Part II

Part II
Immunology
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ANTIGEN–ANTIBODY
REACTIONS

ANTIGEN-ANTIBODY REACTIONS
• Antigens - antibody - combine - specifically

- observable manner
• Body - Basis of antibody-mediated
immunity - infectious disease
• Tissue injury - hypersensitivity -
autoimmune disease
• Laboratory - Diagnosis of infection

• Epidemiology - Identification of infectious/

non-infectious agents
• General - Detection and quantitation of
antigen, antibody

• Antigen – Functionally recognised and bound

by antibody or T cell antigen receptor
• Antigen - any size - Proteins
Carbohydrate
Nucleic acid
Lipid
• Ag-Ab reactions - NEVER COVALENT so always
reversible

Reaction – Three stages
Primary Stage
• Initial interaction
• Without visible effect
• Low temperature
• Physical chemistry
• Thermodynamic reaction reversible
• Van der Waal’s forces ionic bond and hydrogen
bonding
• Detection - markers - radio isotopes; fluorescent
dye, ferritin

Secondary Stage
• Demonstrable events
• Precipitation
• Agglutination
• Lysis of cell
• Neutralisation of toxin
• Complement fixation
• Enhancement of phagocytosis
Tertiary Reaction
Ag-Ab reaction - vivo chain reaction –
neutralisation, destruction of injurious Ag,
tissue damage

GENERAL FEATURES
• Reaction-specific - homologous antibody

specificity not absolute - cross reactivity
• Entire molecules react
• No denaturation of antigen or antibody
• Combination on surface antigens -
immunologically relevant
• Combination firm - reversible
• Firmness - affinity and avidity

AFFINITY
• Intensity of attraction between antigen and

antibody molecules
• Closeness of fit between epitope and
paratope
• Binding strength between an antibody and
an epitope

AVIDITY
• Strength of bond after the formation of

antigen–antibody complexes
• Measure of binding capacity
• Pentameric IgM lower affinity than IgG, but
higher avidity of IgM is due to its higher
valency, which enables it to bind effectively
to the antigen

MEASUREMENT OF BINDING
Measurement (1) Mass

(2) Antibody titre-highest dilution of serum
shows an observable reaction with antigen
Titre, influenced by
- Nature and quantity of antigen
- Type and condition of test

PARAMETERS
Sensitivity: Ability to detect minute quantities

of antigen/antibody
Specificity: Ability to detect homologous
antigen and no other

SEROLOGICAL REACTIONS
• Precipitation Reaction

PRECIPITATION
Soluble antigen and antibody electrolytes

Suitable temperature and pH
• Insoluble precipitate - precipitation
• Suspended - floccules - flocculation

PRECIPITATION
• Prozone - Zone of antibody excess

• Zone of equivalence
• Postzone - Zone of antigen excess

PRECIPITATION - MECHANISM
Lattice hypothesis

APPLICATIONS
• Qualitative or quantitative test sensitive in

detection of antigen, 1 µg of protein - can
be detected
• Forensic application - Detection of
blood/serum stains
• Testing - Food adulterants

APPLICATIONS
• Grouping of Streptococci – Lancefield

technique
• VDRL test for syphilis
• To standardise toxins and toxoids
• To test toxigenicity in diphtheria
bacillus

TYPES OF PRECIPITATION TESTS
Ring test Slide test Tube test

Immunodiffusion (pptn in gel)
• Single diffusion one dimension
• Double diffusion one dimension
• Single diffusion two dimension
• Double diffusion two dimension
immunoelectrophoresis electro immunodiffusion
- CIE
• Rocket electrophoresis
RING TEST
• Simplest precipitation test – Consists

of layering antigen solution over
column of antiserum in a narrow tube
• Precipitate at the junction of two
liquids
Example: Ascoli’s thermoprecipitin test
Lancefield grouping of streptococci

SLIDE TEST
• Drop of antigen and antiserum on a slide –

mixed by shaking – floccules appear
Example: VDRL slide test – syphilis

TUBE TEST
Quantitative tube flocculation test

• Standardisation of toxin and toxoid
• Serial dilutions of toxin/toxoid are added to
tubes with fixed quantity of antitoxin,
amount of toxin/toxoid that flocculates one
unit of antitoxin - Lf dose
Example: Kahn test for syphilis

IMMUNODIFFUSION (PRECIPITATION IN GEL)
• Visible, distinct band of precipitation

• Different antigens observed
• Cross-reaction and non-identity between
different antigens

IMMUNODIFFUSION
Single diffusion - one dimension - Oudin

procedure
• Antibody - agar gel - test tube
• Antigen solution layered over it
• Antigen diffuses towards the agar gel,
forming a line of precipitation

