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Clinical Biochem > Immunoassays > Flashcards

Immunoassays Flashcards

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
Q

What is a labelled immunoassay

A

a ligand or antibody is labelled by attaching a molecule that either produces a signal directly or indirectly

26
Q

What are the common types of labels

A

chemiluminescent molecules, enzyme and fluorophores

27
Q

What are examples of enzymes used as labels

A

horseradish peroxidase, alkaline phosphatase, G6PD, B-D-galactosidase

28
Q

What detection methods are used with enzymes

A

photometer, fluorometer, luminometer

29
Q

What are chemiluminescence methods used as labels

A

luminol, acridinium esters, ruthenium complexes, dioxetane

30
Q

What detection methods are used with chemiluminescence

A

luminometers

31
Q

What fluorescence methods are used as labels

A

rhodamine B, fluorescein, europium

32
Q

What detection methods are used with fluorescence

A

fluorometer

33
Q

What occurs in a competitive assay

A

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
Q

What are the two methods of competitive assays

A

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
Q

What is the non-competitive assay

A

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
Q

What are common interferences in immunoassays

A

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
Q

What is the prozone (hook effect)

A

excess concentrations of antigens from sample interferes in sandwich assays by saturating capture and label antibodies preventing sandwich formation

38
Q

What are homogenous assays

A

physical separation of the bound and unbound labelled ligands is not required, signal is altered when the labelled ligands are bound

39
Q

What are heterogenous assays

A

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
Q

What is solid phase separation

A

polystrene wells, membrane and magnetic beads, a reaction vessel holds the antibodies in place while unbound materials are washed away

41
Q

What is adsorption separation

A

small particles are used to trap small antigens

42
Q

What are lateral flow immunoassays

A

a combination of sandwich assay and planar affinity chromatography

43
Q

How do lateral flow immunoassays work

A

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
Q

What is the non-competitive electro-chemiluminescent immunoassay

A

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
Q

What is the relationship between the signal and the sample concentration in non-competitive electro-chemiluminescent immunoassay

A

directly proportional

46
Q

What is the competitive electro-chemiluminescent immunoassay

A

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
Q

How is the signal related to the amount of ligand in the sample in competitive electro-chemiluminescent immunoassay

A

indirectly proportional

48
Q

What is the two step competitive electro-chemiluminescent immunoassay

A

the same as the competitive but the patient sample is incubated first before the labelled ligand is added

49
Q

What is the chemiluminescent immunoassay sandwich assay

A

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
Q

How is the signal related to the sample concentration in the chemiluminescent immunoassay sandwich assay

A

directly proportional

51
Q

What is the chemiluminescent immunoassay competitive assay

A

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
Q

What is the relationship between signal and analyte concentration in chemiluminescent immunoassay competitive assay

A

inversely proportional

53
Q

What is the enzyme multiplier immunoassay technique

A

a homogeneous, competitive assay

54
Q

What is the relationship between signal and ligand concentration in the enzyme multiplier immunoassay technique

A

proportional

55
Q

What is the enzyme multiplier immunoassay technique used for

A

low molecular weight analytes found in high concentrations

56
Q

How does the enzyme multiplier immunoassay technique work

A

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
Q

What are interferences with enzyme multiplier immunoassay

A

anything that interferes with enzyme activity or absorbs light at the same wavelength

58
Q

What is florescent polarization immunoassay

A

a homogeneous, competitive assay

59
Q

What is the relationship between the signal and ligand concentration in the florescent polarization immunoassay

A

inversely proportional

60
Q

How does the florescent polarization immunoassay work

A

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
Q

What is the florescent polarization immunoassay suitable for

A

small ligands like hormones and drugs

62
Q

What are interferences in florescent polarization immunoassay

A

hemolysis, bilirubin and lipemia, sample viscosity, light scatter from particles

Clinical Biochem (15 decks)

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