AGE-elastin was obtained via the incubation of human aortic α-elastin (1.33 mg/mL) with 100 mmol/L glucose for 30 days, as described by Baydanoff et al. [27 (link)]. A blocking ELISA was used for the detection of IgM and IgG autoantibodies to AGEs of vascular EL. The 96-well plates were coated with AGE-elastin (5 μg/mL) and incubated with 100 μL of human sera (diluted 1:20) for 1 h at 37 °C. Then, 100 µL of goat anti-human IgM Ab, Fc5µ, HRP conjugate (AP114P, Sigma-Aldrich, St. Louis, MO, USA) and goat anti-human IgG Ab, Fc, HRP conjugate (AP113P, SigmaAldrich, St. Louis, MO, USA), respectively, were added to each well. Immunoconjugates were diluted 1:10,000 and ortho-phenylenediamine was used as the chromogen. The reaction was stopped by adding 50 μL/well of sulfuric acid (4 M H2SO4), and the optical density was measured on a Coulter Microplate Reader UV Max (Molecular Devices Corp., Menlo Park, CA, USA) at a wavelength of 492 nm. All samples were tested in triplicate.
Ap113p
Manufactured by Merck Group
Sourced in United States
The AP113P is a laboratory equipment designed for general laboratory use. It features a compact and durable construction. The core function of the AP113P is to perform basic tasks required in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Ap114p, by Merck Group (2 mentions)
Human civ, by Merck Group (1 mentions)
96 well flat bottom plate, by Thermo Fisher Scientific (1 mentions)
Envision 2102, by PerkinElmer (1 mentions)
Nunc maxisorp microtiter plates, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg h l horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Rituximab, by InvivoGen (1 mentions)
5 protocols using ap113p
1
AGE-Elastin Autoantibody Detection
2
ELISA for CIV-specific IgM and IgG
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To measure the ACIVAbs IgM and ACIVAbs IgG concentrations, a sandwich ELISA was used. The assays were performed as follows: microtiter 96-well polystyrene plates were coated with 100 μL of 10 μg/mL of human CIV (Sigma-Aldrich, St. Louis, MO, USA), followed by an overnight incubation at 4 °C. Then, 100 μL serum sample (diluted 1:10) was placed in each well of the microtiter plates and incubated for 1 h at 37 °C. After washing, 100 μL of goat anti-human IgM Ab, Fc5µ, HRP conjugate (AP114P, Sigma-Aldrich, St. Louis, MO, USA), or goat anti-human IgG Ab, Fc, and HRP conjugate (AP113P, Sigma-Aldrich, St. Louis, MO, USA), respectively, were added to each well for 1 h at 37 °C. All immunoconjugates were diluted 1:10,000. Then, 100 μL of ortho-phenylenediamine (4 mg/mL in 0.05 M citrate buffer) was added as a colorimetric substrate for 30 min. The reaction was stopped by adding 50 μL of 4 M H2SO4 to each well, and the optical density was measured with a micro-ELISA plate reader (Coulter Microplate Reader UV Max, Molecular Devices Corp., Menlo Park, CA, USA) at a wavelength of 492 nm.
3
SARS-CoV-2 ACE2-Fc Binding Assay
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ELISAs were performed with the human ACE2-Fc chimera protein (GenScript). Flat-bottom, 96-well plates (Thermo Fisher Scientific) were coated with 416 nM of each polyP. After blocking with 5% nonfat dry milk in phosphate-buffered saline (PBS) for 1 hour at 37°C, the plates were incubated with the ACE2-Fc chimeric protein used at increasing concentrations (0.1 to 2 μM) in 3% bovine serum albumin (BSA)–PBS for 1 hour at room temperature with gentle shaking. In parallel assays, the ACE2-Fc chimeric protein was tested under the same conditions on uncoated wells after blocking, as a negative control. The plates were then washed with PBS and incubated with an anti-human HRP-conjugated antibody (AP113P, Sigma-Aldrich, USA) for 1 hour at room temperature with gentle shaking. The remaining steps were performed as previously described (51 (link)). Absorbance at 450 nm was measured with a plate reader (EnVision; 2102, PerkinElmer), and the binding values are reported as the means of at least three determinations.
4
ELISA-based Vaccine Antibody Assay
5
Quantifying Serum Levels of Biotherapeutics
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Plasma levels of hG-CSF were analyzed using the hG-CSF Quantikine enzyme-linked immunosorbent assay (ELISA) kit (SCS50; R&D Systems). Serum rituximab levels were analyzed using the rituximab ELISA kit (IG-AB106; Eagle Biosciences). For anti–hG-CSF, 5J8, and anti–IL-5 mAb ELISAs, immunoassay plates were coated with antigen (BioLegend 578606 and Sino Biologicals 11085-V08H and 15673-HNCE, respectively) and blocked with 3% bovine serum albumin. After binding of expressed serum proteins, detection was carried out with enzyme-linked anti-human IgG and 3,3′,5,5′-tetramethylbenzidine substrate (AP113P; Millipore). For the anti-rituximab ELISA, the coat protein was rituximab from InvivoGen, and the detection antibody was goat anti-mouse IgG H+L horseradish peroxidase from Thermo Fisher.
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