IMMUNODIFFUSION
Double diffusion - one dimension

Oakley–Fulthorpe procedure
• Antibody incorporated in gel
• Above this column of plain agar
• Antigen layered on top of this
• Antigen and antibody move towards each
other

IMMUNODIFFUSION
Single and double diffusion in one dimension

RADIAL IMMUNODIFFUSION
(single diffusion in two dimensions)
• Antiserum in gel - slide/Petri dish

• Antigen added to wells cut on surface
• Diffusion radially from well
• Ring-shaped bands of precipitation

OUCHTERLONY PROCEDURE
(double diffusion - two dimensions)
Most widely employed

• Agar gel on a slide
• Wells cut using a template
• Antiserum in central well
• Antigen in surrounding wells
• Example: Elek’s gel precipitation test for
C.diphtheriae

DOUBLE DIFFUSION –TWO DIMENSIONS
Double diffusion in two dimensions

IMMUNOELECTROPHORESIS
• Electrophoretic separation of a composite

antigen into its constituent proteins
• Followed by immunodiffusion against its
antiserum
• Result - Separate precipitation lines
between each protein and its antibody

IMMUNOELECTROPHORESIS
Immunoelectrophoresis
ELECTROIMMUNODIFFUSION
1) Counter immunoelectrophoresis (CIE)

• Simultaneous electrophoresis of antigens
and antibody in gel in opposite directions
Example: -fetoprotein, cryptococcal antigen

COUNTERCURRENT IMMUNOELECTROPHORESIS (CIE)
Counterimmunoelectrophoresis

One dimensional, single

electroimmunodiffusion (rocket
electrophoresis)
• Quantitative estimation of antigens
• Antigen - Increasing concentration placed in
wells - punched in set gel
• Antigen electrophoresed into antibody
containing agarose
• Pattern of immunoprecipitation - Rocket

ROCKET ELECTROPHORESIS
Rocket electrophoresis

• Laurell’s two-dimensional electrophoresis
• Antigen mixture electrophoretically
separated in a direction perpendicular to the
final rocket
Laurell’s two-dimensional electrophoresis

AGGLUTINATION
Particulate antigen + Antibody + Electrolytes

- Suitable temperature
Agglutination optimal - Ag + Ab -
Equivalence

APPLICATIONS
Slide Agglutination
• Uniform suspension of particulate antigen
and appropriate antiserum
• Positive result - Clumping of particles and
clearing of the drop
• Typing of bacterial isolates
• Blood groups and typing

TUBE AGGLUTINATION
• Standard quantitative method

• Particulate antigen and equal volume of
serial dilution of antiserum
• Agglutination titre
• Diagnosis - Typhoid, brucellosis, typhus
fever

TUBE AGGLUTINATION
Widal Test: ‘H’ - flagellar antigen - large,

loose, fluffy clumps
‘O’ - somatic antigen - tight compact
deposits
Tube agglutination – Brucella - prozone

HETEROPHILE AGGLUTINATION
Weil–Felix - Typhus
Typhus, rickettsiae and Proteus
Streptococcus MG agglutination - Primary
atypical pneumonia
Paul-Bunnel test – Infectious mononucleosis

AGGLUTINATION USING RBC
Paul-Bunnel Test: Infectious mononucleosis

adsorbed by ox RBC not by guinea pig
Cold Agglutination Test: Mycoplasma
pneumoniae agglutinate human O group at
4°C reversible at 3°C

ANTIGLOBULIN (COOMBS) TEST
• Coombs, Mourant and Race (1945) detection of

anti-Rh antibodies that do not agglutinate Rh-
positive erythrocytes in saline
• Serum - Incomplete anti-Rh antibodies mixed with
Rh-positive red cells
• Antibody globulin coats the surface of erythrocytes
(not agglutinated)
• Erythrocytes coated with antibody washed free of
unattached protein treated with rabbit antiserum
against human gamma globulin (Coomb’s serum),
cells agglutinate

COOMBS TEST
Direct or Indirect
Direct: Sensitisation of erythrocytes in vivo
Example: Hemolytic disease of newborn
Indirect: Sensitisation of erythrocytes in vitro

PASSIVE AGGLUTINATION TEST
Attach antigen to surface of carrier particles

Carrier particles - Red cells, latex, bentonite
Passive hemagglutination - Rose-Waaler -
Rheumatoid arthritis - RA factor
Antigen-sheep erythrocytes sensitised with
subagglutinating dose of rabbit antisheep
erythrocyte antibody (amboceptor)

PASSIVE AGGLUTINATION
• Latex agglutination test – polysterene latex

• Example: ASO, CRP, RA factor
• Co-agglutination – Protein A bearing
Staphylococci coated with any IgG antiserum
will be agglutinate if mixed with
corresponding antiserum

