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WO2007102442A1 - Anticorps monoclonal, hybridome, et procédé de quantification de lipopolysaccharide - Google Patents

Anticorps monoclonal, hybridome, et procédé de quantification de lipopolysaccharide Download PDF

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Publication number
WO2007102442A1
WO2007102442A1 PCT/JP2007/054094 JP2007054094W WO2007102442A1 WO 2007102442 A1 WO2007102442 A1 WO 2007102442A1 JP 2007054094 W JP2007054094 W JP 2007054094W WO 2007102442 A1 WO2007102442 A1 WO 2007102442A1
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WIPO (PCT)
Prior art keywords
lps
monoclonal antibody
lpsp
xanthomonas
lipopolysaccharide
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Ceased
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PCT/JP2007/054094
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English (en)
Japanese (ja)
Inventor
Gen-Ichiro Soma
Hiroyuki Inagawa
Chie Kohchi
Takashi Nishizawa
Teruko Honda
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Biomedical Research Group Inc
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Biomedical Research Group Inc
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Priority to JP2008503835A priority Critical patent/JP5216575B2/ja
Publication of WO2007102442A1 publication Critical patent/WO2007102442A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • G01N33/56911—Bacteria
    • 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
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • 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
    • G01N2400/50—Lipopolysaccharides; LPS

Definitions

  • the present invention utilizes a monoclonal antibody for identifying, quantifying, or capturing various lipopolysaccharides that are cell wall constituents of Gram-negative bacteria, a hyperdoma that produces the monoclonal antibody, and the monoclonal antibody.
  • the present invention relates to a method for quantifying lipopolysaccharide.
  • Lipopolysaccharide is a substance that is localized on the cell surface of gram-negative bacteria called lipopolysaccharide (LPS).
  • the basic structure is a glycolipid in which a sugar chain (core polysaccharide, O antigen polysaccharide, etc.) is bound to a lipid called lipid A (Non-patent Document 1).
  • the type of sugar in the sugar chain and the number of repetitions vary depending on the species. It is known that the lipid A moiety also has a different structure depending on the bacterial species. That is, LPS has a different structure depending on the microorganism from which it is derived.
  • Non-patent Document 1 LPS is known to have biological activities such as immunostimulation (Non-patent Document 1). However, reflecting the fact that the structure of LPS varies depending on the microorganism from which it is derived, it is known that its biological activity also varies greatly depending on the type of LPS (Non-patent Document 1). In recent years, attention has been focused on achieving disease prevention and health maintenance using the biological activity of LPS (Non-patent Document 2). It has also been reported that LPS, which is conventionally ingested or exposed, has important significance for maintaining health (Non-patent Document 3).
  • LPS LPS
  • Conventional LPS The Limulus reaction is used for general measurements.
  • the Limulus reaction is an application of coagulation of blood cells lysate of Kabutoga ni to LPS and coagulation, and a kit with improved convenience is commercially available (Non-patent Document 4).
  • Rimlas reaction Does not reflect the fine structure of LPS, and does not react only with specific LPS with very low specificity, such as reacting with glycolipids such as cardiolipin.
  • a monoclonal antibody is one of substances that can recognize only a specific structure with high specificity (Non-patent Document 5). So far, monoclonal antibodies that recognize the lipid part of Escherichia coli LPS are known, and other monoclonal antibodies against LPS such as Salmonella are known. However, it has been found that monoclonal antibodies can be obtained accidentally for individual substances, and it is impossible to predict the biological properties of such monoclonal antibodies. Therefore, the fact that monoclonal antibodies have been obtained against E. coli and Salmonella LPS does not lead to estimation of the structure and biological properties of monoclonal antibodies against LPS derived from other microorganisms.
  • the present inventors pay attention to microorganisms symbiotic to edible materials or microorganisms used for food production or edible, especially gram-negative bacteria.
  • LPS present in these microorganisms is orally It has been reported that it is a safe and safe substance by transdermal administration, and exhibits an extremely excellent immune immunity activation effect (Patent Document 1).
