before washing and addition AEC substrate solutions
(BD Biosciences).
10. Carefully monitor spot formation and stop the reaction by
gently washing the plate with tap water when development
appears to slow.
11. Take the base off the plates and air-dry in dark.
12. Spots are counted by ImmunoSpot S6 ELISPOT analyzer
(CTL, USA) and analyzed using ImmunoSpot Software.
13. Spots in negative control wells are subtracted from the number
of spots in sample wells and the results are expressed as antibody secreting cells (ASC) per 10
6 BM cells or spots per 10
6
splenocytes.
4 Notes
1. Most of the freely available tools are suitable for this purpose,
given that ideal codon optimization is not necessary for a DNA
vaccine. Antigen sequence selection plays a more important
role and has to be tried for each individual case either empirically or facilitated by computer modeling.
2. Because there are inconsistent data on effects of presence and
frequency of CG motifs in DNA vaccine vectors, we do not
routinely check or change this in our DNA vaccine constructs.
For potential human trials, researchers can select vectors with
the kanamycin resistance gene rather than ampicillin to avoid
potential side effects.
3. In our experience, TOP10 is good for cloning and Giga-prep of
most of our DNA vaccine constructs. If some constructs contain difficult sequences (e.g., secondary structure or high AT or
GC content), other strains can be tried (e.g., DH5α or Stbl).
Also, some constructs might show a degree of toxicity to
bacteria (e.g., the JEV construct in our hands), which will
yield lower amounts of plasmid DNA.
4. If using other brands kit for purification, follow the standard
manual. Manual prep using the PEG6000 method is also possible, but endotoxin levels should be checked. Certain brands
may have more RNA contamination in the final plasmid product, so agarose gel analysis is also recommended.
5. A careful consideration should be given to the mice legs to
make sure the procedure was done properly without extreme
pain and other side effects.
6. C57BL/6 mice are more susceptible to JEV virus infection
than BALB/c mice if challenge studies are desired.
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Lei Li et al.
(BD Biosciences).
10. Carefully monitor spot formation and stop the reaction by
gently washing the plate with tap water when development
appears to slow.
11. Take the base off the plates and air-dry in dark.
12. Spots are counted by ImmunoSpot S6 ELISPOT analyzer
(CTL, USA) and analyzed using ImmunoSpot Software.
13. Spots in negative control wells are subtracted from the number
of spots in sample wells and the results are expressed as antibody secreting cells (ASC) per 10
6 BM cells or spots per 10
6
splenocytes.
4 Notes
1. Most of the freely available tools are suitable for this purpose,
given that ideal codon optimization is not necessary for a DNA
vaccine. Antigen sequence selection plays a more important
role and has to be tried for each individual case either empirically or facilitated by computer modeling.
2. Because there are inconsistent data on effects of presence and
frequency of CG motifs in DNA vaccine vectors, we do not
routinely check or change this in our DNA vaccine constructs.
For potential human trials, researchers can select vectors with
the kanamycin resistance gene rather than ampicillin to avoid
potential side effects.
3. In our experience, TOP10 is good for cloning and Giga-prep of
most of our DNA vaccine constructs. If some constructs contain difficult sequences (e.g., secondary structure or high AT or
GC content), other strains can be tried (e.g., DH5α or Stbl).
Also, some constructs might show a degree of toxicity to
bacteria (e.g., the JEV construct in our hands), which will
yield lower amounts of plasmid DNA.
4. If using other brands kit for purification, follow the standard
manual. Manual prep using the PEG6000 method is also possible, but endotoxin levels should be checked. Certain brands
may have more RNA contamination in the final plasmid product, so agarose gel analysis is also recommended.
5. A careful consideration should be given to the mice legs to
make sure the procedure was done properly without extreme
pain and other side effects.
6. C57BL/6 mice are more susceptible to JEV virus infection
than BALB/c mice if challenge studies are desired.
420
Lei Li et al.