INCB 024360_1204669-58-8_DataSheet_MedChemExpress

第一三共在日本提交CS-8958的新药申请
无
【期刊名称】《国外药讯》
【年(卷),期】2010(000)003
【摘要】日本第一三共公司表示已将一种有专利权的抗流感新药CS-8958的申请提交到日本厚生省。

CS-8958是一种长效神经氨酸酶抑制剂laniflamivir的前体药物，这种药物是由第一三共公司和其澳大利亚合作伙伴Biota公司合作完全为日本市场开发的，该药物会以吸入剂的方式直接作用于呼吸道。

【总页数】1页(P21)
【作者】无
【作者单位】无
【正文语种】中文
【中图分类】R951
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NATURE NEUROSCIENCENAT REV CANCER NATURE REVIEWS CANCERNAT REV DRUG DISCOV NATURE REVIEWS DRUG DISCOVERYNAT REV IMMUNOL NATURE REVIEWS IMMUNOLOGYNAT REV NEUROSCI NATURE REVIEWS NEUROSCIENCENEUROLOGY NEUROLOGYNEURON NEURONNEUROPSYCHOPHARMACOL NEUROPSYCHOPHARMACOLOGYNEUROSCI BIOBEHAV R NEUROSCIENCE AND BIOBEHAVIORAL REVIEWSNEW ENGL J MED NEW ENGLAND JOURNAL OF MEDICINEOBES REV Obesity ReviewsONCOGENE ONCOGENEPHARMACOL REV PHARMACOLOGICAL REVIEWSPHARMACOL THERAPEUT PHARMACOLOGY & THERAPEUTICSPHYSIOL REV PHYSIOLOGICAL REVIEWSPHYSIOLOGY PHYSIOLOGYPLOS MED PLOS MEDICINEPROG NEUROBIOL PROGRESS IN NEUROBIOLOGYPROG RETIN EYE RES PROGRESS IN RETINAL AND EYE RESEARCHPSYCHOL BULL PSYCHOLOGICAL BULLETINPSYCHOL REV PSYCHOLOGICAL REVIEWREV MED VIROL REVIEWS IN MEDICAL VIROLOGYSCHIZOPHRENIA BULL SCHIZOPHRENIA BULLETINSEMIN CANCER BIOL SEMINARS IN CANCER BIOLOGYSEMIN IMMUNOL SEMINARS IN IMMUNOLOGYSTEM CELLS STEM CELLSSTROKE STROKETHORAX THORAXTRENDS COGN SCI TRENDS IN COGNITIVE SCIENCESTRENDS ENDOCRIN MET TRENDS IN ENDOCRINOLOGY AND METABOLISMTRENDS IMMUNOL TRENDS IN IMMUNOLOGYTRENDS MOL MED TRENDS IN MOLECULAR MEDICINETRENDS NEUROSCI TRENDS IN NEUROSCIENCESTRENDS PHARMACOL SCI TRENDS IN PHARMACOLOGICAL SCIENCESWHO TECH REP SER WHO TECHNICAL REPORT SERIESAM J CARDIOL AMERICAN JOURNAL OF CARDIOLOGYAM J EPIDEMIOL AMERICAN JOURNAL OF EPIDEMIOLOGYAM J PATHOL AMERICAN JOURNAL OF PATHOLOGYAM J PHYSIOL-HEART C AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULA ANTIMICROB AGENTS CH ANTIMICROBIAL AGENTS AND CHEMOTHERAPYBRIT J CANCER BRITISH JOURNAL OF CANCERCANCER-AM CANCER SOC CANCERCHEST CHESTCNS NEUROL DISORD-DR CNS & Neurological Disorders-Drug Targets ENDOCRINOLOGY ENDOCRINOLOGYEUR J NEUROSCI EUROPEAN JOURNAL OF NEUROSCIENCEFREE RADICAL BIO MED FREE RADICAL BIOLOGY AND MEDICINEINFECT IMMUN INFECTION AND IMMUNITYINT J CANCER INTERNATIONAL JOURNAL OF CANCERINT J RADIAT ONCOL INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOL INVEST OPHTH VIS SCI INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCEJ APPL PHYSIOL JOURNAL OF APPLIED PHYSIOLOGYJ CLIN ENDOCR METAB JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM J CLIN MICROBIOL JOURNAL OF CLINICAL MICROBIOLOGYJ COMP NEUROL JOURNAL OF COMPARATIVE NEUROLOGYJ IMMUNOL JOURNAL OF IMMUNOLOGYJ INFECT DIS JOURNAL OF INFECTIOUS DISEASESJ MED CHEM JOURNAL OF MEDICINAL CHEMISTRYJ NEUROCHEM JOURNAL OF NEUROCHEMISTRYJ NEUROPHYSIOL JOURNAL OF NEUROPHYSIOLOGYJ NUTR JOURNAL OF NUTRITIONJ PHARMACOL EXP THER JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPE J PHYSIOL-LONDON JOURNAL OF PHYSIOLOGY-LONDONJ UROLOGY JOURNAL OF UROLOGYJ VIROL JOURNAL OF VIROLOGYKIDNEY INT KIDNEY INTERNATIONALNEUROIMAGE NEUROIMAGENEUROSCIENCE NEUROSCIENCEPEDIATRICS PEDIATRICSRADIOLOGY RADIOLOGYTRANSPLANTATION TRANSPLANTATIONVIROLOGY VIROLOGYNATURE NATUREP NATL ACAD SCI USA PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES SCIENCE SCIENCEANN NY ACAD SCI ANNALS OF THE NEW YORK ACADEMY OF SCIENCESISSN大类名称复分大类分区是否top期刊2009年影响因子1680-7316地学1Y 4.881 0003-0007地学1Y 6.123 0930-7575地学1Y 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2.874 0928-4249农林科学1Y 3.579 0044-8486农林科学2Y 1.925 0706-652X农林科学2Y 1.951 0378-1127农林科学2Y 1.950 0003-1488农林科学2Y 1.714 0032-079X农林科学2Y 2.517 1526-5161社会科学1Y 4.000 0012-9682社会科学1Y 4.000 0304-4076社会科学2Y 1.902 0002-9297生物1Y12.303 0066-4154生物1Y29.875 1056-8700生物1Y18.955 1936-122X生物1Y19.304 1081-0706生物1Y19.571 1543-592X生物1Y8.190 0066-4170生物1Y11.271 0066-4197生物1Y13.235 1527-8204生物1Y11.568 0066-4227生物1Y12.804 0066-4286生物1Y11.212 1543-5008生物1Y23.460 0092-8674生物1Y31.152 1931-3128生物1Y13.021 1550-4131生物1Y17.350 1934-5909生物1Y23.563 1040-9238生物1Y10.216 0960-9822生物1Y10.992 0955-0674生物1Y14.153 0959-437X生物1Y8.987 1369-5266生物1Y10.333 0959-440X生物1Y9.344 1534-5807生物1Y13.363 0890-9369生物1Y12.075 1088-9051生物1Y11.342 0021-9525生物1Y9.575 1092-2172生物1Y12.585 1097-2765生物1Y14.608 1744-4292生物1Y12.125 1465-7392生物1Y19.527 1552-4450生物1Y16.058 1061-4036生物1Y34.284 1548-7091生物1Y16.874 1471-0056生物1Y27.822 1740-1526生物1Y17.644 1471-0072生物1Y42.198 1545-9985生物1Y12.2731040-4651生物1Y9.293 1544-9173生物1Y12.916 1553-7390生物1Y9.532 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Product Data SheetTrigonox BDi-tert-butyl peroxideTrigonox® B is a pure peroxide in liquid form.CAS number110-05-4EINECS/ELINCS No. 203-733-6TSCA statuslisted on inventory Molecular weight 146.2Active oxygen contentperoxide10.94%SpecificationsAppearance Clear liquidAssay≥ 99.0 %ApplicationsTrigonox® B (Di-tert-butyl peroxide) can be used for the market segments: polymer production, polymer crosslinking and acrylics production with their different applications/functions. For more information please check our website and/or contact us.Half-life dataThe reactivity of an organic peroxide is usually given by its half-life (t½) at various temperatures. For Trigonox® B in chlorobenzene half-life at other temperatures can be calculated by using the equations and constants mentioned below:0.1 hr at 164°C (327°F)1 hr at 141°C (286°F)10 hr at 121°C (250°F)Formula 1kd = A·e-Ea/RTFormula 2t½ = (ln2)/kdEa153.46 kJ/moleA 4.20E+15 s-1R8.3142 J/mole·KT(273.15+°C) KThermal stabilityOrganic peroxides are thermally unstable substances which may undergo self-accelerating decomposition. The lowest temperature at which self-accelerating decomposition may occur with a substance in the packaging as used for transport is the Self-Accelerating Decomposition Temperature (SADT). The SADT is determined on the basis of the Heat Accumulation Storage Test.SADT80°C (176°F)Method The Heat Accumulation Storage Test is a recognized test method for thedetermination of the SADT of organic peroxides (see Recommendations on theTransport of Dangerous Goods, Manual of Tests and Criteria - United Nations, NewYork and Geneva).StorageDue to the relatively unstable nature of organic peroxides, a loss of quality will occur over a period of time. To minimize the loss of quality, Nouryon recommends a maximum storage temperature (Ts max. ) for each organic peroxide product.Ts Max.40°C (104°F) andTs Min.-30°C (-22°F) to prevent crystallizationNote When stored according to these recommended storage conditions, Trigonox® Bwill remain within the Nouryon specifications for a period of at least 6 months afterdelivery.Packaging and transportIn North America Trigonox® B is packed in non-returnable, five gallon polyethylene containers of 30 lb net weight and steel drums of 100 or 340 lb net weight. In other regions the standard packaging is a 30-liter HDPE can (Nourytainer®) for 20 kg peroxide. Delivery in a 200 l steel drum for 150 kg peroxide is also possible in a number of countries. Both packaging and transport meet the international regulations. For the availability of other packed quantities consult your Nouryon representative. Trigonox® B is classified as Organic peroxide type E; liquid, Division 5. 2; UN 3107.Safety and handlingKeep containers tightly closed. Store and handle Trigonox® B in a dry well-ventilated place away from sources of heat or ignition and direct sunlight. Never weigh out in the storage room. Avoid contact with reducing agents (e. g. amines), acids, alkalis and heavy metal compounds (e. g. accelerators, driers and metal soaps). Please refer to the Safety Data Sheet (SDS) for detailed information on the safe storage, use and handling of Trigonox® B. This information should be thoroughly reviewed prior to acceptance of this product. The SDS is available at /sds-search.Major decomposition productsAcetone, Methane, tert-ButanolAll information concerning this product and/or suggestions for handling and use contained herein are offered in good faith and are believed to be reliable.Nouryon, however, makes no warranty as to accuracy and/or sufficiency of such information and/or suggestions, as to the product's merchantability or fitness for any particular purpose, or that any suggested use will not infringe any patent. Nouryon does not accept any liability whatsoever arising out of the use of or reliance on this information, or out of the use or the performance of the product. Nothing contained herein shall be construed as granting or extending any license under any patent. Customer must determine for himself, by preliminary tests or otherwise, the suitability of this product for his purposes.The information contained herein supersedes all previously issued information on the subject matter covered. The customer may forward, distribute, and/or photocopy this document only if unaltered and complete, including all of its headers and footers, and should refrain from any unauthorized use. Don’t copythis document to a website.Trigonox® and Nourytainer are registered trademarks of Nouryon Functional Chemicals B.V. or affiliates in one or more territories.Contact UsPolymer Specialties Americas************************Polymer Specialties Europe, Middle East, India and Africa*************************Polymer Specialties Asia Pacific************************2022-6-30© 2022Polymer crosslinking Trigonox B。

