WO2005108979A1 - 末梢血単核球の多糖結合能測定キット - Google Patents
末梢血単核球の多糖結合能測定キット Download PDFInfo
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- WO2005108979A1 WO2005108979A1 PCT/JP2005/007306 JP2005007306W WO2005108979A1 WO 2005108979 A1 WO2005108979 A1 WO 2005108979A1 JP 2005007306 W JP2005007306 W JP 2005007306W WO 2005108979 A1 WO2005108979 A1 WO 2005108979A1
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- polysaccharide
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- immunostimulatory
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- fluorescently
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
- G01N33/56972—White blood cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/975—Kit
Definitions
- Peripheral blood mononuclear cell polysaccharide binding ability measurement kit Peripheral blood mononuclear cell polysaccharide binding ability measurement kit
- the present invention provides a polysaccharide binding ability measurement kit effective for simply measuring whether or not which immunostimulating polysaccharide is effective as an immunostimulant for which individual, and the effectiveness as an immunostimulant It relates to a measuring method.
- Lentinan and other immunostimulatory polysaccharides are known to be effective as immunostimulants and antineoplastic agents, but it is also known that the effects vary greatly between individuals (Non-Patent Reference 1).
- immunostimulatory polysaccharides bind to the surface of peripheral blood mononuclear cells of mammals such as human and mouse, for example, monocytes, granulocytes and lymphocytes (Non-Patent Documents 2 and 3).
- the binding ability of immunostimulatory polysaccharide to peripheral blood mononuclear cells varies between individuals and mouse strains, and it is highly likely that the expression of the effect of immunostimulatory polysaccharide and the binding ability are correlated.
- the ability of immunostimulatory polysaccharides to bind to peripheral blood mononuclear cells is based on the known method of using immunostimulatory polysaccharide antibodies such as anti-lentinan antibody.
- the anti-lentinan antibody is IgM and is difficult to use.
- Lentinan antibodies are produced in-house and there is a problem that it is difficult to constantly supply a constant titer. Furthermore, since the leukocyte fraction is separated from the blood and subjected to the reaction in the presence of serum, there is a problem that the operation is complicated.
- This method is a method that can measure the binding ability of a labeled immunostimulatory polysaccharide to peripheral blood mononuclear cells much more efficiently and with higher precision than a method using an anti-lentinan antibody or the like.
- this method measured non-specific binding in addition to specific binding to the immunostimulatory polysaccharide. Even if this is the case, it does not mean that only the specific binding ability of the tested individual to the immunostimulatory polysaccharide is measured, and that the binding ability of the tested individual and the effect may not always be correlated. I found out.
- Non-Patent Document 1 Cancer Res. 44: 5132 (1984)
- Non-Patent Document 2 50th Annual Mtg.Proc.Jpn.Cancer Assoc.p259 (1991)
- Non-Patent Document 3 Int.J. Immunopharmac. 18: 211 (1996)
- Non-Patent Document 4 J. Clin. Invest. Vol. 98, No. 1, July 1996, pp50-61
- Non-Patent Document 5 J. Exp.Med.Vol. 196, No. 3, August 5, 2002, pp407-412
- An object of the present invention is to provide a kit for measuring the polysaccharide binding ability of peripheral blood mononuclear cells, which enables determination with higher prediction accuracy.
- Another object of the present invention is to provide a method for determining the effectiveness of an immunostimulatory polysaccharide as an immunostimulant, which has higher prediction accuracy.
- the present invention provides, in addition to using a detection reagent containing a fluorescently labeled immunostimulatory polysaccharide, a standard reagent containing a fluorescently labeled substance of a polysaccharide different from the fluorescently labeled immunostimulatory polysaccharide, or a fluorescently labeled immunostimulatory polysaccharide.
- the present invention provides a detection reagent containing a fluorescently labeled immunostimulating polysaccharide, a fluorescently labeled polysaccharide different from the fluorescently labeled immunostimulating polysaccharide, or the same polysaccharide as the fluorescently labeled immunostimulating polysaccharide; And a standard reagent containing an immunostimulatory polysaccharide which has not been subjected to the method.
