain antigenicity . They become
immunogenic when combined with a larger protein molecule
called carrier . They are of 2 types :
 Complex haptens : Contain 2 or more epitopes .
 Simple haptens : Usually contain only one epitope .
• Antigen-Host relationship :
 Self / autoantigens : They belong to the host itself and due to
immunological tolerance they are not immunogenic . But if
they are biologically altered , they can become
immunogenic ,e.g. in autoimmune diseases .
 Non-self or foreign antigens : They are immunogenic and are
of 3 types based on their phylogenetic distance to the host –
 Alloantigens – species specific antigens .
 Isoantigens – present only in subsets of a species e.g. blood
group antigens .
 Heteroantigens – antigens belonging to 2 different species e.g.
Heterophile antigen , exists in unrelated species like – Weil-
Felix reaction, Paul-Bunnell test and cold agglutination test .
• Factors influencing immunogenicity :
1. Size and chemical nature of antigen
2. Susceptibility of antigen to tissue enzymes
3. Structural complexity and foreignness to the host
4. Genetic factor
5. Optimal dose or route of antigen administration
6. Repeated doses of antigen
7. Multiple antigens ( like the use of Adjuvants )
8. Effect of prior administration of antibody
• Adjuvants : Any substance that enhances the immunogenicity
of an antigen . These are used in vaccines to enhance the
immunogenicity of vaccine antigens .e.g. LPS of Bordetella
pertussis acts as an excellent adjuvant for diphtheria and
tetanus toxoids .
• Biological Classes of antigens :
1. T- dependent antigen : most of the normal antigens .
2. T-independent antigen : like bacterial capsule , flagella and
LPS .
3. Superantigens : like toxic shock syndrome toxin ( TSST-1),
Streptococcal pyrogenic exotoxin (SPE)- A and C ,etc.
• Superantigens :
 The variable beta region of T cell receptor is the receptor for
superantigens
 They can activate T cells directly without being processed by
antigen presenting cells . Nonspecific activation of T cells
leads to polyclonal B cell activation leading to
hypergammaglobulinemia .
 Diseases associated with superantigens are : Toxic shock
syndrome ,food poisoning ,scalded skin syndrome , atopic
dermatitis ,kawasaki syndrome ,etc.
 ANTIBODY

• Definition : Antibody or immunoglobulin is a specialized

glycoprotein produced from activated B cells (plasma cells) in
response to an antigen ,and capable of combining with the
antigen that triggered its production .
• There are 5 classes of immunoglobulins recognized –
IgG ,IgA,IgM ,IgD and IgE ( their respective heavy chain types
includes – gamma, alpha ,mu,delta ,epsilon respectively ).
• An antibody molecule is a Y shaped heterodimer composed of
4 polypeptide chains – 2 identical light chains (25000 Da) and
2 identical heavy chains ( 50000 Da or more).
• Paratope : It is the site on the hypervariable region of antibody
that makes actual contact with the epitope of an antigen .
• Hinge region :In heavy chain (gamma, alpha and delta) the
junction formed between CH1 and CH2 domain constitutes the
hinge region . This region is rich in proline and cysteine , it
makes the molecule quite flexible , allowing immunoglobulin
molecule reach towards the antigen by assuming different
positions .
 This hinge region is sensitive to various enzymatic digestion .
 In IgM and IgE , mu and epsilon do not have hinge region but
their constant region has an additional domain .
• Functions of immunoglobulins :
1. Antigen binding by Fab region
2. Effector functions by Fc region
 Fixation of complement
 Binding to various cell types ( like
phagocytes ,lymphocytes ,platelets ,mast cells ,NK cells ,
eosinophils and basophils bearing Fc region ) and also
binding to placental trophoblasts resulting in transfer of IgG
across the placenta .
 IMMUNOGLOBULIN CLASSES :

