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ELISA

Manufactured by Alpha Diagnostic
Sourced in United States

ELISA (Enzyme-Linked Immunosorbent Assay) is a sensitive analytical technique used to detect and quantify specific substances, such as proteins, hormones, or antibodies, in a sample. The core function of ELISA is to measure the concentration of a target analyte by using antibodies or antigens that are linked to an enzyme. When the target analyte is present, the enzyme catalyzes a reaction that produces a measurable signal, allowing for the quantification of the analyte.

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46 protocols using ELISA

HepG2.2.15 and HepG2-H1.3 cell culture supernatants were passed through 0.22 μm Millex® GP filter (Merck Millipore) and assayed for HBsAg by ELISA (Alpha Diagnostic International) according to the manufacturer’s instructions. Samples were analyzed on a Tecan M200 plate reader using Magellan 6.5 software (Tecan).
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Mice were bled at times indicated and serum was collected. ANAs were quantified using ELISA (Alpha Diagnostic). Total serum immunoglobulin was measured by ELISA (BD Bioscience). Serum creatinine measured using the Indiko system as per manufacturer’s instructions. Urine was collected weekly at indicated ages and urine albumin measured by ELISA as per the manufacturer’s instructions (Ebioscience).
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Blood plasma was analyzed for blood urea nitrogen (BUN), creatinine, calcium, and phosphorus using colorimetric assays (Point Scientific, Canton, MI, USA, or BioAssay Systems, Hayword, CA, USA). Intact PTH and serum C-terminal FGF23 were determined by ELISA kits (Quidel, San Diego, CA, USA). Serum levels of an oxidative stress marker, 8-hydroxy-2’-deoxyguanosine (8-OHdG) were measured using an ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA) and TBARS were measured using TBARS assay (TCA method) kit (Cayman Chemical, Ann Arbor, MI, USA). Serum inflammation marker C-reactive protein (CRP) was measured by ELISA (Alpha Diagnostic International, San Antonio, TX, USA). Serum AGE levels were determined by OxiSelect Advanced End Glycation product Competitive ELISA (Cell BIOLABS, San Diego, CA, USA). Total serum iron levels and total iron binding capacity (TIBC) were determined by the Endocrinology core lab at the Indiana University School of Medicine.
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Levels of albumin and creatine were assessed in pooled urine using commercially available kits: Albuwell M (Exocell) and Creatinine Parameter Assay Kit (R & D Systems) respectively. IgG specific to dsDNA was quantified by ELISA (Alpha Diagnostics). C3 levels were quantified by ELISA (Quidel).
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Urinary albumin was monitored weekly by dipstick analysis (Albustix; Siemens). Animals were considered positive for albuminuria at ≥100 mg/dL18 (link), 30 (link), 31 (link), 33 (link). Urinary albumin excretion rate (mg/day) was assessed by ELISA (Alpha Diagnostic International) using overnight urine samples collected at the conclusion of the study as previously described18 (link), 30 (link), 31 (link), 33 (link). Glomerulosclerosis scoring was assessed by investigators blinded to the sample as previously described by our laboratory18 (link), 19 .
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Anti-dsDNA antibody levels were measured in 1:5000 dilutions of sera by ELISA (Alpha Diagnostics, San Antonio, TX, USA) according to the manufacturer’s instructions. Total IgG and IgG2a levels were measured in 1:100,000 dilutions of sera by ELISA (Bethyl Laboratories, Montgomery, TX, USA) according to the manufacturer’s instructions. IFN-γ levels were measured in 1:10 dilutions of sera by ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.
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Twenty-four-hour urine samples collected were measured for daily urinary albumin excretion rate (mg/day) using ELISA (Alpha Diagnostics, Burlington, North Carolina, USA). Urinary albumin-to-creatinine ratio was calculated as urine albumin/urine creatinine (μg/mg), and urinary creatinine measured using an ELISA kit for mouse albumin (Exocell, Philadelphia, Pennsylvania, USA).
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Urinary albumin was used as a marker of renal injury and was measured weekly by dipstick assay (Albustix; Siemens). At the conclusion of the study, overnight urine samples were used to measured urinary albumin excretion rate (mg/day) by ELISA (Alpha Diagnostic International, San Antonio, TX) as previously described (Mathis et al. 2011, 2012, 2013, 2014). Additional markers of renal injury, kidney injury molecule‐1 (KIM‐1) and neutrophil gelatinase‐associated lipocalin (NGAL), were measured by ELISA (R&D Systems, Minneapolis, MN) as per the manufacturer's instructions. Kidney sections were prepared for glomerulosclerosis scoring with hemotoxylin and eosin staining by Histology Core laboratory services for the Department of Physiology and Biophysics at UMMC, and investigators blinded to the samples scored the sections as previously described by our laboratory (Venegas‐Pont et al. 2009).
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Splenocytes from infected animals were stimulated in vitro with 5 ng/ml of IL-12 or 20 ug/ml of STAg for 72 h and supernatants were assayed for IFN-gamma by ELISA (eBiosciences, San Diego, CA). IFN-gamma and other cytokines were measured from serum collected from the tail vein by a multiplex bead assay (Bio-Rad, Hercules, CA). Anti-dsDNA was measured by ELISA from murine serum monthly to assess autoimmune status (Alpha Diagnostic International, San Antonio, TX). Urine was collected overnight in metabolic cages once a month and proteinuria was measured to monitor lupus-disease progression. Anti-PD-L1 (gift from Gordon Freeman Dana Farber Research Institute, Boston, MA) was administered intraperitoneally at 200 mg starting at day −1 before infection and every third day thereafter.
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Alpha Diagnostic International (ELISA) was employed to identify the expression levels of IFs, such as IL-1, IL-1β, IL-6, IL-10, TGF-β and IFN-γ, in renal tissues. For details, refer to the kit specification.
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