19. Refrigerated centrifuge.
20. Mini PROTEAN Tetra system.
21. Semidry transblot system.
22. Heater.
2.4 Detection
of Native HBV Antigens
1. ELISA—Monolisa HBsAg ULTRA kit (Bio-Rad) (see Note 5).
2. Multichannel pipette.
3. 96-well plate absorbance reader.
2.5 Antigen
Purification
1. Sucrose solutions: 15%, 20%, 25%, 35%, 45%, and 60% sucrose
in water. All solutions are heat-sterilized and stored at 4
C (see
Note 6).
2. Ultracentrifuge (SW32Ti and SW41Ti rotors).
3. Ultracentrifuge tubes.
4. Vortex.
5. Dialysis membranes, 10 MWCO.
6. Liquid nitrogen.
7. Lyophilizer.
3 Methods
Perform all steps at room temperature, unless otherwise indicated.
3.1 Small-Scale HBV
Antigen Expression
1. Seed 5 Â 10
5 HEK293T cells in 6-well plates; add 2 mL of
complete DMEM and grow in a CO 2 incubator for 24 h.
2. Replace cell media with 2 mL of fresh DMEM and transfect
cells with either pCi-S or pCi-S/preS1
21–47 plasmids, using a
mixture of 200 μL TB, 6 μL TR, and 2 μg plasmid DNA per
well (see Note 7). Keep nontransfected HEK293T cells as a
control (see Note 8).
3.2 Biochemical
Characterization
of HBV Antigens
1. Harvest the HEK293T cells and supernatants at 48 h
posttransfection.
2. Briefly collect cells by centrifugation at 1200 Â g for 2 min then
incubate resulting pellets with lysis buffer, for 30 min, on ice.
3. Clarify lysates by centrifugation at 10,000 Â g, for 10 min, at
4
C. Determine the total protein concentration using the BCA
kit, as indicated by the supplier.
4. To monitor the N-glycosylation status of HBV proteins, treat
samples either with PNG-ase F or EndoH enzymes, following
the protocol provided by the supplier (see Note 9).
5. Heat-denature sample volumes (equivalent of 30 μg of total
protein/lane) and molecular standards (equivalent of 2 μg protein/lane) in the presence of either nonreducing or reducing
SDS sample loading buffer, at 95
C, for 5 min (see Note 10).
HBV Surface Antigens in Mammalian Cells
87
20. Mini PROTEAN Tetra system.
21. Semidry transblot system.
22. Heater.
2.4 Detection
of Native HBV Antigens
1. ELISA—Monolisa HBsAg ULTRA kit (Bio-Rad) (see Note 5).
2. Multichannel pipette.
3. 96-well plate absorbance reader.
2.5 Antigen
Purification
1. Sucrose solutions: 15%, 20%, 25%, 35%, 45%, and 60% sucrose
in water. All solutions are heat-sterilized and stored at 4
C (see
Note 6).
2. Ultracentrifuge (SW32Ti and SW41Ti rotors).
3. Ultracentrifuge tubes.
4. Vortex.
5. Dialysis membranes, 10 MWCO.
6. Liquid nitrogen.
7. Lyophilizer.
3 Methods
Perform all steps at room temperature, unless otherwise indicated.
3.1 Small-Scale HBV
Antigen Expression
1. Seed 5 Â 10
5 HEK293T cells in 6-well plates; add 2 mL of
complete DMEM and grow in a CO 2 incubator for 24 h.
2. Replace cell media with 2 mL of fresh DMEM and transfect
cells with either pCi-S or pCi-S/preS1
21–47 plasmids, using a
mixture of 200 μL TB, 6 μL TR, and 2 μg plasmid DNA per
well (see Note 7). Keep nontransfected HEK293T cells as a
control (see Note 8).
3.2 Biochemical
Characterization
of HBV Antigens
1. Harvest the HEK293T cells and supernatants at 48 h
posttransfection.
2. Briefly collect cells by centrifugation at 1200 Â g for 2 min then
incubate resulting pellets with lysis buffer, for 30 min, on ice.
3. Clarify lysates by centrifugation at 10,000 Â g, for 10 min, at
4
C. Determine the total protein concentration using the BCA
kit, as indicated by the supplier.
4. To monitor the N-glycosylation status of HBV proteins, treat
samples either with PNG-ase F or EndoH enzymes, following
the protocol provided by the supplier (see Note 9).
5. Heat-denature sample volumes (equivalent of 30 μg of total
protein/lane) and molecular standards (equivalent of 2 μg protein/lane) in the presence of either nonreducing or reducing
SDS sample loading buffer, at 95
C, for 5 min (see Note 10).
HBV Surface Antigens in Mammalian Cells
87