Lecture 6.2

Question 1

What element is commonly used in time-resolved fluorescence assay?

Answer 1

a lanthanide, such as europium, with an extraordinarily long fluorescent lifetime

Question 2

What is the principle of time-resolved fluorescence assay?

Answer 2

The fluorescence of a sample is monitored as a function of time after excitation by a flash of light

Question 3

Why time-resolved fluorescence?

Answer 3

for precise detection of the fluorescence signal with minimal background noise.

Question 4

What is the full form of DELFIA?

Answer 4

dissociation-enhanced lanthanide fluoroimmuno assay

Question 5

Explain the principle of DELFIA?

Answer 5

a type of time-resolved fluorescence immunoassay that utilizes lanthanide chelates as labels for detection

–> dissociating the chelate to measure fluorescence after binding

Question 6

What is the upside of DELFIA?

Answer 6

robust and very sensitive (10^-17 moles/well)

Question 7

Why is DELFIA not ideal for HTS?

Answer 7

it involves several binding, incubation and wash steps

Question 8

Apart from dissociation of lanthanide chelates from ligands, how else could lanthanides be used in DELFIA?

Answer 8

binding of europium-labelled ligand (donor) to the allophycocyanine (APC)-labelled receptor (acceptor) brings the donor-acceptor pair into close proximity and energy transfer takes place resulting in emission at 665nm.

–>protein-protein interaction

Question 9

What is the full from of FRET?

Answer 9

Fluorescence Resonance Energy Transfer (FRET)

Question 10

What is the principle of FRET (fluorescence resonance energy transfer)?

Answer 10

Distance-dependent interaction
between the electronic excited
states of two dye molecules in
which excitation is transferred
from a donor molecule to an
acceptor molecule without
emission of a photon

Question 11

What is the principle of fluorescence polarization (FP) based on?

Answer 11

fluorescent molecules excited by light polarized in one plane will emit a signal in a polarized fashion

–> when tracer bound to larger molecule, less rotation+tumbling, emission remains polarized

Question 12

What are the 2 parameters that are essential in fluorescence polarization assay?

Answer 12

1. fluorescence lifetime of the excited fluorescent probe (τfl)
2. the rotational correlation time θ of the molecule it is bound to (θrot)

Question 13

In a fluorescence polarization assay, what happens when τfl&raquo_space; θrot?

Answer 13

molecules randomize in solution during the emission
–> emitted light of the fluorescent probe depolarized

Question 14

In a fluorescence polarization assay, what happens when τfl &laquo_space;θrot?

Answer 14

excited molecules stay aligned during emission
–> emission polarized

Question 15

In a fluorescence polarization assay, what is meant by ‘rotational correlation time θ of the molecule’?

Answer 15

it is a parameter that describes how fast a molecule tumbles in solution

Question 16

In FP, if the molecule has short rotational time, and the fluorescence lifetime of probe is longer than that, then light will be depolarized more likely.

True or false

Answer 16

True

Question 17

Explain the principle of competitive FP assay kinase activity related experiments.

Answer 17

1. Bound tracer rotates slowly and light remains polarized
2. Phosphopeptide competes with
   tracer for antibody and frees a
   significant portion of bound
   tracer.
3. Unbound tracer rotates rapidly and light becomes depolarized.

–> kinase acitivity is detected as a decrease in polarization

Question 18

What is AlphaScreen?

Answer 18

bead-based chemistry used to study biomolecular
interactions in a microplate format

Question 19

What is the principle of AlphaScreen?

Answer 19

Binding of molecules captured on the beads leads to an energy transfer from one bead to the other

–> luminescent/fluorescent signal.

Question 20

What types of beads does AlphaScreen assay contain?

Answer 20

Donor beads and Acceptor beads

Question 21

What are the characteristics of donor beads and acceptor beads in AlphaScreen assay?

Answer 21

1. coated with a hydrogel –> minimizes non- specific binding and self-aggregation
2. provides reactive aldehyde groups for conjugating biomolecules to the bead surface
3. Beads are latex-based, 250 nm in diameter
4. too small to sediment
5. larger surface area
6. large enough to be centrifuged/filtered –> no chormatographic seperation needed

Question 22

Why are beads in AlphaScreen assay coated with a hydrogel?

Answer 22

to minimize non- specific binding and self-aggregation

Question 23

Why prefer AlphaScreen beads over SPA beads?

Answer 23

1. too small to sediment, no clogging of small tips during
   dispensing
2. larger surface area
   3.large enough to be centrifuged/filtered –> no
   chormatographic seperation needed

Question 24

What does ‘Alpha’ in AlphaScreen stand for?

Answer 24

Amplified Luminescent Proximity Homogeneous Assay

Question 25

In AlphaScreen, laser light of which wavelength is used for illumination?

Answer 25

680nm

Question 26

What is the science behind AlphaScreen?

Answer 26

--> illumination with laser light at 680 nm --> photosensitizer in donor bead converts ambient oxygen to singlet state oxygen -->If acceptor bead is close proximity, singlet-state oxygen molecules reacts with chemiluminescent groups in the acceptor beads --> energy transfer --> emission at 520-620nm

Question 27

In an AlphaScreen assay, when donor beads and acceptor beads are not in close proximity, what happens when the beads are illuminated with a laser light at 680nm?

Answer 27

excited singlet-state oxygen molecules diffuse approximately 250 nm (one bead diameter) before rapidly decaying

Question 28

What are the advantages of cell-based assays over in vitro assays?

