ANTIGEN - ANTIBODY

REACTIONS

Dr.Gomathi M.D.,
Prof.
Dept of Microbiology
SRM MCH & RC
 Ag-Ab reactions - USES
(i) In vivo (In the body)

• Protection against microbes and their products.

• Forms the basis of Ab mediated immunity in infectious diseases.

• Forms the basis of tissue injury in some types of hypersensitivity &

auto-immune diseases.

(ii) In vitro (in the lab-serological tests)

• To detect the presence of Ab or Ag.

• Vital role in diagnosing diseases.

• Monitoring the level of the humoral immune response

• Identify molecules of biological / Medical interest.

 Ag-Ab interaction
1.Primary stage :
No visible effect.
Reaction is rapid.
Reaction is reversible.
Occurs by ionic bonds, H2 bonding.
Free & bound Ag/Ab can be estimated by physical or
chemical methods
2.Secondary stage: Leads to Precipitation, Agglutination, lysis
of cells, Neutralisation of toxins , Complement fixation test,
Immobilisation of microbes, enhancement of phagocytosis –
demonstratable.
3.Tertiary reaction: some Ag-Ab reactions leads to chain
reactions resulting in neutralisation or destruction of injurious
antigens or to tissue damage.
 General characters of Ag-Ab
reactions
• Highly specific. (In some cases Ab elicited by one Ag
can cross react with an unrelated Ag due to antigenic
similarity)
• Both Ag & Ab participate in lock & key arrangement.
• No Denaturation of Ag/Ab during the reaction.
• Surface antigens are most relevant during combination.
• Entire molecules react (Not fragments).
• Combination is firm but reversible, firmness depends
upon the affinity (attraction) & avidity (Strength of bond).
• There is no chemical alteration in either the Ab or Ag
• Combine in varying proportions.
 Ag – Ab Measurement
• Measurement of Ag / Ab is in units / titer

• Titer is the reciprocal of the highest

dilution of the serum which shows and
observable reaction
 Parameters of serological tests
Sensitivity:

• Ability to identify correctly all those who have the disease.

• True positives
• False negatives – low / nil.

Specificity:

• Ability to identify correctly all those who do not have the

disease
• True negatives
• False positive – low / nil.
 Types of Ag-Ab reaction
1. Agglutination reactions.
2. Precipitation reactions.
3. Complement fixation test (CFT)
4. Neutralisation test (NT)
5. Opsonisation
6.Immunofluorescence (IF)
7. Radioimmunoassay (RIA)
8. Enzyme linked immunosorbent assay (ELISA)
9. Immuno electroblot techniques.
10. Immunochromatographic tests
11.Immunoelectronmicroscopic tests.
 AGGLUTINATION-GENERAL
CHARACTERS
• Antigen is particulate (insoluble)

• Present on the surface of cell

• In presence of electrolytes

• Optimal proportion, temp & pH

• More sensitive than precipitation because of the

direct nature of antigen-antibody interaction
 AGGLUTINATION
Visible clumping of particles
Better with IgM
TYPES OF AGGLUTINATION
• Slide agglutination
• Tube agglutination
• Antiglobulin (Coomb’s) test
• Heterophile agglutination test
• Passive agglutination (indirect)
Latex agglutination,
Haemagglutination,
Co-agglutination
 SLIDE AGGLUTINATION TEST

Uses:
To identify bacterial isolates within 30-60 seconds
Method of Blood grouping & cross matching
Rapid & convenient
 Blocking antibodies

• Occasionally antibodies are

formed that will react with the
antigenic determinants on a cell
but will not cause agglutination.
Ex Anti Rh antibodies and anti
brucella antibodies
TUBE AGGLUTINATION TEST
Uses
• Quantitative methods for detection of Abs
• Serological diagnosis of
Enteric fever - Widal test
Brucellosis - Brucella agglutination test
Rickettsial fevers- Weil Felix test
Infectious mononucleosis- Paul Bunnell test
Primary atypical pneumonia -
Cold agglutination test
Streptococcal MG agglutination test

U U U U UU U U U
HETEROPHILE AGGLUTINATION
TEST
• Due to common Ag sharing in unrelated
species
Weil Felix test
Paul Bunnell test
Cold agglutination test
Streptococcal MG agglutination test
PASSIVE AGGLUTINATION TEST
• Precipitation reaction converted into
agglutination

• Soluble Ag is attached to the surface of

carrier particles

• Latex, bentonite & RBC(s)

 REVERSE PASSIVE
AGGLUTINATION
• Ab is absorbed to carrier particle

• For estimation of Ag

• Eg: Detection of HBsAg using RPHA

LATEX AGGLUTINATION TEST
• Latex particles can be coated with Ag/Ab
• Eg: Anti Streptolysin O
C Reactive Protein
Rheumatoid Factor
LATEX AGGLUTINATION TEST
HAEMAGGLUTINATION TEST
• RBC(s) senstised with Ag are used for detection of Abs
• Eg: Rose-Waaler test
CO-AGGLUTINATION TEST USE
• For detection of bacterial Ag in serum, urine & CSF
• Neisseriae, Str.B, C, D, F, G & N & H.influenzae Ags A-F
 NEUTRALISATION TEST
Principle:
• When antitoxin combines with a toxin, the
biological effect of toxin gets neutralised
• Since toxin is an Ag in solution, it is
precipitated
• Eg: Viral neutralisation tests
Neutralisation of bacterial toxins
VIRAL NEUTRALISATION TESTS

Test serum + Normal serum +

viral suspension viral suspension

Embryonated egg / Embryonated egg /

tissue culture tissue culture

No evidence of infection Evidence of infection

NEUTRALISATION OF BACTERIAL
TOXINS
• In vivo
Schick test: To demonstrate immunity to
Diphtheria intradermally
• In vitro
Nagler reaction: To demonstrate lecithinase
production by Clostridium perfringens
ASO (antistreptolysin) test
NAGLER REACTION

Lecithinase Antiserum
 COMPLEMENT FIXATION TEST
Principle:
• When complement binds to an Ag – Ab
complex it becomes “fixed” and “used up”

• CFT involves two steps and five reagents

(Ag ,Ab , compliment, sheep cells and
amboceptor
 CFT – Controls
• Ag & serum control to ensure that they are
not anti complementary
• Complement control to ensure that desired
amount of complement is added
• Cell control to see that they do not
undergo lysis in the options of the
complement
COMPLEMENT FIXATION TEST

Uses:
• Retrospective diagnosis of viral, Fungal , Rickettsial,
Chlamydial, Protozoan infections
• Wassermann’s reaction for syphilis
COMPLEMENT DEPENDANT TESTS
• Immobilisation test ( eg. TPI)
Motile treponemes treated with patient’s
serum in presence of complement

• Immune adherence (eg.V.cholerae)

Ag-Ab complex adhere to particulate
materials (RBC/ platelet/macrophage) in
presence of complement

Contd…
 COMPLEMENT DEPENDANT
TESTS Contd…
• Cytolytic or cytocidal reaction
Live bacteria when mixed with specific Ab in
presence of complement will produce
bacteriolysis
Eg: Vibrio cholerae
 Indirect CFT
• Certain sera do not fix g.pig complement
• Setup test in duplicate
• After first step standard antiserum known
to fix complement is added to one set
• If test serum is positive , antigen would
have been used up. There fore standard
serum will not fix complement
• Therefore hemolysis indicates a positive
result in the indirect CFT
Conglutinating complement
absorption test