Journal: bioRxiv

Article Title: Early Binding of Anti-Amyloid Antibodies to CAA Drives Complement Activation, Inflammation and ARIA in Mice

doi: 10.64898/2026.03.04.709591

Figure Lengend Snippet: a, Schematic of 7-week treatment in 16.5-month-old APP/PS1dE9;hAPOE4 mice (n = 7-8/group) with 3D6 or IgG2a control mAb. b, Prussian blue-labeled hemosiderin deposits in cortex indicating microhemorrhage (asterisks). Scale bar: 50 µm. c, Quantification of Prussian blue–positive area (%) in the whole sagittal brain section; Mann-Whitney test, p < 0.0005. d, Amylo-Glo (AG)–positive fibrillar amyloid area (%) in cortex; unpaired t-test, p < 0.0005. e, Prussian blue staining with eosin counterstain in cortex showing hemosiderin deposits (asterisks) and red-orange cytoplasm of extravasated RBCs (arrows). Scale bar: 100 µm. f, Immunostaining with anti-mouse IgG2a fluorescently-tagged secondary antibody, C1q and AG in brain sections from 3D6- and IgG2a-treated mice. Scale bar: 50 µm. Representative immunofluorescence staining in the g, cortex and h, cerebellum showing C1q and activated C3 fragments C3b/iC3b/C3c deposition near AG-positive plaques (asterisks) and vascular amyloid (arrow), Scale bar in g & h : 250 µm. Quantification of % immunoreactivity (IR) for i, C1q; j, C1q + CD31 + AG + colocalized area; k, activated C3 fragments C3b/iC3b/C3c; and l, activated C3 fragments + CD31 + AG + colocalization in cerebellum. m, Levels of complement C3 in the plasma following 7-weeks of passive immunization. Unpaired t-test in j, k and Mann-Whitney test for i, l . *p<0.05 and **p<0.005, ***p<0.0005. Data are expressed as mean ± SEM.

Article Snippet: Following washes in PBS, sections were blocked either in 10% normal goat serum (Gibco, #16210064), or heat inactivated goat serum (Novus Biologicals #S13110H), or normal donkey serum (Sigma, #S30-M) for 1 hour at room temperature followed by overnight 4°C incubation with primary antibodies against C1q (1:500, Abcam #ab182451), C3b/iC3b/C3c (1:300, HycultBiotech #HM1078), C5b-9 (1:300, Bioss #bs-2673R), CD31 (1:500, R&D Systems #AF3628), C3 (1:200, HycultBiotech #HM1045), GFAP (1:1000, Abcam # ab5804), MMR/CD206 (1:300,R&D #AF2535) MMP-9 (1:500, Bioss #bs-0397R), Iba-1 (1:1000; Dako, Wako #019-19741), Iba-1 (1:500, Synaptic Systems, #234308), CD68 (1:500, Bio-Rad #MCA1957), S97 Aβ (pan-Aβ rabbit polyclonal, 1:2000, gift Dr. Dominic Walsh, BWH, USA), CD31 (1:500, R&D #AF3628), Collagen-IV (1:300, Abcam #6586), Alexa Fluor 488 anti-β-Amyloid, aggregated antibody (1: 200, clone A17171C, Biolegend # 856508), Von Willebrand Factor Antibody (1:500, Novus Biologicals #NB600-586).

Techniques: Control, Labeling, MANN-WHITNEY, Staining, Immunostaining, Immunofluorescence, Clinical Proteomics

