(a) Adjust settings and gates by using blank and highest
standards to optimally differentiate between infected
(fluorescing) and noninfected cells.
(b) Plot the amount of fluorescing cells against the virus
concentration and calculate sample concentration from
the linear range of the curve.
(c) Determine the total infective virus content in each fraction using the sample volume of the SXC.
(d) Calculate the recovery in % by comparing the total infective virus amount in the individual fractions with the feed
solution (see Note 45).
3.2.6 Size Determination
Using Dynamic Light
Scattering
1. Transfer 500 μL of the samples into individual semi-micro
cuvettes (see Note 46).
2. Start a new manual measurement in the Zetasizer Nano ZS90
software with the following conditions:
(a) Dispersant refractive index: 1.45.
(b) Viscosity of the dispersant: 0.954 cP.
(c) 90
angle
(d) Number of measurements: 3.
(e) Data processing: multiple narrow modes.
3. Afterward, check the detected size populations and record the
mean values and standard deviations.
4. If necessary, prepare different sample dilutions in PBS to
exclude buffer effects, and repeat the measurement (see Note
47).
3.2.7 Quantification
of Total Protein Amounts
1. Prepare the standard calibration samples according to the manufacturer’s instructions (see Note 48).
2. Mix working reagent A and B in the ratio 50:1 (A:B) in a fresh
50-ml centrifuge tube. Use that solution within 1 h.
3. Transfer 25 μL of each sample and standard into a clear 96-well
flat bottom microplate using two wells per sample (duplicate
measurements).
4. Add 200 mL of the reaction mix to each well, gently shake
plate, and cover it with a lid.
5. Incubate the plate at 37
C for 30 min (see Notes 49 and 50).
6. Use a plate reader to detect the absorbance at 562 nm.
7. Calculate the mean for each duplicate measurement after blank
subtraction.
8. Prepare the standard calibration curve by plotting the blanked
absorbance versus the protein concentration, and use it to
calculate the concentration of your samples (see Notes 51–53).
Viral Nanoplex Vaccine Production
237
standards to optimally differentiate between infected
(fluorescing) and noninfected cells.
(b) Plot the amount of fluorescing cells against the virus
concentration and calculate sample concentration from
the linear range of the curve.
(c) Determine the total infective virus content in each fraction using the sample volume of the SXC.
(d) Calculate the recovery in % by comparing the total infective virus amount in the individual fractions with the feed
solution (see Note 45).
3.2.6 Size Determination
Using Dynamic Light
Scattering
1. Transfer 500 μL of the samples into individual semi-micro
cuvettes (see Note 46).
2. Start a new manual measurement in the Zetasizer Nano ZS90
software with the following conditions:
(a) Dispersant refractive index: 1.45.
(b) Viscosity of the dispersant: 0.954 cP.
(c) 90
angle
(d) Number of measurements: 3.
(e) Data processing: multiple narrow modes.
3. Afterward, check the detected size populations and record the
mean values and standard deviations.
4. If necessary, prepare different sample dilutions in PBS to
exclude buffer effects, and repeat the measurement (see Note
47).
3.2.7 Quantification
of Total Protein Amounts
1. Prepare the standard calibration samples according to the manufacturer’s instructions (see Note 48).
2. Mix working reagent A and B in the ratio 50:1 (A:B) in a fresh
50-ml centrifuge tube. Use that solution within 1 h.
3. Transfer 25 μL of each sample and standard into a clear 96-well
flat bottom microplate using two wells per sample (duplicate
measurements).
4. Add 200 mL of the reaction mix to each well, gently shake
plate, and cover it with a lid.
5. Incubate the plate at 37
C for 30 min (see Notes 49 and 50).
6. Use a plate reader to detect the absorbance at 562 nm.
7. Calculate the mean for each duplicate measurement after blank
subtraction.
8. Prepare the standard calibration curve by plotting the blanked
absorbance versus the protein concentration, and use it to
calculate the concentration of your samples (see Notes 51–53).
Viral Nanoplex Vaccine Production
237