Journal: Archives of Razi Institute

Article Title: Application and Validation of SARS-CoV-2 RBD Neutralizing ELISA Assay

doi: 10.22092/ARI.2021.356677.1890

Figure Lengend Snippet: Schematic representation of the principle of SARS-CoV-2-RBD neutralizing ELISA assay2.2. Assay Optimization

Article Snippet: To immobilize the hACE2 protein at 400 ng/well in 100 μl of 1X working Coating Buffer on the microtiter plates wells, a calculated volume from Stock solution vial of Recombinant His-Tag-Human ACE2 protein in a concentration of 2.8 mg/ml (Raybiotech) was diluted immediately before usage with the prepared 1X working coating buffer from 5X stock ELISA Plate Coating Buffer (Elabscience) to a required volume at the appropriate concentration (4 ng/μl), which was determined in the initial experiments.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Archives of Razi Institute

Article Title: Application and Validation of SARS-CoV-2 RBD Neutralizing ELISA Assay

doi: 10.22092/ARI.2021.356677.1890

Figure Lengend Snippet: Standard curve of anti-SARS-COV-2-RBD nAbs for the in-house developed SARS-COV-2-RBD neutralizing ELISA assay

Article Snippet: To immobilize the hACE2 protein at 400 ng/well in 100 μl of 1X working Coating Buffer on the microtiter plates wells, a calculated volume from Stock solution vial of Recombinant His-Tag-Human ACE2 protein in a concentration of 2.8 mg/ml (Raybiotech) was diluted immediately before usage with the prepared 1X working coating buffer from 5X stock ELISA Plate Coating Buffer (Elabscience) to a required volume at the appropriate concentration (4 ng/μl), which was determined in the initial experiments.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Archives of Razi Institute

Article Title: Application and Validation of SARS-CoV-2 RBD Neutralizing ELISA Assay

doi: 10.22092/ARI.2021.356677.1890

Figure Lengend Snippet: Mean ± standard deviation of OD values of different anti-SARS-COV-2-RBD neutralizing antibody standards generated by the designed neutralizing ELISA kit

Article Snippet: To immobilize the hACE2 protein at 400 ng/well in 100 μl of 1X working Coating Buffer on the microtiter plates wells, a calculated volume from Stock solution vial of Recombinant His-Tag-Human ACE2 protein in a concentration of 2.8 mg/ml (Raybiotech) was diluted immediately before usage with the prepared 1X working coating buffer from 5X stock ELISA Plate Coating Buffer (Elabscience) to a required volume at the appropriate concentration (4 ng/μl), which was determined in the initial experiments.

Techniques: Standard Deviation, Generated, Enzyme-linked Immunosorbent Assay, Concentration Assay, Negative Control, Positive Control

Journal: Archives of Razi Institute

Article Title: Application and Validation of SARS-CoV-2 RBD Neutralizing ELISA Assay

doi: 10.22092/ARI.2021.356677.1890

Figure Lengend Snippet: Cut-off value results of the in-house neutralizing ELISA; A) Optimized ROC analysis cut-off value of positive and B) Concentration and log concentration limits for the optimal established cut-off in this assay

Article Snippet: To immobilize the hACE2 protein at 400 ng/well in 100 μl of 1X working Coating Buffer on the microtiter plates wells, a calculated volume from Stock solution vial of Recombinant His-Tag-Human ACE2 protein in a concentration of 2.8 mg/ml (Raybiotech) was diluted immediately before usage with the prepared 1X working coating buffer from 5X stock ELISA Plate Coating Buffer (Elabscience) to a required volume at the appropriate concentration (4 ng/μl), which was determined in the initial experiments.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Archives of Razi Institute

Article Title: Application and Validation of SARS-CoV-2 RBD Neutralizing ELISA Assay

doi: 10.22092/ARI.2021.356677.1890

Figure Lengend Snippet: Receiver operating characteristic curve analysis for the optimal cut-off value for detecting the positive anti-SARS-COV-2-RBD neutralizing antibodies using the in-house developed neutralizing ELISA kit

Article Snippet: To immobilize the hACE2 protein at 400 ng/well in 100 μl of 1X working Coating Buffer on the microtiter plates wells, a calculated volume from Stock solution vial of Recombinant His-Tag-Human ACE2 protein in a concentration of 2.8 mg/ml (Raybiotech) was diluted immediately before usage with the prepared 1X working coating buffer from 5X stock ELISA Plate Coating Buffer (Elabscience) to a required volume at the appropriate concentration (4 ng/μl), which was determined in the initial experiments.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Archives of Razi Institute

