rhcd46  (R&D Systems)

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: Molecular immunology

Article Title: Autoantibodies to CD59, CD55, CD46 or CD35 are not associated with atypical haemolytic uraemic syndrome (aHUS)

doi: 10.1016/j.molimm.2014.07.017

Figure Lengend Snippet: (a) Newcastle preparation, after dialysis (Lane 1, 20 µl and lane 2, 10 µl), or St. Louis prep (lane 5), were loaded in non-reducing SDS–PAGE buffer (irrelevant lanes are not shown). (b) 400 ng London (1), St. Louis (2) and Newcastle (3) rhCD46 prep., or BSA (4) were run on 15% SDS–PAGE and blotted. rhCD46 was detected using GB24 and shee panti-mouse – HRPO (both 1:1000) and a 1 min exposure is shown for each blot. (c) Again 400 ng of each preparation and BSA (1–4 as above) were loaded onto two identical15% gels and blotted. The Nitrocellulose was divided in two and then probed with GB24 or P1 sera (1/300) or P1 IgG (1/50) or BDC sera (1/300) as indicated. The four ‘strips’ were aligned and exposed for 5 min. (d) Coomassie stained gel of GB24 purified Newcastle produced rhCD46. “Page ruler markers” are indicated throughout.

Article Snippet: Testing and purification of E. coli produced recombinantCD46 (membrane cofactor protein, MCP) We originally received a batch of recombinant human CD46 (rhCD46) protein, generated in E. coli from Dr Claudia Kemper’s group (London, UK) and used this to screen our BDC and aHUS cohorts for the presence of autoantibodies to CD46.

Techniques: SDS Page, Staining, Purification, Produced

Journal: Molecular immunology

Article Title: Autoantibodies to CD59, CD55, CD46 or CD35 are not associated with atypical haemolytic uraemic syndrome (aHUS)

doi: 10.1016/j.molimm.2014.07.017

Figure Lengend Snippet: (a) A standard curve based on the mAb GB24 (1 µg/ml) binding to rhCD46 using ELISA is shown, allowing RU values to be assigned to test samples in the ELISA. (b) Indicates the relative unit (RU) values of 100 healthy blood donor controls (BDC) and 89 aHUS patient samples. Mean RU values are indicated by the solid line in each cohort and a dashed line representing the 97.5 percentile of the BDC group is also indicated. (c) The highest reacting BDC and aHUS samples, as indicated, were applied to nitrocellulose strips from a Western blotted rhCD46 prep gel. Each strip (delineated by vertical bars) was aligned prior to exposure for 20 min. Irrelevant intervening strips have been removed in this picture. GB24 was used as a positive control for protein loading and position. Molecular weight markers are shown and results are representative of two experiments.

Article Snippet: Testing and purification of E. coli produced recombinantCD46 (membrane cofactor protein, MCP) We originally received a batch of recombinant human CD46 (rhCD46) protein, generated in E. coli from Dr Claudia Kemper’s group (London, UK) and used this to screen our BDC and aHUS cohorts for the presence of autoantibodies to CD46.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Stripping Membranes, Positive Control, Molecular Weight