重组水蛭素Ⅲ多克隆抗体的制备

目的:制备重组水蛭素Ⅲ(rHV3)多克隆抗体。方法:分别用戊二醛法与碳二亚胺法处理抗原,电泳检测rHV3自身交联及与牛血清白蛋白(BSA)交联结果;分别免疫家兔、豚鼠和大鼠后取血清,双向免疫扩散试验测定效价。结果:rHV3交联BSA后作为抗原免疫豚鼠和家兔产生了抗体,抗体效价1∶16。结论:rHV3交联BSA作为rHV3抗原免疫家兔、豚鼠可以产生rHV3多克隆抗体。     

MBS和戊二醛连接hPTH载体特异抗体及载体抗体的比较研究*

目的:比较间-马来酰亚胺苯甲酸-N-羟基琥珀酰亚胺酯(MBS)和戊二醛连接人甲状旁腺激素(hPTH)载体后抗体的产生效果。方法:MBS法和戊二醛一步法分别将hPTH(1-84)与载体牛血清白蛋白(BSA)连接后作为免疫原免疫家兔;间接ELISA法比较两组兔中特异性hPTH(1-84)抗体和BSA抗体的产生,并使用免疫吸收和亲和层析吸附清除BSA抗体,对清除情况进行比较。结果:MBS组抗hPTH(1-84)抗体效价总体上高于戊二醛组,抗载体BSA抗体效价低于戊二醛组;MBS组在同一时间hPTH(1-84)抗体效价高于BSA抗体,戊二醛组获取的抗血清中BSA抗体效价则明显高于hPTH(1-84)抗体。MBS组经亲和层析吸附BSA抗体被清除完全,戊二醛组经免疫吸收和亲和层析吸附仍有残留BSA抗体。结论:采用MBS法连接半抗原载体制备hPTH(1-84)抗体,降低了载体抗体的影响,提高了hPTH(1-84)抗体的特异性,可应用于hPTH药理作用和代谢途径等研究工作中。     

雌酮人工抗原的制备与鉴定

目的 制备雌酮人工抗原. 方法 采用碳化二亚胺法将雌酮半抗原与载体蛋白牛血清清蛋白连接制备免疫耦联物, 用紫外分光光度法测定耦联率, 并测定兔抗体血清效价. 结果 测得耦联率比为12：1, 血清抗体效价为6.4×103.结论 该 方法 简便、 结果 准确可靠, 可应用于雌酮免疫原合成.     

黄芩苷完全抗原的合成鉴定及其多克隆抗体的制备

目的:研究黄芩苷作为一种半抗原分子,通过与载体蛋白偶联形成完全抗原后,使免疫动物血清中产生特异性抗体的可能性,为进一步建立黄芩苷的免疫分析测定方法提供实验依据。方法:采用活性酯法将黄芩苷(BAL)与牛血清白蛋白(BSA)偶联,制备黄芩苷完全抗原(BAL-BSA),并采用红外吸收光谱法、SDS-PAGE法、MALDI-TOF-MS法对其进行鉴定。用此完全抗原免疫动物制备抗血清,通过间接ELISA法检测其抗体效价。结果:完全抗原BAL-BSA中BAL与BSA的偶联比为3∶1;用此完全抗原免疫的家兔产生针对BAL的多克隆抗体,抗体效价最高可达1∶8000。结论:成功合成了BAL的完全抗原BAL-BSA,且该抗原具有良好的反应原性与免疫原性。     

氰基胍由氰基硫脲简便合成

氰基胍(4)是抗溃疡药西咪替丁(1)和抗高血压药吡那地尔(2)的重要组成部分,通常由相应的硫脲/脲制备,但多数要求分离碳化二亚胺或S-烷基异硫脲鎓盐中间体。本文报道应用水溶性的碳化二亚胺(WSC)由氰基硫脲(3)一步合成4的简便方法。 3和伯或仲胺于DMF中室温下加入1-(3-二甲氨基丙基)-3-乙基碳化二亚胺盐酸盐(WSC)(加料顺序对反应收率影响很大),搅拌反应30分钟,得相应4,7例收率60～85%。该法优于DCC法,而且3也并不都须分离,如异硫氰酸苯酯与氰氨基钠反应得3     