- the present invention also relates to a method of contacting a test blood with a detection reagent containing a fluorescence-labeled immunostimulatory polysaccharide to obtain a target peripheral blood mononuclear cell fraction in blood, measuring the fluorescence intensity thereof, and measuring the fluorescence.
- the test blood is brought into contact with a standard reagent containing a fluorescent label of a polysaccharide different from the fluorescently labeled immunostimulatory polysaccharide used in the detection reagent to obtain a target peripheral blood mononuclear cell fraction in the blood, and the fluorescence intensity is measured. Determining the amount of binding (A) between the fluorescent label of the different polysaccharide and the target peripheral blood mononuclear cell, except that the unlabeled polysaccharide contained in the standard reagent or another This step can be omitted when the contact is carried out in the presence of a label with a fluorescent dye.
- a method for determining the effectiveness of an immunostimulating polysaccharide as an immunostimulating agent comprising:
- the present invention also relates to a method in which a test blood is brought into contact with a detection reagent containing a fluorescently labeled immunostimulating polysaccharide to obtain a target peripheral blood mononuclear cell fraction in blood, and the fluorescence intensity is measured.
- the test blood is contacted with the standard reagent containing the immunostimulatory polysaccharide, which is the same polysaccharide as the fluorescence-labeled immunostimulatory polysaccharide used in the detection reagent and is fluorescently labeled, and the detection reagent containing the fluorescence-labeled immunostimulatory polysaccharide.
- the standard reagent containing the immunostimulatory polysaccharide which is the same polysaccharide as the fluorescence-labeled immunostimulatory polysaccharide used in the detection reagent and is fluorescently labeled
- the detection reagent containing the fluorescence-labeled immunostimulatory polysaccharide Obtaining a target peripheral blood mononuclear cell fraction in the blood and measuring the fluorescence intensity to determine the amount of binding (A) between the fluorescently labeled immunostimulatory polysaccharide and the target peripheral blood mononuclear cell;
- a method for determining the effectiveness of an immunostimulating polysaccharide as an immunostimulating agent comprising:
- the fluorescence-labeled immunostimulatory polysaccharide used in the present invention includes a polysaccharide capable of introducing a reactive group such as CHO, -COOH, -NH2, -NHS, -CN, or an epoxy group into a glucose residue of the polysaccharide. Anything can be used. Examples thereof include ⁇ -glucan, OC-glucan, heteropolysaccharide (preferably heterodalkane), protein-bound glucan, and the like, preferably j8-glucan, more preferably lentinan, zymozan, malanized malan, schizophyllan, Pustulin, Scleroglucan and Lihenan. The molecular weight of these polysaccharides is not particularly limited.
- Examples of the fluorescent substance for introducing a fluorescent label into the polysaccharide include a fluorescent substance having a reactive group such as NH2-, COOH- or imide in the molecule (a secondary substance such as a fluorescent labeled avidin such as biotin).
- a fluorescent substance e.g., Shin Seikagaku Jikken Lecture 3 carbohydrate 1 (top) pl 9 ⁇ , ⁇ 95 ⁇ (1 "0) Tokyo Kagaku Dojin, Tokyo, the sugar Quality 1 (below), p605 ⁇ , p743 ⁇ and carbohydrate II, p283 ⁇
- a fluorescent substance having --NH2 (including imide) or a conjugated product of Piotinii Is preferably a wavelength that can be detected by flow cytometry analysis such as FACS! /, Preferably, a fluorescein-based compound.
- 5- thiosemicarbazide, FIT and (Fluorescein isothiocyanate) are discouraged force 21 such.
- the fluorescent-labeled polysaccharide used as a standard reagent and different from the fluorescent-labeled immunostimulatory polysaccharide used in the detection reagent is different from the fluorescent-labeled immunostimulatory polysaccharide used in the detection reagent in the binding mode to peripheral blood mononuclear cells.
- Any polysaccharide can be used as long as it is fluorescently labeled with the same fluorescent dye.
- standard reagents for the measurement of the binding ability of j8-1,3-glucan are ⁇ -glucan, heteropolysaccharide (preferably heterodarcan) and the like fluorescently labeled with the same fluorescent substance.
- the molecular weight is not particularly limited, and may be a monosaccharide or a disaccharide, but is preferably 0.01 to 100 times (preferably 0.1 to 10 times, more preferably about the same) as the polysaccharide to be measured.
