Lipase Assay Kit, BioAssay(TM)

Product Overview

• Article Name: Lipase Assay Kit, BioAssay(TM)
• Biozol Catalog Number: USB-L2496-08
• Supplier Catalog Number: L2496-08
• Alternative Catalog Number: USB-L2496-08-96
• Manufacturer: US Biological
• Category: Kits/Assays

Product Description

Lipase catalyzes the hydrolysis of ester bonds on the glycerol backbone of a lipid substrate. In humans, pancreatic lipase is the key enzyme responsible for breaking down fats in the digestive system by converting triglycerides to monoglycerides and free fatty acids. Human pancreatic lipase and its related protein 2 are the main lipases secreted by the pancreas. In acute pancreatitis, lipase levels can rise 5 to 10-fold within 24 to 48 hours. Increased activities have also been associated with pancreatic duct obstruction, pancreatic cancer, kidney disease, salivary gland inflammation, bowel obstruction, and other pancreatic diseases. Decreased levels may indicate permanent damage to lipase-producing cells in the pancreas. Simple, direct and automation-ready procedures for measuring lipase activity are very desirable. This Lipase assay is based on an improved dimercaptopropanol tributyrate (BALB) method, in which SH groups formed from lipase cleavage of BALB react with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) to form a yellow colored product. The color intensity, measured at 412 nm, is proportional to the enzyme activity in the sample. Applications: Direct assays of lipase activity in serum, plasma, saliva, urine and other biological samples. Key Features: Sensitive and accurate. Linear detection range 40 to 1600 U/L lipase activity in 96-well plate assay. Convenient and high throughput. The procedure involves adding a single working reagent, and reading the optical density at 10 min and 20 min at room temperature or 37C. Can be automated to process thousands of samples per day. Kit Components: 100 tests in 96-well plate L2496-08A: Assay Buffer (pH 8.5), 15ml L2496-08B: Color Reagent, 1ml. Contains 530 mg BALB Reagent L2496-08C: Calibrator, 2ml (equivalent to 735 U/L) Storage and Stability: Store all components at 4C. Shelf life: 12 months after receipt. Materials Required But Not Supplied: Pipeting (multi-channel) devices Clear-bottom 96-well plates (e.g. Corning Costar) and plate reader. Sample Preparation: Lipase inhibitors (EDTA, and certain detergents like Tween-20, NP-40), mercaptoethanol and dithiothreitol interfere with this assay and should be avoided in sample preparation. Samples can be stored frozen for at least one month, if not assayed immediately. Tissue and cell lysates can be obtained by homogenization in cold PBS buffer and centrifugation (e.g. 5 min at 14,000 rpm). Preparation of Working Reagent: Mix Color Reagent into Assay Buffer and shake vial to mix. Add 0.8ml BALB Reagent (sufficient for 100 assays). Alternatively for partial reconstitution: for each well of reaction, mix 5mg Color Reagent, 140ul Assay Buffer and 8ul BALB Reagent. The Working Reagent should be prepared freshly and used within one hour. Important: This assay is based on a kinetic reaction, addition of the Working Reagent should be quick. Use of a multi-channel pipettor is recommended. Assay Procedure: 1. Transfer 150ul H2O and 150ul Calibrator into wells of a clear- bottom 96-well plate. Pipette 10ul samples into separate wells. 2. Add 140ul Working Reagent to each sample well. Tap plate briefly to mix reaction mixture. Note: If the assay is to be performed at another temperature (e.g. 37C), warm up the Working Reagent to this temperature prior to adding to the sample. 3. Read absorbance at 412nm on a plate reader at 10 min (OD10min) and at 20 min (OD20min). Calculations: Lipase activity is calculated as follows: Activity = [(OD20min -OD10min)/(ODcalibrator - ODH20))] X 735 (U/L) where: OD20min and OD10min are the OD412nm values of the sample at 20 min and 10 min, respectively. ODCalibrator and ODH2O are the OD412nm values of the Calibrator and water at 20 min. The number ''735 is the equivalent activity (U/L) of the calibrator under the assay conditions. Note: If the calculated activity is higher than 1600 U/L, dilute sample in water and repeat assay. Multiply the results by the dilution factor (n). For Assays in Cuvette: For assay