  • Patent Document 2 products aimed at various disease prevention and health maintenance can be provided.
  • the LPS contained in each product is identified and the content is measured to clarify the product's effect or to identify the combination Needless to say, it is necessary to make sure that the LPS quality and quantity are sufficient and sufficient to achieve the desired effect.
  • the present inventors have established microorganisms symbiotic to edible materials, preferably Pantoea, Pantoea, Dispersa, Acetobacter, Acetopacter, Darconobacter, Fraturia, Darconic acid bacteria, Xanthomonas, Zymomonas, etc., or microorganisms used for food production or edible, preferably Pantoea, Pantoea dispersa, acetic acid bacteria, acetopacter, darconobacter, fraturia, dalconic acid bacteria, xanthmona Succeeded in creating a monoclonal antibody against a specific LPS, paying attention to Gram-negative bacteria such as S.
  • Patent Document 1 # 112005-342971
  • Patent Document 2 WO2005Z030938
  • Non-patent literature 1 Christian A, Ulrich Z, Trends in Glycoscience ana Glycotechnologyl4, 69-86 (2002)
  • Non-Patent Document 2 Genichiro Tsuji Genji Progress Research Report on Pharmaceutical Research 16: 7—22 (2000)
  • Non-Patent Document 3 Kure, K. Yamazaki, Zen Moh, Taro Shirakawa Chemistry and Biology 44 21 -26 (2006)
  • Non-Patent Document 4 Nakamura T, et al. Eur. J. Biochem. 176, 89-94 (1988)
  • Non-Patent Document 5 Ed Harlow, David Lane, Antibodies Alaboratory manual Chapter6 pl4
  • the present invention provides a monoclonal antibody for specifying the quality and quantity of LPS contained or conjugated, a hyperidoma producing the monoclonal antibody, and a lipopolysaccharide capable of specifying the quality and quantity of the LPS.
  • the purpose is to provide a quantitative method.
  • the monoclonal antibody of the present invention is present in microorganisms that animals are orally ingested orally. It is characterized by recognizing lipopolysaccharide lipid A, core polysaccharide or O antigen polysaccharide.
  • the lipopolysaccharide lipid A, core polysaccharide or O antigen polysaccharide is present in microorganisms symbiotic to edible plants.
  • the lipopolysaccharide lipid A, core polysaccharide or O antigen polysaccharide is characterized in that it is present in microorganisms used for food production or edible.
  • the microorganism is characterized in that it is Pantoea, acetic acid, darconic acid, Xanthomonas, or Zymomonas.
  • the Pantoea bacterium is Pantoea 'Agglomerans or Pantoea' disperser.
  • the acetic acid bacterium is acetopacter, dalconobacter, or fraturia.
  • the high-pridoma of the present invention is characterized in that the receipt number is NITE ABP-325, NITE ABP-326, NITE ABP-327, NITE ABP-328, NITE ABP-329 or NITE ABP-330. To do.
  • the monoclonal antibody of the present invention is characterized by being produced by the above-mentioned hyperidoma.
  • the method for quantifying lipopolysaccharide of the present invention is characterized by using the monoclonal antibody.
  • LPSp present in Bacterial Pantoea agglomerans
  • the second purpose of this example is to specifically react to LPSp.
  • LPSp can be specifically quantified even in the presence of Escherichia coli LPS, which is used as a representative of other LPS not only with high-purity LPSp. It was clarified that LPSp can be quantified even in the extract from rice bran where LPSp is to be added. [0019] The monoclonal antibody obtained this time is expected to be useful for specific detection of LPSp in various experimental systems.
  • ELISA quantification method neutralization of LPSp biological activity in vivo and in vitro, localization of LPSp on cell surface, intracellular, tissue, etc., search for factors that specifically bind to LP Sp, Pantoea agglomerans Detection is considered.
