monoclonal antibody anti mouse mbl c  (Hycult Biotech)

Journal: Acta Neuropathologica Communications

Article Title: Targeted deletions of complement lectin pathway genes improve outcome in traumatic brain injury, with MASP-2 playing a major role

doi: 10.1186/s40478-020-01041-1

Figure Lengend Snippet: Summary table

Article Snippet: The brain coronal sections were incubated overnight at 4 °C with primary monoclonal antibody anti-mouse MBL-C (1 µg/ml; Hycult Biotechnology, Uden, The Netherlands) followed by a secondary biotinylated antibody against rat IgG.

Techniques:

Journal: Acta Neuropathologica Communications

Article Title: Targeted deletions of complement lectin pathway genes improve outcome in traumatic brain injury, with MASP-2 playing a major role

doi: 10.1186/s40478-020-01041-1

Figure Lengend Snippet: Experimental design. a WT or KOs mice (including: MASP-2 −/− , MBL −/− , FCN-A −/− , CL-11 −/− , MBL-C −/− , MASP-1/3 −/− and MBL-A −/− ) were subjected to TBI or sham injury. Sensorimotor deficits were assessed weekly by neuroscore and beam walk test. The sum of 4-week performances of each mouse genotype has been used to calculate the health score. b MBL brain presence and residual LP activity in plasma was assed in MASP-2 −/− and WT TBI mice 30′ after surgery. Histopathological analysis was done for MASP-2 −/− and WT mice at 6 weeks after TBI

Article Snippet: The brain coronal sections were incubated overnight at 4 °C with primary monoclonal antibody anti-mouse MBL-C (1 µg/ml; Hycult Biotechnology, Uden, The Netherlands) followed by a secondary biotinylated antibody against rat IgG.

Techniques: Activity Assay

Journal: Acta Neuropathologica Communications

Article Title: Targeted deletions of complement lectin pathway genes improve outcome in traumatic brain injury, with MASP-2 playing a major role

doi: 10.1186/s40478-020-01041-1

Figure Lengend Snippet: Brain MBL-C deposition and plasmatic LP activation 30′ after TBI in WT or MASP-2 −/− mice. a Representative low-magnification images of MBL-C immunolabeling at 30′ after TBI or sham surgery (the cortical edge is outlined in yellow). MBL-C quantification was done over an area of 350 µm from the contusion edge (Fig. ). Scale bars 50 μm. b MBL-C deposition in brains of MASP-2 −/− mice was similar to that of WT. Data is shown as a scatter dot plot, line at mean ± SEM ( n = 2–4). Two-way Anova followed by Sidak’s post hoc test, ** p <0.01 compared with Sham MASP-2 −/− , *** p < 0.001 compared with Sham WT. c In vitro assay for MBL-driven LP activation on mannan—plasma from MASP-2 −/− lack C4 convertase activity, resulting in minimal C4b deposition compared to WT mice. The data is shown as a scatter dot plot, line at mean ± SEM ( n = 2–4), Two-way Anova followed by Sidak’s post hoc test, *** p <0.001 compared with Sham or TBI WT

Article Snippet: The brain coronal sections were incubated overnight at 4 °C with primary monoclonal antibody anti-mouse MBL-C (1 µg/ml; Hycult Biotechnology, Uden, The Netherlands) followed by a secondary biotinylated antibody against rat IgG.

Techniques: Activation Assay, Immunolabeling, In Vitro, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Fibrinogen-specific antibody induces abdominal aortic aneurysm in mice through complement lectin pathway activation

doi: 10.1073/pnas.1315512110

Figure Lengend Snippet: Activity of anti-fibrinogen mAb glycovariants in complement activation. (A) Schematic depiction of different glycan chains linked to asparagine 297 (Asn297) of the CH2 domain of IgG heavy chain. Gal, galactose; GLcNAc, N-acetyl glucosamine; man, mannose. (B) Blotting with Erythrina cristagalli lectin (ECL) detects the level of terminal galactosylation. Coomassie blue-stained gel is shown for protein loading control. (C) Mouse serum (as a source of MBL, at the indicated percentage of dilution) was incubated with plate-bound C9 or G10 mAb (10 μg/mL); MBL-C binding to the mAb was detected with an anti–MBL-C antibody. Data shown represent mean ± SEM of triplicate values derived from three independent experiments. *P < 0.01, **P < 0.001. (D) Aortas from elastase-perfused, G10-reconstituted μMT mice were probed for MBL-C (blue) and G10 (red) deposition. Representative sections showing colocalization of MBL-C and fibrinogen (detected with G10 mAb). Elastic fibers (EF) autofluoresce in green and blue spectra. (Scale bar, 15 μm.) (E) μMT mice were perfused with elastase and immediately injected with 250 μg of isotype control or the indicated mAb. Aortas were harvested at 30 min and examined for MBL-C (red) or C3 (red) deposition as evidence of complement activation. Representative photomicrographs are shown. (Scale bar, 50 μm.) (F) Quantitative analysis of MBL-C binding and C3 deposition in abdominal aortas 30 min after elastase perfusion and mAb transfer. n = 3 aortas per treatment, six to nine sections per aorta. Values represent fold increase in integrated OD (IntDen) ± SEM compared with isotype (iso) control (set at 1). *P < 0.05, **P < 0.01, ***P < 0.002, NS, not significant.

