人禽流感HSN1病毒HA1蛋白抗原表位及主要免疫功能区特征的研究

目的 确定人禽流感H5N1病毒HA1蛋白的单克隆抗体(McAb)抗识别表位及主要免疫功能区.方法 将16条人禽流感H5N1病毒HA1基因重叠片段克隆入真核表达载体pDisplay,构建表达HA1蛋白的重叠肽段重组质粒,转染HeLa细胞后制备细胞抗原片.利用灭活的H5N1病毒免疫BALB/C小鼠,制备病毒特异单克隆抗体(单抗)和多克隆抗体(多抗),采用间接免疫荧光法分析单抗、多抗与HA1肽段的反应性.确定HA1蛋白的单抗识别表位及主要免疫功能区.结果 成功克隆、表达了16条不同长度的H5N1肽段,并制备、获得30株分泌抗H5N1型禽流感病毒McAb的杂交瘤细胞株,其中18株为抗HA的单抗,18株中有6株单抗具有中和病毒活性.利用表达的H5N1肽段对单抗识别表位进行分析,初步确定3B5、3D1、3132、M6、M3等18株McAb识别表位区域分别位于氨基酸残基aa133-143、aa155-165、aa166-196、aa166-176、aa34-66及aa296-328之间,其中3B5、3D1、3B2、M6等McAb识别表位为序列依赖型表位,M3和M7等具有中和活性的McAb识别表位为构象性中和表位.结论 利用制备的H5N1病毒特异性McAb和表达的病毒HA1蛋白重叠肽抗原,初步确定了18株抗HA McAb的识别表位和HA1蛋白的主要免疫功能区,为进一步研究开发新型疫苗提供了重要理论依据.     

脑梗塞患者脑脊液中三种阿片肽含量变化

用放射免疫法对144例脑梗塞患者及24例对照者脑脊液中β—内啡肽(β—EP)、亮脑啡肽(LEK)、强啡肽A_(1-13)(D_(yn)A_(1-13))含量进行检测,结果显示脑梗塞急性期3天内β—EP、LEK含量较对照组显著增高.D_(yn)A_(1-13)较对照组显著降低,一周后逐渐接近或达到对照组水平。同时也发现基底节区脑梗塞患者脑脊液中β-EP、LEK较其它部位梗塞为高,D_(yn)A_(1-13)较其它部位梗塞为低,与对照组相比差异显著。提示脑脊液中二种阿片肽含量变化与脑梗塞时缺血性应激反应有关。三者不平衡变化,参与了急性脑梗塞时缺血性脑水肿等病理生理过程。也提示脑梗塞急性期(尤其3天内)适量应用阿片受体拮抗剂或激动剂,有利于减轻缺血性脑水肿,缓解神经受损症状。     

低剂量X射线诱导离体人淋巴细胞染色体损伤的适应性反应

适应性反应的存在已被广泛证实,但主要是通过染色单体型畸变或SCE(姐妹染色单体互换)指标证实的.本文试图对低剂量(简称D_1)辐射如何影响DNA复制前大剂量(D_2)辐射诱发体型染色体畸变进行观察.实验用健康成年人静脉血淋巴细胞,分成三组.(1)G_0期(加PHA前)照射D_1后间隔0、3、5、7和9h再照射D_2;(2)G_1期照射D_1后间隔0,3,5,7和9h再照射D_2.(1)、(2)组用的D_1和D_2分别为0.02Gy和3.0Gy X射线;(3)用标记BrdU(5-溴脱氧尿嘧啶核苷)法分析G_0和G_1期受0.01～0.10Gy X射线照射后细胞动力学变化.结果:1.染色单体型畸变率在G_0和G_1期受D_1     

用单克隆抗体BA法快速检出细胞培养物中的支原体

口腔支原体是实验室细胞培养物的主要污染源之一.为解决支原体的快速诊断问题,1987年我们建立了7株分泌抗口腔支原体单克隆抗体(McAb)的杂交瘤细胞株.进而我们应用McAb-BA(biotin-avidin)法检查了7株细胞培养物,其中4份阳性,3份阴性,获得了满意结果.     