- the fluorescence intensity was measured! / It is good to be 0.01 to 100 times (preferably 0.1 to 10 times, more preferably the same degree) in terms of the converted intensity per glucose as compared with the fluorescence-labeled polysaccharide (detection reagent).
- the same polysaccharide as the fluorescently labeled immunostimulatory polysaccharide used in the detection reagent, which is fluorescently labeled can be used as the standard reagent.
- both the detection reagent and the standard reagent are liquid and housed in separate containers.
- the detection reagent or the standard reagent is added to the aqueous solution in an amount of 1 to 10,000 g / mL (preferably 10 to 1,000 g / mL, more preferably 100 to 500 g / mL) and a suitable preservative (Ajiy Sodium hydroxide, etc.) and a reagent for suppressing the fluidity of the cell membrane (such as sodium azide sodium and cytochalasin B).
- the kit of the present invention further comprises a hemolytic 'fixing solution, a washing' analysis solution, and a blood collection tube.
- the material for the blood collection tube is not particularly limited, but is preferably made of glass, polyethylene, polypropylene, polystyrene, or the like, and is preferably an anticoagulant treated with heparin, citric acid, EDTA, or the like.
- the blood collection volume is preferably 0.01 to 10 mL, particularly preferably 1 to 5 mL. It is.
- a standard reagent solution (an unlabeled polysaccharide different from the fluorescently labeled immunostimulating polysaccharide or a labeled product with another fluorescent dye coexists in the blood collection tube)
- the detection reagent solution (Solution B) in a volume ratio of 1/100 to 1/2 (preferably 1/10 to 1/20) (final concentration of polysaccharide is Ol ⁇ g-1,000 ⁇ g / mL, preferably 1-100 ⁇ g / mL, more preferably 10-50 ⁇ g / mL) for 5 to 180 minutes (preferably 15 to 90 minutes, more preferably Leave for 30-60 minutes).
- the leaving temperature is preferably 0 to 50 ° C.
- this operation can also be performed after the hemolysis / fixing solution treatment described below.
- the fixative may be a hypotonic solution such as water that can lyse erythrocytes.
- the added amount is 1 to 100 times (preferably 2 to 50 times, more preferably 10 to 40 times) the blood solution to be treated, mixed with blood, and 0 ° C to 40 ° C (preferably room temperature).
- the mixture is allowed to stand for 1 to 60 minutes (preferably 5 to 30 minutes, more preferably 10 to 15 minutes) to lyse the red blood cells, and centrifuged to remove the supernatant. If more than 24 hours are required for measurement, it is desirable to fix the cells.
- red blood cells are lysed and centrifuged as in the above, and the supernatant is removed. Wash if necessary. • After washing with the analysis solution, leave at 0 ° C to room temperature (preferably refrigerated storage) and measure within 72 hours (preferably within 48 hours).
- FIX & PERM Reagent A manufactured by Caltag
- formalin-based fixing agent formaldehyde or the like
- the washing solution may be an isotonic solution such as a physiological saline solution, for example, a commercially available PBS (phosphate buffered saline) -based reagent or the like. If necessary, those containing BSA (bovine serum albumin), sodium azide, ethylene glycol, etc. may be used (for example, washing buffer (PBS (-) + 0.1% BSA + 0.1% sodium azide) etc). It is advisable to add 0.5 to 10 times (preferably 1 to 4 times) the amount of added blood to the processed blood, mix, and then centrifuge to remove the supernatant. Perform this operation one to several times, add 0.5 to 10 times (preferably 1 to 4 times) the volume of the treated blood, mix, and analyze by flow cytometry. If measurement requires more than 24 hours, use a preservative or cell membrane fluidity inhibitor (for example, It is desirable to use one containing cytochalasin B).
- PBS phosphate buffered saline
- BSA bovine serum albumin
- the blood used in the kit of the present invention can be used either immediately after collection, in ice after collection, stored in a refrigerator for a short time, or stored frozen at -30 ° C or lower.
- the blood stored frozen may be thawed at the time of use by a usual method.