BD Transduction Laboratories™Technical Data SheetBioimaging Certified ReagentPurified Mouse Anti-PaxillinProduct InformationMaterial Number:610569Size:150 µg Concentration:250 µg/ml Clone: 177/PaxillinImmunogen:Chicken Paxillin aa. 1-557Isotype:Mouse IgG1QC Testing: HumanTested in Development: Bovine, Chicken, Dog, Mouse, Rat Reactivity:68 kDaTarget Molecular Weight:Aqueous buffered solution containing BSA, glycerol, and ≤0.09% sodium azide.Storage Buffer:DescriptionPaxillin, a focal adhesion protein, is a substrate for several tyrosine kinases such as src, FAK, and p120BRC/ABL. The tyrosinephosphorylation of paxillin is affected by conditions that change cell-cell adhesion. This is consisent with the possibility that paxillin isinvolved in the regulation of cell morphology. Additionally, because of its SH3-binding domain, paxillin associates tightly with FAK and Crk in an extracellular matrix-independent manner. Although paxillin was initially detected in fibroblasts, its phosphorylation may be important during neurite extension during differentiation.This antibody is routinely tested by western blot and immunofluorescent imaging. Other applications were tested at BD Biosciences Pharmingen during antibody development only or reported in the literature.Western blot analysis of Paxillin on a humanendothelial lysate. Lane 1: 1:1000, lane 2: 1:2000, lane 3: 1:4000 dilution of the anti-Paxillin antibody.Immunofluorescent staining of HeLa cells. Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~10,000 cells per well. After overnight incubation, cells were stained using the methanol fix/perm protocol (below) and the anti-Paxillin antibody. The second step reagent was Alexa Fluor® 555 conjugated anti-mouse Ig(Invitrogen). The image was taken on a Pathway 850 imager using a 20x objective. This antibody also stained A549 and U2OS cells and worked with both the Triton X100 and methanol fix/perm protocols (see Recommended Assay Procedure).Preparation and StorageThe monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.Store undiluted at -20° C.Application NotesApplicationBioimaging Routinely TestedWestern blot Routinely TestedImmunofluorescence Tested During DevelopmentImmunoprecipitation Not RecommendedImmunohistochemistry Not RecommendedRecommended Assay Procedure:Methanol Procedure for a 96 well plate:Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and add 100 µl/well 90% methanol. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well blocking buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1 hour at RT. Wash three times with PBS. Flick out PBS and add second step reagent. Incubate for 1 hour at RT. Wash three times with PBS. Image sample.Triton-X 100 Procedure for a 96 well plate:Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and add 100 µl/well 0.1% Triton-X 100. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well blocking buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1 hour at RT. Flick out and wash three times with PBS. Flick out and add second step reagent. Incubate for 1 hour at RT. Flick out and wash three times with PBS. Image sample.Suggested Companion ProductsNameCatalog Number Size Clone 353219BD Falcon™ 96-well Imaging Plate 1 plate(none) 611450Human Endothelial Cell Lysate500 µg(none) 554656Stain Buffer (FBS)500 ml(none) 554655Fixation Buffer100 ml(none) 558050Perm Buffer III125 ml(none) Product Notices1.Since applications vary, each investigator should titrate the reagent to obtain optimal results.2.Please refer to /pharmingen/protocols for technical protocols.3.Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water beforediscarding to avoid accumulation of potentially explosive deposits in plumbing.4.Source of all serum proteins is from USDA inspected abattoirs located in the United States.ReferencesKu H, Meier KE. Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascade in EL4 thymoma cells. J Biol Chem. 2000;275(15):11333-11340.(Biology: Immunoprecipitation, Western blot)Leventhal PS, Feldman EL. Tyrosine phosphorylation and enhanced expression of paxillin during neuronal differentiation in vitro. J Biol Chem. 1996;271(11):5957-5960.(Biology)Salgia R, Li JL, Lo SH, et al. Molecular cloning of human paxillin, a focal adhesion protein phosphorylated by P210BCR/ABL. J Biol Chem. 1995;270(10):5039-5047.(Biology)Takayama Y, Tanaka S, Nagai K, Okada M. Adenovirus-mediated overexpression of C-terminal Src kinase (Csk) in type I astrocytes interferes with cell spreading and attachment to fibronectin. Correlation with tyrosine phosphorylations of paxillin and FAK. J Biol Chem. 1999; 274(4):2291-2297.(Biology: Immunofluorescence, Immunoprecipitation, Western blot)Turner CE, Glenney JR Jr, Burridge K. Paxillin: a new vinculin-binding protein present in focal adhesions. J Cell Biol. 1990; 111(3):1059-1068.(Biology)。

DOI：10.16658/ki.1672-4062.2023.04.025血清转铁蛋白、尿液转铁蛋白、尿液视黄醇结合蛋白、胱抑素C检测对糖尿病肾病的临床应用价值分析袁金，陈琼，张雯福州市第八医院检验科，福建福州350000[摘要]目的分析血清转铁蛋白、尿液转铁蛋白、尿液视黄醇结合蛋白联合胱抑素C检测对糖尿病肾病诊断的应用价值。