Article Snippet: After washes, the plates were incubated with rat anti-mouse MBL-C monoclonal antibody (0.4 μg/mL; HM 1038, clone 14D12; Hycult Biotech) followed by incubation with anti-rat-HRP (0.16 μg/mL; 112-035-167; Jackson ImmunoResearch Laboratories).

Techniques: Activity Assay, Activation Assay, Staining, Incubation, Binding Assay, Derivative Assay, Injection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Fibrinogen-specific antibody induces abdominal aortic aneurysm in mice through complement lectin pathway activation

doi: 10.1073/pnas.1315512110

Figure Lengend Snippet: Effect of galactose removal on C9 mAb activity. (A) Untreated and β-galactosidase–treated C9 mAbs were blotted with Erythrina cristagalli lectin (ECL) to confirm depletion of terminal galactose residues in C9(G0). Coomassie blue-stained gel is shown for loading control. (B) Binding of MBL-C to untreated C9 and galactose-depleted C9(G0) mAb. Data shown represent mean ± SEM of triplicate values derived from two independent experiments. *P < 0.01. (C) μMT mice were perfused with elastase and administered 250 μg of C9 or C9(G0) mAb i.v. At 30 min, aortas were harvested and examined for MBL-C (red) or C3 (red) deposition as evidence of complement activation. Representative photomicrographs are shown. (Scale bar, 50 μm.) (D) Quantitative analysis of MBL-C and C3 deposition in abdominal aortas 30 min after elastase perfusion and mAb transfer. n = 3 aortas per treatment, six to nine sections per aorta. Values represent fold increase in integrated OD (IntDen) ± SEM compared with isotype control (set at 1). *P = 0.0089, **P = 0.0008. Pathogenicity of C9(G0) mAb in μMT mice. (E) Day 14 AD, expressed as percentage, in WT, untreated or μMT mice treated i.v. with 250 μg of the indicated mAb. Values represent mean ± SEM; each symbol represents an individual mouse. *P < 0.01, **P < 0.001, NS, not significant.

Article Snippet: After washes, the plates were incubated with rat anti-mouse MBL-C monoclonal antibody (0.4 μg/mL; HM 1038, clone 14D12; Hycult Biotech) followed by incubation with anti-rat-HRP (0.16 μg/mL; 112-035-167; Jackson ImmunoResearch Laboratories).

Techniques: Activity Assay, Staining, Binding Assay, Derivative Assay, Activation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Fibrinogen-specific antibody induces abdominal aortic aneurysm in mice through complement lectin pathway activation

doi: 10.1073/pnas.1315512110

Figure Lengend Snippet: The LP contribution to elastase-induced AAA. (A) Mice were perfused with elastase on day 0 and day 14. AD was measured and expressed as increase in percentage (%). Mice deficient in MBL-A and MBL-C (MBL-A/C−/−) were as resistant to elastase-induced AAA as the AP factor B-deficient (fB−/−) mice. Each symbol represents an individual mouse. Values represent mean ± SEM *P < 0.001; NS, not significant. (B) IgG (red) binding to elastase-perfused aorta is independent of MBL-A/C. In contrast, there was minimal properdin (red) deposition in MBL-A/C−/− perfused aorta, whereas properdin was found in abundance in WT perfused aorta. Armenian hamster IgG served as isotype control for anti-properdin mAb (H4). (Scale bar, 50 μM.) (C) Quantitative analysis of properdin deposition in abdominal aortas 30 min after elastase perfusion. n = 3 aortas per genoytpe, six to nine sections per aorta. Values represent fold increase in integrated OD (IntDen) ± SEM in WT compared with MBL-A/C−/− (set at 1). *P = 0.0004. (D) Schematic of the order of events in murine elastase-induced AAA. Elastase perfusion triggers local injury, inducing fibrinolysis/fibrin clot formation and revealing neoepitopes that are recognized by natural antibodies. MBL binds antibodies and the immune complex initiates LP activation and properdin-stabilized convertase (C3bBbP) assembly, with subsequent amplification by the AP. Complement activation leads to the generation of anaphylatoxins (C3a, C5a) that attract neutrophils, setting up the inflammatory cascade and culminating in the eventual aneurysmal development.

Article Snippet: After washes, the plates were incubated with rat anti-mouse MBL-C monoclonal antibody (0.4 μg/mL; HM 1038, clone 14D12; Hycult Biotech) followed by incubation with anti-rat-HRP (0.16 μg/mL; 112-035-167; Jackson ImmunoResearch Laboratories).

Techniques: Binding Assay, Activation Assay, Amplification