Methods

3.1. Reagent This protocol describes the preparation of working dilutions of

Preparation NA-Star® Substrate and drugs (NAIs) for use in consecutive proto cols. Note: the NA-Star® Buffer and NA-Star® Accelerator do not require further preparation.

1. Prepare a 1:1,000 working dilution of NA-Star® Substrate as follows:

• Briefly spin the tube containing undiluted NA-Star® Substrate in a microcentrifuge to settle the contents.

• To make 20 ml of diluted substrate, for example, add 20 ml NA-Star® Substrate to 19.98 ml NA-Star® Buffer.

Dilute appropriate amounts of substrate depending on the number of viruses to be tested (see Note 1).

2. Prepare 10 ml of 20 mM stock solutions of zanamivir and oseltamivir as follows:

• Weigh 66.5 mg of zanamivir (MW: 332.31) or 70.66 mg GR121167X (MW: 350.331); 56.9 mg of oseltamivir carboxylate (MW: 284.35) or 77.3 mg of d-tartrate salt of oseltamivir carboxylate (MW: 386.44) (see Note 2).

• Transfer weighed NAI powder into a 15-ml conical tube, labeled appropriately.

• Add 10 ml of molecular grade distilled water to each tube containing respective NAIs.

• Agitate each tube until the NAIs are dissolved. This will be the initial stock solution of 20 mM for each NAI.

• Filter solution through 0.22-mm filter.

• Aliquot 20 mM NAI solution in 1 ml volumes and store at -20°C.

3. Prepare a 50 mM working solution of each NAI by diluting the 20 mM stock solution of NAI as follows.

• Add 25 ml of the 20 mM NAI stock solution into 9.975 ml of molecular grade distilled water.

• Aliquot the 50 mM stock solution in 1 ml volumes.

4. Preparation of 2x half-log10 dilutions of the NAIs as follows:

• Serially dilute the 50 mM working concentrations of zanamivir and oseltamivir carboxylate in NA-Star® Buffer as shown in Table 1. If higher (or lower) volumes of the inhibitors are prepared, the respective volumes of buffer and NAI shown in Table 1 can be scaled up (or down) by a common factor.

• Transfer the NAI dilutions to columns 1-10 ofa 12-column reagent reservoir. Add NA-Star® Buffer to columns 11 and 12. Seal the reservoir with an adhesive plate seal.

• The diluted NAIs can be stored at 4°C for up to 1 month.

The background activity of reagents used in a particular luminom-eter (plate reader) and 96-well plate should be determined prior to testing viruses in the chemiluminescent NI assay. Background activity needs to be determined only once for each plate reader/ 96-well plate combination and the value determined can be used for all consecutive runs using the respective plate reader/96-well

3.2. Determining Background Activity for the Plate Reader

Table 1

Preparation of 2x half-log10 dilutions of NAI

Dilution no.

Buffer volume (ml)

NAI volume

Resulting NAI concentration (nM)

Final NAI

concentration (nM)a

1

21.6

900 ml of 50 mM NAI

2,000

1,000

2

16.2

7.5 ml from dilution 1

633

316

3

16.2

7.5 ml from dilution 2

200

100

4

16.2

7.5 ml from dilution 3

63.4

31.7

5

16.2

7.5 ml from dilution 4

20

10.0

6

16.2

7.5 ml from dilution 5

6.3

3.18

7

16.2

7.5 ml from dilution 6

2

1.01

8

16.2

7.5 ml from dilution 7

0.64

0.32

9

16.2

7.5 ml from dilution 8

0.20

0.10

10

16.2

7.5 ml from dilution 9

0.06

0.03

a The final concentration of NAI in the assay reaction volume, which accounts for the twofold dilution (25 ml of the virus dilution combined with 25 ml of NAI). The final concentration does not account for 10 ml of NA-Star substrate and 60 ml of NA-Star accelerator. However, some laboratories do account for the 10 ml of NA-Star substrate when calculating the final concentration of NAI in the reaction mix a The final concentration of NAI in the assay reaction volume, which accounts for the twofold dilution (25 ml of the virus dilution combined with 25 ml of NAI). The final concentration does not account for 10 ml of NA-Star substrate and 60 ml of NA-Star accelerator. However, some laboratories do account for the 10 ml of NA-Star substrate when calculating the final concentration of NAI in the reaction mix plate combination. This will allow calculation of the approximate NA activity required to be within the linear range for the chemilu-minescent NI assay.

1. Prepare to determine the background activity for a particular plate reader and 96-well plate as follows:

• Remove NA-Star® Buffer and NA-Star® Accelerator from 4°C storage and allow reagents to reach room temperature.

• Prepare 1:1,000 dilution of NA-Star® Substrate (see Subheading 3.1, step 1).

2. Add 50 ml NA-Star® Buffer to all wells of the 96-well plate.

3. Using a 200 ml 8-channel pipette or electronic repeater pipette, add 10 ml of the 1:1,000 dilution of NA-Star® Substrate to each of the 12 columns.

• Since all wells are identical and only contain NA-Star® Buffer, the same tips can be used to dispense the substrate. Ensure that the pipette tips are in the bottom of each well and monitor the volume in the pipette tip to guarantee correct dispensation of the substrate. After the addition of the substrate, tap the plate gently on each side to mix the buffer and substrate.