用免疫荧光法筛选抗人μ链单克隆抗体的研究

用人血清IgM球蛋白重链——μ链免疫的BALB/C小鼠脾细胞与鼠骨髓瘤SP2/0细胞在聚乙二醇(PEG)作用下融合,用改进的免疫荧光技术筛选出6株分泌单克隆抗体(McAb)的杂交瘤细胞,其中5株分泌物确定为抗人μ链McAb,1株未定.杂交瘤细胞经4次克隆,半年多传代,接种BALB/C鼠可稳定地产生腹水抗体.     

用单克隆抗体分析鉴定登革3型病毒的不同抗原决定簇

登革病毒(DEN)抗原成分复杂,与许多虫媒披膜病毒都有严重的血清学交叉反应。杂交瘤单克隆抗体(Mcab)技术建立之后,Dittmar等人首先制备出了抗DEN-3病毒的McAb,可进行DEN-3病毒的鉴定分型。随后,Henchal等人制备出了抗DEN1-4型病毒的McAb,用这些McAb进行DEN病毒抗原决定簇研究,依据间接免疫荧光染色(IFA)证实了DEN病毒的四种不同抗原决定簇,即:黄病毒属特异的、DEN-1-4型病毒特异的、DEN-1、3型病毒特异的和DEN病毒各型型特异的。阎国珍等人在研究抗DEN-4病毒McAb时,获得了抗DEN-2、4型病毒特异的McAb,证明DEN-2和DEN-4病毒间有共同特异抗原决定簇。本文介绍通过用抗DEN-3病毒McAb对不同披膜病毒抗原交叉反应测定来分析鉴定     

APAAP桥联酶标技术检测支原体和虫媒病毒

APAAP免疫酶标技术(即碱性磷酸酶-抗碱性磷酸酶桥联酶标显色技术)是最近几年随着单克隆抗体(McAb)技术进展而发展起来的一新兴组化染色技术.此项技术在国内首先由杨志刚等同志建立并推广应用.我们将这一方法应用于支原体和虫媒病毒抗原检测,获得了满意结果.材料与方法支原体:口腔支原体引自生物制品检定所,猪鼻支原体(BTS-7CF8)引自中监所.病毒:辛德比斯病毒引自生物制品检定所,森林脑炎病毒(森张株)引自长春生物制品所.上述支原体和病毒感染敏感细胞一定时间后按常规法进行细胞滴片,经丙酮固定后,作为感染抗原片.抗体:我们用口腔支原体和猪鼻支原体免     

103株支原体药敏试验结果分析

我们对103株支原体(静脉支原体Ｕｕ与人型支原体Ｍｈ)进行了三类6种抗生素药物敏感试验。现报告如下。1　材料与方法11　支原体株来源　1997年6月～1998年12月本院性病门诊384例泌尿生殖道感染者的生殖系拭子样品中检出的支原体103株(Ｕｕ Ｍｈ),均为首次检出株。12　试剂与方法　酵母浸出液常规配制,分装于常规试管中固体培养基加入14ｇ/Ｌ琼脂,分装于无菌平皿中。将收采集的标本接种于培养基中,置37℃5%ＣＯ2培养箱培养,观察到接种管已变红色,即取02～03ｍｌ培养液接种于固体培养基平皿内继续培养3～5ｄ,用低培镜观察菌落生长情况。2　结…     

亲和素促排减轻荷瘤裸鼠放免治疗毒副作用的实验研究

本研究将1 88Re标记生物素化单克隆抗体 (McAb Bt)和亲和素 (Av)用于荷人结肠癌裸鼠模型 ,评价其在肿瘤放免治疗 (RIT)中的价值 ,现报道如下。一、材料与方法1.抗癌胚抗原 (CEA)单抗McAb的生物素化及1 88Re标记参照文献 [1,2 ]。2 .荷瘤裸鼠RIT实验。建立荷人结肠癌裸鼠模型。将荷瘤裸鼠随机分为 3组 ,每组 5只。①Av促排组 :先尾静脉注射1 88Re McAb Bt11 1MBq (2 0 μg) ,2 4h后再注射Av 80 μg;②未实施Av促排组 :尾静脉注射1 88Re McAb Bt 11 1MBq (2 0μg) ;③对照组 :尾静脉注射生理盐水 10 0μl。肿瘤体积的检测采用双…     

