polyclonal goat anti-factor b a235  (CompTech Computer Technologies)

Journal: bioRxiv

Article Title: Mucosally sourced complement factor B modulates the host response to colitis

doi: 10.64898/2025.12.31.697141

Figure Lengend Snippet: ( A ) Dot plot illustrating the expression of complement genes representing the classical (CP), lectin (LP) and alternative (AP) pathway in intestinal tissue biopsies from uninflamed and non-inflamed regions of patients with ulcerative colitis (UC, Single Cell Portal: SCP259). ( B ) Plots depict CFB expression across denoted colonic cell types in the same dataset from intestinal tissue of patients with UC and healthy controls. * P <0.05, *** P <0.001, ns, not significant, Wilcoxon rank test. ( C ) Immunofluorescence staining for Factor B (red), E-cadherin (green) and DAPI (blue) in colonic tissue specimens from patients with IBD versus controls (non-IBD). (Scale bar = 90 μ m). ( D ) Quantification of the relative intensity of Factor B expression across tissue type. ( E ) Representative spatial transcriptomic slides (Visium) showing CFB expression in intestinal tissues obtained from patients with UC compared to healthy controls (HC, GSE189184). Graph depicts quantification of CFB expression from 3 HC and 7 UC specimens. ( F ) Representative spatial transcriptomic slides showing DUOX2-CFB co-localization spots in HC (left) and UC tissue (right) from the same dataset. Bar plot represents average co-expression across tissue samples. Error bars represent standard error of the mean (SEM). * P < 0.05, unpaired t-test.

Article Snippet: The sections were then blocked with 3% Bovine Albumen (Thermo Fisher, #J10857.22) diluted in phosphate buffered saline (PBS) at room temperature for 1 h. For primary incubation the sections were stained with a 1:50 dilution of polyclonal rabbit anti-goat Factor B antibody (Quidel Ortho, # A311) and a 1:100 dilution of mouse anti-E-Cadherin (BD Sciences, Cat. 610181) overnight at 4°C.

Techniques: Expressing, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Mucosally sourced complement factor B modulates the host response to colitis

doi: 10.64898/2025.12.31.697141

Figure Lengend Snippet: ( A ) Immunoblot denoting Factor B (FB) in plasma of global Cfb knockouts ( Cfb −/− ), mice deficient in liver-derived FB ( Cfb f/f AlbCre +/− ) and their littermates ( Cfb f/f AlbCre −/− ). MM: molecular marker. The same volume of plasma was loaded per well at 1:20 dilution. ( B ) Cfb expression on qRT-PCR in the colons from indicated mice, values normalized to Hprt1 . ( C ) Quantification of the relative intensity of Factor B expression in colon tissue from indicated mice. ( D ) Representative immunofluorescence images of colonic tissue from Cfb −/− , Cfb f/f AlbCre +/− and Cfb f/ f AlbCre −/− mice (n=4/group), stained with Factor B (red), E-cadherin (green) and DAPI (blue). Scale bars = 70 μ m. ( E ) Graph depicts serum CD14 concentration. Serum from mice challenged with lipopolysaccharide (LPS) were used as a positive control for barrier leak. Each dot represents an individual mouse. Bar graphs show the mean ± SEM, * P < 0.05; ** P < 0.01, ns, non-significant, unpaired t test. ( F ) Dot plot illustrating the expression of complement genes representing the classical (CP), lectin (LP) and alternative (AP) pathway across cell types indicated along the Y axis, based on single-cell RNA-seq data from the large intestines of non-injured adult C57BL/6 mice ( Cfb f/f ), housed in specific pathogen-free conditions. Cells pooled from n=2 mice.

Article Snippet: The sections were then blocked with 3% Bovine Albumen (Thermo Fisher, #J10857.22) diluted in phosphate buffered saline (PBS) at room temperature for 1 h. For primary incubation the sections were stained with a 1:50 dilution of polyclonal rabbit anti-goat Factor B antibody (Quidel Ortho, # A311) and a 1:100 dilution of mouse anti-E-Cadherin (BD Sciences, Cat. 610181) overnight at 4°C.

