Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Patent
1995-01-31
1998-06-16
Leary, Louise
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
435 4, 4352401, 4352402, 422 50, 422 55, 422 681, 436 34, 436164, 436805, 436809, 356 401, C12Q 102, C12Q 100, C12N 500, G01N 3300
Patent
active
057668752
DESCRIPTION:
BRIEF SUMMARY
FIELD OF INVENTION
This invention generally relates to a method for monitoring the metabolism of cells, e.g. metabolic extracellular acid production. More particularly, this invention comprises the steps of placing the cells in a buffer containing an acid/base indicator and placing the cell/buffer solution in wells of a multiassay plate, heating the solution to about 37 degrees centigrade, mixing the solution in the multiassay plate, measuring the ratio of the optical density of the acid/base indicator at more than one different wavelength of light passing vertically through the solution, and repeating the mixing and measuring steps to kinetically monitor the rate of change in extracellular pH with extremely high precision.
BACKGROUND OF THE INVENTION
A variety of techniques and devices are commercially available for the detection and measurement of substances present in fluid or other translucent samples by determining the light transmittance of the sample. Similarly, absorptive and fluorescent acid/base indicator dyes have been used to indicate the pH of aqueous sample compartments as small as individual living cells (Arnost Kotyk and Jan Slavik, Assays of Intracellular pH Using Chemical Probes: Absorption Spectroscopy, Intracellular pH and Its Measurement, pp. 69-85, CRC Press, Bocca Raton, Fla., 1989.) However, the pH of samples can change with a change in temperature, and can cause inaccuracies in measurement. An example of how the pH of pure water is different at temperatures of 25.degree. C. and 37.degree. C. is shown as follows.
It is well known that the pH of pure water at 25.degree. C. is 7.0. The necessary equation to calculate the pH of pure water at 37.degree. C. is:
The ionization constant of water is 10.sup.-14 at 25.degree. C., its .DELTA.H.degree.=13.5 ##EQU1##
This is the ionization constant of water at 37.degree. C. The H.sup.+ and OH.sup.- in pure water is surely low enough so that the activities of the
Similarly, the pK.sub.a values of both buffering systems and acid-base indicators change with temperature. For example, the change in absorbance of Cresol Red, at 575 nanometers, in a TRIS temperature changes (K. Schilling, H. Hoppe and A. Horn, Uberprufung der Termperierung von Mikrotiterplatten in Thermaostatisierbaren Readern mit einem hochauflosenden optischen Thermometer, BL Journal (3) 77-80, 1992; T. D. O'Leary, J. L. Badennoch and R. Bais, Optical Methods for Monitoring Temperature in Spectrophotometric Analysers, Ann. Clin. Biochem. 20, 153-157, 1983; L. Bowie, F. Esters, J. Bolin and N. Gochman, Development of an Aqueous Temperature-Indicating Technique and Its Application to Clinical Laboratory Instrumentation, Clin. Chem. 22/4, 449-455, 1976).
The present invention incorporates by reference the "Background of the Invention" for U.S. Pat. Nos. 4,968,148 and 5,112,134. As discussed in U.S. Pat. Nos. 4,968,148 and 5,112,134, the prior art has many problems and limitations. Although the vertical beam absorbance reader, taught in U.S. Pat. Nos. 4,968,148 and 5,112,134, solves or diminishes these problems and limitations, it has been discovered that buffer and pH indicator dye temperature effects and temperature fluctuation and light scatter interferences in the pH monitoring of cells in microplates can obscure the measurement of metabolic extracellular acid production.
SUMMARY AND OBJECTS OF THE INVENTION
It is the primary objective of this invention to provide an improved method of using the inventions of U.S. Pat. No. 4,968,148 and 5,112,134. More particularly, the present invention comprises steps that balance both buffer and pH indicator dye temperature effects. The present invention also compensates for temperature fluctuation, light scatter interferences, and light absorbance path length fluctuation in pH monitoring of cells in microplates. These inventive steps result in a more accurate measurement of metabolic extracellular acid production and also provide an improved method for measuring temperature of solutions spectrophotometrically.
The present invention uses a
REFERENCES:
patent: 4448534 (1984-05-01), Wertz et al.
patent: 4456380 (1984-06-01), Kondo et al.
patent: 4568186 (1986-02-01), Yoshimura et al.
patent: 4968148 (1990-11-01), Chow et al.
patent: 4986665 (1991-01-01), Yamanashi et al.
patent: 5112134 (1992-05-01), Chow et al.
Satoh et al. (1990), "Simultaneous Assay for Megakaryocyte Colony-Stimulating Factor and Megakaryocyte Potentiator and Its Application," J. Lab. Clin. Med., vol. 116, pp. 162-171. Month not available.
