3. Antibiotics (e.g., ampicillin; working concentration:
20–50 μg/ml), depending on the antibiotic resistance carried
by the used vector (see Note 2).
4. Endotoxin-free plasmid DNA purification Kit which ensures
endotoxin level <0.1 EU/μg DNA.
5. PBS, pH 7.4.
6. Culture tubes, flasks, and dishes.
7. 37
C shaking incubator.
8. Refrigerated centrifuge.
9. Pyrogen- or endotoxin-free plastic tubes and tips.
10. Glassware (see Note 3).
2.4 Carriers Activity
Testing In Vivo
1. Purified vaccine plasmid—recombinant expression vector (e.g.,
pCI, Promega) carrying cDNA encoding appropriate antigen
(e.g., hemagglutinin (HA) from influenza virus).
2. Animals to be vaccinated (e.g., mice, chickens).
3. Syringes with needles, and tubes for blood collection (e.g., 1.5
or 2 ml Eppendorf tubes).
4. 96-well flat-bottom plates: MediSorp Surface (Nunc) for
detection of antibodies in chicken sera, MaxiSorp Surface
(Nunc) for mouse sera.
5. Antigens for plates coating.
6. Enzyme-labeled anti-host secondary antibodies: goat antichicken IgY (Fc-specific)-horseradish peroxidase (HRP)
(Pierce/Thermo Scientific), anti-mouse IgG-alkaline phosphatase (AP)-conjugated (Sigma-Aldrich).
7. 3,3
0 ,5,5
0 -Tetramethylbenzidine (TMB) for HRP-based procedure, P-Nitrophenyl-phosphate (pNPP) for AP-based
procedure.
8. PBS, pH 7.4.
9. PBST: 0.05% (v/v) Tween 20 in PBS.
10. 0.1% Tween 20 (v/v) in PBS.
11. Blocking buffer: 2% BSA (v/v) in PBS.
12. 0.5 M H 2 SO 4 , 3 M NaOH.
13. Sealing tape for 96-well plates (ThermoFisher).
14. Multichannel pipettes.
15. Automated microplate washer (optional).
16. Microplate reader.
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20–50 μg/ml), depending on the antibiotic resistance carried
by the used vector (see Note 2).
4. Endotoxin-free plasmid DNA purification Kit which ensures
endotoxin level <0.1 EU/μg DNA.
5. PBS, pH 7.4.
6. Culture tubes, flasks, and dishes.
7. 37
C shaking incubator.
8. Refrigerated centrifuge.
9. Pyrogen- or endotoxin-free plastic tubes and tips.
10. Glassware (see Note 3).
2.4 Carriers Activity
Testing In Vivo
1. Purified vaccine plasmid—recombinant expression vector (e.g.,
pCI, Promega) carrying cDNA encoding appropriate antigen
(e.g., hemagglutinin (HA) from influenza virus).
2. Animals to be vaccinated (e.g., mice, chickens).
3. Syringes with needles, and tubes for blood collection (e.g., 1.5
or 2 ml Eppendorf tubes).
4. 96-well flat-bottom plates: MediSorp Surface (Nunc) for
detection of antibodies in chicken sera, MaxiSorp Surface
(Nunc) for mouse sera.
5. Antigens for plates coating.
6. Enzyme-labeled anti-host secondary antibodies: goat antichicken IgY (Fc-specific)-horseradish peroxidase (HRP)
(Pierce/Thermo Scientific), anti-mouse IgG-alkaline phosphatase (AP)-conjugated (Sigma-Aldrich).
7. 3,3
0 ,5,5
0 -Tetramethylbenzidine (TMB) for HRP-based procedure, P-Nitrophenyl-phosphate (pNPP) for AP-based
procedure.
8. PBS, pH 7.4.
9. PBST: 0.05% (v/v) Tween 20 in PBS.
10. 0.1% Tween 20 (v/v) in PBS.
11. Blocking buffer: 2% BSA (v/v) in PBS.
12. 0.5 M H 2 SO 4 , 3 M NaOH.
13. Sealing tape for 96-well plates (ThermoFisher).
14. Multichannel pipettes.
15. Automated microplate washer (optional).
16. Microplate reader.
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395