Journal: iScience

Article Title: Chimpanzee adenovirus-mediated multiple gene therapy for age-related macular degeneration

doi: 10.1016/j.isci.2023.107939

Figure Lengend Snippet: In vivo expression of eGFP, PEDF, sFlt-1, and sCD59 following IVT administration of AdCs (A) Representative fundus images of the retina from individual mice following IVT injection of 1.5×10 9 vp and 7.5×10 9 vp AdC68-eGFP. The eGFP signal could be detected from 48 h to 35 days post-injection. The dotted circles represent the edge of mouse retina. (B–D) Assessment of PEDF, sFlt-1, and sCD59 mRNA expression in retina-choroid-sclera complexes isolated from 10 mice. In each mouse, one eye was injected with AdC68-PFC (five mice for 1.5×10 9 vp and five mice for 7.5×10 9 vp) whereas the contralateral, un-injected eye served as control (only five eyes were used for analysis). At 4 days post-injection, RNA was purified from the retina-choroid complexes and real-time qPCR was conducted. Absolute number of mRNA copies were calculated using the standard curve method. (E–H) Images of western blot and quantification of the PEDF, sFlt-1, and sCD59 protein amount expressed in retina-choroid complexes of five mice. In each mouse, one eye was injected with AdC68-PFC (7.5×10 9 vp) whereas the contralateral, un-injected eye served as control. Total protein was obtained from retina-choroid-sclera complexes isolated from AdC68-PFC-treated (7.5×10 9 vp) and un-injected eyes 7 days post-injection. Antibodies against GAPDH were used for the internal control. The relative expression of PEDF, sFlt-1, and sCD59 in the un-injected eyes was set to 1. Data are expressed as mean ± SEM, and analyzed using one-way ANOVA multiple comparisons with Tukey’s method among groups in (B) and Student’s t test (two-tailed) in (C) (∗p < 0.05, ∗∗p < 0.01). PEDF, pigment epithelium-derived factor; sFlt-1, soluble fms-like tyrosine kinase-1; sCD59, soluble forms of CD59; IVT, intravitreal.

Article Snippet: Rabbit polyclonal anti-CD59 , Sino Biological , Cat#12474-RP02.

Techniques: In Vivo, Expressing, Injection, Isolation, Control, Purification, Western Blot, Two Tailed Test, Derivative Assay

Journal: iScience

Article Title: Chimpanzee adenovirus-mediated multiple gene therapy for age-related macular degeneration

doi: 10.1016/j.isci.2023.107939

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-CD59 , Sino Biological , Cat#12474-RP02.

Techniques: Virus, Recombinant, Transfection, Protease Inhibitor, Plasmid Preparation, Gel Extraction, Software

Journal: International Journal of Molecular Sciences

Article Title: Bispecific mAb 2 Antibodies Targeting CD59 Enhance the Complement-Dependent Cytotoxicity Mediated by Rituximab

doi: 10.3390/ijms23095208

Figure Lengend Snippet: mAb 2 bispecific molecules contain antigen binding Fc fragments (Fcabs), which can be derived from yeast Fcab libraries: ( a ) Model of a mAb 2 antibody: Fab fragment in light teal, Fc in gray, and modified residues in the Fc-CH3 domain highlighted in blue (AB loop) and red (EF loop). The Figure was prepared using PyMOL version 2.5 (Schrödinger LLC.) based on PDB:1HZH; ( b ) Scheme of selection and pool expansion of CD59-reactive Fcabs (EU numbering scheme for antibody amino acids is used).

Article Snippet: Polyclonal anti-CD59 RP-02 antibody (12474-RP02, SinoBiological, Beijing, China) was detected with anti-rabbit-HRP (Sigma-Aldrich, St. Louis, MO, USA) at a dilution of 1:2 500 in 2.5% BSA-PBS.

Techniques: Binding Assay, Derivative Assay, Modification, Selection

Journal: International Journal of Molecular Sciences

Article Title: Bispecific mAb 2 Antibodies Targeting CD59 Enhance the Complement-Dependent Cytotoxicity Mediated by Rituximab

doi: 10.3390/ijms23095208

Figure Lengend Snippet: Anti-CD59 antigen-binding Fc fragments (Fcabs) from initial selections of Fcab libraries: ( a ) HPLC-SEC chromatography of derived mAb 2 molecules in native conditions (MWS: molecular weight standard); ( b ) ELISA with coated CD59; ( c ) Competition of biotinylated HuMax-20 (HX)-BER3 mAb 2 with HX-BER1, HX-BER2 and HX-BER3; ( d ) Biolayer interferometry measurement of response to immobilized CD59: HX-BER1 to 3 were applied in 2-fold dilution steps starting from 1 µM. RX: rituximab, TRA: trastuzumab.

