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°)