CHEM Immunoassays Flashcards
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]
- ruthenium labeled antigen (CHEMILUMINESCENCE) competes with sample (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 ?
WASH STEP removes unbound reagents (more sensitive and specific than one-step)
RECALL: non-competitive = sandwich assays
Differentiate competitive vs non-competitive immunoassays
Competitive: patient analyte and labeled-ligand compete for the same Ab binding site
Non-competitive (sandwich): patient analyte binds to capture Ab first, and then labeled-ligand binds
What is the difference between simultaneous and sequential competitive assays ?
Simultaneous (one step) - labeled ligand and unlabeled (patient) analyte added/ compete for Ab at the same time
Sequential (two step) - unlabeled (patient) analyte is incubated with excess Ab first, THEN labelled ligands added
- more sensitive and enhances the detection limit of assay
Describe the competitive CLIA immunoassay
Chemiluminescent Immunoassay:
- sample analyte is mixed with antigen-specific Ab, magnetic beads coated with capture Ab, and enzyme-labeled ligand
- sample analyte competes with enzyme-labeled ligand for binding sites
- magnet holds particle complex; unbound substances are washed away
- chemiluminescence is INVERSE to [antigen]
what is the CLIA sandwich assay ?
- sample analyte mixed with magnetic beads coated with Ab, and enzyme-labeled Ab
- analyte is sandwiched between magnetic bead-Ab and enzyme-Ab = immune complex
- magnet holds complex; unbound substances are washed away
- chemiluminescence is PROPORTIONAL to [analyte]
What is stokes shift in fluorescently labelled immunoassays ?
difference b/w max absorption and max emission wavelength
what is EMIT?
Enzyme Multiplier Immunoassay Technique:
- homogeneous, competitive assay
- patient analyte and labeled ligand compete for binding sites
- when enzyme-labeled ligand is bound = steric hinderance = no enzymatic activity
- detects UNBOUND ENZYME-labeled ligand = signal is PROPORTIONAL to [analyte]
NOTE: 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
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
- sample analyte travels across surface by capillary action (buffers and liquids control flow rate)
- conjugate pad holds the detector Ab labeled with colloidal gold
- detection zone is made of capture Ab that bind to analyte and labeled-antibody complex
- detection control will bind ONLY labeled antibody (without analyte of interest)
Detection line AND Control line = POS
Control line ONLY = POS
No lines = Invalid result
What happens to the lattices if excess antigen is present ?
Lattices saturate and separate both capture Ab and labeled Ab (Prozone/ Hook effect) = FALSE NEG
Describe principle of noncompetitive assays
- capture Ab are immobilized on solid substrate
- sample analyte bind to capture Ab
- labeled Ab bind to different epitope on sample ligand = sandwich
- can be done simultaneously (one-step) or sequentially (two-step)
What is the principle of competitive assays ?
unlabeled patient analyte and labeled 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
Advantage of a larger stokes shift
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 in immunoassays ?
- “human anti-mouse antibodies”
- interfere in sandwich immunoassays as they bind both capture and labeled antibody = FALSE POS
What can improve sensitivity of turbidimetry and nephelometry lattice formation ?
coupling of latex particles to antibody
What are 3 commonly used labels for labeled immunoassays ?
enzymes, chemiluminescent molecules, fluorophores
What are the 3 types of light scatter ?
Rayleigh: side and forward scatter
Rayleigh-DeBye: little side; mostly forward scatter
Mie: ONLY forward
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 EMIT
HIL
what are rheumatoid factors ?
- IgM autoantibodies that can BIND TO REAGENT ANTIBODIES = INTERFERENCE
- in patients with rheumatoid arthritis, lupus erythematosus, hepatitis, and leukemia
What are interferences in turbidimetry ?
large particles that scatter light (dust and lipoproteins)
what are heterophile antibodies and how do they interfere in immunoasssays ?
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 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?
Electro-chemiluminescence Immunoassay:
- sample analyte, 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 is INVERSE to [analyte]
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 ?
Electro-chemiluminescence Immunoassay:
- sample analyte is incubated with biotinylated Ab
-streptavidin-magnetic particle added and biotinylated Ab binds = complex
- unbounds are washed away
- voltage + TPA = chemiluminescence measured with photomultiplier
- signal PROPORTIONAL to [analyte]
What causes chemiluminescence in labeled immunoassays ?
Chemical reaction = emission of light
- oxidation of labels = gain energy
- return to ground state = produce light
how do we reduce HAMA interference ?
adding animal specific sera to test reagent = neutralizes HAMAs
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 labeled immunoassays ?
- absorbs light at one wavelength and emits light at a LONGER wavelength
- no background interferences = 1000x more sensitive than spec + photometry
- signal = PROPORTIONAL to [excited fluorophores]
- fluorometer at 90° to excitation light
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
Lattice requirements for light scatter assays
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