Article Snippet: Polyclonal anti-CD59 RP-02 antibody (12474-RP02, SinoBiological, Beijing, China) was detected with anti-rabbit-HRP (Sigma-Aldrich, St. Louis, MO, USA) at a dilution of 1:2 500 in 2.5% BSA-PBS.

Techniques: Binding Assay, Chromatography, Derivative Assay, Molecular Weight, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Bispecific mAb 2 Antibodies Targeting CD59 Enhance the Complement-Dependent Cytotoxicity Mediated by Rituximab

doi: 10.3390/ijms23095208

Figure Lengend Snippet: Number of CD20 and CD59 molecules per cell of Raji, ARH-77, Daudi, and SK-BR-3 cell lines determined with QIFIKIT ® .

Article Snippet: Polyclonal anti-CD59 RP-02 antibody (12474-RP02, SinoBiological, Beijing, China) was detected with anti-rabbit-HRP (Sigma-Aldrich, St. Louis, MO, USA) at a dilution of 1:2 500 in 2.5% BSA-PBS.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Bispecific mAb 2 Antibodies Targeting CD59 Enhance the Complement-Dependent Cytotoxicity Mediated by Rituximab

doi: 10.3390/ijms23095208

Figure Lengend Snippet: Cell assays with anti-CD20/anti-CD59 bispecific antibodies: ( a ) Complement-dependent cytotoxicity assay in rituximab (RX)-(left) and HuMax-20 (HX)-(right) format on Raji cells and RX-based mAb 2 on ARH-77 cells (center). Sample size was n = 3 for Raji and n = 2 for ARH-77 cells. One-way ANOVA was used to determine the significance (* 0.01 ≤ p < 0.05, ** 0.001 ≤ p < 0.01, *** 0.0001 ≤ p < 0.001, **** p ≤ 0.0001); ( b ) SK-BR-3 cell staining of RX-BER mAb 2 molecules (left panel), the interaction of RX-BER2 and RX with SK-BR-3 cells with and without phospholipase C (PLC)-treatment (central panel), MEM-43-staining of SK-BR-3 cells with and without PLC treatment (right panel). 2nd only: cells stained with fluorescent conjugate only, without primary antibody. Presented are mean and S.D.

Article Snippet: Polyclonal anti-CD59 RP-02 antibody (12474-RP02, SinoBiological, Beijing, China) was detected with anti-rabbit-HRP (Sigma-Aldrich, St. Louis, MO, USA) at a dilution of 1:2 500 in 2.5% BSA-PBS.

Techniques: CDC Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Bispecific mAb 2 Antibodies Targeting CD59 Enhance the Complement-Dependent Cytotoxicity Mediated by Rituximab

doi: 10.3390/ijms23095208

Figure Lengend Snippet: Characterization of shuffled BER1x3-clone based mAb 2 molecules: ( a ) SEC-HPLC profiles in native conditions of rituximab (RX)-, HuMax-20 (HX)- and 4420-based mAb 2 molecules (MWS: molecular weight standard); ( b ) ELISA response of RX-based bispecific antibodies with parental Fcabs and the shuffled clone to immobilized CD59; ( c ) Biolayer interferometry-determined binding of RX-BER1x3 to immobilized CD59, antibody in 2-fold dilutions starting with 300 nM; ( d ) Complement-dependent cytotoxicity (CDC) effect of RX-BER1, -3 and -1x3 for Raji and RX-BER1x3 for ARH-77 and Daudi cells. Sample size was n = 3 for Raji and n = 2 for ARH-77 and Daudi cells. One-way ANOVA was used to determine the significance (n.s. (not significant) 0.05 ≤ p , * 0.01 ≤ p < 0.05, ** 0.001 ≤ p < 0.01, *** 0.0001 ≤ p < 0.001, **** p ≤ 0.0001), mean and S.D. are presented; ( e ) 1st panel: CDC effect on Raji cells of RX, mix of RX and 4420 antibody, mix of RX and 4420-BER1x3 mAb 2 and RX-BER1x3 mAb 2 ( n = 2), 2nd panel: CDC effect on Raji cells of RX, RX-BER1x3, and mix of RX and RX-BER1x3 ( n = 3). One-way ANOVA was used to determine the significance (n.s. (not significant) 0.05 ≤ p , ** 0.001 ≤ p < 0.01, *** 0.0001 ≤ p < 0.001, **** p ≤ 0.0001), 3rd panel: Raji cell staining with RX and RX-BER1x3 at 4 °C and at 37 °C ( n = 2), 4th panel: the effect of RX and RX-BER1x3 on Raji cells with and without complement ( n = 2). Mean and S.D. are presented.