- the kit of the present invention can be applied to staining not only blood mononuclear cells but also other cell suspensions. Suspension of various cell lines (cancer cell line, leukocyte cell line, etc.), cell suspension of cancer tissue and various organs (lymph node (Example), spleen, lung, liver, intestinal tract, skin, etc.) (EDTA treatment ⁇ It can be applied to staining of enzyme (trypsin, collagenase, etc.) treated single cell suspension! /,). The procedure was performed using a single cell suspension prepared at 1 ⁇ 10 3 to 10 8 cells / mL (preferably 1 ⁇ 10 4 to 10 7 cells / mL, more preferably 1 ⁇ 10 5 to 10 6 cells / mL). If there is, it can be measured by the same operation as above.
- the kit of the present invention can be applied to not only single color analysis but also multi-color analysis. That is, the analysis can be performed in combination with flowcytometry using an antibody against a specific substance inside or outside a certain cell (for example, a surface antigen or the like) or an intracellular substance (site force or the like).
- a specific substance inside or outside a certain cell for example, a surface antigen or the like
- an intracellular substance site force or the like.
- measurement can be easily performed by coexisting an antibody against the cell surface substance.
- an antibody labeled with biotin or the like may be used, and after washing, treated with avidin or the like labeled with another fluorescent labeling substance!
- a secondary antibody against the antibody for example, an antibody labeled with a fluorescent labeling substance different from the fluorescent labeling substance or a biotin, etc.
- treatment with avidin or the like labeled with another fluorescent labeling substance Even if the antibody is not coexistent during the operation of adding the standard reagent solution or the detection reagent solution to the blood collection tube, it is also possible to perform the measurement by treating the antibody with the antibody after the hemolysis operation.
- test blood is brought into contact with a detection reagent containing a fluorescence-labeled immunostimulatory polysaccharide to obtain a target peripheral blood mononuclear cell fraction in blood, and the fluorescence intensity is measured. Then, the amount (B) of the conjugate of the fluorescence-labeled immunostimulatory polysaccharide and the target peripheral blood mononuclear cell was determined, and
- the test blood is brought into contact with a standard reagent containing a fluorescent label of a polysaccharide different from the fluorescently labeled immunostimulatory polysaccharide used in the detection reagent to obtain a target peripheral blood mononuclear cell fraction in the blood, and the fluorescence intensity is measured.
- a standard reagent containing a fluorescent label of a polysaccharide different from the fluorescently labeled immunostimulatory polysaccharide used in the detection reagent to obtain a target peripheral blood mononuclear cell fraction in the blood, and the fluorescence intensity is measured.
- the binding amount (A) between the fluorescently labeled substance and the target peripheral blood mononuclear cells the unlabeled polysaccharide different from the fluorescently labeled immunostimulatory polysaccharide or the labeled substance with another fluorescent dye coexists in the detection reagent
- the amount of the conjugate or the amount of the conjugate can be the number (ratio) of cells to be conjugated (the same applies hereinafter).
- test blood is contacted with a detection reagent containing a fluorescently-labeled immunostimulating polysaccharide to obtain a target peripheral blood mononuclear cell fraction in blood, and the fluorescence intensity is measured to determine the fluorescently-labeled immunostimulating polysaccharide and the target peripheral blood monosaccharide.
- test blood is contacted with the same polysaccharide as that used in the detection reagent, which is fluorescently labeled, and is contacted with a standard reagent containing the immunostimulatory polysaccharide and a detection reagent containing the fluorescence-labeled immunostimulatory polysaccharide.
- a peripheral blood mononuclear cell fraction was obtained, and its fluorescence intensity was measured to determine the binding amount (A) between the fluorescence-labeled immunostimulatory polysaccharide and the target peripheral blood mononuclear cell,
- the amount of nonspecific polysaccharide binding can be removed, and the specific binding of the immunostimulatory polysaccharide to be measured can be measured. Furthermore, by using the above two determination methods in combination, it is possible to remove the non-specific amount of the antitumor polysaccharide, and to confirm that the binding mode to the cell is not changed by the fluorescent labeling of the immunostimulatory polysaccharide. The specific binding of the immunostimulatory polysaccharide used can be measured, and the effectiveness of the immunostimulatory polysaccharide as an immunostimulant can be predicted with high accuracy.
