Antibody Production for a Rapid Fluorescence Polarization Immunoassay of Estrone

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HIF-1α_纳米抗体的制备及其抑制黑素瘤生长的作用

山西农业科学 2023，51（12）：1435-1441Journal of Shanxi Agricultural Sciences HIF-1α纳米抗体的制备及其抑制黑素瘤生长的作用李佳敏1，贾琼1，秦蓉芬1，迟志端1，王富明2，范瑞文1（1.山西农业大学动物医学学院，山西太谷 030801；2.晋中市庄子乡综合便民服务中心，山西晋中 030600）摘要：缺氧诱导因子1α（Hypoxia inducible factor 1α，HIF-1α）参与低氧微环境相关疾病的发生等过程，具有控制肿瘤生长和发展的功能。

黑色素瘤是一种发生于人和动物恶性程度较高的肿瘤。

为探明HIF-1α纳米抗体对黑色素瘤的影响，研究利用前期保存的羊驼源黑色素瘤细胞噬菌体文库筛选HIF-1α纳米抗体，经原核表达与纯化后，通过Western Blot和免疫组织化学验证HIF-1α纳米抗体与抗原的结合性；分别通过CCK-8法、划痕试验以及Western Blot法检测其对B16黑素瘤细胞的增殖和迁移能力及其相关分子表达的影响。

结果表明，经表达和纯化获得的HIF-1α纳米抗体分子质量约为16 ku，没有跨膜结构，具有亲水性。

通过Western Blot和免疫组织化学验证了其具有良好的抗原结合性。

在增殖试验和划痕试验中，与对照组相比，HIF-1α纳米抗体抑制了B16细胞的增殖和迁移，下调了靶基因VEGF的表达，并使细胞增殖和迁移相关蛋白Ras、ERK、RAC和RAF的表达量下调。

预测HIF-1α纳米抗体进入B16细胞内，与抗原结合，通过下游靶基因VEGF下调RAs、ERK、RAC、RAF的表达，从而对细胞增殖和迁移起抑制作用，可作为黑色素瘤治疗的新靶点。

关键词：HIF-1α；纳米抗体；B16细胞；Western Blot法；CCK-8法；细胞增殖；细胞迁移中图分类号：R739.5　文献标识码：A 文章编号：1002‒2481（2023）12‒1435‒07Effect on Preparation of HIF-1α Nano-Antibody and ItsInhibition of Melanoma GrowthLI Jiamin1，JIA Qiong1，QIN Rongfen1，CHI Zhiduan1，WANG Fuming2，FAN Ruiwen1（1.College of Veterinary Medicine，Shanxi Agricultural University，Taigu 030801，China；2.Jinzhong City Zhuangzi Integrated Convenient Service Center，Jinzhong 030600，China）Abstract：The hypoxia inducible factor 1α(HIF-1α) is involved in the occurrence of diseases related to hypoxia microenvironment and has the function of controlling tumor growth and development. As we known, melanoma is a highly malignant tumor occurring in animals and humans. To explore the effect of HIF-1α nano-antibody on melanoma, in this study, the phage library of alpaca-drived melanoma cells previously preserved in our laboratory was used to screen HIF-1α nano-antibodies. After prokaryotic expression and purification, the binding of HIF-1α nano-antibody and its antigen was verified by Western blot and immunohistochemistry. The effects of HIF-1α nano-antibody on the proliferation and migration of B16 melanoma cells and the expression of related molecules were detected by CCK-8, wound healing test, and Western blot methods. The results showed that HIF-1α nano-antibody obtained by expression and purification was hydrophilic protein without transmembrane structure and had a molecular weight of about 16 ku. Western blot and immunohistochemistry results showed that it had good antigenic binding. In the proliferation and wound healing experiments, HIF-1α nano-antibody inhibited the proliferation and migration of B16 cells, down-regulated the expression of target gene VEGF and the proliferation and migration related proteins Ras, ERK, RAC, and RAF, comparing with the control group. In Conclusion, it was predicted that HIF-1α nano-antibody entered B16 cells and combined with antigens and down-regulated the expression of RAs, ERK, RAC, RAF through the downstream target gene VEGF, which inhibited cell proliferation and migration, and could be used as a new target for melanoma treatment.Key words：HIF-1α; nano-antibody; B16 cells; Western Blot method; CCK-8 method; cell proliferation; cell migration氧是生命活动中所必需的物质，且在其中起重要作用[1]。

biontech lpx成分

Biontech LPX 成分Biontech LPX 是一种新型的基因疗法药物，它的主要成分是一种特殊的脂质纳米颗粒。

这种脂质纳米颗粒是由多种生物大分子组成，包括磷脂、胆固醇和 PEG 脂质。

这些成分在制备 Biontech LPX 时起到了非常重要的作用，它们为药物提供了良好的稳定性和生物相容性，使得 Biontech LPX 能够有效地传递基因药物到靶细胞内。

1. 磷脂磷脂是构成细胞膜的主要成分，它具有双亲性的化学性质，既能够与水相互作用，又能够与脂肪相互作用。

在 Biontech LPX 中，磷脂主要起到了包裹和保护 mRNA 分子的作用。

由于 mRNA 分子本身非常容易被降解，所以需要通过包裹在磷脂纳米颗粒内来提高其稳定性和细胞摄取率。

2. 胆固醇胆固醇是一种重要的生物大分子，在细胞膜的结构和功能上发挥着非常重要的作用。

在 Biontech LPX 中，胆固醇的加入可以增加纳米颗粒的稳定性和生物相容性，使得纳米颗粒能够更好地在体内循环并传递基因药物到靶细胞内。

3. PEG 脂质PEG 脂质是一种水溶性的聚合物，它具有良好的生物相容性和生物降解性。

在 Biontech LPX 中，PEG 脂质的加入可以增加纳米颗粒在体内的循环时间，并减少免疫反应的发生，从而提高基因药物的传递效率和安全性。

总结Biontech LPX 是一种基因疗法药物，其主要成分是一种特殊的脂质纳米颗粒。

这种纳米颗粒由磷脂、胆固醇和 PEG 脂质组成，它们能够为基因药物的传递提供良好的稳定性和生物相容性。

通过了解 Biontech LPX 的成分和作用机制，我们可以更好地理解其在基因治疗领域的应用前景和潜在的临床效果。

Biontech LPX 成分对于药物的效果有着重要影响，值得相关研究者和医学工作者进一步深入研究和探讨。

Biontech LPX 的发展和应用将为基因疗法领域的发展带来新的希望和机遇。

Biontech LPX 成分的研究和开发将有助于推动基因疗法领域的进步，为更多患者带来健康和幸福。

破伤风类毒素蛋白纯化液名词解释

破伤风类毒素蛋白纯化液名词解释下载提示：该文档是本店铺精心编制而成的，希望大家下载后，能够帮助大家解决实际问题。

文档下载后可定制修改，请根据实际需要进行调整和使用，谢谢！本店铺为大家提供各种类型的实用资料，如教育随笔、日记赏析、句子摘抄、古诗大全、经典美文、话题作文、工作总结、词语解析、文案摘录、其他资料等等，想了解不同资料格式和写法，敬请关注！Download tips: This document is carefully compiled by this editor. I hope that after you download it, it can help you solve practical problems. The document can be customized and modified after downloading, please adjust and use it according to actual needs, thank you! In addition, this shop provides you with various types of practical materials, such as educational essays, diary appreciation, sentence excerpts, ancient poems, classic articles, topic composition, work summary, word parsing, copy excerpts, other materials and so on, want to know different data formats and writing methods, please pay attention!一、定义破伤风类毒素蛋白纯化液是一种用于纯化破伤风类毒素蛋白的液体溶液。

从毒蛇毒液中发现新型生物活性分子

从毒蛇毒液中发现新型生物活性分子毒蛇毒液中不仅包含着致命的神经毒素和血液毒素，还蕴藏着无数与人类健康息息相关的生物活性分子。

在毒蛇毒液中探索和发现新型生物活性分子，成为了当今生物医药领域的重要研究方向之一。

在众多毒蛇毒液中，一种名为罗氏角腹蛇的毒蛇毒液被科学家们视为“活黄金”般的宝藏。

这种毒液来源于澳大利亚东海岸地区的罗氏角腹蛇，被广泛应用于医药领域。

其中，最为著名的神奇成分莫过于ACE抑制剂——Captopril。

Captopril是一种降压药，为一种ACE（Angiotensin-Converting Enzyme，血管紧张素转化酶）类似物，通过抑制ACE逆转肾素-血管紧张素系统发挥作用，在全球治疗高血压和心血管疾病方面发挥了极其重要的作用。

Captopril被称为“ACE的神经毒素”，因为这一分子首次被发现于罗氏角腹蛇毒液中，并且其结构中的功能团绝大多数来源于毒液中的天然氨基酸。

罗氏角腹蛇毒液中的Captopril结构可以说，毒蛇毒液是新型生物活性分子的重要来源。

因此，科学家们越来越注重从毒蛇毒液中发现新型药物和医学用途的生物活性分子，如针对癌症、神经系统疾病、心血管疾病和自身免疫性疾病等领域的治疗药物。

针对罗氏角腹蛇毒液中的Captopril这一事例，科学家们进一步研究发现，不同种类的毒蛇毒液中存在不同种类的ACE抑制剂。

对于蛇类而言，ACE抑制剂可以起到降血压和促进心脏功能的作用。

但几乎所有的ACE抑制剂都是由天然氨基酸结构组成，这意味着它们有很大的局限性，不太可能发展出优异性、高特异性的分子。

因此，科学家们研究的方向需要向更宏大开发。

从毒蛇毒液中寻找新型ACE抑制剂并非最终的研究目标，只是用以启迪科学家们对天然界中更多潜在的新型药物和疗法探索的一部分。

毒蛇毒液中蕴藏的新型生物活性分子，是科学家们无尽的研究源泉。

在今后的研究当中，科学家们会继续把探索毒蛇毒液中新型生物活性分子的研究视为一项重要的方向。

《2024年穿龙薯蓣皂苷对胶原诱导性关节炎小鼠CD4~+T细胞亚群平衡的影响及其调控机制研究》范文

《穿龙薯蓣皂苷对胶原诱导性关节炎小鼠CD4~+T细胞亚群平衡的影响及其调控机制研究》篇一摘要：本研究探讨了穿龙薯蓣皂苷对胶原诱导性关节炎（CIA）小鼠模型中CD4~+T细胞亚群平衡的影响及其可能的调控机制。

结果表明，穿龙薯蓣皂苷可以有效地调整CD4~+T细胞亚群的失衡，改善小鼠关节炎的病理过程，且该效应可能与其调节细胞信号转导、促进抗炎和抗增殖的作用相关。

一、引言关节炎是一种常见的慢性自身免疫性疾病，其发病机制复杂，涉及免疫系统紊乱。

穿龙薯蓣皂苷作为一种天然植物提取物，具有多种生物活性，包括抗炎、抗氧化和免疫调节等作用。

本研究旨在探讨穿龙薯蓣皂苷对胶原诱导性关节炎（CIA）小鼠模型中CD4~+T细胞亚群平衡的影响及其调控机制。

二、材料与方法1. 实验材料：穿龙薯蓣皂苷、CIA小鼠模型、相关试剂与仪器等。

2. 实验方法：（1）建立CIA小鼠模型；（2）分组及给药：将小鼠随机分为正常对照组、模型组、穿龙薯蓣皂苷治疗组；（3）检测各组小鼠CD4~+T细胞亚群平衡；（4）分析穿龙薯蓣皂苷对CIA小鼠的病理过程的影响；（5）研究穿龙薯蓣皂苷的调控机制。

三、实验结果1. 穿龙薯蓣皂苷对CIA小鼠CD4~+T细胞亚群平衡的影响：穿龙薯蓣皂苷治疗组小鼠的CD4~+T细胞亚群平衡得到显著改善，与模型组相比，治疗组中Th1/Th2比例趋于正常，Th17细胞数量减少。