Journal: bioRxiv

Article Title: Early Binding of Anti-Amyloid Antibodies to CAA Drives Complement Activation, Inflammation and ARIA in Mice

doi: 10.64898/2026.03.04.709591

Figure Lengend Snippet: a, Schematic of anti-Aβ immunization with 500 µg/wk 3D6 IgG2a mAb and an IgG2a isotype control for 13 weeks from 17 to 20 months in aged male APP/PS1dE9;hAPOE4 AD-like mouse model. For baseline data, baseline control mice were used. For spontaneous hemorrhage detection, 1x PBS-injected aged controls were used. b, T2*-weighted FLASH MRI of 20-month-old male APP/PS1dE9;hApoE4 mice after 13 weeks of passive immunization with 3D6 mAb (500 µg/week), showing cerebral microbleeds as hypointense spots (arrowheads), compared to 1× PBS–treated aged controls. c, Frequency of cerebral microbleeds detected by T2* MRI in 3D6–treated (n = 3) and PBS-treated (n = 4) mice. d, Quantification of Prussian blue–positive (% hemosiderin-stained) area in IgG2a and 3D6–treated mice. Unpaired t-test: **p < 0.005, ***p < 0.0005. Data represent mean ± SEM. e, Representative Prussian blue–stained sections showing hemosiderin deposits (arrowheads, with selected examples in insets) in cortex, hippocampus, thalamus, and cerebellum from 1× PBS (n=4), IgG2a isotype control (n = 5), and 3D6 IgG2a–treated mice (n = 5). Scale bar: 200 µm. f, Immunofluorescence staining in the cerebellum showing 3D6 mAb localization (anti-Ms IgG2a) and C1q deposition at sites of vascular and parenchymal amyloid labeled with Amylo-Glo (AG). g, Detection of activated complement fragments C3b/iC3b/C3c at vascular amyloid sites associated with 3D6 mAb. h, Localization of membrane attack complex (MAC/C5b-9) along vascular amyloid (arrows) and plaques (asterisk) in 3D6–treated mice. Minimal complement staining is observed in IgG2a-treated controls. Scale bars: f–h, 25 µm.

Article Snippet: Following washes in PBS, sections were blocked either in 10% normal goat serum (Gibco, #16210064), or heat inactivated goat serum (Novus Biologicals #S13110H), or normal donkey serum (Sigma, #S30-M) for 1 hour at room temperature followed by overnight 4°C incubation with primary antibodies against C1q (1:500, Abcam #ab182451), C3b/iC3b/C3c (1:300, HycultBiotech #HM1078), C5b-9 (1:300, Bioss #bs-2673R), CD31 (1:500, R&D Systems #AF3628), C3 (1:200, HycultBiotech #HM1045), GFAP (1:1000, Abcam # ab5804), MMR/CD206 (1:300,R&D #AF2535) MMP-9 (1:500, Bioss #bs-0397R), Iba-1 (1:1000; Dako, Wako #019-19741), Iba-1 (1:500, Synaptic Systems, #234308), CD68 (1:500, Bio-Rad #MCA1957), S97 Aβ (pan-Aβ rabbit polyclonal, 1:2000, gift Dr. Dominic Walsh, BWH, USA), CD31 (1:500, R&D #AF3628), Collagen-IV (1:300, Abcam #6586), Alexa Fluor 488 anti-β-Amyloid, aggregated antibody (1: 200, clone A17171C, Biolegend # 856508), Von Willebrand Factor Antibody (1:500, Novus Biologicals #NB600-586).

Techniques: Control, Injection, Staining, Immunofluorescence, Labeling, Membrane

Journal: bioRxiv

Article Title: Early Binding of Anti-Amyloid Antibodies to CAA Drives Complement Activation, Inflammation and ARIA in Mice

doi: 10.64898/2026.03.04.709591

Figure Lengend Snippet: a, Representative immunofluorescence staining in the cerebellum area showing activated C3 fragments (C3b/iC3b/C3c) and TCC/MAC complex (C5b-9) along with fibrillar amyloid (Amylo-Glo dye, AG) in IgG2a isotype and 3D6 mAb passively immunized mice. Arrows indicate iC3b or C5b-9 immunoreactivity along CAA + vessels stained with AG. Scale bar 50 µm. Quantification of % area immunoreactivity for b, iC3b c, C5b-9 per brain section. Approximately 22 to 25 Amylo-Glo + CAA vessels were outlined to quantify d, CAA + iC3b + colocalization & e, CAA + C5b9 + colocalization area. f, Serum TCC/MAC levels measured by ELISA g, % CAA estimated by AG + CD31 + colocalization data. h, Representative double IHC staining for iC3b (DAB), S97 Aβ (Vector red), with Prussian blue combination for microhemorrhages detection in the 3D6 treated mice showed iC3b-associated microhemorrhages in leptomeningeal or penetrating vessels (arrows) and signs of vascular amyloid clearance (arrowheads) in vessels with complement deposition. In IgG2a-treated mice, vascular amyloid/plaque staining is prominent with minimal complement and Prussian blue staining (arrows), Scale bar=20 µm. N=5/group, Parametric unpaired t-test, *p<0.05 and **p<0.005, ****p<0.00005. Data are expressed as mean ± SEM.