Article Title: Application and Validation of SARS-CoV-2 RBD Neutralizing ELISA Assay

doi: 10.22092/ARI.2021.356677.1890

Figure Lengend Snippet: Serum panels using the in-house designed SARS-COV-2-RBD neutralizing ELISA assay; A) Positive control samples for the assay validation and B) Negative control samples for the assay validation

Article Snippet: To immobilize the hACE2 protein at 400 ng/well in 100 μl of 1X working Coating Buffer on the microtiter plates wells, a calculated volume from Stock solution vial of Recombinant His-Tag-Human ACE2 protein in a concentration of 2.8 mg/ml (Raybiotech) was diluted immediately before usage with the prepared 1X working coating buffer from 5X stock ELISA Plate Coating Buffer (Elabscience) to a required volume at the appropriate concentration (4 ng/μl), which was determined in the initial experiments.

Techniques: Enzyme-linked Immunosorbent Assay, Positive Control, Biomarker Discovery, Negative Control, Concentration Assay

Journal: Archives of Razi Institute

Article Title: Application and Validation of SARS-CoV-2 RBD Neutralizing ELISA Assay

doi: 10.22092/ARI.2021.356677.1890

Figure Lengend Snippet: Agreement of the results between the in-house SARS-COV-2-RBD neutralizing ELISA assay and gold standard test at the in-house established cut-off

Article Snippet: To immobilize the hACE2 protein at 400 ng/well in 100 μl of 1X working Coating Buffer on the microtiter plates wells, a calculated volume from Stock solution vial of Recombinant His-Tag-Human ACE2 protein in a concentration of 2.8 mg/ml (Raybiotech) was diluted immediately before usage with the prepared 1X working coating buffer from 5X stock ELISA Plate Coating Buffer (Elabscience) to a required volume at the appropriate concentration (4 ng/μl), which was determined in the initial experiments.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Archives of Razi Institute

Article Title: Application and Validation of SARS-CoV-2 RBD Neutralizing ELISA Assay

doi: 10.22092/ARI.2021.356677.1890

Figure Lengend Snippet: Validation parameters of the in-house SARS-COV-2-RBD neutralizing ELISA assay

Article Snippet: To immobilize the hACE2 protein at 400 ng/well in 100 μl of 1X working Coating Buffer on the microtiter plates wells, a calculated volume from Stock solution vial of Recombinant His-Tag-Human ACE2 protein in a concentration of 2.8 mg/ml (Raybiotech) was diluted immediately before usage with the prepared 1X working coating buffer from 5X stock ELISA Plate Coating Buffer (Elabscience) to a required volume at the appropriate concentration (4 ng/μl), which was determined in the initial experiments.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: VISTA is an activating receptor in human monocytes

doi: 10.1084/jem.20201601

Figure Lengend Snippet: Binding and specificity of novel anti-VISTA mAbs. (A) VISTA mAb1 and VISTA mAb2 binding to plate-bound recombinant human (hu) PD-L1, huB7H5, or murine (mu) B7H5 by ELISA. (B) Binding kinetics of mAb1 and mAb2 to human and murine VISTA by SPR. No binding (N.B.) was observed for mAb2 to murine VISTA. (C) Representative Biacore sensorgram plots from simultaneous binding assay with mAb1 or mAb2 captured on the surface. Antigen binding to surface mAb is shown, followed by injection of solution mAb (soln). RU, reference units. (D) Summarized FACS staining results that quantify the percentage of VISTA + cells in human whole blood. n = 6 independent donors. T N , naive T cells; T Ag-Ex , antigen-experienced T cells. (E) FACS-based median fluorescence intensity (MFI) of isotype control, mAb1, or mAb2 binding to CD14 + PBMCs while in the presence of titrated recombinant His-tagged VISTA (VISTA-His; green triangles or blue circles) or recombinant His-tagged PD-L1 (PD-L1 His; purple triangles or red squares). A–E are representative of three independent experiments.

Article Snippet: The ELISA Substrate Reagent Pack (DY999), ELISA plate-coating buffer (DY006), reagent diluent concentrate 2 (DY995), and Stop Solution (DY994) were purchased from R&D Systems.

