4. Collect the cells by centrifugation (9000 Â g for 10 min), and
wash five times with 0.5–1.0 mL of CFD. The last wash fraction
must be collected.
5. Elute proteins bound to the surface of leptospires by adding
100 μL of 0.1 M glycine-HCl to the sedimented bacteria. After
resuspension, incubate for 5 min.
6. Collect the supernatant by centrifugation (9000 Â g for
10 min).
7. Add 5 μL of sample buffer (5Â concentrated) to 20 μL of the
eluate fractions as well as 20 μL of the last wash fraction, and
incubate at 95
C for 5 min for complete disruption of molecular interactions. Apply the samples, and run the gel at a
constant current of 25 mA until the bromophenol blue dye
reaches the bottom of the gel. Turn off the power supply, and
keep the gel in running buffer until ready to transfer.
8. Soak nitrocellulose membrane and wet the sponges and filter
papers in Transfer buffer. Assemble sandwich as follows: black
side transfer—sponge—filter paper—gel—membrane—filter
paper—sponge—red side transfer. Remove air bubbles
between layers by rolling a pipette over the “sandwich.” Fill
apparatus with Transfer buffer. For a Mini-Transblot, transfer
for 1 h at 350 mA at 4
C.
9. Block nonspecific binding sites by incubating the membranes
with Blocking buffer overnight at 4
C.
10. Incubate the membranes with primary antibodies (anti-FH,
anti-C4BP, or anti-vitronectin) diluted according to manufactures’ recommendations in Blocking buffer for 1 h at room
temperature on a rocking platform. Wash the membranes three
times with Wash buffer for 5 min each.
11. Add the secondary peroxidase-conjugated antibodies diluted
according to manufactures’ recommendations in Blocking
buffer for 1 h at room temperature. Wash the membranes
three times with Wash buffer for 5 min each.
12. Incubate the membranes with a chemiluminescent reagent
according to manufactures’ recommendations for 1 min to
detect proteins bound to peroxidase-conjugated antibodies.
Positive signals can be detected by either a chemiluminescence
imaging system or using a X-ray film in a darkroom.
3.3 Direct Cleavage
of Complement
Proteins by
Leptospira-Secreted
Proteases
1. Prepare SDS polyacrylamide gel prior to the assay as described
in Subheading 3.2.
2. Spin down Leptospira cultures at 3200–5400 Â g for 25 min at
4
C, and wash the pellet three times with PBS. Resuspend in
modified EMJH medium, and determine the number of
leptospires.
194
Lourdes Isaac and Angela Silva Barbosa
wash five times with 0.5–1.0 mL of CFD. The last wash fraction
must be collected.
5. Elute proteins bound to the surface of leptospires by adding
100 μL of 0.1 M glycine-HCl to the sedimented bacteria. After
resuspension, incubate for 5 min.
6. Collect the supernatant by centrifugation (9000 Â g for
10 min).
7. Add 5 μL of sample buffer (5Â concentrated) to 20 μL of the
eluate fractions as well as 20 μL of the last wash fraction, and
incubate at 95
C for 5 min for complete disruption of molecular interactions. Apply the samples, and run the gel at a
constant current of 25 mA until the bromophenol blue dye
reaches the bottom of the gel. Turn off the power supply, and
keep the gel in running buffer until ready to transfer.
8. Soak nitrocellulose membrane and wet the sponges and filter
papers in Transfer buffer. Assemble sandwich as follows: black
side transfer—sponge—filter paper—gel—membrane—filter
paper—sponge—red side transfer. Remove air bubbles
between layers by rolling a pipette over the “sandwich.” Fill
apparatus with Transfer buffer. For a Mini-Transblot, transfer
for 1 h at 350 mA at 4
C.
9. Block nonspecific binding sites by incubating the membranes
with Blocking buffer overnight at 4
C.
10. Incubate the membranes with primary antibodies (anti-FH,
anti-C4BP, or anti-vitronectin) diluted according to manufactures’ recommendations in Blocking buffer for 1 h at room
temperature on a rocking platform. Wash the membranes three
times with Wash buffer for 5 min each.
11. Add the secondary peroxidase-conjugated antibodies diluted
according to manufactures’ recommendations in Blocking
buffer for 1 h at room temperature. Wash the membranes
three times with Wash buffer for 5 min each.
12. Incubate the membranes with a chemiluminescent reagent
according to manufactures’ recommendations for 1 min to
detect proteins bound to peroxidase-conjugated antibodies.
Positive signals can be detected by either a chemiluminescence
imaging system or using a X-ray film in a darkroom.
3.3 Direct Cleavage
of Complement
Proteins by
Leptospira-Secreted
Proteases
1. Prepare SDS polyacrylamide gel prior to the assay as described
in Subheading 3.2.
2. Spin down Leptospira cultures at 3200–5400 Â g for 25 min at
4
C, and wash the pellet three times with PBS. Resuspend in
modified EMJH medium, and determine the number of
leptospires.
194
Lourdes Isaac and Angela Silva Barbosa