活泼酯法合成绿原酸人工抗原及其鉴定

目的人工合成绿原酸(chlorogenic acid,CGA)完全抗原并进行鉴定,为制备绿原酸单克隆抗体奠定基础。方法采用活泼酯法,将绿原酸分别与牛血清白蛋白(bovine serum albumin,BSA)和卵白蛋白(ovalbumin,OVA)进行偶联,用紫外扫描法、SDS-PAGE电泳法进行鉴定;间接ELISA法测定免疫小鼠血清抗体效价。结果经紫外扫描法和电泳法检测,CGA与载体蛋白成功偶联,免疫小鼠后获得的多抗血清效价达到1×106。结论成功合成绿原酸完全抗原,可用于绿原酸单克隆抗体的制备。     

两种方法制备的A群脑膜炎球菌多糖-破伤风类毒素结合物的物理化学及免疫学特性

作者采用两种化学偶联方法制备了A群脑膜炎球菌多糖(APS)-破伤风类毒素(π)结合物,检测其物理化学及免疫学特性,并用小鼠进行免疫原性研究。偶联法Ⅰ以溴化氰作偶联剂一步完成(简称1.2号),方法Ⅱ先在APS上引入甘氨酸(Gly)或6-氨基正己酸(6-aha)作隔离基团,并以N-乙基-N′-(3-二甲氨基丙基)碳化二亚胺(EDC)作偶联剂,分两步完成APS与π的共价结合,前者为3、4号,后者为5、     

制备百日咳无细胞菌苗的简易方法

作者应用染色配体层析法从百日咳杆菌的培养上清中提取百日咳毒素(PT)和丝状血凝素(FHA),并研究了用水溶性1-乙基-3-(3-二甲氨丙基)-碳化二亚胺(EDAC)进行类毒化的条件。应用SS培养基加环糊精和环糊精液体(CL)培养基制备PT和FHA。用Blue Sepharose层析从培养上清中提取抗原。SS培养基加环糊精产生的PT是FHA的2.5倍,     

化学交联抗原组成的无细胞百日咳疫苗的效力

在联合疫苗与多价疫苗研制过程中 ,抗原总量的增加可能是个问题 ,制备高效抗原可使疫苗的抗原含量减至最小。研究发现弱抗原性抗原经化学交联后能诱生抗体。为此作者在小鼠呼吸道感染模型中研究了化学交联抗原组成的无细胞百日咳疫苗的效力。　　作者将百日咳杆菌 Tohama株在改良的S- S液体培养基中静止培养 5天后 ,取上清液提纯百日咳杆菌丝状血凝素 ( FHA)、百日咳毒素 ( PT)、百日咳 6 9k Da外膜蛋白( PRN)。然后将上述三种抗原以 9∶ 2∶ 1的蛋白比混合 ,加入 2或 50 m M 1 -乙基 - 3- ( 3-二甲胺基丙基 )碳化二亚胺 ( EDC)和 0 …     

盐酸美沙酮人工抗原的合成

目的:利用化学方法将美沙酮半抗原与载体蛋白偶联制备美沙酮人工抗原。方法:结构修饰后的美沙酮类似物与载体BSA蛋白经过混合酸酐法或碳二亚胺法连接合成美沙酮人工抗原,利用紫外光谱扫描法、蛋白质电泳法和胶体金免疫层析法进行检测。结果:美沙酮人工合成抗原连接成功,胶体金免疫层析检测结果显示合成抗原与抗体有特异性反应。结论:该方法合成的美沙酮抗原可作为美沙酮免疫用抗原和美沙酮免疫检测方法建立所用包被抗原。     

Synthesis of stable-isotope-labeled N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide and N-(3-dimethylaminopropyl)-N′-ethylurea

N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) is a carbodiimide coupling reagent commonly used for the preparation of amides from carboxylic acids and amines. Because of initial concerns regarding the genotoxicity of EDC and its use in GMP syntheses at Bristol Myers Squibb, the quantitation of residual EDC and its by-product N-(3-dimethylaminopropyl)-N′-ethylurea (EDU) by liquid chromatography–mass spectrometry (LCMS) impurity analysis was required. These analyses required the use of stable-isotope-labeled EDC and EDU to serve as internal standards. To meet this need, stable-isotope-labeled EDC 9 and EDU 10 were prepared from [1,2-¹³C₂] ethylene glycol and [¹³C,¹⁵N] potassium cyanide in overall yields of 6% and 8%, respectively.     

