Evaluation of ” Caterina assay”: An Alternative Tool
Evaluation of ” Caterina assay“: An Alternative Tool to the Commercialized Kits Used for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Identification
Here we describe the first molecular test developed in the early stage of the pandemic to diagnose the first cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Sardinian patients in February-March 2020, when diagnostic certified methodology had not yet been adopted by clinical microbiology laboratories. The “Caterina assay” is a SYBR®Green real-time reverse-transcription polymerase chain reaction (rRT-PCR), designed to detect the nucleocapsid phosphoprotein (N) gene that exhibits high discriminative variation RNA sequence among bat and human coronaviruses.
The molecular method was applied to detect SARS-CoV-2 in nasal swabs collected from 2110 suspected cases. The study article describes the first molecular test developed in the early stage of the declared pandemic to identify the coronavirus disease 2019 (COVID-19) in Sardinian patients in February-March 2020, when a diagnostic certified methodology had not yet been adopted by clinical microbiology laboratories.
The assay presented high specificity and sensitivity (with a detection limit ≥50 viral genomes/μL). No false-positives were detected, as confirmed by the comparison with two certified commercial kits. Although other validated molecular methods are currently in use, the Caterina assay still represents a valid and low-cost detection procedure that could be applied in countries with limited economic resources.
Description: The Fluorogenic HDAC Assay Kit is a complete assay system_x000D_designed to measure histone deacetylase (HDAC) class 1 activity for screening_x000D_and profiling applications. The kit comes in a convenient 96-well format, with all the_x000D_reagents necessary for 100 fluorescent HDAC activity measurements. In_x000D_addition, the kit includes purified HDAC2 enzyme and a potent HDAC inhibitor,_x000D_Trichostatin A, for use as a positive and negative control. The Fluorogenic HDAC_x000D_Assay Kit is based on a unique fluorogenic substrate and developer combination._x000D_This assay method eliminates dealing with the radioactivity, extraction, and_x000D_chromatography aspects of traditional assays. Using this kit, only two simple_x000D_steps on a microtiter plate are needed to analyze the HDAC activity level. First,_x000D_the HDAC fluorometric substrate, containing an acetylated lysine side chain, is_x000D_incubated with purified HDAC enzyme. The deacetylation sensitizes the_x000D_substrate so subsequent treatment with the Lysine Developer produces a_x000D_fluorophore that can then be measured using a fluorescence reader at 485 nm_x000D_(excitation)/528 nm (emission)._x000D__x000D_Our other HDAC kit (#50033) contains a substrate that is excited at wavelengths 350-380 nm and fluoresces at wavelengths 440-460 nm. However, the substrate in this kit fluoresces at longer (green) wavelengths: 485 nm(excitation)/528 nm (emission). It is most useful when the sample or inhibitor fluoresces at wavelengths that overlap those of our other HDAC substrate
Description: For sensitive and high-throughput phosphate determination. Key Features: Reagent very stable. Due to our innovative formulation, no precipitation of reagent occurs. Therefore no filtration of reagent is needed prior to assays, as is often required with other commercial kits. High sensitivity and wide detection range: detection of as little of 1.6 pmoles of phosphate and useful range between 0.02 µM and 40 µM phosphate. Fast and convenient: homogeneous "mix-and-measure" assay allows quantitation of free phosphate within 20 minutes. Compatible with routine laboratory and HTS formats: assays can be performed in tubes, cuvettes or microplates, on spectrophotometers and plate readers. Robust and amenable to HTS: Z factors of 0.7 to 0.9 are observed in 96-well and 384-well plates. Can be readily automated on HTS liquid handling systems. Method: OD620nm (malachite green). Samples: Biological, environment etc. Species: All. Procedure: Assay takes 30 min. Kit size: 2500 tests. Detection limit: 0.02 µM.
Description: The OxiSelect ROS Assay Kit is a cell-based assay for measuring hydroxyl, peroxyl, and other reactive oxygen species activity within a cell. The assay employs the cell-permeable fluorogenic probe DCFH-DA, which diffuses into cells and is deacetylcated by cellular esterases into the non-fluorescent DCFH (Figure 1). In the presence of ROS, DCFH is rapidly oxidized to highly fluorescent DCF. Fluorescence is read on a standard fluorometric plate reader.
