Lecture 6.1 Flashcards
What is high-throughput screening (HTS)?
An enabling technology that aims to rapidly assess the activity of a large number of compounds or extracts on a given target
What is the difference bwteen HTS and uHTS?
HTS: 96- and 384-well plates
uHTS: 1536-well plates with minuscule volumes (uL to nL
scale)
–> peak throughput rates of over 100,000
compounds/day
HTS usually takes weeks of engineering and fine-tuning to achieve sufficient __________, ________, and ______________
speed, robustness, and cost-effectiveness
What are the challenges associated with developing a HTS assay?
- Reagent stability and cost
- Environmental factors (temp/oxidation/agitation)
- statistic –> signal-to-noise ratios, quality measures
What are some low-throughput assays?
- filtration
- ELISA
- centrifugation
What is the problem associated with filtration as an assay?
time consuming and waste generating
What is the problem associated with ELISA as an assay?
too many assay steps and washes
What is the problem associated with centrifugation as an assay?
time consuming
Define drug screening.
the search for a chemical entity that interacts selectively with a defined biological target
Why did the invention of 96-well microtiter plate mark the beginnig of HTS?
parallel processing, miniaturization and applicable to automation
What is the standard solvent for samples for HTS?
dimethylsulfoxide (DMSO)
–> most compounds are soluble in DMSO
–> DMSO is compatible in small quantites (1%) with most biological targets
Why is DMSO an ideal solvent for compound shipment?
high melting point (18.5C)
What precaution should be taken when using DMSO as a solvent for samples?
DMSO is hygroscopic, samples must be stored in a dry environment.
How are samples for HTS usually stored?
in microtiter plates in a dry, inert, and cold environment (-20°C to 4°C) to prevent compound degradation (oxidation) and precipitation
What kind of a storage system may be required if the compound library is large?
an automated liquid storage system (e.g., REMP AG)
–> store more than a million microtiter plates in a
temperature- and humidity-controlled environment and provide robotized access
Apart from being stored in microtiter plates, how else could compound libraries be stored?
as dry films
What is the advantage of storing compound libraries as dry films?
- reduced likelihood of sample oxidation
- reconstitution in an appropriate solvent
What is the disadvantage of storing compound libraries as dry films?
uncertainty that all of the compounds will be redissolved in the assay medium in the absence of a solubilizer
Explain the principle of mother and daughter plates?
The transition of samples from compound storage to HTS requires the preparation of intermediate dilution plates
Mother plate –> intermediate dilution –> daugher plates —> HTS assay platforms
How is sample stored in the mother plate?
in a frozen DMSO solution
Why is compound management facility needed?
to manage various sources of compound libraries, ranging from historical collection of synthetic compounds to natural product extract libraries
Why barcodes on microtiter plates?
to track them and to provide a unique identification number for each compound
What is the advantage of using barcodes for identification of microtiter plates and their compounds?
inexpensive, offer exceptional security, minimize errors, and provide real-time data exchange
Barcodes are frequently printed with readable text, which allows for simultaneous human and automatic decoding.
True or false
True
What are the 3 types of codes commonly used in HTS?
- Code 39
- Interleaved 2-of-5
- Code 128
What are the characteristics of code 39 and code 128?
incorporate alpha-numeric characters and allow bi-directional scanning
How many characters do Code 39 and 128 encode?
Code 39: 43 digits (does not include a check digit)
Code 128: 128 characters (includes check digits)
What is the characteristic of interleaved 2-of-5?
- incorporates only numeric characters
- includes start and stop codes and an optional check digit
- programmed in two dimensions –> store larger amounts of information
Give an example of inerleaved 2-of-5.
Code PDF417
–> consists of a multi-row symbology and may encode up to 1850 text characters
Give 2 examples of ‘biological barcodes’
- DNA barcode
- Fluorescence barcode
What is DNA barcode?
A DNA barcode is a short, standardized DNA sequence used for identifying species or organisms
What are some usages of DNA barcodes?
