Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2001-01-04
2002-10-22
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S174000, C435S183000, C435S188000, C435S235100, C435S287100, C435S288100, C435S810000, C436S536000, C436S810000
Reexamination Certificate
active
06468734
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a container for measurement of cell functions involved in immune and inflammatory reactions, a kit for measurement of the cell functions and a method for measuring the cell functions, and more particularly to a container for measurement of cell functions which is carried out through determination of physiologically active substances, such as cytokines, produced by blood cells including granulocytes, monocytes, macrophages, lymphocytes or the like, for example, kit and method for such a measurement of cell functions.
DESCRIPTION OF PRIOR ART
Leukocytes, such as granulocytes, monocytes, macrophages and lymphocytes, play various rolls in diversified bioprotective reactions including immune and inflammatory reactions in blood or respective organs. It is known that these cells exhibit important functions in a variety of morbidities including infectious diseases; inflammatory diseases such as hepatitis and nephritis; immune allergic diseases such as rheumatoid arthritis and asthma; and cancer, and the functions of these cells are either suppressed or enhanced as the morbidity varies.
It is also known that a variety of drugs, such as anti-inflammatory drugs, immunosuppressants, immunoenhancers and anticancer drugs, are useful in the therapy of these diseases, wherein the functions of these cells are either suppressed or enhanced concurrently. It is therefore important to examine the functions of these cells whereby the morbidities of various diseases, effects and side-effects of drugs can be identified to determine therapeutic schemes, doses of the drugs and timings of the drug administration.
In view of the above-described reasons, a granulocyte phagocytic activity test, a granulocyte bactericidal activity (active oxygen producing capacity) test, a lymphocyte transformation test and the like have been conventionally conducted at hospital examining rooms or centers in order to measure such cell functions. Also in recent years, a surface antigen test has been carried out which utilizes a flow cytometer and fluorescence-labelled monoclonal antibodies against respective surface antigens of various immunocompetent cells. However, the conventional testing methods have required such specialized techniques as separation and culture of cells, microscopic measurement or the like, to consequently necessitate time-consuming measurements, RI facilities and expensive equipments.
Also, monocytes in blood, as well as macrophages into which the monocytes moved into tissues differentiates and matures, have a wide spectrum of functions, for example, as coming into play in foreign body exclusion through phagocytosis and immune formation through antigen presentation, or as secreting various physiologically active substances, such as cytokine and prostaglandin, to thereby regulate an inflammatory or immune reaction. Like granulocytes and lymphocytes, these monocytes and macrophages play important rolls also in a variety of morbidities. It is therefore very important to identify the functions of these cells. Particularly in infectious diseases, unlike the granulocytes and lymphocytes, the monocytes and macrophages exhibit slight changes in terms of the number of cells and primarily amplify their functions, so that the measurement of changes in cell functions becomes more important (“Macrophages”, written by Tohru Tokunaga, Kodansha Scientific, 1st Ed. published in 1986). Tumor necrosis factor &agr; (hereinafter referred to as TNF&agr;), interleukin-1&bgr; (hereinafter referred to as IL-1&bgr;), and interleukin-6 (hereinafter referred to as IL-6), all called as monokine, are cytokines which are produced mainly by leukocytes including monocytes and macrophages, among blood cells, and which come into play in various inflammatory and immune reactions.
A variety of methods reported to date examines the above-described cytokine-producing functions of blood or leukocytes separated from blood. For example, in gazettes of Patent Laying-open Nos. Hei 2-196961 and Hei 3-285692, methods are disclosed which react lipopolysaccharide (LPS) or lectin with blood to induce production of cytokines, such as TNF&agr; or IL-1&bgr;, which are subsequently quantitatively determined. Also, in gazettes of Patent Laying-open Nos. Hei 6-209992 and Hei 7-67955, methods are disclosed which react blood with polymer materials having a specific surface roughness or chemical structures to induce production of TNF&agr;. Also, in gazettes of Patent Laying-open Nos. Hei 7-299732 and Hei 7-151752, bioreaction tests are disclosed which react blood with polymer materials having a specific surface roughness to determine the amount of produced TNF&agr; or IL-1&bgr;. Also, in a gazette of Tokkohyo No. Hei 7-500905, a method is disclosed which measures immunoactivity of a tested substance by determining the production of cytokines, such as TNF&agr; or IL-1&bgr;, induced from human peripheral blood leukocytes.
However, the above-described methods for measurement of cell functions which have been conventionally carried out at hospital examining rooms or centers, as well as the methods disclosed in the gazettes of the above-listed laid-open patents, have the following problems. That is, these tests all require specialized operations, such as an operation of collecting blood from an examined human using an injector and thereafter manually transferring the blood to various reactors as by pipetting, cell separation for separating leukocytes and the others, cell culture for the purpose of measuring cell functions or the like. This carries a risk for an examining person to acquire various infectious diseases, such as hepatitis and AIDS, when the person contacts the blood. Also, there is a possibility that various bacteria or dusts are accidentally incorporated into a specimen blood during such operations. There exists another risk of adversely affecting the measurement results when those contaminants or operations physically stimulate the cells in blood without necessity.
Particularly in the conventional methods which employ a specific reactor to collect blood therein and determine the production of cytokines, specifically of TNF&agr; or IL-1&bgr; induced from leukocytes, there has existed an occasion that the endotoxin, such as LPS derived from gram-negative bacteria, have been originally incorporated in a blood collecting equipment, such as an injector, or in a reactor. Since even a very slight amount of endotoxin can induce production of TNF&agr; or IL-1&bgr; from leukocytes, it was impossible to obtain reliable measurement results when, for example, entry of a slight amount of dusts during a manufacturing process or contamination through employed cleaning water resulted in incorporation of a small amount of endotoxin in the above-described blood collecting equipment or reactor.
In view of the above-described problems, a method for measurement of cell functions is sought which is more simplified in its operations, less risky and more accurate than conventional methods.
In another aspect, anticoagulants have been conventionally utilized when measuring various physiologically active substances in blood, functions of blood cells, surface antigens of blood cells or the like.
However, there exists no general standard for endotoxin contents in anticoagulant, other than a guideline given by “Endotoxin testing method” in the dispensatory of 13th revised Japanese Pharmacopeia, which, for anticoagulants employed as an injection drug, officially sets 5 EU/Kg as a standard for a specification of minimal pyrogenic dose to a rabbit.
Endotoxin is lipopolysaccharide constituting a cell-wall outer membrane of gram-negative bacteria, and a very slight amount thereof suffices to stimulate blood cells, such as leukocytes, to produce physiologically active substances such as a variety of cytokines including TNF&agr;, IL-1&bgr;, IL-6, or granulocyte-macrophage colony-stimulating factors. They exhibit various physiological actions such as pyrogenic activity and endotoxin shock (Nippon Igaku-kan “Inflammation and cytokin
Kobayashi Koji
Kuriyama Kiyoshi
Setoguchi Yuji
Chin Christopher L.
Sekisui Kagaku Kogyo Kabushiki Kaisha
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