相思子毒素单克隆抗体的制备纯化及鉴定

目的制备相思子毒素单克隆抗体并鉴定其特性。方法以甲醛处理的相思子毒素毒蛋白为抗原免疫BALB/c小鼠;取免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞融合,经间接ELISA法筛选、融合细胞有限稀释法克隆、克隆化杂交瘤细胞株的亚类鉴定等方法筛选出单克隆抗体杂交瘤细胞株,用杂交瘤细胞株诱生小鼠腹水,应用蛋白A亲和层析法进行单抗的纯化,并对单克隆抗体的特异性进行鉴定。结果获得了4株可稳定分泌单克隆抗体的杂交瘤细胞2D3、4E6、1C8和1E5,诱生的腹水效价分别为1∶1×107、1∶1×106、1∶1×105、1∶1×106,亚类鉴定表明2D3为IgG1,其余3株均为IgG2b;特异性鉴定显示它们与多种毒素均无交叉反应,经过亲和层析,获得了纯化的单抗。结论获得了特异性的相思子毒素单克隆抗体,为建立相思子毒素的检测及纯化方法奠定了基础,其中4E6的效价最高,可作为检测相思子毒素的核心试剂。     

抗哇巴因单克隆抗体的制备及初步鉴定

目的制备抗哇巴因单克隆抗体。方法以半抗原哇巴因与蛋白质载体卵清蛋白(OVA)的耦联物为抗原,免疫BALB/C小鼠,取脾细胞与SP2/0细胞融合,获得两株分泌抗哇巴因的单克隆抗体的细胞株(OUA1,OUA2)。结果两株细胞染色体数目均为100条左右,证实为杂交瘤细胞;所分泌抗体为小鼠IgG1亚类;小鼠腹水效价测定分别为5×106,8×106;和地高辛交叉反应率分别为1.342%和2.323%;ELISA相加试验表明,两株单克隆抗体可能针对同一抗原决定簇。结论成功制备出小分子物质哇巴因的单克隆抗体。     

人心肌肌钙蛋白Ⅰ单克隆抗体的制备与鉴定

为了建立抗人心肌肌钙蛋白Ⅰ(CTnI)单克隆抗体(McAb)的杂交瘤细胞株。以重组表达并纯化后的CTnI免疫BalB/C小鼠,采用传统单克隆抗体技术筛选能稳定分泌抗CTnI McAb的杂交瘤细胞株。结果:建立了3株稳定分泌抗CTnI McAb的杂交瘤细胞株2E3,3D4和3E6,腹水效价分别为4×10-5,1×10-6,1×10-5,亲和常数分别为4.8×108,5.2×109,3.6×108,最低检测浓度分别为20,40,20μg/L,其中2E3和3D4可以识别CTnI的不同表位。     

抗重组人骨唾液酸蛋白单克隆抗体的制备及鉴定

目的研制重组人骨唾液酸蛋白(rhBSP)单克隆抗体(mAb),并鉴定其特性。方法以纯化的rhBSP免疫Balb/c小鼠,采用杂交瘤技术制备抗rhBSP mAb;用亚型鉴定试剂条鉴定IgG亚类;ELISA鉴定mAb的特异性和效价。结果获得2株能稳定分泌特异性mAb的抗rhBSP的杂交瘤细胞系AHB1和AHB5,Ig亚类分别为IgG2a和IgG1,轻链均为κ型,其效价分别为1×10-3和1×10-7。腹水mAb经Protein A亲和色谱柱纯化后,纯度达92%以上。结论获得抗rhBSP的mAb,为进一步研究BSP的生物学功能和用于临床诊断实验研究创造了条件。     

