Butyrylcholinesterase - B-Bridge International, Inc.
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1. there are few interferants that will affect the readings obtained 1 Sample or standard added to well 2 The reaction is initiated with the addition of the Reaction Mix containing BChE Substrate and Thio Detection 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 B Bridge International Inc Butyrylcholinesterase 110811 KIT COMPONENTS Black 96 well plates 2 plates Butyrylcholinesterase Standard BChE 225 ul 200 mU ml Thio Detection Reagent 2 vials Stored in desiccator BChE Substrate 2 vials Butyrylthiocholine iodide freeze dried with stabilizers Dimethyl Sulfoxide Solvent DMSO 14 ml DMSO will freeze at 2 8 C Store tightly capped at room temperature 10X Assay Buffer 28 ml Store all components at 2 8 C except DMSO Store DMSO tightly capped at room temperature MATERIALS REQUIRED BUT NOT SUPPLIED Deionized or distilled water Fluorescence 96 well plate reader capable of reading fluorescent emission at 510 nm with excita tion at 390 nm Software for converting raw relative fluorescent unit FLU readings from the plate reader and carrying out four parameter logistic curve 4PLC fitting PRECAUTIONS This kit should only be used b2. variety of species This assay is species independent It will also measure BChE in extracted tissue samples and cell lysates BACKGROUND 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 BChE preferentially acts on butyrylcholine but also hydrolyzes acetylcholine BChE f Pr Ne PL a n N wN Ny Butyrylcholine Choline 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 neuromuscular 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 physiological reaction to them As an activator enzyme BChE converts administered prodrugs into functional 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 hyd
3. 5 1 25 0 625 and 0 313 mU ml Use all standards within 2 hours of preparation Standard Standard Standard Standard Standard Standard Standard Reagent cea EG EE ey ee Assay Buffer Volume 450 ul 250 ul 250 ul 250 ul 250 ul 250 ul 250 ul BChE Standard 50 ul Standard 1 250 ul Standard 2 250 ul Standard 3 250 ul Standard 4 250 ul Standard 5 250 ul Standard 6 250 ul Final Concentration mU mL 20 10 5 2 5 1 25 0 625 0 313 B Bridge International Inc Butyrylcholinesterase 110811 SAMPLE PREPARATION Store serum or plasma on ice until assaying or freeze in aliquots for later use Samples containing visible particulate should be centrifuged prior to using Samples must be diluted in 1X Assay Buffer prior to assaying Human serum and plasma typically have to be diluted 1 300 to read in the assay Any samples with BChE activity outside the standard curve range should be diluted further with 1X Assay Buffer to obtain readings within the standard curve Use all samples within 2 hours of dilution ASSAY PROTOCOL Standards and samples should be run in duplicate 1 Use a plate layout sheet to properly identify samples and standards 2 Pipet 100 ul of samples or standards into duplicate wells in the plate 3 Pipet 100 ul of 1X Assay Buffer into duplicate wells as a zero standard 4 Add 50 ul of prepared Reaction Mix to each well using a repeater or multi cha
4. B Bridge International Inc wA Butyrylcholinesterase Fluorescent Activity Kit User Manual Catalog K3016 1 B Bridge International Inc Butyrylcholinesterase 110811 TABLE OF CONTENTS Intended Use Background Assay Principle Kit Components Materials Required Precautions Reagent Preparation Sample Preparation Assay Protocol Calculations NOD Oo fF A FW 0 W Typical Standard Curve Example Notice to Purchaser This product is to be used for Research Purposes Only It is not to be used for Drug or Diagnostic Purposes nor is it intended for Human Use B Bridge products may not be resold modified for resale or used to manufacture commercial products without the express written consent of B Bridge International Inc EXCEPT AS OTHERWISE EXPRESSLY SET FORTH IN THIS USER MANUAL B BRIDGE DOES NOT MAKE ANY REPRESENTATION OR WARRANTIES OR CONDITIONS OF ANY KIND EITHER EXPRESSED OR IMPLIED WITH RESPECT TO THE PRODUCTS OR INFORMATION DISCLOSED HEREUNDER INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY FIT FOR A PARTICULAR PURPOSE OR NONINFRINGEMENT OF THE INTELLECTUAL PROPERTY RIGHTS OF THIRD PARTIES B Bridge International Inc All Rights Reserved B Bridge International Inc Butyrylcholinesterase 110811 INTENDED USE The B Bridge Butyrylcholinesterase Fluorescent Activity Kit cat K3016 1 quantitatively measures Butyrylcholinesterase activity in serum and EDTA and heparin plasmas from a