方法选取2019年1月—2022年2月福州市第八医院治疗的60例糖尿病肾病患者为研究对象，依据患者的尿白蛋白排泄量/肌酐比值差异将其分为A组（22例，蛋白尿正常）、B组（21例，微量蛋白尿）、C 组（17例，大量蛋白尿），另选同期的20名体检健康者设为对照组。

所有患者均接受血清转铁蛋白、尿液转铁蛋白、尿液视黄醇结合蛋白联合胱抑素C检测，分析4组检测结果。

结果4组受检者血清转铁蛋白、尿液转铁蛋白、尿液视黄醇结合蛋白、胱抑素C检测结果比较，差异有统计学意义（F=538.653、10.125、197.153、416.223，P<0.05）。

结论血清转铁蛋白、尿液转铁蛋白、尿液视黄醇结合蛋白、胱抑素C检测对于糖尿病肾病患者有一定的参考价值。

[关键词] 血清转铁蛋白；尿液转铁蛋白；尿液视黄醇结合蛋白；胱抑素C；糖尿病肾病[中图分类号] R59 [文献标识码] A [文章编号] 1672-4062（2023）02（b）-0025-04Analysis of Serum Transferrin, Urine Transferrin, Urine Retinol-binding Protein, Cystatin C Clinical Application Value of Diabetes NephropathyYUAN Jin, CHEN Qiong, ZHANG WenDepartment of Clinical Laboratory, Fuzhou Eighth Hospital, Fuzhou, Fujian Province, 350000 China[Abstract] Objective To analyze the diagnostic value of serum transferrin, urine transferrin, urine retinol-binding protein combined with cystatin C in diabetic nephropathy. Methods 60 patients with diabetes nephropathy who were treated in Fuzhou Eighth Hospital from January 2019 to February 2022 were selected as the study subjects. According to the difference of urinary albumin excretion/creatinine ratio, the patients were divided into group A (22 cases, nor⁃mal proteinuria), group B (21 cases, microalbuminuria), and group C (17 cases, massive proteinuria), another 20 healthy individuals who underwent physical examination during the same period were selected as the control group. All patients received serum transferrin, urine transferrin, urine retinol-binding protein combined with cystatin C detec⁃tion. The test results were analyzed between the four groups. Results In the detection results of serum transferrin, urine transferrin, urine retinol binding protein, and cystatin C among the four groups of subjects, the difference was statistically significant (F=538.653, 10.125, 197.153, 416.223, P<0.05). Conclusion The detection of serum transfer⁃rin, urine transferrin, urinary retinol binding protein and cystatin C has certain reference value for patients with diabe⁃tes nephropathy.[Key words] Serum transferrin; Urine transferrin; Urine retinol-binding protein; Cystatin C; Diabetic nephropathy糖尿病肾病若未能及时诊治，一旦发展至终末期治疗的难度会显著提高[1]。

核准日期：2017年3月10日修改日期：2017年5月26日2017年11月29日2018年7月24日2020年2月13日2021年2月26日2022年1月17日2022年2月22日维莫非尼片说明书请仔细阅读说明书并在医师指导下使用【药品名称】通用名：维莫非尼片商品名：佐博伏®,英文商品名Zelboraf®英文名：Vemurafenib film-coated tablets汉语拼音：Weimofeini Pian【成份】本品主要活性成分为维莫非尼，以维莫非尼和琥珀酸醋酸羟丙甲纤维素固体分散体存在。

化学名称：丙烷-1-磺酸{3-[5-(4-氯苯基)-1H- 吡咯并[2,3-b]吡啶-3-羰基]-2,4-二氟代苯基}- 酰胺。

化学结构式：分子式：C23H18ClF2N3O3S分子量：489.93【性状】两面凸起、粉白色至橙白色的薄膜衣片。

【适应症】佐博伏®适用于治疗经CFDA批准的检测方法确定的BRAF V600突变阳性的不可切除或转移性黑色素瘤。

【规格】240 mg【用法用量】患者必须经由CFDA批准的检测方法确定的证明肿瘤为BRAF V600突变阳性，才可使用佐博伏®治疗。

佐博伏®不能用于BRAF野生型黑色素瘤患者。

首剂药物应在上午服用，第二剂应在此后约12小时，即晚上服用。

每次服药均可随餐或空腹服用。

用一杯水送服药物，服药时整片吞下佐博伏®片剂。

不应咀嚼或碾碎佐博伏®片剂。

标准剂量佐博伏®的推荐剂量为960 mg（四片240 mg片剂），每日两次。

治疗持续时间建议佐博伏®治疗应持续至疾病进展或发生不可接受的毒性反应（参见表1和表2）。

漏服如果漏服一剂计划的药物，可在下一剂服药4小时以前补服漏服的药物，以维持每日两次的给药方案。

不应同时服用两剂药物。

呕吐如果佐博伏®服药后发生呕吐，患者不应追加剂量，而应按常规剂量继续治疗。

Product Name:Ampicillin CAS No.:69-53-4Cat No :HY-B0522Product Data SheetCat. No.:HY B0522MWt:349.40Formula:C16H19N3O4S Purity :>98%Solubility:Mechanisms:Biological Activity:Pathways:Anti-infection; Target:Antibacterial DMSOAmpicillin is an orally active broad-spectrum antibiotic.Target: Antibacterial Ampicillin is an antibiotic useful for the treatment of a number of bacterial infections. It is a beta-lactam antibiotic that is part of the aminopenicillin family and is roughly equivalent to its successor,amoxicillin in terms of spectrum and level of activity. Belonging to the penicillin group of beta-lactam antibiotics, ampicillin is able to penetrate Gram-positive and some Gram-negative bacteria. It differs from penicillin G, or benzylpenicillin, only by the presence of an amino group. That amino group helps the drug penetrate the outer membrane of Gram-negative bacteria. Ampicillin acts as an irreversible inhibitor of the enzyme transpeptidase which is needed by bacteria to make their cell References:[1]. /wiki/Ampicillin irreversible inhibitor of the enzyme transpeptidase, which is needed by bacteria to make their cell walls. It inhibits the third and final stage of bacterial cell wall synthesis in binary fission...Caution: Not fully tested. For research purposes onlyMedchemexpress LLC18 W i l k i n s o n W a y , P r i n c e t o n , N J 08540,U S AE m a i l : i n f o @m e d c h e m e x p r e s s .c o m W e b : w w w .m e d c h e m e x p r e s s .c o m。

碧云天生物技术/Beyotime Biotechnology订货热线：400-168-3301或800-8283301订货e-mail：******************技术咨询：*****************网址：碧云天网站微信公众号MONOMAC1 (人急性单核白血病细胞)产品编号产品名称包装C6604 MONOMAC1 (人急性单核白血病细胞) 1支/瓶产品简介：Organism Tissue Morphology Culture Properties Homo sapiens (Human) Peripheral blood Round Suspension本细胞株详细信息如下:General InformationCell Line Name MONOMAC1 (Human Acute Monocytic Leukemia Cells)Synonyms MONO-MAC-1; Mono Mac 1; Monomac-1; MonoMac1; Mono-Mac-1; MM1Organism Homo sapiens (Human)Tissue Peripheral bloodCell Type －Morphology RoundDisease －Strain －Biosafety Level* 1Age at Sampling 64 yearsGender MaleGenetics －Ethnicity －Applications －Category Cancer cell line* Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.CharacteristicsKaryotype －Virus Susceptibility －Derivation －Clinical Data －Antigen Expression －Receptor Expression －Oncogene －Genes Expressed －Gene expression databases ArrayExpress: E-MTAB-2770; E-MTAB-3610; GEO: GSM482510; GSM887337; GSM888413; GSM1670129Metastasis －Tumorigenic －Effects －Comments －Culture MethodDoubling Time 30~50 hrsMethods for Passages Split cells to new flask with fresh medium. No need to treat them enzymatically to detach them from the surface of the culture vessel.Medium 90% RPMI 1640+10% h.i. FBS+2mM L-glutamine+1x non-essential amino acids+1mM sodium pyruvateSpecial Remarks －Medium Renewal －Subcultivation Ratio －Growth Condition 95% air+ 5% CO2, 37ºCFreeze medium DMEM (high glucose)+20% FBS+10% DMSO，也可以订购碧云天的细胞冻存液(C0210)。

