polyclonal rabbit anti-human apoe  (Thermo Fisher)

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Serum factors create species-specific barriers to hepatic gene transfer by lipid nanoparticles in liver-humanized mice

doi: 10.1016/j.omtm.2025.101470

Figure Lengend Snippet: Comparative histopathology and putative LNP receptor expression in chimeric NSG-PiZ and FRG mouse livers Livers from liver-humanized NSG-PiZ or FRG mice at 24- or 48-h post treatment with PBS, or LNP1–3 formulated with DasherGFP mRNA (A and B). Paraffin-embedded sections from representative livers were stained with H&E (A). Scale bar, 100 μm. Frozen sections from representative livers were stained with Oil red O and counterstained with hematoxylin (B). Scale bars, 1 mm (inset image) and 100 μm (main image). Dashed lines indicate approximate borders between human and mouse hepatocytes. H, human hepatocytes; M, mouse hepatocytes. snRNA-seq and scRNA-seq analysis of putative LNP receptor mRNA levels in hepatocytes of chimeric FRG and NSG-PiZ mice (C). (D) Detection of ALB , A PO E , LDLR , ASGR1 , and ASGR2 mRNA in human and mouse hepatocytes from NSG-PiZ and FRG mice. (E) The percentage of total reads per cell for ALB , APOE , LDLR , ASGR1 , and ASGR2 in human (H) or mouse (M) hepatocytes determined by scRNA-seq (NSG-PiZ) or snRNA-seq (FRG). For the scRNA-seq analysis, cells were pooled from four low engrafted chimeric NSG-PiZ mice prior to single-cell analysis. For the snRNA-seq analysis, reads were pooled from two highly engrafted chimeric FRG mice or two low engrafted chimeric FRG mice from a previously published dataset (Bioproject: PRJNA857105; GEO: GSM6319631 , GSM6319632 , GSM6319637 , GSM6319638 ; Cabanes-Creus et al. ). FRG reads were processed as previously described (Cabanes-Creus et al. ) and scRNA-seq reads were processed as described in the methods. (C) was created using Biorender.

Article Snippet: Serum from chimeric NSG-PiZ mice (HuAlb >1 mg/mL) was depleted of ApoE via immunoprecipitation using a rabbit anti-human ApoE polyclonal antibody (cat # VPA00665 , Bio-Rad, Hercules, CA) and protein G spin trap columns (Cat #28903134, Marlborough, MA) according to the manufacturer’s instructions.

Techniques: Histopathology, Expressing, Staining, Single-cell Analysis

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Serum factors create species-specific barriers to hepatic gene transfer by lipid nanoparticles in liver-humanized mice

doi: 10.1016/j.omtm.2025.101470

Figure Lengend Snippet: NSG-PiZ serum factors impact LNP transfection of human hepatocytes (A) Flow cytometry and immunofluorescence analysis of GFP expression in mouse and human (HLA+ or hCK18+) hepatic cells from chimeric NSG-PiZ mice 24 h after intravenous delivery of SM-102 LNPs (1.3 mg/kg). Chimeric NSG-PiZ mice were administered PBS ( n = 2) or SM-102 LNPs ( n = 4). Representative scatterplots and histograms show relative GFP expression in human or mouse cells for two PBS- and three SM-102 LNP-treated mice along with mouse HuAlb concentrations prior to LNP administration. Representative immunofluorescence staining of paraffin sections for the fourth SM-102 LNP-treated mouse with antibodies specific for GFP (green) and hCK18 (red). (B) serum impacts transfection of PHH in vitro . SM-102 LNP was preincubated with the indicated serum for 15 min at room temperature (RT) prior to addition to media. GFP expression was imaged 6 h after after LNP administration. (C) Inhibition of PHH transfection by chimeric NSG-PiZ mouse serum. SM-102 LNPs, LP01 LNPs, or ALC0315 LNPs were pre-treated with normal mouse serum for 15 min at RT prior to addition to media containing 2% chimeric serum. GFP expression was imaged 6 h after after LNP administration. (D) Inhibition of PHH transfection by recombinant human ApoE. SM-102 LNP was pre-treated with normal mouse serum for 15 min at RT prior to addition to media containing recombinant human ApoE at the indicated concentration. (E) Inhibition of PHH transfection by ApoE depleted chimeric NSG-PiZ mouse serum. Western blot for ApoE using chimeric NSG-PiZ serum, anti-ApoE depleted chimeric NSG-PiZ serum, or control anti-Flag depleted chimeric NSG-PiZ serum (left). SM-102 LNP was pre-treated with normal mouse serum for 15 min at RT prior to addition to media containing 2% chimeric NSG-PiZ serum deleted of ApoE. GFP expression was imaged 6 h after after LNP administration. (F) Inhibition of PHH transfection by naive NSG-PiZ mouse serum. SM-102 LNP was pre-treated with normal mouse serum for 15 min at RT prior to addition to media containing 2% chimeric or naive NSG-PiZ serum. GFP expression was imaged 6 h after after LNP administration. (G) NSG mouse serum supports SM-102 transfection of PHH. SM-102 LNP was pre-treated with NSG serum or chimeric NSG-PiZ serum for 15 min at RT prior to addition to media. GFP expression was imaged 6 h after LNP administration. The experiments shown in (C–F) were performed concurrently and for comparison, the same representative image for SM-102 transfection of PHH after pre-incubation with normal mouse serum is shown as the upper left image within each panel.

Article Snippet: Serum from chimeric NSG-PiZ mice (HuAlb >1 mg/mL) was depleted of ApoE via immunoprecipitation using a rabbit anti-human ApoE polyclonal antibody (cat # VPA00665 , Bio-Rad, Hercules, CA) and protein G spin trap columns (Cat #28903134, Marlborough, MA) according to the manufacturer’s instructions.