• IgG :
1. Constitutes 70-80% of total Ig in the body .
2. It can cross placenta so provides immunity to the fetus and
new born .
3. It can activate the classical pathway of complement system
4. Plays role in phagocytosis by binding to the Fc receptors on
phagocytes .
5. Raised IgG levels represents chronic or past infections
6. Plays major role in neutralization of toxins
7. IgG subclass ( except IgG3) mediates coagglutination reaction
by binding to protein-A of S.aureus .
• IgM :
1. It has highest molecular weight and maximum sedimentation
coefficient . It is pentameric ( 10 Fab regions , 10 valencies ).
2. Present only in intravascular compartment , not in body fluids or
secretions . Protection against blood invasion by harmful
microorganisms .
3. Raised in primary immune response ( in acute infection )
4. Most potent activator of classical complement pathway .
5. Acts as an opsonin so enhances phagocytosis
6. First antibody to be synthesized in fetal life ( 20 weeks) , so
provides immunity to the fetus .
7. Mediates agglutination .
• IgA :
1. Exists in 2 forms : serum IgA(monomeric ) and secretory
IgA( dimeric ). Constitutes 10-15% of total serum Ig .
2. Serum IgA plays role in antibody-dependent cell mediated
cytotoxicity (ADCC) and degranulation of immune cells .
3. Secretory IgA is predominant antibody found in body
secretions , mediates local or mucosal immunity .
4. Breast milk is rich in secretory IgA , provides good protection
to the immunologically immature infant’s gut .
• IgE :
1. Lowest serum concentration, shortest half life and minimum
daily production .
2. It is heat labile ( inactivated at 56 degree centigrade for 1 hr)
3. Has affinity for surface tissue cells of the same species
(homocytotropism )
4. Mainly extravascular in distribution
5. It is highly potent and mediates type 1 hypersensitivity
reactions , its response is seen in asthma , anaphylaxis ,etc.
6. It is elevated in helminthic infections .
• IgD :
1. It is found as membrane Ig on the surface of B cells and acts
as a B cell receptor along with IgM .
2. It has the highest carbohydrate content among all Ig .
• Antigenic determinants of immunoglobulins :
1. Isotypes : Vary from each other in amino acid sequences of
constant region of their heavy chains . E.g. 5 classes of Ig . It
is similar in all members of same species .
2. Idiotypes : The unique amino acid sequence present in
paratope region of one member of a species acts as antigenic
determinant to other members of the same species .
3. Allotypes : The antigenic determinants present in the isotype
genes in constant region of H and L chains , encoded by
multiple alleles called as allotypes .
 Examples of abnormal immunoglobulins :

• Bence Jones proteins :

1. Produced in multiple myeloma ( neoplastic condition of
plasma cells )
2. Cancerous plasma cells produce excess of light chains which
are accumulated in patient’s serum and excreted in urine .
3. They characteristically get coagulated at 50 degree and
redissolves again at 70 degree centigrade .
• Cryoglobulinemia : ( in multiple myeloma and hepC inf)
1. They usually constitute of IgM directed against Fc of IgG .
2. Precipitate at low temperature but redissolves again if blood
is heated .
 MONOCLONAL ANTIBODY :

• These are antibodies derived from a single clone of plasma

cells ; all having the same antigen specificity i.e. produced
against a single epitope of an antigen .
• They are produced by Hybridoma technique , developed by
G Kohler and C Milstein .
• Principle : 1) Produces monoclonal antibody of same antigen
specificity due to B cell component
2) Multiplies indefinitely producing clone of identical cells due
to immortal myeloma cell component .
• Types :
1. Mouse mAb : 100% mouse derived proteins .
2. Chimeric mAb : 34% mouse and 66% human proteins
3. Humanized mAb : 10% mouse and 90% human derived .
4. Human mAb : 100% human derived amino acids .
• Applications :
1.Widest application is in detection of antigen .e.g.
hepB ,serogrouping of streptococci ,pregnancy detection
test ,etc.
2.Post exposure prophylaxis against hepatitisB ,rabies ,tetanus.
3. Used in treatment of various inflammatory ds, allergic ds and
cancers .
4. Used as immunotoxins and enzymes .
 CLASS SWITCH OVER :

