2 Materials
2.1 Vaccine
Preparation
1. Phosphate-buffered saline (PBS): 1.5 mM KH 2 PO 4 ,
155.2 mM NaCl, 2.7 mM Na 2 HPO 4 ·7H 2 O, and pH 7.2.
Add about 100 ml water to a glass beaker. Weigh 210 mg of
KH 2 PO 4 , 9 g of NaCl, and 726 mg of Na 2 HPO 4 ·7H 2 O and
transfer to the beaker. Add water to bring volume to 1 l. Mix
and adjust pH to 7.2 with HCl. Autoclave and then store at
room temperature (RT).
2. 0.1–1 mg/ml purified leptospiral protein in PBS (see Note 1).
3. Complete Freund’s adjuvant (CFA) (see Notes 2 and 3).
4. Incomplete Freund’s adjuvant (IFA).
5. 5 ml glass syringes.
6. Three-way plastic stopcock with Luer lock.
Fig. 1 Survival of LigA-immunized hamsters after lethal challenge. Groups of
hamsters were immunized subcutaneously three times with LigA (100 μg), PBS,
or PBS with adjuvant. Animals were challenged intraperitoneally with 1 Â 10
4
L. interrogans serovar Copenhageni strain Fiocruz L1-130 at day 0 and were
monitored daily for 28 days [12]
Fig. 2 IgG response to immunization with purified recombinant LigA protein.
Hamster sera were collected weekly during the immunization protocol. Anti-LigA
antibody levels (IgG) were measured in triplicate by ELISA. For each sample, the
IgG level is read at OD 655, and the pre-immune read is subtracted to it. Each data
point represents the mean of IgG response of groups of hamsters immunized
with LigA, PBS, or PBS with adjuvant at different time points. Error bars indicate
standard deviation. Vertical dotted lines show the immunization days
Leptospirosis Vaccine Studies in Hamsters
259
2.1 Vaccine
Preparation
1. Phosphate-buffered saline (PBS): 1.5 mM KH 2 PO 4 ,
155.2 mM NaCl, 2.7 mM Na 2 HPO 4 ·7H 2 O, and pH 7.2.
Add about 100 ml water to a glass beaker. Weigh 210 mg of
KH 2 PO 4 , 9 g of NaCl, and 726 mg of Na 2 HPO 4 ·7H 2 O and
transfer to the beaker. Add water to bring volume to 1 l. Mix
and adjust pH to 7.2 with HCl. Autoclave and then store at
room temperature (RT).
2. 0.1–1 mg/ml purified leptospiral protein in PBS (see Note 1).
3. Complete Freund’s adjuvant (CFA) (see Notes 2 and 3).
4. Incomplete Freund’s adjuvant (IFA).
5. 5 ml glass syringes.
6. Three-way plastic stopcock with Luer lock.
Fig. 1 Survival of LigA-immunized hamsters after lethal challenge. Groups of
hamsters were immunized subcutaneously three times with LigA (100 μg), PBS,
or PBS with adjuvant. Animals were challenged intraperitoneally with 1 Â 10
4
L. interrogans serovar Copenhageni strain Fiocruz L1-130 at day 0 and were
monitored daily for 28 days [12]
Fig. 2 IgG response to immunization with purified recombinant LigA protein.
Hamster sera were collected weekly during the immunization protocol. Anti-LigA
antibody levels (IgG) were measured in triplicate by ELISA. For each sample, the
IgG level is read at OD 655, and the pre-immune read is subtracted to it. Each data
point represents the mean of IgG response of groups of hamsters immunized
with LigA, PBS, or PBS with adjuvant at different time points. Error bars indicate
standard deviation. Vertical dotted lines show the immunization days
Leptospirosis Vaccine Studies in Hamsters
259