2. 穿龙薯蓣皂苷对CIA小鼠病理过程的影响：穿龙薯蓣皂苷治疗组小鼠的关节炎症状得到明显缓解，关节肿胀程度减轻，关节滑膜炎症反应减弱。

3. 穿龙薯蓣皂苷的调控机制研究：（1）穿龙薯蓣皂苷可能通过调节细胞信号转导途径，如NF-κB、MAPK等，从而影响CD4~+T细胞的活化与分化；（2）穿龙薯蓣皂苷可能通过促进抗炎和抗增殖的作用，抑制关节炎症反应和关节损伤；（3）穿龙薯蓣皂苷还可能通过调节免疫相关基因的表达，如IL-17、IFN-γ等，进一步影响CD4~+T细胞的亚群平衡。

TUNEL细胞凋亡检测试剂盒罗氏

For research purposes only. Not for use for in vitro diagnosticprocedures for clinical diagnosis.In Situ Cell Death Detection Kit, PODKit for immunohistochemical detection and quantification of apop-tosis (programmed cell death) at single cell level, based on labeling of DNA strand breaks (TUNEL technology): Analysis by light microscopy.Cat. No. 1 684 817Store at Ϫ15 to Ϫ25°C 1 Kit (50 tests)Instruction ManualVersion 3, January 20031. Preface1.1Table of contentsP reface (2)1.1.1Table of contents (2) (3)1.2 Kitcontents (5)2. Introduction2.1Product overview (5) (8)2.2 Backgroundinformation3. Procedures and required materials (10)3.1Flow chart (10)3.2Preparation of sample material (11)3.2.1Adherent cells, cell smears and cytospin preparations (11) (12)sections3.2.2 Tissue3.2.2.1 Treatment of paraffin-embedded tissue (12)3.2.2.2Treatment of cryopreserved tissue (14)3.3Labeling protocol (15)3.3.1 Before you begin (15)3.3.2Labeling protocol for adherent cells, cell smears, cytospin preparations,and tissues (16)3.3.3 Labeling protocol for difficult tissue (17) (18)conversion3.4 Signal (19)4. Appendix (19)4.1 Trouble-shooting (22)4.2 References (23)4.3 Relatedproducts1.2 KitcontentsCaution The Label solution contains cacodylate, toxic by inhalation and swal-lowed, and cobalt dichloride, which may cause cancer by inhalation.Avoid exposure and obtain special instructions before use.When using do not eat, drink or smoke. After contact with skin, washimmediately with plenty of water. In case of accident or if you feelunwell seek medical advice immediately (show label where possible).Collect the supernatants from the labeling reactions in a tightly closed,non-breakable container and indicate contents. Discard as regulatedfor toxic waste.Kit contents Please refer to the following table for the contents of the kit.Vial/CapLabel Contents1 blue Enzyme Solution•Terminal deoxynucleotidyl transferasefrom calf thymus (EC 2.7.7.31), recom-binant in E. coli, in storage buffer•10× conc.•5×50␮l2 violet Label Solution•Nucleotide mixture in reaction buffer•1×conc.• 5 × 550 ␮l3 yellow Converter-POD•Anti-fluorescein antibody, Fab frag-ment from sheep, conjugated withhorse-radish peroxidase (POD)•Ready-to-use• 3.5mlAdditional equipment required In addition to the reagents listed above, you have to prepare several solutions. In the table you will find an overview about the equipment which is needed for the different procedures.Detailed information is given in front of each procedure.Procedure Equipment Reagents Preparation of sample material (section 3.2)•Adherent cells, cell smears and cytospinpreparations (section3.2.1)•Cryopreserved tissue (section 3.2.2.2)•Washing buffer: Phosphate buffered saline(PBS*)•Blocking solution: 3% H2O2 in methanol•Fixation solution: 4% Paraformaldehyde inPBS, pH 7.4, freshly prepared •Permeabilisation solution: 0.1% Triton X-100 in 0.1% sodium citrate, freshly pre-pared (6)Paraffin-embedded tissue (section 3.2.2.1)•Xylene and ethanol (absolute, 95%, 90%, 80%, 70%, diluted in double distilled water)•Washing buffer: PBS*•Proteinase K*, nuclease, working solution: [10-20 µg/ml in 10 mM Tris/HCl, pH 7.4-8] Alternative treatments•Permeabilisation solution: (0.1% Triton1) X–100, 0.1% sodium citrate) , freshly prepared •Pepsin* (0.25% - 0.5% in HCl, pH 2) or trypsin*, 0.01 N HCl, nuclease free•0.1 M Citrate buffer, pH 6 for microwave irradiationLabeling protocol (section 3.3)Positive control (section 3.3.1)•Micrococcal nuclease or •DNase I, grade I *Adherent cells, cell smears, cytospin preparations, and tissues (section 3.3.2) •Parafilm orcoverslips•HumidifiedchamberWashing buffer: PBS*Difficult tissue (section 3.3.3)•Plastic jar•Microwave•Humidifiedchamber•Citrate buffer, 0.1 M, pH 6.0.•Washing buffer: PBS*•Tris-HCl, 0.1 M pH 7.5, containing 3% BSA*and 20% normal bovine serumSignal conversion (section 3.4)•Humidified chamber •Parafilm or coverslip •Washing buffer: PBS*•DAB Metal Enhanced Substrate Set* or alternative POD substrates •Mounting medium for light microscopy1.2 Kitcontents,continued2. Introduction2.1Product overviewTest principle Cleavage of genomic DNA during apoptosis may yield double-stranded, low molecular weight DNA fragments (mono- and oligonu-cleosomes) as well as single strand breaks (“nicks”) in high molecularweight DNA.Those DNA strand breaks can be identified by labeling free 3’-OH ter-mini with modified nucleotides in an enzymatic reaction.Fig. 1: Test principleApplication The In Situ Cell Death Detection Kit is designed as a precise, fast and simple, non-radioactive technique to detect and quantify apoptotic celldeath at single cell level in cells and tissues. Thus, the In Situ CellDeath Detection Kit can be used in many different assay systems.Examples are:•Detection of individual apoptotic cells in frozen and formalin fixedtissue sections in basic research and routine pathology.•Determination of sensitivity of malignant cells to drug induced apo-ptosis in cancer research and clinical oncology.•Typing of cells undergoing cell death in heterogeneous populationsby double staining procedures (6).Specificity The TUNEL reaction preferentially labels DNA strand breaks gener-ated during apoptosis. This allows discrimination of apoptosis fromnecrosis and from primary DNA strand breaks induced by cytostaticdrugs or irradiation (3, 4).Test interference False negative results: DNA cleavage can be absent or incomplete in some forms of apoptotic cell death (37). Sterical hindrance such asextracellular matrix components can prevent access of TdT to DNAstrand breaks. In either case false negative results can be obtained.False positive results: Extensive DNA fragmentation may occur in latestages of necrosis (4, 38).DNA strand breaks may also be prominent in cell populations withhigh proliferative or metabolic activity. In either case false positiveresults may be obtained. To confirm apoptotic mode of cell death, themorphology of respective cells should be examined very carefully.Morphological changes during apoptosis have a characteristic pattern.Therefore evaluation of cell morphology is an important parameter insituations where there is any ambiguity regarding interpretation ofresults.Sample material•Cytospin and cell smear preparations•Adherent cells cultured on chamber slides (31)•Frozen or formalin-fixed, paraffin-embedded tissue sections (1, 25,26, 29, 30, 32–34, 36, 39)Assay time2–3 hours, excluding culture, fixation and permeabilisation of cells and preparation of tissue sections.Number of tests The kit is designed for 50 tests.Kit storage/ stability The unopened kit is stable at Ϫ15 to Ϫ25°C through the expiration date printed on the label.Reagent Storage and stabilityTUNEL reaction mixture The TUNEL reaction mixture should be pre-pared immediately before use and shouldnot be stored.Keep TUNEL reaction mixture on ice untiluse.Converter-POD Once thawed the Converter-POD solutionshould be stored at 2–8°C (maximum stabil-ity 6 months).Note: Do not freeze!Advantage Please refer to the following table.Benefit FeatureSensitive Detection of apoptotic cell death at singlecell level at very early stages (1, 2, 6).Specific Preferential labeling of apoptosis versusnecrosis (3, 4).Fast Short assay time (2-3 h).Convenient•Reagents are provided in stable, opti-mized form.•No dilution steps required.Flexible•Suitable for fixed cells and tissue. Thisallows accumulation, storage and trans-port of samples (2, 5).•Double staining enables identification oftype and differentiation state of cellsundergoing apoptosis (6).Function-tested Every lot is function-tested on apoptoticcells in comparison to a master lot.2.2 BackgroundinformationCell death Two distinct modes of cell death, apoptosis and necrosis, can be distin-guished based on differences in morphological, biochemical andmolecular changes of dying cells.Programmed cell death or apoptosis is the most common form ofeukaryotic cell death. It is a physiological suicide mechanism that pre-serves homeostasis, in which cell death naturally occurs during normaltissue turnover (8, 9). In general, cells undergoing apoptosis display acharacteristic pattern of structural changes in nucleus and cytoplasm,including rapid blebbing of plasma membrane and nuclear disintegra-tion. The nuclear collapse is associated with extensive damage tochromatin and DNA-cleavage into oligonucleosomal length DNA frag-ments after activation of a calcium-dependent endogenous endonu-clease (10, 11). However, very rare exceptions have been describedwhere morphological features of apoptosis are not accompanied witholigonucleosomal DNA cleavage (37).Apoptosis Apoptosis is essential in many physiological processes, includingmaturation and effector mechanisms of the immune system (12, 13),embryonic development of tissue, organs and limbs (14), developmentof the nervous system (15, 16) and hormone-dependent tissueremodeling (17). Inappropriate regulation of apoptosis may play animportant role in many pathological conditions like ischemia, stroke,heart disease, cancer, AIDS, autoimmunity, hepatotoxicity and degen-erative diseases of the central nervous system (18–20).In oncology, extensive interest in apoptosis comes from the observa-tion, that this mode of cell death is triggered by a variety of antitumordrugs, radiation and hyperthermia, and that the intrinsic propensity oftumor cells to respond by apoptosis is modulated by expression ofseveral oncogenes and may be a prognostic marker for cancer treat-ment (21).Identification of apoptosis Several methods have been described to identify apoptotic cells (22– 24). Endonucleolysis is considered as the key biochemical event of apoptosis, resulting in cleavage of nuclear DNA into oligonucleosome-sized fragments. Therefore, this process is commonly used for detec-tion of apoptosis by the typical “DNA ladder“ on agarose gels during electrophoresis. This method, however, can not provide information regarding apoptosis in individual cells nor relate cellular apoptosis to histological localization or cell differentiation.This can be done by enzymatic in situ labeling of apoptosis induced DNA strand breaks. DNA polymerase as well as terminal deoxynucle-otidyl transferase (TdT) (1-6, 25-36, 41) have been used for the incor-poration of labeled nucleotides to DNA strand breaks in situ. The tailing reaction using TdT, which was also described as ISEL (in situ end labeling) (5, 35) or TUNEL (TdT-mediated dUTP nick end labeling) (1, 6, 31, 33) technique, has several advantages in comparison to the in situ nick translation (ISNT) using DNA polymerase:•Label intensity of apoptotic cells is higher with TUNEL compared to ISNT, resulting in an increased sensitivity (2, 4).•Kinetics of nucleotide incorporation is very rapid with TUNEL com-pared to the ISNT (2, 4).•TUNEL preferentially labels apoptosis in comparison to necrosis, thereby discriminating apoptosis from necrosis and from primary DNA strand breaks induced by antitumor drugs or radiation (3, 4).2.2 Backgroundinformation,continued3. Procedures and required materialsThe working procedure described below has been developed andpublished by R. Sgonc and colleagues (6). The main advantage of thissimple and rapid procedure is the use of fluorescein-dUTP to labelDNA strand breaks. This allows the detection of DNA fragmentationby fluorescence microscopy directly after the TUNEL reaction priorto the addition of the secondary anti-fluorescein-POD-conjugate.3.1Flow chartAssay procedure The assay procedure is explained in the following flow chart.Adherent cells, cell smears and cytospin preparations Cryopreservedtissue sectionsParaffin-embeddedtissue sections↓↓↓Fixation •Dewaxation •Rehydration •ProteasetreatmentPermeabilisation of samples↓Addition of TUNEL reaction mixtureOPTIONAL: Analysis of samples by fluorescence microscopy↓Addition of Converter-PODAddition of Substrate solution↓Analysis of samples by light microscopy3.2Preparation of sample material3.2.1Adherent cells, cell smears and cytospin preparationsAdditional solutions required •Washing buffer: Phosphate buffered saline (PBS)•Blocking solution: 3% H2O2 in methanol•Fixation solution: 4% Paraformaldehyde in PBS, pH 7.4, freshly pre-pared•Permeabilisation solution: 0.1% Triton1) X-100 in 0.1% sodium citrate, freshly prepared (6)Procedure In the following table describes the fixation of cells, blocking of endo-genous peroxidase and cell permeabilisation.Note: Fix and permeabilisate two additional cell samples for the nega-tive and positive labeling controls.Step Action1Fix air dried cell samples with a freshly prepared Fixationsolution for 1 h at 15-25°C.2Rinse slides with PBS.3Incubate with Blocking solution for 10 min at 15-25°C.4Rinse slides with PBS.5Incubate in Permeabilisation solution for 2 min on ice (2-8°C).6Proceed as described under 3.3.3.2.2 Tissue sections3.2.2.1 Treatment of paraffin-embedded tissuePretreatment of paraffin embedded tissue Tissue sections can be pretreated in 4 different ways. If you use Pro-teinase K the concentration, incubation time and temperature have to be optimized for each type of tissue (1, 29, 33, 36, 40, 42).Note: Use Proteinase K only from Roche Applied Science, because it is tested for absence of nucleases which might lead to false-positive results!The other 3 alternative procedures are also described in the following table (step 2).Additional solutions required •Xylene and ethanol (absolute, 95%, 90%, 80%, 70%, diluted in dou-ble distilled water)•Washing buffer: PBS•Proteinase K, nuclease free (Cat. No. 745 723), working solution: [10-20 ␮g/ml in 10 mM Tris/HCl, pH 7.4-8]Alternative treatments•Permeabilisation solution: 0.1% Triton1) X–100, 0.1% sodium citrate, freshly prepared•Pepsin* (0.25% - 0.5% in HCl, pH 2) or trypsin*, 0.01 N HCl, nuclease free•0.1 M Citrate buffer, pH 6 for the microwave irradiationProcedure In the following table the pretreatment of paraffin-embedded tissue with Proteinase K treatment and 3 alternative procedures aredescribed.Note: Add additional tissue sections for the negative and positivelabeling controls.Step Action1Dewax and rehydrate tissue section according to standardprotocols (e.g. by heating at 60°C followed by washing inxylene and rehydration through a graded series of ethanoland double dist. water) (1, 33, 36).2Incubate tissue section for 15-30 min at 21–37°C with Pro-teinase K working solution.Alternatives:Treatment:1. Permeabilisa-tion solutionIncubate slides for 8 min.2. Pepsin* (30, 40)or trypsin*15-60 min at 37°C.3. Microwave irradiation •Place the slide(s) in a plastic jar containing 200 ml 0.1 M Citrate buffer, pH6.0.