- a standard reagent containing a fluorescent label of a polysaccharide different from the fluorescence-labeled immunostimulatory polysaccharide is used (non-specific different If unlabeled different polysaccharides or labeled with another fluorescent dye coexist in the detection reagent, the nonspecific binding amounts of different polysaccharides can be removed from the former measurement results, /! Since the specific binding ability of the immunostimulatory polysaccharide can be measured, there is an advantage that the effectiveness of the immunostimulatory polysaccharide as an immunostimulant can be predicted with high accuracy.
- the immunoreactivity is increased. Fluorescent labeling of the activating polysaccharide can be used to confirm that the binding mode to cells has changed, and the measured binding ability of the immunostimulating polysaccharide itself can be accurately measured, and as an immunostimulating agent for the immunostimulating polysaccharide. Can be predicted with high accuracy.
- a first standard reagent containing a fluorescently labeled substance of a polysaccharide different from the fluorescently labeled immunostimulating polysaccharide (a polysaccharide different from the detection reagent)
- the first standard reagent can be omitted when an unlabeled substance or a labeled substance with another fluorescent dye is used together) and the same polysaccharide as the fluorescent-labeled immunostimulatory polysaccharide used in the detection reagent, and
- the second standard reagent containing no immunostimulatory polysaccharide is used, the binding ability of the immunostimulatory polysaccharide itself can be measured more accurately, and the effectiveness of the immunostimulatory polysaccharide as an immunostimulant can be further improved. Predictability is high.
- the kit of the present invention can quickly and easily combine the fluorescence intensity shift (difference), ie, individual immunostimulatory polysaccharide, by combining with a detection reagent, a standard reagent, hemolysis, fixative, washing, analysis solution, and blood collection tube. Can be measured.
- Disperse lentinan bulk powder (manufactured by Ajinomoto Co.) in distilled water while kneading in a mortar, 120 ° C, 20 minutes The solution dissolved in the autoclave was used as a solution.
- Heart blood was collected from ICR mice (purchased from CLEA Japan, female, 5 to 8 weeks old) under ether anesthesia using a syringe containing henolin. Under ice-cooling, heparin-treated blood was dispensed into Falcon2054 tubes at 90 ⁇ l / tube, and Fluorescein-labeled lentinan was added at 10 ⁇ l / tube.
- MC monocyte fraction
- GC granulocyte fraction
- LC lymphocyte fraction
- the target of Fluorescein-labeled lentinan is FITC-labeled dextran (min.
- FITC-labeled dextran min.
- the same analysis was performed using 2 million particles (Sigma) (the results are shown in the FD section of Fig. 1, for example).
- Lewis rats purchased from Nippon Charls River Co., Ltd., 9 to 10 weeks of age, male
- a syringe containing heparin were collected using a syringe containing heparin, and tested for binding inhibition using blood frozen in a -30 ° C freezer. Test was carried out.
- Fluorescein-labeled lentinan was reacted with whole blood of ICR mice for 1 hour at a final concentration of 5, 10, 20, 40 ⁇ g / ml (FL5, FL10, FL20, FL40), and the results of FACS analysis of Fluorescein-labeled lentinan-binding cells were analyzed.
- Figures 1-3 show. In the monocyte fraction, concentration-dependent binding of fluorescein-labeled lentinan was observed. At 40 g / ml, about 40% of monocytes bound to fluorescein-labeled lentinan (Fig. 1).
- a concentration-dependent binding was also observed in the granulocyte fraction, and about 20% of the granulocytes were bound with 40 g / ml of fluorescein-labeled lentinan (FIG. 2). Although concentration-dependent binding was observed in the lymphocyte fraction, the ratio of the bound cells was less than 1% even with Fluorescein-labeled lentinan at 40 ⁇ g / ml (FIG. 3).
- Fluorescein-labeled lentinan When Fluorescein-labeled lentinan is reacted with whole blood of ICR mice at a final concentration of 5, 10, 20, or 40 g / ml for 1 hour, 50 ⁇ g / ml lentinan bulk solution is added (FL5 + L50, FL10 + L50, FL20 + L50, FL40 + L50) inhibited the binding of Fluorescein-labeled lentinan (Figs. 1-3).