  • Monoclonal antibodies against edible gram-negative bacteria such as acetic acid bacteria-specific LPS and Xanthomonas-specific LPS or gram-negative bacteria-specific LPS symbiotic to edible plants were established using the same method.
  • Pantoea agglomerans cells were suspended in physiological saline to a concentration of 1 ⁇ 10 9 Zml and heated at 80 ° C. for 30 min.
  • P3U1 cells 8-azaguanine resistant mouse (BALBZc-derived) myeloma cell line P3U1 cells were used. P3U1 cells were previously subcultured every 2 days in RPMI1640 medium containing 10% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • P3U1 cells on the first day of subculture were transferred to a 50 ml centrifuge tube and centrifuged at 1000 rpm at room temperature for 7 minutes. Remove the supernatant and resuspend the precipitated P3U1 cells in 10 ml of RPMI1640 medium. It was. The P3U1 cells prepared by the above operation were used for the next cell fusion.
  • Example 1, 2 The spleen was aseptically removed and placed in a plastic petri dish having a diameter of 6 cm. 5 ml of RPMI 1640 medium was poured into this plastic petri dish, and the extracted spleen was washed. Separately, two 6 cm diameter plastic dishes containing 5 ml of RPMI1640 medium were prepared and washed twice more in this. The above washing operation was further repeated in a clean bench. The spleen was crushed in the last transferred flask and the splenocytes were suspended in RPMI 1640 medium.
  • This cell suspension was transferred to a 15 ml centrifuge tube, centrifuged at 1000 rpm at room temperature for 7 minutes, and the supernatant was removed.
  • the precipitated cells were suspended in 10 ml of RPMI 1640 medium and allowed to stand at room temperature for 3 minutes.
  • the cell suspension was transferred to a 50 ml centrifuge tube so that the tissue piece sinking to the bottom of the centrifuge tube was not sucked and removed.
  • the sensitized cell group prepared by the above operation was used for the next cell fusion.
  • the cell suspension was mixed gently to make the inner solution homogeneous, and centrifuged at 1000 rpm for 5 minutes at room temperature. The supernatant was removed, and the cells were suspended in a medium containing hypoxanthine, aminopterin, and thymidine (2 ⁇ 10 6 sensitized cells / ml). This cell suspension was spread on a 96-well flat-bottom plate so as to be 0.2 mlZ. 7 days while standing at 37 ° C, 5% CO
  • step 3 The following operations were performed on the cells obtained in step 3: the hybridoma cells.
  • PBS phosphate buffered saline
  • PBS-T a solution containing 05% polyoxyethylene (20) sonorebitan monolaurate (manufactured by Wako Pure Chemical Industries, Ltd., equivalent to Tween20)
  • BSA bovine serum albumin
  • the monoclonal antibody-producing hybridoma obtained above was cloned using a known and commonly used limiting dilution method.
  • the obtained clones were selected using the same ELISA method as described in 4. As a result, 8 types of monoclonal antibody-producing cells were obtained. It was clarified that 6 out of 8 obtained were monoclonal antibodies that react specifically with LPSp. Of these six monoclonal antibodies, four (32-G2, 4-E11 (reception number NITE ABP-329), 20-A8, 49-F2) were included. The remaining two (86-Fl, 34-G2 (reception number NITE ABP-330)) were IgM. Of the 8 types, the remaining 2 types (29-F7, 103 C3) showed the same level of response to all LPS containing synthetic lipid A. Presumed that both antibodies are IgM I
  • the monoclonal antibody 34-G2 obtained in 3: of Example 1 was placed in a 96-well immunoplate in 0.05 ml Zwell and allowed to stand at 4 ° C. After washing 3 times with PBS-T, add 3% BSA and leave at room temperature for 1 hour to prevent nonspecific adsorption of antibodies and other proteins. Then, after washing 3 times with PBS-T, 0.05 ml of PBS solution containing high-purity LP Sp serially diluted in wells was added and allowed to stand at room temperature for 1 hour.