专利名称：Humanized anti-CEA T84.66 antibody and uses thereof发明人：Paul J. Yazaki,Mark A. Sherman,John E.Shively,Andrew A. Raubitschek,Anna M. Wu 申请号：US11077978申请日：20050311公开号：US07273608B2公开日：20070925专利内容由知识产权出版社提供摘要：Embodiments of the present invention utilize a more efficient CDR grafting technique to generate humanized versions of the T84.66 antibody. The technique used to generate these antibodies utilizes crystallographic structural data to select an immunoglobulin framework having maximum structural overlap with a non-human donor molecule. This technique was used to develop humanized T84.66 antibodies exhibiting in vitro binding affinity and specificity for carcinoembryonic antigen (CEA) nearly identical to that of T84.66 and the ability to specifically target tumors expressing CEA in vivo.申请人：Paul J. Yazaki,Mark A. Sherman,John E. Shively,Andrew A. Raubitschek,Anna M. Wu地址：Glendale CA US,Pasadena CA US,Arcadia CA US,San Marino CA US,Sherman Oaks CA US国籍：US,US,US,US,US代理机构：Perkins Coie LLP代理人：Michael J. Wise更多信息请下载全文后查看。

Guidance for Industry Bioanalytical Method ValidationU.S. Department of Health and Human ServicesFood and Drug AdministrationCenter for Drug Evaluation and Research (CDER)Center for Veterinary Medicine (CVM)May 2001BPGuidance for Industry Bioanalytical Method ValidationAdditional copies are available from:Drug Information Branch (HFD-210)Center for Drug Evaluation and Research (CDER)5600 Fishers Lane, Rockville, MD 20857 (Tel) 301-827-4573Internet at /cder/guidance/index.htmorCommunications Staff (HFV-12)Center for Veterinary Medicine (CVM)7500 Standish Place, Rockville, MD 20855 (Tel) 301–594-1755Internet at /cvmU.S. Department of Health and Human ServicesFood and Drug AdministrationCenter for Drug Evaluation and Research (CDER)Center for Veterinary Medicine (CVM)May 2001BPTable of ContentsI.INTRODUCTION (1)II.BACKGROUND (1)A.F ULL V ALIDATION (2)B.P ARTIAL V ALIDATION (2)C.C ROSS-V ALIDATION (3)III.REFERENCE STANDARD (4)IV.METHOD DEVELOPMENT: CHEMICAL ASSAY (4)A.S ELECTIVITY (4)B.A CCURACY, P RECISION, AND R ECOVERY (5)C.C ALIBRATION/S TANDARD C URVE (5)D.S TABILITY (6)E.P RINCIPLES OF B IOANALYTICAL M ETHOD V ALIDATION AND E STABLISHMENT (8)F.S PECIFIC R ECOMMENDATIONS FOR M ETHOD V ALIDATION (10)V.METHOD DEVELOPMENT: MICROBIOLOGICAL AND LIGAND-BINDING ASSAYS (11)A.S ELECTIVITY I SSUES (11)B.Q UANTIFICATION I SSUES (12)VI.APPLICATION OF VALIDATED METHOD TO ROUTINE DRUG ANALYSIS (13)A CCEPTANCE C RITERIA FOR THE R UN (15)VII.DOCUMENTATION (16)A.S UMMARY I NFORMATION (16)B.D OCUMENTATION FOR M ETHOD E STABLISHMENT (17)C.A PPLICATION TO R OUTINE D RUG A NALYSIS (17)D.O THER I NFORMATION (19)GLOSSARY (20)GUIDANCE FOR INDUSTRY1Bioanalytical Method ValidationI.INTRODUCTIONThis guidance provides assistance to sponsors of investigational new drug applications (INDs), new drug applications (NDAs), abbreviated new drug applications (ANDAs), and supplements in developing bioanalytical method validation information used in human clinical pharmacology, bioavailability (BA), and bioequivalence (BE) studies requiring pharmacokinetic (PK) evaluation. This guidance also applies to bioanalytical methods used for non-human pharmacology/toxicology studies and preclinical studies. For studies related to the veterinary drug approval process, this guidance applies only to blood and urine BA, BE, and PK studies.The information in this guidance generally applies to bioanalytical procedures such as gas chromatography (GC), high-pressure liquid chromatography (LC), combined GC and LC mass spectrometric (MS) procedures such as LC-MS, LC-MS-MS, GC-MS, and GC-MS-MS performed for the quantitative determination of drugs and/or metabolites in biological matricessuch as blood, serum, plasma, or urine. This guidance also applies to other bioanalytical methods, such as immunological and microbiological procedures, and to other biological matrices, such as tissue and skin samples.This guidance provides general recommendations for bioanalytical method validation. The recommendations can be adjusted or modified depending on the specific type of analytical method used. II.BACKGROUND1 This guidance has been prepared by the Biopharmaceutics Coordinating Committee in the Center for Drug Evaluation and Research (CDER) in cooperation with the Center for Veterinary Medicine (CVM) at the Food and Drug Administration.This guidance has been developed based on the deliberations of two workshops: (1) Analytical Methods Validation: Bioavailability, Bioequivalence, and Pharmacokinetic Studies (held on December 3B5, 19902 ) and (2) Bioanalytical Methods Validation C A Revisit With a Decade of Progress (held on January 12B14, 20003).Selective and sensitive analytical methods for the quantitative evaluation of drugs and their metabolites (analytes) are critical for the successful conduct of preclinical and/or biopharmaceutics and clinical pharmacology studies. Bioanalytical method validation includes all of the procedures that demonstrate that a particular method used for quantitative measurement of analytes in a given biological matrix, such as blood, plasma, serum, or urine, is reliable and reproducible for the intended use. The fundamental parameters for this validation include (1) accuracy, (2) precision, (3) selectivity, (4) sensitivity, (5) reproducibility, and (6) stability. Validation involves documenting, through the use of specific laboratory investigations, that the performance characteristics of the method are suitable and reliable for the intended analytical applications. The acceptability of analytical data corresponds directly to the criteria used to validate the method.Published methods of analysis are often modified to suit the requirements of the laboratory performing the assay. These modifications should be validated to ensure suitable performance of the analytical method. When changes are made to a previously validated method, the analyst should exercise judgment as to how much additional validation is needed. During the course of a typical drug development program, a defined bioanalytical method undergoes many modifications. The evolutionary changes to support specific studies and different levels of validation demonstrate the validity of an assay’s performance. Different types and levels of validation are defined and characterized as follows:A.Full Validation•Full validation is important when developing and implementing a bioanalytical method for the first time.•Full validation is important for a new drug entity.• A full validation of the revised assay is important if metabolites are added to an existing assay for quantification.B.Partial ValidationPartial validations are modifications of already validated bioanalytical methods. Partial validation can range from as little as one intra-assay accuracy and precision determination to a nearly full2 Workshop Report: Shah, V.P. et al., Pharmaceutical Research: 1992; 9:588-592.3 Workshop Report: Shah, V.P. et al., Pharmaceutical Research: 2000; 17:in press.validation. Typical bioanalytical method changes that fall into this category include, but are not limited to:•Bioanalytical method transfers between laboratories or analysts•Change in analytical methodology (e.g., change in detection systems)•Change in anticoagulant in harvesting biological fluid•Change in matrix within species (e.g., human plasma to human urine)•Change in sample processing procedures•Change in species within matrix (e.g., rat plasma to mouse plasma)•Change in relevant concentration range•Changes in instruments and/or software platforms•Limited sample volume (e.g., pediatric study)•Rare matrices•Selectivity demonstration of an analyte in the presence of concomitant medications•Selectivity demonstration of an analyte in the presence of specific metabolitesC.Cross-ValidationCross-validation is a comparison of validation parameters when two or more bioanalytical methods are used to generate data within the same study or across different studies. An example of cross-validation would be a situation where an original validated bioanalytical method serves as thereference and the revised bioanalytical method is the comparator. The comparisons should be done both ways.When sample analyses within a single study are conducted at more than one site or more than one laboratory, cross-validation with spiked matrix standards and subject samples should be conducted at each site or laboratory to establish interlaboratory reliability. Cross-validation should also be considered when data generated using different analytical techniques (e.g., LC-MS-MS vs.ELISA4) in different studies are included in a regulatory submission.All modifications should be assessed to determine the recommended degree of validation. The analytical laboratory conducting pharmacology/toxicology and other preclinical studies for regulatory submissions should adhere to FDA=s Good Laboratory Practices (GLPs)5 (21 CFR part 58) and to sound principles of quality assurance throughout the testing process. The bioanalytical method for human BA, BE, PK, and drug interaction studies must meet the criteria in 21 CFR 320.29. The analytical laboratory should have a written set of standard operating procedures (SOPs) to ensure a complete system of quality control and assurance. The SOPs should cover all aspects of analysis from the time the sample is collected and reaches the laboratory until the results of the analysis are reported. The SOPs also should include record keeping, security and chain of sample custody4 Enzyme linked immune sorbent assay5 For the Center for Veterinary Medicine, all bioequivalence studies are subject to Good Laboratory Practices.