Techniques: Western Blot, Clinical Proteomics, Derivative Assay, Marker, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Concentration Assay, Positive Control, RNA Sequencing

Journal: bioRxiv

Article Title: Mucosally sourced complement factor B modulates the host response to colitis

doi: 10.64898/2025.12.31.697141

Figure Lengend Snippet: ( A-B ) Human epithelial Caco-2 cells were incubated in serum-free media alone (non-treated, NT), media with rIL-1β (50ng/ml) or TNF-α (50ng/ml). ( A ) Supernatant was collected after 24 hours, concentrated 50-fold and analyzed by immunoblotting for Factor B. ( B ) Human primary fibroblasts were incubated in serum-free media alone (NT), media with rIL-1β (50ng/ml) or TNF-α (50ng/ml). Immunoblot depicts CFB in supernatant at the 24 h time point. Graph depicts Factor B protein quantification relative to the loaded control. The same volume of supernatant was loaded in each well. Each dot represents an individual replicate. Bar graphs show the mean ± SEM. All immunoblots were repeated at least twice. Statistics performed using an ordinary one-way ANOVA test adjusted for multiple comparisons.

Article Snippet: The sections were then blocked with 3% Bovine Albumen (Thermo Fisher, #J10857.22) diluted in phosphate buffered saline (PBS) at room temperature for 1 h. For primary incubation the sections were stained with a 1:50 dilution of polyclonal rabbit anti-goat Factor B antibody (Quidel Ortho, # A311) and a 1:100 dilution of mouse anti-E-Cadherin (BD Sciences, Cat. 610181) overnight at 4°C.

Techniques: Incubation, Western Blot, Control

Journal: bioRxiv

Article Title: Mucosally sourced complement factor B modulates the host response to colitis

doi: 10.64898/2025.12.31.697141

Figure Lengend Snippet: ( A ) SPF-housed global knockout ( Cfb −/− ), liver-deficient ( Cfb f/f AlbCre +/− ) and littermate control ( Cfb f/f AlbCre −/− ) mice were administered 2.5% DSS in drinking water for 7 days, followed by conventional water for 3 days. Mice were euthanized on Day 11. Body weights were monitored daily during treatment. ( B ) Colon lengths evaluated at end of study. ( C ) Colonic tissue was homogenized for measuring TNF-α and MPO levels, normalized to weight (per g) of tissue. ( D ) Fecal lipocalin-2 (LCN-2) levels compared at the end of the study. ( E ) Complement activity in the colonic tissue measured using C3bBbP (alternative pathway convertase). ( F ) Differentially regulated pathways upregulated in Cfb −/− versus Cfb f/f AlbCre +/− based on bulk RNA-seq of colonic tissue at end of treatment. Pathway analysis conducted using Enrichr (MSigDB). ( G ) Adult C57BL/6 mice housed in SPF conditions were given 2.5% DSS in drinking water for a week. Starting day 5, mice were treated with a daily oral gavage of a Factor B inhibitor, iptacopan (20 mg/kg), or vehicle control. Body weights were monitored daily. ( H ) Colon lengths compared at end of the experiment. ( I ) IL-1β in colonic tissue lysate and ( J ) LCN-2 in fecal samples. Bar graphs show the mean ± SEM, * P < 0.05; ** P < 0.01, ns, non-significant. Unpaired t test when analyzing a two-group categorical comparison, and 2-way ANOVA adjusted for multiple comparisons when analyzing longitudinal data.

Article Snippet: The sections were then blocked with 3% Bovine Albumen (Thermo Fisher, #J10857.22) diluted in phosphate buffered saline (PBS) at room temperature for 1 h. For primary incubation the sections were stained with a 1:50 dilution of polyclonal rabbit anti-goat Factor B antibody (Quidel Ortho, # A311) and a 1:100 dilution of mouse anti-E-Cadherin (BD Sciences, Cat. 610181) overnight at 4°C.