Effects of amounts of additives on peptide coupling mediated by a water-soluble carbodiimide in alcohols

Abstract: The optimal amounts of 1-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt) and 1-hydroxy-7-azabenzotriazole (HOAt) for enhancement of peptide coupling mediated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride in alcoholic solvents were found to be less than equimolar against the carboxyl component or the carbodiimide. In comparison with the use of equimolar additives, the use of less equimolar ones was more effective in suppressing the competitive ester formation and in increasing the yield of desired peptides. EDC hydrochloride/around 0.1 equimolar HOAt or HOOBt were efficient reagents for peptide synthesis in the media.     

电子数据捕获系统在新药临床试验中的影响

随着计算机和网络技术的发展,电子数据捕获（EDC）系统在新药临床试验中的优势不断显现,并受到新药临床试验研究机构的青睐。EDC凭借其技术上的优势,简化了传统以纸张病例报告表为载体的临床试验流程,克服了其数据录入和数据清理滞后的缺点;研究者、临床监查员、数据管理人员等在应用EDC系统情况下的角色和职责分工也发生了相应的改变;同时,其为新的试验设计和统计方法,如适应性设计方法的应用实施提供的有利的平台。     

Reaction of carbodiimide for the introduction of p-bis(2-chloroethyl)amino-L-phenylalanine to lysozyme

In the synthesis of new prodrugs with inhibitoring action of tumour growth, a new nitrogen mustard derivative was obtained, proceeding of the coupling between an egg-white lysozyme with an antitumor amine nucleophile, the methyl ester of p-bis-(2-chloroethyl)amino-L-phenylalanine (Melphalan), catalyzed by 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide (EDC), at pH 5.0 and room temperature. In that case, the mechanism for the modification isn't selective of Asp101 in lysozyme. As in cases of histamine and D-glucosamine [3], it is evident that Melphalan is one type of amine who doesn't cause a selective modification of Asp101 but causes somewhat random reaction, because Asp101 is modified followed by modifications of other carboxyls. In this case, we suggest that the amine (Melphalan) may also bind to the substrate binding site in competition with EDC. With this type of amine, enzyme-nucleophile interactions predominate, and the selective activation of Asp101 by EDC is reduced to lead a more random reaction.     

Pharmacokinetics and macromolecular interactions of ethylene dichloride in rats after inhalation or gavage

Ethylene dichloride (EDC) induces tumors in rats and mice when administered chronically by gavage. However, chronic inhalation of EDC vapor failed to induce any treatment-related tumors. To help understand the consequences of environmental exposure to EDC by either route, [¹⁴C]EDC was administered to male Osborne-Mendel rats by gavage (150 mg/kg in corn oil) or inhalation (150 ppm, 6 hr). EDC was extensively metabolized following either exposure. No significant differences were observed in the route of excretion of nonvolatile metabolites. In each case, ～85% of the total metabolites appeared in the urine, with 7 to 8, 4, and 2% found in the CO₂, carcass, and feces, respectively. The major urinary metabolites were thiodiacetic acid and thiodiacetic acid sulfoxide, suggesting a role for glutathione in biotransformation of EDC. Gross macromolecular binding (primarily protein binding) was studied after inhalation or gavage. No marked differences were noted between the two routes, or between “target” and “nontarget” tissues, after in vivo administration of EDC. Covalent alkylation of DNA by EDC was studied in Salmonella typhimurium and rats. DNA alkylation in S. typhimurium was directly related to the frequency of mutation in these bacteria. However, when DNA was purified from the organs of rats exposed in vivo to EDC, very little alkylation was observed after either gavage or inhalation (2 to 20 alkylations per million nucleotides). DNA alkylation after gavage was two to five times higher than after inhalation, but no marked differences were noted between target and nontarget organs. Pharmacokinetic studies indicated that peak blood levels of EDC were approximately five times higher after gavage than after inhalation. When pharmacokinetic data were modeled, it appeared that the elimination of EDC may become saturated when high blood levels are produced and that such saturation is more likely to occur when equivalent doses are administered by gavage versus inhalation. Since toxicity often occurs when the normal detoxification pathways are overwhelmed, this toxicity may represent the most reasonable explanation for the apparent differences between the two bioassays.     