•Apply 350 W microwave irradiation for 5 min.3Rinse slide(s) twice with PBS.4Proceed as described under 3.3.3.2.2.1 Treatment of paraffin-embedded tissue, continued3.2.2.2Treatment of cryopreserved tissueAdditional solutions required •Fixation solution: 4% Paraformaldehyde in PBS, pH 7.4, freshly pre-pared•Washing buffer: PBS•Blocking solution: 3% H2O2 in methanol•Permeabilisation solution (0.1% Triton1) X–100, 0.1% sodium citrate), freshly preparedCryopreserved tissue In the following table the pretreatment of cryopreserved tissue is described.Note: Fix and permeabilisate two additional samples for the negative and positive labeling controls.Step Action1Fix tissue section with Fixation solution for 20 min at 15–25°C.2Wash 30 min with PBS.Note:For storage, dehydrate fixed tissue sections 2 min inabsolute ethanol and store at Ϫ15 to Ϫ25°C.3Incubate with Blocking solution for 10 min at 15–25°C.4Rinse slides with PBS.5Incubate in Permeabilisation solution for 2 min on ice (2–8°C).6Proceed as described under 3.3.3.3Labeling protocol 3.3.1Before you beginPreparation of TUNEL reaction mixtureOne pair of tubes (vial 1: Enzyme Solution, and vial 2: Label Solution) is sufficient for staining 10 samples by using 50 ␮l TUNEL reaction mix-ture per sample and 2 negative controls by using 50 ␮l Label Solution per control.Note : The TUNEL reaction mixture should be prepared immediately before use and should not be stored. Keep TUNEL reaction mixture on ice until use.Additionalreagents required •Micrococcal nuclease or •DNase I, grade I (Cat. No. 104 132)ControlsTwo negative controls and a positive control should be included in each experimental set up.Step Action1Remove 100 ␮l Label Solution (vial 2) for two negative con-trols.2Add total volume (50 ␮l) of Enzyme solution (vial 1) to the remaining 450 ␮l Label Solution in vial 2 to obtain 500 ␮l TUNEL reaction mixture.3Mix well to equilibrate components.Negative control:Incubate fixed and permeabilized cells in 50 ␮l/well Label Solution (without terminal transferase) instead of TUNEL reaction mixture.Positive control:Incubate fixed and permeabilized cells with micro-coccal nuclease or DNase I, grade I (3000 U/ml– 3 U/ml in 50 mM T ris-HCl, pH 7.5, 10 mM MgCl 2 1mg/ml BSA) for 10 min at 15-25°C to induce DNA strand breaks, prior to labeling procedures.3.3.2Labeling protocol for adherent cells, cell smears, cytospin preparations andtissuesAdditional equipment and solutions required •Washing buffer: PBS •Humidified chamber •Parafilm or coverslipProcedure Please refer to the following table.Step Action1Rinse slides twice with PBS.2Dry area around sample.3Add50␮l TUNEL reaction mixture on sample.Note: For the negative control add 50 ␮l Label solution each.To ensure a homogeneous spread of TUNEL reaction mixtureacross cell monolayer and to avoid evaporative loss, samplesshould be covered with parafilm or coverslip during incuba-tion.4Add lid and incubate for 60 min at 37°C in a humidified atmo-sphere in the dark.5Rinse slide 3 times with PBS.6Samples can be analyzed in a drop of PBS under a fluores-cence microscope at this state. Use an excitation wavelengthin the range of 450–500 nm and detection in the range of515–565 nm (green).3.3.3 Labeling protocol for difficult tissueAdditional equipment and solutions required •Citrate buffer, 0.1 M, pH 6.0.•Washing buffer: PBS•Tris-HCl, 0.1 M pH 7.5, containing 3% BSA and 20% normal bovine serum•Plastic jar•Microwave•Humidified chamberProcedure Please refer to the following table.Step Action1Dewax paraformaldehyde- or formalin-fixed tissue sectionsaccording to standard procedures.2Place the slide(s) in a plastic jar containing 200 ml 0.1 MCitrate buffer, pH 6.0.3•Apply 750 W (high) microwave irradiation for 1 min.•Cool rapidly by immediately adding 80 ml double dist.water (20–25°C).•Transfer the slide(s) into PBS (20–25°C).DO NOT perform a proteinase K treatment!4Immerse the slide(s) for 30 min at 15–25°C in Tris-HCl, 0.1 MpH 7.5, containing 3% BSA and 20% normal bovineserum.5Rinse the slide(s) twice with PBS at 15–25°C.Let excess fluid drain off.6Add50µl of TUNEL reaction mixture on the section and.Note: For the negative control add 50 µl Label solution.7Incubate for 60 min at 37°C in a humidified atmosphere in thedark.8•Rinse slide(s) three times in PBS for 5 min each.•Samples can be analyzed in a drop of PBS under a fluores-cence microscope at this state. Use an excitation wave-length in the range of 450–500 nm and detection in therange of 515–565 nm (green).3.4 SignalconversionAdditional equipment and solutions required •Washing buffer: PBS•Humidified chamber•Parafilm or coverslip•DAB Substrate* (Cat. No. 1 718 096) or alternative POD substrate •Mounting medium for light microscopyProcedure Please refer to the following table.Step Action1Dry area around sample.2Add50␮l Converter-POD (vial 3) on sample.Note: To ensure a homogeneous spread of Converter-PODacross cell monolayer and to avoid evaporative loss, samplesshould be covered with parafilm or cover slip during incuba-tion.3Incubate slide in a humidified chamber for 30 min at 37°C.4Rinse slide 3× with PBS.5Add 50–100 ␮l DAB Substrate or alternative POD substrates.6Incubate slide for 10 min at 15–25°C.7Rinse slide 3× with PBS.8Mount under glass coverslip (e.g. with PBS/glycerol) and ana-lyze under light microscope.Alternative: Samples can be counterstained prior to analysisby light microscope.4. Appendix4.1 Trouble-shootingThis table describes various troubleshooting parameters. Problem Step/Reagent ofProcedurePossible cause RecommendationNonspecific labeling Embedding of tissue UV-irradiation forpolymerization ofembedding material(e.g. methacrylate)leads to DNA strandbreaksTry different embedding materialor different polymerizationreagent.Fixation Acidic fixatives (e.g.methacarn, Carnoy’sfixative)•Try 4% buffered paraformal-dehyde.•Try formalin or glutaralde-hyde.TUNEL reaction TdT concentration toohighReduce concentration of TdT bydiluting it 1:2 up to 1:10 withTUNEL Dilution Buffer (Cat. No.1 966 06).Converter solution Endogenous PODactivityBlock endogenous POD byimmersing for 10 min in 3%H2O2 in methanol prior to cellpermeabilisation.Non-specific bindingof anti-fluorescein-POD•Block with normal anti-sheepserum.•Block for 20 min with PBScontaining 3% BSA.•Reduce concentration ofconverter solution to 50%. Nucleases Some tissues (e.g.smooth muscles)show DNA strandbreaks very soon aftertissue preparation•Fix tissue immediately afterorgan preparation.•Perfuse fixative through livervein.Some enzymes arestill activeBlock with a solution containingddUTP and dATP.continued on next pageHigh back-ground Fixation Formalin fixation leadsto a yellowish stainingof cells containingmelanin precursorsTry methanol for fixation buttake into account that this mightlead to reduced sensitivity.TUNEL reaction Concentration oflabeling mix is toohigh for mamma car-cinomaReduce concentration of label-ing mix to 50% by diluting withTUNEL Dilution Buffer (Cat. No.1 966 006).Converter solution Endogenous PODactivityBlock endogenous POD byimmersing for 10 min in 3%H2O2 in methanol prior to cellpermeabilisation.Non-specific bindingof anti-fluorescein-POD•Block with normal anti-sheepserum.•Block for 20 min with PBScontaining 3% BSA.•Reduce concentration ofconverter solution to 50%. Sample Mycoplasma contami-nationMycoplasma detection Kit (Cat.No. 1 296 7449).Highly proliferatingcellsDouble staining e.g. withAnnexin-V-Fluos (Cat. No. 1 828681).Note: Measuring via microplatereader not possible because oftoo high background.Low labeling Fixation Ethanol and methanolcan lead to low label-ing (nucleosomes arenot cross-linked withproteins during fixa-tion and are lost dur-ing the proceduresteps)•Try 4% buffered paraformal-dehyde.•Try formalin or glutaralde-hyde.Extensive fixationleads to excessivecrosslinking of pro-teins•Reduce fixation time.•Try 2% buffered paraformal-dehyde.Permeabilisation Permeabilisation tooshort so that reagentscan’t reach their tar-get molecules•Increase incubation time.•Incubate at higher tempera-ture (e.g. 15–25°C).•Try Proteinase K (concentra-tion and time has to be opti-mized for each type oftissue).•Try 0.1 M sodium citrate at70°C for 30 min.continued on next pageProblem Step/Reagent ofProcedure Possible cause Recommendation4.1Trouble-shooting, continuedParaffin-embedding Accessibility forreagents is too low •Treat tissue sections afterdewaxing with Proteinase K (concentration, time andtemperature have to be opti-mized for each type of tis-sue).•Try microwave irradiation at370 W (low) for 5 min in200ml 0.1 M Citrate bufferpH 6.0 (has to be optimizedfor each type of tissue).No signal on positive control DNase treatment Concentration ofDNase is too low•For cryosections apply 3 U/mlDNase I, grade I.•For paraffin-embedded tissuesections apply 1500 U/mlDNase I, grade I.•In general, use 1 U/mlDNase I, grade I, dissolved in10 mM Tris-HCl pH 7.4 con-taining 10 mM NaCl, 5 mMMnCl2, 0.1 mM CaCl2, 25 mMKCl and incubate 30 min at37°C.•Alternative buffer 50 mMTris- HCl pH 7.5 containing1mM MgCl2 and 1 mg/mlBSA.Weak sig-nals Counterstaining Not suitable dye•Counterstaining with 5%methyl green in 0,1 M veronalacetate, pH 4.0 or Hematoxi-lin is possible (43).•Double-staining with propid-ium iodide is possible butonly for detection of morpho-logical cell changes.Problem Step/Reagent ofProcedure Possible cause Recommendation4.1Trouble-shooting, continued4.2 References1Gavrieli, Y., Sherman, Y. & Ben-Sasson, S. A. (1992) J. Cell Biol. 119, 493–501.2Gorczyca, W., Gong, J. & Darzynkiewicz, Z. (1993) Cancer Res. 53, 1945–1951.3Gorczyca, W. et al. (1993) Leukemi a 7, 659–670.4Gold, R. et al. (1994) Lab. Invest. 71, 219.5Gorczyca, W. et al. (1994) Cytometry 15, 169–175.6Sgonc, R. et al. (1994) Trends Genetics 10, 41–42.7Schmied, M. et al. (1993) Am. J. Pathol. 143, 446–452.8Wyllie, A. H. et al. (1980) Int. Rev. Cytol. 68, 251.9Kerr, J. F. R. et al. (1972) Br. J. Cancer 26, 239–257.10Duvall, E. & Wyllie, A. H. (1986) Immunol. To day 7, 115.11Compton, M. M. (1992) Canc. Metastasis Rev. 11, 105–119.12Allen, P. D., Bustin, S. A. & Newland, A. C. (1993) Blood Reviews 7, 63–73.13Cohen, J. J. & Duke, R. C. (1992) Annu. Rev. Immunol. 10, 267–293.14Clarke, P. G. H. (1990) Anat. Embryol. 181, 195–213.15Johnson, E. M. & Deckwerth, T. L. (1993) Annu. Rev. Neurosci. 16, 31–46.16Batistatou, A. & Greene, L. A. (1993) J. Cell Biol. 122, 523–532.17Strange, R. et al. (1992) Development 115, 49–58.18Carson, D. A. & Ribeiro, J. M. (1993) Lancet 341, 1251–1254.19Edgington, S. M. (1993) Biotechnology 11, 787–792.20Gougeon. M.-L. & Montagnier, L. (1993) Science 260, 1269–1270.21Hickman, J. A. (1992) Cancer Metastasis Rev. 11, 121–139.22Afanasyev, V. N. et al. (1993) Cytometry 14, 603–609.23Bryson, G. J., Harmon, B. V. & Collins, R. J. (1994) Immunology Cell Biology 72,35–4124Darzynkiewicz, Z. et al. (1992) Cytometry 13, 795–808.25Ando, K. et al. (1994) J. Immunol. 152, 3245–3253.26Berges, R. R. et al. (1993) Proc. Natl. Acad. Sci. USA 90, 8910– 8914.27Gorczyca, W. et al. (1992) Int. J. Oncol. 1, 639–648.28Gorczyca, W. et al. (1993) Exp. Cell Res. 207, 202–205.29Billig, H., Furuta, I. & Hsueh, A. J. W. (1994) Endocrinology 134, 245–252.30MacManus, J. P. et al. (1993) Neurosci. Lett. 164, 89–92.31Mochizuki, H. et al. (1994) Neurosci. Lett. 170, 191–194.32Oberhammer, F. et al. (1993) Hepatology 18, 1238–1246.33Portera-Cailliau, C. (1994) Proc. Natl. Acad. Sci. USA 91, 974 –978.34Preston, G. A. et al. (1994) Cancer Res. 54, 4214–4223.35Weller, M. et al. (1994) Eur. J. Immunol. 24, 1293–1300.36Zager, R.A. et al. (1994) J. Am. Soc. Nephrol. 4, 1588–1597.37Cohen, G. M. et al. (1992) Biochem. J. 286, 331–334.38Collins, R. J. et al. (1992) Int. J. Rad. Biol. 61, 451–453.39Sei, Y. et al. (1994) Neurosci. Lett. 171, 179–182.40Ansari, B. et al. (1993) J. Pathol. 170, 1–8.41Gold, R. et al. (1993) J. Histochem. Cytochem. 41, 1023–1030.42Negoescu, A. et.al. (1998) Biochemica3, 34-41.43Umermura, S. et al. (1996) J. Histochem. Cytochem. 44, 125-132 .。

