BIOO LIFE SCIENCE PRODUCTS

Transcription

1 BIOO LIFE SCIENCE PRODUCTS FOR REFERENCE PURPOSES This manual is for Reference Purposes Only. DO NOT use this protocol to run your assays. Periodically, optimizations and revisions are made to the kit and protocol, so it is important to always use the protocol included with the kit. Amylase Assay Kit Manual Catalog #: BIOO Scientific Corp. 2011

2 TABLE OF CONTENTS GENERAL INFORMATION... 1 Product Description... 1 Introduction... 1 Procedure Overview... 1 Required Materials Not Provided With the Kit... 2 Warnings and Precautions... 2 SAMPLE PREPARATION... 3 AMYLASE DETERMINATION TEST PROTOCOL... 3 Reagent Preparation... 3 Sample Test Procedure... 4 Determination of Amylase Activity in Serum Samples... 4 Standard Curve Construction (Optional)... 4 RELATED PRODUCTS... 5 The MaxDiscovery Amylase Assay Kit is intended for laboratory use only, unless otherwise indicated. This product is NOT for clinical diagnostic use. MaxDiscovery is a trademark of Bioo Scientific Corporation.

3 GENERAL INFORMATION Product Description The MaxDiscovery Amylase Assay Kit is a plate-based kinetic assay for the determination of amylase in serum and urine samples. The kit uses a spectrophotometric assay to detect amylase activity directly from serum and urine samples. The unique features of the kit are: GENERAL INFORMATION High sensitivity and low detection limit (15 U/L) A rapid (5 minutes) and robust enzyme-based assay which does not require expensive instrumentation High reproducibility Introduction Amylase is an enzyme that catalyzes the breakdown of starch into sugars. The pancreas makes alpha amylase to hydrolyze dietary starch into disaccharides and trisaccharides which are converted by other enzymes to glucose to supply the body with energy. Blood serum amylase is often measured for purposes of medical diagnosis. While elevated levels of amylase in serum can reflect one of several medical conditions, it is most commonly measured for the diagnosis of acute pancreatitis. The MaxDiscovery Amylase Assay Kit uses a coupled enzymatic assay scheme using a chromophore-linked starch analogue called p-nitrophenyl-d-maltoheptaoside (PNPG7) to detect amylase. In the first step, amylase selectively degrades the PNPG7 substrate to form maltotriose and p-nitrophenyl maltotetraoside (PNPG4). Next the PNPG4 is successively cleaved by glucoamylase and glucosidase to form yellow-colored p-nitrophenol product, which absorbs 405 nm light. Therefore, the absorbance at 405 nm is proportional to the amylase activity of the sample. A p-nitrophenol standard is included with the kit to verify assay performance. Procedure Overview After preparing the sera, the assay is performed by adding Amylase Reagent into microplate wells containing 5 µl sera. Using a plate reader, the absorbance of each well at 405 nm is measured. The concentration of amylase in each sample is directly determined from calculating the change in absorbance over 5 min. BIOO LIFE SCIENCE PRODUCTS 1

4 Kit Contents, Storage and Shelf Life The MaxDiscovery Amylase Assay Kit has the capacity for 96 determinations or testing of 42 samples in duplicate (using 12 wells for standards). Store the kit as described in the table below. The shelf life is 6 months after receipt when the kit is properly stored. Kit Contents Amount Storage Microtiter Plate One Room temp Amylase Reagent 1 vial 4 C pnp standard 0.8 ml 4 C pnp Dilution Buffer 2 x 1.8 ml 4 C Required Materials Not Provided With the Kit Microtiter plate reader (405 nm) Centrifuge to prepare serum samples Deionized or distilled water (dh2o) PBS (phosphate buffered saline, ph 7.3) 1.5 ml microfuge tubes Multichannel pipet or repeating pipettor (recommended but not required) Warnings and Precautions BIOO strongly recommends that you read the following warnings and precautions to ensure your full awareness of the techniques and other details you should pay close attention to when running the assays. Periodically, optimizations and revisions are made to the kit and manual. Therefore, it is important to follow the protocol coming with the kit. If you need further assistance, you may contact your local distributor or BIOO at techsupport2@biooscientific.com. Add standards to plate only in the order from low concentration to high concentration, as this will minimize the risk of compromising the standard curve. Anticoagulants such as citrate and EDTA bind calcium, which is needed for amylase activity. Plasma with any anticoagulant other than heparin should not be used. Hemolysis and lipemia will increase the amylase values. Macroamylasemia increases the activity of pancreatic amylase in serum Insulin and bacteria can also interfere with or cause increased amylase activity. BIOO makes no warranty of any kind, either expressed or implied, except that the materials from which its products are made are of high quality. There is no warranty of merchantability of this product, or of the fitness of the product for any purpose. BIOO shall not be liable for any damages, including special or consequential damage, or expense arising directly or indirectly from the use of this product. BIOO LIFE SCIENCE PRODUCTS 2

