title: Functional assessment of rat complement pathway activities and quantification of soluble C5b-9 in an experimental model of renal ischemia/reperfusion injury.
abstract: There is a growing interest in the monitoring of complement activation, not only in clinical settings but also in experimental models. However, for rodents only a limited number of tools are available to assess complement activity and activation. Here we describe three ELISAs for the measurement of rat classical (CP), MB-lectin (LP) and alternative (AP) pathway activities in serum and plasma. Moreover, we optimised a soluble C5b-9 (sC5b-9) ELISA for the detection of low level complement activation in rat. We determined the conditions for correct sample handling and showed that the assays had low inter- and intra-assay variation. We applied these assays to monitor complement activation in an experimental rat model of renal ischemia/reperfusion injury. We did not observe major complement consumption following reperfusion in CP or LP, and only minor AP consumption at 24h post reperfusion. However, MBL depletion prior to ischemia/reperfusion using a monoclonal antibody, transiently and specifically inhibited 75% of LP activity and ameliorated the AP consumption at 24h. To further assess complement activation during renal IRI, we monitored serum sC5b-9 and found that it was only significantly increased 72h post-reperfusion, but not when rats were pre-treated with anti-MBL or after sham surgery. In conclusion the described assays enable sensitive, reproducible and comprehensive assessment of complement activation in experimental rat models.
authors: Dept of Nephrology, Leiden University Medical Center, Leiden, The Netherlands; Hycult Biotech, Uden, The Netherlands.
authors: J
authors: J
authors: Kotimaa
authors: Dept of Nephrology, Leiden University Medical Center, Leiden, The Netherlands.
authors: P
authors: P
authors: van der Pol
authors: Hycult Biotech, Uden, The Netherlands.
authors: S
authors: S
authors: Leijtens
authors: Dept of Nephrology, Leiden University Medical Center, Leiden, The Netherlands.
authors: N
authors: N
authors: Klar-Mohammad
authors: Hycult Biotech, Uden, The Netherlands.
authors: G
authors: G
authors: Schilders
authors: Dept of Nephrology, Leiden University Medical Center, Leiden, The Netherlands.
authors: M R
authors: MR
authors: Daha
authors: Hycult Biotech, Uden, The Netherlands.
authors: H
authors: H
authors: Rutjes
authors: Dept of Nephrology, Leiden University Medical Center, Leiden, The Netherlands. Electronic address: Kooten@lumc.nl.
authors: C
authors: C
authors: van Kooten
mesh_terms: D000818:Animals; D019138:Antibodies, Blocking / Q000008:administration & dosage; D015938:Complement Membrane Attack Complex / Q000378:metabolism*; D003170:Complement Pathway, Alternative / Q000276:immunology*; D003171:Complement Pathway, Classical / Q000276:immunology*; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D007668:Kidney / Q000098:blood supply* / Q000378:metabolism / Q000473:pathology; D008297:Male; D037601:Mannose-Binding Lectin / Q000037:antagonists & inhibitors / Q000276:immunology*; D015588:Observer Variation; D051381:Rats; D011917:Rats, Inbred Lew; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000276:immunology*
chemical_list: D019138:Antibodies, Blocking; D015938:Complement Membrane Attack Complex; D037601:Mannose-Binding Lectin
keywords: Complement pathway activity; Functional complement activity ELISA; MBL; Renal ischemia/reperfusion injury; sC5b-9