用改进的高效液相色谱法测定维生素D2与D3

本文叙述了用高效液相色谱[HPLC]测定强化食品中维生素D_2和D_3的方法,包括样品的皂化、抽提、浓缩、残渣、用甲醇溶解并用半制备色谱分离含有维生素D的部分,收集浓缩后再用ODS柱和预柱将维生素D再分为D_2和D_3。我们将正相柱(硅胶柱)改为反相柱(ODS柱.4.6×250mm 5μm),并参照George,w等人的方法,在样品处理、流动相的比例方面做适当的改进,使分离效果明显改善,定量分析时,D_2和D_3的检测限度各为0.2个IU。在已知的样品中加入标准D_2和D_3,测定其回收率,D_2平均为87.3%(范围80～104%),D_3平均为103.3%(范围92.2～109.6%),重复性的标准误差D_2和D_3分别为5.1%和6.4%。     

低剂量X射线全身照射诱导小鼠胸腺细胞CD_3分子表达的适应性反应

采用免疫荧光流式细胞术（ＦＣＭ）观察了昆明种小鼠胸腺细胞ＣＤ３分子表达的变化。首次报道了０．０７５ＧｙＸ射线单次全身照射可诱导小鼠胸腺细胞ＣＤ３分子表达对其后１．５Ｇｙ攻击剂量照射的适应性反应。证实，１．５Ｇｙ全身照射后胸腺ＣＤ３阳性细胞明显减少，照后１８小时仅为假照射组的３３．７４％。当１．５Ｇｙ照射前６小时预先照射０．０７５Ｇｙ时，可明显减轻其后攻击剂量对ＣＤ３分子表达的抑制作用，此时，ＣＤ３阳性细胞数显著高于单纯攻击剂量对照组（Ｐ＜０．０１）。对其机理进行了讨论。     

登革1型病毒的单克隆抗体

我们用SP2/0-Ag14小鼠骨髓瘤细胞与用登革1型病毒免疫的BALB/C小鼠脾细胞在融合剂PEG-1000的诱导下进行融合,获得了15株产生抗登革1型病毒单克隆抗体的杂交瘤细胞系。其中14株属登革病毒型特异,1株属登革病毒亚群特异,它们的荧光效价均在10,240以上。15株杂交瘤均无血凝活性,3株有补体结合活性。1D_4,1B_9的IgG亚类为IgG_1,1F_(11)为IgG_2a,轻链均为K型。1株杂交瘤(1D_4)的染色体数为87～115。用SDS-PAGE测定1D_4分子量,重链为54,000,轻链为24,000。15株杂交瘤连续传代3～6个月,冻存复苏后抗体水平未见下降。     

抗A型肉毒毒素鼠源性单克隆抗体的制备和鉴定

目的：制备与鉴定抗A型肉毒神经毒素重链C端（ heavy chain of botulinum neurotoxin serotype A, BoNT／AHc）特异性鼠单克隆抗体。方法通过纯化BoNT／AHc抗原、免疫BALB／c小鼠并建立杂交瘤细胞以制备鼠单抗,用ELISA、Western印迹实验和抗体分型试剂盒进行分析鉴定,并通过ELISA检测鼠单抗与BoNT／AHc突变体结合,初步鉴定其在BoNT／AHc的结合表位。结果得到纯度较高的BoNT／AHc抗原,制备了4株特异性鼠单抗1A4、3H3、3H7、5H8。间接ELISA结果显示,单抗细胞上清效价均＞3．0×103;Western印迹检测结果显示,单抗均能与BoNT／AHc特异性结合;抗体亚型鉴定结果为1A4、3H7属于IgG1（Κ）,3H3属于IgM（Κ）,而5H8属于IgG2b （Κ）;叠加ELISA实验表明,4株单抗抗原识别表位相近;用ELISA检测单抗与BoNT／AHc突变体结合实验结果初步确定了4株单抗与BoNT／AHc的结合表位。结论对抗A型肉毒毒素鼠源性抗体完成了制备和鉴定,为肉毒毒素中和抗体的开发与阐明中和抗体的表位奠定了基础。     