(accountability systems that ensure integrity of test articles), sample preparation, and analytical tools such as methods, reagents, equipment, instrumentation, and procedures for quality control and verification of results.The process by which a specific bioanalytical method is developed, validated, and used in routine sample analysis can be divided into (1) reference standard preparation, (2) bioanalytical method development and establishment of assay procedure, and (3) application of validated bioanalytical method to routine drug analysis and acceptance criteria for the analytical run and/or batch. These three processes are described in the following sections of this guidance.III.REFERENCE STANDARDAnalysis of drugs and their metabolites in a biological matrix is carried out using samples spiked with calibration (reference) standards and using quality control (QC) samples. The purity of the reference standard used to prepare spiked samples can affect study data. For this reason, an authenticated analytical reference standard of known identity and purity should be used to prepare solutions of known concentrations. If possible, the reference standard should be identical to the analyte. When this is not possible, an established chemical form (free base or acid, salt or ester) of known purity can be used. Three types of reference standards are usually used: (1) certified reference standards (e.g., USP compendial standards); (2) commercially supplied reference standards obtained from a reputable commercial source; and/or (3) other materials of documented purity custom-synthesized by an analytical laboratory or other noncommercial establishment. The source and lot number, expiration date, certificates of analyses when available, and/or internally or externally generated evidence of identity and purity should be furnished for each reference standard.IV.METHOD DEVELOPMENT: CHEMICAL ASSAYThe method development and establishment phase defines the chemical assay. The fundamental parameters for a bioanalytical method validation are accuracy, precision, selectivity, sensitivity, reproducibility, and stability. Measurements for each analyte in the biological matrix should be validated. In addition, the stability of the analyte in spiked samples should be determined. Typical method development and establishment for a bioanalytical method include determination of (1) selectivity, (2) accuracy, precision, recovery, (3) calibration curve, and (4) stability of analyte in spiked samples.A.SelectivitySelectivity is the ability of an analytical method to differentiate and quantify the analyte in thepresence of other components in the sample. For selectivity, analyses of blank samples of theappropriate biological matrix (plasma, urine, or other matrix) should be obtained from at leastsix sources. Each blank sample should be tested for interference, and selectivity should be ensured at the lower limit of quantification (LLOQ).Potential interfering substances in a biological matrix include endogenous matrix components, metabolites, decomposition products, and in the actual study, concomitant medication and other exogenous xenobiotics. If the method is intended to quantify more than one analyte, each analyte should be tested to ensure that there is no interference.B.Accuracy, Precision, and RecoveryThe accuracy of an analytical method describes the closeness of mean test results obtained by the method to the true value (concentration) of the analyte. Accuracy is determined by replicate analysis of samples containing known amounts of the analyte. Accuracy should be measured using a minimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentrations is recommended. The mean value should be within 15% of the actual value except at LLOQ, where it should not deviate by more than 20%. The deviation of the mean from the true value serves as the measure of accuracy.The precision of an analytical method describes the closeness of individual measures of an analyte when the procedure is applied repeatedly to multiple aliquots of a single homogeneous volume of biological matrix. Precision should be measured using a minimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentrations is recommended. The precision determined at each concentration level should not exceed 15% of the coefficient of variation (CV) except for the LLOQ, where it should not exceed 20% of the CV. Precision is further subdivided into within-run, intra-batch precision or repeatability, which assesses precision during a single analytical run, and between-run, inter-batch precision or repeatability, which measures precision with time, and may involve different analysts, equipment, reagents, and laboratories.The recovery of an analyte in an assay is the detector response obtained from an amount of the analyte added to and extracted from the biological matrix, compared to the detector response obtained for the true concentration of the pure authentic standard. Recovery pertains to the extraction efficiency of an analytical method within the limits of variability. Recovery of the analyte need not be 100%, but the extent of recovery of an analyte and of the internal standard should be consistent, precise, and reproducible. Recovery experiments should be performed by comparing the analytical results for extracted samples at three concentrations (low, medium, and high) with unextracted standards that represent 100% recovery.C.Calibration/Standard CurveA calibration (standard) curve is the relationship between instrument response and known concentrations of the analyte. A calibration curve should be generated for each analyte in thesample. A sufficient number of standards should be used to adequately define the relationship between concentration and response. A calibration curve should be prepared in the same biological matrix as the samples in the intended study by spiking the matrix with known concentrations of the analyte. The number of standards used in constructing a calibration curve will be a function of the anticipated range of analytical values and the nature of theanalyte/response relationship. Concentrations of standards should be chosen on the basis of the concentration range expected in a particular study. A calibration curve should consist of a blank sample (matrix sample processed without internal standard), a zero sample (matrix sample processed with internal standard), and six to eight non-zero samples covering the expected range, including LLOQ.1.Lower Limit of Quantification (LLOQ)The lowest standard on the calibration curve should be accepted as the limit ofquantification if the following conditions are met:C The analyte response at the LLOQ should be at least 5 times the responsecompared to blank response.C Analyte peak (response) should be identifiable, discrete, and reproducible witha precision of 20% and accuracy of 80-120%.2.Calibration Curve/Standard Curve/Concentration-ResponseThe simplest model that adequately describes the concentration-response relationshipshould be used. Selection of weighting and use of a complex regression equation should be justified. The following conditions should be met in developing a calibration curve:C#20% deviation of the LLOQ from nominal concentrationC#15% deviation of standards other than LLOQ from nominal concentrationAt least four out of six non-zero standards should meet the above criteria, including the LLOQ and the calibration standard at the highest concentration. Excluding thestandards should not change the model used.D.StabilityDrug stability in a biological fluid is a function of the storage conditions, the chemical properties of the drug, the matrix, and the container system. The stability of an analyte in a particular matrix and container system is relevant only to that matrix and container system and should not be extrapolated to other matrices and container systems. Stability procedures should evaluate the stability of the analytes during sample collection and handling, after long-term (frozen at theintended storage temperature) and short-term (bench top, room temperature) storage, and after going through freeze and thaw cycles and the analytical process. Conditions used in stability experiments should reflect situations likely to be encountered during actual sample handling and analysis. The procedure should also include an evaluation of analyte stability in stock solution.All stability determinations should use a set of samples prepared from a freshly made stock solution of the analyte in the appropriate analyte-free, interference-free biological matrix. Stock solutions of the analyte for stability evaluation should be prepared in an appropriate solvent at known concentrations.1.Freeze and Thaw StabilityAnalyte stability should be determined after three freeze and thaw cycles. At least three aliquots at each of the low and high concentrations should be stored at the intendedstorage temperature for 24 hours and thawed unassisted at room temperature. Whencompletely thawed, the samples should be refrozen for 12 to 24 hours under the sameconditions. The freeze–thaw cycle should be repeated two more times, then analyzedon the third cycle. If an analyte is unstable at the intended storage temperature, thestability sample should be frozen at -700C during the three freeze and thaw cycles.2.Short-Term Temperature StabilityThree aliquots of each of the low and high concentrations should be thawed at roomtemperature and kept at this temperature from 4 to 24 hours (based on the expectedduration that samples will be maintained at room temperature in the intended study) and analyzed.3.Long-Term StabilityThe storage time in a long-term stability evaluation should exceed the time between the date of first sample collection and the date of last sample analysis. Long-term stabilityshould be determined by storing at least three aliquots of each of the low and highconcentrations under the same conditions as the study samples. The volume of samples should be sufficient for analysis on three separate occasions. The