  • Example 2 the well was washed 5 times with PB S-T, and the monoclonal antibody 4-E11 obtained in 3: of Example 1 was placed in 0.05 ml Zwell and allowed to stand at room temperature for 1 hour. Subsequently, the well was washed 5 times with PBS-T, and alkaline phosphatase-conjugated anti-mouse IgG immunoglobulin antibody (manufactured by Sigma) diluted with 1% BSA was placed in 0.05 ml Zwell and allowed to stand at room temperature for 1 hour.
  • the LPSp content of the sample can be measured based on the absorbance data of the unknown sample solution.
  • LP Sp contained in feed feed premix FFP-EA2 supplemented with fermented wheat extract containing LPSp
  • feed premix FFP-EA2 feed premix FFP-EA2 supplemented with fermented wheat extract containing LPSp
  • distilled water was added to 2 g of feed premix FFP-EA2 (containing LPSp), and 30 seconds of stirring (vortex mixer) was performed 10 times, followed by sonication for 5 minutes.
  • vortex mixer stirring
  • (Ultrasonic cleaner AIWA AU-16C) was performed to prepare 20 ml suspension.
  • the lml was dispensed into a microphone tube and centrifuged (room temperature, 5000 rpm, 5 minutes) with a microcentrifuge to obtain a supernatant (sample solution).
  • the obtained sample solution was serially diluted with a 1% BSA-containing PBS solution up to 320-fold and measured by ELISA as in Example 2.
  • an LPSp-added sample solution (added amount LPSp concentration: 4. O / z gZml) prepared by mixing the above sample solution with the standard LPSp solution was prepared and used for measurement. Furthermore, in order to confirm that LPSp was extracted from feed premix FFP-EA2, add 10 ⁇ g LPSp to lg feed premix FFP-EA2, and extract in the same way. The sample solution for extraction confirmation was prepared. When 100% is extracted, the amount of LPSp in the base sample (sample solution) increases by 1 ⁇ g / ml.
  • the LPSp concentration of the sample solution with adjusted feed power was calculated to be 1.1 / z gZml, and the amount of LPSp contained in the premix FFP-EA2 for feed was 11 ⁇ gZg.
  • the LPSp loading capacity of the LPSp-added caro sample solution was 4 ⁇ gZml, and the LPSp content originally contained was 1.1 ⁇ g / ml, so the theoretical LPSp content was 5. 1 ⁇ g / ml.
  • the actual measured value was 5.1 ⁇ gZml, which was consistent with the theoretical value.
  • the LPSp concentration of the sample solution for confirmation of extraction was 2.1 gZml.
  • LPS is converted from gel to polyvinylidene fluoride (PVDF) membrane (manufactured by BIO-RAD) using a semi-wet transfer device (Semi-drive lottery device: SB-160, Taitec, Japan) at 80 mA. The current was transferred for 90 minutes. After transfer, the PVDF membrane was blocked (blocked) for nonspecific reaction in a 3% bovine serum albumin (BSA) -containing PBS solution at room temperature for 30 minutes. Thereafter, washing was performed 5 times with 0.05% Tween 20-containing Tris buffered saline (TBS: 20 mM Tris HC1 pH 7.5, 150 mM NaCl).
  • BSA bovine serum albumin
  • Acetobacter aceti cells were suspended in physiological saline to a concentration of 1 ⁇ 10 9 Zml and heated at 80 ° C. for 30 minutes to be heat-killed bacteria.
  • the monoclonal antibody-producing hyperpridoma obtained above was cloned in the same manner as in Example 1.
  • the obtained clones were selected using the same ELISA method as described in 4. As a result, it was clarified that 10 types of monoclonal antibody-producing cells were monoclonal antibodies that specifically reacted with Acetobacter aceti LPS. Of these 10 monoclonal antibodies, one (5A-4 / A-8.G-12) was IgG.
