Performance of the QPLEX™ Alz plus assay, a novel multiplex kit for screening cerebral amyloid deposition
Background: Alzheimer’s disease (AD) is an irreversible neurodegenerative disease characterized by the hallmark finding of cerebral amyloid deposition. Many researchers have tried to predict the existence of cerebral amyloid deposition by using easily accessible blood plasma samples, but the effectiveness of such strategies remains controversial.
Methods: We developed a new multiplex kit, the QPLEX™ Alz plus assay kit, which uses proteomics-based blood biomarkers to prescreen for cerebral amyloid deposition. A total of 300 participants who underwent Pittsburgh compound B (PiB)-positron emission tomography (PET) which allows imaging of cerebral amyloid deposition were included in this study. We compared the levels of QPLEX™ biomarkers between patients who were classified as PiB-negative or PiB-positive, regardless of their cognitive function. Logistic regression analysis followed by receiver operating characteristic (ROC) curve analysis was performed. The kit accuracy was tested using a randomized sample selection method.
Results: The results obtained using our assay kit reached 89.1% area under curve (AUC) with 80.0% sensitivity and 83.0% specificity. Further validation of the QPLEX™ Alz plus assay kit using a randomized sample selection method showed an average accuracy of 81.5%.
Conclusions: Our QPLEX™ Alz plus assay kit provides preliminary evidence that it can be used as blood marker to predict cerebral amyloid deposition but independent validation is needed.
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
Collagen Triple Helix Repeat Containing Protein 1 (CTHRC1) Magnetic Luminex Assay Kit
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.
Analysis of Cerebrospinal Fluid Extracellular Vesicles by Proximity Extension Assay: A Comparative Study of Four Isolation Kits
There is a lack of reliable biomarkers for disorders of the central nervous system (CNS), and diagnostics still heavily rely on symptoms that are both subjective and difficult to quantify. The cerebrospinal fluid (CSF) is a promising source of biomarkers due to its close connection to the CNS.
Extracellular vesicles are actively secreted by cells, and proteomic analysis of CSF extracellular vesicles (EVs) and their molecular composition likely reflects changes in the CNS to a higher extent compared with total CSF, especially in the case of neuroinflammation, which could increase blood-brain barrier permeability and cause an influx of plasma proteins into the CSF. We used proximity extension assay for proteomic analysis due to its high sensitivity.
We believe that this methodology could be useful for de novo biomarker discovery for several CNS diseases. We compared four commercially available kits for EV isolation: MagCapture and ExoIntact (based on magnetic beads), EVSecond L70 (size-exclusion chromatography), and exoEasy (membrane affinity).
The isolated EVs were characterized by nanoparticle tracking analysis, ELISA (CD63, CD81 and albumin), and proximity extension assay (PEA) using two different panels, each consisting of 92 markers. The exoEasy samples did not pass the built-in quality controls and were excluded from downstream analysis.
The number of detectable proteins in the ExoIntact samples was considerably higher (~150% for the cardiovascular III panel and ~320% for the cell regulation panel) compared with other groups. ExoIntact also showed the highest intersample correlation with an average Pearson’s correlation coefficient of 0.991 compared with 0.985 and 0.927 for MagCapture and EVSecond, respectively.
The median coefficient of variation was 5%, 8%, and 22% for ExoIntact, MagCapture, and EVSecond, respectively. Comparing total CSF and ExoIntact samples revealed 70 differentially expressed proteins in the cardiovascular III panel and 17 in the cell regulation panel. To our knowledge, this is the first time that CSF EVs were analyzed by PEA. In conclusion, analysis of CSF EVs by PEA is feasible, and different isolation kits give distinct results, with ExoIntact showing the highest number of identified proteins with the lowest variability.
A simple and rapid algal assaykit to assess toxicity of heavy metal-contaminated water
This study presents a novel algal-based toxicity test suitable for simple and rapid assessment of heavy metal (Hg2+, Cr6+, Cd2+, Pb2+, or As3+)-contaminated water. A closed-system kit-type algal assay was developed using Chlorella vulgaris. Toxicity was assessed by oxygen evolution in the gaseous phase of the assay kits, which was measured via a needle-type oxygen sensor. Initial cell density, light intensity, and exposure time that enabled favorable test performance for the algal assay kits were 103 cells/mL, 250 μmol m-2s-1, and 18 h, respectively.
Results from the heavy metal toxicity tests demonstrate that Hg2+, Cr6+, Cd2+, and Pb2+ are more toxic in inhibiting algal photosynthetic activity than As3+. The 18 h half-maximum effective concentrations (EC50) for Hg2+, Cr6+, Cd2+, Pb2+, and As3+ were determined to be 31.3 ± 0.5, 179.6 ± 7.5, 301.3 ± 6.1, 476.1 ± 10.5, and 2184.1 ± 31.1 μg/L, respectively. A strong correlation between oxygen concentrations in the headspace of the assay kits and chlorophyll a production indicates that oxygen evolution in the gaseous phase is able to represent algal photosynthetic activity and serve as the end-point in algal toxicity tests. High test sensitivity and reproducibility as well as an easy test protocol and rapid processing time make the algal assay kit a suitable tool for simple and rapid toxicity testing of heavy metal-contaminated water.
Sperm chromatin structure assay versus sperm chromatin dispersion kits: Technical repeatability and choice of assisted reproductive technology procedure
Objective: The sperm DNA fragmentation index (DFI) guides the clinician’s choice of an appropriate assisted reproductive technology (ART) procedure. The DFI can be determined using commercially available methodologies, including sperm chromatin dispersion (SCD) kits and sperm chromatin structure assay (SCSA). Currently, when DFI is evaluated using SCD kits, the result is analyzed in reference to the SCSA-derived threshold for the choice of an ART procedure. In this study, we compared DFI values obtained using SCSA with those obtained using SCD and determined whether the difference affects the choice of ART procedure.
Methods: We compared SCSA to two SCD kits, CANfrag (n=36) and Halosperm (n=31), to assess the DFI values obtained, the correlations between tests, the technical repeatability, and the impact of DFI on the choice of ART.
Results: We obtained higher median DFI values using SCD kits than when using SCSA, and this difference was significant for the CANfrag kit (p<0.001). The SCD kits had significantly higher coefficients of variation than SCSA (p<0.0001). In vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) would be chosen for a significantly higher proportion of patients if a decision were made based on DFI derived from SCD rather than DFI determined using SCSA (p=0.003).
Conclusion: Our results indicate that SCD kit-specific thresholds should be established in order to avoid the unnecessary use of IVF/ICSI based on sperm DNA damage for the management of infertility. Appropriate measures should be taken to mitigate the increased variability inherent to the methods used in these tests.
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.
