抗体夹心酶联免疫吸附法测定重组E.coli L-天冬酰胺酶及药代动力学研究

目的建立大鼠血浆中重组E.coli L-天冬酰胺酶的抗体夹心酶联免疫吸附测定法,并进行药代动力学研究.方法应用重组E.coli L-天冬酰胺酶免疫家兔,分离IgG,用DEAE-纤维素柱色谱纯化,辣根过氧化物酶标记抗体,建立抗体夹心酶联免疫吸附法,测定大鼠血浆中重组E.coli L-天冬酰胺酶浓度.结果方法的线性范围为1～64 U*L-1,血药浓度与时间的关系符合二房室模型,初期和末端的T1/2分别为0.50～0.57 h和2.45～3.02 h,AUC与剂量成正比.结论建立的抗体夹心酶联免疫吸附法在灵敏度、特异性、线性范围、精密度和回收率等方面,满足药代动力学研究要求.实验方法和重组E.coli L-天冬酰胺酶在大鼠中的药代动力学参数为临床研究提供了手段和依据.     

抗体夹心酶联免疫吸附法测定重组E.coli L-天冬酰胺酶及药代动力学研究

目的建立大鼠血浆中重组E.coli L-天冬酰胺酶的抗体夹心酶联免疫吸附测定法,并进行药代动力学研究。方法应用重组E.coli L-天冬酰胺酶免疫家兔,分离IgG,用DEAE-纤维素柱色谱纯化,辣根过氧化物酶标记抗体,建立抗体夹心酶联免疫吸附法,测定大鼠血浆中重组E.coli L-天冬酰胺酶浓度。结果方法的线性范围为1～64 U·L^-1,血药浓度与时间的关系符合二房室模型,初期和末端的T_1/2分别为0.50～0.57 h和2.45～3.02 h,AUC与剂量成正比。结论建立的抗体夹心酶联免疫吸附法在灵敏度、特异性、线性范围、精密度和回收率等方面,满足药代动力学研究要求。实验方法和重组E.coli L-天冬酰胺酶在大鼠中的药代动力学参数为临床研究提供了手段和依据。     

抗体夹心酶免疫吸附法测定重组E.coli水蛭素HV3研究

将重组E. coli水蛭素HV3和BSA交联后免疫豚鼠和家兔,DEAE-纤维素柱层析纯化IgG.用这两种抗体和酶标二抗羊抗兔IgG-HRP建立三抗体夹心酶联免疫吸附法,测定重组E. coli水蛭素HV3的含量.本测定方法的线性范围为0～2 ng/ml,灵敏度为0.04 ng/ml.建立的三抗体夹心酶联免疫吸附法测定重组E.coli水蛭素HV3具有良好的精密度、回收率、灵敏度和特异性.     

重组溶葡萄球菌酶中宿主菌残余蛋白含量的测定

制备球形芽孢杆菌菌体蛋白作为免疫抗原，免疫家免制备抗血清，建立了一种测定重组溶葡萄球菌酶中残余宿主菌菌体蛋白含量的双抗体夹心酶联免疫吸附测定法（Enzyme-linked immunosorbent assay，ELISA）。结果表明，建立的双抗体夹心ELISA法快速、简便、灵敏，检测范围宽，能检测出制品中的微量菌体蛋白；对3批重组溶葡萄球菌酶原液的检测合格，制品中残余宿主菌蛋白含量小于万分之二。此方法可以用于重组溶葡萄球菌酶中残余宿主菌菌体蛋白含量的测定。     

人血浆血管抑素ELISA检测方法的建立及初步应用

目的 建立一种检测人血浆中血管抑素(angiostatin,AS)含量的方法,并探讨其在肿瘤中测定的意义.方法 利用基因重组人AS免疫动物,制备特异性抗人AS多克隆抗体,建立人血浆AS酶联免疫吸附检验(ELISA)方法.并检测了73例恶性肿瘤患者血浆AS含量.结果 该方法检测人血浆AS的线性范围为3.13～100 mg/L,灵敏度为5 mg/L.73例肿瘤患者血浆AS水平均显著高于正常对照组(27.16±7.32) mg/L,(P<0.05或P<0.01).结论 以基因重组AS建立的人血浆AS含量ELISA检测方法,灵敏度高,重复性好,可为恶性肿瘤诊断提供一种新检测技术.     

人乳头瘤病毒重组蛋白疫苗酶联免疫检测法的建立及其应用

目的建立人乳头瘤病毒重组蛋白疫苗(HPV16 L2E6E7)酶联免疫的检测方法,用于疫苗发酵过程中的重组蛋白表达的监控。方法用常规方法制备兔抗HPV16 L2E6E7多克隆抗体作为包被抗体,商品化小鼠抗HPV16 E7单克隆抗体为检测抗体,夹心ELISA方法检测HPV16 L2E6E7抗原参考品,建立抗原标准曲线,确定线性范围及检测限,同时验证该方法的特异性。结果建立了检测HPV16 L2E6E7抗原含量的夹心ELISA方法,抗原参考品系列浓度在19.53~1 250 ng·m L-1内有很好的线性(R2>0.99)和回收率(90%~110%);特异性好,不受发酵液中宿主菌蛋白的干扰。结论建立了人乳头瘤病毒重组蛋白疫苗重组抗原含量的测定方法,为该疫苗的发酵工艺的质控提供了有效技术手段。     