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Injection, Staining, Fluorescence, Control

Journal: The Journal of Experimental Medicine

Article Title: VISTA is an activating receptor in human monocytes

doi: 10.1084/jem.20201601

Figure Lengend Snippet: Characterization of VISTA.COMP and PD-L1.COMP binding on human PBMCs. Related to . (A) Left unstained (gray) or stained with PD-L1.COMP (blue) or VISTA.COMP (red) protein and markers that identify immune cell subtypes, including T cells (CD3 + ), NK cells (CD56 + ), NK T cells (CD3 + CD56 + ), and monocytes (CD14 + SSC hi ) in the presence of 0.5% paraformaldehyde. FSC, forward scatter; SSC, side scatter. (B) Gating strategy for PBMC immune cell identification and T cell subtyping. (C) PD-L1.COMP and VISTA.COMP (negative control) binding to plate-bound, recombinant human (hu) PD1-Fc by ELISA. A and C were performed in two independent experiments.

Article Snippet: The ELISA Substrate Reagent Pack (DY999), ELISA plate-coating buffer (DY006), reagent diluent concentrate 2 (DY995), and Stop Solution (DY994) were purchased from R&D Systems.

Techniques: Binding Assay, Staining, Negative Control, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: A Subregion of Reelin Suppresses Lipoprotein-Induced Cholesterol Accumulation in Macrophages

doi: 10.1371/journal.pone.0136895

Figure Lengend Snippet: (A) ELISA plates were coated with c-myc peptide, purified R5-6 or R5-6C, or null eluent from empty-vector transformed bacteria as a control (Ctrl), and then incubated with RAW 264.7 cell lysates. The apoER2 and VLDLR in RAW 264.7 cell lysates bound to ELISA plates were detected using antibodies against apoER2 and VLDLR. (B) RAW 264.7 cells were transfected with empty pcDNA3.1B vector or apoER2-GFP expression plasmid, and then incubated with 30 μg/ml of apoB-carrying and apoE-free lipoproteins (+LP) or without lipoproteins (-LP) in the presence or absence of 0.2 μg/ml purified R5-6 protein. The amount of R5-6 bound to the cell surface was determined by ELISA using an antibody against c-myc epitope. The background binding was determined using a normal mouse IgG. (C-D) RAW 264.7 cells were treated with 0.2 μg/ml of purified R5-6 protein or culture medium alone (Ctrl). The protein level of ABCA1 was determined by immunoblotting and quantified relative to β-actin. (E) RAW 246.7 cells were treated as described in plane B. The mRNA level of ABCA1 was determined by quantitative real-time RT-PCR and normalized relative to GAPDH mRNA. (F) RAW 264.7 cells were labeled with 0.25 μCi/ml of 3 H-cholesterol, followed by incubation with 5 μg/ml c-myc peptide, 0.2 μg/ml purified R5-6 protein or vehicle control (Ctrl). Cholesterol efflux was determined in cells incubated with or without apoAI treatment, and was expressed as the percentage of radioactivity in the medium compared to the total radioactivity in the cells and medium. ApoAI-mediated cholesterol efflux was calculated as the difference of efflux from cells in the presence and absence of apoAI treatment. Data represent the mean ± SEM of four or more independent experiments. * p < 0.05 vs . Ctrl; † , p <0.05 vs . c-myc-immobilized ELISA plates or treated cells; ‡ , p < 0.05 vs . cells transfected with the same plasmids and untreated with R5-6; $ , p < 0.05 vs . cells transfected with empty vector and treated with R5-6; and # , p < 0.05 vs . cells transfected with apoER2-GFP-expression plasmid and untreated with lipoproteins.

Article Snippet: PI3K inhibitor LY294002 (sc-201426), Sp1 inhibitor mithramycin A (sc-200909), ELISA plate (sc-204463), scrambled siRNA, and siRNAs specific for VLDLR or apoER2, antibodies against ABCA1 (sc-58219), β-actin (sc-47778), apoER2 (sc-20746), VLDLR (sc-18824), and mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Plasmid Preparation, Transformation Assay, Bacteria, Control, Incubation, Transfection, Expressing, Binding Assay, Western Blot, Quantitative RT-PCR, Labeling, Radioactivity