1. Once B cell is stimulated by an antigen ,heavy chain gene

undergoes rearrangement where VDJ segment combines
with one of the CH gene segments ;first with C(mu) forming
IgM followed by others .This process is called class switching
or isotype switching .
2. Each CH region genes except C(delta) , contains a highly
conserved DNA flanking sequence containing tandem
repeats called as switching site .
3. Regulated by specific cytokines secreted by helper T cells .
 ANTIGEN-ANTIBODY REACTION :

• General properties of antigen-antibody reactions :

1. Specific
2. Noncovalent interactions
3. Strength :
• Affinity – It refers to the sum total of noncovalent
interactions between a single epitope of an antigen with its
corresponding paratope present on antibody .
• Avidity – Describes the affinities of all the binding sites
when multivalent antibody reacts with a complex antigen
carrying multiple epitopes .
• The diagnostic tests based on antigen-antibody reactions are
called as immunoassays . They may be quantitative or
qualitative .
• Qualitative assays : The undiluted specimen containing the
antibody directly mixed with suspension of antigen or vice
versa . Here , test can be reported as Positive or Negative ,
based on presence or absence of antigen or antibody .
• Quantitative assays : When the qualitative test turns
positive , the exact amount of antibody in serum can be
known by serial dilutions of patient’s serum mixed with a
known quantity of antigen . Antibody titer can be calculated .
• Antibody Titer of a serum : It is the reciprocal of highest
dilution that shows an observable reaction with the antigen .
• Marrack ( 1934) proposed the lattice hypothesis to explain 2
phenomena –
1. Prozone phenomenon : In the earlier test tubes of serial
dilutions ,antibodies are in excess so Ag-Ab reaction fails to
occur .
2. Postzone phenomenon : In the later test tubes of serial
dilutions , antigens are in excess so Ag-Ab reaction fails to
occur .
Ag-Ab reaction always occurs at the Zone of equivalence .
 TYPES OF ANTIGEN-ANTIBODY REACTIONS :

• CONVENTIONAL TECHNIQUES :
1. Precipitation reaction
2. Agglutination reaction
3. Complement fixation test
4. Neutralization test
• NEWER TECHNIQUES :
1. ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
2. Enzyme-linked fluorescent assay (ELFA) – Ag-Ab complex
detected by fluorometric methods .
3. Immunofluorescence assay (IFA)
4. Radioimmunoassay (RIA)
5. Chemiluminescence –linked immunoassay (CLIA)
6. Immunohistochemistry
7. Rapid tests –
 Lateral flow assay ( immunochromatographic test )
 Flow through assay
8. Western blot
9. Immunoassays using electron microscope
 PRECIPITATION REACTION :

• Definition : When a soluble antigen reacts with its antibody

in the presence of optimum temperature, pH and electrolytes,
it leads to formation of the antigen-antibody complex in the
form of –
 Insoluble precipitate band when gel containing media used
 Insoluble floccules when liquid medium is used .
• Precipitation in liquid medium e.g. Ascoli’s thermoprecipitin
test done for anthrax ( ring test ) , VDRL –Venereal Disease
Research Laboratory and RPR –Rapid Plasma Reagin for
Syphillis (slide flocculation test ) , Kahn test used previously
for syphillis (tube flocculation test ) .
• Precipitation in gel e.g. Elek’s test for detecting toxin of
Corynebacterium diphtheriae and Eiken test to detect toxin
of Escherichia coli .
• Precipitation in gel in presence of electric current :
1. Electroimmunodiffusion (EID)
2. Countercurrent immunoelectophoresis (CIEP) e.g. detection
of alpha fetoprotein in serum .
 AGGLUTINATION REACTION :