抗delta-like-1单克隆抗体的制备与鉴定

目的 制备抗delta-like-1的单克隆抗体并鉴定其特异性.方法 用delta-like-1多肽-BSA蛋白复合物作为免疫原免疫Balb/c小鼠,取其脾细胞与小鼠骨髓瘤细胞(SP2/0)融合,经多次筛选及克隆化,建立可以稳定分泌抗delta-like-1单抗的杂交瘤细胞株.用ELISA、Western blot、细胞免疫组化对此抗体特性进行鉴定.结果 筛选到两株能稳定分泌抗delta-like-1单抗的细胞株3H11C11A9E3和2D5D11F8F1,亚类鉴定两株均为IgG1,轻链为kappa链;ELISA法测定两株细胞腹水抗体效价分别为1∶4.096×105和1∶1.024×105,抗体亲和常数分别为3.98×107、3.28 × 107L·mol-1;Western blot显示此单抗能特异性识别神经胶质瘤细胞株U251中的天然delta-like-1蛋白;细胞免疫组化进一步显示delta-like-1分布于细胞核中.结论 成功制备了抗delta-like-1单克隆抗体,可为研究delta-like-1与脑胶质瘤等神经系统疾病的关系奠定基础.     

抗SARS病毒融合蛋白单克隆抗体的制备与初步鉴定

用基因工程技术表达的SARS-CoV S蛋白片段和N蛋白片段的融合蛋白作抗原免疫Balb/c小鼠,将免疫鼠脾细胞与Sp2/0细胞融合,经ELISA法筛选阳性克隆及测定效价,用免疫印迹法进行特异性鉴定。最终获得2株(1F11和3D2)能稳定分泌抗SARS-CoV融合蛋白单克隆抗体的杂交瘤细胞。其培养上清的ELISA效价分别为1∶1024和1∶512;腹水效价分别为:1×10-6,2×10-5。1F11株识别融合蛋白上的N抗原表位,3D2识别其上的S抗原表位。该单抗可望成为快速诊断SARS-CoV感染的特异性抗体。     

猪戊型肝炎病毒重组蛋白ORF2-V1单克隆抗体的制备及抗原表位差异分析

目的：开发猪戊型肝炎病毒单克隆抗体并探究其抗原识别位点对病毒的分型,疫苗开发研究和疾病的鉴别诊断具有重要意义。结果：本研究用纯化的SHEV大庆株（DQ）重组蛋白ORF2-V1免疫BALB／c小鼠,取免疫小鼠脾细胞与SP2／0骨髓瘤细胞进行融合,经3次克隆和间接ELISA筛选,获得了αC11、αC12、γH1、γF8、BC4和CH86株稳定分泌单克隆抗体的杂交瘤细胞株。通过间接ELISA测定单抗效价分别为：细胞培养上清1：1．60×10^3～1：3．20×10^3,腹水为1：1．28×10^6～1：2．56×10^6：经ELISA法测定,6株单克隆抗体均与重组蛋白ORF2-V1反应,而不与ORF2其它部分片段的蛋白反应;分别用制备的6株单克隆抗体和SHEV多克隆抗血清对感染SHEV的A549细胞培养物进行了间接免疫荧光（IFA）病原的检测,同时用猪的阳、阴性血清、SP2／0细胞培养上清做对照,结果表明均呈阳性反应,说明这6株单抗可以与天然病毒发生反应;与用多克隆抗体的阳性对照相比,McAb鉴定的SHEV感染细胞培养物间接免疫荧光染色结果特异性强,排除了高背景的非特异荧光影响,对结果的判定更为准确。而以多克隆抗体作为检测抗体,IFA有一定的非特异性荧光反应。单抗的亚类鉴定结果表明,所有McAbs均为IgG1型,且所有单抗的轻链均为K链。     

ApoAⅡ单克隆抗体对抗原决定簇的测定及应用研究

目的　研究载脂蛋白AⅡ (ApoAⅡ )单克隆抗体 (McAb)对其抗原的决定簇。方法　应用淋巴细胞杂交瘤技术 ,建立抗人ApoAⅡ杂交瘤细胞株 (BI1 ,BI2 ,BI3,BI4 ,BI5,BI6 ,BI7) ,以制备的McAb用双向免疫扩散法和ELISA对其免疫学特性进行了测定 ,用单抗相加试验和双抗体竞争试验对其抗原决定簇进行分析。结果　Ig亚类测定 :BI1 、BI2 、BI5和BI7为IgG1 ,而BI3、BI4 和BI6 均为IgG2b,腹水效价BI1与BI6 为 1× 10 - 6 ,BI2 、BI3、BI4 、BI5和BI7均达到 1× 10 - 5。特异性测定 :与载脂蛋白AⅠ、B、CⅠ、CⅡ、CⅢ、E、脂蛋白 (a)、人血清白蛋白及纤维蛋白溶酶原 (pg)没有交叉反应。单抗相加试验和双抗体竞争试验结果证实 ,BI1 、BI4 和BI5为识别apoAⅡ上同一抗原决定簇的McAb、BI2 、BI3、BI6 和BI7则为针对apoAⅡ上另一抗原决定簇的McAb。结论　ApoAⅡ的McAb对抗原决定簇的测定为预测动脉粥样硬化危险性提供了理论依据     