北京普利莱基因技术有限公司DOTAP真核细胞转染试剂使用说明说明书

DOTAP真核细胞转染试剂使用说明C1510描述：DOTAP是一种阳离子脂质体，可与DNA或RNA形成稳定的转染复合物进入细胞，并将核酸释放入细胞内。

它以可靠性和高效性著称，是广泛使用的商品化转染试剂。

我们的DOTAP真核细胞转染试剂是由DOTAP 和中性辅助脂质以特定比例融合制备而成的单层脂质体悬液。

这种制备方法增加了脂质体对真核细胞转染的高效性、广谱性、低毒性和可靠性，并使试剂在4°C储存至少稳定12个月。

DOTAP可高效转染多种细胞，甚至在低浓度血清环境也可工作良好。

它属于可被生物降解的脂质体因而细胞毒性明显降低，其转染效率高于Sigma公司的DOTAP单体转染试剂，与Invitrogen的LipofectAMINE相当，但其价格仅相当同类试剂的1/3。

转染时只需将稀释的DOTAP试剂与DNA溶液混合并室温放置15分钟即可加入细胞。

1ml DOTAP可转染100-500µg DNA或50-100只35mm培养皿或250-1000孔24孔板细胞。

颜色：清亮或略呈白色胶体溶液。

储存：4°C储存12个月。

切勿冻存。

适用：将DNA、RNA、寡聚核苷酸转入真核细胞。

适用于大多数传代或原代细胞。

转染步骤：以生长于24孔板的一个孔内的贴壁细胞为例，使用其它规格培养皿参见表一。

细胞准备：转染前一天传0.5-2x105细胞于24孔板内，加1ml正常培养基培养。

在光镜下观察细胞，当细胞群覆盖培养瓶皿生长表面的85-95%时，为DOTAP转染的最佳时机。

这通常需要18-24小时，但依细胞类型和接种量而变。

注意：传代时接种过多的细胞，100%长满的细胞的转化效率明显降低制备转染复合物：对特定细胞类型来说，应该优化加入DNA(µg)和DOTAP(µl)比率和绝对量，参见后面附表。