Article Snippet: Following washes in PBS, sections were blocked either in 10% normal goat serum (Gibco, #16210064), or heat inactivated goat serum (Novus Biologicals #S13110H), or normal donkey serum (Sigma, #S30-M) for 1 hour at room temperature followed by overnight 4°C incubation with primary antibodies against C1q (1:500, Abcam #ab182451), C3b/iC3b/C3c (1:300, HycultBiotech #HM1078), C5b-9 (1:300, Bioss #bs-2673R), CD31 (1:500, R&D Systems #AF3628), C3 (1:200, HycultBiotech #HM1045), GFAP (1:1000, Abcam # ab5804), MMR/CD206 (1:300,R&D #AF2535) MMP-9 (1:500, Bioss #bs-0397R), Iba-1 (1:1000; Dako, Wako #019-19741), Iba-1 (1:500, Synaptic Systems, #234308), CD68 (1:500, Bio-Rad #MCA1957), S97 Aβ (pan-Aβ rabbit polyclonal, 1:2000, gift Dr. Dominic Walsh, BWH, USA), CD31 (1:500, R&D #AF3628), Collagen-IV (1:300, Abcam #6586), Alexa Fluor 488 anti-β-Amyloid, aggregated antibody (1: 200, clone A17171C, Biolegend # 856508), Von Willebrand Factor Antibody (1:500, Novus Biologicals #NB600-586).

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Plasmid Preparation

Journal: Analytical biochemistry

Article Title: A Quantitative Lateral Flow Assay to Detect Complement Activation in Blood

doi: 10.1016/j.ab.2015.01.024

Figure Lengend Snippet: Half-lives of C3 and C3 split-products

Article Snippet: The measurement range of the Minineph is 0.275-4.44 g/L (using the recommended 1:11 sample dilution). iC3b ELISA For the in-house iC3b ELISA, Immunlon 1B microtiter plates (Thermo-Scientific) were coated with a mouse anti-human iC3b monoclonal antibody (Quidel) for capture while goat anti-C3 polyclonal HRP antibody was employed as the detection antibody.

Techniques:

Journal: Analytical biochemistry

Article Title: A Quantitative Lateral Flow Assay to Detect Complement Activation in Blood

doi: 10.1016/j.ab.2015.01.024

Figure Lengend Snippet: A. Schematic diagram of LFA design; see Materials and Methods for a more detailed explanation. B and C. Standard curves of total C3 and iC3b LFAs. Purified proteins were serially diluted in LFA buffer and analyzed in triplicate. Results are expressed as reflectance units (RU) (mean ± SD) and are representative of three separate experiments.

Article Snippet: The measurement range of the Minineph is 0.275-4.44 g/L (using the recommended 1:11 sample dilution). iC3b ELISA For the in-house iC3b ELISA, Immunlon 1B microtiter plates (Thermo-Scientific) were coated with a mouse anti-human iC3b monoclonal antibody (Quidel) for capture while goat anti-C3 polyclonal HRP antibody was employed as the detection antibody.

Techniques: Purification

Journal: Analytical biochemistry

Article Title: A Quantitative Lateral Flow Assay to Detect Complement Activation in Blood

doi: 10.1016/j.ab.2015.01.024

Figure Lengend Snippet: C3 and iC3b levels in 10 normal donors

Article Snippet: The measurement range of the Minineph is 0.275-4.44 g/L (using the recommended 1:11 sample dilution). iC3b ELISA For the in-house iC3b ELISA, Immunlon 1B microtiter plates (Thermo-Scientific) were coated with a mouse anti-human iC3b monoclonal antibody (Quidel) for capture while goat anti-C3 polyclonal HRP antibody was employed as the detection antibody.

