Butyrylcholinesterase Fluorescent Activity Kit, 2 Plate

Produktnummer ABIN577659

Produktdetails

Target: Butyrylcholinesterase (BCHE)

Reaktivität: Human, Ratte, Maus, Schwein, Hund, Affe, Huhn

Nachweismethode: Fluorometric

Untere Nachweisgrenze: 0.012 mU/mL

Applikation: Activity Assay (AcA)

Verwendungszweck: The DetectX® Butyrylcholinesterase Activity kit is designed to quantitatively measure butyrylcholinesterase (BChE) activity in a variety of samples.

Marke: DetectX®

Proben: Serum, Plasma

Analytische Methode: Quantitative

Spezifität: Species Independent. Samples Types validated: Serum and Plasma

Sensitivität: 0.018 mU/mL

Produktmerkmale: The Butyrylcholinesterase (BChE) Fluorescent Activity kit is a simple, sensitive and stable kit that measures BChE activity in a wide variety of samples. The kit contains everything needed to measure activity, including the microtiter plate. Diluted samples are added to the wells. The reaction is initiated by addition of a reaction mix containing the butyrylthiocholine substrate and ThioStar®. After a 20 minute incubation, the signal is read at 510nm with excitation at 390nm. The activity can also be read kinetically. A stable human BChE standard solution is provided to generate a standard curve for the assay. The assay shows almost zero reactivity with AChE. Butyrylcholinesterase (BChE) belongs to the same structural class of proteins as acetylcholinesterase (AChE). The 440kDa tetrameric glycoprotein is predominantly found in blood, kidneys, intestine, liver, lung, heart and the central nervous system. Many species, such as human, horse and mice exhibit high BChE activity in plasma, whereas rats have higher acetylcholinesterase activity in plasma. A deficiency of BChE can result in delayed metabolism of various drugs, such as cocaine, and treatment with doses of BChE can help in overcoming the physiological reaction to them. In Alzheimer's disease therapeutics, the use of potent cholinesterase inhibitors protect the limited acetylcholine levels, acting on both AChE and BChE. BChE may have roles in attention, executive function, emotional memory and behaviour. Other intentional or environmental methods of impairment are organophosphates and carbamates with anticholinergic properties, which are used as insecticides worldwide or as warfare agents.

Bestandteile: Black 96 Well Plates 2 Plates
Butyrylcholinesterase Standard 225 μL Butyrylcholinesterase (BChE) at 200 mU/mL in a special stabilizing solution. CAUTION: This material is isolated from human serum. Treat as potentially infectious.
ThioStar® Detection Reagent 2 vials ThioStar thiol detection substrate stored in a desiccator pouch. Reconstitute with dry DMSO.
BChE Substrate 2 vials Butyrylthiocholine iodide freeze dried with stabilizers.
Dry DMSO 14 mL Dry Dimethyl sulfoxide solvent over molecular sieves. May be stored at room temperature.
Assay Buffer Concentrate 28 mL A 10x concentrated Tris buffer containing detergents and stabilizers.

Benötigtes Material: Distilled or deionized water.
Fluorescence 96 well plate reader capable of reading fluorescent emission at 510 nm, with excita- tion at 390 nm.
Contact your plate reader manufacturer for correct filter sets.
Set plate param- eters for a 96-well Corning Costar 3650 plate.
See: http://www.ArborAssays.com/resources/lit.asp for plate dimension data.
Software for converting raw relative fluorescent unit (FLU) readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting.

Anwendungsinformationen

Applikationshinweise: This assay has been validated for serum and EDTA and heparin plasmas from a variety of species.
Samples containing visible particulate should be centrifuged prior to using.

Kommentare:

Sample values: A variety of serum and plasma samples were tested in the assay, including chicken, mouse, rat, dog, monkey, pig and human samples.
Values averaged 4,565 mU/mL.
The average for 23 human serum and plasma samples was 6,268 ± 2,506 mU/mL.
Five rat serum and plasma samples had low activity levels of between 293 and 365 mU/mL.

Testdauer: 1 h

Plattentyp: Uncoated

Protokoll: A human BChE standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve.
The kit utilizes a proprietary non- fluorescent molecule, ThioStar®, that covalently binds to the thiol product of the reaction between the BChE Substrate and BChE in the standards or samples, yielding a fluorescent product read at 510 nm in a fluorescent plate reader with excitation at 390 nm.
The kit is suitable for measuring BChE activity in appropriately diluted serum or plasma from a number of species.
It will also measure BChE in extracted tissue samples and cell lysates.
Because the readout of BChE activity is purely chemical, there are few interferants that will affect the read- ings obtained.
REACTION OVERVIEW
1.
Sample or standard added to well.
2.
The reaction is initiated with the addition of the Reaction Mix containing BChE Substrate and ThioStar® Reagent.
3.
Incubate for 20 minutes and read fluorescent signal.
Calculate BChE activity from standard curve.
4.
Alternatively samples can be read kinetically.
Follow steps 1 and 2 above.
Add Reaction Mix and read signal at 510 nm over time.
Compare rates for samples and standards to determine sample BChE activity.