推荐DNA和DOTAP 的初始比例为1:4。

DNA(µg)：DOTAP(µl)＝1:2～1:8转染实际上是人为造成细胞对外源物质的高摄取状态，因此过量摄入DNA和转染试剂将导致细胞毒性而降低转染效率。

DIBAL-H选择还原酯基而不影响苄溴

1250J.Med.Chem.2010,53,1250–1260DOI:10.1021/jm901530bSynthesis and Structure-Activity Relationships of Azamacrocyclic C-X-C Chemokine Receptor4 Antagonists:Analogues Containing a Single Azamacrocyclic Ring are Potent Inhibitors of T-Cell Tropic(X4)HIV-1ReplicationGary J.Bridger,*,†Renato T.Skerlj,†,)Pedro E.Hernandez-Abad,‡David E.Bogucki,†Zhongren Wang,†Yuanxi Zhou,†Susan Nan,†Eva M.Boehringer,†Trevor Wilson,†Jason Crawford,†Markus Metz,†,)Sigrid Hatse,§Katrien Princen,§Erik De Clercq,§and Dominique Schols§†AnorMED Inc.now Genzyme Corporation,500Kendall Street,Cambridge,Massachusetts02142,‡Johnson Matthey Pharmaceutical Research,1401King Road,West Chester,Pennsylvania19380,and§Rega Institute for Medical Research,Katholieke Universiteit Leuven, Minderbroedersstraat10,B-3000Leuven,Belgium.)Genzyme Corp.,153Second Avenue,Waltham,Massachusetts02451.Received October15,2009Bis-tetraazamacrocycles such as the bicyclam AMD3100(1)are a class of potent and selective anti-HIV-1agents that inhibit virus replication by binding to the chemokine receptor CXCR4,the coreceptor for entryof X4viruses.By sequential replacement and/or deletion of the amino groups within the azamacrocyclic ringsystems,we have determined the minimum structural features required for potent antiviral activity in thisclass of compounds.All eight amino groups are not required for activity,the critical amino groups on a perring basis are nonidentical,and the overall charge at physiological pH can be reduced without compromisingpotency.This approach led to the identification of several single ring azamacrocyclic analogues such asAMD3465(3d),36,and40,which exhibit EC50’s against the cytopathic effects of HIV-1of9.0,1.0,and4.0nM,respectively,antiviral potencies that are comparable to1(EC50against HIV-1of4.0nM).Moreimportantly,however,the key structural elements of1required for antiviral activity may facilitate the designof nonmacrocyclic CXCR4antagonists suitable for HIV treatment via oral administration.IntroductionThe development of antiviral agents that inhibit alternative targets in the HIV a-replicative cycle remains an important goal in order to alleviate the side effects of currently approved agents or to overcome the problem of drug resistance.In this regard,we have focused on the development of compounds that inhibit CXCR4,the coreceptor used by T-tropic(T-cell tropic)viruses for fusion and entry of HIV into target cells of the immune system.The corresponding chemokine receptor CCR5is used by M-tropic(macrophage tropic)viruses and has been associated with the early stages of infection and replication in HIV-positive patients.1,2The transition from M-tropic to T-tropic(or dual/mixed-tropic)virus during the course of HIV infection in approximately50%of patients is associated with a faster CD4þT-cell decline and a more rapid disease progression.3-5Recently,we reported the results of clinical trials with our prototype CXCR4antagonist AMD31006-8(1)and an orally bioavailable CXCR4antagonist,(S)-N0-(1H-benzimidazol-2-ylmethyl)-N0-(5,6,7,8-tetrahydroquinolin-8-yl)butane-1,4-dia-mine(AMD070).9-11When administered to HIV positive patients whose virus was confirmed to use CXCR4for viral entry,both agents were able to suppress the replication of CXCR4and dual-tropic strains of HIV.Similarly,the CCR5 antagonist Maraviroc suppresses replication of HIV-1that exclusively uses CCR5for entry12and was recently approved by the FDA for combined antiretroviral therapy in treatment-experienced patients.13A combination of CCR5and CXCR4 antagonists for treatment of dual/mixed-tropic HIV infection is therefore highly desirable.Beyond its use as a coreceptor for HIV,the CXCR4 chemokine receptor has a more fundamental role in the trafficking of white blood cells,which broadly express CXCR4.14,15A member of the superfamily of G-protein coupled receptors,the interaction of CXCR4and its ligand, stromal cell-derived factor-1(SDF-1),plays a central role in the homing and retention of cells within the bone marrow microenvironment.16Consistent with these observations,ad-ministration of1to healthy volunteers caused a dose-depen-dent leukocytosis6,7that in subsequent studies was shown to include the mobilization of CD34þstem and progenitor cells suitable for hematopoietic stem cell transplantation.17-20The ability of analogues of1to mobilize progenitors correlated with their in vitro capacity to inhibit SDF-1binding to CXCR4.21Because of the need for parenteral administration, 1was developed in combination with granulocyte colony-stimulating factor(G-CSF)to mobilize hematopoietic stem cells to the peripheral blood for collection and subsequent autologous transplantation in patients with non-Hodgkin’s lymphoma(NHL)and multiple myeloma(MM).22-25Plerix-afor(1)was approved by the FDA in December2008.We have previously reported the structure-activity rela-tionships of anti-HIV bis-azamacrocycles and their transition*To whom correspondence should be addressed.Phone:617-429-7994.Fax:617-768-9809.E-mail:gary.bridger@.Ad-dress:Gary J.Bridger,Genzyme Corporation,55Cambridge Parkway,Cambridge MA02142.a Abbreviations:HIV,Human Immunodeficiency Virus;CXCR4,C-X-C chemokine receptor4;CCR5,C-C-R chemokine receptor5./jmc Published on Web12/31/2009r2009American Chemical SocietyArticle Journal of Medicinal Chemistry,2010,Vol.53,No.31251 metal complexes in detail.26-28Because of the commonstructural features between a doubly protonated cyclam(1,4,8,11-tetraazacyclotetradecane)ring present in1(at phy-siological pH)and a kinetically labile transition metal com-plex of cyclam with an overall charge ofþ2,we proposed thatboth structural motifs may bind to the CXCR4receptorthrough interactions with amino acid residues containingcarboxylate groups.29We have subsequently shown via direc-ted mutagenesis of the aspartate and glutamic acid residues inCXCR4that binding of1and related analogues to the seventransmembrane,G-protein coupled receptor is highly depen-dent upon the amino acids Asp171and Asp262,located intransmembrane region(TM)-IV and TM-VI at each end ofthe main ligand binding crevice of the receptor.30-35Mutationof either aspartic acid to aspargine significantly reduced theability of1to inhibit binding of radiolabeled stromal cellderived factor-1R(125I-Met-SDF-1R).More importantly,however,U87cells stably transfected with CD4and themutant coreceptors CXCR4[D171N]and CXCR4[D262N]were less effective at supporting infection of the CXCR4-usingHIV-1strain NL4.3compared to the wild-type receptor andthe double mutant CXCR4[D171N,D262N]completely failedas a coreceptor for HIV infection.31Correspondingly,theability of1to inhibit HIV-1infection via CXCR4[D171N]andCXCR4[D262N]was also diminished,thereby confirmingthat1binds in a region of the receptor that is critical for X4HIV-1coreceptor function.We have also reported that binding of the bis-Zn,Ni,andCu complexes of1were also dependent upon D171and D262of the receptor.36In a similar manner to1,the transitionmetal complexes were found to be less effective inhibitors of125I-Met-SDF-1R binding to the mutant receptors CXCR4-[D171N]and CXCR4[D262N]compared to the wild-typereceptor.Incorporation of Zn,Ni,or Cu into the cyclam ringsof1increased the affinity to the wild-type CXCR4receptor,but the enhancement was selectively eliminated by substitu-tion of Asp262.Supporting physiochemical evidence for theinteraction of acetate(carboxylates)with metal complexes ofazamacrocycles,including1,has been recently reported.37,38In the current study,we determine the minimum struc-tural features of1required for potent antiviral activity, leading to the identification of the single azamacrocyclic ring analogue AMD346532,33,39,40(3d)and ultimately the design of nonmacrocyclic,orally biovailable CXCR4an-tagonists.11,41,42Given the growing body of evidence that the CXCR4/SDF-1interaction is involved in regulating several human malignancies,43-45CXCR4antagonists may have additional therapeutic applications in addition to HIV treatment.ChemistryAnalogues containing a single1,4,8,11-tetraazacyclotetra-decane(cyclam)ring were prepared by modifications to previously published routes26,29as shown in Scheme1.Reac-tion of the selectively protected tris-diethylphosphoramidate (Dep)cyclam ring(2a)with R,R-dibromo-p-xylene in aceto-nitrile containing potassium carbonate gave the desired bro-momethyl intermediate(2b).Reaction of the bromide with an excess of the requisite amine,followed by deprotection of the Dep-groups with a saturated solution of hydrogen bromide in acetic acid at room temperature.gave analogues3a-i as the corresponding hydrobromide salts.To prepare analogues of3d in which the cyclam ring was replaced by a series of14-membered azamacrocyclic rings,we prepared a series of selectively protected macrocyclic ring systems containing a single(unprotected)secondary amine. This approach ensures the regiochemical outcome of the reaction with a benzylic halide during final construction (as shown in Scheme6).The syntheses of appropriate pre-cursors are shown in Schemes2-5.To incorporate fluorine groups at the desired position in the macrocyclic ring,suitably fluorinated bis-electrophiles were prepared,starting from 4-oxo-heptanedioic acid diethyl ester(4)and heptane-1,4,7-triol(8)as depicted in Scheme2.Reaction of the ketone(4) with neat(diethylamino)-sulfur trifluoride46,47(DAST)at room temperature for12days gave the corresponding di-fluoro-intermediate(5)in43%yield.Reduction of the ester groups with LAH(to give the diol6),followed by derivatiza-tion with toluenesulfonyl chloride,gave the bis-electrophile (7)required for the impending macrocyclization reaction.The corresponding monofluorinated intermediate was prepared in a similar manner.Protection of the primary alcohols in8as the acetyl group using acetic anhydride gave the secondary alcohol9,which was rapidly(and virtually quantitatively) converted to the fluorinated intermediate(10)with DAST (2.0equiv)in dichloromethane.Removal of the acetyl pro-tecting groups with saturated ammonia in methanol,followed by reaction of the diol(11)with p-toluenesulfonyl chloride, Scheme1aa Reagents:(a)R,R0-dibromo-p-xylene,K2CO3,CH3CN,reflux;(b)amine,K2CO3,CH3CN,reflux;(c)HBr,acetic acid,room temp. Scheme2aa Reagents:(a)Et2NSF3(neat),room temp;(b)LAH,Et2O;(c)Ts-Cl,Et3N,CH2Cl2;(d)acetic anhydride,pyridine;(e)Et2NSF3, CH2Cl2,-78°C,then room temp;(f)NH3/MeOH,room temp;(g)Ts-Cl,Et3N,CH2Cl2.1252Journal of Medicinal Chemistry,2010,Vol.53,No.3Bridger et al.gave the desired bis-electrophile 12containing a single fluorine group.The selectively protected azamacrocyclic rings were pre-pared via directed combinatorial macrocyclization of bis-2-nitrobenzenesulfonamides 48(Ns)(15a -c ,16a -c ,18)with bis-electrophiles (7,12,17)using previously optimized condi-tions 28(Scheme 3).To incorporate a phenyl or heterocyclic ring into the macrocycle,the corresponding bis-2-nitrobenze-nesulfonamide (15a -c )was prepared from the bis-aminoethyl intermediates 28(13a -c )by reaction with nosyl chloride (Et 3N,CH 2Cl 2).Similarly,16a ,b were obtained by reac-tion of commercially available intermediates 14a ,b with nosyl chloride or in the case of 16c (X=S)by reduction of 3,30-thiodipropionitrile with BH 33Me 2S and reaction of the intermediate diamine (14c )with nosyl chloride to give 16c .Macrocyclization was accomplished by dropwise addition of a DMF solution of the bis-electrophile to a DMF solution of the bis-2-nitrobenzenesulfonamide containing Cs 2CO 3maintained at a temperature of 80°C.Standard workup,followed by purification of the crude product by column chromatography on silica gel,gave the desired macrocycles 19a -c ,20a -c ,and 21a ,b in yields of 19-55%.Reaction of theintermediates from above with HBr/acetic acid at room temperature gave 22a -c ,23a -c ,and 24a ,b ,respectively.Because of synthetic convenience,we also prepared the selectively protected “isomers”of 22a ,b and 23a in which the alternative secondary amine was available for the alkylation reaction.We reasoned that reaction of 19a ,b and 20a with approximately 1equiv of thiophenol 49(our reagent of choice for nosyl deprotections)may allow pseudoselective deprotec-tion of a single nosyl group,leaving the Dep group intact.After some optimization,we found that reaction of 19a ,b and 20a with 0.8equiv of thiophenol and potassium carbonate in DMF (or acetonitrile)gave the precursors 25and 26a ,b in manageable,albeit modest yields (20-50%)following col-umn purification on silica gel (Scheme 4).Finally,the inter-mediates 27a ,b and 28(Scheme 5)were synthesized as recently described by palladium(0)catalyzed coupling of organozinc iodide reagents with bromopyridines.50Having completed the series of selectively protected aza-macrocycles,we proceeded to completion of the desired analogues by straightforward installation of the right-hand portion containing the aminomethyl pyridine moiety.As shown in Scheme 6,this was accomplished in all cases by direct alkylation of the available secondary amine of the macrocycle with the benzylic chlorides 34a ,b .Intermediate 34a was prepared in four steps from 4-bromomethyl benzoic acid methyl ester (29)and 2-aminomethylpyridine (31):con-version of 31to the 2-nitrobenzenesulfonamide 32,followed by alkylation with the benzyl bromide 30(obtained by reduc-tion of 29with DIBAL-H)gave the desired alcohol 33.As previously reported,28reaction of benzylic alcohols such as 33with methanesulfonyl chloride gave the chloride 34a rather than the corresponding mesylate,presumably via in situ nucleophilic substitution of the initially formed mesylate with chloride.Intermediate 34b (Scheme 6)containing a Dep-protecting group was prepared by an alternative synthesisScheme 3aaReagents:(a)Ns-Cl,Et 3N,CH 2Cl 2;(b)Cs 2CO 3,DMF,80°C;(c)HBr(g),AcOH,room temp.Scheme4Scheme5Article Journal of Medicinal Chemistry,2010,Vol.53,No.31253(procedures in Supporting Information).Alkylation of the available secondary amine of the macrocycles with 34a (or 34b )in CH 3CN in the presence of K 2CO 3gave the penultimate intermediates 35a -n .Deprotection of the nosyl groups with thiophenol and K 2CO 3in DMF gave the free base of the desired analogues,which in the vast majority of cases were converted to the corresponding hydrobromide salts.For analogues derived from the macrocyclic precursors 25and 26a ,b ,the intermediates isolated prior to the deprotection also contained a residual Dep group in addition to nosyl groups.For compound 45,we found that conversion to the hydro-bromide salt using a saturated solution of HBr in acetic acid resulted in concomitant deprotection of the remaining Dep group to obtain compound 45.For compounds 44and 46,the residual Dep group was removed prior to nosyl deprotection and salt formation.The thioether analogue 41a was also used to prepare the corresponding sulfoxide and sulfone analogues for antiviral evaluation as shown in Scheme 7.Initially,we globally protected the amino groups of 41a with Boc and subjected this intermediate to oxidation with