temperature for a maximum of 30 min, and then centrifuge at
4
C for 600–700 Â g for 15 min. After withdrawn the serum,
split it in aliquots and immediately transfer to À70
C. Store
serum aliquots at this temperature until use, and avoid repeated
freeze/thaw cycles.
3. To avoid ongoing in vitro Complement activation, maintain
buffers on ice until use. Thaw serum samples and purified
Complement proteins/fragments immediately before use
keeping the vials on ice as well.
4. It is recommended to evaluate the specific proteolytic activity in
the Leptospira supernatants by using different concentrations
of proteins from supernatants of Leptospira cultures
(0.6–0.06 μg) and higher concentrations (3 μg) of Complement proteins.
5. If necessary, check for the presence of LPS by using Limulus
Amebocyte Lysate kit.
Acknowledgments
This work was supported by the Sa ˜o Paulo Research Foundation
(FAPESP) grants # 2017/12924-3 (LI and ASB) and 2018/
12896-2 (ASB) and the National Council for Scientific and Technological Development (CNPq) grants # 307780/2017-1
(LI) and 305114/2017-4 (ASB).
References
1. Hajishengallis G, Reis ES, Mastellos DC et al
(2017) Novel mechanisms and functions of
complement.
Nat
Immunol
18
(12):1288–1298. https://doi.org/10.1038/
ni.3858
2. Johnson RC, Muschel LH (1965) Antileptospiral activity of normal serum. J Bacteriol
89:1625–1626
3. Barbosa AS, Isaac L (2018) Complement
immune evasion by spirochetes. Curr Top
Microbiol Immunol 415:215–238. https://
doi.org/10.1007/82_2017_47
4. Meri T, Murgia R, Stefanel P et al (2005)
Regulation of complement activation at the
C3-level by serum resistant leptospires. Microb
Pathog 39:139–147
5. Barbosa AS, Abreu PA, Vasconcellos SA et al
(2009) Immune evasion of Leptospira species
by acquisition of human complement regulator
C4BP. Infect Immun 77:1137–1143. https://
doi.org/10.1128/IAI.01310-08
6. Castiblanco-Valencia MM, Fraga TR, Silva LB
et al (2012) Leptospiral immunoglobulin-like
proteins interact with human complement regulators factor H, FHL-1, FHR-1, and C4BP. J
Infect Dis 205:995–1004. https://doi.org/
10.1093/infdis/jir875
7. da Silva LB, Miragaia LS, Breda LC et al (2015)
Pathogenic Leptospira species acquire factor H
and vitronectin via the surface protein LcpA.
Infect Immun 83:888–897. https://doi.org/
10.1128/IAI.02844-14
8. Fraga TR, Courrol DS, Castiblanco-Valencia
MM et al (2014) Immune evasion by pathogenic Leptospira strains: the secretion of proteases that directly cleave complement
proteins. J Infect Dis 209:876–886. https://
doi.org/10.1093/infdis/jit569
9. Amamura TA, Fraga TR, Vasconcellos SA et al
(2017) Pathogenic Leptospira secreted proteases target the membrane attack complex: a
potential role for thermolysin in complement
inhibition. Front Microbiol 8:958. https://
doi.org/10.3389/fmicb.2017.00958
Complement Resistance Assays
197
4
C for 600–700 Â g for 15 min. After withdrawn the serum,
split it in aliquots and immediately transfer to À70
C. Store
serum aliquots at this temperature until use, and avoid repeated
freeze/thaw cycles.
3. To avoid ongoing in vitro Complement activation, maintain
buffers on ice until use. Thaw serum samples and purified
Complement proteins/fragments immediately before use
keeping the vials on ice as well.
4. It is recommended to evaluate the specific proteolytic activity in
the Leptospira supernatants by using different concentrations
of proteins from supernatants of Leptospira cultures
(0.6–0.06 μg) and higher concentrations (3 μg) of Complement proteins.
5. If necessary, check for the presence of LPS by using Limulus
Amebocyte Lysate kit.
Acknowledgments
This work was supported by the Sa ˜o Paulo Research Foundation
(FAPESP) grants # 2017/12924-3 (LI and ASB) and 2018/
12896-2 (ASB) and the National Council for Scientific and Technological Development (CNPq) grants # 307780/2017-1
(LI) and 305114/2017-4 (ASB).
References
1. Hajishengallis G, Reis ES, Mastellos DC et al
(2017) Novel mechanisms and functions of
complement.
Nat
Immunol
18
(12):1288–1298. https://doi.org/10.1038/
ni.3858
2. Johnson RC, Muschel LH (1965) Antileptospiral activity of normal serum. J Bacteriol
89:1625–1626
3. Barbosa AS, Isaac L (2018) Complement
immune evasion by spirochetes. Curr Top
Microbiol Immunol 415:215–238. https://
doi.org/10.1007/82_2017_47
4. Meri T, Murgia R, Stefanel P et al (2005)
Regulation of complement activation at the
C3-level by serum resistant leptospires. Microb
Pathog 39:139–147
5. Barbosa AS, Abreu PA, Vasconcellos SA et al
(2009) Immune evasion of Leptospira species
by acquisition of human complement regulator
C4BP. Infect Immun 77:1137–1143. https://
doi.org/10.1128/IAI.01310-08
6. Castiblanco-Valencia MM, Fraga TR, Silva LB
et al (2012) Leptospiral immunoglobulin-like
proteins interact with human complement regulators factor H, FHL-1, FHR-1, and C4BP. J
Infect Dis 205:995–1004. https://doi.org/
10.1093/infdis/jir875
7. da Silva LB, Miragaia LS, Breda LC et al (2015)
Pathogenic Leptospira species acquire factor H
and vitronectin via the surface protein LcpA.
Infect Immun 83:888–897. https://doi.org/
10.1128/IAI.02844-14
8. Fraga TR, Courrol DS, Castiblanco-Valencia
MM et al (2014) Immune evasion by pathogenic Leptospira strains: the secretion of proteases that directly cleave complement
proteins. J Infect Dis 209:876–886. https://
doi.org/10.1093/infdis/jit569
9. Amamura TA, Fraga TR, Vasconcellos SA et al
(2017) Pathogenic Leptospira secreted proteases target the membrane attack complex: a
potential role for thermolysin in complement
inhibition. Front Microbiol 8:958. https://
doi.org/10.3389/fmicb.2017.00958
Complement Resistance Assays
197