Article Snippet: Polyclonal anti-CD59 RP-02 antibody (12474-RP02, SinoBiological, Beijing, China) was detected with anti-rabbit-HRP (Sigma-Aldrich, St. Louis, MO, USA) at a dilution of 1:2 500 in 2.5% BSA-PBS.

Techniques: Molecular Weight, Enzyme-linked Immunosorbent Assay, Binding Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Bispecific mAb 2 Antibodies Targeting CD59 Enhance the Complement-Dependent Cytotoxicity Mediated by Rituximab

doi: 10.3390/ijms23095208

Figure Lengend Snippet: Affinity-matured BER1x3-derived clones from libraries BER1x3_4NNK (“BUD” clones) and BER1x3_5NNK (“TH” clones): ( a ) Complement-dependent cytotoxicity effect of rituximab (RX)-based mAb 2 molecules on Raji cells, compared with parental RX-BER1x3, in 2-fold dilutions starting from 30 nM, decreasing concentrations are presented from dark to light blue (sample size n = 2, mean and S.D. are presented); ( b ) HPLC-SEC in native conditions of RX-based mAb 2 molecules (MWS: molecular weight standard); ( c ) ELISA with CD59 and mouse serum albumin as a control antigen (sample size n = 2, mean and S.D. are shown).

Article Snippet: Polyclonal anti-CD59 RP-02 antibody (12474-RP02, SinoBiological, Beijing, China) was detected with anti-rabbit-HRP (Sigma-Aldrich, St. Louis, MO, USA) at a dilution of 1:2 500 in 2.5% BSA-PBS.

Techniques: Derivative Assay, Clone Assay, Molecular Weight, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Bispecific mAb 2 Antibodies Targeting CD59 Enhance the Complement-Dependent Cytotoxicity Mediated by Rituximab

doi: 10.3390/ijms23095208

Figure Lengend Snippet: BER1x3-derived clones from libraries BER1x3_4NNK and BER1x3_5NNK, selected with mammalian cells-derived CD59 antigen: ( a ) ELISA response of rituximab (RX)-based mAb 2 molecules to HEK-expressed CD59 and mouse serum albumin as a control antigen; ( b ) Complement-dependent cytotoxicity (CDC) assay of RX-based bispecific antibodies on Raji cells, compared with parental clone RX-BER1x3, in 2-fold dilutions starting from 5 nM, decreasing concentrations are presented from dark to light blue (sample size n = 2, mean and S.D. are shown); ( c ) Profiles of RX-derivates in SEC-HPLC in native conditions (MWS: molecular weight standard); ( d ) Biolayer interferometry-measured response of RX-BER5-1-3 to bacterially expressed and refolded CD59 (left panel) and mammalian cells expressed CD59 (right panel), in 2-fold dilutions starting from 300 nM; ( e ) Comparison of the CDC effect of RX and RX-BER5-1-3 on Raji cells (left panel), ARH-77 cells (central panel) and Daudi cells (right panel) (sample size n = 3 for experiments done with Raji and n = 2 for ARH-77 and Daudi, one-way ANOVA was used to determine the significance (n.s. (not significant) 0.05 ≤ p , * 0.01 ≤ p < 0.05, ** 0.001 ≤ p < 0.01, *** 0.0001 ≤ p < 0.001), mean and S.D. are shown; ( f ) SK-BR-3 cell surface binding of RX-based mAb 2 molecules at 2 µM concentration (left panel) and the CDC (in)activity of RX and the mAb 2 derivates on this cell line, sample size n = 2 (right panel). Mean and S.D. are presented.

Article Snippet: Polyclonal anti-CD59 RP-02 antibody (12474-RP02, SinoBiological, Beijing, China) was detected with anti-rabbit-HRP (Sigma-Aldrich, St. Louis, MO, USA) at a dilution of 1:2 500 in 2.5% BSA-PBS.

