TITLE: Flaviviruses (West Nile, Zika, Dengue) NS2B/NS3 Fluorescence Dose Response
AUTHORS: Haim Barr, Noa Lahav

[DESCRIPTION]
This is a functional, biochemical assay used to identify treatments for viral infectious diseases related to viral Flaviviridae infection, (specifically West Nile, Zika, and Dengue) and targets the conserved NS2B/NS3 protein.

Utilizing a direct enzyme activity measurement method, the experiment was performed in a 384-well plate reading the fluorescence intensity. This assay tested the mode of action of inhibition.   

It was developed at the Weizmann Institute of Science, as a part of the ASAP Drug Discovery Consortium. 
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Experiment Concentrations (From Stock to Assay) 
 

Assay Buffer
 
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Compound Plate Design for Dose Response:
        Total assay volume: 20 µL
        Compounds top assay concentration: 60 µM
        Dilution factor: 2
        Dose response points: 12
        Number of replicates: 2
        Backfill with DMSO: yes

Compounds Plate Design for 2-Point Assay:
        Total Assay Volume: 16 µL
        Compounds Assay Concentration: 100 µM and 50 µM
        Dilution Factor: 2
        Dose Response Points: 2
        Number of Replicates: 2
        Backfill with DMSO: Yes


[BEFORE_START]
Note: Inhibitor compounds stock concentration is 20 mM. Compounds are pre-dispensed into 384 plates and stored at -200˚C until use.

[STEPS]
SECTION: Determine which Flavivirus is needed and prepare solutions
1. Determine which Flavivirus is needed and prepare solutions based on the materials section.
     ReagentStockLoaded into GNFFinal in assay plateunitsDENV NS2B/NS3217000200100nMZIKV NS2B/NS3225000200100nMWNV NS2B/NS3222000200100nMSubstrate10000 105uMAssay buffer    HEPES pH=7.3201010mMNaCl1005050mMGlycerol5055%Igepal100.050.05%TCEP100011mM

SECTION: Prepare 384-well Plate for experiment
2. OPEN the EQUIcon Software and SELECT the "Flavivirus dispense 7,8 C" Program
SECTION: Prepare 384-well Plate for experiment
3. PRIME the GNF Washer/Dispenser II (GNF) with 3 mL and 3 mL 
SECTION: Prepare 384-well Plate for experiment
4. CONFIRM that the GNF had accurately dispensed Ethanol and Water
SECTION: Prepare 384-well Plate for experiment
4.1. WEIGH the plate and RECORD
SECTION: Prepare 384-well Plate for experiment
4.2. DISPENSE  3 mL and 3 mL into a plate 
SECTION: Prepare 384-well Plate for experiment
4.3. WEIGH the plate and RECORD. Determine if the GNF Washer/Dispenser II had accurately dispensed  3 g and 2.367 g 
SECTION: Prepare 384-well Plate for experiment
5. CONNECT Assay Buffer to 7C and your Flavivirus NS2B/NS3 to position 8C of the GNF Washer/Dispenser II.
SECTION: Prepare 384-well Plate for experiment
5.1. PRIME the GNF with 300 µL and with 300 µL200 nanomolar (nM) respectively. 

SECTION: Prepare 384-well Plate for experiment
6. DISPENSE 10 µL to columns 1 and 23 using the 7C position of the GNF
Note: These columns will be the inhibitor control columns (Containing: substrate + assay buffer + DMSO, no compounds)
SECTION: Prepare 384-well Plate for experiment
7. DISPENSE 10 µL 200 nanomolar (nM) to columns 2 through 22 and column 24 using the 8C position of the GNF.
 Note: 200 nanomolar (nM) is two times the assay concentration. The final concentration of the Flavivirus NS2B/NS3 is 100 nanomolar (nM) during the assay.
Columns 2 and 24 are neutral control columns (Contain: Enzyme + substrate + DMSO, no compounds)

SECTION: Prepare 384-well Plate for experiment
8. CENTRIFUGE 1500 rpm, 1 min  plate to remove bubbles

SECTION: Prepare 384-well Plate for experiment
9. INCUBATE plate 120 min at Room temperature
⚠ Make sure the plate is protected from light!

During Incubation: PREPARE the GNF to dispense the Flavivirus Substrate
SECTION: Prepare 384-well Plate for experiment
9.1. EMPTY 7C of the GNF. 
SECTION: Prepare 384-well Plate for experiment
9.2. WASH 7C tubing in Assay Buffer. Discard used Assay buffer
SECTION: Prepare 384-well Plate for experiment
9.3. PRIME 7C of the GNF with 300 µL 10 micromolar (µM) 

SECTION: Prepare 384-well Plate for experiment
10. DISPENSE 10 µL 10 nanomolar (nM) to Columns 1 through 23 (the full plate)
 Note: 10 nanomolar (nM) is two times the assay concentration. The final concentration of the Flavivirus Substrate is 5 nanomolar (nM) during the assay.
SECTION: Prepare 384-well Plate for experiment
11. CENTRIFUGE 1500 rpm, 1 min  plate to remove bubbles

SECTION: Prepare 384-well Plate for experiment
12. INCUBATE plate for 30 min at Room temperature 
⚠ Make sure the plate is protected from light!

Recommended: Clean GNF during incubation
SECTION: Read Plate Flourescence
13. READ and RECORD the plate Relative fluorescence units (RFU) via the "Flavivirus protocol" on the PHERAstar FS Control Software.
 
 

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