ANTIGEN - ANTIBODY

REACTIONS

Dr.Gomathi M.D.,
Prof.
Dept of Microbiology
SRM MCH & RC
 Introduction
• Serological reactions: Ag- Ab reactions in vitro.

• Differences between agglutination and

precipitation.

• Prozone phenomenon.

• There is no irreversible chemical alteration in

either the antibody or the antigen (reversible).
 Ag-Ab reactions - USES
(i) In vivo (In the body)

• Protection against microbes and their products.

• Ab mediated immunity in infectious diseases.
• Tissue injury in some types of hypersensitivity & auto-immune
diseases.

(ii) In vitro (in the lab)

• 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.

2.Secondary stage: Precipitation, Agglutination, lysis of cells,

Neutralisation test, Complement fixation test,Immobilisation
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 relavent during combination.
• Entire molecules react (Not fragments).
• Combination is firm but reversible, firmness depends
upon the affinity (attraction) & avidity (Strength of bond).
• Combine in varying proportions.
 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.
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)
• In presence of electrolytes
• Optimal proportion, temp & pH
• Visible clumping of particles
• Better with IgM
TYPES OF AGGLUTINATION
• Slide agglutination
• Tube agglutination
• Antiglobulin (Coomb’s) test
• Heterophile agglutination test
• Passive agglutination
Latex agglutination,
Haemagglutination,
Co-agglutination
SLIDE AGGLUTINATION TEST

Uses:
To identify bacterial isolates
Blood grouping & cross matching
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 blood, urine & CSF
• N.gonorrhoeae, Strep.pyogenes & H.influenzae Ags
 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