immunoassays Flashcards
what are immunoassays?
methods which utilise the antigen:antibody immunocomplex in order to detect the presence of a specific analyte within a sample
which immunoglobin is used in most reagents?
IgG
what are monoclonal antibodies?
antibodies produced from a single cell line using mouse hybridoma technology
how are polyclonal antibodies produced?
immunise an animal with the target Ag
the animals B cells with then produce polyclonal antibodies as a result
antiserum is then collected which will contain the polyclonal antibodies
what are the advantages of polyclonal antibodies?
wider range of antibodies produced so can be used to test for multiple epitopes on a single Ag
what are the disadvantages of polyclonal antibodies?
if the animal producing the antiserum dies, may lose the ability to produce the correct antiserum
ethical implications due to the use of animals
what are the advantages of monoclonal antibodies?
highly specific to single epitope
grows easily in the lab
the hybridoma cell line is potentially immortal so can produce the Ab constantly and indefinately.
what are the disadvantages of monoclonal antibodies?
the high specificity means a small change in the epitope can make it unuseful
what is the definition of a label?
a molecule which will react as a part of the assay causing the production of a signal which can then be detected
what are the four categories of immunoassays?
homogenous
heterogenous
competitive
non competitive
what are the principles of radio immunoassays?
Ab or Ag is labelled with a radioactive isotope
can be either competitive or non-competitive
the amount of radioactivity is measured
what are advantages of radio immunoassays?
high sensitivity
simple
what are the disadvantages of radio immunoassays?
need specialised equipment which may not be available or may be expensive
health implications
what does ELISA stand for?
enzyme-linked immunosorbent assay
what kind of label is used in ELISA?
enzyme
what are examples of enzyme labels used in ELISA?
alkaline phosphatase
horseradish peroxidase
beta galactosidase
what are the principles of ELISA?
enzyme label reacts as part of the assay to produce a colour change which can be detected by eye, spectrophotometer or colorimeter
what are the principles of indirect ELISA?
heterogenous
solid phase Ag used to measure levels of Ab in serum
what does EMIT stand for?
enzyme-multiplied immunoassay
what are the principles of EMIT?
enzyme label
homogenous
competitive
enzyme label is chemically conjugated to analyte
added to the sample along with specific Ab
this conjugate is inhibited when bound to the Ab
colour change is proportional to analyte concentration
more inhibition = greater colour change
what does CEDIA stand for?
cloned enzyme donor immunoassay
what are the principles of CEDIA?
enzyme label
homogenous
competitive
enzyme modified to produce 2 subunits which must combine to be active
the analyte Ag is bound to one subunit
therefore in the absence of the analyte in the sample, the Ag-enzyme conjugate binds to Ab and the enzyme is inactivated
in the presence of the analyte in the sample the Ag in the sample binds to the Ab and the two sub units are able to combine and become active
colour change is proportional to analyte concentration in sample
what does KIMS stand for?
kinetic microparticle immunoassay
what are the principles of KIMS?
competitive
specific Ab is covalently coupled to microparticles
target Ag is linked to a macromolecule
in the absence of the analyte in the sample, Ag on the surface of microparticles binds to the Ab molecules and form particle aggregates
these block light transmission
in the presence of the analyte in the sample the Ag binds to the Ab and prevents agglutination
increase in absorption is indirectly proportional to [Ag]
what enzyme is commonly used in CEDIA?
beta galactosidase
what does FPIA stand for?
fluorescence polarisation immunoassay
what are the principles of FPIA?
uses polarised light to excite rotating molecules
when excited, the rotating molecules will emit fluoresence in the same plane when returning to ground state
Ab and florescent tagged Ag mixed with patient sample
in the presence of Ag in the sample, the Ab binds to it leaving the tagged Ag free to rotate freely
in the absence of the Ag in the patient sample, the Ab binds to the tagged Ag causing it to rotate more slowly giving off increased polarised light
intensity of polarised light is inversely proportional to [Ag]
what are the principles of chemiluminescence labelled immunoassays?
emission of light occurs when a substrate decays from an excited state to a ground state
energy is generated from a chemical reaction
what are the principles of electrochemiluminescence?
one Ab is labelled with biotin and one Ab is labelled with ruthenium
these two Ab form a sandwich complex with the Ag in the sample
microbeads coated with steptavidin added which bind to the biotin
the complex is then transferred to a measuring cell
beads bind to the magnetised surface of the cell
unbound molecules washed away
the wash contains TPA and a voltage triggers the ECL reaction
the reaction releases light which can be detected by a photomultiplier
how do lateral flow immunoassays work?
non-competitive and sandwich
sample added to the sample pad which acts like a sponge
once soaked the sample migrates to the release pad which contains Ab’s conjugated to coloured particles
capture Ab’s on the test line specific to the Ag bind to the Ab/Ag conjugate
the control line contains Ab’s specific to the conjugated Ab
are lateral flow tests qualitative or quantitative?
qualitative
what determines the sensitivity of an immunoassay?
the affinity of the Ab for the Ag
what are advantages of immunoassays?
some are highly sensitive ( physio-chemical methods, RIA and chemiluminescence)
simplicity ( small volumes, no extraction needed, easily automated)
universal application ( can produce Ab’s to a wide variety of substances )
Ag/Ab highly specific
what are disadvantages of immunoassays?
cross reactivity
high dose hook effect
presence of human anti mouse Ab due to cancer treatment or handling mice could give a false positive or negative
what is the high dose hook effect?
very high [Ag] saturates all the Ab sites and prevents sandwich formation
this causes the concentration to be falsely lowered
what is cross reactivity?
when a substance which is structurally similar to the analyte being tested interferes with the assay providing a false positive result
