sheep polyclonal anti nephrin  (R&D Systems)

Journal: Journal of Translational Autoimmunity

Article Title: Afucosylated IgG in idiopathic nephrotic syndrome patients with anti-nephrin autoantibodies correlate with disease activity

doi: 10.1016/j.jtauto.2025.100307

Figure Lengend Snippet: Schematic overview of the workflow for the characterization of anti-nephrin autoantibodies . Panel A. Paired serum and urine samples from 20 healthy donors, 100 NS patients were analyzed using an ELISA assay to detect anti-nephrin autoantibodies. Panel B. The same paired samples were analyzed using a pull-down assay to verify whether the identified anti-nephrin positive samples could also immunoprecipitate the extracellular domain of human nephrin. Panel C. The fucose content of IgG from the same serum samples was measured using an ELISA assay using the fucose-binding lectins. INS, Idiopathic nephrotic syndrome; FSGS, focal segmental glomerulosclerosis; MCD, minimal change disease; MN, membranous nephropathy; LN, lupus nephritis; SDNS, steroid-dependent NS; MDNS, multidrug-dependent NS; MRNS, multidrug-resistant NS.

Article Snippet: Membranes were blocked overnight (ON) at 4 °C with 3 % BSA, washed three times with PBST, and incubated ON at 4 °C with sheep anti-nephrin polyclonal antibody (R&D Systems Cat# AF4269, 1:1000 in blocking buffer).

Techniques: Enzyme-linked Immunosorbent Assay, Pull Down Assay, Binding Assay

Journal: Journal of Translational Autoimmunity

Article Title: Afucosylated IgG in idiopathic nephrotic syndrome patients with anti-nephrin autoantibodies correlate with disease activity

doi: 10.1016/j.jtauto.2025.100307

Figure Lengend Snippet: Circulating antibodies against the extracellular domain of nephrin in idiopathic nephrotic syndrome (INS) patients . Panel A shows the levels of circulating anti-nephrin antibodies measured by ELISA in 10 healthy donors and 100 INS patients stratified based on biopsy. FSGS, focal segmental glomerulosclerosis; MCD, minimal change disease; MN, membranous nephropathy; LN, lupus nephritis. Additionally, in patients with FSGS and MCD, anti-nephrin levels were further stratified based on the type of INS. SDNS, steroid-dependent NS; MDNS, multidrug-dependent NS; MRNS, multidrug-resistant NS. Panel B shows the titer of immunoprecipitating anti-nephrin antibodies in the serum and urine of the same subjects as in panel A. Panel C shows the prevalence of circulating anti-nephrin autoantibodies in the investigated cohorts of children and young adults with INS, with red and light gray colors representing positive and negative percentages, respectively. Panel D shows the prevalence of circulating anti-nephrin autoantibodies in FSGS and MCD samples stratified based on their pharmacological response. The dotted line indicates the statistically significant threshold.

Article Snippet: Membranes were blocked overnight (ON) at 4 °C with 3 % BSA, washed three times with PBST, and incubated ON at 4 °C with sheep anti-nephrin polyclonal antibody (R&D Systems Cat# AF4269, 1:1000 in blocking buffer).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Translational Autoimmunity

Article Title: Afucosylated IgG in idiopathic nephrotic syndrome patients with anti-nephrin autoantibodies correlate with disease activity

doi: 10.1016/j.jtauto.2025.100307

Figure Lengend Snippet: Correlation between anti-nephrin antibody titers and urinary proteinuria in patients with anti-nephrin-associated MCD or FSGS . Panels A and B show the correlation between anti-nephrin antibody titers in serum, measured by ELISA (A) or immunoprecipitation (B), and 24-h proteinuria. Panels C and D show the corresponding correlation between urinary anti-nephrin antibody titers, measured by ELISA (C) or immunoprecipitation (D), and proteinuria. R = Spearman's correlation coefficient.

Article Snippet: Membranes were blocked overnight (ON) at 4 °C with 3 % BSA, washed three times with PBST, and incubated ON at 4 °C with sheep anti-nephrin polyclonal antibody (R&D Systems Cat# AF4269, 1:1000 in blocking buffer).

