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3006 Clin. Biochem > Immunoassays > Flashcards

Immunoassays Flashcards

1
Q

Capture Ab (or Ag) vs Reporter Ab (or Ag)

A
  • Capture Ab (/Ag): attach to something solid

- Reporter Ab (/Ag): gives signal via colour change / fluorescence

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2
Q

Competitive vs non-competitive

A
  • 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
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3
Q

Homogenous vs non-homozygous (heterogenous)

A
  • homogenous: everything the same (soluble). Easier, faster bc no wash step
  • heterogenous: (l) & (s) (insoluble). More sensitive
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4
Q

analyte

A

something you measure - can be Ag, Ab, Enzymes

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5
Q

E.g. of Low & Hi molecular weight proteins

A

Low (Small): Steroids, T4/T3, Drugs, Catecholamines

High (Big): TSH, FSH, Ig

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6
Q

monoclonal vs polyclonal

A
  • monoclonal: made from hybridoma cell line in humans=immortal, binds to same epitope
  • polyclonal: made from animals = dies, binds to many Ab
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7
Q

Radioimmunoassays (RIA) vs Immuno-radiometric assay (IRMA)* slide 9

A
  • RIA: competitive = Ab limiting. > sml & big molecules = use 3H labels
  • IRMA: noncompetitive = Ag limiting > big molecules
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8
Q

turbidmitry vs Nephlometry

A
  • Turbidimetry: measures transmitted light (decrease bc of light scattered)
  • Nephlometry: measures scattered light
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9
Q

3 types of Ag measured

A
  • NATURALLY present (e.g. thyroid hormone)
  • BODY PRODUCES not usually present (e.g cancer)
  • NOT NATURAL(e.g. drugs)
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10
Q

Radial immunodiffusion, immunonephelometry, immunoturbidimetry, and precipatitaion use _clonal Ab (or _) as it relies on forming large complexes

A

polyclonal Ab

OR a mix of monoclonal Ab

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11
Q

DELFIA and europium

A
  • 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
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12
Q

chemiluminescence vs chemifluorescence

A
  • ..ilumines.: enzyme reaction to generate light

- ..fluores.: fluorescent molecule EXCITED = light emitted

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13
Q

dis & advantages for non-isotopic labels (compared to isotopic (radiolabels) labels)

A 
ADV
- longer shelf life
- higher sensitivity
- less Hazardous
DIS
- not as much assays developed ( isotopic = more assays)
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14
Q

describe chemiluminescence assay (CLIA)

A
  • use Ag* or Ab* w/ labels - can produce light : HRP, ALP or luminol
  • luminol oxidised by hydrogen peroxide in alkaline cond. => blue light
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15
Q

advantages of CLIA vs fluorescent

A
  • longer lasting

- higher sensitivity

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16
Q

principle of Immulite 1000, chemiluminiescent light

A
  • competitive assay
  • beaad covered by Ab* or Ag*
  • enzyme ALP (reagent)
  • substrate: dioxetane
17
Q

Describe Enzyme Multiplied Immunoassay Technique (EMIT) how works

A
  • 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
18
Q

what is Fluorescein

A
  • fluorescnet label

- absorbs light @ 490nm => release energy at 520nm

19
Q

fluorescein is used to distinguish

A
  • sml & lrg molecules

- bc larger molecules (Ab-AgF) rotate slower than sml molecules (AgF) = distinguishable

20
Q

type of assay Fluorescence Polarization Immunoassay (FPIA) is

A

homogeneous competitive fluorescence immunoassay.

21
Q

FPIA is used to provide

A

accurate and sensitive measurements of small toxicological analytes (drugs)

22
Q

Chemiluminescent Magnetic Nanopartices (CMIA) uses chemiluminescent label that produces light when _ with a trigger reagent.

A

combined/irradiated

23
Q

hook effect gives & solution

A
  • falsely low values due to v. high Ag added = affecting binding capacity in one-tep rxn
  • solve w/ 2-step w/ washing step
24
Q

Heterophile antibodies have their maximum effect is when the analyte is in a ————-concentration. For example, ———.

A

a) low

b) troponin

25
Q

What is the principle or best approach to minimize heterophile interference or to check for it?

A
  • heterophile blocking reagents (HBR)

- immunoglobulin inhibiting reagents (IIR)

26
Q

Dis & Advantages of immunoassays

A

ADV: highly sensitive; automated; less hazardous (if no radioactive labels used)
DIS: Cross rxn (Aby bind to similar Ag) ; difficult to analyse multiple solutes

3006 Clin. Biochem (13 decks)

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