Sputum samples were processed through dilution with 8 times volume PBS followed by centrifugation at 3000×g for 15 min. The repeatability of three different NE assays was tested: 1) an activity-based fluorescence resonance energy transfer (FRET) assay according to the protocol of Korkmaz et al. [24 (link)], 2) an activity-based immunoassay (ProteaseTag ELISA; ProAxsis, Belfast, UK) [25 (link)] and 3) a total NE ELISA (AssayMax; Assaypro, St Charles, MO, USA), which measures total protein rather than activity. For the FRET assay, the intra-assay coefficient of variation was 5.6% and the interassay coefficient of variation was 8.8%. For the total NE assay, the intra-assay coefficient of variation was 14.5% and interassay coefficient of variation was 10.6%. For the activity-based immunoassay, the intra-assay coefficient of variation was 7.1% and interassay coefficient of variation was 7%.
Assaymax
Manufactured by Assaypro
Sourced in United States
The AssayMax is a versatile laboratory equipment designed for precise and reliable sample analysis. It utilizes advanced technology to perform a wide range of assays, ensuring accurate and reproducible results. The core function of the AssayMax is to facilitate efficient sample processing and measurement, enabling researchers and scientists to gather valuable data for their investigations.
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5 protocols using assaymax
1
Comparative Analysis of Neutrophil Elastase Assays
2
Oral Glucose Tolerance Test and Metabolic Markers
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OGTT was done with 75-gram standard anhydrous glucose. The glucose was ingested in 300 ml of water after overnight fasting. Blood samples were drawn initially and two hours after ingestion. Plasma fasting glucose, serum total cholesterol, high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), and triglyceride levels were determined by spectrophotometry method (Advia 1800 Clinical Chemistry System Siemens, USA). The fasting serum insulin levels were assessed by chemiluminescence method (Beckman-Coulter Inc., USA). Homeostatic model assessment of IR (HOMA-IR) was calculated by the formula:
After centrifugation, blood samples were stored at -85°C until analysis. The fasting serum levels of the adiponectin, retinol-binding protein 4 (RBP-4), tumour necrosis factor-a (TNF-a), leptin, resistin (AssayMax, Assaypro, USA), vaspin, and visfatin (RayBiotech Inc., USA) were determined by ELISA method.
After centrifugation, blood samples were stored at -85°C until analysis. The fasting serum levels of the adiponectin, retinol-binding protein 4 (RBP-4), tumour necrosis factor-a (TNF-a), leptin, resistin (AssayMax, Assaypro, USA), vaspin, and visfatin (RayBiotech Inc., USA) were determined by ELISA method.
3
Plasma Corticosterone Measurement by ELISA
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After thawing, the plasma corticosterone concentrations (ng/ml) were measured. The
corticosterone concentrations were determined by an enzyme-linked immunoassay (ELISA) kit
(AssayMax; AssayPro, Saint Charles, USA) according to the manufacturer’s instructions.
Details of the assay are described in Zhang et al. [44 (link)].
corticosterone concentrations were determined by an enzyme-linked immunoassay (ELISA) kit
(AssayMax; AssayPro, Saint Charles, USA) according to the manufacturer’s instructions.
Details of the assay are described in Zhang et al. [44 (link)].
4
Quantifying Extracellular BMP4 Levels
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Culture supernatants were harvested and used to measure the extra cellular concentration of BMP4 by a commercial, sensitive, enzyme-linked immuno sorbent assay (ELISA) kit (AssayMax; AssayPro LLC, Winfield, MO). The total protein content of cell monolayers was estimated by a Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA) and used to normalize the results.
5
Plasma Biomarkers in Hyperbilirubinemia
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Plasma biomarkers (prothrombin time, INR, antithrombin III, bilirubin, aspartate transaminase-AST, alanine transaminase-ALT, albumin, alkaline phosphatase-ALP, gamma glutamyl transpherase-GGT, lactate levels) were measured within 24 h after enrolment in the study (day 1) and on consecutive days 3, 5, 7, and 14. An abdominal ultrasound was performed to exclude mechanical causes of hyperbilirubinemia. At the same time, blood samples were drawn for the analysis of novel biomarkers: endothelin-1 (ET-1), hepcidin, plasminogen activator inhibitor 1 (PAI-1), the thrombin-antithrombin (TAT) complex, and interferon-gamma inducible protein 10 kDa (IP-10).
Blood samples were collected and centrifuged at 3000 rpm at room temperature in EDTA tubes; consecutively, samples were frozen at −28 °C within 30 min of collection. Levels of human hepcidin IP-10, PAI-1, and ET-1 were measured using Quantikine ELISA tests (R&D Systems Inc., Minneapolis, MN, USA). Human TAT complex levels were measured using Assay Max (Assaypro LLC, St. Charles, MO, USA). All assays employ the quantitative sandwich enzyme immunoassay technique. Tests were performed and interpreted according to the manufacturer’s instructions.
Blood samples were collected and centrifuged at 3000 rpm at room temperature in EDTA tubes; consecutively, samples were frozen at −28 °C within 30 min of collection. Levels of human hepcidin IP-10, PAI-1, and ET-1 were measured using Quantikine ELISA tests (R&D Systems Inc., Minneapolis, MN, USA). Human TAT complex levels were measured using Assay Max (Assaypro LLC, St. Charles, MO, USA). All assays employ the quantitative sandwich enzyme immunoassay technique. Tests were performed and interpreted according to the manufacturer’s instructions.
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