8. Laminar flow hood.
9. For large-scale production: 1700 cm
2 ribbed-surface roller
bottles and Roll-In CO 2 control incubator.
2.2 Cell Transfection
1. Dissolve plasmids pCi-S (encoding for the wild-type HBV-S
protein) and pCi-S/preS1
21–47 (encoding for the chimeric
HBV S/L protein) in 10 mM Tris–HCl, pH 8.5 at final concentrations higher than 1 μg DNA/μL (see Note 2).
2. Transfection reagent (TR): 1 mg/mL polyethylenimine (PEI),
pH 7.
3. Transfection buffer (TB): Opti-MEM I-GlutaMAX-I.
2.3 Detection
of Denatured HBV
Antigens
1. Cell lysis buffer: 0.01 M Tris–HCl, pH 7.5, 0.002 M EDTA,
0.150 M NaCl, 0.5% Triton X-100 and protease inhibitor
cocktail (1Â) (see Note 3).
2. Reducing SDS sample loading buffer (5Â): 0.25 M Tris–HCl
(pH 6.8), 10% SDS, 25% β-mercaptoethanol, 0.1% bromophenol blue, 50% glycerol. Store aliquots at À20
C.
3. Nonreducing SDS sample loading buffer (5Â): 0.25 M Tris–
HCl (pH 6.8), 10% SDS, 0.1% bromophenol blue, 50% glycerol. Store aliquots at À20
C.
4. SDS 10% polyacrylamide (PAA) gels.
5. SDS-PAA gel electrophoresis (SDS-PAGE) running buffer:
0.025 M Tris–HCl, pH 8.2, 0.190 M glycine, 0.1% SDS.
6. Peptide: N-glycosidase F (PNG-ase F) and Endoglycosidase H
(Endo H) (see Note 4).
7. Nitrocellulose membrane.
8. Western-blot semidry transfer buffer: 0.025 M Tris–HCl,
0.192 M glycine, 20% methanol.
9. Phosphate buffer saline (PBS; 10Â): 1.4 M NaCl, 0.027 M
KCl, 0.1 M Na 2 HPO 4 , 0.018 M KH 2 PO 4 , pH 7.4.
10. Western-blot blocking solution: 10% nonfat milk in PBS (1Â).
11. Western-blot washing solution: 0.1% Tween in PBS (1Â).
12. Primary mouse anti-preS1 antibody, secondary anti-mouseHRP antibody.
13. Antibody dilution solution: 1% nonfat milk, 0.1% Tween in
PBS (1Â).
14. Enhanced Chemiluminescence detection kit.
15. Purified HBV-L protein for the standard curve.
16. BCA protein assay kit.
17. Prestained molecular weight standards.
18. Autoradiography films.
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Mihaela-Olivia Dobrica et al.
9. For large-scale production: 1700 cm
2 ribbed-surface roller
bottles and Roll-In CO 2 control incubator.
2.2 Cell Transfection
1. Dissolve plasmids pCi-S (encoding for the wild-type HBV-S
protein) and pCi-S/preS1
21–47 (encoding for the chimeric
HBV S/L protein) in 10 mM Tris–HCl, pH 8.5 at final concentrations higher than 1 μg DNA/μL (see Note 2).
2. Transfection reagent (TR): 1 mg/mL polyethylenimine (PEI),
pH 7.
3. Transfection buffer (TB): Opti-MEM I-GlutaMAX-I.
2.3 Detection
of Denatured HBV
Antigens
1. Cell lysis buffer: 0.01 M Tris–HCl, pH 7.5, 0.002 M EDTA,
0.150 M NaCl, 0.5% Triton X-100 and protease inhibitor
cocktail (1Â) (see Note 3).
2. Reducing SDS sample loading buffer (5Â): 0.25 M Tris–HCl
(pH 6.8), 10% SDS, 25% β-mercaptoethanol, 0.1% bromophenol blue, 50% glycerol. Store aliquots at À20
C.
3. Nonreducing SDS sample loading buffer (5Â): 0.25 M Tris–
HCl (pH 6.8), 10% SDS, 0.1% bromophenol blue, 50% glycerol. Store aliquots at À20
C.
4. SDS 10% polyacrylamide (PAA) gels.
5. SDS-PAA gel electrophoresis (SDS-PAGE) running buffer:
0.025 M Tris–HCl, pH 8.2, 0.190 M glycine, 0.1% SDS.
6. Peptide: N-glycosidase F (PNG-ase F) and Endoglycosidase H
(Endo H) (see Note 4).
7. Nitrocellulose membrane.
8. Western-blot semidry transfer buffer: 0.025 M Tris–HCl,
0.192 M glycine, 20% methanol.
9. Phosphate buffer saline (PBS; 10Â): 1.4 M NaCl, 0.027 M
KCl, 0.1 M Na 2 HPO 4 , 0.018 M KH 2 PO 4 , pH 7.4.
10. Western-blot blocking solution: 10% nonfat milk in PBS (1Â).
11. Western-blot washing solution: 0.1% Tween in PBS (1Â).
12. Primary mouse anti-preS1 antibody, secondary anti-mouseHRP antibody.
13. Antibody dilution solution: 1% nonfat milk, 0.1% Tween in
PBS (1Â).
14. Enhanced Chemiluminescence detection kit.
15. Purified HBV-L protein for the standard curve.
16. BCA protein assay kit.
17. Prestained molecular weight standards.
18. Autoradiography films.
86
Mihaela-Olivia Dobrica et al.