5. nnel pipet 5 Gently tap the sides of the plate to mix the reagents 6 Incubate at room temperature for 20 minutes 7 Read the fluorescent emission at 510 nm with excitation at 370 410 nm CALCULATIONS Average the duplicate fluorescent unit FLU readings for each standard and sample Create a standard curve by reducing the data using the 4PLC 4 parameter logistic curve fitting routine on the plate reader after subtracting the mean FLU for the zero standard The sample activity obtained should be multiplied by the dilution factor to obtain neat sample values B Bridge International Inc Butyrylcholinesterase 110811 TYPICAL STANDARD CURVE EXAMPLE ONLY Standard curves vary with each assay Always run your own standard curves for calculation of results do not use this data 60 000 50 000 40 000 30 000 Net FLU 20 000 10 000 o 5 10 15 20 BChE Activity mU mL B Bridge International Inc Butyrylcholinesterase 110811
6. rolyzed 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 activity Potent cholinesterase 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 protein BChE positive neurons project to the frontal cortex portion of the brain BChE may have roles in attention executive function emotional memory and behavior 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 progression or target for future therapies ASSAY PRINCIPLE The Butyrylcholinesterase Fluorescent Activity kit is designed to quantitatively measure butyrylcholinesterase BChE activity in a variety of samples The kit utilizes a proprietary non fluorescent molecule Thio 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 readout of BChE activity is purely chemical
7. te prior to assaying 1X Assay Buffer Prepare a 1X Assay Buffer by diluting 1 part 10X Assay Buffer with 9 parts deionized water 1X Assay Buffer is stable for up to 3 months at 4 C 10X Thio Detection Reagent Make a 10X Thio Detection Reagent by adding 700 ul of DMSO to 1 vial of Thio Reagent vortex thoroughly Store any unused reconstituted Detection Reagent at 4 C in the desiccator and use within 2 weeks 10X Butyrylcholinesterase Substrate Make a 10X BChE Substrate Reagent by adding 700 ul of DMSO to 1 vial of BChE Substrate vortex thoroughly Store any unused reconstituted BChE Substrate at room temperature and use within 2 weeks Reaction Mix Follow the table below to make the appropriate volume of Reaction Mix Use the Reaction Mix within 1 hour of preparation Protect from light Hat Plate Whole Plate 10X BChE Substrate 300 ul 550 ul 10X Thio Detection Reagent 300 ul 550 ul DMSO 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 the vial Pipet 450 ul of 1X Assay Buffer into tube 1 and 250 ul into tubes 2 to 7 Carefully add 50 ul of the BChE Standard to tube 1 and vortex completely Take 250 ul 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
8. y qualified personnel who have had laboratory safety instruction The complete User Manual should be read and understood before using this product Dimethyl sulfoxide DMSO is a powerful aprotic organic solvent that has been shown to enhance the rate of skin absorption of skin permeable substances Wear protective gloves safety glasses and clothing when working with DMSO Consult your institution s safety procedures for working with hazardous 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 Protective clothing gloves and safety glasses should be worn Consult your institution s safety procedures for working with human samples Thio Detection Reagent should be stored at 4 C in the desiccator Allow the desiccator to warm to room temperature prior to opening Thio Detection Reagent will react with strong nucleophiles Buffers containing the preservatives sodium azide Proclin and Kathon will react with the substrate B Bridge International Inc Butyrylcholinesterase 110811 REAGENT PREPARATION Allow the kit reagents to come to room temperature for 30 minutes We recommend that all standards and samples be run in duplicate to accurately determine BChE activity Ensure that all samples have reached room temperature and have been diluted as appropria
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