concentrations of allthe stability samples should be compared to the mean of back-calculated values for the standards at the appropriate concentrations from the first day of long-term stabilitytesting.4.Stock Solution StabilityThe stability of stock solutions of drug and the internal standard should be evaluated at room temperature for at least 6 hours. If the stock solutions are refrigerated or frozenfor the relevant period, the stability should be documented. After completion of thedesired storage time, the stability should be tested by comparing the instrumentresponse with that of freshly prepared solutions.5.Post-Preparative StabilityThe stability of processed samples, including the resident time in the autosampler, should be determined. The stability of the drug and the internal standard should be assessedover the anticipated run time for the batch size in validation samples by determiningconcentrations on the basis of original calibration standards.Although the traditional approach of comparing analytical results for stored samples with those for freshly prepared samples has been referred to in this guidance, other statistical approaches based on confidence limits for evaluation of an analyte=s stability in abiological matrix can be used. SOPs should clearly describe the statistical method andrules used. Additional validation may include investigation of samples from dosedsubjects.E.Principles of Bioanalytical Method Validation and Establishment•The fundamental parameters to ensure the acceptability of the performance of a bioanalytical method validation are accuracy, precision, selectivity, sensitivity,reproducibility, and stability.• A specific, detailed description of the bioanalytical method should be written. This can be in the form of a protocol, study plan, report, and/or SOP.•Each step in the method should be investigated to determine the extent to which environmental, matrix, material, or procedural variables can affect the estimation of analyte in the matrix from the time of collection of the material up to and including the time ofanalysis.•It may be important to consider the variability of the matrix due to the physiological nature of the sample. In the case of LC-MS-MS-based procedures, appropriate steps should be taken to ensure the lack of matrix effects throughout the application of the method,especially if the nature of the matrix changes from the matrix used during method validation.• A bioanalytical method should be validated for the intended use or application. All experiments used to make claims or draw conclusions about the validity of the methodshould be presented in a report (method validation report).•Whenever possible, the same biological matrix as the matrix in the intended samples should be used for validation purposes. (For tissues of limited availability, such as bone marrow, physiologically appropriate proxy matrices can be substituted.)•The stability of the analyte (drug and/or metabolite) in the matrix during the collection process and the sample storage period should be assessed, preferably prior to sampleanalysis.•For compounds with potentially labile metabolites, the stability of analyte in matrix from dosed subjects (or species) should be confirmed.•The accuracy, precision, reproducibility, response function, and selectivity of the method for endogenous substances, metabolites, and known degradation products should beestablished for the biological matrix. For selectivity, there should be evidence that thesubstance being quantified is the intended analyte.•The concentration range over which the analyte will be determined should be defined in the bioanalytical method, based on evaluation of actual standard samples over the range,including their statistical variation. This defines the standard curve.• A sufficient number of standards should be used to adequately define the relationship between concentration and response. The relationship between response and concentration should be demonstrated to be continuous and reproducible. The number of standards used should be a function of the dynamic range and nature of the concentration-responserelationship. In many cases, six to eight concentrations (excluding blank values) can define the standard curve. More standard concentrations may be recommended for nonlinear than for linear relationships.•The ability to dilute samples originally above the upper limit of the standard curve should be demonstrated by accuracy and precision parameters in the validation.•In consideration of high throughput analyses, including but not limited to multiplexing, multicolumn, and parallel systems, sufficient QC samples should be used to ensure control of the assay. The number of QC samples to ensure proper control of the assay should be determined based on the run size. The placement of QC samples should be judiciously considered in the run.•For a bioanalytical method to be considered valid, specific acceptance criteria should be set in advance and achieved for accuracy and precision for the validation of QC samples over the range of the standards.F.Specific Recommendations for Method Validation•The matrix-based standard curve should consist of a minimum of six standard points, excluding blanks, using single or replicate samples. The standard curve should cover the entire range of expected concentrations.•Standard curve fitting is determined by applying the simplest model that adequately describes the concentration-response relationship using appropriate weighting and statistical tests for goodness of fit.•LLOQ is the lowest concentration of the standard curve that can be measured with acceptable accuracy and precision. The LLOQ should be established using at least five samples independent of standards and determining the coefficient of variation and/orappropriate confidence interval. The LLOQ should serve as the lowest concentration on the standard curve and should not be confused with the limit of detection and/or the low QC sample. The highest standard will define the upper limit of quantification (ULOQ) of an analytical method.•For validation of the bioanalytical method, accuracy and precision should be determined using a minimum of five determinations per concentration level (excluding blank samples).The mean value should be within ±15% of the theoretical value, except at LLOQ, where it should not deviate by more than ±20%. The precision around the mean value should not exceed 15% of the CV, except for LLOQ, where it should not exceed 20% of the CV.Other methods of assessing accuracy and precision that meet these limits may be equally acceptable.•The accuracy and precision with which known concentrations of analyte in biological matrix can be determined should be demonstrated. This can be accomplished by analysis ofreplicate sets of analyte samples of known concentrations C QC samples C from anequivalent biological matrix. At a minimum, three concentrations representing the entire range of the standard curve should be studied: one within 3x the lower limit of quantification (LLOQ) (low QC sample), one near the center (middle QC), and one near the upperboundary of the standard curve (high QC).•Reported method validation data and the determination of accuracy and precision should include all outliers; however, calculations of accuracy and precision excluding values that are statistically determined as outliers can also be reported.•The stability of the analyte in biological matrix at intended storage temperatures should be established. The influence of freeze-thaw cycles (a minimum of three cycles at twoconcentrations in triplicate) should be studied.•The stability of the analyte in matrix at ambient temperature should be evaluated over a time period equal to the typical sample preparation, sample handling, and analytical run times.•Reinjection reproducibility should be evaluated to determine if an analytical run could be reanalyzed in the case of instrument failure.•The specificity of the assay methodology should be established using a minimum of six independent sources of the same matrix. For hyphenated mass spectrometry-basedmethods, however, testing six independent matrices for interference may not be important.In the case of LC-MS and LC-MS-MS-based procedures, matrix effects should beinvestigated to ensure that precision, selectivity, and sensitivity will not be compromised.Method selectivity should be evaluated during method development and throughout methodvalidation and can continue throughout application of the method to actual study samples.•Acceptance/rejection criteria for spiked, matrix-based calibration standards and validation QC samples should be based on the nominal (theoretical) concentration of analytes.Specific criteria can be set up in advance and achieved for accuracy and precision over therange of the standards, if so desired.V.METHOD DEVELOPMENT: MICROBIOLOGICAL AND LIGAND-BINDING ASSAYSMany of the bioanalytical validation parameters and principles discussed above are also applicable to microbiological and ligand-binding assays. However, these assays possess some unique characteristics that should be considered during method validation.A.Selectivity IssuesAs with chromatographic methods, microbiological and ligand-binding assays should be shown to be selective for the analyte. The following recommendations for dealing with two selectivity issues should be considered:1.Interference From Substances Physiochemically Similar to the Analyte•Cross-reactivity of metabolites, concomitant medications, or endogenouscompounds should be evaluated individually and in combination with the analyteof interest.•When possible, the immunoassay should be compared with a validated reference method (such as LC-MS) using incurred samples and predetermined criteria foragreement of accuracy of immunoassay and reference method.。

