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Clinical Biochem > Immunoassays > Flashcards

Immunoassays Flashcards

(62 cards)

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1
Q

When does rayleigh scatter occur

A

if the oarticles diameter is less than 1/10 the side of the incident wavelength

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2
Q

How does light scatter in rayleigh scatter

A

light scatters symmetrically forwards and backwards with minimal scatter at 90 degrees

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3
Q

When does Mie scatter occur

A

if the particle diameter is greater than ten times the size of the incident light

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4
Q

How does light scatter in Mie scatter

A

most of the light scatters forward

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5
Q

When does Rayleigh-debye scatter occur

A

if the particle and the wavelength of incident light are approximately the same

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6
Q

How does light scatter in Rayleigh-debye scatter

A

most of the light scatters forward but there is also detectable side and backscatter

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7
Q

How does turbidimetry work

A

a turbid solution decreases the intensity of the incident light

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8
Q

Where is the detector positioned in turbidimetry

A

180 degrees from the incident light

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9
Q

What type of wavelengths does turbidimetry use

A

short

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10
Q

What are common interferences in turbidimetry

A

large particles such as dust and lipoproteins

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11
Q

How do we minimize interferences in turbidimetry

A

bichromatic incident light, individual sample blanks and kinetic measurement

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12
Q

How does nephelometry work

A

measuring scattered light at an angle other than 180 degrees

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13
Q

Where is the detector placed in nephelometry

A

30 to 90 degrees relative to the incident light

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14
Q

What is a lattice

A

a three-dimensional structure formed by the reaction of antigens and antibodies

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15
Q

What occurs at antigen and antibody equivelence

A

large complex lattices are formed

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16
Q

What occurs if there is excess antigen

A

the size of the lattices decreases which decreases light scatter

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17
Q

How can we improve sensitivity of lattice formation assays

A

by coupling latex particles

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18
Q

When are light scatter assays measured

A

when the lattice is large enough to scatter light but not large enough to precipitate

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19
Q

What is the particle enhanced turbidimetric immunoassay

A

reagent antibodies are coupled to particles which are composed of latex or polystyrene which facilitates the formation of larger lattices

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20
Q

What type of assay is the particle enhanced turbidimetric immunoassay

A

homogenous, non-competitive

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21
Q

How does turbidity relate to analyte concentration in the particle enhanced turbidimetric immunoassay

A

it is proportional

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22
Q

What is the particle enhanced turbidimetric inhibition immunoassay

A

an antibody to the analyte of interest and latex particles coated in the antibody of interest are used. Free analyte competes with the latex bound particles for the antibody binding sites. The patient analyte binds with antibodies and blocks lattice formation

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23
Q

What type of assay is particle enhanced turbidimetric inhibition immunoassay

A

homogeneous, competitive

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24
Q

How is turbidity related to analyte concentration in particle enhanced turbidimetric inhibition immunoassay