  • the remaining nine types (1A-5 / E-1 O'G-1, 1G-10 / B-11 ⁇ ⁇ -1, 1, 4 ⁇ -9 / F-11 ⁇ ⁇ -5 (reception number ⁇ ⁇ — 325), 5 ⁇ -5 / ⁇ -10 ⁇ ⁇ 10, 5A-6 / C-3 'D-11, 5E-7 / E-11' C-12, 6A-7 / H-3 ⁇ ⁇ -12, 6C-9 / F-7 ⁇ ⁇ -11 (reception number NITE ABP-326), 6G-6 / H-3'C-12) were IgM.
  • Acetopacter aceti LPS was added to defatted rice bran and conditioned rice bran to prepare a hot water extracted liquid, and the amount of Acetopacter aceti LPS was measured.
  • Acetopacter vaseti LPS was not detected in the defatted rice bran heated extract and the adjusted rice bran heated extract.
  • almost 100% of the added sample was recovered. Based on the above, it was possible to specifically quantify Acetobacter vulgaris LPS even when other types of LPS and Acetobacter vulgaris LPS coexist (Table 5).
  • Example 1 Specifically, it was carried out in the same manner as in Example 1 using feed premix FFP-EA2 (containing Acetobacter 1. Aceti LPS).
  • Table 6 shows the measurement results of the Acetopacter aceti LPS by ELISA.
  • the Acetopacter aceti LPS concentration of the adjusted sample solution was calculated to be 1.0 gZml, and the amount of acetopacter aceti LPS contained in the premix FFP-EA2 for feed was 10 / z gZg.
  • the amount of Acetobacter's LPS added sample solution is 4 g / ml, which is 1.0 ⁇ g / ml.
  • the theoretical value of the Acetobacter LPS LPS is 5.0 ⁇ gZml.
  • the actual measured value was 5.0 gZml, which was consistent with the theoretical value.
  • the concentration of Acetopacter aceti LPS in the sample solution for confirmation of extraction was 2.0 g / ml. Since the concentration is base sample ⁇ . ⁇ / z gZml, the amount extracted is 1.0 / z gZml. Therefore, it was shown that the extraction efficiency (actually extracted extracted acetopactor LPS amount Z added acetopactor acetyl LPS amount X 100) was 100%.
  • Xanthomonas cells were suspended in physiological saline to 1 ⁇ 10 9 Zml and heated at 80 ° C. for 30 minutes to be heat-killed bacteria.
  • Example 2 The same procedure as in Example 1 was performed using cells of Xanthomonas.
  • Hybridomas producing antibodies against Xanthomonas LPS were selected in the same manner as in Example 1 for the cells obtained in Step 3 above.
  • the monoclonal antibody-producing hyperpridoma obtained above was cloned in the same manner as in Example 1.
  • the obtained clones were selected using the same ELISA method as described in 4. As a result, it was clarified that 22 types of monoclonal antibody-producing cells were monoclonal antibodies that specifically reacted with Xanthomonas LPS. Of these 22 monoclonal antibodies, 6 (1-1A1H9F9, 1-2F5E11F5, 2-2D4G5C1 (reception number NITE ABP-328), 2-4B2G1E6, 2-10D1D1E4, 2-10A10G2B2) were IgG.
  • the remaining 16 types (1-2G5A6G1, 1-3B6C10H1, 1- 3D11G6C2, 1- 5F9F11F1, 1-7A1E12F5 (Receipt number NITE ABP-327), 1-7C6C10G6, 1-7C9C9A8, 1-9F5A9C1, 1-9F11E 2-1H8F4B1, 2-3A11F9E11, 2-7G2E1C3, 2- 7G10B8A6, 2-8F7F4G4, 2-10A3G6 E5, 2-10E12C11C6) were IgM.
  • Example 7 the same procedure as in Example 1 was performed using monoclonal antibodies 1-7A1E12F5 and 2-4B2G1E6 obtained in 3: of Example 2 on a 96-well immunoplate.
  • Xanthomonas LPS had a concentration of lOngZml or more (Table 7). This data also shows the relationship between Xanthomonas LPS content and absorbance.