COMPLEMENT FIXATION TEST
Complement - Ag - Ab lyses erythrocytes

Kills/lyse bacteria, immobilises motile organism
Promotes phagocytosis
Immune adherence
Tissue damage

CFT
Ag-Ab complex fix complement

Versatile, sensitive
CFT - Two steps
Five reagents - Ag
Ab
Complement
Sheep RBC
Amboceptor

CFT
Wasserman reaction
1st step: Inactivated serum - 37°C
+
Wasserman antigen
+
Guinea pig serum (complement)
Ag + Ab - complement utilised

CFT
2nd Step: Testing for complement
- Sensitised RBC
+
Amboceptor
Lysis - Negative CFT
Absence of lysis - Positive CFT
(complement utilised)

CFT
Complement fixation test

CFT
Complement fixation test—Wassermann reaction

CONGLUTINATING COMPLEMENT ABSORPTION TEST
• Systems that do not fix complement

• Horse complement – Non-hemolytic
• Indicator system - Sensitised sheep RBC
+
Bovine serum
Bovine serum - beta globulin

(conglutinin)

CONGLUTINATING COMPLEMENT ABSORPTION TEST
Acts as antibody to complement

Conglutinin causes agglutination of sensitised
sheep RBC

COMPLEMENT DEPENDENT TESTS
Bacteria + antibody + complement +

particulate matter (erythrocytes, platelets)
Bacteria aggregate  immune adherence
Immobilisation test – TPI
Cytolytic test- live bacteria + antibody +
complement bacteria lysed and killed

NEUTRALISATION TESTS
Virus Neutralisation Tests

Neutralisation of viruses
Neutralisation of bacteriophages

Plaque inhibition
Specific antiphage serum
Toxin Neutralisation
Bacterial exotoxins - Tetanus, diphtheria
Diphtheria toxin - Schick test
NEUTRALISATION
Anti streptolysin O (ASO) test – Streptococcal

O hemolysin
Nagler’s reaction – Clostridium perfringens
alpha toxin

OPSONISATION
• ‘Opsonin’ – Wright - 1903

• Heat-labile substance - Facilitates
phagocytosis (complement)
• Heat-stable serum factor – ‘bacteriotrophin’
• ‘Opsonic index’- Progress of resistance -
ratio of phagocytic activity of patient’s
blood for given bacterium to phagocytic
activity of blood from normal individual

RADIOIMMUNOASSAY(RIA)
• Radioisotopes conjugated to antigen or

antibody
• Binder ligand assay
• Analyte or ligand – Substance whose
concentration is to be determined
• Binder – Binder protein which binds to the
ligand
• RIA – Berson and Yallow, 1959

RIA
• RIA – Competitive binding assay

• Fixed amount of antibody and radiolabelled
antigen react in the presence of unlabelled
antigen
• Labelled and unlabelled antigens compete
for limited binding sites on the antibody
• After the reaction ‘free’ and ‘bound’
fractions of antigen separate and their
radioactive counts are measured

RIA
Radioimmunoassay procedure

RIA STANDARD CURVE
• The concentration of test antigen can be

calculated using the ‘standard dose
response’ or ‘calibrating curve’
Radioimmunoassay standard curve

ENZYME IMMUNOASSAY
• Enzyme-labelled conjugates
• Homogenous EIA – Does not require bound
and free fractions to be separated
• Heterogenous EIA – Requires the separation
of free and bound fractions either by
centrifugation or absorption on solid
surfaces and washing

ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)
• Involves the use of immunosorbent material

for one of the components of the reaction:
antigen or antibody
• Principle illustrated by outlining its
application for detection of Rotavirus
antigen in feces

ELISA
• Microtitre wells coated with goat antirotavirus
antibody
• Fecal samples added after washing
• Incubated, wells washed
• Guinea pig antirotavirus antiserum labelled
with alkaline phosphatase added
• Incubated, wells washed
• Substrate added
• Positive – colour change
• Negative – no colour change
ELISA: TYPES
• Sandwich ELISA – Antigen adsorbed onto

microassay plate wells
• Test serum added, incubated, washed
• Goat anti-human immunoglobulin antibody
conjugated with enzyme is added
• Wash, substrate added
• Positive – colour change
• Negative – no colour change

ELISA: TYPES
• Competitive ELISA – Similar to RIA, unknown

antigen (sample) and known antigen
(standard) compete with each other for
fixed antibody
• Cylinder or cassette ELISA – Each sample
tested in a separate disposable cassette, in-
built controls, result read visually

ELISA: TYPES
Enzyme linked immunosorbent assay (EL ISA)

USES OF ELISA
• Detection of infectious diseases – HIV,

Hepatitis, EBV, Dengue, TORCH panel etc
• Food toxins – aflatoxins
• Food adulterants
• Mycobacterial antibody detection