Product Information Presentation, Storage and StabilityThe Incucyte® Fabfluor-pH Antibody Labeling Reagents for antibody internalization are supplied as lyophilized solids in sufficient quantity to label 50 μg of test antibody, when used at the suggested molar ratio (1:3 of test antibody to labeling Fab). The lyophilized solid can be stored at 2-8° C for one year. Once re-hydrated, any unused reagent should be aliquoted and stored at -80° C for up to one year. Avoid repeated freeze-thaw cycles.Incucyte® Fabfluor-pH Antibody Labeling ReagentsFor Antibody Internalization AssaysAntibody Labeling Reagent Rehydrated: -80° C *Excitation and Emission maxima were determined at a pH of 4.5.Fabfluor_quick_guideBackgroundIncucyte ® Fabfluor-pH Antibody Labeling Reagents are designed for quick, easy labeling of Fc-containing test antibodies with a Fab fragment-conjugated pH-sensitive fluorophore. The pH-sensitive dye based system exploits the acidic environment of the lysosomes to quantify in-ternalization of the labeled antibody. As Fabfluor labeled antibodies reside in the neutral extracellular solution (pH 7.4), they interact with cell surface specific antigens and are internalized. Once in the lysosomes, they enter an acidic environment (pH 4.5–5.5) and a substantial in-crease in fluorescence is observed. In the absence of ex-pression of the specific antigen, no internalization occurs and the fluorescence intensity of the labeled antibodies remains low. With the Incucyte ® integrated analysis soft-ware, background fluorescence is minimized. These reagents have been validated for use with a number of different antibodies in a range of cell types. The Incucyte ® Live-Cell Analysis System enables real-time, kinetic eval -uation of antibody internalization.Recommended UseWe recommend that the Incucyte ® Fabfluor-pH Antibody Labeling Reagents are prepared at a stock concentration of 0.5 mg/mL by the addition of 100 μL of sterile water and triturated (centrifuge if solution not clear). The reagent may then be diluted directly into the labeling mixture with test antibody. Do NOT sonicate the solution.Additional InformationThe Fab antibody was purified from antisera by a combination of papain digestion and immunoaffinity chromatography using antigens coupled to agarose beads. Fc fragments and whole IgG molecules have been removed.Human Red (Cat. No. 4722) or Human Orange (Cat. No. 4812)—Based on immunoelectrophoresis and/ or ELISA, the antibody reacts with the Fc portion of human IgG heavy chain but not the Fab portion of human IgG. No antibody was detected against human IgM, IgA or against non-immunoglobulin serum proteins. The anti-body may cross-react with other immunoglobulins from other species.Mouse IgG1 (Cat. No. 4723), IgG2a (Cat. No. 4750) or IgG2b (Cat. No. 4751)—Based on antigen-binding assay and/or ELISA, the antibody reacts with the Fc portion of mouse IgG, IgG2a or IgG2b, respectively, but not the Fab portion of mouse immunoglobulins. No antibody was detected against mouse IgM or against non–immunoglobulin serum proteins. The antibody may cross-react with other mouse IgG subclasses or with immunoglobulins from other species.Rat (Cat. No. 4737)—Based on immunoelectrophoresis and/or ELISA, the antibody reacts with the Fc portion of rat IgG heavy chain but not the Fab portion of rat IgG. No antibody was detected against rat IgM, IgA or against non-immunoglobulin serum proteins. The antibody may cross-react with other immunoglobulins from other species.A.B.C.D.R e d O b j e c t A r e a (x 105 μm 2 p e r w e l l )Time (hours)A U C x 106 (0–12 h )log [α–CD71] (g/mL)Example DataFigure 1: Concentration-dependent increase in antibody internalization of Incucyte ® Fabfluor labeled-α-CD71 in HT1080 cells. α-CD71 and mouse IgG1 isotype control were labeled with Incucyte ® Mouse IgG1 Fabfluor-pH Red Antibody Labeling Reagent. HT1080 cells were treated with either Fabfluor-α-CD71 or Fabfluor-IgG1 (4 μg/mL); HD phase and red fluorescence images were captured every 30 minutes over 12 hours using a 10X magnification. (A) Images of cells treated with Fabfluor-α-CD71 display red fluorescence in the cytoplasm (images shown at 6 h). (B) Cells treated with labeled isotype control display no cellular fluorescence. (C) Time-course of Fabfluor-α-CD71 internalization with increasing concentrations of Fabfluor-α-CD71 (progressively darker symbols). Internalization has been quantified as the red object area for each time-point. (D) Concentration response curve to Fabfluor-α-CD71. Area under the curve (AUC) values have been determined from the time-course shown in panel C (0-12 hours) and are presented as the mean ± SEM, n=3 wells.CD71-FabfluorIgG-FabfluorProtocols and ProceduresMaterialsIncucyte® Fabfluor-pH Antibody Labeling ReagentTest antibody of interest containing human, mouse, or rat IgG Fc region (at known concentration)Target cells of interestTarget cell growth mediaSterile distilled water96-well flat bottom microplate (e.g. Corning Cat. No. 3595) for imaging96-well round black round bottom ULA plate (e.g. Corning Cat. No. 45913799) or amber microtube (e.g. Cole Parmer Cat. No. MCT-150-X, autoclaved) for conjugation step0.01% Poly-L-Ornithine (PLO) solution (e.g. Sigma Cat. No. P4957), optional for non-adherent cells Recommended control antibodiesIt is strongly recommended that a positive and negative control is run alongside test antibodies and cell lines. For example, CD71, which is a mouse anti-human antibody, is recommended as a positive control for the mouse Fab.Anti-CD71, clone MEM-189, IgG1 e.g. Sigma Cat. No. SAB4700520-100UGAnti-CD71, clone CYG4, IgG2a e.g. BioLegend Cat. No. 334102Isotype controls, depending on isotype being studied—Mouse IgG1, e.g. BioLegend Cat. No. 400124, Mouse IgG2a e.g. BioLegend Cat. No. 401501Preparation of Incucyte® Antibody Internalization Assay 1. Seed target cells of interest1.1 Harvest cells of interest and determine cell concentra-tion (e.g. trypan blue + hemocytometer).1.2 Prepare cell seeding stock in target cell growth mediawith a cell density to achieve 40–50% confluence be-fore the addition of labeled antibodies. The suggested starting range is 5,000–30,000 cells/well, although the seeding density will need to be optimized for each cell type.Note: For non-adherent cell types, a well coating may be required to maintain even cell distribution in the well. For a 96-well flat bottom plate, we recommend coating with 50 μL of either 0.01% Poly-L-Or-nithine (PLO) solution or 5 μg/mL fibronectin diluted in 0.1% BSA.Coat plates for 1 hour at ambient temperature, remove solution from wells and then allow the plates to dry for 30-60 minutes prior to cell addition.1.3 Using a multi-channel pipette, seed cells (50 µL perwell) into a 96-well flat bottom microplate. Lightly tapplate side to ensure even liquid distribution in well. Toensure uniform distribution of cells in each well, allowthe covered plate sit on a level surface undisturbed at room temperature in the tissue culture hood for 30minutes. After cells are settled, place the plate insidethe Incucyte® Live-Cell Analysis System to monitor cell confluence.Note: Depending on cell type, plates can be used in assay once cells have adhered to plastic and achieved normal cell morphology e.g.2-3 hours for HT1080 or 1-2 hours for non-adherent cell types. Some cell types may require overnight incubation.2. Label Test Antibody2.1 Rehydrate the Incucyte® Fabfluor-pH Antibody Label-ing Reagent with 100 µL sterile water to result in a final concentration of 0.5 mg/mL. Triturate to mix (centrifuge if solution is not clear).Note: The reagent is light sensitive and should be protected fromlight. Rehydrated reagent can be aliquoted into amber or foilwrapped tubes and stored at -80° C for up to 1 year (avoid freezing and thawing).2.2 Mix test antibody with rehydrated Incucyte® Fabfluor–pH Antibody Labeling Reagent and target cell growth media in a black round bottom microplate or ambertube to protect from light (50 µL/well).a. Add test antibody and Incucyte® Fabfluor–pH Anti-body Labeling Reagent at 2X the final concentration.We suggest optimizing the assay by starting with afinal concentration of 4 µg/mL of test antibody or theFabfluor-pH Antibody Labeling Reagent (i.e. 2Xworking concentration = 8 µg/mL).Note: A 1:3 molar ratio of test antibody to Incucyte® Fabfluor-pHAntibody Labeling Reagent is recommended. The labeling re-agent is a third of the size of a standard antibody (50 and 150KDa, respectively). Therefore, labeling equal quantities will pro-duce a 1:3 molar ratio of test antibody to labeling Fab.b. Make sufficient volume of 2X labeling solution for50 µL/well for each sample. Triturate to mix.c. Incubate at 37° C for 15 minutes protected from light.Note: If performing a range of concentrations of test antibody,e.g. concentration response-curve, it is recommended to createthe dilution series post the conjugation step to ensure consistentmolar ratio. We strongly recommend the use of both a negativeand positive control antibody in the same plate.3. Add labeled antibody to cells3.1 Remove cell plate from incubator.3.2 Using a multi-channel pipette, add 50 µL of 2X labeledantibody and control solutions to designated wells.Remove any bubbles and immediately place plate in the Incucyte® Live-Cell Analysis System and start scanning.Note: To reduce the risk of condensation formation on the lid priorto first image acquisition, maintain all reagents at 37° C prior toplate addition.4. Acquire images and analyze4.1 In the Incucyte® Software, schedule to image every15-30 minutes, depending on the speed of the specific antibody internalization.a Scan on schedule, standard. If the Incucyte® Cell-by-Cell Analysis Software Module (Cat. No. 9600-0031)is available, adherent cell-by-cell or non-adherentcell-by-cell scan types can be selected.b Channel selection: select “phase” and “red” or“phase” and "orange” (depending on reagent used).c Objective: 10X or 20X depending on cell types used,generally 10X is recommended for adherent cells,and 20X for non-adherent or smaller cells.NOTE: The optional Incucyte® Cell-by-Cell Analysis SoftwareModule enables the classification of cells into sub-populationsbased on properties including fluorescence intensity, size andshape. For further details on this analysis module and its appli-cation, please see: /cell-by-cell.4.2 To generate the metrics, user must create an AnalysisDefinition suited to the cell type, assay conditions andmagnification selected.4.3 Select images from a well containing a positiveinternalization signal and an isotype control well(negative signal) at a time point where internalizationis visible.4.4 In the Analysis Definition:Basic Analyzer:a. Set up the mask for the phase confluence measurewith fluorescence channel turned off.b. Once the phase mask is determined, turn the fluores-cence channel on: Exclude background fluorescencefrom the mask using the background subtractionfeature. The feature “Top-Hat” will subtract localbackground from brightly fluorescent objects withina given radius; this is a useful tool for analyzing ob-jects which change in fluorescence intensity overtime.i The radius chosen should reflect the size of thefluorescent object but contain enough backgroundto reliably estimate background fluorescence inthe image; 20-30 μm is often a useful startingpoint.ii The threshold chosen will ensure that objectsbelow a fluorescence threshold will not bemasked.iii Choose a threshold in which red or orange objectsare masked in the positive response image but lownumbers in the isotype control, negative responsewell. For a very sensitive measurement, for example,if interested in early responses, we suggest athreshold of 0.2.NOTE: The Adaptive feature can be used for analysis but maynot be as sensitive and may miss early responses. If interestedin rate of response, Top-Hat may be preferable.Cell-by-Cell (if available):a. Create a Cell-by-Cell mask following the softwaremanual.b. There is no need to separate phase and fluorescencemasks. The default setting of Top-Hat No Mask forthe fluorescence channel will enable backgroundsubtraction without generation of a mask. Ensurethat the Top-Hat radius is set to a value higher thanthe radius of the larger clusters to avoid excess back-ground subtraction.c. The threshold of fluorescence can be determined inCell-by-Cell Classification.Specifications subject to change without notice.© 2020. All rights reserved. Incucyte, Essen BioScience, and all names of Essen BioScience prod -ucts are registered trademarks and the property of Essen BioScience unless otherwise specified. Essen BioScience is a Sartorius Company. Publication No.: 8000-0728-A00Version 1 | 2020 | 04Sales and Service ContactsFor further contacts, visit Essen BioScience, A Sartorius Company /incucyte Sartorius Lab Instruments GmbH & Co. KGOtto-Brenner-Strasse 20 37079 Goettingen, Germany Phone +49 551 308 0North AmericaEssen BioScience Inc. 300 West Morgan Road Ann Arbor, Michigan, 48108USATelephone +1 734 769 1600E-Mail:***************************EuropeEssen BioScience Ltd.Units 2 & 3 The Quadrant Newark CloseRoyston Hertfordshire SG8 5HLUnited KingdomTelephone +44 (0) 1763 227400E-Mail:***************************APACEssen BioScience K.K.4th floor Daiwa Shinagawa North Bldg.1-8-11 Kita-Shinagawa Shinagawa-ku, Tokyo 140-0001 JapanTelephone: +81 3 6478 5202E-Mail:*************************5. Analysis GuidelinesAs the labeled antibody is internalized into the acidic environment of the lysosome, the area of fluorescence intensity inside the cells increases.This can be reported in two ways:Ways to Report Basic AnalyzerCell-by-Cell Analysis* To correct for cell proliferation, it is advisable to normalize the fluorescence area to the total cell area using User Defined Metrics.For Research Use Only. Not For Therapeutic or Diagnostic Use.LicensesFor non-commercial research use only. Not for therapeutic or in vivo applications. Other license needs contact Essen BioS cience.Fabfluor-pH Red Antibody Labeling Reagent: This product or portions thereof is manufactured under license from Carnegie Mellon University and U.S. patent numbers 7615646 and 8044203 and related patents. This product is licensed for sale only for research. It is not licensed for any other use. There is no implied license hereunder for any commercial use.Fabfluor-pH Orange Antibody Labeling Reagent: This product or portions thereof is manufactured under a license from Tokyo University and is covered by issued patents EP2098529B1, JP5636080B2, US8258171, and US9784732 and related patent applications. This product and related products are trademarks of Goryo Chemical. Any application of above mentioned technology for commercial purpose requires a separate li -cense from: Goryo Chemical, EAREE Bldg., SF Kita 8 Nishi 18-35-100, Chuo-Ku, Sapporo, 060-0008 Japan.SupportA complete suite of cell health applications is available to fit your experimental needs. Find more information at /incucyte Foradditionalproductortechnicalinformation,************************************************************/incucyte。