- Encoding/decoding of binary info in/from DNA sequences
- Single-cell sequencing technology –> to label individual cells
- evaluating therapeutic potency of DNA-labeled drug formulations by barcode ratio in dead and live cells –> in vivo breast cancer
- for the HTS of delivery vehicles in vivo –> evaluate delivery efficiency
What is meant by ‘fluorescent barcode’?
the use of fluorescent labels or markers to create a unique signature or pattern for identification or tracking purposes.
What are some usages of fluorescent barcode?
- Cell screening by flow cytometry
- Spectra of different fluorescent dyes used as
fluorescence barcodes - High-throughput fluorescence-activated cell
sorting (FACS) of fluorescence- labeled cells - In situ hybridization probe
What is ‘in situ hybridization probe’?
labels a library of target RNAs in intact biological
samples at subcellular resolution
How does PRISM incroportae DNA barcode system to allow screening of large numbers of compounds against large numbers of cell lines?
pooled screening of mixtures of cancer cell
lines by labeling each cell line with 24-nucleotide
barcodes
How are tagged cells in PRISM identified?
through Luminex microsphere system where the number of cells in each cell strain can be accurately quantified
In addition to the 96-well format, what other microtiter plates have been developed?
384-, 762-, 864-, and 1536-well
the 384- and 1536-well formats are now standard
What issues are raised with increasing density of HTS essay?
accuracy of liquid dispensing
The accuracy of common liquid handling equipment in the 96- and 384-well format is limited at low volumes, but 0.5 ml may be dispensed with reasonable
reliability.
True or False
True
What is the normal range of DMSO in most assays?
0.1-1%
How to ensure assay robustness?
- check parameters
–> stability of reagents, assay kinetics, tolerance of solvents - spike samples with known inhibitors in different conc, determine well-to-well CV
- Z’ factor
What does the screen validation ratio (Z’ factor) take into account?
the variability of both the baseline data and maximal response signal.
How is Z’ calculated?
1 - ( 3* SDsignal + 3* SDbaseline)/ (MEANsignal - MEANbaseline)
SDsignal is SD of maximal response unit
SDbaseline is basal response
What value of Z’ is plausible?
computed values <0 indicate implausible
assays while those >0 are plausible
*Z’ factors >0.5 indicate excellent assays
What are the characteristics of HTS?
96- ,384-, 1536-well and higher format
Minimum number of assay steps
No separation (homogeneous)
Minimum number of wash steps
High signal to noise ratio
Amenable to automation
Incubation time < 1 hour
Ambient incubation temperature
Name some HTS assay technologies.
- Radioactive proximity assays
–> scintillation proximity assay (SPA)
–> ScintiPlates; FlashPlates; LeadSeeker - TIme-resolved fluorescence assays
- Fluorescence polarization
- Amplification via chemical rxn
(ALPHAScreen) - Cell-based assays
–> reporter gene; direct mRNA
detection; FLIPR
What are some cell-based assays?
- reporter gene
- direct mRNA detection
- FLIPR
What are some radioactive heterogeneous format assays?
- filtration
- adsorption
- precipitation
- radioimmunoassays
What is the principle of scintillation proximity assay?
- scintillant incorporated into small fluomicrospheres to which target molecules attach
- radioactive molecule binds to target
- scintillant is stimulated to produce light
- light is detected by photomultiplier tube of scintillation counters or CCD imagers
What kind of beads are used in scintillation proximity assays?
polymer beads of ~5 mm diameter are coated with antibodies, streptavidin, receptors or enzymes
What are the limitations of scintillation proximity assay (SPA)?
- sensitivity to colour quench by test compounds
- variable efficiency of scintillation counting due to sedimentation of beads
- limited sensitivity
- long readout times of detection instruments
What are the advantages of scintillation proximity assay?
- assay flexibility
- uses less radioactive label
- convenience
- bead assortment