Release of ciprofloxacin from poloxamer-graft-hyaluronic acid hydrogels in vitro

Recently, in situ gel formation has extensively been studied to enhance ocular bioavailability and duration of the drug activity. In this study, we report grafting of poloxamer onto the hyaluronic acid for application of tissue engineering oriented ophthalmic drug delivery system. Graft copolymers were prepared by coupling mono amine-terminated poloxamer (MATP) with hyaluronic acid (HA) backbone using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxylsuccinimide (NHS) as coupling agents. The coupling of MATP with HA was clarified by 1H NMR and FT-IR spectroscopy. The gelation temperature of graft copolymers was dependent on the content of HA and the concentration of poloxamer. From drug release studies in vitro, ciprofloxacin was sustainedly released from the poloxamer-g-hyaluronic acid hydrogel due to the in situ gel formation of the copolymer and viscous properties of HA.     

Effect of disulfiram pretreatment on the tissue distribution, macromolecular binding, and excretion of [U-1,2-14C]dichloroethane in the rat

The effect of disulfiram (DSF) pretreatment on the distribution of [14C]ethylene dichloride (EDC) in selected organs and/or tissues of control and EDC-pretreated rats was studied. The presence of EDC metabolites and their binding to an acid-insoluble extract of the tissues, as well as purified protein and DNA, were evaluated. Dietary DSF was found to modulate the distribution, excretion, and macromolecular binding of EDC and/or its metabolites at 4 and 24 hr following ip administration. The urinary excretion of [14C]EDC metabolites was not affected by subchronic inhalation exposure to nonradiolabeled EDC. However, DSF pretreatment increased the fat deposition of EDC and decreased the urinary excretion of its metabolites. DSF also increased the binding of EDC metabolites to DNA and decreased the binding to protein in the liver, kidneys, spleen, and testes. However, prior exposure to EDC alone increased the binding of its metabolites to DNA in the kidneys only.     

阿霉素与Annexin V的偶联及其对乳腺癌细胞的杀伤作用

目的研究将阿霉素与Annexin V偶联的方法,以及偶联物对乳腺癌细胞的杀伤作用。方法使用EDC/sulfo-NHS为偶联剂,将阿霉素与Annexin V偶联,使用紫外可见分光光度法对偶联物进行了表征,用MTT方法检测偶联物对过氧化氢处理的乳腺癌细胞系MDA-MB-231的肿瘤细胞杀伤作用。结果在过量的EDC存在下,提高sulfo-NHS的浓度,有助于提高偶联比。偶联物的浓度为1μmol/L时,MDA-MB-231肿瘤细胞的存活率只有27.7%;而单独使用阿霉素,浓度要达到1.8μmol/L时,才能达到相同的抑制率;Annexin V在3.88μmol/L时,细胞存活率为63.9%。结论偶联物对MDA-MB-231肿瘤细胞的杀伤作用要强于两者分别单独使用时的效果,这为进一步研究抗肿瘤药物提供了依据。     

Early developmental actions of endocrine disruptors on the hypothalamus, hippocampus, and cerebral cortex

Sex steroids and thyroid hormones play a key role in the development of the central nervous system. The critical role of these hormonal systems may explain the sensitivity of the hypothalamus, the cerebral cortex, and the hippocampus to endocrine-disrupting chemicals (EDC). This review examines the evidence for endocrine disruption of glial-neuronal functions in the hypothalamus, hippocampus, and cerebral cortex. Focus was placed on two well-studied EDC, the insecticide dichlorodiphenyltrichloroethane (DDT) and polychlorinated biphenyls (PCB). DDT is involved in neuroendocrine disruption of the reproductive axis, whereas polychlorinated biphenyls (PCB) interact with both the thyroid hormone- and sex steroid-dependent systems and disturb the neuroendocrine control of reproduction and development of hippocampus and cortex. These results highlight the impact of EDC on the developing nervous system and the need for more research in this area.     

电子数据采集：临床试验的未来之路

 电子数据采集（electronic data capture,EDC）可以提高临床试验效率、保障数据质量、缩短研究周 期、降低研究成本。随着互联网的普及和临床试验行业对EDC认识的提高,临床试验电子化趋势将不可避免。本文首先 介绍了临床试验数据管理的现状及EDC的概念,然后对应用EDC系统带来的益处和面临的困难进行了分析,最后对药品研 发者应用EDC系统提供了建议。