360 - Immunoassays Flashcards
these factors influence light scatter
solution particle size and number
three types of light scatter
Rayleigh, Mie, and Rayleigh-DeBye
describe Rayleigh scatter
light scatters symmetrically forwards and backwards from the particle with minimal scatter at 90 to the incident light
particle diameter is less than 1/10 the size of the incident wavelength of light
describe Mie scatter
- occurs if the particle diameter is greater than ten times the size of the incident light wavelength
- most of the light is scattered forwards from the particle
describe Rayleigh-Debye scatter
- occurs if the particle and the wavelength of the incident light are approximately the same sizes
- most of the light is scattered forwards, but there is also detectable side and backscatter
these two methodologies measure light scatter
turbidimetry and nephelometry
in this method, the detector is positioned 180° from the incident light
turbidimetry
- turbidimeter measures transmitted light
T or F. Turbidimetry uses short wavelengths of the incident light, as shorter wavelength light provides higher energy for light scatter
T!
Turbidimetric measurements are subject to interference from …
large particles that scatter light such as dust and lipoproteins
NOTE: dilutions may overcome interference; other ways = bichromatic incident light, individual sample blanks, kinetic measurement
if the background colour of the solution is minimal, this can be used to measure turbidimetry
spec
these measure scattered light at an angle other than 180°
nephelometers
in nephelometry, at what angle(s) is/are the detector(s) positioned relative to the incident light?
30 to 90 degrees
NOTE: positioning of the detector also minimizes error from coloured specimens and increases sensitivity; sample blank should have NO measurable scatter compared to the rxn
Turbidimetric & nephelometric immunoassays rely on ______________ formation
lattice
this describes the relationship between the concentration of antigen, antibody, and precipitation
Heidelberger and Kendall immunoprecipitation curve
- As the concentration of antigen increases, lattices are formed and increase in size
T or F. Lattices of sufficient size and complexity can precipitate out of solution
T!
what happens if there is excess antigen compared to antibody (lattice formation)?
- the size of the lattices decreases, as does precipitation
- in sample blank, the unbound antigen will scatter light symmetrically forward and backwards
- in rxn, the antigen-antibody complex scatters light forward
how do we enhance the sensitivity of these assays
may be enhanced by coupling latex particles to the antibody
when are light scatter assays measured??
when the lattice is large enough to scatter light but not large enough to precipitate
describe PETIA
- particle enhanced turbidimetric immunoassay
- reagent antibodies are coupled to particles
- particles usually composed of latex or polystyrene
- coupling of the reagent antibodies to the latex particle => formation of larger immunocomplex lattices when the analyte of interest is present
- analyte of interest must be complex that it can be bound by more than one reagent Ab
- homogeneous, non-competitive
- turbidity proportional to analyte
describe PETINA
- particle-enhanced turbidimetric inhibition immunoassay
- two reagents: an antibody to the analyte of interest and latex particles coated in the analyte of interest
- Free analyte in the patient sample competes with the reagent analyte bound to the latex particles for binding with the reagent antibody
- absence of patient analyte = two reagents form a lattice and the turbidity of the reaction increases
- presence = lattice formation is inhibited and the turbidity of the reaction does not increase to the same degree
- small molecules; haptens
- homogeneous, competitive
- turbidity inversely proportional to analyte
describe PETINA
- particle-enhanced turbidimetric inhibition immunoassay
- two reagents: an antibody to the analyte of interest and latex particles coated in the analyte of interest
- free analyte in the patient sample competes with the reagent analyte bound to the latex particles for binding with the reagent antibody
- ABSENCE of patient analyte = 2 reagents form lattice; turbidity increased
- PRESENCE = lattice formaiton INHIBITTED = turbidity does not increase
- suitable for small molecules/haptens
- homogenous, competitive
- turbidity inversely related to analyte concentration
three commonly used labels
enzymes
chemiluminescent molecules
fluorophores
Enzymes can be used to label both ______ and __________.
ligands and antibodies
commonly used enzymes for immunoassays
alkaline phosphatase
horseradish peroxidase
glucose-6-phosphate dehydrogenase
how do enzymes act in immunoassays?
they act on their corresponding substrates and changes in absorbance can be measured
NOTE: chemiluminescent and fluorescent substrates can be used and provide higher sensitivity of detection (100-1000X) than chromogenic substrates
T or F. Fluorophores absorb light at one wavelength an emit light at a longer wavelength
T!
define Stokes shift
in fluorescence, the difference between the maximum excitation wavelength and the maximum emission wavelength
- the larger the Stoke shift, the lower the backgorund interference
fluorometers vs specs
similar to one another
BUT most fluorometers measure emission light at 90° to the excitation light
approximately one thousand times more sensitive than spectrophotometric methods
intensity of the fluorescent signal is directly proportional to the intensity of the excitation light
fluorometry is also more specific than photometry, as there is a negligible background
the emission of light as the result of a chemical rxn
chemiluminescence
how does chemiluminescence work?
