revision lec Flashcards
lineage of DCs
myeloid (they are innate cells)
role of DCs
relay information (antigen-presentation) to the adaptive immune system (CD4+ T cells) with complementary ligand
complement proteins are the
soluble components of serum (30 of them)
benefits of lectin pathway
direct recognition –> no antibodies involved
important in recognition of fungal residues
benefits of pentraxins
can activate classical complement pathway independent of antibodies
how does antibody binding initiate classical pathway
activates C1q/r/s complex
importance of b fragment of subunits
forms the next protease
amplification loop of alternative pathway
C3b required to bind directly to microbial surface to activate alternative pathway
more C3b made when C3 is broken down by C3 convertase into C3a + C3b
final component of complement
C3
C3a importance
inflammatory mediator
attracts phagocytes
C3b importance
binds to complement receptors on phagocytes (alternative pathway)
causes opsonisation of phagocytes
removal of immune complexes
formation of MAC complex
after late events of complement
C3b combines with C3 convertase to make C5 convertase
cascade of proteolytic events –> MAC
importance of phage display
alternative method to make really highly specific monoclonal antibodies
allows you to make a combinatorial library
method of phage display to make combinatorial library
take B cell source e.g. spleen from animal
extract mRNA from the B cells
convert mRNA to cDNA
PCR all the heavy and light chains
all the heavy and light chains from different B cells are mixed up and form many different combinations
controlling localisation of antibody expression on phage
express them at specific proteins e.g. pVIII or pIII
why use biopanning?
identify specific antibodies expressed on phages
method of biopanning
introduce phage population to microtitre plate containing wells of antigen
wash away any unbound antibody
UV source kills any other unbound antibodies
(bound antibodies identified by marker)
elute just antibodies of interest (bound)
introduce specific antibody to E.Coli and allow phage s to infect and multiply
what is immunofluorescence good for
good at looking at/identifying localisation of surface antigens (extracellular)
good at picking up antigens from live cells (not dormant cells) –> important diagnostic marker
identifying antibodies at a higher resolution
use immunogold electron microscopy
role of SDS
applies negative charge to molecules
gives molcules the same charge to mass ratio
SDS-PAGE in 1D
separation on basis of molecular weight
highest molecular weight at top of gel
why do we denature the polypeptides in western blotting
break protein up into small components to see better localisation of where the antigens are and where the antibody will bind
SDS-PAGE in 2D
separate peptides on basis of isoelectric point
then apply SDS and separate on basis of molecular charge
reason for 2D
allows further separation of peptides e.g. even dimers can be separated
how do you get the proteins out the gel in western blotting
sandwich the gel
expose to an electric current which fixes the proteins permanently to a membrane
can then probe proteins using enzyme-linked antibodies
what is DAS-ELISA good for
double antibody sandwich
good for measuring components within human serum
method of DAS-ELISA
capture antibody bound to microtitre plate by its Fc domain
bathe plate in solution containing antigen of interest
antigen binds to Fab domain of capture antibody
add reporter antibody
binds to epitope of antigen
features of reporter antibody in DAS-ELISA
must bind to different epitope on the antigen than the capture antibody
otherwise capture antibody will bind competitively to antigen and there will be no binding sites left for the reporter antibody
why are glycoconjugate vaccines developed
there are many limitations of polysaccharide only vaccines
limitations of polysaccharide vaccines
do not induce memory
induce T-cell independent immune response
immune response is immature (only IgM)
vaccines do not work well in very young or elderly (populations that most require the vaccine)
example of protein conjugate used in chemical coupling
CRM197 diptheria genetic toxoid protein
benefits of glyconjugate vaccines
immune response against protein/polysaccharides fully mature
immune memory induced
long-lasting
example glycoconjugate vaccine and against what disease
prenvar 7
against streptococcus pneumoniae
