immunoassays Flashcards
give some examples of immunoassays?
pregnancy tests
lateral flows
why is the use of antibodies in research, diagnostics and treatment widespread?
because of their ability to specifically bind to antigens and their ability to fix complement
how are antibodies produced?
theyre produced through matured b cells and are then clonally expanded and with the help of t cells, the b cells become plasma and start to secrete antibodies
how specific do you want antbodies to be for the purpose of recognising analyte if it has high avidity?
you want them to be really specific
what does analyte mean?
a substance whose chemical constituents are being identified and measured.
what tells the b cells what isotype of antibodies to make?
the signals around them
what does it mean if an antibody is specific and has high avidity?
this means that theyve got a high ability to bind
what does multiple immunisations increase?
it increases the amount and affinity of the antibody in the serum of immunised animals
what is the first isotype of antibody produced?
IgM
how do IgG antibodies get produced after immunisations?
the first type of antibodies produced are IgM then they undergo class switching to IgG which is highly specific and is produced after immunisations
if your making antibodies for use in humans, are they produced in humans first?
no, as they’ll target the own body, you make them in another animal
what is the process for producing antibodies for use?
o Once the amimals have been immunologically primed (mounted an antibody repsinse), they have high levels of specific antibody in the serum
o This means they can therefore collect the blood, separate serum and use this antiserum for future tests, treatments ect
o You can repeatedly bleed antimals, allowing a plentiful source of antiserum
You can collect several liters of antiserum from an immunised cow each month
o this is releatively cheap and easy and allows for the generation of polyconal antibosies
what is the problem with using serum as a way of making antibodies ?
it contains many other proteins which may cause problems to sensitive assays
why is it better to purify antibodies?
for improved storage and higher concentraion
it also allows you to only have helpful antibodies
what are the steps of the purification of antibodies from antiserum?
- the b cell binds the virus through the viral coat of the protein
- then the virus particle is internalised and degraded
- the peptides from internal proteins of the virus are presented to the t cell, which activates the B cell
- activated B cell produces antibody against viral coat protein
what does multiple immunisations do for the amount and affinity of antibodies in the serum of immunised animals?
it increases both the amount and affinity of antibodies in the serum of immunised animals
what does multiple immunisations also result in? [in terms of the type of antibody]
it results in class switching with the antibodies more likely to be IgG or IgA
what do you need to consider when generating antibodies for use?
antigen- so you make an antibody actually specific to the antigen
adjuvant - you want one thats effective at enhancing the body’s immune response to the antigen
kenetics- dunno why, just know theyre important
what does it mean when animals are immunologically ‘primed’?
they mount an antibody response
how do you get antiserum?
you immunise an animal until it gets an antibody response then collect the blood, separate the serum (antiserum)
what can antiserum be used for?
future tests
treatments
what is a relatively cheap way of producing polycolonal antibodies? and why?
antiserum
as you can repeatedly bleed animals allowing for plentiful source of antiserum and you can collect several litres of antiserum of an immunised cow every month
what are the cons of generating antibodies through serum?
the serum contains many other proteins which may cause problems in sensitive assays e.g. cross reactivity
therefore its better to purify improved storage and higher concentration
how do you purify antibodies for use?
the animal is immunised, then the serum is extracted which includes antibodies then the antibodies are bound to a protein and the serum proteins are washed off and elute the bound antibodies