2.2 Acquisition
of Complement
Regulatory Proteins by
Leptospira
1. Leptospira cultures (as described in Subheading 2.1).
2. Filtered Complement fixation diluent buffer (CFD): 4 mM
sodium barbitone, 0.145 M NaCl, 0.83 mM MgCl 2 ,
0.25 mM CaCl 2 , pH 7.3.
3. NHS-EDTA: NHS containing 10 mM EDTA pH 8.0 (see Note
2).
4. Glycine buffer: 0.1 M glycine-HCl, pH 2.0.
5. Primary antibodies: anti-FH, anti-C4BP, or anti-vitronectin
produced in mouse, rabbit, goat, or sheep.
6. Secondary antibodies: horseradish peroxidase (HRP) conjugated antibodies with specificity for the antibody species and
isotype of the primary antibody being used (e.g., anti-mouse
IgG-HRP, anti-rabbit IgG-HRP, anti-goat IgG-HRP, or antisheep IgG-HRP).
7. Centrifuge with appropriate adaptors for 50 mL tubes/a
microcentrifuge for 1.5–2.0 mL tubes.
8. Bacterial counting chamber [cell depth: 0.02 mm (1/50 mm)].
9. Water bath/dry block incubator.
2.3 Direct Cleavage
of Complement
Proteins by
Leptospira-Secreted
Proteases
1. Leptospira cultures (as described in Subheading 2.1).
2. A pool of NHS: aliquot and maintain at À80
C until use (see
Note 2).
3. Purified human Complement proteins: keep at À80
C until
use (see Note 3).
4. Primary antibodies: anti-C3, anti-C4, anti-C5, anti-C6, antiC7, anti-C8, or anti-C9 produced in rabbit, goat, or sheep.
5. Secondary antibodies: HRP-conjugated antibodies with specificity for the antibody species and isotype of the primary antibody being used (e.g., anti-rabbit IgG-HRP, anti-goat
IgG-HRP, or anti-sheep IgG-HRP).
6. Refrigerated centrifuge with appropriate adaptors for 50 mL
tubes or for microcentrifuge tubes.
7. Bacterial counting chamber [cell depth: 0.02 mm (1/50 mm)].
8. Water bath/dry block incubator.
9. 0.22 μm sterile syringe filters.
10. A kit to determine the total protein concentration.
2.4 Inhibition
of Complement
Activation by
Proteases Secreted by
Pathogenic Leptospira
1. Leptospira cultures (as described in Subheading 2.1).
2. A pool of NHS: aliquot and maintain at À80
C until use (see
Note 2).
3. Escherichia coli lipopolysaccharide.
4. Human IgG or human IgM.
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Lourdes Isaac and Angela Silva Barbosa
of Complement
Regulatory Proteins by
Leptospira
1. Leptospira cultures (as described in Subheading 2.1).
2. Filtered Complement fixation diluent buffer (CFD): 4 mM
sodium barbitone, 0.145 M NaCl, 0.83 mM MgCl 2 ,
0.25 mM CaCl 2 , pH 7.3.
3. NHS-EDTA: NHS containing 10 mM EDTA pH 8.0 (see Note
2).
4. Glycine buffer: 0.1 M glycine-HCl, pH 2.0.
5. Primary antibodies: anti-FH, anti-C4BP, or anti-vitronectin
produced in mouse, rabbit, goat, or sheep.
6. Secondary antibodies: horseradish peroxidase (HRP) conjugated antibodies with specificity for the antibody species and
isotype of the primary antibody being used (e.g., anti-mouse
IgG-HRP, anti-rabbit IgG-HRP, anti-goat IgG-HRP, or antisheep IgG-HRP).
7. Centrifuge with appropriate adaptors for 50 mL tubes/a
microcentrifuge for 1.5–2.0 mL tubes.
8. Bacterial counting chamber [cell depth: 0.02 mm (1/50 mm)].
9. Water bath/dry block incubator.
2.3 Direct Cleavage
of Complement
Proteins by
Leptospira-Secreted
Proteases
1. Leptospira cultures (as described in Subheading 2.1).
2. A pool of NHS: aliquot and maintain at À80
C until use (see
Note 2).
3. Purified human Complement proteins: keep at À80
C until
use (see Note 3).
4. Primary antibodies: anti-C3, anti-C4, anti-C5, anti-C6, antiC7, anti-C8, or anti-C9 produced in rabbit, goat, or sheep.
5. Secondary antibodies: HRP-conjugated antibodies with specificity for the antibody species and isotype of the primary antibody being used (e.g., anti-rabbit IgG-HRP, anti-goat
IgG-HRP, or anti-sheep IgG-HRP).
6. Refrigerated centrifuge with appropriate adaptors for 50 mL
tubes or for microcentrifuge tubes.
7. Bacterial counting chamber [cell depth: 0.02 mm (1/50 mm)].
8. Water bath/dry block incubator.
9. 0.22 μm sterile syringe filters.
10. A kit to determine the total protein concentration.
2.4 Inhibition
of Complement
Activation by
Proteases Secreted by
Pathogenic Leptospira
1. Leptospira cultures (as described in Subheading 2.1).
2. A pool of NHS: aliquot and maintain at À80
C until use (see
Note 2).
3. Escherichia coli lipopolysaccharide.
4. Human IgG or human IgM.
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Lourdes Isaac and Angela Silva Barbosa