Aufbereitung der Reagenzien:

Allow the kit reagents to come to room temperature for 30 minutes.
We recommend that all stan- dards and samples be run in duplicate to allow the end user to accurately determine BChE activ- ity.
Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit.
Assay Buffer Prepare the Assay Buffer by diluting one part of the 10x Assay Buffer Concentrate with nine parts deionized water for a 1:10 dilution.
It is stable for up to 3 months when stored at 4 °C.
ThioStar® Detection Reagent Remove a vial of ThioStar Reagent from the desiccator pouch and add 700 μL of the provided DMSO to the vial.
Vortex thoroughly.
This is a 10X concentrate of the ThioStar Detection Reagent.
Seal any unused reconstituted Detection Reagent in the vial in the desiccator pouch.
Store at 4 °C and use within 2 weeks. ® www.ArborAssays.com 7 WEB INSERT 150921 reagent preparatiOn cOntinued Butyrylcholinesterase Substrate Add 700 μL of the provided DMSO to the BChE Substrate vial and vortex thoroughly.
This is a 10x concentrate of the substrate.
Store any unused reconstituted BChE Substrate at room tempera- ture and use within 2 weeks.
Reaction Mix Dilution Table 1/2 Plate Full Plate 10X BChE Substrate Concentrate 300 μL 550 μL 10X ThioStar® Concentrate 300 μL 550 μL DMSO 2.4 mL 4.4 mL Standard Preparation BChE Standards are prepared by labeling seven test tubes as #1 through #7.
Briefly spin vial of standard in a microcentrifuge to ensure contents are at bottom of vial.
Pipet 450 μL of Assay Buf- fer into tube #1 and 250 μL into tubes #2 to #7.
Carefully add 50 μL of the BChE Standard to tube #1 and vortex completely.
Take 250 μL of the BChE solution in tube #1 and add it to tube #2 and vortex completely.
Repeat these serial dilutions for tubes #3 through #7.
The activity of BChE in tubes 1 through 7 will be 20, 10, 5, 2.5, 1.25, 0.625 and 0.313 mU/mL.
Use all Standards within 2 hours of preparation.

Aufbereitung der Proben:

Serum & Plasma Store separated serum or plasma on ice until assaying or freeze in aliquots for later use. Samples must be diluted in Assay Buffer prior to running in the kit. Any samples with BChE activity outside the standard curve range should be diluted further with Assay Buffer to obtain readings within the standard curve. Human serum and plasma typically have to be diluted ≥ 1:300 to read in the assay. Use all samples within 2 hours of dilution.

Testdurchführung:

1. Use the plate layout sheet on the back page of the insert to aid in proper sample and standard identification. Set plate parameters for a 96-well Corning Costar 3650 plate. .
   2. Pipet 100 μL of samples or standards into duplicate wells in the plate.
   3. Pipet 100 μL of Assay Buffer into duplicate wells as a Zero standard.
   4. Add 50 μL of the prepared Reaction Mix to each of the wells using a repeater pipet.
   5. Gently tap the sides of the plate to ensure adequate mixing of the reagents.
   6. Incubate at room temperature for 20 minutes.
   7. Read the fluorescent emission at 510 nm with excitation at 370-410 nm. Please contact your plate reader manufacturer for suitable filter sets.

Ergebnisberechnung:

Average the duplicate FLU readings for each standard and sample.
Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean FLUs for the zero standard.
The sample activity obtained should be multiplied by the dilution fac- tor to obtain neat sample values.
Or use the online tool from http://www.myassays.com/arbor-assays-butyrylcholinesterase-fluorescent-activity-kit.assay to calculate the data. *The MyAssays logo is a registered trademark of MyAssays Ltd.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Vorsichtsmaßnahmen: As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
The complete insert should be read and understood before attempting to use the product.
Dimethyl sulfoxide is a powerful aprotic organic solvent that has been shown to enhance the rate of skin absorption of skin-permeable substances.
Wear protective gloves when using the solvent especially when it contains dissolved chemicals.
The Butyrylcholinesterase Standard is derived from human blood.
It has been extensively tested for viral contamination, but all human blood products should be treated as potentially infectious and adequate precautions taken.
ThioStar® Detection Reagent should be stored at 4°C in the desiccator.
Allow the desiccator to warm to room temperature prior to opening.
ThioStar will react with strong nucleophiles.
Buffers containing the preservatives sodium azide, Proclin™ and Kathon™ will react with the substrate.

