Chemistry: analytical and immunological testing – Biological cellular material tested
Reexamination Certificate
2000-01-24
2002-09-17
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Biological cellular material tested
C436S068000, C436S164000, C436S165000, C436S172000, C435S002000, C435S004000, C435S029000, C422S052000
Reexamination Certificate
active
06451608
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for measuring an amount of active oxygen produced by leukocytes and oxidative stress, more specifically to a method for measuring an amount of active oxygen produced by leukocytes and oxidative stress, and a method for assessing an effect of a substance selected from the group consisting of drugs, foods, and substances made from their ingredients to enhance or suppress an amount of active oxygen produced by leukocytes and oxidative stress.
BACKGROUND OF THE INVENTION
Production of active oxygen by leukocytes is inevitable for killing bacteria. A high active oxygen-productive capability of leukocytes is very important for protection against infection. Active oxygen is, however, known to damage tissues and DNA. For example, ischemia reperfusion injury is thought to be caused by active oxygen, which would probably be produced by leukocytes.
In order to prevent diseases, it is very important to measure an amount of active oxygen produced by leukocytes, and, to develop a method for increasing or decreasing, case by case, the amount of produced active oxygen.
Luminol enhancing light emission is widely used at the laboratory level for measurement of an amount of active oxygen produced by leukocytes. This method is performed by measuring faint light emitted from luminol when it is oxidized by various active oxygen species. Several substances other than luminol are also used for enhancement. Here, the term “luminol enhancing light emission” also includes light emission obtained by enhancement by substances other than luminol.
This method is simple, but requires to separate leukocytes from erythrocytes since light absorbance by erythrocytes must be prevented. The method for separating these blood cells is complicated and time-consuming. Furthermore, leukocytes largely change their state during the separation procedure, which raises the problem that the results of the measurement are not reliable.
To solve this problem, a method of measuring whole blood diluted to about to is proposed (Takayama, Eto, and Yamanaka, Standardization of Measurement of Oxidative Stress of Blood Samples by Detection of Luminol Enhancing Chemiluminescence—aiming at monitoring time course of in vivo oxidative stress—, Journal of Japanese Pharmacology 111(3), 177-186). However, dilution itself is thought to cause changes of the state of leukocytes. Furthermore, the dilution inevitably leads to considerable reduction of enhancement. The conventional methods cannot thus be effective for clinical and diagnostic use due to the above problems.
SUMMARY OF THE INVENTION
An objective of the present invention is to provide a method for measuring luminol enhancing light emission without separating leukocytes and erythrocytes, that is, using whole blood. This method is able to solve the problems attributable to the procedure for separating leukocytes from erythrocytes, to remarkably increase reliability of the measurement, and to considerably improve the measuring efficiency.
Considering protection by leukocytes against infection and damages of tissues, namely oxidative stress, the problems to be solved include not only the amount of active oxygen produced by leukocytes but also (1) how many leukocytes gather at the site of infection or are trapped by the capillary bed at that site, or (2) how long leukocytes take to pass through the capillary bed. In other words, a product of the amount of active oxygen produced by each leukocyte and the above (1) or (2) is considered to be a good index for ability to protect against infection and oxidative stress.
However, no effective method has been proposed so far for measuring the number of leukocytes trapped by the capillary bed or the time required for leukocytes to pass through the capillary bed.
Another object of the present invention is to provide a method for measuring a product of the amount of active oxygen produced by each leukocyte and the number of leukocytes trapped by the capillary bed or the time required for leukocytes to pass through the capillary bed to thereby provide an effective index for ability of leukocytes to protect against infection and oxidative stress caused by leukocytes.
A final object of the present invention is to improve the measurement efficiency so as to provide an effective measuring method which can be applied to clinical and diagnostic use.
A first aspect of the invention provides a method for measuring an amount of active oxygen produced by leukocytes and oxidative stress, wherein said method comprises allowing an anticoagulated whole blood sample collected from a human or an animal to flow in a microchannel array comprising a substrate with one or more fine channel arrays arranged on its surface and a transparent substrate which is adhered onto said substrate by contact bonding, and measuring a light emission level from whole leukocytes passing through said microchannel array through said transparent substrate to use the measured light emission level as an index of an amount of active oxygen produced by leukocytes and oxidative stress.
A second aspect of the invention provides the method according to the first aspect of the invention, wherein said light emission level is measured with or without stopping the flow of the whole blood.
A third aspect of the invention provides the method according to the first or second aspects of the invention, wherein a time-integral value of the measured light emission level is used as an index of an amount of active oxygen produced by leukocytes and of oxidative stress.
A fourth aspect of the invention provides the method according to any one of the first to third aspects of the invention, wherein a substance that amplifies light emission is added to the whole blood sample, and said whole blood sample is allowed to flow to measure the light emission level.
A fifth aspect of the invention provides the method according to any one of the first to fourth aspects of the invention, wherein a leukocyte-stimulating substance, a leukocyte-stimulating cell, or a leukocyte-stimulating particle is added to the whole blood sample, and said whole blood sample is allowed to flow to measure the light emission level.
A sixth aspect of the invention provides the method according to any one of the first to fifth aspects of the invention, wherein the light emission level is measured using a photomultiplier in combination with a direct current amplifier.
A seventh aspect of the invention provides the method according to any one of the first to fifth aspects of the invention, wherein the light emission level is measured using a photomultiplier in combination with a photoelectronic counter.
An eight aspect of the invention provides the method according to any one of the first to seventh aspects of the invention, wherein said method comprises providing a rotating stage on which plural microchannel array holders each of which is capable of holding the microchannel array can be mounted, allowing whole blood samples to flow in the plural microchannel arrays in turn, transferring said microchannel arrays onto said rotating stage to measure the light emission level in turn, and measuring the light emission level repeatedly from the first microchannel array when the measurement is done for the last microchannel array.
A ninth aspect of the invention provides a method for assessing an effect of a substance selected from the group consisting of drugs, foods, and substances made from their ingredients to enhance or suppress an amount of active oxygen produced by leukocytes and oxidative stress, wherein said method comprises allowing an anticoagulated whole blood sample collected from a human or an animal to flow in a microchannel array, measuring a light emission level from whole leukocytes passing through said microchannel array through said transparent substrate, allowing a whole blood sample to which said substance selected from the group has been added to flow in the array, measuring a light emission level from whole leukocytes passing through said microchannel array through
Director of National Food Research Institute, Ministry of Agricu
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Wallenhorst Maureen M.
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