抗重组人巨细胞病毒单克隆抗体的制备与鉴定

为了获得抗人巨细胞病毒(HCMV)单克隆抗体(McAb).采用重组HCMV(rhCMV)gp52蛋白片段免疫Balb/c小鼠,将免疫鼠脾细胞与Sp2/0细胞融合,经ELISA法筛选阳性克隆及测定效价,用免疫印迹法进行特异性鉴定.最终获得4株(3E9,5A6,4G12,2H2)能稳定分泌高效价抗rhCMV McAb的杂交瘤细胞.其培养上清的ELISA效价分别为12048,11024,11024,1512;腹水效价分别为1×10-7,1×10-7,1×10-6,2×10-6.它们分别识别gp52蛋白上的2个不同的抗原表位,2H2识别gp52蛋白上的一个表位,3e9,5A6和4G12识别另一个表位.该单抗可作为快速诊断CMV感染的特异性抗体.     

单株抗人肝脏微粒体蛋白及细胞色素P450单克隆抗体的制备及初步鉴定

目的:制备抗正常人肝脏微粒体(HLM)及细胞色素P450蛋白的单克隆抗体.方法:采用人肝脏微粒体蛋白作为免疫原免疫Balb/C小鼠,按常规方法融合、克隆,经间接ELISA法筛选抗体阳性杂交瘤细胞株,通过单抗亚类、Western blot对抗体进行初步鉴定.结果:成功地制备了一株抗肝微粒体蛋白的单克隆抗体(MAb),命名为MAb 10C7.用鼠单抗亚类快速鉴定纸条鉴定10C7为IgG2b.Western blot分析显示,MAb 10C7可特异识别相对分子量为46kD的人肝脏微粒体蛋白.结论:MAb 10C7抗正常人肝脏微粒或细胞色素P450蛋白的特异性强,对相关蛋白质的功能研究有较好的应用前景.     

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.     

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.     

Alzheimer’s disease – current trends in basic and clinical research. Highlights from the 16th ECNP

Advances in the molecular biological knowledge of neuronal nicotinic acetylcholine receptors (nAChRs) have led to a growing interest by the pharmaceutical industry in the development of novel compounds that selectively modulate nAChR function. The ability of (-)-nicotine, an activator of nAChRs, to enhance attentional aspects of cognition in animals and humans, to exert neuroprotective and anxiolytic-like effects, and presumably to mediate the negative correlation between smoking and Alzheimer's (and Parkinson's) Disease, has focused interest on the potential therapeutic utility of modulators of nAChR function for treatment of some of the deficits associated with these progressive, neurodegenerative conditions. Numerous compounds are known which activate nAChRs and which might serve as lead compounds toward the development of such agents. The pharmacologic diversity of neuronal nAChR subtypes suggests the possibility of developing selective compounds which would have more favourable side-effect profiles than existing agents. This broader class of agents, collectively called cholinergic channel modulators (ChCMs), is anticipated to encompass compounds which would have more favourable side-effect profiles than existing agents, which generally exhibit low selectivity. This selectivity may be achieved by preferentially activating some subtypes of nAChRs (i.e., Cholinergic Channel Activators, ChCAs) or inhibiting the function of other subtypes (Cholinergic Channel Inhibitors, ChCIs). An overview of the biology of nAChRs and the rationale for the use of ChCMs for the treatment of dementia related to neurodegenerative diseases are presented, followed by a discussion of lead compounds and compounds under consideration for clinical evaluation.     

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.