United States Patent ( 10 ) Patent No . : US 7479 , 375 B 2
(54) METHOD FOR CLASSIFYINGA 5,372,946 A 12/1994 Cusak et al. MCROORGANISM IN A BIOLOGICAL 5,389,547 A 2f1995 Tanaka et al. SAMPLE 5,401,647 A 3, 1995 Tanaka et al. 5,474,984 A 12/1995 Tanaka et al. (75) Inventors: Norman R. Wainwright, Falmouth, MA 5,504,011 A 4, 1996 Gavin et al. 5,518,006 A 5/1996 Mawhirt et al. (US); Dana M. Nutter, Charleston, SC 5.534.226 A 7/1996 Gavin etal (US) 5,550,030 A 8/1996 Tanaka et al. 5,574,023 A 11/1996 Shibata et al. (73) Assignee: Charles River Laboratories, Inc., 5,591.403 A 1/1997 Gavin et al. Wilmington, MA (US) 5,591,628 A 1/1997 Baek et al. 5,605,806 A 2/1997 Tanaka et al. (*) Notice: Subject to any disclaimer, the term of this 5,637,474 A 6/1997 Takaoka et al. patent is extended or adjusted under 35 D380,555 S 7/1997 Kurosaki et al. U.S.C. 154(b) by 264 days. 5,681,710 A 10/1997 Tanaka et al. 5,695,948 A 12/1997 Tanaka et al. (21) Appl. No.: 11/331,600 5,702,882 A 12/1997 Tamura et al. D390,661 S 2/1998 Foggia (22) Filed: Jan. 13, 2006 (Continued)
A new kinetic single-stage Limulus amoebocyte lysate method for the detection of endotoxin in water and plasma.
A convenient kinetic chromogenic Limulus amoebocyte lysate (LAL) method, based on components originally intended for a two-stage end-point method, has been developed. The components (LAL and chromogenic substrate) can be pooled and subsequently used in a single-stage kinetic procedure adapted to microplates. With the help of a kinetic software, it is possible to measure over the three log range 0.005-12 EU/ml in one test run--a range considerably wider than the range of the end-point procedure. A good linearity of the log-log standard curve, reflected by coefficients of regression between 0.997 and 1.0, is shown as well as a high-resolution (> 700 s) between the negative control and the lowest standard point. Moreover a good precision (C.V. < 10%) is obtained in both water and plasma, showing the usefulness of the method in different applications. Finally, a strong correlation (r = 0.95-0.99) to other LAL methods is demonstrated.
Purification and characterization of a prophenoloxidase activating enzyme from crayfish blood cells
Abstract A proteinase was purified from crayfish haemocytes by affinity chromatography on heparin-sepharose and phenyl-sepharose, followed by DEAE-cellulose ion-exchange chromatography. This proteinase could mediate the conversion of prophenoloxidase (proPO) to its active form, phenoloxidase (PO), and its was therefore designated a prophenoloxidase activating enzyme, ppA. The purified ppA had a molecular mass of about 36,000 Da. Since ppA was a proteinase able to cleave chromogenic peptide substrates of trypsin, and serine proteinase inhibitors were strongly inhibitory towards ppA activity, the enzyme appeared to be a serine type proteinase. It exhibited maximal enzyme activity at neutral and slightly alkaline pH, and was sensitive to heat inactivation at 58°C.