- Fluorescein-labeled lentinan was reacted with whole rat blood after freezing and thawing at a final concentration of 150 ⁇ g / ml (FL150) for 1 hour, and Fluorescein-labeled lentinan-binding cells were analyzed by FACS. Was observed (FIG. 4).
- lentinan Presumably binds to mouse monocytes, which suggests that lentinan is effective as an immunostimulant for the mouse.
- Human blood collected in a henolin blood collection tube was dispensed at 250 1 / tube, and one of the blood samples was supplemented with the same Fluorescein-labeled lentinan as used in Example 1 so as to be 100 g / ml.
- a PE-labeled antibody against human CD14 manufactured by CALTAG was added, and after culturing at 37 ° C for 30 minutes, a 0.83% ammonium chloride solution (0.1% sodium bicarbonate, 0.0037% (Containing EDTA '4Na) at 10 ml / tube and hemolyzed.
- FITC-labeled dextran molecular weight: 2 million; Sigma
- Fluorescein-labeled lentinan standard reagent 1
- Percentage force of Fluorescein-labeled lentinan-binding cells The ratio of FITC-labeled dextran-binding cells was subtracted, and the resulting value was taken as the ratio of lentinan-binding positive cells (shown in FIGS. 5 and 6 and shown as FL in FIG. 7). .
- blood stored at room temperature or refrigerated until the day after blood collection, and blood collected on another day were also examined.
- lentinan bulk solution (standard reagent 2) to human blood at a concentration of 100 g / ml, incubate at 37 ° C for 15 minutes, then add Fluorescein-labeled lentinan at a concentration of 10 / zg / ml, and further add at 37 ° C. Incubated for 30 minutes. Then, a PE-labeled antibody against human CD14 (manufactured by CALTAG) was added, and the mixture was further cultured at 37 ° C for 30 minutes. Then, 2 ml / ml of FACS Lysing Solution (manufactured by Hemolysis' fixative BD, diluted 10-fold with distilled water) was added.
- FACS Lysing Solution manufactured by Hemolysis' fixative BD, diluted 10-fold with distilled water
- Figures 5 and 6 show the results of two examples (Example 1 and Example 2). Individual differences were observed in monocytes, granulocytes, and lymphocytes, and the strength was similar in all fractions (Figure 5). The results are shown for blood stored at room temperature or refrigerated for one day and blood collected on another day. The results were the same on the day of blood collection under both refrigerated and room temperature storage conditions. Similar results were obtained when blood was collected on another day ( Figure 6).
- Lentinan binding inhibition test Figure 7 shows the results of two examples (Example 3 and Example 4). Binding of Fluorescein-labeled lentinan was inhibited when 100 ⁇ g / ml of lentinan bulk powder solution was co-existed with Fluorescein-labeled lentinan before reacting with blood at a final concentration of 10 g / ml.
- the binding amount of Fluorescein-labeled lentinan (FL) to monocytes (MC) force
- the binding amount of FITC-labeled dextran (A1) was subtracted from the binding amount (FIG.
- Lentinan binds to human monocytes in Example 1, Example 2, Example 3 and Example 4; Is estimated to be more effective as an immunostimulant.
- the amount of binding (A2) to unlabeled lentinan and Fluorescein-labeled lentinan [LNT + FL] was determined from the amount of binding of Fluorescein-labeled lentinan (FL) to monocytes (MC).
- Lentinan specifically binds to human monocytes in Examples 3 and 4 because of the large amount of binding! / Lentinan is an immunostimulant for humans in Example 3 as well. Is estimated to be more effective.
- ICR mice purchased from Clea Japan, Inc., 4-6 weeks old, female
- BALB / c mice purchased from Japan Charls River, Inc., 11 ⁇ 13 weeks old, female
- C57BL / 6 mouse purchasedd from CLEA Japan, Inc., 10 ⁇ : L 1 week old, male
- C3H / HeN mouse purchased from CLEA Japan
- C3H / HeJ mouse purchasedd from CLEA Japan
- heparin-treated blood was dispensed into Falcon2054 tubes at 90 ⁇ l / tube, and fluorescein-labeled lentinan was added at 10 ⁇ l / tube.
