Immunoassays Flashcards
When does rayleigh scatter occur
if the oarticles diameter is less than 1/10 the side of the incident wavelength
How does light scatter in rayleigh scatter
light scatters symmetrically forwards and backwards with minimal scatter at 90 degrees
When does Mie scatter occur
if the particle diameter is greater than ten times the size of the incident light
How does light scatter in Mie scatter
most of the light scatters forward
When does Rayleigh-debye scatter occur
if the particle and the wavelength of incident light are approximately the same
How does light scatter in Rayleigh-debye scatter
most of the light scatters forward but there is also detectable side and backscatter
How does turbidimetry work
a turbid solution decreases the intensity of the incident light
Where is the detector positioned in turbidimetry
180 degrees from the incident light
What type of wavelengths does turbidimetry use
short
What are common interferences in turbidimetry
large particles such as dust and lipoproteins
How do we minimize interferences in turbidimetry
bichromatic incident light, individual sample blanks and kinetic measurement
How does nephelometry work
measuring scattered light at an angle other than 180 degrees
Where is the detector placed in nephelometry
30 to 90 degrees relative to the incident light
What is a lattice
a three-dimensional structure formed by the reaction of antigens and antibodies
What occurs at antigen and antibody equivelence
large complex lattices are formed
What occurs if there is excess antigen
the size of the lattices decreases which decreases light scatter
How can we improve sensitivity of lattice formation assays
by coupling latex particles
When are light scatter assays measured
when the lattice is large enough to scatter light but not large enough to precipitate
What is the particle enhanced turbidimetric immunoassay
reagent antibodies are coupled to particles which are composed of latex or polystyrene which facilitates the formation of larger lattices
What type of assay is the particle enhanced turbidimetric immunoassay
homogenous, non-competitive
How does turbidity relate to analyte concentration in the particle enhanced turbidimetric immunoassay
it is proportional
What is the particle enhanced turbidimetric inhibition immunoassay
an antibody to the analyte of interest and latex particles coated in the antibody of interest are used. Free analyte competes with the latex bound particles for the antibody binding sites. The patient analyte binds with antibodies and blocks lattice formation
What type of assay is particle enhanced turbidimetric inhibition immunoassay
homogeneous, competitive
How is turbidity related to analyte concentration in particle enhanced turbidimetric inhibition immunoassay
inversely proportional
What is a labelled immunoassay
a ligand or antibody is labelled by attaching a molecule that either produces a signal directly or indirectly
What are the common types of labels
chemiluminescent molecules, enzyme and fluorophores
What are examples of enzymes used as labels
horseradish peroxidase, alkaline phosphatase, G6PD, B-D-galactosidase
What detection methods are used with enzymes
photometer, fluorometer, luminometer
What are chemiluminescence methods used as labels
luminol, acridinium esters, ruthenium complexes, dioxetane
What detection methods are used with chemiluminescence
luminometers
What fluorescence methods are used as labels
rhodamine B, fluorescein, europium
What detection methods are used with fluorescence
fluorometer
What occurs in a competitive assay
labelled and unlabelled ligands compete for a limited number of binding sites, the proportion of labelled ligands binding the antibody is inversely proportional to the amount of unlabelled ligands in the sample
What are the two methods of competitive assays
simultaneous - the labelled and unlabelled ligands are added at the same time
sequential - the unlabelled ligands are added first and incubated before the addition of the labelled ligand
What is the non-competitive assay
antibodies are immobilized on a solid substrate and sample ligands bind to the antibodies, labelled antibodies for the sample ligand are added and then a wash step occurs to wash away excess
What are common interferences in immunoassays
hyperlipidemia (increased sample turbidity), rheumatoid factors (binds reagents can cause increase or decrease in signal), biotin (increase or decrease), human anti-mouse antibodies (increase or decrease), heterophile antibodies, prozone (false negative)
What is the prozone (hook effect)
excess concentrations of antigens from sample interferes in sandwich assays by saturating capture and label antibodies preventing sandwich formation
What are homogenous assays
physical separation of the bound and unbound labelled ligands is not required, signal is altered when the labelled ligands are bound
What are heterogenous assays
bound labelled ligands and unbound ligands cannot be distinguished from one another and require physical separation separation may be performed by chromatography or precipitation
What is solid phase separation
polystrene wells, membrane and magnetic beads, a reaction vessel holds the antibodies in place while unbound materials are washed away
What is adsorption separation
small particles are used to trap small antigens
What are lateral flow immunoassays
a combination of sandwich assay and planar affinity chromatography
How do lateral flow immunoassays work
analyte is added and flows to where antibodies are conjugated to a tag, it then continues to flow to where the test line has antibodies that also bind the analyte, any of the conjugated antibodies that do not have analyte attached flow through to the control line where they bind to IgG antibodies
What is the non-competitive electro-chemiluminescent immunoassay
monoclonal antibodies are labelled with ruthenium, an applied voltage oxidizes the ruthemium which causes light to be emitted a magnetic streptavidin micropartical attaches which allows the bound particles to remain stuck in the reaction chamber during the wash step
What is the relationship between the signal and the sample concentration in non-competitive electro-chemiluminescent immunoassay
directly proportional
What is the competitive electro-chemiluminescent immunoassay
sample ligand and ligand labelled with the ruthenium complex are incubated with ligand specific biotinylated antibodies and compete for binding sites on the antibody, then the streptavidin labelled magnetic particles are added and bind the biotinylated antibody washing and analysis occurs as normal
How is the signal related to the amount of ligand in the sample in competitive electro-chemiluminescent immunoassay
indirectly proportional
What is the two step competitive electro-chemiluminescent immunoassay
the same as the competitive but the patient sample is incubated first before the labelled ligand is added
What is the chemiluminescent immunoassay sandwich assay
paramagnetic particles are directly or indirectly coated with the capture antibody, the analyte and added conjugate form immune complexes that bind to the particles then a magnet binds the particles and a wash step occurs then dioxetane-P is added and a reaction happens that results in chemiluminescence
How is the signal related to the sample concentration in the chemiluminescent immunoassay sandwich assay
directly proportional
What is the chemiluminescent immunoassay competitive assay
the paramagnetic particles are either directly or indirectly coated with the capture antibody it is then mixed with an antigen-specific antibody and an enzyme labelled analyte they compete for binding sites, the rest of the reaction continues as normal
What is the relationship between signal and analyte concentration in chemiluminescent immunoassay competitive assay
inversely proportional
What is the enzyme multiplier immunoassay technique
a homogeneous, competitive assay
What is the relationship between signal and ligand concentration in the enzyme multiplier immunoassay technique
proportional
What is the enzyme multiplier immunoassay technique used for
low molecular weight analytes found in high concentrations
How does the enzyme multiplier immunoassay technique work
enzyme labelled ligand and patient ligand compete for binding sites on the antibody, binding of the labelled ligand causes steric hinderance of the enzymes activity, only unbound enzyme reacting ligands can react with the substrate to create a signal
What are interferences with enzyme multiplier immunoassay
anything that interferes with enzyme activity or absorbs light at the same wavelength
What is florescent polarization immunoassay
a homogeneous, competitive assay
What is the relationship between the signal and ligand concentration in the florescent polarization immunoassay
inversely proportional
How does the florescent polarization immunoassay work
a fluorophore labelled ligand and the sample ligand compete for antibody binding sites, when the fluorophore labelled ligand binds an antibody it spins slower forming a stronger signal
What is the florescent polarization immunoassay suitable for
small ligands like hormones and drugs
What are interferences in florescent polarization immunoassay
hemolysis, bilirubin and lipemia, sample viscosity, light scatter from particles
