5. Aspirate or decant solution from wells and discard the liquid.
Wash wells three times with 200 μl of PBS.
6. Add 100 μl of hamster sera (pre-immune or immune sera,
1:64,000 dilution in PFB) and incubate for 1 h at 37
C (see
Note 15).
7. Aspirate or decant solution from wells and discard the liquid.
Wash wells three times with 200 μl of PBS.
8. Add 100 μl of HRP-conjugated goat anti-Syrian hamster IgG
(1:5000 dilution in PFB) and incubate for 30 min at RT.
9. Aspirate or decant solution from wells and discard the liquid.
Wash wells three times with 200 μl of PBS.
10. Add 100 μl of 3,3
0 ,5,5
0 -TMB substrate solution. Incubate plate
for 15 min at RT in the dark (see Note 16).
11. Read the absorbance of each well at 655 nm with an ELISA
plate reader.
12. The antibody response can be expressed as the relative antiantigen level, determined by subtracting the absorbance of the
pre-immune sera (baseline or background read) from the
immune sera collected at different time points (Fig. 2).
3.5 Evaluation
of Kidney Colonization
by Culture
1. Pulverize the kidney by passing it through a 3 ml syringe
directly into a 15 ml tube containing 10 ml semisolid EMJH
supplemented with 100 μg/ml 5-FU (see Note 17).
2. Make a 1:100 dilution into a new 15 ml tube containing 10 ml
semisolid EMJH supplemented with 5-FU.
3. Incubate tube at 30
C for 4 weeks.
4. Check cultures weekly by dark-field microscopy for up to
28 days. A positive culture appears in 7–14 days. A Dinger’s
ring may become visible ~0.5 cm under the surface of the agar.
3.6 Evaluation
of Blood and Tissue
Colonization by qPCR
1. Extract DNA from kidney and liver tissues. Dice 50–80 mg of
the kidney or liver. Place the diced tissue in a 1.5 ml microcentrifuge tube with 500 μl PBS (see Note 18). Homogenize
tissue with tissue disrupter for 1 min at 5 movements per
second. Transfer volume corresponding to 25 mg of tissue
(see Note 19) into a microcentrifuge tube and perform the
DNA extraction by using the DNeasy Blood and Tissue Kit
(Qiagen) following the manufacturer’s instructions. Elute
DNA in 100 μl elution buffer.
2. Extract DNA from 100 μl blood collected on EDTA using the
same DNA extraction kit (see Note 20). Elute the DNA in
100 μl elution buffer.
3. Extract DNA from 5 ml L. interrogans culture at a density of
4 Â 10
5 spirochetes/ml using the same DNA extraction kit,
264
Karen V. Evangelista and Kristel Lourdault
Wash wells three times with 200 μl of PBS.
6. Add 100 μl of hamster sera (pre-immune or immune sera,
1:64,000 dilution in PFB) and incubate for 1 h at 37
C (see
Note 15).
7. Aspirate or decant solution from wells and discard the liquid.
Wash wells three times with 200 μl of PBS.
8. Add 100 μl of HRP-conjugated goat anti-Syrian hamster IgG
(1:5000 dilution in PFB) and incubate for 30 min at RT.
9. Aspirate or decant solution from wells and discard the liquid.
Wash wells three times with 200 μl of PBS.
10. Add 100 μl of 3,3
0 ,5,5
0 -TMB substrate solution. Incubate plate
for 15 min at RT in the dark (see Note 16).
11. Read the absorbance of each well at 655 nm with an ELISA
plate reader.
12. The antibody response can be expressed as the relative antiantigen level, determined by subtracting the absorbance of the
pre-immune sera (baseline or background read) from the
immune sera collected at different time points (Fig. 2).
3.5 Evaluation
of Kidney Colonization
by Culture
1. Pulverize the kidney by passing it through a 3 ml syringe
directly into a 15 ml tube containing 10 ml semisolid EMJH
supplemented with 100 μg/ml 5-FU (see Note 17).
2. Make a 1:100 dilution into a new 15 ml tube containing 10 ml
semisolid EMJH supplemented with 5-FU.
3. Incubate tube at 30
C for 4 weeks.
4. Check cultures weekly by dark-field microscopy for up to
28 days. A positive culture appears in 7–14 days. A Dinger’s
ring may become visible ~0.5 cm under the surface of the agar.
3.6 Evaluation
of Blood and Tissue
Colonization by qPCR
1. Extract DNA from kidney and liver tissues. Dice 50–80 mg of
the kidney or liver. Place the diced tissue in a 1.5 ml microcentrifuge tube with 500 μl PBS (see Note 18). Homogenize
tissue with tissue disrupter for 1 min at 5 movements per
second. Transfer volume corresponding to 25 mg of tissue
(see Note 19) into a microcentrifuge tube and perform the
DNA extraction by using the DNeasy Blood and Tissue Kit
(Qiagen) following the manufacturer’s instructions. Elute
DNA in 100 μl elution buffer.
2. Extract DNA from 100 μl blood collected on EDTA using the
same DNA extraction kit (see Note 20). Elute the DNA in
100 μl elution buffer.
3. Extract DNA from 5 ml L. interrogans culture at a density of
4 Â 10
5 spirochetes/ml using the same DNA extraction kit,
264
Karen V. Evangelista and Kristel Lourdault