chemiluminescent substances, like luminol, gain energy during an oxidation reaction and emit light when they return to ground state
- oxidation of these labels requires an oxidizing agent and a catalyst
- most assays have no background noise or interference
- specific and sensitive
- labels are stable and can be automated
- some reagents are toxic (Acridinium esters)
describe competitive assays
- labelled and unlabelled ligands compete for a limited number of binding sites
- proportion of labelled ligands binding the antibody is inversely proportional to the amount of unlabelled ligands in the sample
- simultaneous (one-step) or sequentially (two-step)
simultaneous competitive assay
the labelled and unlabelled ligands are added to the antibodies at the same time
sequential competitive assay
the unlabelled ligands are incubated with excess antibodies and then labelled ligands are added
sequential method is more sensitive than simultaenous and enhances the detection limit of the assay
sequential competitive assay
the unlabelled ligands are incubated with excess antibodies and then labelled ligands are added
sequential method is more sensitive than simultaneous and enhances the detection limit of the assay
describe non-competitive (sandwich) assay
- antibodies are immobilized on a solid substrate
- sample ligands bind to the immobilized antibodies
- labelled antibodies which binds to a different epitope on the ligand are subsequently added
- can be performed simultaneously or sequentially
- sequential method has wash steps to remove unbound reagents; it is more sensitive and specific than the one-step method
hyperlipidemia
an increase in one or more of the following: triglycerides, free cholesterol and cholesterol esters
= sample turbidity
rheumatoid factors
IgM autoantibodies that bind to the Fc portion of antibodies
- in 70% of rheum arthritis
- SLE, hepatitis, leukemia
- these interfere w immunoassays by binding reagent Abs
biotin
- vitamin H
- fruits, veggies, some meats
- included in most multivitamins
- has strong affinity or streptavidin (similar to Ab-Ag rxn)
- excess biotin is present in the sample = will compete with the reagent biotin for binding with the streptavidin
- depending on the immunoassay format, excess biotin can falsely increase or decrease the analyte concentration
HAMA
- human anti-mouse antibodies
- formed in ppl exposed to mouse antigens
- may interfere in sandwich immunoassays; they bind both the capture and labelled antibody to give a false pos rxn
- HAMA interference may be reduced by adding animal-specific sera to the test reagent to neutralize heterophilic Abs
heterophile abs
- antibodies formed in individuals after exposure to foreign antigens (animals, bacteria, viruses, and blood transfusions)
- these Abs may interfere in sandwich immunoassays as they may bind both capture and labelled Ab to give a false pos rxn
prozone
- aka Hook effect
- excess concentrations of antigen from the sample interferes in sandwich assays by saturating capture and label Abs = prevent sandwich formation
= false neg rxn - prozone can be overcome by diluting patient sample
homogenous competitive assays
- physical separation of bound and unbound labelled ligands is not required
- measured signal is altered when the labelled ligands are bound
(ex: turbidimetry, FPIA)
heterogenous competitive assays
- bound labelled ligands and unbound ligands cannot be distinguished from one another; require physical separation
- Ab is immobilized onto a surface typically to facilitate separation by washing, decanting, and centrifugation
- if Ab not immobilized, separation may be performed by chromatography or separation
separation techniques
solid phase, adsorption
solid phase separation technique
polystyrene wells, membranes and magnetic beads
automated systems commonly use magnetic beads
Abs coupled to magnetic beads, and the application of a magnet to the reaction vessel holds the antibodies in place while unbound materials are washed away
adsorption separation technique
this separation method uses small particles to trap small antigens
commonly used materials = charcoal and dextran, silica, Sephadex, ion-exchange resins
describe lateral flow immunoassays
- aka immunochromatographic assays
- POCT
- combines a sandwich immunoassay and planar affinity chromatography
- testing surface composed of overlapping membranes supported by a plastic or cardboard backing
- test surface is composed of multiple areas or zones: sample pad, conjugate, detection, and absorbent pad
- liquid sample is applied to sample pad; migrates across test surface by capillary action to the absorbent pad at the opposite end
- sample pad evenly distributes the sample across the width of the test surface; may have pores to filter the sample
- may contain buffers and liquids to control the flow rate
- conjugate pad holds the detector particles
- particles are typically monoclonal antibodies labelled with colloidal gold
- detection zone is characterized by a line of immobilized antibodies to the analyte; the immobilized antibodies bind the analyte bound to the labelled antibody
- second line of Abs may be present in detection zone; bine labelled Abs from conjugate pad = control
colloidal gold
has an intense colour and does not require a development process for visualization
- used in lateral flow immunoassay
how to increase the sensitivity of lateral flow immunoassay
increasing capacity of absorbant pad as larger sample volumes can be used
ECLIA
- electrochemiluminescent immunoassay
