IMMUNOASSAYS Flashcards
what type of assay is PETINA?
Particle Enhanced Turbidimetric Inhibition Immunoassay:
- homogeneous, competitive immunoassay
- turbidity of the reaction is INVERSE to [analyte]
ie. analyte disrupts antibody-antigen complexes = turbidity decreases
What technique is used to measure turbidemtry if the background colour of the solution is minimal ?
spectrophotometery
what methods are used to overcome interferences ?
- sample dilutions
- bichromatic incident light
- individual sample blanks
- kinetic measurement
Describe the two-step competitive assay in ECLIA
- sample (ligand) is incubated with corresponding biotinylated Ab
- ruthenium-labeled ligand is added and binds sample-biotin-Ab complex
- magnetic streptavidin-coated microparticles simultaneously complexes with biotin-sample-Ab-ligand-ruthenium complex
- unbound substances are washed away
- voltage + TPA = chemiluminescent of ruthenium complex
- Photomultiplier detects light = INVERSELY proportional to [ligand]
What is the relationship between [antigen] and lattice formation ?
increase in [antigen] = increase in lattices and size
what is the prozone (hook effect) ?
- excess [antigen] saturates capture and label antibodies = FALSE NEG
- can be overcome by dilution
what is the difference in the sequential method of non competitive assays (eg. turbidometry) ?
WASH STEP removes unbound reagents (more sensitive and specific than one-step)
RECALL: non-competitive = sandwich assays
Differentiate competitive vs non-competitive
Competitive: patient ligand and labeled-ligand compete for the same Ab binding site
Non-competitive: patient ligand binds first, and then labeled-ligand binds to bound patient ligand (sandwich)
What is the difference between simultaneous and sequential competitive assays ?
Simultaneous (one step) - labelled and unlabeled ligands added/ compete for Ab at the same time
Sequential (two step) - unlabeled patient ligands are incubated with excess Ab, THEN labelled ligands added
- more sensitive and enhanced detection limit of assay
Describe the competitive CLIA immunoassay
Chemiluminescent Immunoassay:
- sample is mixed with magnetic Ab-particles, antigen-specific Ab, and enzyme-labeled (ALP) analyte
- sample analyte competes with enzyme-analyte for binding sites
- magnet holds particle complex; unbound substances are washed away
- dioxetane P is added = converted to dioxetane by ALP = chemiluminescence = INVERSE to [antigen]
what is the CLIA sandwich assay ?
- sample mixed with magnetic Ab-particles and enzyme-labeled Ab
- sample is sandwiched between magnetic particle-Ab and enzyme-Ab = immune complex
- magnet holds complex; unbound substances are washed away
- dioxetane P is added = converted to dioxetane by ALP = chemiluminescence = PROPORTIONAL to [antigen]
What is stokes shift in fluorescently labelled immunoassays ?
difference b/w max absorption and max emission wavelength
Differentiate solid phase and adsorption separation
Solid phase:
ie. polystyrene wells, membranes and magnetic beads
- antibody sticks to magnetic beads and unbounds are washed
Adsorption:
ie. charcoal, dextran, silica, sephadex, ion exchange resins
- small particles trap small antigens
What is Rayleigh-Debye scattering?
- particle and wavelength of incident light are of EQUAL SIZE
- MOSTLY FORWARD SCATTER BUT side and backscatter also detected
What is Mie scattering?
- PARTICLE SIZE GREATER than 10x the size of incident light
- OPTIMAL FORWARD SCATTER detected
what is FPIA ?
FLOURESCENT POLARIZATION IMMUNOASSAY:
- homogenous, competitive assay
- fluorophore and sample ligand compete for Ab binding site
- signal is INVERSE to [ligand]
- amount of polarized fluorescence is inverse to speed of rotation of ligand-antibody complex (more light “passes through polarization” bc complex rotates slower than free ligand)
- good for small hormones/ drugs but not larger proteins
what is EMIT?
