Journal: Heliyon

Article Title: Proteolytic shedding of CD46 from human hepatocytes indicates liver stress

doi: 10.1016/j.heliyon.2024.e40841

Figure Lengend Snippet: Establishing and validating an ELISA for soluble CD46 (sCD46) . (A) Signal:noise (S:N) values for different concentrations of CD46 capture and detection antibodies in Reagent Diluent 2 (n = 2). (B) Titration curve using recombinant human CD46 (rhCD46) as the analyte. Lower limit of quantification (LLOQ), upper limit of quantification (ULOQ) and limit of detection (LOD) are indicated (n = 3). (C) Accuracy (spike recovery) of human serum samples (n = 5). (D) Intra-assay precision, inter-assay precision and reproducibility of human serum sample at two different dilution factors. (E) Analysis of assay linearity (n = 4, Pearson correlation, indicating 90 % prediction bands). (F) Linear regression of the bias plotted against nominal titres (n = 4, Pearson correlation). (G) Robustness based on incubation timing (n = 4, one sample t -test to a mean of 1). (H) Stability of sCD46 (pg/ml) in representative cell culture supernatants over multiple freeze/thaw cycles depending on the storage temperature and the application of a protease inhibitor. (I) rhCD46 titration in ELISA and competitive FACS-based assay (n = 3). (J) Performance of commercial CD46 ELISA kits in detecting serum sCD46 compared to our in-house ELISA. Samples dilutions were based on manufacturers' recommendations.

Article Snippet: Next, 100 μl/well of samples, the corresponding 1:2 serial diluted standards of rhCD46 (2 ng/ml to 31.25 pg/ml, 10256-CD, R&D Systems) and a blank control are added and then incubated for 2 h. Then, 100 μl/well detection antibody (50 ng/ml, BAF2005, R&D Systems) is added followed by another incubation for 2 h. Next, 100 μl/well of Streptavidin-HRP (DY998, R&D Systems) is added followed by 100 μl/well of substrates (DY999, R&D Systems) for 20 and 30 min, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Titration, Recombinant, Intra Assay, Inter Assay, Incubation, Cell Culture, Protease Inhibitor

Journal: Heliyon

Article Title: Proteolytic shedding of CD46 from human hepatocytes indicates liver stress

doi: 10.1016/j.heliyon.2024.e40841

Figure Lengend Snippet: Establishing and validating an ELISA for soluble CD46 (sCD46) . (A) Signal:noise (S:N) values for different concentrations of CD46 capture and detection antibodies in Reagent Diluent 2 (n = 2). (B) Titration curve using recombinant human CD46 (rhCD46) as the analyte. Lower limit of quantification (LLOQ), upper limit of quantification (ULOQ) and limit of detection (LOD) are indicated (n = 3). (C) Accuracy (spike recovery) of human serum samples (n = 5). (D) Intra-assay precision, inter-assay precision and reproducibility of human serum sample at two different dilution factors. (E) Analysis of assay linearity (n = 4, Pearson correlation, indicating 90 % prediction bands). (F) Linear regression of the bias plotted against nominal titres (n = 4, Pearson correlation). (G) Robustness based on incubation timing (n = 4, one sample t -test to a mean of 1). (H) Stability of sCD46 (pg/ml) in representative cell culture supernatants over multiple freeze/thaw cycles depending on the storage temperature and the application of a protease inhibitor. (I) rhCD46 titration in ELISA and competitive FACS-based assay (n = 3). (J) Performance of commercial CD46 ELISA kits in detecting serum sCD46 compared to our in-house ELISA. Samples dilutions were based on manufacturers' recommendations.

Article Snippet: Briefly, 3 × 10 5 CD46-expressing MOLT-4 cells were stained with a PE-conjugated CD46 antibody (REA312, Miltenyi Biotec) after sequential incubation with ViaKr-808 (Beckman Coulter, USA) and 10 % human FcR block (Miltenyi Biotec).

Techniques: Enzyme-linked Immunosorbent Assay, Titration, Recombinant, Intra Assay, Inter Assay, Incubation, Cell Culture, Protease Inhibitor

Journal: Heliyon

Article Title: Proteolytic shedding of CD46 from human hepatocytes indicates liver stress

doi: 10.1016/j.heliyon.2024.e40841

Figure Lengend Snippet: Fat-induced shedding of CD46 involves the prostaglandin E2 (PGE2) pathway and matrix metalloproteinases (MMPs) . (A) Concentration of serum sCD46 in healthy (n = 112) and steatotic (n = 46) patients (Welch two sample t -test). (B) Fat-loading (FL) of HepaRG increased the concentration of sCD46 in the culture supernatants compared to unloaded (UL) HepaRG (n = 7, paired two-sample t -test). (C) The effect of MMP inhibitor TAPI-1 on CD46 shedding measured via ELISA (n = 3, Pearson correlation). (D) qPCR results expressed as fold-change in mRNA expression levels of FL/UL HepaRG (n = 6, one-sample Wilcoxon signed-rank test). (E) PGE2 treatment induces the shedding of CD46 in FL HepaRG in a dose-dependent manner (n = 3). (F) qPCR results expressed as fold-change in gene expression after PGE2 treatment (1 mM) compared to control DMSO (n = 6, Wilcoxon signed-rank test). (G) MMP1 ELISA results of FL HepaRG treated with 1 mM PGE2 or control supernatants (n = 7, Wilcoxon signedrank test).

Article Snippet: Briefly, 3 × 10 5 CD46-expressing MOLT-4 cells were stained with a PE-conjugated CD46 antibody (REA312, Miltenyi Biotec) after sequential incubation with ViaKr-808 (Beckman Coulter, USA) and 10 % human FcR block (Miltenyi Biotec).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Gene Expression, Control