  • the Xanthomonas LPS content of the sample can be measured based on the absorbance data of the unknown sample solution.
  • Xanthomonas LPS contained in feed premix FFP-EA2 for feed supplemented with fermented wheat extract containing Xanthomonas LPS
  • the quantifiable power was examined.
  • Example 1 Specifically, the same procedure as in Example 1 was performed using feed premix FFP-EA2 (containing Xanthomonas LPS).
  • Table 9 shows the measurement results of Xanthomonas LPS by ELISA. The formula used to calculate Xanthomonas LPS is
  • the Xanthomonas LPS concentration of the sample solution with adjusted feed power was calculated to be 1.0 ⁇ gZml, and the amount of Xanthomonas LPS contained in the feed premix FFP-EA2 was 10 ⁇ g / g.
  • the Xanthomonas LPS addition sample of the sample solution containing Xanthomonas LPS was 4 g / ml, and the Xanthomonas LPS contained in the sample was 1.0 ⁇ g / ml. Is 5.0 g / ml. The actual measured value was 5.0 g / ml, which was consistent with the theoretical value.
  • the Xanthomonas LPS concentration of the sample solution for extraction confirmation was 2.0 / z gZml. Since the basic sample force is .O / z gZml, the amount extracted is 1.0 / z gZml. Therefore, it was shown that the extraction efficiency (actually extracted Xanthomonas LPS amount Z-added Caro Xanthomonas LPS amount X 100) was 100%.
  • Xanthomonas LPS could be detected by Western blotting using the monoclonal antibody of the present invention (antibody No. 1-7 Al E12 F5) was examined in the same manner as 2 in Example 1 (3). As a result, a blue band was observed in the lane in which Xanthomonas LPS was migrated, and no blue band was observed in E. coli LPS and Pantoea 'agglomerans LPS used as controls. From the above results, it was shown that the monoclonal antibody (antibody number 1-7 Al E12 F5) specifically binds to Xanthomonas LPS and can specifically detect Xanthomonas LPS by Western blotting. All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

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Abstract

Pour déterminer la qualité ou la quantité d'un LPS contenu dans ou mélangé à un produit, on s'intéresse à un micro-organisme présent en symbiose avec la substance alimentaire, un micro-organisme utilisé pour produire un aliment ou un micro-organisme utilisé comme aliment, en particulier une bactérie à Gram négatif. L'invention concerne un anticorps monoclonal capable de reconnaître le lipide A dans un lipopolysaccharide, un polysaccharide de noyau ou un polysaccharide d'antigène O contenu dans le micro-organisme et donc capable de reconnaître un LPS spécifique.
PCT/JP2007/054094 2006-03-05 2007-03-02 Anticorps monoclonal, hybridome, et procédé de quantification de lipopolysaccharide Ceased WO2007102442A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009031642A1 (fr) 2007-09-05 2009-03-12 Pola Pharma Inc. Composition pharmaceutique
GB2481267A (en) * 2010-06-15 2011-12-21 Univ Leicester Methods for assessing ability of ingestible substances to cause inflammation
JP2018038296A (ja) * 2016-09-06 2018-03-15 有限会社バイオメディカルリサーチグループ ハイブリドーマ、モノクローナル抗体及びリポ多糖の定量方法

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009031642A1 (fr) 2007-09-05 2009-03-12 Pola Pharma Inc. Composition pharmaceutique
GB2481267A (en) * 2010-06-15 2011-12-21 Univ Leicester Methods for assessing ability of ingestible substances to cause inflammation
GB2481267B (en) * 2010-06-15 2016-04-20 Univ Leicester Assay for determining the risk of a toll-like receptor containing foodstuff for causing systemic inflammation in vivo
JP2018038296A (ja) * 2016-09-06 2018-03-15 有限会社バイオメディカルリサーチグループ ハイブリドーマ、モノクローナル抗体及びリポ多糖の定量方法

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