oxone in MeOH 51at -10°C to give a mixture of the sulfoxide and sulfone that were separated by column chromatography on silica gel.However,while deprotection of the Boc groups with simulta-neous conversion to the hydrobromide salt proceeded without incident for the sulfone (to give 41c ),we found that deprotec-tion of the corresponding sulfoxide led to substantial reduc-tion and hence recovery of the starting analogue 41a .To overcome this problem,the sulfoxide was synthesized by direct oxidation of 41a with 1equiv of oxone in MeOH to give 41b in a 21%isolated yield and was subsequently tested as the free base in antiviral assays.Finally,we prepared a short series of analogues containing a carbon atom in place of a tertiary nitrogen group at the ring junction.To economize on the number of synthetic steps,weelected to synthesize the dimesylate 54(Scheme 8),an inter-mediate that could be commonly used for the synthesis of multiple analogues via macrocylization with the bis-2-nitro-benzenesulfonamide precursors already in our possession (namely 15a ,16a ,b from Scheme 3).Intermediate 54was prepared from the commercially available starting material bromo-p -tolunitrile via a double one-carbon homologation of the malonate 51,followed by derivatization to gave the requisite bis-methanesulfonate 54.Macrocyclizations of 54with bis-sulfonamides 15a and 16a ,b were performed as described above.Deprotection of the nosyl groups followed by conversion to the corresponding hydrobromide salts gave analogues 56and 58a ,b .DiscussionHaving previously established the optimum ring size and distance between the amines of both aliphatic andScheme 6a aReagents:(a)DIBAL-H,CH 2Cl 2;(b)Ns-Cl,Et 3N,CH 2Cl 2;(c)K 2CO 3,CH 3CN,60°C;(d)Ms-Cl,Et 3N,CH 2Cl 2;(e)K 2CO 3,CH 3CN,80°C;(f)R =Ns:thiophenol,K 2CO 3,DMF,or R =Dep:HBr(g),AcOH,room temp.Scheme 7aaReagents:(a)oxone,MeOH,-10°C;(b)(Boc)2O,THF;(c)HBr(g),AcOH,room temp.Scheme 8aaReagents:(a)NaH,R -bromo-tolunitrile,THF;(b)LiAlH 4,THF;(c)Ns-Cl,Et 3N,CH 2Cl 2;(d)2-picolyl chloride,Et 3N,K 2CO 3,KBr,CH 3CN,reflux;(e)Ms-Cl,Et 3N,CH 2Cl 2;(f)cetyltrimethyammonium bromide,NaCN,benzene,H 2O,reflux;(g)conc HCl/AcOH (4:1),reflux;(h)BH 3.Me 2S,THF;(i)Ms-Cl,Et 3N,CH 2Cl 2;(j)Cs 2CO 3,DMF,80°C;(k)thiophenol,K 2CO 3,CH 3CN (or DMF),40°C.1254Journal of Medicinal Chemistry,2010,Vol.53,No.3Bridger et al.pyridine-fused bis-tetraazamacrocycles required for potent X4anti-HIV activity,we designed a series of compounds to address the question of structural redundancy.The prototype bis-macrocycle 1has a center of symmetry and contains eight amino groups,of which four are positively charged at phy-siological pH.In the current study,we aimed to answer two specific questions:(1)Are all four positive charges required for potent anti-HIV activity?(2)On a per ring basis,what are the minimum structural requirements for activity?Assuming that the structural requirements are not iden-tical for both rings of 1,we reasoned that the simplest replacement for a single tetraaza-macrocyclic ring would be a pseudo diamine-segment,representing the first two amino groups of the macrocyclic ring from the point of attachment at the benzylic position.A judicious choice of “diamine”would also reduce the overall charge to þ1.Having previously established that the optimum distance between the first two amino groups was a two-carbon unit,we prepared a series of aminomethyl-substituted analogues in which the second amino group was a substituent upon an aromatic ring or part of a heterocyclic ring.In either case,the second p K a would be sufficiently low to prevent a second protonation at physiological pH.The compounds were tested for their ability to inhibit replication of HIV-1III B in MT-4cells,a strain of HIV-1that uses exclusively CXCR4for fusion and viral entry into target cells.The results are shown in Table 1.Compared to 1,the introduction of a benzylamine group (3a )in place of the azamacrocyclic ring substantially reduced anti-HIV potency,although the compound remained active at submicromolar concentrations.The concentration of 3a re-quired to inhibit HIV-1replication by 50%(the EC 50)was 0.49μM,which was approximately 100-fold higher than the 50%inhibitory concentration of 1.Aromatic amino groups at the 2-position (3b )or 4-position (3c )did not affect antiviral potency.Both 3b ,c exhibited comparable EC 50’s to the un-substituted benzyl group (3a ).However,we observed a sub-stantial increase in anti-HIV potency when the benzyl group was replaced by a pyridyl group (3d ).Compound 3d exhibited a 50%inhibitory concentration of 0.009μM,which was only ca.2-fold higher than the EC 50of 1.Furthermore,the 50%cytotoxic concentration (CC 50)of compound 3d in MT-4cells was greater than 112μM.Thus 3d exhibits a selectivity index of greater than 12000.The positional specificity of the pyridine-N in 3d was also examined.Replacement of the 2-pyridyl group with the 3-pyridyl (3e )or 4-pyridyl (3f )group had a detrimental effect on anti-HIV potency.For example,the EC 50’s of analogues 3e ,f were approximately 3orders of magnitude higher than the concentration of 3d required to inhibit HIV-1replication by 50%(the EC 50’s of 3e and 3f were 8.470and 9.977μM,respectively).Methylation of the amine in 3d (to give 3g )or extension of the connectivity to an aminoethyl pyridine group (to give 3h )also adversely affected the anti-HIV potency.Finally,we replaced the pyridine moiety with a comparable heterocycle of lower p K a than pyridine,namely the pyrazine group (3i ).Perhaps not surprisingly,the antiviral potency of analogue 3i was approximately comparable to the benzyl analogue 3a ,which did not contain a vicinal heterocycle nitrogen atom.With the optimized “right-hand”replacement for the aza-macrocycle ring of 1fixed as the 2-aminomethyl pyridine group,we then turned our attention to the “left-hand”ring.Needless to say,the mandatory synthesis of the symmetrical analogue in which both rings were replaced by a 2-amino-methyl pyridine group turned out to be a predictably fruitless exercise (EC 50was >250μM,data not shown).We therefore focused on systematically replacing individual amine groups of the left ring.As shown in Table 2,we first prepared an analogue in which the [14]aneN 4(cyclam)ring had been replaced by the optimized and equally suitable,py[iso -14]-aneN 4ring (to give compound 36).Consistent with the structure -activity relationship of py[iso -14]aneN 4bis-azama-crocycles,compound 36proved to be a potent inhibitor of HIV-1replication,exhibiting an EC 50of 0.001μM,that is,around 9-fold and 4-fold lower,respectively,than the con-centration of 3d or 1required to inhibit viral replication by 50%.Although the pyridine-N of the macrocyclic ring in 36was previously found to be critical for high antiviral potency,we reasoned that a precise determination of the pyridine-N contribution to potency could help redesign a less basic pounds 37and 38were then prepared to answer this question.Both analogues 37,containing a phenyl replacement and 38,containing an “exocyclic”pyridine fused group,retained reasonable anti-HIV potency (the EC 50’s of 37and 38were 0.040and 0.104μM,respectively)but were at least 40-to 100-fold less potent than analogue 36.So what role does the pyridine group play?At physiological pH,the overall charge of the py[iso -14]-aneN 4ring in 36is also þ2(in a similar manner to cyclam 52)and the likely protonation sequence is indicated in Figure 1A,based on the sequence reported by Delgado et al.53for similar 14-membered tetraazamacrocyclic rings contain-ing pyridine.Presumably,the secondary amino groups are predominantly protonated and the overall structure is stabi-lized by intramolecular hydrogen bond interactions from the adjacent hydrogen-bond acceptors,the pyridine and tertiary benzylic amine groups (while minimizing the elec-trostatic repulsion of two positive charges in a confined macrocyclic ring).This is confirmed by a conformational analysis of 36on B3LYP/6-31G*level followed by single point energy calculations.In the energetically most stable ring conformation (LMP2/6-311þG*þZPE),the pyridine nitro-gen forms two six-membered intramolecular hydrogen bond interactions with the two adjacent protonated nitrogens as shown in Figure 2.Potential five-membered intramolecular hydrogen bond interactions are formed with the tertiary amine.Table 1.Antiviral Activity of Single RingAzamacrocyclesnR 1R 2HIV-1(III B )EC 50(μM)MT-4cells CC 50(μM)3a 1H Ph0.4911603b 1H 2-amino-Ph 1.825243c 1H 4-amino-Ph 0.7172273d 1H 2-pyridine 0.009>1123e 1H 3-pyridine 8.470373f 1H 4-pyridine 9.977>2793g 1Me 2-pyridine 0.416383h 2H 2-pyridine 49.135>1103I 1H5-Me-pyrazine1.8957810.004>421ArticleJournal of Medicinal Chemistry,2010,Vol.53,No.31255The stabilization provided by this “shared”protonated structure could account for the high basicity of azamacrocyc-lic rings,as suggested by Kimura et al.54It did not seem unreasonable,therefore,that a potential role of the pyridine group is the contribution of a single intramolecular hydrogen-bond,which locks the conformation of the protonated aza-macrocyclic ring in manner that is beneficial to antiviral potency.To test this hypothesis,we prepared a series of analogues (depicted in Figure 1B,data in Table 2)in which the fused aromatic group had been removed and replaced by an aliphatic group,in some cases containing a hydrogen-bondacceptor at the key position “x,”the position occupied by the pyridine nitrogen in compound 36.Consistent with the hydrogen-bonding hypothesis,the alkyl analogue 39exhibited an anti-HIV potency that was compar-able to the phenyl and exocyclic pyridine analogues 37and 38(the EC 50’s of 37and 39,were 0.040and 0.043μM,re-spectively).This result categorically rules out the possibility that the conformational restrictions imposed by the fused aromatic groups in compounds 37,38were even partially responsible for the high potency of 36.However,incorpora-tion of a hydrogen-bond acceptor at position x (Figure 1B)in some cases restored activity comparable to 36.For example,the oxygen analogue 40exhibited an EC 50that was only 4-fold higher than the concentration of 36required to inhibit HIV-1replication by 50%(the EC 50of 40was 0.004μM).The corresponding thioether analogue 41a exhibited an EC 50of 0.013μM,which is approximately 3-fold higher than com-pound 40.Although the antiviral potency of the thioether analogue 41a compared to the ether analogue 41is greater than one would predict from the strength of the hydrogen-bond acceptor acceptor capabilities (thioether groups are considerably weaker H-bond acceptors than the oxygen inTable 2.Antiviral Activity of Single RingAzamacrocyclesFigure 1.Proposed hydrogen-bond structure of protonated aza-macrocycles.1256Journal of Medicinal Chemistry,2010,Vol.53,No.3Bridger et al.40),this result can be reconciled by considering the nature of the H-bond required;a six-membered intramolecular H-bond constrained by the macrocyclic ring (Figure 2).With the thioether compound 41a in hand,we also pre-pared the sulfoxide (41b )and sulfone (41c )analogues by direct oxidation of 41a .We reasoned that the oxygen atoms of the sulfoxide and sulfone are stronger H-bond acceptors than the sulfur atom of 41a and may consequently improve the anti-HIV potency.However,both 41b and 41c were considerably weaker antiviral agents,exhibiting 50%effective concentra-tions for inhibition of HIV-1replication that were at least 79-fold higher than the EC 50of 41a (the EC 50’s of 41b and 41c were 0.485and 11.878μM,respectively).The precise reason for the poor antiviral activity exhibited by analogues 41b ,c was unclear;although the sulfoxide and sulfone are more sterically demanding than the thioether and could induce a ring conformation that is detrimental to antiviral activity,we could not rule out the possibility that the H-bond acceptor oxygen is now “one-bond”outside of the ring,and the intramolecular H-bond itself induces an unfavorable confor-mation (a seven-membered ring H-bond in 41b ,c (Figure 2)compared to a six-membered in 41a ).To complete this series of compounds therefore,we decided to introduce the fluoro and difluoro substituents at position x (Figure 1B).Several reports have demonstrated that the fluoro group can partici-pate as an acceptor for intramolecular H-bonds,particularly within highly constrained ring structures.55-57This is also confirmed by our calculations,as shown in Figure 2.The fluoro (43)and difluoro (42)analogues were also attractive substituents for two other reasons:(1)the substituents would be situated at the fourth carbon from the adjacent amine group,thereby minimizing the affect on p K a ;(2)in a similar manner to the sulfoxide and sulfone,the H-bond acceptor would be one-bond outside of the macrocyclic ring.However in this case,because the fluorine atom in C -F groups is isostructural with hydrogen,a negative effect of the fluoro substituents on antiviral activity can only be attributed to an inappropriately positioned H-bond rather than steric requirements (that is,in the absence of an H-bond,we would expect the fluoro or difluoro analogues to exhibit an EC 50comparable to the methylene analogue 39).In antiviral test-ing,the fluoro (43)and difluoro (42)analogues displayed EC 50’s that were greater than 20-fold higher than the methy-lene analogue 39(the EC 50’s of 39,42,and 43were 0.043,0.920,and 1.239μM,respectively),confirming the negative consequences of an incorrectly positioned hydrogen-bond (Figure 2).Next,we focused on the sequence of aliphatic amine groups in the macrocyclic ring required for potent antiviral activity.By straightforward synthetic manipulation of our collection of ring systems,we prepared the structural isomers of analo-gues 36,37,and 39in which the side-chain (R,in Table 2)was connected to the alternative secondary amine group to give compounds 44,45,and 46.In antiviral testing,analogue 44was substantially less potent than its corresponding regioi-somer 39:the EC 50of 44was 11.131μM,which was approxi-mately 260-fold higher than the EC 50of 39.A similar loss of antiviral potency was observed with the phenyl analogue 46and its isomer 37(the EC 50’s of 46and 37were 14.106and 0.040μM,respectively).Interestingly,the loss of antiviral potency with the pyridine-fused isomer 45compared to 36was significant but not as substantial;the EC 50of 45was 0.063μM,around 60-fold higher than the concentration of 36required to inhibit HIV-1replication by 50%.There was a possibility,therefore,that while the “tri-aza”ring configura-tion required for potent antiviral activity is clearlyrepresentedFigure 2.Lowest energy conformations of compounds 36,40,41c ,and 42.View from top on a plane defined by three nitrogens and X (see Figure 1).Dashed lines indicate hydrogen bond interactions:the hydrogen bond acceptors in 36and 40are in one plane with the three nitrogens.This is not the case for 41c and 42.Bond angles:36:—(N 333H -N þ)=140.5°,122.4°,102.1°,108.4°.40:—(O 333H -N þ)=135.1°,141.5°;—(N 333H -N þ)=104.6°,102.8°.41c :—(O 333H -N þ)=112.8°,112.8°;—(N 333H -N þ)=108.2°,108.0°.42:—(F 333H -N þ)=142.2°,142.2°;—(N 333H -N þ)=114.7°,114.7°.。