Answer 28

1. the cells self-replicate, no need to specially prepare target 2. targets &readouts examined in biological context --> mimics physiological situation 3. insights about bioavailability and cytotoxicity 4. can be used when molecular target is unknown 5. cytotoxic compounds detected and eliminated

Question 29

What are some typical cell-based assay?

Answer 29

◼ Direct mRNA detection ◼ FLIPR assays ◼ Luciferase assays ◼ Radioactive binding assays

Question 30

What are the disadvantages of cell-based high-throughput drug screening?

Answer 30

1. require specially engineered cell lines 2. cells liable to become detached from support 3. high rate of false +ves due to non-specific effects 4. difficult to miniature 5. assay conditions (e.g. use of solvents, pH) limited by cell viability 6. require high-capacity cell culture facilities

Question 31

What are primary cells and immortalized cells?

Answer 31

Primary cells: directly isolated from living organisms, tissues, or organs, in natural state, not altered or transformed Immortalized cells: been modified or transformed in the laboratory to have an indefinite lifespan

Question 32

What are some advantages of using immortalized cells over primary cells in drug screening?

Answer 32

1. unlimited abundance 2. homogenous cell type 3. simple handling 4. low assay background 5. low cost

Question 33

What are some advantages of using primary cells over immortalized cells in drug screening?

Answer 33

1. cell encironment resembles in vivo 2. high relation to physiology 3. cell phenotype: tissue selective

Question 34

Name 2 direct mRNA detection technologies

Answer 34

1. Chromagen: High performance signal amplification (HPSA) technology --> mRNA detection using chromogenic or fluorescent detection systems 2. Tropix: Xpress-Screen - mRNA Quantitation assay

Question 35

Describe how a chromagnic detection system would detect mRNA.

Answer 35

1. microwell with capture probe 2. sample mRNA attached to capture probe 3. detection probe, with enzyme attached, attach with sample mRNA that is captured 4. enzyme activated, converts substrate present in solution into fluorogenisis

Question 36

Name an enzyme that could be used in chromagnic detection system to detect mRNA

Answer 36

alkaline phosphatase

Question 37

Explain the principle of Tropic: Xpress Screen (mRNA quantitation assay).

Answer 37

1. biotinylated capture probe capture mRNA of interest 2. Antibody/Alk phos conjugate (specific fo RNA/DNA hybrids) attached to mRNA of interest 3. Alk phos acts on proprietory substrate 4. chemiluminescence

Question 38

What are the benefits of mRNA quantitation technology?

Answer 38

1. quantitative and sensitive detection of specific mRNA 2. Requires no purification or amplification of mRNA of interest 3. Applicable to almost any cell lines --> no transfection required --> rapid assay development 4. No foreign proteins and no over-expression

Question 39

What is the full form of FLIPR?

Answer 39

Fluorometric Imaging Plate Reader

Question 40

What is the use of FLIPR?

Answer 40

perform quantitative optical detection of 1. receptor/ion channel-mediated changes in cellular membrane potential or 2. intracellular calcium using fluorescent indicator dyes.

Question 41

Apart from HTS, what else is FLIPR used for?

Answer 41

target identification, assay development, and follow-up pharmacological analysis of compound specificity and selectivity.

Question 42

Many receptors and ion channels coupled to intracellular signaling events involving changes in ___________, _____________ (e.g., calcium), or __________ can be studied using the FLIPR with appropriately selected fluorescent dyes.

Answer 42

cell membrane potential intracellular cation concentrations intracellular pH

Question 43

What is the FLIPR system comprised of?

Answer 43

1. multiwell fluid addition 2. simultaneous kinetic data measurement 3. optics system for fluorescent illumination and detection

Question 44

What are the major advantages of FLIPR system?

Answer 44

◼ real-time analysis of whole-cell signaling events ◼ relatively rapid measurement (e.g., 1-sec intervals) of signaling events ◼ simultaneous detection in 96 wells (or 384 wells) offering HTS capabilities ◼ adaptability for assaying G protein-coupled receptors and ion channels

Question 45

What are the general applications of the FLIPR?

Answer 45

1. Identification, characterization, and screening of cell lines 2. Functional SAR analysis of receptor/ion channel modulators 3. High-throughput screening assays of compound libraries 4. Electrical stimulation of cells-analysis of calcium and other second messengers

Question 46

What are the 2nd generation and 3rd generation FLIPR called?

Answer 46

2nd gen: The Tetra 3rd gen: The Penta

Question 47

What is depolarization?

Answer 47

a change in the membrane potential of a cell, where the membrane potential becomes less negative (moves towards zero) --> influx of positively charged ions (commonly sodium ions) into the cell

Question 48

What is hyperpolarization?

Answer 48

increase in the membrane potential, making the inside of the cell more negative than at the resting state -->efflux of positively charged ions (e.g., potassium ions) or an influx of negatively charged ions

Question 49

What molecule is used to study whether an inhibitor or activator hperpolarizes or depolarizes the cell?

Answer 49

DiBAC4 -->With depolarization, DiBAC4 further partitions into the cell, leading to an increase in fluorescence. --> hyperpolarization results in dye extrusion and thus, a decrease in fluorescence

Question 50

In FLIPR-based assay using DiBAC4, what happens to fluorescence upon addition of a potassium channel opener?

Answer 50

increased efflux of potassium ions (K+) --> decrease in fluorescence

Question 51

What is DiBAC4?

Answer 51

fluorescent membrane potential-sensitive dye commonly used in biological research to monitor changes in membrane potential in cells