Urine and plasma samples were collected from the patients before renal biopsy, and samples from the healthy controls were stored at −80°C until analysis. Activated complement components, including Bb, C4d, C3a, C5a, and SC5b-9, were detected by ELISA kits (Quidel, USA). Urinary levels of MBL were detected by ELISAs using a commercial MBL Oligomer ELISA kit (Bioporto, Hellerup, Denmark). All assays were conducted according to the manufacturer’s instructions. Urine and plasma samples had only one freeze/thaw cycle before analysis. Frozen specimens were thawed rapidly at 37°C and immediately moved to ice to prevent complement activation prior to dilution. After dilution, the samples were loaded into the microassay wells as rapidly as possible.
Elisa kit
Manufactured by Quidel
Sourced in United States
ELISA (Enzyme-Linked Immunosorbent Assay) kits are laboratory equipment used for the detection and quantification of various biomolecules, such as proteins, antibodies, and hormones, in a sample. These kits utilize the principles of immunoassay technology to provide accurate and reliable results.
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Lab products found in correlation
Elisa kit, by Enzo Life Sciences (2 mentions)
Elisa kit, by Hycult Biotech (2 mentions)
Dri chem7000, by Heska (2 mentions)
Modp analyzer, by Roche (2 mentions)
Rapidlab 348ex machine, by Siemens (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Tbars tca method assay kit, by Cayman Chemical (1 mentions)
Elisa, by Alpha Diagnostic (1 mentions)
Qiaamp dna blood maxi kit, by Qiagen (1 mentions)
Elisa, by Hycult Biotech (1 mentions)
Elisa kit, by Abcam (1 mentions)
Sensolyte pnpp alp assay kit, by AnaSpec (1 mentions)
Eia kit, by Biomedical Technologies (1 mentions)
Vacuette, by Greiner (1 mentions)
Vettest analyzer, by IDEXX (1 mentions)
Elisa kit, by Immundiagnostik (1 mentions)
Elisa kit, by Cusabio (1 mentions)
Elisa kit, by USCN (1 mentions)
Luminex human discovery assay plates, by R&D Systems (1 mentions)
Luminex 200 analyzer, by Thermo Fisher Scientific (1 mentions)
50 protocols using elisa kit
1
Quantification of Complement Activation
2
Comprehensive Metabolic and Oxidative Profiling
3
Assessing Bone Metabolism in AI Therapy
4
Assay for MASP-2 Inhibitor Evaluation
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Example 9
The following assay is used to test whether a MASP-2 inhibitory agent blocks the classical pathway by analyzing the effect of a MASP-2 inhibitory agent under conditions in which the classical pathway is initiated by immune complexes.
Methods:
To test the effect of a MASP-2 inhibitory agent on conditions of complement activation where the classical pathway is initiated by immune complexes, triplicate 50 μl samples containing 90% NHS are incubated at 37° C. in the presence of g/ml immune complex (IC) or PBS, and parallel triplicate samples (+/−IC) are also included which contain 200 nM anti-properdin monoclonal antibody during the 37° C. incubation. After a two hour incubation at 37° C., 13 mM EDTA is added to all samples to stop further complement activation and the samples are immediately cooled to 5° C. The samples are then stored at −70° C. prior to being assayed for complement activation products (C3a and sC5b-9) using ELISA kits (Quidel, Catalog Nos. A015 and A009) following the manufacturer's instructions.
5
Urine Biomarker Sampling and Analysis
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One 10-ml urine sample was collected from the catheter tubing, or a clean catch was requested directly from the patient within a few hours of the routine morning blood draw that indicated the patient had AKI. Samples were immediately refrigerated and then centrifuged at 5000 × g for 10 minutes at 4°C within 4 hours of collection. Samples were stored in separated, 1 mL aliquots without additives at -80°C till biomarker measurements. Urine YKL-40 levels were assessed in the last quarter of 2012 using enzyme-linked immunosorbent assay (ELISA) kits as previously described (Quidel, San Diego, CA)
[13 (link)]. Population based urine levels have not been defined for YKL-40, but published data for 22 non-diabetic patients with normal renal function indicate urine YKL-40 is typically undetectable or less than 0.2 ng/ml in healthy individuals
[17 (link)]. Urine NGAL levels were measured via ELISA by the Devarajan laboratory between 2008 and 2009 as reported in our prior publication
[8 (link)]. Population mean levels of urine NGAL in healthy adults are between 10–23 ng/ml
[18 (link)]. Note, the documented intra-assay and inter-assay coefficients of variation for both YKL-40 and NGAL ELISA methods are less than 10%. All laboratory measurements were performed by personnel blinded to patient information.
[13 (link)]. Population based urine levels have not been defined for YKL-40, but published data for 22 non-diabetic patients with normal renal function indicate urine YKL-40 is typically undetectable or less than 0.2 ng/ml in healthy individuals
[17 (link)]. Urine NGAL levels were measured via ELISA by the Devarajan laboratory between 2008 and 2009 as reported in our prior publication
[8 (link)]. Population mean levels of urine NGAL in healthy adults are between 10–23 ng/ml
[18 (link)]. Note, the documented intra-assay and inter-assay coefficients of variation for both YKL-40 and NGAL ELISA methods are less than 10%. All laboratory measurements were performed by personnel blinded to patient information.
