Methods of analyzing samples for bacteria using whole cell capture and ATP analysis
A whole-cell, bacterial technology, used in analytical materials, instruments, measuring devices, etc.
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[0161] The invention has now been described in connection with several specific embodiments foreseen by the inventors, given enabling descriptions. Insubstantial modifications of the invention, including modifications not presently foreseen, may nevertheless constitute equivalents thereof. Therefore, the scope of the present invention should not be limited by the details and structures described herein, but only by the following claims and their equivalents.
[0162] All parts, percentages, ratios, etc. in the examples, as well as in the remainder of the specification, are on a molar basis unless otherwise indicated. All solvents and reagents not identified suppliers were purchased from Aldrich Chemical (Milwaukee, WI). Water was purified using a U-V Milli-Q water purifier (Millipore, Bedford MA) with a resistivity of 18.2 Mohms / cm.
[0163] Acronym
[0164]
preparation example 1
[0165] Preparation Example 1 - Preparation of Antibody Functionalized Magnetic Beads
[0166] Murine anti-Protein A monoclonal antibody MAb-107 is described in U.S. Patent Application Serial No. 11 / 562,747 and PCT Patent Publication No. WO 2008 / 143697, filed November 22, 2006 (both titled "ANTIBODY WITH PROTEIN A SELECTIVITY" (Antibodies Selective for Protein A)) are described. Murine anti-Protein A monoclonal antibody MAb-107 was biotinylated with EZ-Link NHS-PEO4-Biotin from Pierce (Product No. 21330) according to the manufacturer's instructions. Streptavidin-coated magnetic particles (1 μm Dynal T1 ) were obtained from Invitrogen, Inc. (Carlsbad, CA). All reactions and washes were performed in PBS L-64 buffer (phosphate buffered saline containing 0.2% w / v PLURONIC L64) unless otherwise indicated. Washing procedures consisted of three consecutive washes unless otherwise indicated. The washing procedure involves placing a magnet close to the tube to attract particles to ...
preparation example 2
[0168] Preparation example 2-phosphate-buffered saline (PBS-L64) containing PLURONIC L64 buffer buffer) preparation
[0169] Phosphate-buffered saline (PBS) solutions were prepared by diluting ten-fold 10x PBS concentrate (commercially available from EMD Biosciences (San Diego CA)). This gave a PBS buffer solution with the following salt composition: 10 mM sodium phosphate, 137 mM sodium chloride, 2.7 mM potassium chloride. The PBS buffer has a pH of 7.5 at 25°C. To prepare PLURONIC L64-containing phosphate-buffered saline (PBS-L64 buffer), 0.2% (w / v) PLURONIC L64 surfactant (available from BASF Corporation, (Mount Olive, NJ)) was added to PBS buffer. The PBS-L64 buffer has a pH of 7.5 at 25°C.
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