Techniques: Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: Journal of Translational Autoimmunity

Article Title: Afucosylated IgG in idiopathic nephrotic syndrome patients with anti-nephrin autoantibodies correlate with disease activity

doi: 10.1016/j.jtauto.2025.100307

Figure Lengend Snippet: Evaluation of fucose in IgG by enzyme-linked immunosorbent assay (ELISA) with biotinylated lectins. Panels A and B show the levels of core-fucosylated IgG (α1,6-linked fucose) and antennary-fucosylated IgG (α1,2-linked fucose), respectively, measured using AAL and UEA-I lectins in the serum of healthy donors and NS patients, stratified by circulating anti-nephrin autoantibodies. Panels C and D display the inverse correlation between 24-h proteinuria and serum IgG fucosylation levels, as measured by AAL (Panel C) and UEA-I (Panel D), in patients with anti-nephrin–associated MCD or FSGS. Panels E and F show the corresponding levels of core and antennary fucosylation in IgG from urine samples, measured by AAL and UEA-I, and stratified by anti-nephrin antibody status. Red dots indicate patients positive for anti-nephrin autoantibodies, as confirmed by both ELISA and immunoprecipitation.

Article Snippet: Membranes were blocked overnight (ON) at 4 °C with 3 % BSA, washed three times with PBST, and incubated ON at 4 °C with sheep anti-nephrin polyclonal antibody (R&D Systems Cat# AF4269, 1:1000 in blocking buffer).

Techniques: Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: Clinical and Experimental Nephrology

Article Title: Co-localization of IgG with nephrin in immune-mediated idiopathic nephrotic syndrome

doi: 10.1007/s10157-025-02741-5

Figure Lengend Snippet: Representative double-immunofluorescence staining for nephrin and IgG. Double-immunofluorescence staining of frozen sections of biopsies obtained from patients with idiopathic nephrotic syndrome. First column, nephrin staining; second column, IgG staining; third column, merged image; fourth column, magnified image of the boxed area in the merged image. Nephrin in the glomeruli is labeled red, and IgG in the glomeruli is labeled green. When these images are overlaid, nephrin and IgG co-localization appear yellow. A Case 3 (active phase) shows a co-localization-positive pattern. When the image is magnified, the red nephrin and the green IgG clearly overlap, and the co-localization appears yellow. B Case 18 (active phase) shows a co-localization-negative pattern. In magnified image, the red nephrin and the green IgG regions clearly do not overlap

Article Snippet: An Alexa 546-labeled anti-nephrin antibody was produced by incubating Alexa FluorTM 546 NHS Ester (A20002; Invitrogen, Carlsbad, CA, USA) with anti-human nephrin (N) rabbit polyclonal IgG (Cat. No. 29050) (Immuno-Biological Laboratories, Fujioka, Japan) and purified using an antigen-specific column and its specificity is supported by the observation that it shows negative staining in kidney tissues from patients with NPHS1 null variants, where nephrin protein is absent.

Techniques: Double Immunofluorescence Staining, Staining, Labeling

Journal: Clinical and Experimental Nephrology

Article Title: Co-localization of IgG with nephrin in immune-mediated idiopathic nephrotic syndrome

doi: 10.1007/s10157-025-02741-5

Figure Lengend Snippet: Comparison of nephrin/IgG co-localization between active and remission phases in the same idiopathic nephrotic syndrome case. Merged images of nephrin and IgG staining in Case 3 ( A ) and Case 21 ( B ) in both active phase and remission phase. The pathological findings on light microscopy were MCD in Case 3 and FSGS in Case 21. Nephrin and IgG colocalize in the active phase (left columns). However, nephrin and IgG no longer colocalize during remission (right columns). MCD minimal change disease; FSGS focal segmental glomerulosclerosis

Article Snippet: An Alexa 546-labeled anti-nephrin antibody was produced by incubating Alexa FluorTM 546 NHS Ester (A20002; Invitrogen, Carlsbad, CA, USA) with anti-human nephrin (N) rabbit polyclonal IgG (Cat. No. 29050) (Immuno-Biological Laboratories, Fujioka, Japan) and purified using an antigen-specific column and its specificity is supported by the observation that it shows negative staining in kidney tissues from patients with NPHS1 null variants, where nephrin protein is absent.

Techniques: Comparison, Staining, Light Microscopy

Journal: bioRxiv

Article Title: Vascularized Bioengineered Kidney Using Decellularized Scaffold Recellularized with human Placenta-Derived Angiogenic stem Cells and Kidney Organoids

doi: 10.1101/2025.07.10.664023

Figure Lengend Snippet: (A), The photograph of bioengineered kidney 14 days post transplantation; (B), hPASCs promote angiogenesis: (a), Immunofluorescence of CD31 staining; (b), Statistical analysis of the density of vascular structures; (c) The immunofluorescence co-staining results of EHD3, α-SMA, and CD31 with hPASCs. (C), HE staining analysis of Glomerulus-like structure: (a), Native group; (b), DKS+hPASCs+KIO implant group. Scale bar 200 μm (up); 50 μm (down); (D), HE staining analysis of Tubule-like structure: (a), Native group; (b), DKS+hPASCs+KIO implant group. Scale bar 200 μm (up); 50 μm (down); (E), Fluorescent staining of implant: (a), The native tissue was as control; (b), Surface markers of glomerulus (NPHS1, NPHS2), collecting tube (AQP2), distal tube (NKCC2, CDH1), and proximal tube (LRP2); Scale bar 20 µm.

