3. Incubate 1 Â 10
9 leptospires in fresh modified EMJH medium
for 4 h at 37
C. Harvest supernatants after centrifugation at
3200–5400 Â g for 25 min at 4
C, and pass through a 0.22 μm
sterile syringe filter.
4. Determine total protein concentration in the supernatants.
5. Incubate 0.25 μg of each purified Complement protein (e.g.,
C3, fragment C3b, C6) or NHS (use a volume corresponding
to 0.25 μg of the selected Complement protein) with 3 μg of
proteins from the leptospiral supernatants for 1 h at 37
C (see
Note 4).
6. Analyze the cleavage products (25 μL of reaction mixture) by
SDS-PAGE and Western blot as described in Subheading 3.2.
After electrophoresis in 12–15% SDS-polyacrylamide gels,
transfer proteins to nitrocellulose membranes for 1 h at
350 mA.
7. Block nonspecific binding sites by incubating the membranes
with Blocking buffer overnight at 4
C.
8. Incubate the membranes with primary antibodies (anti-C3,
anti-C4, anti-C5, anti-C6, anti-C7, anti-C8, or anti-C9)
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.
9. 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.
10. 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.
3.4 Inhibition
of Complement
Activation by
Proteases Secreted by
Pathogenic Leptospira
1. Spin down the cultures at 3200–5400 Â g for 25 min at 4
C,
and wash the pellet three times using PBS. Resuspend in modified EMJH medium, and determine the number of leptospires.
2. Incubate 1 Â 10
9 leptospires in fresh modified EMJH medium
for 4 h at 37
C. Harvest supernatants after centrifugation at
3200–5400 Â g for 25 min at 4
C, and pass through a 0.22 μm
sterile syringe filter.
3. Determine total protein concentration in the supernatants (see
Note 5).
4. Coat 96-well plates with 100 μL (1 μg/well) of Escherichia coli
LPS (for the Alternative Pathway), human IgG or human IgM
(0.4 μg/well; for the Classical Pathway), or mannan from
Complement Resistance Assays
195
9 leptospires in fresh modified EMJH medium
for 4 h at 37
C. Harvest supernatants after centrifugation at
3200–5400 Â g for 25 min at 4
C, and pass through a 0.22 μm
sterile syringe filter.
4. Determine total protein concentration in the supernatants.
5. Incubate 0.25 μg of each purified Complement protein (e.g.,
C3, fragment C3b, C6) or NHS (use a volume corresponding
to 0.25 μg of the selected Complement protein) with 3 μg of
proteins from the leptospiral supernatants for 1 h at 37
C (see
Note 4).
6. Analyze the cleavage products (25 μL of reaction mixture) by
SDS-PAGE and Western blot as described in Subheading 3.2.
After electrophoresis in 12–15% SDS-polyacrylamide gels,
transfer proteins to nitrocellulose membranes for 1 h at
350 mA.
7. Block nonspecific binding sites by incubating the membranes
with Blocking buffer overnight at 4
C.
8. Incubate the membranes with primary antibodies (anti-C3,
anti-C4, anti-C5, anti-C6, anti-C7, anti-C8, or anti-C9)
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.
9. 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.
10. 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.
3.4 Inhibition
of Complement
Activation by
Proteases Secreted by
Pathogenic Leptospira
1. Spin down the cultures at 3200–5400 Â g for 25 min at 4
C,
and wash the pellet three times using PBS. Resuspend in modified EMJH medium, and determine the number of leptospires.
2. Incubate 1 Â 10
9 leptospires in fresh modified EMJH medium
for 4 h at 37
C. Harvest supernatants after centrifugation at
3200–5400 Â g for 25 min at 4
C, and pass through a 0.22 μm
sterile syringe filter.
3. Determine total protein concentration in the supernatants (see
Note 5).
4. Coat 96-well plates with 100 μL (1 μg/well) of Escherichia coli
LPS (for the Alternative Pathway), human IgG or human IgM
(0.4 μg/well; for the Classical Pathway), or mannan from
Complement Resistance Assays
195