- fluorescein-labeled lentinan was added at 10 ⁇ l / tube.
- ⁇ -labeled antibody against mouse monocyte surface antigen F4 / 80 ⁇ -labeled anti-mouse F4 / 80 antibody; Serotec
- FACS Lysing Solution hemolysis ⁇ fixative BD; 10-fold diluted with distilled water
- the ratio of Fluorescein-positive cells to PE-positive cells in the monocyte fraction was analyzed by developing to Fluorescein / PE.
- Figure 8 shows the average value of the ratio of Fluorescein-positive cells in F4 / 80-positive monocytes.
- the strain at BALB / c was higher than that of the other strains (Fig. 8A).
- FITC-labeled dextran was reacted, almost no binding was observed (FIG. 8C).
- Non-Patent Documents 1 and 6 there is a result of examining the antitumor effect of lentinan by subcutaneously transplanting S180 mouse sarcoma cells into mice of different strains. According to this, the antitumor effect of lentinan is strong in the S180 subcutaneous transplant model of ICR and BALB / c. The antitumor effect using C57BL / 6 is moderate, and the antitumor effect using C3H is medium. Fluorescein-labeled lentinan had a similar tendency to the amount of binding of lentinan.
- FIG. 1 shows the results of a Fluorescein-labeled lentinan binding test, the results of a FITC-labeled dextran binding test, and the results of a binding test when unlabeled lentinan coexists with Fluorescein-labeled lentinan in the monocyte fraction.
- FIG. 2 shows the results of a Fluorescein-labeled lentinan binding test, the results of a FITC-labeled dextran binding test, and the results of a binding test when unlabeled lentinan coexists with Fluorescein-labeled lentinan in the granulocyte fraction.
- FIG. 3 shows the results of a Fluorescein-labeled lentinan binding test, the results of a FITC-labeled dextran binding test, and the results of a binding test in the case where unlabeled lentinan coexists with Fluorescein-labeled lentinan in the lymphocyte fraction.
- FIG. 4 shows the results of a Fluorescein-labeled lentinan binding test in the monocyte fraction of unfrozen-thawed blood and frozen-thawed blood.
- FIG. 5 shows the results of a Fluorescein-labeled lentinan binding test in a CD14-positive monocyte fraction, a granulocyte fraction, and a lymphocyte fraction.
- FIG. 6 shows the results of a Fluorescein-labeled lentinan binding test using monocyte fractions using blood collected on the day after blood collection stored refrigerated or at room temperature, and blood collected on another day.
- FIG. 7 shows the results of a binding inhibition test when unlabeled lentinan coexists with fluorescein-labeled lentinan in the monocyte fraction.
- FIG. 8 shows the results of a Fluorescein-labeled lentinan binding test and an FITC-labeled dextran binding test result of an F4 / 80-positive monocyte fraction.
- A is Fluorescein-labeled lentinan at 20 g / ml
- B is Fluorescein-labeled lentinan at 5 g / ml
- C is FITC-labeled dextran at 20 g / ml.
- FL Fluorescein-labeled lentinan
- FD Fluorescein-labeled dextran
- LNT unlabeled lentinan
- + L is This shows the case where unlabeled lentinan coexists with fluorescein-labeled lentinan
- the numerical value is the concentration (unit: microgram / milliliter).