Article Snippet: Primary Antibodies: CD31 (Abcam; ab281583, diluted 1:100);α-SMA (Abcam; ab7817, diluted 1:150); human podocalyxin antibody (PODXL) (R&D Systems; AF1658, diluted 1:250); Lotus tetragonolobus lectin (LTL) fluorescein (Vector Laboratories; FL-1321-2, diluted 1:100-1:400);Purified mouse anti-E-cadherin (CDH1) (BD Biosciences; 610182, diluted 1:50); Rabbit anti-pax2 antibody (Abcam; ab79389, diluted 1:50);Anti-Lrp2 (Abcam; ab76969, diluted 1:200);NKCC2 polyclonal antibody (Proteintech; 18970-1-AP, diluted 1:100-1:200); Aquaporin 2 (AQP2) (Santa Cruz; sc-515770, diluted 1:250); NPHS1 Polyclonal antibody (R & D system; AF4269-SP, diluted 1:400-1:500); NPHS2 Polyclonal antibody (Proteintech; 20384-1-AP, diluted 1:400-1:500); WT1 (Santa Cruz; sc-7385, diluted 1:400); EHD Polyclonal antibody (Proteintech; 25320-1-AP, diluted 1:200); CNN1 (CST;17819T, diluted 1:500); CDH5 (Abcam; ab313632, diluted 1:500); MECOM (ATLAas antibodies; HPA046537, diluted 1:500); PAI 1 (Abcam; ab222754, diluted 1:1000).

Techniques: Transplantation Assay, Immunofluorescence, Staining, Control

Journal: bioRxiv

Article Title: Vascularized Bioengineered Kidney Using Decellularized Scaffold Recellularized with human Placenta-Derived Angiogenic stem Cells and Kidney Organoids

doi: 10.1101/2025.07.10.664023

Figure Lengend Snippet: (A), HE staining analysis of Glomerulus-like structure: (a), Native group; (b), DKS+hPASCs+KIO implant group. Scale bar 200 µm (up); 25 µm (down). (B), HE staining analysis of Tubule-like structure: (a), Native group; (b), DKS+hPASCs+KIO implant group.200 µm (up); 25 µm (down). (C), Fluorescent staining of implant: (a), The native tissue was as control; (b), Implant were positive for markers of glomerulus (NPHS1, NPHS2, EHD3), collecting tube (AQP2), distal tube (NKCC2, CDH1), and proximal tube (LRP2). Scale bar 20 µm. (D), ScRNA-seq analysis of implant of DKS+hPASCs+KIO using human genome: (a), Human cell types; (b), Marker genes of each cell type; (c), Specific markers. (E), The MapQuery function predicts the original cell types; (a), Vascular and immune cells were derived from hPASCs; (b), Kidney parenchymal cells originated from KIO.

Article Snippet: Primary Antibodies: CD31 (Abcam; ab281583, diluted 1:100);α-SMA (Abcam; ab7817, diluted 1:150); human podocalyxin antibody (PODXL) (R&D Systems; AF1658, diluted 1:250); Lotus tetragonolobus lectin (LTL) fluorescein (Vector Laboratories; FL-1321-2, diluted 1:100-1:400);Purified mouse anti-E-cadherin (CDH1) (BD Biosciences; 610182, diluted 1:50); Rabbit anti-pax2 antibody (Abcam; ab79389, diluted 1:50);Anti-Lrp2 (Abcam; ab76969, diluted 1:200);NKCC2 polyclonal antibody (Proteintech; 18970-1-AP, diluted 1:100-1:200); Aquaporin 2 (AQP2) (Santa Cruz; sc-515770, diluted 1:250); NPHS1 Polyclonal antibody (R & D system; AF4269-SP, diluted 1:400-1:500); NPHS2 Polyclonal antibody (Proteintech; 20384-1-AP, diluted 1:400-1:500); WT1 (Santa Cruz; sc-7385, diluted 1:400); EHD Polyclonal antibody (Proteintech; 25320-1-AP, diluted 1:200); CNN1 (CST;17819T, diluted 1:500); CDH5 (Abcam; ab313632, diluted 1:500); MECOM (ATLAas antibodies; HPA046537, diluted 1:500); PAI 1 (Abcam; ab222754, diluted 1:1000).

Techniques: Staining, Control, Marker, Derivative Assay