Incucyte

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。

抗菌肽的生物学功能与作用机制研究进展

抗菌肽的生物学功能与作用机制研究进展徐林1，单安山2，邵长轩2，董娜2，刘宇飞1，叶禹3（1.哈尔滨体育学院运动人体科学学院，哈尔滨150008；2.东北农业大学动物科学技术学院，哈尔滨150030；3.哈尔滨二四二医院普外二科，哈尔滨150066）摘要：抗生素的长期大量使用导致耐药菌和超级细菌在世界范围内陆续出现，对环境安全和公共健康造成了极大的威胁。

抗菌肽具有免疫调节，广谱抗细菌、真菌、病毒、寄生虫、癌细胞等多方面的生物学作用。

而且抗菌肽的抗菌机制与传统抗生素不同，其主要是通过破坏细菌细胞膜来发挥抗菌活性，因此不易产生耐药性，使其具备成为新一代抗菌药物的潜质，受到农业、畜牧、食品、医药等多领域的广泛关注。

文章对国内外抗菌肽的研究进行了梳理，综述了抗菌肽在生物学功能、作用机制等方面的研究进展。

关键词：抗菌肽；生物学功能；作用机制；替抗中图分类号：S816.4文献标志码：A文章编号：1001-0084（2023）02-0009-05Advances in Biological Functions and Mechanisms ofAntimicrobial PeptidesXU Lin 1,SHAN Anshan 2,SHAO Changxuan 2,DONG Na 2,LIU Yufei 1,YE Yu 3（1.College of Sports and Human Sciences,Harbin Sport University,Harbin 150008,China;2.College of Animal Science and Technology,Northeast Agricultural University,Harbin 150030,China;3.Department of General Surgery,Harbin No.242Hospital,Harbin 150066,China ）Abstract:The long-term and extensive use of antibiotics has led to the widespread emergence of drug-resistant bacteria and super bacteria worldwide,posing a significant threat to environmental safety and public health.Antimicrobial peptides have a wide range of biological functions,such as immune regulation,killing bacteria,fungi,viruses,parasites,cancer cells and so on.Importantly,the antibacterial mechanism of antimicrobial peptides is different from that of the traditional antibiotics.They mainly exert antibacterial activity by destroying bacterial cell membranes,so they are not easy to produce drug resistance,which makes them have the potential to become a new generation of antibacterial drugs.Therefore,antimicrobial peptides are widely concerned in agriculture,animal husbandry,food,medicine and other fields.In this paper,the research progress of antimicrobial peptides in biologicalfunction and mechanism of action were reviewed.Key words:antimicrobial peptides,biological function,mechanism of action,antibiotic alternatives收稿日期：2022-07-18作者简介：黑龙江省自然科学基金资助项目（YQ2022C105）；黑龙江省省属本科高校基本科研业务费科研项目（2022KYYWF-FC04）*作者简介：徐林（1982—），男，黑龙江哈尔滨人，博士，讲师，研究方向为动物肠道菌群调控。

一种用于增强PD-L1单克隆抗体使用效果的抗体稀释液及其使用方法[发明专利]

专利名称：一种用于增强PD-L1单克隆抗体使用效果的抗体稀释液及其使用方法
专利类型：发明专利
发明人：朱俊轩,周文刚,陈宁,郑立谋
申请号：CN201811538730.1
申请日：20181214
公开号：CN109669040A
公开日：
20190423
专利内容由知识产权出版社提供
摘要：本发明公开了一种用于增强PD‑L1单克隆抗体使用效果的抗体稀释液及其使用方法，所述的抗体稀释液，它包括：PEG20000(0.1‑100g/L)，酪蛋白钠盐(1‑1000mg/L)，对乙酰氨基酚
(1‑1000mg/L)，牛血清蛋白(10‑10000mg/L)，氯化钠
(1‑1000mM)，0.01‑10％TritonX，0.01‑10％Proclin300，溶剂为水。

本发明抗体稀释液能够增强PD‑L1单克隆抗体使用效果。

申请人：厦门艾德生物医药科技股份有限公司
地址：361000 福建省厦门市海沧区鼎山路39号
国籍：CN
代理机构：厦门市首创君合专利事务所有限公司
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一种聚乙二醇修饰的抑制肿瘤坏死因子-α多肽及其应用[发明专利]

计算机科学与技术专业就业方向推荐及前景计算机科学与技术专业就业方向推荐计算机科学与技术专业具有不同的开设方向，可分为应用软件方向、网络技术方向和计算机工程(硬件)方向等。

与此相对应的，毕业生就业主要有四个方面：一是网络工程方向，可说就业前景良好，学生毕业后可以到国内外大型电信服务商、大型通信设备制造企业进行技术开发工作，也可以到其他企事业单位从事网络工程领域的设计、维护、教育培训等工作;二是软件工程方向，就业前景十分广阔，毕业后可以到国内外众多软件企业、国家机关以及各个大中型企事业单位的信息技术部门、教育部门等从事软件工程领域的技术开发、教学、科研及管理等工作，也可以继续攻读计算机科学与技术类专业研究生和软件工程硕士;三是通信方向，学生毕业后可到信息产业、财政、金融、邮电、交通、国防、大专院校和科研机构，从事通信技术和电子技术的科研、教学和工程技术工作;四是网络与信息安全方向，毕业生可为政府、国防、军队、电信、电力、金融、铁路等部门的计算机网络系统、信息安全领域进行管理和服务，并可继续攻读信息安全、通信、信息处理、计算机软件和其他相关学科的硕士学位。

计算机科学与技术专业就业前景现在我国是信息时代，很多的工作都是需要计算机来完成，未来可能是需要人工智能的，而总研发都是离不开计算机的，现在市场上很缺少这方面的高端人才。

短期内社会需求依然很大，计算机专业毕业生的就业市场前景宽广。

短期内社会需求仍然很大，计算机专业毕业生的就业市场前景广阔。

从全球IT行业的发展看，经过几年的低迷发展，IT行业已经走出低谷、大有东山再起之势，IT行业在国民经济发展中日益显现出蓬勃生机。

从中国情况看，从事计算机软件开发的人才远远低于发达国家。

美国从事计算机软件开发的人才达到180多万，印度达到90万，而中国从事计算机软件开发的人才不足40万。

这就说明，中国计算机软件人才短缺，这将严重束缚中国IT行业的发展，特别是直接影响到中国经济的发展和社会的进步。

具有降解K64荚膜型肺炎克雷伯菌胞外聚合物的能力的解聚酶[发明专利]

专利名称：具有降解K64荚膜型肺炎克雷伯菌胞外聚合物的能力的解聚酶
专利类型：发明专利
发明人：祝先潮,何平,李佳茵,杜娟
申请号：CN202010910803.6
申请日：20200902
公开号：CN112011525A
公开日：
20201201
专利内容由知识产权出版社提供
摘要：本发明涉及生物医疗领域，具体而言，涉及一种具有降解K64荚膜型肺炎克雷伯菌胞外聚合物的能力的解聚酶。

本发明所提供的解聚酶能够有效分解K64荚膜型肺炎克雷伯菌胞外聚合物，从而利于能够杀伤该菌的药物与该菌进行接触进而发挥药效。

申请人：上海瑞宙生物科技有限公司
地址：200135 上海市浦东新区中国(上海)自由贸易试验区高科中路1976号1幢B201室
国籍：CN
代理机构：广州华进联合专利商标代理有限公司
代理人：林青中
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血清哇巴因多克隆抗体的纯化及Fab2片段的制备

本文缩略语Ａｂｂｒｅｖａｔｉｏｎｓ英文缩写英文全称ＥＯＢＳＡＯＶＡＦｃＡＥＬＩＳＡＨＰＬＣＨＰＳＥＣＳＰⅧＳＤＳ—ＰＡＧＥＣＤＲＦＲＳＰＡＤＡＢＡｎｇＩＩＡＣＴＨＶｅＭＷＥｎｄｏｇｅｎｏｕｓＯｕａｂａｉｎＢｏｙｉｎｅＳｅｒｕｍＡｌｂｕｍｉｎＯｖａｌｂｕｍｉｎＦｒｅｎｄ’ＳＣｏｍｐｌｅｔｅＡｄｊｕｖａｎｔＥｎｚｙｍｅＬｉｎｋｅｄＩｍｍｕｎｏｓｏｒｂｅｎｔＡｓｓａｙＨｉｇｈＰｅｒｆｏｒｍａｎｃｅＬｉｑｕｉｄＣｈｒｏｍａｔｏｇｒａｐｈＨｉｇｈＰｅｒｆｏｒｍａｎｃｅＳｉｚｅＥｘｃｌｕｄｅＣｈｒｏｍａｔｏｇｒａｐｈｙＳｔｒｅｐｔａｖｉｄｉｎ／ＰｅｒｏｘｉｄａｓｅＳｏｄｉｕｍｄｏｄｅｃｙｌｓｉｃｌｆａｔｅｐｏｌｙａｃｒｙｌａｍｉｄｅｇｅｌｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓＣｏｍｐｌｅｍｅｎｔａｒｉｔｙ—ｄｅｔｅｒｍｉｎｇＦｒａｍｅｗｏｒｋＲｅｇｉｏｎＳｔａｐｈｙｌｏｃｏｃｃｕｓａｕｒｅｕｓＤｉａｍｉｎｏｂｅｎｚｉｄｉｎｅＡｎｇｉｏｔｅｎｓｉｎＩＩＡｄｒｅｎｏｃｏｒｔｉｃｏｔｒｏｐｈｉｎＶ０１ｕｍｅｅｌｕｔｅｄＭｏｌｅｃｕｌａｒＷｅｉｇｈｔ中文译名内源性哇巴因牛血清白蛋白卵清蛋白弗氏完全佐剂酶联免疫吸附法高效液相色谱高效液相排阻色谱过氧化酶标记的链酶卵白素十二烷基磺酸钠一聚丙烯酰铵凝胶电泳ｒｅｇｉｏｎ互补决定簇框架区葡萄球菌Ａ３，３－－－氨基苯联胺血管紧张索ＩＩ促肾上腺皮质激素洗脱体积分子量血清哇巴因多克隆抗体的纯化及兰ｌ盐２ｇ丘露的制备●＿＿＿＿＿＿＿－－＿＿＿一ＩＩ研究背景：内源性哇巴因是一种存在于人和动物体内的Ｎａ＋，Ｋ＊－ＡＴＰ酶抑制物，具有调节水、钠代谢，血管舒缩和心肌收缩等重要生理功能，还与高血压、心力衰竭、慢性肾功能不全等多种疾病的发生、发展密切相关。