CHEMILUMINESCENCE IMMUNOASSAY (CLIA)
• Chemiluminescent compounds are used in

CLIA as a label to provide signal during
antigen antibody reaction
• The signal (light) can be amplified,
measured and concentration of analyte
calculated

IMMUNOELECTROBLOT (WESTERN BLOT)
• The technique is a combination of three

procedures
• Separation of ligand and antigen by gel
electrophoresis
• Blotting of electrophoresed ligand fraction
• Enzyme immunoassay to detect antibody

IMMUNOFLUORESCENCE
• Fluorescence - Property of absorbing light

rays of one wavelength and emitting rays of
a different wavelength
• Direct immunofluorescence test - Specific
antiserum labelled with a fluorescent dye,
used for identification of antigen

IMMUNOFLUORESENCE
Direct immunofluorescence tests

IMMUNOFLUORESCENCE
• Indirect immunofluorescence test – Uses an

antiglobulin fluorescent conjugate
• Known antigen is placed on a slide
• A drop of test serum is added
• Antibody if present (in serum sample) reacts
with antigen on the slide
• Fluorescent labelled antiserum to human
gamma globulin is added
• Positive – Bright against dark background
IMMUNOFLUORESENCE
Indirect immunofluorescence

IMMUNOFLUORESCENCE
• Immunohistochemical technique – Helps to

visualise antigen antibody reactions in situ
• Flow cytometry –Fluorescence technique
used to identify and enumerate cells bearing
a particular antigen or surface marker
• Cells are made to flow in a single stream in
a flow cell by hydrodynamic focussing

FLOW CYTOMETER
Schematic diagram of a flow cytometer

APPLICATIONS
• Differential leucocyte count

• T cell subsets - CD4 and CD8 counts in HIV
patients
• Diagnosis, prognosis and treatment of
cancer

Part II - Chapter 12 - Antigen-Antibody Reactions

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© Universities Press (India) Private Limited Universities Press

• Antigens - antibody - combine - specifically

© Universities Press (India) Private Limited Universities Press

• Epidemiology - Identification of infectious/

© Universities Press (India) Private Limited Universities Press

• Antigen – Functionally recognised and bound

© Universities Press (India) Private Limited Universities Press

© Universities Press (India) Private Limited Universities Press

© Universities Press (India) Private Limited Universities Press

• Reaction-specific - homologous antibody

© Universities Press (India) Private Limited Universities Press

• Intensity of attraction between antigen and

© Universities Press (India) Private Limited Universities Press

• Strength of bond after the formation of

© Universities Press (India) Private Limited Universities Press

Measurement (1) Mass

© Universities Press (India) Private Limited Universities Press

Sensitivity: Ability to detect minute quantities

© Universities Press (India) Private Limited Universities Press

© Universities Press (India) Private Limited Universities Press

Soluble antigen and antibody electrolytes

© Universities Press (India) Private Limited Universities Press

• Prozone - Zone of antibody excess

© Universities Press (India) Private Limited Universities Press

© Universities Press (India) Private Limited Universities Press

• Qualitative or quantitative test sensitive in

© Universities Press (India) Private Limited Universities Press

• Grouping of Streptococci – Lancefield

© Universities Press (India) Private Limited Universities Press

Ring test Slide test Tube test

• Simplest precipitation test – Consists

© Universities Press (India) Private Limited Universities Press

• Drop of antigen and antiserum on a slide –

© Universities Press (India) Private Limited Universities Press

Quantitative tube flocculation test

© Universities Press (India) Private Limited Universities Press

• Visible, distinct band of precipitation

© Universities Press (India) Private Limited Universities Press

Single diffusion - one dimension - Oudin

© Universities Press (India) Private Limited Universities Press

Double diffusion - one dimension

© Universities Press (India) Private Limited Universities Press

Single and double diffusion in one dimension

© Universities Press (India) Private Limited Universities Press

• Antiserum in gel - slide/Petri dish

© Universities Press (India) Private Limited Universities Press

Most widely employed

© Universities Press (India) Private Limited Universities Press

Double diffusion in two dimensions

© Universities Press (India) Private Limited Universities Press

• Electrophoretic separation of a composite

© Universities Press (India) Private Limited Universities Press

1) Counter immunoelectrophoresis (CIE)

© Universities Press (India) Private Limited Universities Press

© Universities Press (India) Private Limited Universities Press

One dimensional, single

© Universities Press (India) Private Limited Universities Press

© Universities Press (India) Private Limited Universities Press

Laurell’s two-dimensional electrophoresis

Particulate antigen + Antibody + Electrolytes

© Universities Press (India) Private Limited Universities Press

© Universities Press (India) Private Limited Universities Press

• Standard quantitative method