碧云天生物技术/Beyotime Biotechnology订货热线：400-168-3301或800-8283301订货e-mail：******************技术咨询：*****************网址：碧云天网站微信公众号NF-κB p65 Rabbit Polyclonal Antibody产品编号产品名称包装AF0246 NF-κB p65 Rabbit Polyclonal Antibody50μl产品简介：来源用途交叉反应性分子量Rabbit WB, FC, IHC H, M, R65KDa WB, Western blot; IP, Immunoprecipitation; IF, Immunofluorescence; IHC, Immunohistochemistry; ICC,Immunocytochemistry;FC, Flow Cytometry; ELISA, Enzyme-linked Immuno sorbent Assay; Ch IP, C hromatin I mmunoprecipitation Assay.H, Human;M, M ouse; R,R at; C, Chicken; Cw, C ow; D g, D og; Gp, Guinea pig; Hm, Hamster; Hr, Horse; Mk, Monkey; P g, Pig;Rb,R abbit; S, Sheep; Z, Zebrafish; All, all species expected.配套提供了Western一抗稀释液，可以用于Western检测或其它适当用途时的一抗稀释。

建议抗体使用时的稀释比例如下(实际使用时需根据抗原水平的高低作适当调整)：WB IP IF IHC ICC FC ELISA ChIP 1:1000-1:2000 - - 1:100-1:200 - 1:50-1:100 - -抗体详细信息如下:About this AntibodyName NF-κB p65 Rabbit Polyclonal AntibodyCategory Polyclonal antibody(pAb); Primary antibodyIsotype IgGPurification Peptide affinity purifiedAbout the ImmunogenImmunogen This antibody is produced by immunizing rabbits with a synthetic peptide (KLH-coupled) corresponding to N-terminal NF-kB p65.Gene ID5970SwissProt Q04206Synonyms NFKB3; Nuclear factor NF-kappa-B p65 subunit; RELA; TF65; Transcription factor p65; p65 Category Immunology and InflammationBackground NF-kappa-B is a pleiotropic transcription factor present in almost all cell types and is the endpoint of a series of signal transduction events that are initiated by a vast array of stimuli related to many biological processes such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis. NF-kappa-B is a homo- or heterodimeric complex formed by the Rel-like domain-containing proteins RELA/p65, RELB, NFKB1/p105, NFKB1/p50, REL and NFKB2/p52 and the heterodimeric p65-p50 complex appears to be most abundant one. In unstimulated cells, NF-κB is sequestered in the cytoplasm by IκB inhibitory proteins. NF-κB-activating agents can induce the phosphorylation of IκB proteins, targeting them for rapid degradation through the ubiquitin-proteasome pathway and releasing NF-κB to enter the nucleus where it regulates gene expression .包装清单：产品编号产品名称包装AF0246 NF-κB p65 Rabbit Polyclonal Antibody50μlAZ050Western一抗稀释液50ml－说明书1份保存条件：NF-κB p65 Rabbit Polyclonal Antibody -20ºC保存，Western一抗稀释液-20ºC或4ºC保存，一年有效。