4. Incubate the 96-well plate containing NA-Star® Buffer and NA-Star® Substrate for 30 min at room temperature.

5. Set up the plate reader in accordance with the manufacturer's instructions.

6. Place the 96-well plate onto the plate reader and measure luminescence.

7. Average the luminescence values determined for each of the 96 wells of the plate. This is the background activity of NA-Star® Buffer and NA-Star® Substrate in that type of 96-well plate. Multiply this value by 60. This is the target value for NA activity. Selecting a virus dilution in the NA activity portion of the assay which corresponds to this target value will provide a signal-to-background ratio of approximately 30:1 (NA activity: background). The linear range of NA activity for the chemiluminescent NI assay is between 10:1 and 40:1.

3.3. Neuraminidase Measuring the neuraminidase (NA) activity of each virus prior to Activity Assay performing the inhibition portion of the assay (see Subheading 3.5

for Determining Virus and Note 3) improves the reproducibility of IC50 data and allows Dilution the user to select a working virus dilution that provides NA activity within the linear range for the NI assay. Too low or too high level of NA activity may skew IC50 values.

1. Prepare to perform NA activity for a total of eight viruses on a single 96-well plate as follows:

• Remove test and reference viruses from -80°C storage and allow to thaw at room temperature in a class II biological safety cabinet.

• Remove NA-Star® Buffer and NA-Star® Accelerator from 4°C storage and place at room temperature.

• Prepare 1:1,000 dilution ofNA-Star® Substrate in NA-Star® Buffer for the appropriate number of plates to be tested for NA activity and NI assays (see Subheading 3.1, step 1).

• Set up the plate reader in accordance with the manufacturer's instructions.

2. Perform a series of twofold dilutions of test and reference viruses in NA-Star® Buffer (see Fig. 1), within a class II biological safety cabinet, as follows:

• In a 96-well white plate, add 80 ml NA-Star® Buffer to all wells in column 1.

• Add 50 ml NA-Star® Buffer to the remaining wells of the plate (columns 2-12). Column 12 will be a blank control.

• Prepare an initial 1:5 dilution of the batch of eight viruses by adding 20 ml of the virus to 80 ml NA-Star® Buffer

Discn rd final 50 in I

^J) NA-Star buffer

Well

-

2

3

4

5

6

7

8

9

10

11

12

Virus Dilution

5

10

20

40

80

160

320

64«

12S0

2560

5120

Background

Fig. 1. Twofold serial dilution of test and reference viruses (8 viruses per plate).

in the well in column 1, rows A to H, using one row per virus.

• Using an 8-channel 200-ml multichannel pipette set at 50 ml volume, perform serial twofold dilutions from columns 1 to 11. Pipette the virus suspension in column 1 up and down (three times) and transfer 50 ml from the wells in column 1 to the wells in column 2. Repeat this for columns 2-11 and discard the last 50 ml from column 11. Column 12 should only contain 50 ml of NA-Star® Buffer as a blank control (background).

3. Using an 8-channel 200-ml pipette, add 10 ml of NA-Star® Substrate (diluted 1:1,000) to each well, starting from column 12 toward column 1 (see Note 4).

• If using an 8-channel 200-ml electronic repeater pipette, aspirate 120 ml of the NA-Star® Substrate into each pipette tip (enough to dispense 12 times). Visually inspect the pipette tips to ensure that they hold equal the volume of the substrate. Dispense 10 ml of NA-Star® Substrate to each column, starting from column 12 toward column 1.

Ensure that the pipette tips touch the bottom of each well to guarantee correct dispensation of the substrate.

• After the addition of the NA-Star® Substrate, tap the plate gently on each side to mix the virus and substrate.

4. Incubate the plates containing virus and substrate at room temperature (~20-22°C) for 30 min.

5. Set up the plate reader in accordance with the manufacturer's instructions.

6. Following 30 min incubation, place the 96-well plate on the plate reader and measure NA activity (see Note 5).

7. Determine the well with the NA activity (light signal) which corresponds to the target level of NA activity (as determined in Subheading 3.2) for the plate reader/96-well plate combination being used. The virus dilution which is equal to the target signal should be selected for the NI assay.

• For example: If the background activity is 250 relative luminescence units (RLU), 60 times this background is 15,000 RLU, therefore the virus dilution with the NA activity closest to 15,000 RLU should be selected for the NI assay.

3.4. Diluting Viruses

Viruses to be tested in the chemiluminescent NI assay are appropriately diluted prior to the assay, to allow NA activity to be measured within the linear range for the assay.

Perform virus dilution in a class II biological safety cabinet as follows:

1. Using the dilution factor determined in the NA activity assay, dilute each virus to be tested in NA-Star® Buffer, in 1.5 ml centrifuge tubes as shown in Table 2. Prepare a volume of 400 ml of each diluted virus for each NAI to be tested.

2. Vortex centrifuge tubes containing diluted virus suspension for about 5 s and briefly spin down in microcentrifuge.

3. Transfer each diluted virus from centrifuge tubes to corresponding section of a 12-channel V-bottom dilution reservoir.

4. Only add eight viruses to each of the 12-channel reservoirs as only eight viruses are assayed per 96-well plate. Note: If testing is done in duplicate, only four viruses could be tested per plate.

5. Table 2 provides a summary of volumes of virus and buffer needed to prepare dilutions similar to those made for the NA activity assay (see Notes 6-8).

3.5. Chemiluminescent A total of eight viruses may be assayed on a single 96-well white Neuraminidase plate, one virus per row. Inclusion ofNAI-sensitive and NAI-resistant

Inhibition Assay reference viruses is recommended (see Subheading 2.3, step 2).

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