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IMMUNOASSAYS Flashcards

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1
Q

what type of assay is PETINA?

A

Particle Enhanced Turbidimetric Inhibition Immunoassay:

  • homogeneous, competitive immunoassay
  • turbidity of the reaction is INVERSE to [analyte]

ie. analyte disrupts antibody-antigen complexes = turbidity decreases

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2
Q

What technique is used to measure turbidemtry if the background colour of the solution is minimal ?

A

spectrophotometery

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3
Q

what methods are used to overcome interferences ?

A
  • sample dilutions
  • bichromatic incident light
  • individual sample blanks
  • kinetic measurement
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4
Q

Describe the two-step competitive assay in ECLIA

A
  1. sample (ligand) is incubated with corresponding biotinylated Ab
  2. ruthenium-labeled ligand is added and binds sample-biotin-Ab complex
  3. magnetic streptavidin-coated microparticles simultaneously complexes with biotin-sample-Ab-ligand-ruthenium complex
  4. unbound substances are washed away
  5. voltage + TPA = chemiluminescent of ruthenium complex
  6. Photomultiplier detects light = INVERSELY proportional to [ligand]
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5
Q

What is the relationship between [antigen] and lattice formation ?

A

increase in [antigen] = increase in lattices and size

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6
Q

what is the prozone (hook effect) ?

A
  • excess [antigen] saturates capture and label antibodies = FALSE NEG
  • can be overcome by dilution
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7
Q

what is the difference in the sequential method of non competitive assays (eg. turbidometry) ?

A

WASH STEP removes unbound reagents (more sensitive and specific than one-step)

RECALL: non-competitive = sandwich assays

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8
Q

Differentiate competitive vs non-competitive

A

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)

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9
Q

What is the difference between simultaneous and sequential competitive assays ?

A

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

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10
Q

Describe the competitive CLIA immunoassay

A

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]
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11
Q

what is the CLIA sandwich assay ?

A
  • 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]
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12
Q

What is stokes shift in fluorescently labelled immunoassays ?

A

difference b/w max absorption and max emission wavelength

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13
Q

Differentiate solid phase and adsorption separation

A

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

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14
Q

What is Rayleigh-Debye scattering?

A
  • particle and wavelength of incident light are of EQUAL SIZE
  • MOSTLY FORWARD SCATTER BUT side and backscatter also detected
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15
Q

What is Mie scattering?

A
  • PARTICLE SIZE GREATER than 10x the size of incident light
  • OPTIMAL FORWARD SCATTER detected
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16
Q

what is FPIA ?

A

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

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17
Q

what is EMIT?

A

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

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18
Q

Define ECLIA. What is it labeled with ?

A

Electro-chemiluminescent Immunoassay:
- uses monoclonal antibodies labelled with a ruthenium complex

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19
Q

Principle of CLIA

A

Chemiluminescent immunoassay (Beckman coulter DXL):
- uses magnetic beads that bind to antibodies
- chemiluminescence induced by enzymatic oxidation of dioxetane-p to dioxetane

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20
Q

what is biotin also called and where is it found ?

A
  • “vitamin H”
  • found in fruits, veggies, and meats, multivitamins
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21
Q

What is antigen-antibody equivalence ?

A

Ag-Ab lattices can precipitate out of solution

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22
Q

What is another name for noncompetitive assays ?

A

sandwich assays

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23
Q

what is a lateral flow immunoassay (immunochromatographic assays) ?

A
  • POCT that combines sandwich immunoassay and planar affinity chromatography (rapid preg tests)
  • test surface contains: sample pad, conjugate, detection, adsorbent pad
  1. liquid sample travels across surface by capillary action (buffers and liquids control flow rate)
  2. conjugate pad holds the detector particles (monoclonal Ab labelled with colloidal gold)
  3. detection zone is made of immobilized Ab that bind to analyte-labelled antibody complex
  4. detection control will bind ONLY labeled antibody (without analyte of interest)
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24
Q

What happens to the light scatter of the sample blank and reaction analyte with antigen and antibodies ?

A

Sample: unbound antigen will scatter light forwards and backwards (Rayleigh)

Reaction: antigen-antibody complex scatters light MOSTLY FORWARDS (Rayleigh-Debye)

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25
What happens to the lattices if excess antigen is present ?
Lattices decrease and precipitates (Hooking effect) = FALSE NEG
26
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)
27
What is the principle of competitive assays ?
labelled and unlabeled ligands compete for binding sites
28
what does the position of the detector do in nephelometry ?
minimizes error from coloured specimens and increases sensitivity (90°)
29
What do biotinylated Ab have a strong affinity for ?
Strepavidin (microparticles) - eg roche cobas elecsys
30
What does a larger stokes shift indicate ?
lower background interference
31
What does a Heidelberger and Kendall immunoprecipitation curve show ?
the relationship between [antigen], [antibody], and precipitation
32
What do turbidimetric and nephelometric immunoassays rely on?
lattice formation
33
what do HAMAs do
- "human anti-mouse antibodies" - interfere in sandwich immunoassays as they bind both capture and labelled antibody = FALSE POS
34
What can improve sensitivity of turbidimetry and nephelometry lattice formation ?
coupling of latex particlers to antibody
35
What are 3 commonly used labels for labelled immunoassays ?
enzymes, chemiluminescent molecules, fluorophores
36
what are the 2 separation techniques ?
solid phase and adsorption
37
What are the 3 types of light scatter ?
Rayleigh, Rayleigh-DeBye, Mie
38
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
39
List sources of interferences in immunoassays
- hyperlidipidemia - rheumatoid factors - biotin - HAMA - heterophile antibodies - prozone (hook effect)
40
Sources of error in FPIA
- HIL - viscosity - light scatter - quenching - photobleaching
41
Sources of error in EMIT
HIL
42
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
43
What are methodologies that measure light scatter ?
turbidimetry and nephelometry
44
What are interferences in turbidimetry ?
large particles that scatter light (dust and lipoproteins)
45
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
46
what are examples of fluorescence labels and how are they detected ?
- rhodamine B, fluorescein, europium Detected: fluorometer
47
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
48
what are examples of chemiluminescence labels and how are they detected ?
- luminol, acridinium esters, ruthenium complexes, dioxetane Detected: luminometer
49
What angle is the detector positions to the incident light in nephelometry ?
90°
50
pros and cons of chemiluminescence
Pros: - no background noise/ interference - specific and sensitive - stable labels and can be automated Cons: - toxic reagent; acridinium esters
51
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
52
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]
53
How does Rayleigh Scatter work ?
- PARTICLE SIZE is LESS THAN 1/10th of incident wavelength of light - SYMMETRICAL FORWARD, SIDE and BACKWARDS SCATTER
54
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]
55
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
56
how do we reduce HAMA interference ?
adding animal specific sera to test reagent
57
how do we increase sensitivity of the lateral flow immunoassay ?
increase capacity of the absorbent pad as larger volumes can be used
58
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
59
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
60
How are light scatter assays measured in relation to lattices ?
lattice has to be large enough to scatter light but not precipitate
61
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
62
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
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