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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EP05730576.5A EP1736773B1 (en) | 2004-04-16 | 2005-04-15 | Assaying potency of peripheral mononuclear blood cell to bind to polysaccharide |
JP2006512936A JP4577660B2 (ja) | 2004-04-16 | 2005-04-15 | 末梢血単核球の多糖結合能測定キット |
US11/580,936 US7449452B2 (en) | 2004-04-16 | 2006-10-16 | Method for estimating immunostimulant activity of a polysaccharide |
US12/117,999 US7553824B2 (en) | 2004-04-16 | 2008-05-09 | Kit for determining polysaccharide-binding activity of mononuclear cells present in peripheral blood |
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JP2004-121616 | 2004-04-16 | ||
JP2004121616 | 2004-04-16 | ||
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JP2004233513 | 2004-08-10 | ||
JP2005-036312 | 2005-02-14 | ||
JP2005036312 | 2005-02-14 |
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PCT/JP2005/007306 WO2005108979A1 (ja) | 2004-04-16 | 2005-04-15 | 末梢血単核球の多糖結合能測定キット |
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US (2) | US7449452B2 (ja) |
EP (1) | EP1736773B1 (ja) |
JP (1) | JP4577660B2 (ja) |
WO (1) | WO2005108979A1 (ja) |
Cited By (1)
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WO2014054601A1 (ja) * | 2012-10-03 | 2014-04-10 | 国立大学法人弘前大学 | クマリン誘導体が結合した蛍光標識糖誘導体を用いた細胞イメージング方法及びイメージング剤 |
Families Citing this family (2)
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JP4577660B2 (ja) * | 2004-04-16 | 2010-11-10 | 味の素株式会社 | 末梢血単核球の多糖結合能測定キット |
CN105806839A (zh) * | 2016-03-21 | 2016-07-27 | 北京北大明德科技发展有限公司 | 无胚芽大米新陈度检测试剂及方法 |
Citations (2)
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JPH0424555A (ja) * | 1990-05-18 | 1992-01-28 | Ajinomoto Co Inc | 抗レンチナン抗血清及びレンチナンの免疫学的測定法 |
JPH04346791A (ja) * | 1991-05-21 | 1992-12-02 | Taito Kk | 単クローン抗体 |
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US5106616A (en) * | 1988-01-14 | 1992-04-21 | Carrington Laboratories, Inc. | Administration of acemannan |
US5705398A (en) * | 1994-03-02 | 1998-01-06 | The Scripps Research Institute | Methods for identifying inhibitors of LPS-mediated LBP binding |
US6413715B2 (en) * | 1997-01-31 | 2002-07-02 | The Collaborative Group | β(1-3)-glucan diagnostic assays |
US20040132131A1 (en) * | 2001-11-05 | 2004-07-08 | Ofer Markman | Methods for comparitive analysis of carbohydrate polymers and carbohydrate polymers identified using same |
US6956120B2 (en) * | 2000-11-09 | 2005-10-18 | Yasushi Onaka | β-1.3-1.6 glucan (Aureobasidium medium) |
US7507724B2 (en) * | 2001-01-16 | 2009-03-24 | Sloan-Kettering Institute For Cancer Research | Therapy-enhancing glucan |
US7906492B2 (en) * | 2001-01-16 | 2011-03-15 | Sloan-Kettering Institute For Cancer Research | Therapy-enhancing glucan |
JP4577660B2 (ja) * | 2004-04-16 | 2010-11-10 | 味の素株式会社 | 末梢血単核球の多糖結合能測定キット |
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Patent Citations (2)
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JPH0424555A (ja) * | 1990-05-18 | 1992-01-28 | Ajinomoto Co Inc | 抗レンチナン抗血清及びレンチナンの免疫学的測定法 |
JPH04346791A (ja) * | 1991-05-21 | 1992-12-02 | Taito Kk | 単クローン抗体 |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014054601A1 (ja) * | 2012-10-03 | 2014-04-10 | 国立大学法人弘前大学 | クマリン誘導体が結合した蛍光標識糖誘導体を用いた細胞イメージング方法及びイメージング剤 |
JPWO2014054601A1 (ja) * | 2012-10-03 | 2016-08-25 | 国立大学法人弘前大学 | クマリン誘導体が結合した蛍光標識糖誘導体を用いた細胞イメージング方法及びイメージング剤 |
US10288604B2 (en) | 2012-10-03 | 2019-05-14 | Hirosaki University | Method for imaging cell using fluorescence-labeled sugar derivative having coumarin derivative bound thereto, and imaging agent |
Also Published As
Publication number | Publication date |
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US20080220452A1 (en) | 2008-09-11 |
US7553824B2 (en) | 2009-06-30 |
EP1736773B1 (en) | 2014-07-02 |
US7449452B2 (en) | 2008-11-11 |
JP4577660B2 (ja) | 2010-11-10 |
EP1736773A4 (en) | 2008-06-18 |
EP1736773A1 (en) | 2006-12-27 |
US20070031898A1 (en) | 2007-02-08 |
JPWO2005108979A1 (ja) | 2008-03-21 |
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