title: Functional characterization of the lectin pathway of complement in human serum.
abstract: Mannan-binding lectin (MBL) is a major initiator of the lectin pathway (LP) of complement. Polymorphisms in exon 1 of the MBL gene are associated with impaired MBL function and infections. Functional assays to assess the activity of the classical pathway (CP) and the alternative pathway (AP) of complement in serum are broadly used in patient diagnostics. We have now developed a functional LP assay that enables the specific quantification of autologous MBL-dependent complement activation in human serum. Complement activation was assessed by ELISA using coated mannan to assess the LP and coated IgM to assess the CP. Normal human serum (NHS) contains IgG, IgA and IgM antibodies against mannan, as shown by ELISA. These antibodies are likely to induce CP activation. Using C1q-blocking and MBL-blocking mAb, it was confirmed that both the LP and the CP contribute to complement activation by mannan. In order to quantify LP activity without interference of the CP, LP activity was measured in serum in the presence of C1q-blocking Ab. Activation of serum on coated IgM via the CP resulted in a dose-dependent deposition of C1q, C4, C3, and C5b-9. This activation and subsequent complement deposition was completely inhibited by the C1q-blocking mAb 2204 and by polyclonal Fab anti-C1q Ab. Evaluation of the LP in the presence of mAb 2204 showed a strong dose-dependent deposition of C4, C3, and C5b-9 using serum from MBL-wildtype (AA) but not MBL-mutant donors (AB or BB genotype), indicating that complement activation under these conditions is MBL-dependent and C1q-independent. Donors with different MBL genotypes were identified using a newly developed oligonucleotide ligation assay (OLA) for detection of MBL exon 1 polymorphisms. We describe a novel functional assay that enables quantification of autologous complement activation via the LP in full human serum up to the formation of the membrane attack complex. This assay offers novel possibilities for patient diagnostics as well as for the study of disease association with the LP.
authors.affiliation: Department of Nephrology, C3P, Leiden University Medical Center, P.O. Box 9600, 2300 RC, Leiden, The Netherlands. a.roos@lumc.nl
authors.forename: Anja
authors.initials: A
authors.lastname: Roos
authors.forename: Lee H
authors.initials: LH
authors.lastname: Bouwman
authors.forename: Jeric
authors.initials: J
authors.lastname: Munoz
authors.forename: Tahlita
authors.initials: T
authors.lastname: Zuiverloon
authors.forename: Maria C
authors.initials: MC
authors.lastname: Faber-Krol
authors.forename: Francien C
authors.initials: FC
authors.lastname: Fallaux-van den Houten
authors.forename: Ngaisah
authors.initials: N
authors.lastname: Klar-Mohamad
authors.forename: C Erik
authors.initials: CE
authors.lastname: Hack
authors.forename: Marcel G
authors.initials: MG
authors.lastname: Tilanus
authors.forename: Mohamed R
authors.initials: MR
authors.lastname: Daha
mesh_terms: D000906:Antibodies / Q000097:blood / Q000276:immunology; D015922:Complement C1q / Q000276:immunology; D015938:Complement Membrane Attack Complex / Q000502:physiology; D003170:Complement Pathway, Alternative; D003165:Complement System Proteins / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008351:Mannans / Q000276:immunology; D037601:Mannose-Binding Lectin / Q000097:blood / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D009154:Mutation
chemical_list: D000906:Antibodies; D015938:Complement Membrane Attack Complex; D008351:Mannans; D037601:Mannose-Binding Lectin; D015922:Complement C1q; D003165:Complement System Proteins