Description: The OxiSelect ROS Assay Kit is a cell-based assay for measuring hydroxyl, peroxyl, and other reactive oxygen species activity within a cell. The assay employs the cell-permeable fluorogenic probe DCFH-DA, which diffuses into cells and is deacetylcated by cellular esterases into the non-fluorescent DCFH (Figure 1). In the presence of ROS, DCFH is rapidly oxidized to highly fluorescent DCF. Fluorescence is read on a standard fluorometric plate reader.
Description: The OxiSelect Cellular Antioxidant Assay Kit is a cell-based assay for measuring the activity of an exogenous antioxidant compound within adherent cells. Cells are first cultured in a 96-well black fluorescence cell culture plate until confluent. Then the cells are pre-incubated with a cell-permeable DCFH-DA fluorescence probe dye and the bioflavonoid Quercetin, or the antioxidant sample being tested. After a brief incubation, the cells are washed, and the reaction started by adding the Free Radical Initiator. The Free Radical Initiator creates free radicals that convert the probe to highly fluorescent DCF. The Quercetin inhibits the formation of free radicals, and thus DCF formation, in a concentration dependent manner.
Description: The OxiSelect In Vitro ROS/RNS Assay provides a sensitive method to detect total reactive oxygen species (ROS) plus reactive nitrogen species (RNS) in a wide variety of sample types. This assay employs a proprietary fluorogenic probe, DCFH-DiOxyQ; the probe is primed with a dequenching reagent to the highly reactive DCFH form. In the presence of ROS and RNS, the DCFH is rapidly oxidized to the highly fluorescent DCF.
OxiSelect In Vitro ROS/RNS Assay Kit (Green Fluorescence)
Description: The OxiSelect In Vitro ROS/RNS Assay provides a sensitive method to detect total reactive oxygen species (ROS) plus reactive nitrogen species (RNS) in a wide variety of sample types. This assay employs a proprietary fluorogenic probe, DCFH-DiOxyQ; the probe is primed with a dequenching reagent to the highly reactive DCFH form. In the presence of ROS and RNS, the DCFH is rapidly oxidized to the highly fluorescent DCF.
Description: The monitoring of reduced and oxidized glutathione (GSH) in biological samples is essential for evaluating the redox and detoxification status of cells and tissues in relation to the protective role of glutathione against oxidative and free-radical-mediated cell injury.
Description: The OxiSelect ROS Assay Kit is a cell-based assay for measuring hydroxyl, peroxyl, and other reactive oxygen species activity within a cell. The assay employs the cell-permeable fluorogenic probe DCFH-DA, which diffuses into cells and is deacetylcated by cellular esterases into the non-fluorescent DCFH (Figure 1). In the presence of ROS, DCFH is rapidly oxidized to highly fluorescent DCF. Fluorescence is read on a standard fluorometric plate reader.
Description: Acetylcholinesterase, also known as AChE, is an enzyme that degrades (through its hydrolytic activity) the neurotransmitter acetylcholine, producing choline and an acetate group.
Description: The OxiSelect In Vitro ROS/RNS Assay provides a sensitive method to detect total reactive oxygen species (ROS) plus reactive nitrogen species (RNS) in a wide variety of sample types. This assay employs a proprietary fluorogenic probe, DCFH-DiOxyQ; the probe is primed with a dequenching reagent to the highly reactive DCFH form. In the presence of ROS and RNS, the DCFH is rapidly oxidized to the highly fluorescent DCF.
Live or Deadâ„¢ Cell Viability Assay Kit *Green/Red Dual Fluorescence*
Description: This Live or Dead™cell viability uses two fluorogenic indicators: calcein AM for viable cells and a cell-impermeable DNA-binding dye for the cells with compromised membranes.
Live or Deadâ„¢ Cell Viability Assay Kit *Green/Red Dual Fluorescence*
Description: This Live or Dead™cell viability uses two fluorogenic indicators: calcein AM for viable cells and a cell-impermeable DNA-binding dye for the cells with compromised membranes.
Description: Lipid droplets, also referred to as lipid bodies, oil bodies or adiposomes, are lipid-rich cellular organelles that regulate the storage and hydrolysis of neutral lipids.
