rather than the cell monolayer. Check cellular morphology
under a microscope and incubate a little longer if cells are still
attached or not round-shaped yet.
13. This second incubation step is based on our observation that
HEK293T cells still contain an important amount of HBV
antigens at 72 h post-transfection and ensures complete release
of assembled SVPs into the cell medium.
14. We have observed in our laboratory that some of the SVP
sample always remains in the proximity of the pellet as a fine
layer that is lost if the entire sucrose solution is removed. By
leaving the tubes to rest overnight with a small volume of
sample diluted 1:1 in PBS, the pellet gently self-detaches
from the bottom of the tube resulting in a concentrated SVP
solution that contains at most 10% sucrose.
15. Comparing the migration pattern of intra- and extracellular
antigens on SDS-PAGE under reducing conditions provides
valuable information about protein processing along the secretory pathway. Secreted glycosylated HBV antigens will always
migrate slower than their intracellular counterparts due to
trimming of the N-linked glycan to complex structures (as in
Fig. 3). This process can be further confirmed by digestions
with PNGase F and Endo H [10].
16. At this stage the SVP sample contains about 10% sucrose and can
be loaded on top of the first, 15% sucrose-layer of the sucrose
step gradient without additional dilution (see also Note 13).
17. Samples are dialyzed against diluted PBS to avoid concentration of salts in the antigen sample after lyophilization.
18. Lyophilization of the HBV antigens results in a protein powder
that dissolves easily in water. However, we have observed a
slight loss of antigen recognition by the highly conformationdependent monoclonal antibodies of the Monolisa HBsAg
ULTRA kit, suggesting that some epitopes may be denatured
during this procedure.
19. Serially dilute a protein containing preS1 epitope of known
concentration to generate a standard curve (500–20 ng). Proceed as in Subheading 3.2 above, steps 6–11 [10].
20. Typically, this protocol leads to production of 28–30 μg/g and
3.75–4 μg/g cell fresh weight of HBV-S and HBV-S/
preS1
21–47 SVPs, respectively, of about 10–15% purity, which
is compatible with further analysis of the antigenic properties in
animal models [10, 12]. Due to its simplicity and rapidity, this
purification approach is particularly suitable for primary screening of a large number of antigens. It can also be used as a
precursory step in more sophisticated purification protocols
when production of highly pure SVPs might be required.
HBV Surface Antigens in Mammalian Cells
93
under a microscope and incubate a little longer if cells are still
attached or not round-shaped yet.
13. This second incubation step is based on our observation that
HEK293T cells still contain an important amount of HBV
antigens at 72 h post-transfection and ensures complete release
of assembled SVPs into the cell medium.
14. We have observed in our laboratory that some of the SVP
sample always remains in the proximity of the pellet as a fine
layer that is lost if the entire sucrose solution is removed. By
leaving the tubes to rest overnight with a small volume of
sample diluted 1:1 in PBS, the pellet gently self-detaches
from the bottom of the tube resulting in a concentrated SVP
solution that contains at most 10% sucrose.
15. Comparing the migration pattern of intra- and extracellular
antigens on SDS-PAGE under reducing conditions provides
valuable information about protein processing along the secretory pathway. Secreted glycosylated HBV antigens will always
migrate slower than their intracellular counterparts due to
trimming of the N-linked glycan to complex structures (as in
Fig. 3). This process can be further confirmed by digestions
with PNGase F and Endo H [10].
16. At this stage the SVP sample contains about 10% sucrose and can
be loaded on top of the first, 15% sucrose-layer of the sucrose
step gradient without additional dilution (see also Note 13).
17. Samples are dialyzed against diluted PBS to avoid concentration of salts in the antigen sample after lyophilization.
18. Lyophilization of the HBV antigens results in a protein powder
that dissolves easily in water. However, we have observed a
slight loss of antigen recognition by the highly conformationdependent monoclonal antibodies of the Monolisa HBsAg
ULTRA kit, suggesting that some epitopes may be denatured
during this procedure.
19. Serially dilute a protein containing preS1 epitope of known
concentration to generate a standard curve (500–20 ng). Proceed as in Subheading 3.2 above, steps 6–11 [10].
20. Typically, this protocol leads to production of 28–30 μg/g and
3.75–4 μg/g cell fresh weight of HBV-S and HBV-S/
preS1
21–47 SVPs, respectively, of about 10–15% purity, which
is compatible with further analysis of the antigenic properties in
animal models [10, 12]. Due to its simplicity and rapidity, this
purification approach is particularly suitable for primary screening of a large number of antigens. It can also be used as a
precursory step in more sophisticated purification protocols
when production of highly pure SVPs might be required.
HBV Surface Antigens in Mammalian Cells
93