Immunoassays Flashcards
Capture Ab (or Ag) vs Reporter Ab (or Ag)
- Capture Ab (/Ag): attach to something solid
- Reporter Ab (/Ag): gives signal via colour change / fluorescence
Competitive vs non-competitive
- Competitive: non-proportional - signal come from labeled Ag e.g. high labeled Ag = low test analyte (non-labeled)
- non-competitive: proportional - signal come from labeledAb
Homogenous vs non-homozygous (heterogenous)
- homogenous: everything the same (soluble). Easier, faster bc no wash step
- heterogenous: (l) & (s) (insoluble). More sensitive
analyte
something you measure - can be Ag, Ab, Enzymes
E.g. of Low & Hi molecular weight proteins
Low (Small): Steroids, T4/T3, Drugs, Catecholamines
High (Big): TSH, FSH, Ig
monoclonal vs polyclonal
- monoclonal: made from hybridoma cell line in humans=immortal, binds to same epitope
- polyclonal: made from animals = dies, binds to many Ab
Radioimmunoassays (RIA) vs Immuno-radiometric assay (IRMA)* slide 9
- RIA: competitive = Ab limiting. > sml & big molecules = use 3H labels
- IRMA: noncompetitive = Ag limiting > big molecules
turbidmitry vs Nephlometry
- Turbidimetry: measures transmitted light (decrease bc of light scattered)
- Nephlometry: measures scattered light
3 types of Ag measured
- NATURALLY present (e.g. thyroid hormone)
- BODY PRODUCES not usually present (e.g cancer)
- NOT NATURAL(e.g. drugs)
Radial immunodiffusion, immunonephelometry, immunoturbidimetry, and precipatitaion use _clonal Ab (or _) as it relies on forming large complexes
polyclonal Ab
OR a mix of monoclonal Ab
DELFIA and europium
- chemifluorescnence
- capture Ab coated on plate
- Ab* is DELFIA Europium-labeled antibody
- substrate: DELFIA Enhancement Solution =>dissociate europium = form fluorescent chelate
- excited 320nm or 340nm
- measured @ 615nm
chemiluminescence vs chemifluorescence
- ..ilumines.: enzyme reaction to generate light
- ..fluores.: fluorescent molecule EXCITED = light emitted
dis & advantages for non-isotopic labels (compared to isotopic (radiolabels) labels)
ADV - longer shelf life - higher sensitivity - less Hazardous DIS - not as much assays developed ( isotopic = more assays)
describe chemiluminescence assay (CLIA)
- use Ag* or Ab* w/ labels - can produce light : HRP, ALP or luminol
- luminol oxidised by hydrogen peroxide in alkaline cond. => blue light
advantages of CLIA vs fluorescent
- longer lasting
- higher sensitivity
principle of Immulite 1000, chemiluminiescent light
- competitive assay
- beaad covered by Ab* or Ag*
- enzyme ALP (reagent)
- substrate: dioxetane
Describe Enzyme Multiplied Immunoassay Technique (EMIT) how works
- homogenous competitive immunoassay
- Ag bound near active site of enzyme
- Free (test) Ag competes w/ Ag bound to enzy. for Ab
- Lo [Free Ag] = more Ab bind to Ag on enzyme = block active site = substrate can’t bind ≠ colour change
- Hi [Free Ag] = mix bind to free & bound Ag = some active sites open = substrate can bind = colour change
what is Fluorescein
- fluorescnet label
- absorbs light @ 490nm => release energy at 520nm
fluorescein is used to distinguish
- sml & lrg molecules
- bc larger molecules (Ab-AgF) rotate slower than sml molecules (AgF) = distinguishable
type of assay Fluorescence Polarization Immunoassay (FPIA) is
homogeneous competitive fluorescence immunoassay.
FPIA is used to provide
accurate and sensitive measurements of small toxicological analytes (drugs)
Chemiluminescent Magnetic Nanopartices (CMIA) uses chemiluminescent label that produces light when _ with a trigger reagent.
combined/irradiated
hook effect gives & solution
- falsely low values due to v. high Ag added = affecting binding capacity in one-tep rxn
- solve w/ 2-step w/ washing step
Heterophile antibodies have their maximum effect is when the analyte is in a ————-concentration. For example, ———.
a) low
b) troponin
What is the principle or best approach to minimize heterophile interference or to check for it?
- heterophile blocking reagents (HBR)
- immunoglobulin inhibiting reagents (IIR)
Dis & Advantages of immunoassays
ADV: highly sensitive; automated; less hazardous (if no radioactive labels used)
DIS: Cross rxn (Aby bind to similar Ag) ; difficult to analyse multiple solutes