Description: The detection and measurement of free thiol (such as free cysteine, glutathione and cysteine residues in proteins) is one of the essential tasks for investigating biological processes and events in many biological systems.
Description: Our Amplite® Fluorometric 20S Proteasome Assay Kit is a homogeneous fluorescent assay that measures the chymotrypsin-like protease activity associated with the proteasome complex either in cultured cells or cell lysates.
Evaluation of a new semi-automated Hydragel 11 von Willebrand factor multimers assaykit for routine use
Background: Accurate diagnosis and classification of von Willebrand disease (VWD) are essential for optimal management. The von Willebrand factor multimers analysis (VWF:MM) is an integral part of the diagnostic process in the phenotypic classification, especially in discrepant cases. The aim of this study was to evaluate the performance of a new Hydragel 11VWF multimer assay (H11VW).
Methods: Analytical performance characteristics such as repeatability (intra-assay variability, in gel between track variation), reproducibility (inter-assay variability, between gel variation), sensitivity, EQA performance and differences between two commercially available VWF:MM kits (H5VW and H11VW) were analysed in healthy volunteers’ plasmas using in-house prepared reference plasma.
Results: Repeatability and reproducibility results of H11VW demonstrated acceptable and equivalent performance with previously verified H5VW. Participation in EQA was successful. No statistically significant difference was detected between H5VW and H11VW kits for different fractions of multimers: LMWM p=0.807; IMWM p=0.183; HMWM p=0.774.
Conclusions: H11VW demonstrated acceptable analytical performance characteristics. H11VW kit conveniently offers a more significant number of samples on a single gel. H5VW and H11VW kits can be used in daily practice interchangeably.
Developmental validation of VeriFiler™ Plus PCR Amplification Kit: A 6-dye multiplex assay designed for casework samples
The VeriFiler™ Plus PCR Amplification Kit is a 6-dye multiplex assay that simultaneously amplifies a set of 23 autosomal markers (D3S1358, vWA, D16S539, CSF1PO, D6S1043, D8S1179, D21S11, D18S51, D5S818, D2S441, D19S433, FGA, D10S1248, D22S1045, D1S1656, D13S317, D7S820, Penta E, Penta D, TH01, D12S391, D2S1338, and TPOX), a quality indicator system, and two sex-identification markers. Combined, the markers satisfy the requirements of the Chinese National autosomal DNA database as well as expanded CODIS (Combined DNA Index System).
The VeriFiler Plus kit was developed with an improved Master Mix which incorporates the brighter TED™ dye, and accommodates a higher sample loading volume thus allowing for increased sensitivity and enabling maximum information recovery from challenging casework samples including touch, degraded, and inhibited samples.
Here, we report the results of the developmental validation study which followed the SWGDAM (Scientific Working Group on DNA Analysis Methods) guidelines and includes data for PCR-based studies, sensitivity, species specificity, stability, precision, reproducibility and repeatability, concordance, stutter, DNA mixtures, and performance on mock casework samples. The results validate the multiplex design as well as demonstrate the kit’s robustness, reliability, and suitability as an assay for human identification with casework DNA samples.
Performance of Saliva Samples for COVID-19 Diagnosis by Using the Allplex TM 2019-nCoV AssayKit
Background: Although the nasopharyngeal swab (NPS) is the reference sampling method for the detection of SARS-Cov-2, it is not always possible to collect NPS in some patients. Saliva represents an interesting sampling method because it is less invasive and more convenient in patients with nasal or pharyngeal lesions.
Objective: To compare the RT-qPCR test performances of saliva samples with nasal mid-turbinate swab (NMTS) and NPS samples in a cohort of ambulatory patients suspected of having COVID-19.
Study Design: For each of the 112 enrolled patients, NPS, NMTS, and saliva samples were collected and tested for SARS-Cov-2 detection using three different target genes (RdRP, N and E genes) by RT-qPCR.
Results: Among the positive samples (56/112), saliva samples showed a lower percentage of SARS-Cov-2 detection compared to NPS samples, (85.7 vs. 96.4%), while still a lower percentage was observed for NMTS samples (78.6%). In average, saliva samples showed higher Ct values for all tested target genes, compared to those from NPS and NMTS samples.