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Analytical biochemistry

Article Title: A Quantitative Lateral Flow Assay to Detect Complement Activation in Blood

doi: 10.1016/j.ab.2015.01.024

Figure Lengend Snippet: A. Generation of iC3b was assessed with capture time of 15 or 60 min. Shown is the mean ± SEM of four experiments. B. Serum samples were diluted and incubated in ELISA wells coated with anti-iC3b antibody for the capture times indicated. Following washing, iC3b concentrations were determined and compared to a standard curve of purified iC3b. Representative of four similar experiments.

Article Snippet: The measurement range of the Minineph is 0.275-4.44 g/L (using the recommended 1:11 sample dilution). iC3b ELISA For the in-house iC3b ELISA, Immunlon 1B microtiter plates (Thermo-Scientific) were coated with a mouse anti-human iC3b monoclonal antibody (Quidel) for capture while goat anti-C3 polyclonal HRP antibody was employed as the detection antibody.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Purification

Journal: Analytical biochemistry

Article Title: A Quantitative Lateral Flow Assay to Detect Complement Activation in Blood

doi: 10.1016/j.ab.2015.01.024

Figure Lengend Snippet: iC3b levels were determined in samples with known concentrations of iC3b by LFA and ELISA and a regression analysis was performed.

Article Snippet: The measurement range of the Minineph is 0.275-4.44 g/L (using the recommended 1:11 sample dilution). iC3b ELISA For the in-house iC3b ELISA, Immunlon 1B microtiter plates (Thermo-Scientific) were coated with a mouse anti-human iC3b monoclonal antibody (Quidel) for capture while goat anti-C3 polyclonal HRP antibody was employed as the detection antibody.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Analytical biochemistry

Article Title: A Quantitative Lateral Flow Assay to Detect Complement Activation in Blood

doi: 10.1016/j.ab.2015.01.024

Figure Lengend Snippet: iC3b LFA measurement of samples containing C3 or C3 fragments

Article Snippet: The measurement range of the Minineph is 0.275-4.44 g/L (using the recommended 1:11 sample dilution). iC3b ELISA For the in-house iC3b ELISA, Immunlon 1B microtiter plates (Thermo-Scientific) were coated with a mouse anti-human iC3b monoclonal antibody (Quidel) for capture while goat anti-C3 polyclonal HRP antibody was employed as the detection antibody.

Techniques:

Journal: Analytical biochemistry

Article Title: A Quantitative Lateral Flow Assay to Detect Complement Activation in Blood

doi: 10.1016/j.ab.2015.01.024

Figure Lengend Snippet: iC3b levels in plasma and serum stored at different conditions

Article Snippet: The measurement range of the Minineph is 0.275-4.44 g/L (using the recommended 1:11 sample dilution). iC3b ELISA For the in-house iC3b ELISA, Immunlon 1B microtiter plates (Thermo-Scientific) were coated with a mouse anti-human iC3b monoclonal antibody (Quidel) for capture while goat anti-C3 polyclonal HRP antibody was employed as the detection antibody.

Techniques: Clinical Proteomics

Journal: Analytical biochemistry

Article Title: A Quantitative Lateral Flow Assay to Detect Complement Activation in Blood

doi: 10.1016/j.ab.2015.01.024

Figure Lengend Snippet: iC3b levels were measured by LFA in serum from patients with SLE (A) and blood from ICH patients (B) and compared with normal controls. The median and range for each population are shown.

Article Snippet: The measurement range of the Minineph is 0.275-4.44 g/L (using the recommended 1:11 sample dilution). iC3b ELISA For the in-house iC3b ELISA, Immunlon 1B microtiter plates (Thermo-Scientific) were coated with a mouse anti-human iC3b monoclonal antibody (Quidel) for capture while goat anti-C3 polyclonal HRP antibody was employed as the detection antibody.