Techniques: Transfection, Flow Cytometry, Immunofluorescence, Expressing, Staining, In Vitro, Inhibition, Recombinant, Concentration Assay, Western Blot, Control, Comparison, Incubation

Journal: bioRxiv

Article Title: AAV-Delivered Anti-PC-OxPL Antibody Fragments: A Novel Therapeutic Approach to Target ALS

doi: 10.1101/2025.01.22.634350

Figure Lengend Snippet: a. APOE transcript expression in motor neurons exposed to either PSPC or PONPC, as determined by NanoString nCounter analysis. Values are expressed as means ± SEM. b. Upper panel: Elevated APOE profile (% positive cells and average intensity) in wt motor neurons exposed to PONPC according to different washout periods. Values are expressed as means ± SEM. Two-way ANOVA with Šidák’s multiple comparisons test, **p=0.0064, *p = 0.0167, ns, p ≥ 0.05; n = four, two independent experiments. Lower panel: Representative images showing increased APOE profile in wt motor neurons at 24 h PONPC treatment + 24 h washout. Scale bar = 20 µM (APOE, FITC). c. PC-OxPL levels as detected on the surface of different apolipoprotein particles as measured in paired CSF and plasma from ALS patients. Values are expressed as means ± SEM and each dot represents a single individual (n = 15). One-way ANOVA with Dunnett’s multiple comparisons test, ****p < 0.0001, ***p = 0.0007, **p = 0.0015, ns, p ≥ 0.05. d. Upper panel. Representative images of APOE profile in the spinal cord regions (grey and white matter) of NDC and ALS individuals. Scale bar = 100 µM. Lower panel. APOE expression, as determined by infrared (IR) area and mean signal intensity in the spinal cord regions (grey and white matter) of NDC and ALS individuals. Values are expressed as means ± SEM; n = NDC or ALS individuals per group. e. APOE concentrations as detected in paired CSF and plasma from ALS patients compared to HC. Values are expressed as means ± SEM and each dot represents a single individual (n = 15). Mann-Whitney U test, ****p < 0.0001, ns, p ≥ 0.05.

Article Snippet: To detect OxPL-APOE and OxPL-APOC-III, microtiter well plates were coated with polyclonal rabbit anti-human APOE (ThermoFisher Scientific, Waltham, MA) that recognizes all three APOE isoforms at 5 µg/ml and a rabbit monoclonal antibody to APOC- III (Abgent, San Diego, CA) at 2 µg/ml after documenting these concentrations were saturating.

Techniques: Expressing, MANN-WHITNEY

Journal: Molecular Therapy. Nucleic Acids

Article Title: VWA3A -derived ependyma promoter drives increased therapeutic protein secretion into the CSF

doi: 10.1016/j.omtn.2023.07.016

Figure Lengend Snippet: Human VWA3A promoter drives expression in mouse ependyma and protein secretion into the CSF (A) Coronal section of a mouse brain injected i.c.v. with 2.7E10 total viral genomes AAV4.VWA3A.eGFP vector. Zoom region outlines ependymal transduction in the lateral ventricle. Scale bar, 2.5 mm. (B) Experimental paradigm to measure transgene-derived APOE2 in the CSF of ApoE −/− mice. (C) Wes analysis and (D) quantification of vector-derived APOE2 in CSF of ApoE −/− mice. The first iCAG sample lay outside our standard curve and was not quantified. Bars represent group mean.

Article Snippet: A ratio of 1:50 rabbit anti-human ApoE polyclonal antibody (Novus, no. NBP1-31123) and anti-rabbit detection module (Protein Simple, no. DM-001) was used for ApoE detection.

Techniques: Expressing, Injection, Plasmid Preparation, Transduction, Derivative Assay

Journal: Journal of Lipid Research

Article Title: High binding affinity of electronegative LDL to human aortic proteoglycans depends on its aggregation level

doi: 10.1194/jlr.m800318-jlr200

Figure Lengend Snippet: Fig. 4. Apolipoprotein E (apoE) and apoC-III content in LDL(1), total LDL(2), and LDL(2) subfractions. LDL(2) was subfrac- tionated depending on its PG affinity (see Fig. 3A). ApoE and apoC-III were determined as described in Materials and Methods. Results are the mean 6 SD of five independent experiments. * P , 0.05 versus LDL(1); # P , 0.05 versus LDL(2) 1 subfraction; † P , 0.05 versus LDL(2) 2 subfraction; ‡ P , 0.05 versus LDL(2) 3 subfraction.

Article Snippet: Polyclonal rabbit anti-human apoE IgG (R107, IgG purified by Protein G-method) was coupled to CNBr-activated Sepharose CL4B according to the manufacturerʼs instructions (Amersham Pharmacia Biotech).

Techniques:

Journal: Journal of Lipid Research

Article Title: High binding affinity of electronegative LDL to human aortic proteoglycans depends on its aggregation level

doi: 10.1194/jlr.m800318-jlr200

Figure Lengend Snippet: Fig. 5. Analytical PG affinity chromatography of apoE-rich LDL (2) and apoE-poor LDL(2). ApoE-rich and apoE-poor LDL(2) subfractions were obtained by anti-apoE immunoaffinity chroma- tography, as described in Materials and Methods. Samples were concentrated and injected in the PG analytical affinity chroma- tography column. Figure shows a representative experiment. Dashed line indicates NaCl gradient.

Article Snippet: Polyclonal rabbit anti-human apoE IgG (R107, IgG purified by Protein G-method) was coupled to CNBr-activated Sepharose CL4B according to the manufacturerʼs instructions (Amersham Pharmacia Biotech).

Techniques: Affinity Chromatography, Injection