Conclusions: By using the AllplexTM 2019-nCoV Assay Kit, saliva samples showed lower sensitivity for SARS CoV-2 compared to NPS samples; however, the not detected cases had lower viral burden in NPS samples (CT values >33) representing an interesting alternative sampling method in patients in which it is not possible to take a NPS sample.
Application of Lithium Assaykit LS for quantification of lithium in whole blood and urine
A commercially available kit for the quantitation of lithium, the Lithium Assay kit LS, was originally developed to measure lithium in serum or plasma using a conventional microplate reader. We investigated whether use of the kit could be extended to quantify lithium in whole blood and urine samples collected at autopsy.
The calibration curve for whole blood showed good linearity ranging from 0.5 to 20 µg/mL with a coefficient of determination of 0.998 when samples were pretreated with methanol followed by acetonitrile. Moreover, for urine, we obtained excellent linearity with a coefficient of determination of 0.999 without any pretreatment.
The accuracies and precisions were 106.3-174.7% and 1.9-18.1% for whole blood and 83.3-118.8% and 5.7-33.8% for urine. The values in the lower concentration range (0.5-1 µg/mL) were not satisfactory, whereas those in the higher range (2-20 µg/mL) were acceptable. The Lithium Assay kit LS was successfully applied to the measurement of lithium in whole blood and urine samples collected at autopsies. This method appears to be useful for forensic toxicological investigations because of its simplicity and speed.
Description: Description of target: The FAM FLICA™ Caspase 3 & 7 kit uses a quick and easy method to analyze active caspases in apoptotic cells.;Species reactivity: ;Application: FC, IF;Assay info: ;Sensitivity:
Description: The Homogeneous Caspase-3 Assay Kit is a complete assay system designed to measure Caspase-3 activity for screening and profiling applications. It comes in a convenient 96-well format, with all the reagents necessary for 100 fluorescent Caspase-3 activity measurements. In addition, the kit includes purified Caspase-3 enzyme and a potent Caspase-3 inhibitor, Ac-DNLD-CHO, for use as a positive and negative control. Using this kit, only one simple step on a microtiter plate is needed to analyze the Caspase-3 activity level. The fluorogenic substrate, Ac-DEVD-AFC, is incubated with purified Caspase-3 and the enzymatic activity releases AFC fluorophore that can then be measured using a fluorescence reader.
Description: The Homogeneous Caspase-7 Assay Kit is a complete assay system designed to measure Caspase-7 activity for screening and profiling applications. It comes in a convenient 96-well format, with all the reagents necessary for 100 fluorescent Caspase-7 activity measurements. In addition, the kit includes purified Caspase-7 enzyme and a potent Caspase-3/7 inhibitor, Ac-DNLD-CHO, for use as a positive and negative control. Using this kit, only one simple step on a microtiter plate is needed to analyze the Caspase-7 activity level. The fluorogenic substrate, Ac-DEVD-AFC, is incubated with purified Caspase-7 and the enzymatic activity releases AFC fluorophore that can then be measured using a fluorescence reader.
Description: The Homogeneous Caspase-6 Assay Kit is a complete assay system designed to measure Caspase-6 activity for screening and profiling applications. It comes in a convenient 96-well format, with all the reagents necessary for 100 fluorescent Caspase-6 activity measurements. In addition, the kit includes purified Caspase-6 enzyme and a potent Caspase-6 inhibitor, Ac-IETD-CHO, for use as a positive and negative control. Using this kit, only one simple step on a microtiter plate is needed to analyze the Caspase-6 activity level. The fluorogenic substrate, Ac-VEID-AFC, is incubated with purified Caspase-6 and the enzymatic activity releases AFC fluorophore that can then be measured using a fluorescence reader.
Description: The Homogeneous Caspase-8 Assay Kit is a complete assay system designed to measure Caspase-8 activity for screening and profiling applications. It comes in a convenient 96-well format, with all the reagents necessary for 100 fluorescent Caspase-8 activity measurements. In addition, the kit includes purified Caspase-8 enzyme and a potent Caspase-8 inhibitor, Ac-IETD-CHO, for use as a positive and negative control. Using this kit, only one simple step on a microtiter plate is needed to analyze the Caspase-8 activity level. The fluorogenic substrate, Ac-IETD-AFC, is incubated with purified Caspase-8 and the enzymatic activity releases AFC fluorophore that can then be measured using a fluorescence reader.
