Saccharomyces cerevisiae (1 μg/well; for the Lectin Pathway) in
carbonate buffer overnight at 4
C.
5. Before each of the following steps, wash the plates three times
with Wash buffer.
6. Add 200 μL of Blocking buffer to each well and incubate for
2 h at 37
C.
7. Use NHS as a source of Complement proteins. Use NHS
diluted (1:7–1:16) in cold AP buffer to measure Alternative
Pathway activation or NHS diluted (1:50 to 1:120) in cold
GVB
++ to assess Classical and Lectin Pathways activation.
8. Incubate diluted NHS samples with leptospiral culture supernatants (3 μg of total secreted proteins) in 1.5 mL microcentrifuge tubes for 1 h at 37
C. As a negative control, incubate
samples of diluted NHS with PBS under similar conditions.
9. Transfer the reactions to the coated plates (with LPS,
IgG/IgM, or mannan), and incubate for 1 h at 37
C.
10. Evaluate Complement activation by measuring C3b deposition
(Alternative Pathway) or C4b deposition (Classical and Lectin
Pathways) by using polyclonal antibodies against human C3 or
human C4, respectively, both diluted 1:5000 (or according to
the manufactures’ recommendations) for 1 h at 37
C.
11. Wash the plates three times with Wash buffer, and incubate
with peroxidase-conjugated anti-IgG (diluted according to the
manufactures’ recommendations) for 1 h at 37
C. Prepare
OPD solution (4 mg in 10 mL citrate buffer: 0.1 M
Na 3 C 6 H 5 O 7 , 0.2 M NaH 2 PO 4 , pH 5.0.) containing 5 μL of
30% H 2 O 2 . H 2 O 2 should be added to solution immediately
before use. Add 100 μL/well of OPD containing H 2 O 2 .
12. Stop the reaction by adding 50 μL/well of 4 M H 2 SO 4 .
13. Determine the absorbance using a spectrophotometer at
492 nm. Results are expressed as O.D.
14. Repeat the experiments at least three times to compare C3b or
C4b deposition in the presence or absence of leptospiral
secreted proteases (NHS treated only with PBS).
4 Notes
1. Leptospires may also be cultivated in EMJH medium supplemented with Difco™ Leptospira Enrichment EMJH (Becton
Dickinson), which contains albumin, polysorbate 80, and additional growth factors.
2. For obtention of NHS, harvest blood in dry tubes without
additives or with a separator gel. Keep the blood at room
196
Lourdes Isaac and Angela Silva Barbosa
carbonate buffer overnight at 4
C.
5. Before each of the following steps, wash the plates three times
with Wash buffer.
6. Add 200 μL of Blocking buffer to each well and incubate for
2 h at 37
C.
7. Use NHS as a source of Complement proteins. Use NHS
diluted (1:7–1:16) in cold AP buffer to measure Alternative
Pathway activation or NHS diluted (1:50 to 1:120) in cold
GVB
++ to assess Classical and Lectin Pathways activation.
8. Incubate diluted NHS samples with leptospiral culture supernatants (3 μg of total secreted proteins) in 1.5 mL microcentrifuge tubes for 1 h at 37
C. As a negative control, incubate
samples of diluted NHS with PBS under similar conditions.
9. Transfer the reactions to the coated plates (with LPS,
IgG/IgM, or mannan), and incubate for 1 h at 37
C.
10. Evaluate Complement activation by measuring C3b deposition
(Alternative Pathway) or C4b deposition (Classical and Lectin
Pathways) by using polyclonal antibodies against human C3 or
human C4, respectively, both diluted 1:5000 (or according to
the manufactures’ recommendations) for 1 h at 37
C.
11. Wash the plates three times with Wash buffer, and incubate
with peroxidase-conjugated anti-IgG (diluted according to the
manufactures’ recommendations) for 1 h at 37
C. Prepare
OPD solution (4 mg in 10 mL citrate buffer: 0.1 M
Na 3 C 6 H 5 O 7 , 0.2 M NaH 2 PO 4 , pH 5.0.) containing 5 μL of
30% H 2 O 2 . H 2 O 2 should be added to solution immediately
before use. Add 100 μL/well of OPD containing H 2 O 2 .
12. Stop the reaction by adding 50 μL/well of 4 M H 2 SO 4 .
13. Determine the absorbance using a spectrophotometer at
492 nm. Results are expressed as O.D.
14. Repeat the experiments at least three times to compare C3b or
C4b deposition in the presence or absence of leptospiral
secreted proteases (NHS treated only with PBS).
4 Notes
1. Leptospires may also be cultivated in EMJH medium supplemented with Difco™ Leptospira Enrichment EMJH (Becton
Dickinson), which contains albumin, polysorbate 80, and additional growth factors.
2. For obtention of NHS, harvest blood in dry tubes without
additives or with a separator gel. Keep the blood at room
196
Lourdes Isaac and Angela Silva Barbosa