Antigen - antibody reaction based on medical science

microbiology

1. Antigen - Antibody Reaction

2. Antigen-Antibody Reactions-General features
Antigens and antibodies combine specifically in a
observable manner.
Reaction is specific (Ag+ only Homologous Ab &
vice versa). Cross reactions due to antigenic
similarity.
Reaction between entire molecules & not
fragments.
No denaturation of antigen or antibody.

3. Characteristics
Only surface Ag participate in Ag-Ab reactions.
Affinity-intensity of attraction betwn Ag & Ab
molecules
Ag & Ab combine in varying proportions.
Ab-generally bivalent. IgM has 5/more combining
sites.

4. Stages
Ag & Ab reaction occurs in 2 stages:-
Primary stage-
Is initial interaction between Ag & Ab without any visible effects.
Reaction is rapid and reversible – Binding of Ag & Ab by weaker
intermolecular forces
No firm covalent bonding.
Secondary stage –
Has visible effects or demonstrable events like Precipitation,
Agglutination, Complement fixation.
Single Ab-cause different types of Ag-Ab reactions

5. Types of Ag-Ab reactions - Precipitation Reaction
When a soluble Ag combines with its Ab in the presence
of electrolytes at a suitable temperature and pH forms an
insoluble precipitate of Ag-Ab complex-called
precipitation.
Precipitate- usually sediments –at bottom of tube.
Suspended Precipitate-Floccules-reaction is
Flocculation-VDRL test.
Occurs in Liquid Media or Gels – Agar, Agarose and
Polyacrylamide.

6. PROZONE PHENOMENONPROZONE PHENOMENON
The amount of precipitate formed is greatly influenced by the The amount of precipitate formed is greatly influenced by the
relative proportions of Ags & Abs.relative proportions of Ags & Abs.
If increasing quantities of Ags are added to the same amount If increasing quantities of Ags are added to the same amount
of antiserum in different tubes, precipitation is found to occur of antiserum in different tubes, precipitation is found to occur
most rapidly & abundantly in the middle tubes at optimal most rapidly & abundantly in the middle tubes at optimal
proportion.proportion.
–Preceding tubes – Ab excess (Prozone)-false -ve - further Preceding tubes – Ab excess (Prozone)-false -ve - further
dilutions.dilutions.
–Middle tubes – Ag & Ab in equivalent proportions (Zone Middle tubes – Ag & Ab in equivalent proportions (Zone
of equivalenceof equivalence
–Later tubes – Ag excess (Post zone)Later tubes – Ag excess (Post zone)

8. Mechanism of Precipitation
Lattice Hypothesis proposed by Marrack in 1934- Multivalent
antigens combine with bivalent antibodies in varying proportions -
Precipitation results when large lattice formed consisting of
alternating antigen and antibody molecule - occurs in zone of
equivalence.
In the zones of antigen excess, valencies of Ab are fully satisfied-
failure to form large lattice.
In the zones of antibody excess, valencies of Ag are taken up with
Ab - lattice does not enlarge.

9. Marrack’s hypothesis

10. •Blue –Ag
•Orange- Ab
Ab excess-ProzoneAg excess-Postzone
Equivalence – Lattice formation

11. Application of Precipitation reaction
To carry out Qualitative and Quantitative tests.
Sensitive for detecting antigens .
Identification of bacteria .
Detection of Abs for diagnostic purposes-VDRL
in syphilis
Forensic application in identification of human
blood and seminal stains.
Standarisation of toxins & toxoids.
Less sensitive for detection of antibodies.

12. Precipitation Tests in common use
Ring test
 Simplest type.
 Layering antigen solution over a
column of antiserum in a narrow
tube.
 A precipitate forms at the junction
of two liquids.
 Clinical applications only few.
eg :
C-reactive protein test

13. Flocculation test- Slide flocculation test
A drop of antigen solution
and a drop of inactivated
patient serum on a slide
mixed by shaking.
Floccules appear.-
eg : VDRL test (detection of
Ab for syphilis in pt serum)
.

14. Flocculation test- Tube test
eg : Kahn’s test for syphilis-qualitative
Also employed for the standardisation of toxins and
toxoids- quantitative tube flocculation test.- Serial
dilution of toxin/toxoid are added to the tubes
containing a fixed quantity of the antitoxin.

15. Immunodiffusion test ( Precipitation in gel )
Precipitation in gel has advantages over liquid medium.
Performed in a soft (1 %) agar gel.
Easily Visible as a distinct band & can be stained for
preservation. Band is stable.
Different antigens in the reacting mixture can be readily
observed. Each Ag-Ab reactions give rise to a line of
precipitation- helps to identify different Ags.

16. Single diffusion in one dimension-Oudin procedure)
•Antibody incorporated in agar gel in a test tube and antigen
solution layered over it.
•Antigen diffuses downward forming a line of precipitation.
•No. of precipitate bands –indicate no. of different Ags
present.
•It is single diffusion of Ag only and only in one dimension
i.e towards Ab in agar gel.