• Definition : When a particulate or insoluble antigen is mixed

with its antibody in the presence of electrolytes at a suitable
temperature and pH , the particles are clumped or
agglutinated .
• Agglutination is more sensitive than precipitation test and the
clumps are better visualized as compared to bands or
floccules .
• Types : Direct agglutination test , Indirect or passive
agglutination test , reverse passive agglutination test ,
haemaglutination test .
• Direct agglutination test :
1. Slide agglutination– e.g. blood grouping and cross matching
2. Tube agglutination : Antibody titer can be estimated e.g.
Typhoid fever ( Widal test ) –
• Widal test detects antibodies against both H( flagellar) and
O(somatic) antigens of Salmonella Typhi
• H Ag-Ab clumps appear as loose fluffy clumps
• O Ag-Ab clumps appear as chalky white granular dense
deposits .
3. Microscopic agglutination– done in microtiter plate , result
read under microscope e.g. MAT for leptospirosis .
• Indirect or passive agglutination test ( for Ab detection ) :
 Here the precipitation reaction is converted into an
agglutination reaction
 This is possible by coating the soluble antigen on the surface
of a carrier molecule ( e.g. RBC,latex or bentonite ) , so that
the antibody binds to the coated antigen and agglutination
takes place on the surface of the carrier molecule .
 Positive result indicated by formation of visible clumps .
 E.g. Latex agglutination test for antibody detection is used
for detection of ASO ( Antistreptolysin O antibody ).
• Reverse passive agglutination test ( for antigen detection ):
 Here antibody is coated on a carrier molecule which detects
antigen in the patient’s serum .
 Latex agglutination test for antigen detection is used for
detection of CRP(C-reactive protein ),Rheumatoid arthritis
(RA) factor , capsular antigen detection in CSF for
Pneumococcus , Meningococcus and Cryptococcus .
• Direct Hemagglutination test :
 Serum antibodies directly agglutinate with surface antigens
of RBCs to produce a matt .
 E.g. Paul Bunnell test (to detect EBV antibodies in serum )
 Cold agglutination test (to detect Mycoplasma antibodies)
 Blood grouping (ABO and Rh grouping )
 Coombs test or antiglobulin test : It is performed to diagnose
Rh incompatibility by detecting Rh antibody ( blocking
antibodies/ incomplete IgG Ab blocks sites on antigen but no
visible agglutination so Coomb’s reagent added) from
mother’s and baby’s serum .
 COMPLEMENT FIXATION TEST :

• A known Ag( cardiolipin/viralAg/sheep RBCs) is mixed with

inactivated patient’s serum (serum heated at 56 degree
centigrade for 30 mins to destroy complement activity of test
serum and to remove anti-inflammatory effect of some non-
specific inhibitors in the serum ).
• A measured amount of complement (guineapig serum) is
added to the test system .
• Then the test system is incubated at 37degree for 1 hour
• After 1 hour an indicator system (sensitized RBCs) is added to
the test system and again incubated at 37 degree for 30 mins.
• If Ag-Ab matches , they form Ag-Ab complex and utilizes
- complement .
• Results were observed as :
 Positive CFT : If no haemolysis is observed ,it indicates
positive CFT . Ag-Ab reaction and complement fixation
occurs ,so no free complement is available to lyse the RBCs.
 Negative CFT : If haemolysis of RBC is observed , it indicates
negative complement test .No complement fixation occurs ,
so the complement remain free and it haemolyse the RBCs.
• E.g. Wasserman test for diagnosis of syphillis , detection of
some viral infections like arboviruses , rabies ,etc.
 NEUTRALIZATION TEST :

• Less commonly used nowadays

• E.g. Viral neutralization test ( detects presence of neutralizing
antibody in patient’s serum ), plaque inhibition test ,toxin-
antitoxin neutralization test like in Schick test and Nagler’s
reaction ; Haemagglutination inhibition test (HAI) for
diagnosis of various viral diseases e.g. influenza .
 NEWER TECHNIQUES :

• ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) :

 Immunosorbent : Here, an absorbing material like
polystyrene ,polyvinyl are used that specifically absorbs the
Ag or Ab present in serum .
 Enzyme : it is used to label one of the components of
immunoassay i.e. antigen or antibody .
 Substrate –chromogen system : Added at the final step of
ELISA .The enzyme reacts with the substrate , which in turn
activates the chromogen to produce a colour , detected by
spectrophotometry in an ELISA reader . The intensity of colour
in the microtiter plate is proportional to Ag/Ab amount .
 Types of ELISA :
1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA
5. ELISPOT test ( for interferon gamma assay detecting latent
TB)
6. IgG avidity ELISA
 Examples : tests like Dengue NS1 and IgM , HepatitisB,
HepatitisC and HIV-ELISA .
• IMMUNOFLUORESCENCE ASSAY (IFA) –
 Unlike ELISA, fluorescent dye is used instead of enzyme for
labeling of antibody .
 It can detect both cell surface antigens as well as detects
antibodies bound to cell surface antigens .
 2 types : Direct, indirect assays and flow cytometry
 Useful for detection of Antinuclear antibody (ANA) , rabies
antigen detection in corneal smear .
• FLOW CYTOMETRY :
 It is a laser based technology that quantitatively analyses
and separates the cells as they pass through the laser beam .
 Its advanced and upgraded form is called as fluorescence-
activated cell sorting (FACS).
 Examples : used for detecting CD4 T cell count in HIV
infected patients ,detection of leukocytes with specific
markers for the diagnosis of various lymphomas .
• Immunohistochemistry : based on principles of ELISA or IFA ,
used for detection of abnormal cells like tumor cells .
• Radioimmunoassay : principle same as competitive ELISA but
here radioactive molecules are used for labeling and test is
done in liquid medium ,used for detection of HBsAg at a conc.
of <0.01 microgram/ml .
• Chemiluminescence-linked immunoassay (CLIA) : principle
similar to ELISA but here a chemilumenescent compound like
acridinium ester is used to generate light during chemical
reaction instead of chromogenic substance , used for
detecting antibodies to Hepatitis viruses,HIV, TORCH inf. and
biomarkers such as procalcitonin .
• Western Blot :
 Detects specific proteins [antibodies] in a sample containing
mixture of antibodies each targeted against different
antigens of same microbe .
 Eastern blot is a latest modification of it
 As it has excellent specificity , it is often used as a
supplementary test to confirm the result of ELISA .
 Examples – detection of antibody in HIV , Herpes simplex
virus ,hydatid disease, toxoplasmosis, etc .
 RAPID TESTS/POINT OF CARE (POC) TESTS :

• Advantages : very simple method(1 step), rapid (result

between 10-20 minutes ),requires minimal training ,
performed independent of laboratory equipment .
• 2 principles :
1. Immunochromatographic test/ICT (lateral flow assay )
2. Flow through assay
• ICT :
 Based on lateral flow technique
 Useful for detection of hepatitisB,malaria , hepatitisC ,HIV ,
leptospirosis ,Helicobacter pylori , syphilis ,etc.
• Flow through assay :
 It differs from ICT in 2 ways –
1. Protein A is used for labeling antibody instead of gold
conjugate
2. The sample flows vertically through nitrocellulose
membrane as compared to lateral flow in ICT .
 It can be used for both Ag and Ab detection . HIVTRIDOT
test is a classical example , detects antibodies to HIV1 and 2
separately in patient’s serum . Here , proteinA conjugate
binds to Fc portion of the HIV antibodies to give distinct
pinkish purple DOTs , separately for HIV1 and HIV2 Abs .
 IMMUNOELECTRON MICROSCOPY :

• Viral particles appear to be clumped when mixed with specific

antisera and observed under electron microscope .
• This is used for finding hepatitisA virus and rotavirus particles
from stool specimen .
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