Genetic polymorphism of the H1 subunit of inter-alpha-trypsin inhibitor

Inter-alpha-trypsin inhibitor (ITI) consists of 3 subunits (H1, H2 and L) which are encoded by 3 distinct genes. We attempted to prepare the subunit-specific monoclonal antibodies to identify the subunit carrying ITI polymorphism. Three monoclonal antibodies which were specific against each of these 3 subunits (ITI H1, ITI H2 and ITI L) were selected. ITI types could be detected by anti ITI H1 antibody, but not by anti ITI H2 or anti ITI L antibodies. From the result, it was proved that ITI polymorphism originates from the ITIH1 gene products, therefore we propose to call this system ITI H1 polymorphism.     

Localization of pulmonary human sarcoma xenografts in athymic nude mice with indium-111-labeled monoclonal antibodies

In order to study localization of metastatic tumors with a radiolabeled monoclonal antibody, a pulmonary metastases model was devised in athymic mice. Metastatic pulmonary sarcoma colonies were verified by histological examination. A murine monoclonal antibody (MAb 19-24) directed against a human sarcoma antigen was labeled with indium-111 (111In) by use of the linker 1-(p-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (SCN-Bz-DTPA). MAb P3 was similarly labeled as a negative control. In the group given MAb 19-24, the percent injected dose per gram lung tissue bearing tumor colonies (30.1%, 29.6%, and 27.7% on Days 1, 2, and 3, respectively) was significantly (p less than 0.05) higher than in those receiving MAb P3. Hepatic activities of both 111In-MAb 19-24 and 111In-MAb P3 were low. The lungs with tumor colonies demonstrated clearest images on Day 3. The specific binding of 111In-SCN-Bz-DTPA-labeled MAb 19-24 to pulmonary xenografts without appreciable liver uptake indicates that it may be useful in the clinical localization of pulmonic metastatic lesions.     

N，N－二取代甘氨酸－3－羟基－1，4－苯并二氮卓酯制备方法的MNDO研究

应用量子化学的微扰分子轨道理论，对用两种不同合成方法制备Ｎ，Ｎ－二取代甘氨酸－３－羟基－１，４－苯并二氮酯的可行性进行了分析。通过改进的忽略双原子微分重叠法（ＭＮＤＯ）程序计算，得到了原子净电荷、前线轨道电荷及前线轨道系数，代入微扰分子轨道方程后，得到了半定量的理论产率估计，该估计与实验结果基本一致。     

8－氮鸟嘌呤诱变3D_5杂交瘤细胞的初步研究

小鼠杂交瘤３Ｄ５为一株分泌抗人红细胞非凝集性单克隆抗体的杂交瘤细胞株，用８－氮鸟嘌呤（８－ＡＧ）诱变处理可以使之成为具有模拟骨髓瘤性质的次黄嘌呤鸟嘌呤磷酸核糖转移酶缺失的杂交瘤细胞系，并且保留杂交瘤细胞系分泌抗体的能力，在利用自身血细胞凝集试验对各种病原体的快速诊断中应用广泛。实验表明，用不同剂量的８－ＡＧ进行诱变，在不同剂量水平上３Ｄ５杂交瘤细胞主要呈现细胞生长抑制、部分细胞衰老死亡、存活细胞分裂生长成为多细胞克隆及细胞适应８－ＡＧ诱变后恢复正常生长能力等一系列的变化过程。利用间接免疫荧光方法检测３Ｄ５分泌抗体结果表明，诱变后３Ｄ５杂交瘤细胞株仍然保持有分泌抗人红细胞非凝集性单克隆抗体的能力，此细胞系的建立不仅为研制自身血细胞凝集试验的双功能抗体奠定了基础，同时也具有一定理论意义     