Enzyme Multiplier Immunoassay Technique:
- homogeneous, competitive assay
- enzyme labelled ligand bound to Ab = steric hinderance on enzymatic activity
- patient ligand competes for binding sites
- ONLY UNBOUND/ DISPLACED ENZYME-labeled ligands = measurable signal PROPORTIONAL to [ligand]
- best for low MW analytes ie. drugs
Define ECLIA. What is it labeled with ?
Electro-chemiluminescent Immunoassay:
- uses monoclonal antibodies labelled with a ruthenium complex
Principle of CLIA
Chemiluminescent immunoassay (Beckman coulter DXL):
- uses magnetic beads that bind to antibodies
- chemiluminescence induced by enzymatic oxidation of dioxetane-p to dioxetane
what is biotin also called and where is it found ?
- “vitamin H”
- found in fruits, veggies, and meats, multivitamins
What is antigen-antibody equivalence ?
Ag-Ab lattices can precipitate out of solution
What is another name for noncompetitive assays ?
sandwich assays
what is a lateral flow immunoassay (immunochromatographic assays) ?
- POCT that combines sandwich immunoassay and planar affinity chromatography (rapid preg tests)
- test surface contains: sample pad, conjugate, detection, adsorbent pad
- liquid sample travels across surface by capillary action (buffers and liquids control flow rate)
- conjugate pad holds the detector particles (monoclonal Ab labelled with colloidal gold)
- detection zone is made of immobilized Ab that bind to analyte-labelled antibody complex
- detection control will bind ONLY labeled antibody (without analyte of interest)
What happens to the light scatter of the sample blank and reaction analyte with antigen and antibodies ?
Sample: unbound antigen will scatter light forwards and backwards (Rayleigh)
Reaction: antigen-antibody complex scatters light MOSTLY FORWARDS (Rayleigh-Debye)
What happens to the lattices if excess antigen is present ?
Lattices decrease and precipitates (Hooking effect) = FALSE NEG
Describe principle of noncompetitive assays
- Ab are immobilized on solid substrate
- sample ligands bind to immobilized Ab
- labelled Ab bind to different epitope on sample ligand
- can be done simultaneously (one-step) or sequentially (two-step)
What is the principle of competitive assays ?
labelled and unlabeled ligands compete for binding sites
what does the position of the detector do in nephelometry ?
minimizes error from coloured specimens and increases sensitivity (90°)
What do biotinylated Ab have a strong affinity for ?
Strepavidin (microparticles)
- eg roche cobas elecsys
What does a larger stokes shift indicate ?
lower background interference
What does a Heidelberger and Kendall immunoprecipitation curve show ?
the relationship between [antigen], [antibody], and precipitation
What do turbidimetric and nephelometric immunoassays rely on?
lattice formation
what do HAMAs do
- “human anti-mouse antibodies”
- interfere in sandwich immunoassays as they bind both capture and labelled antibody = FALSE POS
What can improve sensitivity of turbidimetry and nephelometry lattice formation ?
coupling of latex particlers to antibody
What are 3 commonly used labels for labelled immunoassays ?
enzymes, chemiluminescent molecules, fluorophores
what are the 2 separation techniques ?
solid phase and adsorption
What are the 3 types of light scatter ?
Rayleigh, Rayleigh-DeBye, Mie
What are the conditions needed in turbidimetry ?
- detector is 180° from incident light (measures transmitted light)
- short wavelength of incident light = higher energy for light scatter
- able to resolve small changes in light intensity
List sources of interferences in immunoassays
- hyperlidipidemia
- rheumatoid factors
- biotin
- HAMA
- heterophile antibodies
- prozone (hook effect)
Sources of error in FPIA
- HIL
- viscosity
- light scatter
- quenching
- photobleaching
Sources of error in EMIT
HIL
what are rheumatoid factors ?
