immunological techniques Flashcards
what is an immunoassay
a biological test that uses 1 or more immunologic products or reagents
what does sensitivity and specificity mean
sensitivity - can detect very small concentrations
specificity - measuring only 1 substance
how does an antibody bind to an antigen
an antibody binds to one molecule on the antigen - the epitope.
once the antigen is eliminated the immune response stops
an antigen with 4 epitopes will need 4 antibodies - each specific to the epitopes
what is a hapten
a small molecule requiring coupling to another molecule to produce an immune response. it needs a carrier molecule to bind to an antibody
what parts of an antibody reacts with what?
Fc interacts with other elements
Fab interacts with ag
what are the classes of antibodies
(Ig) M A D G E
Ig is immunoglobin
what are the types of antibodies that B cells produce
Polyclonal antibody - broad spectrum of antibodies - targets the different epitopes but could have an Ab you dont want
Monoclonal antibody - only recognise the same epitopes - less affinity - used for immunotherapy and in vitro diagnosis
what is an immune response
the body recognising foreign substances with phagoctyes, T cells kill the invaders, B cell produce antibodies
this is regulated by cytokine production
describe immune complexes
antibodies bind to antigen = immune complex
primary - complex forms
secondary - interaction between complex - agglutination
tertiary - complex interacts with the immune cell
measured quantitatively
describe Ag-Ab reactions
Ab + Ag = AgAb
weak = strong
hydrophobic and electrostatic interactions
stable if complementary shape of Ag and binding site of Ab
reversible
what is affinity and avidity
affinity - strength of binding - defined by eqbm constant
avidity - sum of binding strength
K= [AgAb]/[Ag][Ab]
what are the noncellular immunoprecipitation assays
single radial immunodiffusion
rocket immunoelectropheresis
double diffusion
haemagluttination
describe single radial immunodiffusion
SRID
antibody within gel
antigen added to wells in gel
incubate to diffuse
diameter can be graphed against antigen conc
describe rocket immunoelectropheresis
antibody throughout gel
antigen in wells
move antigen by electropheresis
rocket height can be graphed against antigen conc
describe double diffusion
antibody in a well
antigen in other wells
both diffuse - precipitates form line/arc
if antigens are identical - arc forms
if antigens are partly identical - spur forms
if antigens not identical - cross forms
describe agglutination
best results with IgM as it is pentomeric - 5 antibodies
RBC coated in antigen
button - negative reaction
carpet - positive reaction
used for blood grouping, antibody detection
how does cell lysis happen
antigen on cell + antibody + c’ = cell lysis
c’ is an enzyme cascade with 20 serum proteins - ruptures cell membrane
how to find if antibody is in serum via complement fixation assay
add inactive c’ to antigen and c’
complexes will form if antibody in serum is specific for antigen
check for free c’
if c’ fixes - no lysis - positive reaction
if c’ doesnt fix - no antibody - lysis - negative reaction
how does a viral neutralisation test work
serum + virus + cell culture
if there is no change - antibody present - no cytopathic effect
if there is a cytopathic effect - no antibody present
(virus causes cytopathic effect, antibody coats virus so virus cannot enter cell)
what is a ligand and its immunoassays
something that binds to something else eg antibody to HIV envelope
radioimmunoassay (RIA)
ELISA
describe radioimmunoassay
antigens and sample compete for antibody binding site
B/F
bound ag/free ag
tells you if unlabelled antigens present
describe ELISA
enzyme linked immunosorbent assay
antigen attaches to antibody
(possibly secondary antibody with enzyme added)
substrate added - colour change
direct - first antibody has enzyme
indirect - needs secondary antibody
sandwich - antibody binds to plate, then antigen, then secondary antibody
indirect sandwich - antibody, antigen, antibody, secondary antibody
used for antigen detection in infection or presence of antibody eg HIV
what are the uses of cellular assays
identify cells and their functions
organ transplants
disease diagnosis
what is a ficoll hypaque density gradient
ficoll is a polymer of sucrose
hypaque is a dense organic compound
obtains cells for assays via centrifugation eg lymphocytes from blood
what is flow cytometry
uses monoclonal for cell surface antigens
the Fc portion of antibody is removed
label the antibody with fluorescent dye
cells stream, lasers through, each cell is detected
this determines cell size, granularity and surface molecules
what are the cell function assays
lymphocyte transformation mixed lymphocyte culture cytoxicity assays cell movement phagocytosis
describe lymphocyte transformation
measures lymphocytes ability to respond to stimulants eg mitogens
lymphocytes synthesises dna
add radiolabel 3H thymidine - takes up more if stimmed to divide
ficoll hypaque lymphocytes - culture with mitogen - add thymidine - isolate - count
describe mixed lymphocyte culture
measures ability of histoincompatible lymphocyte to stim lymphocyte from someone else
- 1 way - 1 group irradiated - no response
- 2 way - both cell groups respond
used to select kidney donor
describe cytoxicity assays
detects antibody-antigen interaction on cell surface
end point is cell death
lymphocyte + c’ + eosin dye = cell death
the c’ contains antibodies, binds to antigen on lymphocyte surface , ruptures cell membrane, gets dyed red
describe cell movement
capillary tube movement (neutrophils) - migrate out
chemoattractant diffuses towards tube and stims the cells, making the cells diffuse further
micropore filter technique
cells diffuse through filter into filter meshwork, endpoint where 2 cells travel the same distance
describe phagocytosis
determines visible particles that can be counted in the cell
identify ingestible substance that can be extracted and measured
done via spectrophotometer
what is immunohistochemical staining
observe variety of antigens undetected by cytochemical staining eg hormones