6
Acute Toxicity of Revusiran in Rats and Monkeys
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Revusiran was administered to male and female Sprague-Dawley rats (8 weeks old) (five/sex/group) by daily SC injections at 0, 30, 100, or 300 mg/(kg·day) for 5 days at a dose volume of 1.5 mL/kg. Animals were sacrificed 1 day after the final dose. Male cynomolgus monkeys (2–3 years old) (three animals/group) received a single SC dose of revusiran at 0 or 300 mg/kg and were observed for 14 days. Acute toxicity in rats and monkeys was assessed based on clinical signs, injection site observations (monkeys), body weight, qualitative food consumption (monkeys), clinical pathology, gross examinations (rats), organ weights (rats), and microscopic pathology of the liver, spleen, kidney, lung, heart, brain, and injection sites (rats).
In monkeys, blood was collected for analysis of serum TTR concentrations, plasma complement C3a and Bb concentrations, and plasma cytokines/chemokines (CD40 ligand, G-CSF, GM-CSF, IFN-γ, IL-10, IL-12/23 (p40), IL-13, IL-15, IL-17, IL-18, IL-1RA, IL-1β, IL-2, IL-5, IL-6, IL-8, MCP-1, MIP-1α, MIP-1β, TGF-α, TNF-α, and VEGF). Serum TTR was measured by using an enzyme-linked immunosorbent assay (ELISA). Plasma complement was measured by using commercially available ELISA kits (QUIDEL®). Plasma cytokines were measured by using a Milliplex® monkey cytokine kit and BioPlex® analyzer.
In monkeys, blood was collected for analysis of serum TTR concentrations, plasma complement C3a and Bb concentrations, and plasma cytokines/chemokines (CD40 ligand, G-CSF, GM-CSF, IFN-γ, IL-10, IL-12/23 (p40), IL-13, IL-15, IL-17, IL-18, IL-1RA, IL-1β, IL-2, IL-5, IL-6, IL-8, MCP-1, MIP-1α, MIP-1β, TGF-α, TNF-α, and VEGF). Serum TTR was measured by using an enzyme-linked immunosorbent assay (ELISA). Plasma complement was measured by using commercially available ELISA kits (QUIDEL®). Plasma cytokines were measured by using a Milliplex® monkey cytokine kit and BioPlex® analyzer.
7
Biomarkers of Mineral Homeostasis
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At the end of the experiment, rats were sacrificed by aortic puncture and exsanguination under general anesthesia. Blood was collected in heparinized syringes, and plasma was separated by centrifugation and stored at −80 °C until assayed. To determine the levels of the molecules of interest at the moment of sacrifice, ELISA kits were used for the determination of plasma intact PTH (Quidel, San Diego, CA, USA), intact FGF23 (Kainos Laboratories, Tokyo, Japan), c-terminal FGF23 (Quidel), and urine and plasma Klotho (BT Lab, Shanghai, China). Serum and urine calcium, phosphorus, magnesium, and creatinine levels were quantified by spectrophotometry (Biosystems, Barcelona, Spain). The urinary excretion of sodium and potassium was determined with the aid of a Spotlyte Na/K analyzer (Menarini Diagnostics, Barcelona, Spain).
8
Characterizing MASP-2 Inhibition of Classical Pathway
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Example 9
The following assay is used to test whether a MASP-2 inhibitory agent blocks the classical pathway by analyzing the effect of a MASP-2 inhibitory agent under conditions in which the classical pathway is initiated by immune complexes.
Methods:
To test the effect of a MASP-2 inhibitory agent on conditions of complement activation where the classical pathway is initiated by immune complexes, triplicate 50 μl samples containing 90% NHS are incubated at 37° C. in the presence of 10 μg/ml immune complex (IC) or PBS, and parallel triplicate samples (+/−IC) are also included which contain 200 nM anti-properdin monoclonal antibody during the 37° C. incubation. After a two hour incubation at 37° C., 13 mM EDTA is added to all samples to stop further complement activation and the samples are immediately cooled to 5° C. The samples are then stored at −70° C. prior to being assayed for complement activation products (C3a and sC5b-9) using ELISA kits (Quidel, Catalog Nos. A015 and A009) following the manufacturer's instructions.
9
Evaluating MASP-2 Inhibition on Classical Pathway
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Example 9
The following assay is used to test whether a MASP-2 inhibitory agent blocks the classical pathway by analyzing the effect of a MASP-2 inhibitory agent under conditions in which the classical pathway is initiated by immune complexes.
Methods:
To test the effect of a MASP-2 inhibitory agent on conditions of complement activation where the classical pathway is initiated by immune complexes, triplicate 50 μl samples containing 90% NHS are incubated at 37° C. in the presence of 10 μg/ml immune complex (IC) or PBS, and parallel triplicate samples (+/−IC) are also included which contain 200 nM anti-properdin monoclonal antibody during the 37° C. incubation. After a two hour incubation at 37° C., 13 mM EDTA is added to all samples to stop further complement activation and the samples are immediately cooled to 5° C. The samples are then stored at −70° C. prior to being assayed for complement activation products (C3a and sC5b-9) using ELISA kits (Quidel, Catalog Nos. A015 and A009) following the manufacturer's instructions.
10
Bb Quantification Using ELISA Kits
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