Antigen Antibody interaction

The Ag-Ab interaction is due to lots of non-covalent interactions- lock and key!

Affinity- strength of interaction between one

            Ag-binding site and one epitope

•      Low affinity antibodies bind antigen weakly and dissociate easily.

•      High affinity antibody binds antigen more tightly and remain bound longer.

Association constants can be calculated:

At equilibrium,

 the rate of formation = the rate of dissociation, and so k1[Ag][Ab]= k-1[Ab.Ag];

k₁/k_-1= [Ab.Ag]= Ka =  Association or  affinity constant

            [Ag][Ab]

k₁

Ag + Ab  <-> A-^.Ag; 

           k_-1

Avidity

  Binding is often with multiple epitopes to multiple antibodies- the total strength is avidity. Thus, the total binding may be stronger than the individual bindings- there may be co-operativity, etc.  IgM > avidity than IgG with > affinity, b/c of pentameric binding.

Precipitation & Agglutination

Precipitation

When the antigen is Soluble, the reaction of an antibody with the antigen can be   detected by Precipitation (line of ppt.) of the antigen.

Agglutination

When the antigen is particulate, the reaction of an antibody with the antigen can be detected by agglutination (clumping) of the antigen.

Precipitation Serological Tests

• •      One of the easiest of serological tests
• •      Soluble AG & Ab interact and form a lattice that develops into a visible precipitate.
• •      Occur best when antigen and antibody are present in optimal proportions (Equivelance).
• •      Antibodies that aggregate soluble antigens are called precipitins.

 Ag / Ab Precipitation Reactions

Polyclonal antibodies can form lattices, or large aggregates.

However, monoclonal antibody can link only two molecules of antigen and no precipitate is formed.

Ag / Ab Precipitation Reactions

All antigen /antibody bindings are reversible according to Law of Mass Action ‘The rate of any chemical reaction is proportional to the product of the masses of the reacting substances’.

• Free reactants are in equilibrium with bound reactants ---> Equivalent zone
• Excess antibody ----> Prozone
• Excess antigen ---->  Postzone

These zones are demonstrated in the precipitation curve

Precipitation curve

Plots of the amount Ag/Ab complexes precipitated when increasing  Ag concentrations are added to constant  concentration of Ab. It reveals  3 zones:

1. Zone of antibody excess - precipitation is inhibited and antibody not bound to antigen can be detected in the supernatant;

2. Zone equivalence - maximal  precipitation  in  which  antibody  and antigen  form  large  insoluble  complexes  and  neither  antibody  nor antigen can be detected in the supernatant;

3. Zone of antigen excess  -  precipitation  is  inhibited & Ag. not  bound  to Ab. can be detected in the supernatant 

Precipitation Curve

Precipitation Curve