Antigen Antibody Reaction Flashcards
What law governs antigen/antibody reactions
The Law of Mass Action
Formula for Antigen/Antibody rxn
[Ag] + [Ab] -> [AgAb]
* [AgAb] being the antigen-antibody complex
What is the antigen antibody rxn used for in transfusion science
Used in invitro transfusion science testing to determine the presence or absence of antigens or antibodies
Traditionally the visualisation of red cell antigen/ antibody rxns in the lab is achieved by what? (2)
- Haemagglutination
- Haemolysis of red cells
What happens in the first stage of Haemagglutination
Antibodies attach to their corresponding antigen
What is the first stage of haemagglutination known as
Sensitisation or binding or association or coating
What happens during the second stage of haemagglutination
Intracellular bridges are formed as antibodies that are already bound to a red cell antigen bind to another free antigen site on an adjacent red cell
What is the second stage of haemagglutination called
Agglutination
Haemagglutination
Is the first stage reversible or irreversible
Reversible
Haemagglutination
Is the second stage reversible or irreversible
Irreversible
Antibody combining sites are in which region of the antibody
Fab region
Describe the antibody combining sites
Complimentary in shape and size to the antigens that they react specifically with
In the first stage of the antigen antibody rxn, the Fab region and antigens react specifically to form what?
Reversible bonds
What forces and bonds are involved in antibody binding
- Ionic bonds
- Hydrogen bonds
- Hydrophobic bonds
- VDWs forces
- Randomisation of the water
What does the second stage of the antigen antibody reaction involve
The formation of intracellular brides
What 2 types of forces are involved in the second stage of the Ag Ab rxn
- Aggregating forces
- Repulsive forces
How are Aggregating forces involved in the second stage of the Ag Ab reaction
Surface tension charges pulling red cells together
How are Repulsive forces involved in the second stage of the Ag Ab reaction
- The surface of the red cell has a net electronegative charge
- Due to sialic acid and neuraminic acid on surface of red cell
- Each red cell is equally charged and so there is a repulsive force which prevents red cells from adhering to eachother
What is the Zeta potential
The net strength of the forces that exist between red cells when suspended in saline
Zeta potential
The antibody must span what distance to bring about direct agglutination
The antibody must span 20nm distance caused by the zeta potential
Zeta potential
What distance do IgM antibodies have between binding sites and what does this mean
IgM antibodies have a max distance of 30nm between binding sites. This means that they can readily span the gap and directly agglutinate red cells
Zeta potential
What distance do IgG antibodies have between binding sites and what does this mean
IgG antibodies have a max distance of 14 nm between binding sites so usually cannot bring about direct agglutination of red cells
The 4 factors affecting Ag Ab rxns
- Temperature
- pH
- Ionic strength
- Ag and Ab concentrations
Zeta potential
Ways to alter effects of zeta potential (2)
- Reduce the zeta potential and bring red cells into closer proximity to allow agglutination
- Assist IgG antibodies to bridge the gap
Explain how temp affects Ag Ab rxns
- Antibodies react differently at varying temps
- ABO antibodies react best at room temp
- Rh antibodies react best at 37 degrees C
Opt pH range for Ag Ab rxns
pH 6.5 - 6.8
Explain how ionic strength affects Ag Ab rxns
In low ionic strength solutions, ions are able to be more mobile and this favours ionic bonding between Ag and Ab. This speeds up reaction rates and allows shorter incubation periods for some transfusion serology testing
Conditions of excess antigen in a rxn mixture (3) (affects of antigen conc on Ag Ab rxns)
- The use of too strong a red cell suspension
- The addition of too small a volume of serum
- The addition of too large a volume of red cell suspension
How will excess antigen in the rxn mixture affect the rxn
Antibodies are distributed so thinly that the formation of intracellular bridges will be sparse and involve too few cells
Conditions of excess antibody in a rxn mixture (3) (affects of antigen conc on Ag Ab rxns)
- Use of too weak a cell suspension
- Addition of too large a volume of serum
- Addition of too small a volume of cell suspension
How will excess antibody in the rxn mixture affect the rxn
- The majority of antigen sites will be coated by antibodies.
- This leaves few free antigen sites available for attachment to the free binding sites on antibodies that are already attached to adjacent red cells.
- Therefore there is a reduction in intracellular bridge formation and corresponding agglutination
Describe the method of detection, direct agglutination
- red cells suspended in saline are kept apart by a distance of 20nm
- direct agglutination of red cells suspended in saline is usually only observed with IgM antibodies (large enough to span gap between adjacent red cells)
- for this agglutinantion the Ag Ab conc must be optimised -> 3-5% suspension of red cells in saline
Indirect agg
What test detects indirect agglutination
The anti human glubulin (AHG) test
What causes indirect agglutination
IgG antibodies that are too small to cause direct agglutination invitro. The IgG antibody is not large enough to form an intracellular bridge between red cells.
What have IgG antibodies been produced as a result of (2)
- Pregnancy
- Transfusion
How does the AHG test work
- The AHG test is used to detect indirect agglutintion where the IgG antibody is too small to cause direct agglutination invitro
- Another molecule, called anti human globulin (AHG) or Coombs reagent, is added to help bridge the gap
- The AHG test was described by Coombs et al and was originally called to Coombs test
What is the IgG antibosy referred to as
An incomplete antibody