5 SAMPLE PREPARATION SAMPLE PREPARATION 1. Carefully prepare at least 20 µl of sera using standard production procedure (if determinations are performed in singlet then 10 µl is sufficient). 2. Amylase in serum or urine is reported stable for seven days at room temperature (18-30 C) and three months when stored at 4 C and properly protected against evaporation. Note: 1. Samples with values above 800 U/L should be diluted with PBS and re-tested. (Multiply results by dilution factor). 2. Grossly lipemic sera require a sample blank. Add 5 µl of sample to 250 µl PBS, mix, and read the absorbance against water. Subtract this reading value from the absorbance of each serum sample to obtain the corrected reading. AMYLASE DETECTION TEST PROTOCOL AMYLASE DETERMINATION TEST PROTOCOL Reagent Preparation To reconstitute the Amylase Reagent, add exactly 27 ml of deionized or distilled water to the Amylase Reagent mix powder. Mix by swirling or inverting the bottle 10 times. Allow contents to dissolve for 10 minutes at room temperature. Do not shake. Preparation of Creatinine Control Dilutions for Standard Curve 1. Label 6 microfuge tubes: 1, 2, 3, 4, 5, Dilute the pnp Standard using the pnp Dilution Buffer as described in the table below. After dilution, briefly mix each tube before performing the next dilution. (The standard is equivalent to 800 U/L amylase.) Standard Amylase Preparation Tube # (U/L) 1 Add 150 L of pnp Control Add 100 L from Standard Tube # L of pnp Dilution Buffer Add 100 L from Standard Tube # L of pnp Dilution Buffer Add 100 L from Standard Tube # L of pnp Dilution Buffer Add 100 L from Standard Tube # L of pnp Dilution Buffer (Neg) Add 100 L of pnp Dilution Buffer µl of each diluted standard is used per reaction (see below). BIOO LIFE SCIENCE PRODUCTS 3

6 Ave 405 nm absorbance Sample Test Procedure 1. Add 5 µl of each sample or standard (in duplicate) to the microplate wells. 2. Add 245 µl of reagent mix to the wells. 3. Immediately measure the absorbance of each sample at 405 nm. Exactly 5 minutes later, measure the absorbance again. Determination of Amylase Activity in Serum Samples Using the supplied materials and the procedure described above the concentration of amylase (units per liter) can be determined by multiplying the decrease in absorbance in 5 min by For example, if an absorbance decrease of 0.1 is observed over the 5 min interval, the amylase enzyme concentration in the sample would be 1569 x 0.1 = U/L. Standard Curve Construction (Optional) For each calibration point, calculate the average absorbance change. To do this, subtract the average 5 min absorbance value of each point from the average 5 min absorbance value of the Neg (no pnp) point. This calculation should include subtracting the average 5 min absorbance of the Neg value from itself, which is approximately zero. Amylase Standard Curve R 2 = U/L Amylase BIOO LIFE SCIENCE PRODUCTS 4

7 RELATED PRODUCTS Toxicity Assays: Product Catalog Number MaxDiscovery Alanine Transaminase Color Endpoint Assay Kit MaxDiscovery Alanine Transaminase Enzymatic Assay Kit MaxDiscovery Alkaline Phosphatase Color Endpoint Assay Kit MaxDiscovery Alkaline Phosphatase Enzymatic Assay Kit MaxDiscovery Aspartate Transaminase Color Endpoint Assay Kit MaxDiscovery Aspartate Transaminase Enzymatic Assay Kit MaxDiscovery Creatine Kinase Enzymatic Assay Kit MaxDiscovery γ-glutamyl Transferase Enzymatic Assay Kit MaxDiscovery Lactate Dehydrogenase Color Endpoint Assay Kit MaxDiscovery Lactate Dehydrogenase Enzymatic Assay Kit MaxDiscovery Total Bilirubin Assay Kit MaxDiscovery Creatinine Assay Kit Blood Chemistry Assays: Product Catalog Number MaxDiscovery Cholesterol Enzymatic Assay Kit MaxDiscovery Blood Urea Nitrogen (BUN) Enzymatic Assay Kit MaxDiscovery Triglycerides Enzymatic Assay Kit MaxDiscovery LDL Cholesterol Assay Kit MaxDiscovery HDL Cholesterol Assay Kit MaxDiscovery Glucose Assay Kit Cytotoxicity Assays: Product RELATED PRODUCTS Catalog Number MaxDiscovery Aspartate Transaminase (AST) Cytotoxicity Kit MaxDiscovery Lactate Dehydrogenase Cytotoxicity Assay Kit Bioo Scientific Corporation 3913 Todd Lane Suite 312 Austin, TX USA Tel: Fax: (512) Made in USA BIOO Research Products Group BIOO LIFE SCIENCE PRODUCTS 5