- IgM autoantibodies that bind to Fc region of Ab
- patients with rheumatoid arthritis, lupus erythematosus, hepatitis, and leukemia
- ALSO BIND TO REAGENT ANTIBODIES = INTERFERENCE
What are methodologies that measure light scatter ?
turbidimetry and nephelometry
What are interferences in turbidimetry ?
large particles that scatter light (dust and lipoproteins)
what are heterophile antibodies and how do they interfere ?
Ab formed in people after exposure to foreign antigens
- interferes in sandwich assays ; binds both capture and labelled antibodies = FALSE POS
what are examples of fluorescence labels and how are they detected ?
- rhodamine B, fluorescein, europium
Detected: fluorometer
What are examples of enzyme labels and how are they detected ?
- horseradish peroxidase (HRP)
- alakline phosphatase (ALP)
- G6PD
- B-D-galactosidase (BDG)
- Detection: photometer, fluorometer, luminometer
what are examples of chemiluminescence labels and how are they detected ?
- luminol, acridinium esters, ruthenium complexes, dioxetane
Detected: luminometer
What angle is the detector positions to the incident light in nephelometry ?
90°
pros and cons of chemiluminescence
Pros:
- no background noise/ interference
- specific and sensitive
- stable labels and can be automated
Cons:
- toxic reagent; acridinium esters
T or F: In nephelometry, the sample blank should have no measurable scatter compared to the reaction
TRUE: In nephelometry, the sample blank should have no measurable scatter compared to the reaction
how does the one-step competitive immunoassay in ECLIA work?
- sample, biotinylated Ab, and ligand-labeled ruthenium are incubated altogether
- streptavidin-magnetic particles are added and bind to complexes
- unbound particles are washed away
- voltage + TPA = chemiluminescence measured by photomultiplier
- signal INVERSE to [ligand]
How does Rayleigh Scatter work ?
- PARTICLE SIZE is LESS THAN 1/10th of incident wavelength of light
- SYMMETRICAL FORWARD, SIDE and BACKWARDS SCATTER
how does one-step sandwich ECLIA work ?
- sample is incubated with biotinylated Ab
-streptavidin coated magnetic particle added and biotinylated Ab binds =complex - magnetic particle complex is held while unbounds are washed away
- voltage + TPA = chemiluminescence measured with photomultiplier
- signal PROPORTIONAL to [ligand]
how does chemiluminescence function in labelled immunoassays ?
- chemical reaction = emission of light
- oxidation of labels = gain energy
- return to ground state = produce light
- oxidation requires an oxidizing agent and catalyst
how do we reduce HAMA interference ?
adding animal specific sera to test reagent
how do we increase sensitivity of the lateral flow immunoassay ?
increase capacity of the absorbent pad as larger volumes can be used
How does fluorescence function in labelled immunoassays ?
- absorbs light at one wavelength and emits light at LONGER wavelength
- measure emission light at 90° to excitation light
- 1000x more sensitive than spec
- more specific than photometry because no background
- signal = PROPORTIONAL to intensity of excitation
How do enzymes function in labelled immunoassays ?
- labels both ligands and antibodies
- acts on corresponding substrates; changes in absorbance is measured
- chemiluminescent and fluorescent substrates can be used for higher sensitivity of detection
How are light scatter assays measured in relation to lattices ?
lattice has to be large enough to scatter light but not precipitate
Differentiate homogeneous and heterogeneous competitive assays
Homogenous assays = physicals separation of bound and unbound labelled ligands is NOT required (eg. turbidimetry, FPIA)
Heterogenous assays = bound labeled ligands and unbound ligands CANNOT be distinguished form one another (needs physical separation)
- antibody is immobilized on surface and separated by washing
- if antibody is not immobilized, use chromatography or precipitation
What is the correct sequence of components in a direct capture, direct detection ELISA?
1- labeled primary antibody
2 - labeled secondary antibody
3 - unlabeled primary antibody
4 - labeled ligand
5- solid substrate
6 - patient serum
a.
5, 4, 6, 2
b.
5, 6, 1
c.
5, 4, 6, 1
d.
5, 4, 6
b.
5, 6, 1
