the pcDNA™ 3.1(+) plasmid vector is under control of the
cytomegalovirus (CMV) enhancer promoter.
2. Glacial acetic acid is skin corrosive and can cause serious eye
damage. Working with glacial acetic acid requires personal
protective equipment (PPE) and all handling to be performed
in a fume hood.
3. Loosen the lid to prevent the accumulation of steam inside the
bottle and wear heat protective gloves to prevent scalding.
4. Ethidium bromide is a carcinogen which causes DNA mutation; the user must wear PPE and work carefully. Disposal of
used ethidium bromide solution should be in accordance with
institutional regulations. Stock ethidium bromide should be
stored at room temperature, protected from the light.
5. Cool LB broth after autoclaving to room temperature or 4
C
before adding ampicillin to prevent degradation of ampicillin
from the heat. Due to the E protein of flaviviruses being toxic
to bacterial cells, a reduction of ampicillin from the normal
concentration (100 μg/mL) to 50 μg/mL is useful for bacterial
growth.
6. After adding ampicillin, immediately mix the solution by swirling and quickly pour the agar before it sets.
7. Opti-MEM media is required to remove the requirement of
adding fetal bovine serum (FBS) to the cell growth media, as
FBS can significantly affect the subsequent purification and
detection of the VLP.
8. Concentrated HCl is a strong corrosive substance that can
cause serious skin, eye, and respiratory damage. The solution
preparation must be performed in a fume hood with PPE.
9. SDS is acutely toxic via oral and dermal exposure and by
inhalation and can cause skin and eye irritation. The user
must wear a face mask to prevent inhalation of SDS while
weighing the powder. Some protocols recommend weighing
SDS inside a fume hood; however, the user should be aware
this will cause significant air currents that may distribute SDS
around the working environment.
10. This protocol uses a panspecific anti-flavivirus envelope protein
monoclonal antibody that is purified in house from hybridoma
HB112 [14]. Both monoclonal and polyclonal antibodies to
Zika E protein are commercially available.
11. Other suitable secondary antibodies such as a HRP-conjugated
rabbit anti-mouse IgG polyclonal antibody can be substituted
as desired.
12. NaOH is a strong corrosive reagent that can cause skin and eye
irritation. PPE is required.
Zika Virus-Like Particles
201
cytomegalovirus (CMV) enhancer promoter.
2. Glacial acetic acid is skin corrosive and can cause serious eye
damage. Working with glacial acetic acid requires personal
protective equipment (PPE) and all handling to be performed
in a fume hood.
3. Loosen the lid to prevent the accumulation of steam inside the
bottle and wear heat protective gloves to prevent scalding.
4. Ethidium bromide is a carcinogen which causes DNA mutation; the user must wear PPE and work carefully. Disposal of
used ethidium bromide solution should be in accordance with
institutional regulations. Stock ethidium bromide should be
stored at room temperature, protected from the light.
5. Cool LB broth after autoclaving to room temperature or 4
C
before adding ampicillin to prevent degradation of ampicillin
from the heat. Due to the E protein of flaviviruses being toxic
to bacterial cells, a reduction of ampicillin from the normal
concentration (100 μg/mL) to 50 μg/mL is useful for bacterial
growth.
6. After adding ampicillin, immediately mix the solution by swirling and quickly pour the agar before it sets.
7. Opti-MEM media is required to remove the requirement of
adding fetal bovine serum (FBS) to the cell growth media, as
FBS can significantly affect the subsequent purification and
detection of the VLP.
8. Concentrated HCl is a strong corrosive substance that can
cause serious skin, eye, and respiratory damage. The solution
preparation must be performed in a fume hood with PPE.
9. SDS is acutely toxic via oral and dermal exposure and by
inhalation and can cause skin and eye irritation. The user
must wear a face mask to prevent inhalation of SDS while
weighing the powder. Some protocols recommend weighing
SDS inside a fume hood; however, the user should be aware
this will cause significant air currents that may distribute SDS
around the working environment.
10. This protocol uses a panspecific anti-flavivirus envelope protein
monoclonal antibody that is purified in house from hybridoma
HB112 [14]. Both monoclonal and polyclonal antibodies to
Zika E protein are commercially available.
11. Other suitable secondary antibodies such as a HRP-conjugated
rabbit anti-mouse IgG polyclonal antibody can be substituted
as desired.
12. NaOH is a strong corrosive reagent that can cause skin and eye
irritation. PPE is required.
Zika Virus-Like Particles
201