- Roche uses an electro-chemical luminescent immunoassay known as Elecsys
- uses monoclonal Abs labelled w ruthenium complex, Tris (2,2’-bipyridyl) ruthenium (II)-complex or Ru (bpy)2+
- applied voltage oxidizes ruthenium complex and another reagent, tripropylamine (TPA) = oxidized reacts and emits light at 620 nm
- ruthenium complex is recycled, and the TPA is consumed
- variations = one-step sandwich assay and a two-step competitive assay; both use specific interaction bw biotin and streptavidin to partition immune complex from unbound sample and reagents
one-step sandwich ECLIA assay
- sample is incubated with a biotinylated monoclonal ligand‐specific antibody and a monoclonal ligand specific antibody labelled with a ruthenium complex
- sandwich complex is formed with the ligand bound to both a biotinylated and a ruthenium complex labelled antibody
- NOTE: ligand must be sufficiently complex to have more than one epitope for binding
- streptavidin‐coated magnetic microparticles are added, and the biotinylated antibody binds to the streptavidin-coated microparticles
- reaction mixture is transferred to a measuring cell where the microparticles are bound magnetically, and unbound substances are washed away
- presence of TPA = volage applied to ruthenium complexl abelled microparticles to induce chemiluminescence = measure by photomultiplier
- signal directly proportional to amount of ligand present in sample
- non-comp, heterogenous
competitive ECLIA assay
- sample ligand and ligand labelled with the ruthenium complex are incubated with ligand specific biotinylated antibodies and compete for binding sites on the antibody
- streptavidin-labelled magnetic microparticles are added to the reaction mixture and bind the biotinylated antibody
- analyzer transfers the reaction mixture to a measuring cell where the microparticles are bound magnetically, and unbound substances are washed away
- presence of TPA = voltage = chemilumiscence measured by photomultiplier
- signal is INDIRECTLY proportional to amount og ligand present in sample
- competitive, heterogenous
two-step competitive ECLIA assay
- a variation
- first step = patient sample incubated w ligand-specific antibody labelled w biotin
- second step = ruthenium complex labelled ligand is added to rxn and binds to any free ligand specific Ab; simultaneously streptavidin-coated microparticles also added
- instrument is transferring the reaction to a measuring cell
- magnet immobilizes the immune complex bound to the magnetic streptavidin microparticles, and the unbound substances are washed away
- applied voltage induces the chemiluminescent signal of the ruthenium complex, and a photomultiplier detects the light
- signal inversely proportional to concentration of ligand in the sample
Beckman Coulter Dxl
chemilumiscent immunoassay (CLIA)
CLIA
the paramagnetic beads are bound to antibodies, not streptavidin (Roche)
chemiluminescence in the Beckman Coulter system is induced by the enzymatic oxidization of dioxetane-P to dioxetane
in CLIA sandwich assay, the paramagnetic particles are either directly or indirectly coated with the capture antibody
sample is mixed with the particles and an alkaline phosphatase-labelled antibody
sample analyte and conjugate form immune complexes that bind to the particles
magnet binds the paramagnetic particles and washing removes the unbound components
Dioxetane-P is added and is converted to dioxetane by alkaline phosphatase resulting in chemiluminescence
chemiluminescent signal is directly proportional to the amount of antigen in the sample
T or F. CLIA is non-competitive heterogenous
T!
CLIA competitive assay
- paramagnetic particles are either directly or indirectly coated with the capture antibody
- sample is mixed with the particles, an antigen-specific antibody, and an enzyme-labelled analyte
- sample analyte competes with the enzyme labelled analyte for binding sites on the particle-bound antibody
- magnet binds the paramagnetic particles and washing removes the unbound components
- Dioxetane-P is added and is converted to dioxetane by alkaline phosphatase resulting in chemiluminescence
- chemiluminescent signal is inversely proportional to the amount of antigen in the sample
EMIT
enzyme multiplier immunoassay
- homogeneous, competitive assay
- signal is proportional to ligand concentration
- suitable for low molecular weight analytes found in higher concentrations, e.g. therapeutic drugs or drugs of abuse
- enzyme labelled ligand, and patient ligand competes for binding sites on the ligand-specific antibody
- binding of labelled ligand to the antibody causes steric hindrance of the enzyme’s activity
- only unbound enzyme labelled ligand can react with the substrate to create a measurable product
sources of error for EMIT
Anything that interferes with enzyme activity or absorbs at the same wavelength, e.g. hemolysis, bilirubin, lipemia.
FPIA
fluorescent polarization immunoassay
- homogeneous, competitive assay
- signal is inversely proportional to ligand concentration
- many fluorescent labels can polarize light
- ligand-antibody complex rotates slowly compared to free labelled ligand
- amount of polarized fluorescence emitted varies inversely with the speed of rotation; the ligand-antibody complex gives off more polarized fluorescent light than free ligand
- suitable for small ligands like hormones and drugs; NOT suitable for larger proteins
sources of error for FPIA
- hemolysis, bilirubin, and lipemia may potentially interfere with any fluorescent assay
- sample viscosity
- light scatter from particles