Techniques:

Journal: Frontiers in Immunology

Article Title: Functional Analysis of Variants in Complement Factor I Identified in Age-Related Macular Degeneration and Atypical Hemolytic Uremic Syndrome

doi: 10.3389/fimmu.2021.789897

Figure Lengend Snippet: Systemic complement levels in CFI rare variant carriers. (A) FI, (B) FH, and (C) C3bBbP plasma concentrations and (D) the amount of iC3b generated in the C3b degradation assay were determined with ELISA. The C3b degradation was performed three times, and iC3b levels were determined in duplicate. Dotted lines indicate the mean concentrations ± 2SD determined in non-carriers. The mean ± 2SD is (A) 57 µg/ml ± 11 µg/ml for FI, (B) 390 µg/ml ± 64 µg/ml for FH, (C) 12.9 CAU/ml ± 4.1 CAU/ml for C3bBbP, and (D) 8.5 µg/ml ± 1.6 µg/ml for generated iC3b. Solid lines indicate the mean concentration for each group.

Article Snippet: Then, the samples were immediately diluted 40x in PBS with 0.02% Tween and applied to high binding microplates (Greiner, Austria) coated with mouse-anti iC3b (A209, Quidel, USA) 2,500x diluted in coating buffer (15 mM Na 2 CO 3 *10 H 2 O, 35 mM NaHCO 3 , pH 9.6) and blocked with 1% BSA (Sigma, USA).

Techniques: Variant Assay, Clinical Proteomics, Generated, Degradation Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Functional Analysis of Variants in Complement Factor I Identified in Age-Related Macular Degeneration and Atypical Hemolytic Uremic Syndrome

doi: 10.3389/fimmu.2021.789897

Figure Lengend Snippet: CFI variants affect C3b degradation. C3b degradation was performed and generated iC3b was measured with ELISA. 15 µg/ml C3b and 3 µg/ml FH were added to purified FI, and incubated for 3h at 37°C. Th negative control does not contain purified FI, and for the positive control 10 ug/ml serum purified FI was incubated with the C3b/FH mix. As additional control, recombinant WT FI was incubated with 15 µg/ml C3, but FH omitted (WT -FH). The amount of iC3b generated by the variants was normalized to the concentration generated by recombinantly expressed WT FI. Bars and error bars show the mean and SD, individual dots represent the individual measurements. Samples were compared to WT using one-way ANOVA followed by Dunnet’s multiple comparison test. P-values are indicated as ****<0.001.

Article Snippet: Then, the samples were immediately diluted 40x in PBS with 0.02% Tween and applied to high binding microplates (Greiner, Austria) coated with mouse-anti iC3b (A209, Quidel, USA) 2,500x diluted in coating buffer (15 mM Na 2 CO 3 *10 H 2 O, 35 mM NaHCO 3 , pH 9.6) and blocked with 1% BSA (Sigma, USA).

Techniques: Generated, Enzyme-linked Immunosorbent Assay, Purification, Incubation, Negative Control, Positive Control, Control, Recombinant, Concentration Assay, Comparison

Journal: Cell reports

Article Title: Mining HIV controllers for broad and functional antibodies to recognize and eliminate HIV-infected cells

doi: 10.1016/j.celrep.2021.109167

Figure Lengend Snippet:

Article Snippet: Mouse Anti-Human C3 / C3b / iC3b Monoclonal Antibody, FITC Conjugated , CedarLane , CEDARLANE Cat# CL7632F; RRID:AB_10548984.