A

inversely proportional

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25
What is a labelled immunoassay
a ligand or antibody is labelled by attaching a molecule that either produces a signal directly or indirectly
26
What are the common types of labels
chemiluminescent molecules, enzyme and fluorophores
27
What are examples of enzymes used as labels
horseradish peroxidase, alkaline phosphatase, G6PD, B-D-galactosidase
28
What detection methods are used with enzymes
photometer, fluorometer, luminometer
29
What are chemiluminescence methods used as labels
luminol, acridinium esters, ruthenium complexes, dioxetane
30
What detection methods are used with chemiluminescence
luminometers
31
What fluorescence methods are used as labels
rhodamine B, fluorescein, europium
32
What detection methods are used with fluorescence
fluorometer
33
What occurs in a competitive assay
labelled and unlabelled ligands compete for a limited number of binding sites, the proportion of labelled ligands binding the antibody is inversely proportional to the amount of unlabelled ligands in the sample
34
What are the two methods of competitive assays
simultaneous - the labelled and unlabelled ligands are added at the same time sequential - the unlabelled ligands are added first and incubated before the addition of the labelled ligand
35
What is the non-competitive assay
antibodies are immobilized on a solid substrate and sample ligands bind to the antibodies, labelled antibodies for the sample ligand are added and then a wash step occurs to wash away excess
36
What are common interferences in immunoassays
hyperlipidemia (increased sample turbidity), rheumatoid factors (binds reagents can cause increase or decrease in signal), biotin (increase or decrease), human anti-mouse antibodies (increase or decrease), heterophile antibodies, prozone (false negative)
37
What is the prozone (hook effect)
excess concentrations of antigens from sample interferes in sandwich assays by saturating capture and label antibodies preventing sandwich formation
38
What are homogenous assays
physical separation of the bound and unbound labelled ligands is not required, signal is altered when the labelled ligands are bound
39
What are heterogenous assays
bound labelled ligands and unbound ligands cannot be distinguished from one another and require physical separation separation may be performed by chromatography or precipitation
40
What is solid phase separation
polystrene wells, membrane and magnetic beads, a reaction vessel holds the antibodies in place while unbound materials are washed away
41
What is adsorption separation
small particles are used to trap small antigens
42
What are lateral flow immunoassays
a combination of sandwich assay and planar affinity chromatography
43
How do lateral flow immunoassays work
analyte is added and flows to where antibodies are conjugated to a tag, it then continues to flow to where the test line has antibodies that also bind the analyte, any of the conjugated antibodies that do not have analyte attached flow through to the control line where they bind to IgG antibodies
44
What is the non-competitive electro-chemiluminescent immunoassay
monoclonal antibodies are labelled with ruthenium, an applied voltage oxidizes the ruthemium which causes light to be emitted a magnetic streptavidin micropartical attaches which allows the bound particles to remain stuck in the reaction chamber during the wash step
45
What is the relationship between the signal and the sample concentration in non-competitive electro-chemiluminescent immunoassay
directly proportional
46
What is the competitive electro-chemiluminescent immunoassay
sample ligand and ligand labelled with the ruthenium complex are incubated with ligand specific biotinylated antibodies and compete for binding sites on the antibody, then the streptavidin labelled magnetic particles are added and bind the biotinylated antibody washing and analysis occurs as normal
47
How is the signal related to the amount of ligand in the sample in competitive electro-chemiluminescent immunoassay
indirectly proportional
48
What is the two step competitive electro-chemiluminescent immunoassay
the same as the competitive but the patient sample is incubated first before the labelled ligand is added
49
What is the chemiluminescent immunoassay sandwich assay
paramagnetic particles are directly or indirectly coated with the capture antibody, the analyte and added conjugate form immune complexes that bind to the particles then a magnet binds the particles and a wash step occurs then dioxetane-P is added and a reaction happens that results in chemiluminescence
50
How is the signal related to the sample concentration in the chemiluminescent immunoassay sandwich assay
directly proportional
51
What is the chemiluminescent immunoassay competitive assay
the paramagnetic particles are either directly or indirectly coated with the capture antibody it is then mixed with an antigen-specific antibody and an enzyme labelled analyte they compete for binding sites, the rest of the reaction continues as normal
52
What is the relationship between signal and analyte concentration in chemiluminescent immunoassay competitive assay
inversely proportional
53
What is the enzyme multiplier immunoassay technique
a homogeneous, competitive assay
54
What is the relationship between signal and ligand concentration in the enzyme multiplier immunoassay technique
proportional
55
What is the enzyme multiplier immunoassay technique used for
low molecular weight analytes found in high concentrations
56
How does the enzyme multiplier immunoassay technique work
enzyme labelled ligand and patient ligand compete for binding sites on the antibody, binding of the labelled ligand causes steric hinderance of the enzymes activity, only unbound enzyme reacting ligands can react with the substrate to create a signal
57
What are interferences with enzyme multiplier immunoassay
anything that interferes with enzyme activity or absorbs light at the same wavelength
58
What is florescent polarization immunoassay
a homogeneous, competitive assay
59
What is the relationship between the signal and ligand concentration in the florescent polarization immunoassay
inversely proportional
60
How does the florescent polarization immunoassay work
a fluorophore labelled ligand and the sample ligand compete for antibody binding sites, when the fluorophore labelled ligand binds an antibody it spins slower forming a stronger signal
61
What is the florescent polarization immunoassay suitable for
small ligands like hormones and drugs
62
What are interferences in florescent polarization immunoassay
hemolysis, bilirubin and lipemia, sample viscosity, light scatter from particles

Clinical Biochem (15 decks)

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