Techniques: Knock-Out, Control, Activity Assay, RNA Sequencing, Comparison

Journal: bioRxiv

Article Title: Mucosally sourced complement factor B modulates the host response to colitis

doi: 10.64898/2025.12.31.697141

Figure Lengend Snippet: ( A-C ) Data from scRNA-seq of colonic cells from C57BL/6 mice subjected to acute DSS (acute colitis, AC) was re-analyzed for express of complement genes and compared to mice not treated with DSS (non-DSS, healthy controls (HC), GSE264408). ( A ) UMAP with designated cell clusters showing Cfb expression. ( B ) Bar plots depicting changes in the cellular composition of the colonic tissue post-DSS treatment. ( C ) Dot plots showing average gene expression levels of key AP components in the distinct cell clusters of HC (healthy control) mice and those with AC (acute colitis). ( D ) Cfb f/f LysMCre +/− mice and controls were treated with 7 days of 2.5% DSS in drinking water, followed by 3 days of regular water. Body weights were monitored daily. Colon length was determined at the end of the experiment. Colonic tissue was homogenized for comparing MPO and IL-1β levels, normalized to tissue weight. ( E ) Representative RNAScope images depict staining of colonic tissue from Cfb f/f VilCre +/− mice and controls. Scale bar =30μm. ( F ) Cfb f/f VilCre and controls were treated similarly to ( D ). Body weights and the disease activity index (DAI) were measured daily. Mice were sacrificed on day 11. Colons were harvested to compare the colon length, MPO and IL-1β levels, normalized to weight of tissue. ( G ) Cfb f/f Col1a2CreER T2+/− mice and their controls were treated with tamoxifen every other day for 10 days. Colons were harvested and tissue sections were stained for Factor B (red) and vimentin (green). Scale bar =90 μ m. ( H ) Cfb f/f Col1a2CreER T2+/− and littermates were subjected to tamoxifen challenge as detailed in ( G ). 3 days after the last dose of tamoxifen, mice were subjected to DSS challenge as detailed in ( D ). Body weights and DAI scores were monitored daily. Colon tissue was collected at day 11 to measure colon length and cytokines in tissue lysate. Data represent 2 independent experiments. Bar graphs show the mean ± SEM, * P < 0.05; ** P < 0.01, ns, non-significant. Unpaired t test when analyzing a two-group categorical comparison, and 2-way ANOVA adjusted for multiple comparisons when analyzing longitudinal data.

Article Snippet: The sections were then blocked with 3% Bovine Albumen (Thermo Fisher, #J10857.22) diluted in phosphate buffered saline (PBS) at room temperature for 1 h. For primary incubation the sections were stained with a 1:50 dilution of polyclonal rabbit anti-goat Factor B antibody (Quidel Ortho, # A311) and a 1:100 dilution of mouse anti-E-Cadherin (BD Sciences, Cat. 610181) overnight at 4°C.

Techniques: Expressing, Gene Expression, Control, RNAscope, Staining, Activity Assay, Comparison

Journal: Frontiers in Immunology

Article Title: The Role of Properdin in Killing of Non-Pathogenic Leptospira biflexa

doi: 10.3389/fimmu.2020.572562

Figure Lengend Snippet: Binding of properdin to Leptospira. L. biflexa serovar Patoc strain Patoc I (non-pathogenic), L. interrogans serovar Pomona strain Pomona (pathogenic, attenuated), and L. interrogans serovar Kennewicki strain Fromm (pathogenic, virulent) were incubated with (A) 10, 25, and 50% NHS-EDTA or (B) 2.5, 6.25, and 12.5 µg of commercial purified properdin which are equivalent to properdin concentrations found in 10, 25, and 50% NHS used above. The binding was evaluated by ELISA using polyclonal anti-human properdin. Baseline values obtained with PBS were subtracted in (A, B) , respectively for each type of leptospire. Data are expressed as the mean ( ± SD) of three independent experiments each one performed in triplicate. Statistically significant differences (ANOVA one-way test) are indicated. * p ≤ 0.05; confidence interval of 95%. Variance homogeneity was analyzed using Bartlett.

Article Snippet: After five washes, polyclonal goat anti-Factor B at a 1: 2,000 dilution (Complement Technology, Inc.) in PBS-T containing 1% BSA was added and incubation proceeded for 1 h at 37°C.

