SEROLOGY

Precipitation reactions
Serology
• Definition: Study of Ag/Ab rxns in vitro
• Ag/Ab fit in a key lock fashion (reversible rxn)

• Forces include the following non covalent bonds:

hydrophobic, van der waals, H-bonds

• Influencing factors: pH, temperature, ionic

strength, reactants concentration
Serology (cont’d)
Types of Ag/Ab rxns:

• Precipitation: Ag soluble & visible product

• Agglutination: Ag insoluble & visible product

• Reactions with labels: Ag insoluble or soluble &

invisible product
 Precipitation

Definition: soluble Ag reacts with Ab in

optimal proportions  precipitin rxn
 Radial Immunodiffusion (RID)

• Also called single diffusion or Mancini technique, a

quantitave assay.

• Antibody is incorporated into the agar gel : Add Ab in the

dissolved agar let solidify pour in petri-dish

• Antigen is placed in holes punched into the agar

• As the antigen diffuses into the gel, it reacts with the

antibody and when the equivalence point is reached a
white ring of precipitation is formed
Radial Immunodiffusion (RID)
• D2 of ring is proportional to the Ag concentration

• This test is commonly used in the clinical laboratory

for the determination of concentrations of
immunoglobulins and some complement proteins
levels in patient samples.

• Sensitive to IgG, IgA, IgM, C3 & C4 (play the role

of Ags here)
Double diffusion (Ouchterlony)
Qualitative method

• It can be directed against one Ag or a mixture of Ag

• It can be used in identifying relationships between
different antigens.
• Technique:
• Dissolve agar add diluent (PBS) heat pour in
petri-dish or glass slide  cool to RT cut a central
well (Ab) & an adjacent well (Ag)

• Diffusion  white line of precipitation

Double diffusion (Ouchterlony)
In case of a mixture of Ags:
• Aim: detect & compare Ags + check Ag purity

• Central well (Ab) against both or all the Ags

• Interpretation of results:
• If precipitation line is continuous line of identity
 Ag1=Ag2
• If a spur (line of partial-identity) is obtained in
addition  Ags are related but not identical
• Line of non-identity Ags are not related
 Ag 1

The "spur," extending beyond the point of partial identity, is

thought to result from the determinants present in one
.antigen but lacking in the other antigen
Immunoelectrophoresis
Gel is poured on a glass slide

A horizontal well (Ab) and a circular (Ag i.e. serum)

one are cut

Electrophoresis  serum (alkaline pH) moves from –ve

to +ve electrode

Ag/Ab complexes will undergo electroendoosmosis

Method sensitive to IgG, IgA, IgM

 Ab

)
)
)
Ag

- +
 Countercurrent
immunoelectrophoresis
Two wells are cut: -Ab moves to –ve electrode
-Ag moves to +ve electrode
This test only works if conditions can be found where the
antigen and antibody have opposite charges.

Line of precipitation  at optimal proportions

Rapid technique  1 hr instead of 24 hrs

Combination of electrophoresis & double diffusion
Explanation below (in the comment box)
Ph 8.2

+ Ab Ag -
Rocket electrophoresis
Combination of electrophoresis & single diffusion

Ab is fixed in agar

Area under rocket or rocket height is proportional to

Ag concentration