Techniques: Derivative Assay, Clone Assay, Enzyme-linked Immunosorbent Assay, CDC Assay, Molecular Weight, Binding Assay, Concentration Assay, Activity Assay

Journal: Molecular Medicine Reports

Article Title: The effect of anti-HLA class I antibodies on the immunological properties of human glomerular endothelial cells and their modification by mTOR inhibition or GCN2 kinase activation

doi: 10.3892/mmr.2021.11994

Figure Lengend Snippet: Effect of anti-HLAI on ICAM-1, HLA-DR, CD46 and CD59, and the impact of halofuginone or everolimus treatment. (A) Representative experiment for each of the evaluated factors. (B) Cumulative results are presented. Anti-HLAI antibodies upregulated ICAM-1, HLA-DR, CD46 and CD59. Halofuginone or everolimus treatment decreased ICAM-1. Data are presented as the mean ± SEM. *P<0.05 vs. control cells, # P<0.05 vs. anti-HLAI-treated cells, ^ P<0.05 vs. anti-HLAI-treated cells administered halofuginone, + P<0.05 vs. anti-HLAI-treated cells administered everolimus and $ P<0.05 vs. anti-HLAI-treated cells with halofuginone and everolimus. HLAI, human leukocyte antigen class I; ICAM-1, intracellular adhesion molecule-1; Hal, halofuginone; Ever, everolimus; Ctrl, control.

Article Snippet: Blots were incubated at 4°C for 16 h with the primary antibodies specific against activated cleaved caspase-3 (cleaved caspase-3, 1:1,000, cat. no ab13847, Abcam), focal adhesion kinase (FAK, 1:100, cat. no sc-271126, Santa Cruz Biotechnology, Inc.), phosphorylated at Tyr397 FAK (p-FAK, 1:1,000, cat. no 8556, Cell Signaling Technology, Inc.), mTOR (1:100, cat. no sc-517464, Santa Cruz Biotechnology, Inc.), phosphorylated at Ser2448 mTOR (p-mTOR, 1:100, cat. no sc-293133, Santa Cruz Biotechnology, Inc.), p70S6 kinase (p70S6K, 1:100, cat. no sc-8418, Santa Cruz Biotechnology, Inc.), phosphorylated at Thr389 p70S6K (p-p70S6K, 1:1,000, cat. no 9234, Cell Signaling Technology), protein kinase B (Akt, 1:100, cat. no sc-5298, Santa Cruz Biotechnology, Inc.), phosphorylated at Ser474 Akt (p-Akt, 1:1,000, cat. no 4060, Cell Signaling Technology, Inc.), GCN2 kinase (GCN2K, 1:100, cat. no sc-374609, Santa Cruz Biotechnology, Inc.), phosphorylated at Thr899 GCN2K (p-GCN2K, 1:1,000, cat. no ab75836; Abcam), eIF2α (1:100, cat. no sc-133132, Cell Signaling Technology, Inc.), phosphorylated at Ser51 eIF2α (p-eIF2a, 1:1,000, cat. no 9721, Cell Signaling Technology, Inc.), intercellular adhesion molecule 1 (ICAM-1, 1:1,000, cat. no 4915; Cell Signaling Technology), HLA-DR (Ultra-LEAFTM Purified anti-human HLA-DR Antibody, cat. no 307648, Biolegend), CD46 (1:1,000, cat. no CSB-PA923298, Cusabio), CD59 (1:1,000, cat. no CSB-PA004947YA01HU, Cusabio), and β-actin (1:5,000, cat no. 4967, Cell Signaling Technology, Inc.).

Techniques: Control

Journal: Aging (Albany NY)

Article Title: PTX3 modulates the immunoflogosis in tumor microenvironment and is a prognostic factor for patients with clear cell renal cell carcinoma

doi: 10.18632/aging.103169

Figure Lengend Snippet: Complement system factors’ expression and co-localization with PTX3 in renal clear cell carcinoma. Intra-tumoral expression of PTX3 (green) and co-localization with C1q ( A - D ), C5b9 ( E - H ), CD59 ( I - L ), C3aR ( M - P ), C5R ( Q - T ).