Monoclonal antibodies: old and new trends in breast cancer imaging and therapeutic approach

Over the last two decades, various research protocols were applied for scintigraphic imaging, prognosis and treatment of breast cancer, using monoclonal antibodies. Monoclonal antibodies approved by the United States Food and Drug Administration (FDA) include the anti-carcinoembryonic antigen (CEA), and B72.3, prepared against the tumour-associated glycoprotein, TAG-72. The recombinant humanized "cold" anti-HER2 monoclonal antibody (trastuzumab), which targets oncogene receptor HER2 has hitherto been the only monoclonal antibody widely used for the treatment of breast cancer in the USA, with or without chemotherapy. Trastuzumab is constructed against the HER2 oncogene receptor (also known as neu or c-erb-B2), which is overexpressed in 25%-30% of breast cancer cell lines and is associated with poor prognosis. Immuno-lymphoscintigraphy is also applied to guide and monitor the effect of treatment regimes. Radiolabelled, "hot" monoclonal antibodies are currently being applied for the treatment of primary or metastatic breast cancer, in experimental, pre-clinical, or clinical trials, in combination with traditional external beam radiotherapy and/or chemotherapy. Radioimmunotherapy comprises systemically administered monoclonal antibodies, linked to high-energy, beta-emitting radionuclides. Radioactive antibodies, in the form of yttrium-90 (90Y)-BrE-3, 90Y- m170 and 131I- or 90Y- labelled L6 antibody, are applied with adjuvant autologous peripheral blood stem cells transfusion, to prevent myelotoxicity. Partial or rarely complete responses to "hot" antibody treatment, of breast cancer have been reported. Innovative strategies using this combined-modality treatment hold promise for better disease-free and survival rates.     

Antibody guided diagnosis and therapy of brain gliomas using radiolabeled monoclonal antibodies against epidermal growth factor receptor and placental alkaline phosphatase

Twenty-seven patients with brain glioma were scanned using 123I-labeled monoclonal antibodies against epidermal growth factor receptor (EGFR1) or placental alkaline phosphatase (H17E2). Successful localization was achieved in 18 out of 27 patients. Eleven out of 27 patients were also studied using a nonspecific control antibody (11.4.1) of the same immunoglobulin subclass and observable tumor localization was also achieved in five patients. The specificity of targeting was assessed by comparing images obtained with specific and nonspecific antibodies and by examining tumor and normal tissue biopsies after dual antibody administration. Ten patients with recurrent grade III or IV glioma who showed good localization of radiolabeled antibody were treated with 40-140 mCi of 131I-labeled antibody delivered to the tumor area intravenously (n = 5) or by infusion into the internal carotid artery (n = 5). Six patients showed clinical improvement lasting from 6 mo to 3 yr. One patient continues in remission (3 yr after therapy), but the other five who responded initially relapsed 6-9 mo after therapy and died. No major toxicity was attributable to antibody-guided irradiation. Targeted irradiation by monoclonal antibody may be clinically useful and should be explored further in the treatment of brain gliomas resistant to conventional forms of treatment.     

低剂量电离辐射诱发小鼠显性致死突变的适应性反应

须先给予雄性小鼠０．０５Ｇｙ照射，随后给予２．０Ｇｙ照射，观察精子发生不同阶段显性致死突变率，受孕率．结果表明精原干细胞阶段、分化型精原细胞阶段预先照射０．０５Ｇｙ组，平均植入胚胎数、活胎数均明显高于单纯２．０Ｇｙ组，显性致死突变率明显低于单纯２．０Ｇｙ照射组，表现出适应性反应．同时还观察到预先给予小剂量处理组与雌鼠交配的受孕率也高于单纯大剂量组。统计学检验差异有非常显著性．