Understanding of key Ab functional concepts Flashcards
The interaction between an antibody (Ab) and an antigen (Ag) depends on…
four types of non-covalent forces
what are the four types of non covakent forces?
1) ionic bonding
2) hydrogen bonding
3) London dispersion
4) Hydrophobic interaction
where are ionic boning found?
between oppositely charged residues (amino acids)
where are hydrogen bonding found?
the electromagnetic interaction between
hydrogen and two electronegative atoms
where are london dispersion interactions found?
between the outer electron clouds
of two or more atoms
where are hydrophobic interactions found?
where water forces hydrophobic groups
together
The Ab:Ag complex is formed through
weak interactions
Because the Ab:Ag complex is formed through weak
interactions, the complex…
readily dissociates
What happens to the Ab:Ag complex in low pH (<3.5)?
alters protonation and charge depending on residue pKa
What happens to the Ab:Ag complex in high pH (>10.5) ?
alters protonation and charge depending on residue
pKa
High salt concentration (e.g. NaCl > 0.3M) causes
ionic displacement
Chaotrophic agents (e.g. cyanates) interfere with
H-bonding in water
molecules
Strength of total non-covalent interactions between a single Ag-binding site on an Ab and a single epitope is …
…the affinity of the Ab for that epitope
Low affinity Abs bind
weakly and tend to dissociate
High affinity Abs bind
Ag more tightly and remain associated longer
The interaction of an Ab with and Ag at one site will…
…increase the probability of a reaction between Ab and Ag at a different site
The strength of such multiple interactions between a multivalent Ab (e.g.
IgM) and multiple epitopes of an Ag is called…
… avidity
what is valence?
The number of epitopes the antibody interacts with- so IgG has a valence of 2, IgM has a valence of 5 (remember rigid structure doesn’t let more than this bind at once). A single Fab fragment has valence of 1.
Interactions between Ag-Ab are usually ….
… specific
Interactions between Ag-Ab are usually specific but what can occur?
cross reactivbity
Interactions between Ag-Ab are usually specific but cross reactivity can occur with…
Ag’s that share an identical epitope or if the Ab specific for
one epitope also binds to an unrelated epitope possessing similar
chemical properties
Affinity refers to…
… the strength of a signle antibody-antigen interactions. Each IgG antigen binding site typically has high affinity for its target
Avidity refers to…
the strength of all interactions combined. IgM typically has low affinity antigen binding sites but there are ten of the,, so avidity is high
An antibody may react with…
… two different epitopes
IgG, IgA and IgM can precipitate antigen at…
… equivalemce zone. ALlows for the formation of a solid particle, enabling phagocytosis
IgG, IgA and IgM can agglutinate…
…. whole cells at ‘equivalence zone’
What is Haemaglutination
agglutination of erythrocytes
Antibodies interact with chemically distinct
regions of…
… Ag known as epitopes
Antibodies that interact with the same Ag
may do so through…
… distinct epitopes (improves avidity)
In polyclonal antiserum, epitope specific
Ab ‘s recognise…
… the same Ag. (the same Ag, different epitopes recognised.)
Monoclonal antibodies interact with…
the same epitope on the same Ag
Inject rabbit with …
…human Ag of choice (50 - 1000 ug of AgX)
Inject rabbit with human Ag of choice (50
1000 ug of AgX) –
primary response
Inject rabbit with human Ag of choice (50
1000 ug of AgX) –primary response.
Repeat after …
… 30 days – secondary response
Harvest…
… serum and purify the AgX specific antibodies
Mouse immunised with …
… AgX -> harvest spleen cells and fused with
immortalised myeloma cells (hybridoma) ->
expand in cell culture.
Screen each …
… cell clone antibody for AgX reactivity