Lagerung: 4 °C,RT

Informationen zur Lagerung: All components of this kit should be stored at 4°C until the expiration date of the kit. DMSO, when stored at 4°C, will freeze. Can be stored tightly capped at room temperature.

Referenzen

Kuroda, Setoguchi, Uchino, Nagata, Hokonohara: "Effect of rivastigmine on plasma butyrylcholine esterase activity and plasma ghrelin levels in patients with dementia in Alzheimer's disease." in: Geriatrics & gerontology international, (2018) (PubMed).

Zhang, Jain, Tsao, Sinclair, Sun, Hung, Bai, Wu, Jiang: "Butyrylcholinesterase nanocapsule as a long circulating bioscavenger with reduced immune response." in: Journal of controlled release : official journal of the Controlled Release Society, Vol. 230, pp. 73-8, (2016) (PubMed).

Rahhal, Fromen, Wilson, Kai, Shen, Luft, DeSimone: "Pulmonary Delivery of Butyrylcholinesterase as a Model Protein to the Lung." in: Molecular pharmaceutics, Vol. 13, Issue 5, pp. 1626-35, (2016) (PubMed).

Munshaw, Hwang, Torbenson, Quinn, Hansen, Astemborski, Mehta, Ray, Thomas, Balagopal: "Laser captured hepatocytes show association of butyrylcholinesterase gene loss and fibrosis progression in hepatitis C-infected drug users." in: Hepatology (Baltimore, Md.), Vol. 56, Issue 2, pp. 544-54, (2012) (PubMed).

Antigendetails

Target: Butyrylcholinesterase (BCHE)

Andere Bezeichnung: Butyrylcholinesterase (BCHE Produkte)

Synonyme: che Kit, che1 Kit, bche Kit, cholinesterase Kit, CHE1 Kit, CHE2 Kit, E1 Kit, C730038G20Rik Kit, butyrylcholinesterase L homeolog Kit, butyrylcholinesterase Kit, bche.L Kit, bche Kit, BCHE Kit, Bche Kit

Hintergrund: Butyrylcholinesterase (BChE) belongs to the same structural class of proteins as acetylcholinest- erase (AChE). The 440 kDa tetrameric glycoprotein is predominantly found in blood, kidneys, in- testine, liver, lung, heart and the central nervous system. Many species, such as human, horse and mice exhibit high BChE activity in plasma, whereas rats have higher acetylcholinesterase activity in plasma1. BChE preferentially acts on butyrylcholine, but also hydrolyzes acetylcholine. BChE O H O N + N+ O Choline Butyrylcholine BChE serves a few known functions within the body. As a detoxification enzyme, it hydrolyzes ester-containing drugs and scavenges cholinesterase inhibitors, such as succinylcholine, before they have a chance to reach synaptic targets. By doing this, the enzyme minimizes the neuromus- cular effect these agents have. A deficiency of BChE can result in delayed metabolism of various drugs, such as cocaine, and treatment with doses of BChE can help in overcoming the physiologi- cal reaction to them2. As an activator enzyme, BChE converts administered prodrugs into func- tional therapeutics. Bambuterol is a prodrug with anti-asthmatic properties after being converted by BChE. BChE is the only enzyme in human serum that acts on heroin, and its end product, after crossing the blood-brain barrier, is hydrolyzed to morphine by enzymes in the brain. Alzheimer's disease involves the degeneration of cholinergic neurons and loss of cholinergic transmission. The reduction in choline acetyltransferase leads to a decrease in acetylcholine and acetylcholinesterase activity, which appears to cause an increase in BChE activity3. Potent cho- linesterase inhibitor therapeutics protect the limited acetylcholine levels, acting on both AChE and BChE. Selective BChE inhibitors prevent the formation of new beta-amyloid plaques, which are created by BChE cleaving amyloid precursor protein to beta-amyloid protein4. BChE-positive neurons project to the frontal cortex portion of the brain. BChE may have roles in attention, ex- ecutive function, emotional memory and behaviour. As dementia advances, BChE activity has been shown to increase, while AChE activity decreases, leaving the potential for BChE activity to be used as a biomarker for progression5 or target for future therapies