Techniques: Binding Assay, Incubation, Purification, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: The Role of Properdin in Killing of Non-Pathogenic Leptospira biflexa

doi: 10.3389/fimmu.2020.572562

Figure Lengend Snippet: Binding of properdin to Leptospira occurs even in the absence of C3b. The binding of properdin (P) on L. biflexa serovar Patoc strain Patoc I (non-pathogenic), L. interrogans serovar Pomona strain Pomona (pathogenic, attenuated), and L. interrogans serovar Kennewicki strain Fromm (pathogenic, virulent) was quantified by ELISA using polyclonal anti-human properdin (A, B) or anti-human C3 (C) . (A) Leptospira strains were incubated with 40% normal human serum-treated with EDTA (NHS-EDTA) or with C3 depleted serum (C3-DS). (B) Leptospira strains were incubated with purified properdin (P) for 1h at 37°C, washed, and then with C3b (C3b+P) or PBS. (C) Leptospira strains were incubated with purified C3b for 1h at 37°C, washed, and then incubated with P (P + C3b) or PBS. Baseline values obtained without any addition of P or C3b were subtracted respectively for each type of leptospire. Data are expressed as the mean ( ± SD) of three independent experiments each one performed in triplicate.

Article Snippet: After five washes, polyclonal goat anti-Factor B at a 1: 2,000 dilution (Complement Technology, Inc.) in PBS-T containing 1% BSA was added and incubation proceeded for 1 h at 37°C.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Purification

Journal: Frontiers in Immunology

Article Title: The Role of Properdin in Killing of Non-Pathogenic Leptospira biflexa

doi: 10.3389/fimmu.2020.572562

Figure Lengend Snippet: Binding of different properdin oligomers to pathogenic and non-pathogenic Leptospira . Unfractionated commercial properdin (P), high molecular weight aggregate not present in serum (Pn), properdin dimer (P2), trimer (P3), and tetramer (P4) were incubated with L. biflexa serovar Patoc strain Patoc I (non-pathogenic) (A) , L. interrogans serovar Pomona strain Pomona (pathogenic, attenuated) (B) ), and L. interrogans serovar Kennewicki strain Fromm (pathogenic, virulent) (C) . Five µg of each properdin form were used and the binding on the Leptospira surface was evaluated by ELISA using polyclonal anti-human properdin. The mean and standard deviation correspond to three independent experiments, using two independent preparations of P2, P3, P4, or Pn. Baseline values obtained with PBS were subtracted, respectively for each type of leptospire. Data are expressed as the mean ( ± SD) of three independent experiments each one performed in triplicate. Statistically significant differences (ANOVA one-way test) are indicated. * p ≤ 0.05, confidence interval of 95%.

Article Snippet: After five washes, polyclonal goat anti-Factor B at a 1: 2,000 dilution (Complement Technology, Inc.) in PBS-T containing 1% BSA was added and incubation proceeded for 1 h at 37°C.

Techniques: Binding Assay, High Molecular Weight, Incubation, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: The Role of Properdin in Killing of Non-Pathogenic Leptospira biflexa

doi: 10.3389/fimmu.2020.572562

Figure Lengend Snippet: Interaction of outer membrane proteins of Leptospira with properdin. (A) Recombinant proteins of pathogenic L. interrogans serovar Copenhageni 10A (listed in Table 1 ) were immobilized on ELISA plate wells and incubated with 0.5 µg of unfractionated properdin (P). Binding was assessed by ELISA using polyclonal anti-human properdin. (B) Recombinant Lsa30 from pathogenic L. interrogans was immobilized and incubated with 0.5 µg of properdin (P), P2, P3, or P4. Binding was assessed as described above in (A) . These data represent the average ( ± SD) of three independent experiments, after subtracting the basal values obtained with PBS. Each experiment was performed in triplicate, using two independent preparations of P2, P3, P4, and Pn. Statistically significant differences (ANOVA one-way test) are indicated. * p ≤ 0.05, confidence interval of 95%.

Article Snippet: After five washes, polyclonal goat anti-Factor B at a 1: 2,000 dilution (Complement Technology, Inc.) in PBS-T containing 1% BSA was added and incubation proceeded for 1 h at 37°C.