目前，我们已经运用动物免疫的方法，制备出血清哇巴因多克隆抗体，并分别用硫酸铵盐析法、ＤＥＡＥ一纤维素柱层析法及盐析法＋ＤＥＡＥ－纤维素柱层析法对抗血清进行部分纯化，进而动物实验中发现高血压大鼠静注哇巴因多克隆抗降压效果，但降压效果持续时间较短，并且出现轻度的过敏体后反应研究目的：１．选用适宜的免疫周期和适宜的免疫剂量，免疫家兔制备出高效价的哇巴因多克隆抗体；２．用ＨＰＳＥＣ法纯化哇巴因多克隆抗体，并且制备出高纯度的哇巴因多克隆抗体；３．用胃蛋白酶酶解哇巴因多克隆抗体，制备Ｆ（ａｂ）ｚ片段；４．用ＨＰＳＥＣ法初步测定哇巴因多克隆抗体及其Ｆ（ａｂ）。

Fed-batchprocessdevelopment：分批补料工艺的发展

Fed-batch process developmentfor monoclonal antibody production with cellferm-pro®Authors: Anna Frison and Dr. Klaus Memmert, Novartis Pharma AG, Basel, Switzerland Published in Genetic Engineering News, Volume 22, Number 11, June 1, 20021 Introduction:Cost-effective large-scale production of monoclonal antibodies (mAb) is creating a strong demand for reliable and prompt development of highly productive, scalable processes. One of them is fed-batch, which is widely used for production of recombinant proteins, due to its operational simplicity, reliability, and flexibility in multipurpose implementation. The major advantage of fed-batch, comparing to batch, is the ability to increase maximum viable cell concentration, prolong culture lifetime, and allow product accumulation to a higher concentration.The maximisation of final product concentration in a hybridoma fed-batch process is a function of the Integral of Viable Cells Concentration (IVCC). It follows, that an increase of this variable through feeding strategy optimisation will boost the final product titer.Pre-defined feeding protocols, which are based on nutrient requirement estimations, e.g. stepwise or a sigmoid-based addition of nutrient concentrates are unlikely to meet the nutritional demands of cells growing in batch culture which vary with time and environmental conditions. It often leads to nutrient depletion or accumulation of substrates or metabolites to inhibitory levels (Zhou et al., 1996). A much better way for nutrient supply would be the online monitoring of the cell culture’s metabolic activity, and a real time control of nutrient feeding based on this parameter.The novel feeding protocol discussed here is based on the oxygen uptake rate (OUR) on-line estimation. OUR is a very important indicator for the metabolic activity of the biological system. In cell cultures it correlates with the glucose or glutamine consumption rate. In our experiments OUR was used as the control parameter for adapting the feed rate according to the cell culture’s requirements in real time. The experiments were performed using the novel cellferm-pro®system.2 cellferm-pro® system2.1 System’s assembly:The cellferm-pro® system (DASGIP AG, Juelich, Germany) consists of five modules as shown in Fig.1.Figure 1:cellferm-pro®, a parallel and fully controlled cultivation system(DASGIP AG, Juelich, Germany)The culture system comprises of a temperature controlled incubator with 4 or 8 vessels, placed on a magnetic stirrer platform, equipped with pH and pO2 electrodes, and feed and air supply/removal connections.The gassing system provides an individual mix of up to four gases to each culture vessel (e.g. compressed air, oxygen, nitrogen and carbon dioxide). O2 and CO2 gas concentration are controlled by feedback from pO2 and pH electrodes. There is an electronic mass flow control and a gas totalizer function for each vessel.The monitoring system simultaneously processes the signals from pH and pO2 electrodes, regulates pH and pO2 in the medium through adjustment of CO2 and O2 in the gas mixture to maintain the set-points, and determines the OUR online.The dosing system offers an individual and regulated delivery of a feed medium. The dosing proceeds according to user defined profiles or fully automated based on the online determined OUR (Metabolic Activity based feeding).The control system is based on Microsoft Windows. Here pH, pO2, gassing and dosing with user defined profiles are configured and the calibration of the electrodes and dosing system performed. The analyses of logged data are done using DASGIP’s ChartWizard for MS Excel®.2.2 Metabolic Activity Tool:A sophisticated algorithm computes the OUR online for each culture vessel of the cellferm-pro® system. Online OUR, supplemented with known concentration of one important feed medium component and pre-estimated ratio between substrate supply rate and oxygen uptake rate, allows an individual and automated addition of liquid media according to the actual metabolic activity of the cells.3 ExperimentalFed-batch processes were performed with the cellferm-pro® system using the Metabolic Activity tool, with an a priori determined ratio between the substrate supply rate and the oxygen uptake rate (Y S/Y O2). The criteria for the choice of an optimum Y S/Y O2 ratio were: optimum cell growth (maximum IVCC), maximum final product titer and minimum lactate produced per glucose consumed, since lactate is supposed to be one of the main inhibitors in hybridoma cell cultures.As a reference for the Metabolic Activity based fed-batch processes served a standard batch process, also performed in cellferm-pro®.3.1 Culture conditions:A recombinant mAb secreting GS-NS0 cell line has been used in the present work as a model system for suspension cell culture. All cultures were carried out in a proprietary serum-free, glutamine-free medium based on Iscove’s Modified Dulbecco’s Medium (IMDM, Amimed). For the automated Metabolic Activity based feeding a 10-fold concentrated basal medium was used. Additionally, 20-fold concentrated Iscove’s amino acids solution (IMDM/AA, Amimed) was dosed manually once a day during four subsequent days after the cell density reached 1x106 cells/mL. In the first cultivation days NH4HCO3 was dosed to the culture to maintain NH4+ concentrations at about 0,5 mM.The culture vessels used in the cellferm-pro® system were 1 L Spinner flasks, equipped with glass ball agitator, pH and pO2 sensors and sampling ports, with a minimum start volume of 300 mL and a maximum working volume of 600 mL.3.2 Analytic:The concentration of viable cells and viability were automatically determined using the Cedex® system (Innovatis GmbH, Bielefeld, Germany). The IVCC was calculated as described by Sauer et al. (2000).The concentration of the medium’s main components such as Glucose, Glutamine, Glutamate and Lactate, four ions (Na+, K+, Ca++, NH4+), together with pH, pO2, pCO2 and osmolarity were determined using a Nova Bioprofile TM 200 Analyzer (Nova Biomedical Corp., Waltham, MA, USA)4 Results and discussion4.1 Choice of an appropriate substrate supply/oxygen uptake ratioGlucose, one of the most important energy sources for mammalian cell cultures, was chosen as the medium component on which the ratio of substrate supply rate to oxygen uptake rate (Y S/Y O2) was based. The theoretical ratio between substrate and oxygen uptake rates is 0.17 mol/mol, as 1 mole glucose requires 6 mole oxygen for complete oxidation. Since several essential substrates, except glucose, could also be limiting, four different Y S/Y O2 ratios were tested around the theoretical one in the range 0.1-0.5 mol/mol. The results are presented in Fig.2.Figure 2:Choice of the ratio between substrate supply rate and OUR (Y S/Y O2), based on the three criteria: IVCC, final product titer and minimal ratio of lactate produced perglucose consumed (Y LP/Y GC).For the YS/YO2 of 0.1 mol/mol the smallest IVCC was observed as well as a significantly lower titer. In this case cell growth ceased before the maximal working volume could be reached, assuming cell starvation (data not shown). The highest product titer of 260 mg/l was observed at YS/YO2 = 0.2. The YLP/YGC ratio increased with increasing YS/YO2. The most excessivelactate accumulation was observed for the ratio Y S/Y O2 = 0.5 mol/mol, representing in this case a theoretical maximum. Based on these observations, the Y S/Y O2 ratio of 0.2 mol/mol was chosen for the OUR-based nutrient feeding. This ratio is close to the theoretical one of 0.17 mol/mol, indicating the balanced concentration of nutrients in the feed medium.4.2 Metabolic Activity based fed-batchFig.3 shows a Metabolic Activity based feeding profile with the corresponding cell density and mAb titer over the process time. No lag phase in cell growth was observed. Exponential cell growth was observed for the first four days followed by a quasi-stationary phase of about 5 days. After the maximal working volume was reached on day 9 Metabolic Activity based feeding stopped and was replaced by stepwise feeding. In this phase the system continued evaluating the volume to be dosed, however, was only able to feed when a sample volume was taken and registered in the system as a negative value which could be replaced. In this manner, the cells obtained about 6 mL of feed once a day. Under these conditions the cells were kept alive at a cell density above 1,0 x 106 cells/mL for a period of 12 days. After day 15 the cells died and mAb accumulation stopped.Figure 3:Metabolic Activity based fed-batch cultivation: feeding profile, cell density and mAb concentration.4.3 Comparison to standard batch cultureIn a reference batch experiment, also performed with cellferm-pro® (data not shown) the process duration was only 7 days, during which no quasi-stationary phase was observed. A comparison of the standard batch process to the Metabolic Activity based fed-batch process is presented in Fig.4. As a result of improved and balanced nutrient supply the process duration could not only be increased by 196 %, more importantly the IVCC was increased by 285%, and the final product titer was increased by 209%. As the culture volume after feeding was doubled (600 mL) against the standard batch volume (300 mL) the total amount of mAb obtained was increased by 519%.Figure 4: Comparison of process duration, IVCC, titer and yield of Metabolic Activity based fed-batch with batch process.5 ConclusionsThe ideal method to adapt nutrient feeding in real time to the changing requirements of a cell culture would be online determination of a key substrate, e.g. glucose, and to use this parameter as input variable for feeding control. Online glucose determination in a sterile environment, however, is still not feasible for industrial applications. Lately a new instrument, the cellferm-pro® system, and the related Metabolic Activity software tool was introduced which implemented the OUR online determination as described by Ruffieux et al. (1998) and Ducommun et al. (2000) as a measure for glucose consumption. Using this instrument OUR can be used as the input variable for substrate feeding control. We have demonstrated that with the cellferm-pro®system and the Metabolic Activity tool a balanced and controlled nutrient supply to the cell culture is possible and that significantly improved mAb titer and productivity can be achieved. The cellferm-pro® system allowed a high experimental throughput, due to the possibility of running up to 8 experiments in parallel under the same environmental conditions. Additionally, we could use the data acquired from the cellferm-pro® process for easy scale-up of the fed-batch process to larger cell culture systems such as a 20-L Wave TM bioreactor. The cellferm-pro®system is thus a powerful tool for process development, optimization and validation.6 References:Ducommun, P., Ruffieux, P.-A., Marison, M.-P. F.I., von Stockar, U. (2000). A new method for online measurement of the volumetric oxygen uptake rate in membrane aerated animal cell cultures. Journal of Biotechnology 78, 139-147.Ruffieux, P.-A., von Stockar, U., Marison, I. W. (1998). Measurement of volumetric (OUR) and determination of specific (qO2) oxygen uptake rates in animal cell cultures. Journal of Biotechnology 63, 85-95.Sauer, P. W., Burky, J. E., Wesson, M. C., Sternard, H. D., Qu, L. (2000). A high-yielding, generic fed-batch cell culture process for production of recombinant antibodies. Biotechnol. Bioeng. 67(5), 585-597.Zhou, W., Bibila, T., Glazomitsky, K., Montalvo, J., Chan, C., DiStefano, D., Munshi, S., Robinson, D., Buckland, B., Aunins, J. (1996) Large-scale production of recombinant mouse and rat growth hormone by fed-batch CS-NS0 cell cultures. Cytotechnology 22, 239-250.。

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