antigen antibody reactions Flashcards
What governs antigen-antibody reaction?
The law of mass action
What is the equation showing an antibody-antigen reaction?
[Ag] + [Ab] -> (k1 + k2) [AgAb]
What is an application of the antigen-antibody reaction?
It is used in vitro to test for antibodies & antigens
How is the antigen-antibody reaction visualised in transfusion science?
Haemagglutination & haemolysis of RBCs
How many stages are there in haemagglutination?
2
Explain the 1st stage of haemagglutination.
~Known as sensitisation, binding, association, or coating
~Antibodies attach to corresponding antigens
~Antibody combining sites (FAB region) - complimentary in shape & size to antigens - react specifically and form reversible bonds
~bonds in antibody binding include - ionic bonds, hydrogen bonds, hydrophobic bonds, van der Waals,
Explain the second stage of haemagglutination.
~Agglutination
~Intracellular bridges form - antibodies already bound to antigens on one RBC bind to the antigens of others RBCs
~Aggregating forces - surface tension change pulls RBCs together
~Repulsive forces - RBC surface - net - charge due to sialic acid & neuraminic acid - prevents RBCs from adhering to each other
What are Igs composed of?
~2 heavy chains, 2 light chains
~Variable region & constant region
~Heavy chains bound by disulphide bonds
~Y shaped
~2 Antigen combining bond sites on variable region
What is the zeta potential?
Net strength of forces that exist between cells suspended in saline
For direct agglutination to occur what distance must an antibody span & what causes it?
20nm cause by zeta potential
What is the maximum gap between binding sites for IgMs?
30nm - allows direct agglutination
What is the maximum gap between binding sites for IgGs?
14nm - can’t cause direct agglutination
What factors affect antigen-antibody reactions?
Temperature, pH, ionic strength, antigen & antibody conc.
How does temperature affect that antibody-antigen reaction?
React differently at different temperatures - ABO @ RT , Rh @ 37°
How does pH affect that antibody-antigen reaction?
Optimum 6.5-6.8
Little effect between 5.5-8.5
How does ionic strength affect that antibody-antigen reaction?
Low ionic strength solutions - more mobile & favour ionic binding between Ig & antigen - speeds up haemagglutination - e.g. 0.8-0.95% saline
How does excessive antigens affect that antibody-antigen reaction?
Antibodies are dispersed thinly - formation of intracellular bridges sparse and need too much energy - e.g. RBC cell suspension too strong
How does excess antibody affect that antibody-antigen reaction?
Antibodies bind to antigens freely - lack of intracellular bridges as antigens are bound with individual antibodies - (e.g. weak cell suspension)
What is direct agglutination in the transfusion lab?
Direct agglutination of red cells suspended in saline is only observed with IgM antibodies which are large enough to span the gap between adjacent red cells
RBCs in saline kept apart @ 20nm - IgM antibodies - large enough to span the gaps between RBCs - ABO grouping - conc. of antigen & antibody optimised - 1/2 plasma/serum : 1 RBC suspension in saline
What is indirect agglutination?
When the IgG antibody is too small to cause direct agglutination in vitro
IgG antibody isn’t large enough to form intracellular bridges between red cells.
Therefore another molecule is added to help bridge this gap called anti-human globulin or Coombs reagent.
Anti Human Globulin test - IgG antibodies - incomplete antibody as does not form intercellular cellular bridges - AHG/Coomb’s reagent added to help bridge gap - Coombs test
What must be optimised for direct agglutination to occur?
Concentration of antigen and antibody
Mix 1-2 volumes of plasma/serum and a volume of 3-5% suspension of red cells in saline