促红细胞生成素酶联免疫吸附测定法的研究

目的:建立血清促红细胞生成素(erythropoietin, EPO)的酶联免疫检测(ELISA)方法,观察其临床应用价值.方法:制备EPO多克隆抗体,异丙醇洗涤处理酶标板以增强其吸附能力,利用交叉反应法选择出抗原、抗体及酶标抗体的最佳的配对工作浓度.反应后,观察本方法的灵敏度、回收率、特异度、稳定性等指标,观察效果.以正常血清为对照组,测定缺铁性贫血组、乳腺癌组,并与放射免疫检测对比.结果:酶标板结合蛋白能力增强.抗体、酶标抗体、抗原最佳工作浓度分别为1∶1 000,1∶6 000,1∶800.灵敏度为0.46 U/L,与生长激素、铁蛋白交叉反应率低;高浓度、低浓度样品平均回收率分别为96.3%、97.3%,批内和批间变异分别为8.31%、7.82%,稳定性好.乳腺癌组、缺铁性贫血组EPO水平均明显高于正常对照组.放射免疫检测及酶联免疫检测检出结果差别无统计学意义.结论:本血清EPO双抗体夹心ELISA法的建立在诊断相关疾病方面有一定的临床应用价值.     

重组E.coli L-天冬酰胺酶在大鼠中的药物代谢动力学

应用放射性核素示踪技术研究^125I重组E.coliL－天冬酰胺酶在大鼠体内的药物代谢动力学。^125I重组L－天冬氨酶静脉注射后24h内，在尿、粪、胆汁中的排泄量分别占注射剂量的68．95％，4．44％和5．36％。测定血浆中^125I重组E.coliL－天冬酰胺酶浓度，应用聚丙烯酰胺凝胶电泳和生物成像分析系统结合方法评价原药水平，由房室模型评价药物动力学参数，静注后，浓度时间曲线符合二房室模型，初期和末端的t1/2分别为0．52－0．63h和2．39－2．76h，AUFC与剂量成正比。重组E.coliL－天冬酰胺酶的分解代谢产物主要随尿液排泄，重组E.coliL－天冬酰胺酶在大鼠中的药物热力学参数为临床试验提供了有用依据。     

TPPA和酶联免疫吸附法在梅毒诊断中的作用

目的比较分析TPPA与酶联免疫吸附法对诊断梅毒感染的区别,探讨TPPA与酶联免疫吸附法在梅毒诊断中的优势。方法随机选取2011年1月~2013年10月到我中心性病门诊就诊的病患260例作为研究对象,通过随机分组的原则分为实验组和对照组,每组130例研究对象,实验组采取TPPA方法对研究对象进行梅毒螺旋体抗体检测,而对照组采取酶联免疫吸附法对研究对象进行梅毒螺旋体抗体检测。结果在实验组的130例研究对象中,梅毒螺旋体抗体TPPA方法检测的阳性率为26.92%(35/130);在对照组的130例研究对象中,酶联免疫吸附法检测的阳性率为20.00%(26/130)。如果将酶联免疫吸附法作为诊断的金标准,TPPA测定结果的敏感度为93.65%,测定的特异性为97.68%。结论在对梅毒螺旋体抗体检测的敏感度和特异性检测方面,TPPA检测法的特异性比酶联免疫吸附法好,但酶联免疫吸附法的敏感性较TPPA检测法高。在临床中应将酶联免疫吸附法作为梅毒的筛选试验,对于筛选阳性的样本,应用TPPA方法去确诊,只要将两种方法有效的结合起来,才能避免漏检和误诊,具有良好的可重复性等优点,值得临床应用推广。     

应用ELISA法检测门冬酰胺酶中残余大肠杆菌菌体蛋白含量的研究

采用Cygnus公司"大肠杆菌菌体残留蛋白检测试剂盒"检测门冬酰胺酶中残余宿主菌菌体蛋白含量,建立门冬酰胺酶中宿主菌菌体蛋白残留检测方法并进行了相关方法验证。验证结果表明:宿主菌菌体蛋白浓度在0～100 ng/mL范围内呈良好的线性关系,线性相关系数大于0.99,不同实验人员测得的精密度RSD均小于5%,平均回收率为94.34%。通过对8批门冬酰胺酶样品的检测,测得门冬酰胺酶中的残余宿主菌体蛋白含量均小于550×10-6。该方法具有快速,操作安全简便等优点,灵敏度高,重现性好,可用于门冬酰胺酶的宿主菌体蛋白的残留检测。     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Synthesis,Cytotoxicity and Antileishmanial Activity of Some N-(2-(indol-3-yl)ethyl)-7-chloroquinolin-4-amines

We report herein the condensation of 4,7-dichloroquinoline (1) with tryptamine (2) and D-tryptophan methyl ester (3) . Hydrolysis of the methyl ester adduct (5) yielded the free acid (6) . The compounds were evaluated in vitro for activity against four different species of Leishmania promastigote forms and for cytotoxic activity against Kb and Vero cells. Compound (5) showed good activity against the Leishmania species tested, while all three compounds displayed moderate activity in both Kb and Vero cells.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.