Techniques: Membrane, Recombinant, Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay

Journal: Frontiers in Immunology

Article Title: The Role of Properdin in Killing of Non-Pathogenic Leptospira biflexa

doi: 10.3389/fimmu.2020.572562

Figure Lengend Snippet: Properdin is required for AP activation on the surface of L. biflexa serovar Patoc strain Patoc I. Non-pathogenic L. biflexa serovar Patoc strain Patoc I or pathogenic L. interrogans serovar Kennewicki strain Fromm were incubated with properdin-depleted human serum (P-DS), normal human serum (NHS), or heat-inactivated NHS (HI-NHS). Where indicated, leptospires were previously treated with 5 µg of purified properdin (P) before incubating with P-DS [(P + (P-DS)]. The presence of the C3 convertase was evaluated by ELISA using polyclonal anti-Factor B Data are expressed as the mean ( ± SD) of three independent experiments each one performed in triplicate. Statistically significant differences (ANOVA one-way test) are indicated. * p ≤ 0.05; ** p ≤ 0.001, confidence interval of 95%.

Article Snippet: After five washes, polyclonal goat anti-Factor B at a 1: 2,000 dilution (Complement Technology, Inc.) in PBS-T containing 1% BSA was added and incubation proceeded for 1 h at 37°C.

Techniques: Activation Assay, Incubation, Purification, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A) Schematic of Factor I and cofactor cleavage of C3b and iC3b. B) Factor I cleavage assay of C3b. C3b and Factor I (FI) were incubated alone, with Factor H (FH), or C4BP, or with concentrations of Sez6L2-MH ranging from 1 to 10 μg/mL for 2 hours at 37°C. Then samples were analyzed by western blot using antibodies that recognize C3d, a region within the C3α chain (and highlighted by the black rectangle in the schematics in (A)). Coomassie stained gels are also shown. FH and C4BP are known co-factors of FI towards C3b and served as positive controls. Incubation of C3b and FI with Sez6L2-MH also generated the C3 cleavage products α’1 and α’2 showing Sez6L2-MH is a cofactor for Factor I cleavage of C3b. C) Schematic of Factor I + cofactor cleavage of C4b. D) C4b and Factor I (FI) were incubated alone, with FH, C4BP, or with concentrations of Sez6L2-MH ranging from 1 to 10 μg/mL for 2 hours at 37°C. Then samples were analyzed by western blot using a C4 polyclonal antibody or coomassie stained gels. C4 components recognized by the C4 antibody are colored black in the schematic in C. C4BP is a known cofactor of FI for C4b cleavage and served as a positive control. Incubation of C4b and FI with Sez6L2-MH did not result in the appearance of C4b cleavage products.

Article Snippet: Residual Bb on the plate was detected using polyclonal goat anti-Factor B (Comptech, A235, 1:4,000) for 1 hour at room temperature.

Techniques: Cleavage Assay, Incubation, Western Blot, Staining, Generated, Positive Control

Journal: bioRxiv

Article Title: The Sez6 family inhibits complement at the level of the C3 convertase

doi: 10.1101/2020.09.11.292623

Figure Lengend Snippet: A and B) Alternative C3 convertase assay: A 96 well plate coated with C3b was incubated with Factor B and Factor D to form the C3 convertase C3bBb, then incubated with Sez6L2-MH or FH at concentrations ranging from 0 to 500 μg/mL to assess their decay accelerating activity. Factor B remaining bound to the plate (as C3bBb) was detected using an anti-Factor B antibody ELISA in (A) and Bb released into the supernatant is shown via western blot (B). C) Classical C3 convertase assay: A plate coated with C4b was incubated with C2 and C1s-enzyme to form the classical/lectin pathway C3 convertase C4b2a, then incubated with Sez6L2-MH or H-DAF at concentrations ranging from 0 to 500 μg/mL to assess their decay accelerating activity. C2 remaining bound to the plate (presumably as C4b2a) was detected using an anti-C2 antibody ELISA. For A and C ELISAs: N=3 (1 experiment with 3 replicates; representative of 2-3 independent experiments). Statistics: 1-way ANOVAS with Holms-Sidak multiple comparison’s tests were performed for each complement inhibitor with comparisons to 0 ug/mL controls. * p<0.05.

Article Snippet: Residual Bb on the plate was detected using polyclonal goat anti-Factor B (Comptech, A235, 1:4,000) for 1 hour at room temperature.

Techniques: Convertase Assay, Incubation, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Complement Proteins’ Western Blotting Information.

Article Snippet: 6 , CFB , 85 kDa , Goat Anti-factor B Polyclonal antibody # AF2739 (RD Systems) , 1:1000 , Human , Dnk pAb to Goat IgG (HRP) ab97120 (ABCAM) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Western Blot