Article Snippet: To this purpose we employed the following primary antibodies: rat monoclonal IgG2a anti-PTX-3 antibody (clone MNB4, Abcam, Cambridge UK), mouse monoclonal IgG2b anti-C1q (clone JL-1; Abcam); rabbit monoclonal IgG anti-Mannose Binding Lectin (anti-MBL) (clone EPSISR5; Abcam); rabbit polyclonal IgG anti-C3aR (Abcam); mouse monoclonal IgG2a anti-C5R1/CD88 (clone P12/1; Abcam); mouse monoclonal IgG2a anti-C5b-9 (clone aE11; Abcam); rabbit polyclonal IgG anti-CD59 (Sigma-Merck KGaA, Darmstadt, Germany).

Techniques: Expressing

Journal: Chemical research in toxicology

Article Title: 4-Hydroxy-7-oxo-5-heptenoic Acid (HOHA) Lactone is a Potent Inducer of the Complement Pathway in Human Retinal Pigmented Epithelial Cells

doi: 10.1021/acs.chemrestox.8b00028

Figure Lengend Snippet: PCR Primer Sequences and Product Sizes Used for Quantitative Real-Time RT-PCR

Article Snippet: Mouse monoclonal antihuman NRF2 (clone 1E9E3; 66504-1-Ig; observed molecular weight: 110 kDa), rabbit polyclonal complement factor B (10170-1-AP; observed molecular weight: 93–100 kDa), rabbit polyclonal complement factor C3 (21337-1-AP; observed molecular weight: 115 kDa) antibodies, rabbit polyclonal CD59 antibodies (10742-1-AP; observed molecular weight: 18–20 kDa) and HRP-conjugated beta-actin mouse monoclonal antibody (cytosolic proteins loading control, HRP-60008) were procured from Proteintech, Rosemont, IL.

Techniques: Sequencing

Journal: Chemical research in toxicology

Article Title: 4-Hydroxy-7-oxo-5-heptenoic Acid (HOHA) Lactone is a Potent Inducer of the Complement Pathway in Human Retinal Pigmented Epithelial Cells

doi: 10.1021/acs.chemrestox.8b00028

Figure Lengend Snippet: Time-course for up-regulation of complement regulatory proteins: (A) CD46, (B) CD55 and (C) CD59 genes in ARPE-19 cells upon exposure to 0.1 μM HOHA-lactone within 2 h of incubation as measured by RT-qPCR. *-p<0.05; **-p<0.025; ***-p<0.01; ****-p<0.005 and *****-p<0.001 compared to control. For all assays, three independent experiments run in triplicates were conducted.

Article Snippet: Mouse monoclonal antihuman NRF2 (clone 1E9E3; 66504-1-Ig; observed molecular weight: 110 kDa), rabbit polyclonal complement factor B (10170-1-AP; observed molecular weight: 93–100 kDa), rabbit polyclonal complement factor C3 (21337-1-AP; observed molecular weight: 115 kDa) antibodies, rabbit polyclonal CD59 antibodies (10742-1-AP; observed molecular weight: 18–20 kDa) and HRP-conjugated beta-actin mouse monoclonal antibody (cytosolic proteins loading control, HRP-60008) were procured from Proteintech, Rosemont, IL.

Techniques: Incubation, Quantitative RT-PCR, Control

Journal: Chemical research in toxicology

Article Title: 4-Hydroxy-7-oxo-5-heptenoic Acid (HOHA) Lactone is a Potent Inducer of the Complement Pathway in Human Retinal Pigmented Epithelial Cells

doi: 10.1021/acs.chemrestox.8b00028

Figure Lengend Snippet: Levels of CD59 after (A) 2 h and (B) 24 h incubation with various concentrations of HOHA lactone by Western blot. Panels A and B show representative immunoblots of CD59 protein in the cell lysates after 2-hours and 24-hours exposure of ARPE-19 cells, respectively. Graphs in Panels A and B show the results of three independent experiments. *-p<0.05; **-p<0.025.

Article Snippet: Mouse monoclonal antihuman NRF2 (clone 1E9E3; 66504-1-Ig; observed molecular weight: 110 kDa), rabbit polyclonal complement factor B (10170-1-AP; observed molecular weight: 93–100 kDa), rabbit polyclonal complement factor C3 (21337-1-AP; observed molecular weight: 115 kDa) antibodies, rabbit polyclonal CD59 antibodies (10742-1-AP; observed molecular weight: 18–20 kDa) and HRP-conjugated beta-actin mouse monoclonal antibody (cytosolic proteins loading control, HRP-60008) were procured from Proteintech, Rosemont, IL.

Techniques: Incubation, Western Blot