17. Double diffusion in one dimension (Oakley-Fulthrope
procedure)
•Antibody incorporated in
agar gel at bottom of tube, A
column of plain agar above
that and Antigen layered on
top of this.
•The antigen and antibody
move towards each other
thro’ intervening plain agar
and form a precipitate band
at optimum concentration.

19. Single diffusion in two dimensions
(Radial immunodiffusion)
•Antiserum incorporated in agar gel
poured on a slide or petri dish.
•Wells cut on the surface of gel and antigen
added.
•Antigen diffuses radially and forms ring
shaped bands of precipitation (halos)
around the well.
•Diameter of halo gives an estimate of the
concentration of antigen.
•Employed to estimate Immunoglobulin
classes in sera-IgG, IgM, IgA in sera
•Screening sera for antibodies to Influenza
viruses.

21. Double diffusion in two dimensions
(Ouchterlony procedure)
•Widely used.
•Compares different antigens and antisera directly.
•Agar gel poured on a slide and wells are cut.
•Antisera in the central well and different antigens in
the surrounding wells.
•If two adjacent antigens are identical the lines of
precipitate fuse.
•Unrelated antigens cross each other.
•Spur formation indicates Cross reaction or partial identity.
•For diagnosis of small pox.
•Elek’s gel test for Toxigenicity of C.diphtheriae bacilli.

22. Double diffusion in two dimension

23. Double diffusion in two dimensions
Elek’s test
Reaction of identity
Lack of relatedness
Partial identity

24. Immunoelectrophoresis
•Immunodiffusion greatly enhanced.
•Electrophoretic separation of a composite antigen (serum) into its into its
constituent proteinsconstituent proteins followed by immunodiffusion against its against its
antiserum - antiserum - Separate Precipitation lines occur.
•Done on Agar or agarose gel on a slide, antigen well and antibody
trough cut.
•Test serum placed on antigen well and electrophoresed for an hour.
•Antibody against human sera is then placed in the trough.
•Diffusion for 18 - 24 hours.
•Precipitin lines stained and preserved for record.
•Enables identification and approximate quantitation of the various
proteins in the sera (30 different proteins can be identified in human
sera).
•Detection of normal & abnormal proteins like myeloma proteins.
•Useful for testing normal and abnormal proteins in serum and urine.

26. Immunoelectrophoresis

27. Immunoelectrophoresis

28. Electro immunodiffusion
Precipitin lines can be speeded up by electrically driving antigen
and antibody.
Two types Two types
1.1.Counterimmunoelectrophoresis (One dimensional double Counterimmunoelectrophoresis (One dimensional double
electroimmunodiffusion)electroimmunodiffusion)
2.2.Rocket electrophoresis (One dimensional single Rocket electrophoresis (One dimensional single
electroimmunodiffusion)electroimmunodiffusion)

29. 1.1.Counterimmunoelectrophoresis (CIE)Counterimmunoelectrophoresis (CIE)
This involves simultaneous electrophoresis of Ag & Ab This involves simultaneous electrophoresis of Ag & Ab
in gel in opposite directions resulting in precipitation at in gel in opposite directions resulting in precipitation at
a point between thema point between them
Used only when Ag and Ab have opposite charges.Used only when Ag and Ab have opposite charges.
Produce precipitation lines within 30 mins.Produce precipitation lines within 30 mins.
Clinical application: detecting Ags like alphafetoprotein Clinical application: detecting Ags like alphafetoprotein
in serum, Ags of Cryptococcus & Meningococcus in in serum, Ags of Cryptococcus & Meningococcus in
the CSF.the CSF.
It is also applied for detecting hepatitis B antigens and It is also applied for detecting hepatitis B antigens and
antibodiesantibodies

30. Ag Ab
- +
Agarose
gel on
slide
Precipitin line

31. One dimensional single electroimmunodiffusion
(Rocket electrophoresis)
For quantitative estimation of
antigens.
The antiserum to the antigens is
incorporated in agarose gel on
the glass slide.
The antigens in increasing
concentrations is placed in wells
punched in gel.
Antigens electrophoresed into
agarose containing antibody.
•The pattern of The pattern of
immunoprecipitation resembles a immunoprecipitation resembles a
ROCKET (precipitation in cone ROCKET (precipitation in cone
like structures)like structures)
•The length of these rocket like The length of these rocket like
structures corresponds to the structures corresponds to the
concentration of the antigenconcentration of the antigen

32. Rocket electrophoresis

33. Laurell’s two dimensional Immunoelectrophoresis
( Variant of Rocket electrophoresis)
Quantitates each of the several
antigens in a mixture.
First run, antigens mixture is
separated by electrophoresis.
Second run, electrophoresis is
done perpendicular to the first
stage so that it drives the
antigens into the antiserum
containing gel to form
precipitation peaks.
Area of the peak is proportional
to the concentration of antigen.