Techniques: Control, Virus, Recombinant, Reporter Gene Assay, Staining, Labeling, Software

Journal: eNeuro

Article Title: IgM Immunoglobulin Influences Recovery after Cervical Spinal Cord Injury by Modulating the IgG Autoantibody Response

doi: 10.1523/ENEURO.0491-19.2021

Figure Lengend Snippet: ΙgM KO mice have more parenchymal IgG antibodies with enhanced complement-fixing activity. A , IgG deposition in spinal cord sections of IgM-KO and IgM-WT mice at two weeks post-SCI. Scale bar: 100 μm. B , Comparison of the % detected IgG+ immunofluorescent area between groups at two weeks following SCI. See for statistics details. C , Comparison of the % detected IgG+ immunofluorescent area between groups at 10 weeks following SCI. See for statistics details. D , Representative section stained for IgG (red) and complement C3b protein (green). Yellow signal indicates co-localization of IgG and C3b. Scale bar: 10 μm. E–G , Gardner–Altman estimation plots of % immunofluorescent area from deposited IgG, IgG+C3b, and C3b averaged between sections −1200, −600, and +600 μm from the injury epicenter, and normalized per section area ( N = 4–5 animals/group). See for statistics details. H , Gardner–Altman estimation plots of total serum IgG levels in IgM-KO and WT mice at BSL (pre-SCI) and at 2 and 10 weeks post-SCI. There is no difference in IgG levels between IgM-KO and WT mice within each time point (data not shown; see statistical for details about WT vs KO comparisons). Serum IgG levels increase significantly after injury when compared with their BSL levels in IgM-KO and WT mice. See for statistics details. I , Gardner–Altman estimation plots of total serum IgM levels in IgM-KO and WT mice at BSL and at two weeks post-SCI, indicating a significant increase of IgM levels in WT mice, following SCI. See for statistics details. NS = nonsignificant. Graphs B , C : mean ± SEM, * p < 0.05, ** p < 0.01 (see for detailed p values). Graphs E–I : permutation t test, * p < 0.05, ** p < 0.01, *** p < 0.001 (see for detailed p values).

Article Snippet: Next, they were incubated with anti-mouse C3b-FITC antibody (1:100, Cederlane, clone 11H9 recognizing C3/C3b/iC3b) and DAPI (1:250) diluted in 5% goat serum + 1% BSA + 0.3% Triton X-100 in PBS, overnight at 4°C.

Techniques: Activity Assay, Comparison, Staining

Journal: eNeuro

Article Title: IgM Immunoglobulin Influences Recovery after Cervical Spinal Cord Injury by Modulating the IgG Autoantibody Response

doi: 10.1523/ENEURO.0491-19.2021

Figure Lengend Snippet: Estimation statistics table showing effect size (unpaired means difference) with 95% CIs and permutation tests p values as follows: effect size [CI width with lower bound, upper bound]

Article Snippet: Next, they were incubated with anti-mouse C3b-FITC antibody (1:100, Cederlane, clone 11H9 recognizing C3/C3b/iC3b) and DAPI (1:250) diluted in 5% goat serum + 1% BSA + 0.3% Triton X-100 in PBS, overnight at 4°C.

Techniques: Sequencing

Journal: Kidney International

Article Title: Complement receptor 3 mediates renal protection in experimental C3 glomerulopathy

doi: 10.1016/j.kint.2015.11.024

Figure Lengend Snippet: Plasma and glomerular C3 in mice with combined deficiency of factor h (FH) and complement receptor 3. ( a,b ) Plasma C3 levels ( a ) and glomerular C3 staining intensity ( b ) in 8-month-old Cfh –/– .Itgam –/– , Cfh –/– , and Itgam –/– mice housed in specific-pathogen free conditions. Horizontal bars denote median values. AFU, arbitrary fluorescent units. ( c ) Representative images of glomerular C3 immunostaining in the 3 genotypes. Bar = 80 μm. Each symbol represents a mouse. P values derived from Dunn’s multiple comparison test.

Article Snippet: For both iC3b-coated targets the level of iC3b opsonization was checked by flow cytometry using a biotinylated polyclonal antibody that recognizes both human and mouse C3 fragments (clone: 6C9, product no. CL7631B, Cedarlane Laboratories, Burlington, ON, Canada) followed by streptavidin-phycoerythrin (BD Biosciences Pharmingen).

Techniques: Clinical Proteomics, Staining, Immunostaining, Derivative Assay, Comparison