Burstein et al. (1985), "Quantitation of Megakaryocytopoiesis in Liquid Culture by Enzymatic Determination of Acetylcholinesterase," J. Cell. Physiol., vol. 122, pp. 159-165. Month not available.
Wosu (1984), "In Vitro Studies on Feline Panleucopenia Virus. Standardization of Hemagglutination-Inhibition Test for Feline Panleucopenia Virus Antibody," Comp. Immunol. Microbiol. Infect. Dis., vol. 7, pp. 201-206. Month not available.
Hernandez et. al. (1990), "FIA-Spectrophotometric Assay of N-Acetylcysteine by O-Phthalaldehyde Derivatization," Pharmazie (Germany), vol. 45, pp. 745-747. Month not available.
Kulanthaivel et al. (1990), "The ATP-Binding Site of the Human Placental H.sup.+ Pump contains Essential Tyrosyl Residues," Biochemistry, vol. 29, pp. 10807-10813. Month not available.
Sharp et al. (1990), "The Association of Platelet and Red Cell Count with Platelet Impedance Changes in Whole Blood and Light-Scattering Changes in Platelet Rich Plasma: Evidence from the Caerphilly Collaborative Heart Disease Study," Thromb. Haemost., vol. 64, pp. 211-215. Month not available.
Malmer et al. (1990), "Amino Acid Analysis by High-Performance Liquid Chromatography with Methanesulfonic Acid Hydrolysis and 9-Fluorenylmethylchloroformate Derivatization," J. Chromatogr., vol. 514, pp. 227-239. Month not available.
Thiel, (1990), "Determination of Moniliformin by High-Performance Liquid Chromatography," J. Environ. Pathol. Toxicol. Oncol., vol. 10, pp. 162-165. Month not available.
Sethi et al. (1990), "Bb Fragment of Bovine Complement Factor B: Stimulation of the Oxidative Burst in Bovine Monocytes," Can. J. Vet. Res., vol. 54, pp. 410-414. Month not available.
Pape et al. (1990), "Perturbation of Sacroplasmic Reticulum Calcium Release and Phenol Red Absorbance Transients by Large Concentrations of Fura-2 Injected into Frog Skeletal Muscle Fibers," J. Gen. Physiol., vol. 96, pp. 493-516. Month not available.
Hollingworth et al. (1990), "Changes in Phenol Red Absorbance in Response to Electrical Stimulation of Frog Skeletal Muscle Fibers," J. Gen. Physiol., vol. 96, pp. 473-491. Month not available.
Baylor et al. (1990), "Absorbance Signals from Resting Frog Skeletal Muscle Fibers Injected with the pH Indicator Dye, Phenol Red," J. Gen. Physiol., vol. 96, pp. 449-471. Month not available.
Shiau et al. (1990), "Acidic Mucin Layer Facilitates Micelle Dissociation and Fatty Acid Diffusion," Am. J. Physiol., vol. 259, pp. G671-G675. Month not available.
Letellier et al. (1991), "Determination of affinity of Pasteurella multocida Isolates for Porcine Respiratory Tract Mucus, and Partial Characterization of the Receptors," Am. J. Vet. Res., vol. 52, pp. 34-39. Month not available.
Zebrev et al. (1990), "Zavisimoe to Mg2+ Tormozhenie Adgezii Leikotsitov, vyzvannoe Doksitsiklinom," Antibiot. Khimioter, vol. 35, pp. The English translation of the abstract of this article is provided. Month not available.
Fratantoni et al. (1990), "Measuring Platelet Aggregation with Microplate Reader. A New Technical Approach to Platelet Aggregation Studies," Am. J. Clin. Pathol., vol. 94, pp. 613-617. Month not available.
Mayo et al. (1990), "Kinetic Microplate Assay for Superoxide Production by Neutrophils and Other Phagocytic Cells," Meth. Enzymol., vol. 186, pp. 567-575. Month not available.
Bullock et al. (1990), "An Enzyme-Assessed Microplate-Assay for Neutrophil Adherence. I. IgA-Included Adherence of Human PMNs," Inflammation, vol. 14, pp. 427-445. Month not available.
Kobayashi et al. (1990), "Increased Na.sup.+ -H.
Crawford Kimberly L.
Hafeman Dean G.
Sanchez Anthony J.
Wada Henry Garrett
Leary Louise
Molecular Devices Corporation
LandOfFree
Metabolic monitoring of cells in a microplate reader does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Metabolic monitoring of cells in a microplate reader, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Metabolic monitoring of cells in a microplate reader will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1724286