基于胶体金检测方法的蛋白芯片体系的建立

目的 构建一个检测尿液中HCG抗原的蛋白质芯片初级模型;方法 在硝酸纤维素膜上包被HCG单克隆抗体(抗a-HCG),加尿样后,标本中HCG抗原(标准品)与硝酸纤维素膜的抗体特异结合,再加入胶体金标记的单克隆抗体(抗á-HCG),使其特异结合,完成对标本中抗原的染色.最后应用芯片扫描仪对光学信号的灰度值进行分析,从而绘制出标本中不同浓度的HCG对光学信号灰度值的标准曲线;结果 与现有方法 相比,该方法将检测的最低浓度降至25 mIU/ml,具有良好的梯度、线性关系和重复性.结论 该方法 是一种快速、简便、有较强操作性的检测HCG的方法 .     

固相间接免疫测定中酶标二次抗体非特异反应的排除

直接点滴法或免疫转印(Immuno-transfer blot)法在硝酸纤维素膜上检测某一抗原时,常用间接法来进行。单克隆抗体的广泛应用,使得到具有较高特异性的一次抗体不再困难,而待测物与过氧化物酶标记的二次抗体试剂大都为多种物质的混合体。因此,酶标记的抗体中的某些成分     

肺炎支原体蛋白质抗原分析的研究

用SDS-聚丙烯酰胺凝胶电泳和免疫吸印方法,分析肺炎支原体蛋白质抗原成份。SDS-聚丙烯酰胺凝胶电泳经考马斯亮蓝染色可见多条泳带。免疫吸印法转移到硝酸纤维素膜,经酶标记染色可见12条带,其中明显可见者5条带,蛋白质分子量分别为165 000,90 000,65 000,46 000和28 000。其中有个别泳带未见报道,其意义有待进一步研究。     

抗食管癌单克隆抗体及食管癌细胞膜抗原的初步研究

以食管癌“109”细胞株免疫 BALB/C 小鼠,取其脾细胞与小鼠骨髓瘤细胞株SP2/0 融合,经三次克隆化,获得稳定分泌抗食管癌“109”细胞株单克隆抗体的杂交瘤细胞系2GF3。其分泌的单克隆2GF3抗体与外周血淋巴组胞、ABO 型红细胞、癌胚抗原以及3/5胃癌细胞株无反应,与2/5胃癌细胞株有弱(+)反应。与2GF3抗体对应的抗原为2GF3抗原,荧光标记检测表明后者主要表达在细胞膜上。用去污剂提取膜抗原进行电泳后转移至硝酸纤维素膜上进行酶标分析,2GF3抗原是分子量53KD 的糖蛋白,在还原及非还原条件下均显示相同的区带。它与抗癌胚抗原抗体及抗甲胎蛋白抗体均无反应,表明它不同于癌胚抗原和甲胎蛋白。     

紫外线照射日本血吸虫尾蚴疫苗保护性抗原的筛选

目的：分析经紫外线照射处理后日本血吸虫尾蚴蛋白的差异性表达，并从中筛选出有效的保护性抗原组分。方法：收集尾蚴，制备抗原，进行SDS-PAGE电泳后，转印至硝酸纤维素膜上，与照射尾蚴免疫血清和正常感染血清分别进行免疫印渍(Western blot)试验，比较两反应条带的差异性，筛选出惟有免疫血清所能识别的特有的抗原组分，并测定其分子量。结果：在童虫抗原中发现一条能被免疫血清所识别的特异性抗原条带，并测得其分子量为75kDa。结论：紫外线照射致弱尾蚴可诱导宿主产生特异性抗体，被该抗体所识别的抗原分子量为75kDa。     

胃癌相关抗原MG7-Ag模拟肽表位的筛选及测序分析

目的　从噬菌体呈现的随机肽库中筛选胃癌单克隆抗体MG7所识别的胃癌相关抗原的短肽模拟表位 ,为进一步研究胃癌相关抗原与单抗结合的结构基序奠定基础。方法　用胃癌单克隆抗体MG7对呈现于噬菌体衣壳蛋白pⅧ上的 2个九肽库分别进行亲和富集和免疫筛选 ;阳性克隆进一步用荧光标记和硝酸纤维素膜斑点印迹法证实其结合活性 ;经随机抽取部分阳性克隆进行DNA序列分析 ,推导出相应噬菌体呈现肽的氨基酸序列 ,通过序列比较分析相对保守的表位信息 ;运用HLA分子结合分析软件预测已测序短肽与HLA分子结合的可能性。结果　经反复多轮筛选和结合活性检测 ,从pⅧ和pⅧ cys 2个噬菌体肽库中分别得到了 12和 30个阳性克隆 ;通过对部分阳性克隆进行测序分析 ,推导相应的随机肽序列和序列比较分析 ,得到具有相对保守性的表位信息如PLX0 2 S、SAVR、XRMX、YARN等。经计算机预测分析 ,发现它们可与HLA多个分子结合。结论　PLX0 2 S、SAVR、XRMX、YARN等有可能为胃癌单克隆抗体MG7所识别的表位基序。     

斑点酶联免疫吸附试验检测抗独特型单克隆抗体

用斑点酶联免疫吸附试验检测抗独特型单克隆抗体,方法简便、快速,敏感性高,特异性强,所需样品量少,点样后至少可保存一个月;用戊二醛处理硝酸纤维素膜可以增强蛋白吸附。     

胶体金免疫层析法在早孕检测中的应用

目的:免疫层析法是将特异的抗体先固定于硝酸纤维素膜的某一区带,当该干燥的硝酸纤维素膜一端浸入样品(尿液或血清)后,由于毛细管作用,样品沿该膜向前移动,当移动至固定有抗体的区域时,样品中相应的抗原与该抗体发生特异性结合,若用免疫胶体金染色可使该区域显色。胶体金标记稳定、易贮存,常用于定性或半定量的快速免疫检测方法中.在免疫层析快速诊断技术中常用于β-HCG的特异性免疫诊断早孕检测等,具有快速、准确和无污染等优点。     

单克隆抗体酶联免疫吸附测定甲胎蛋白方法的建立

应用二种抗甲胎蛋白(AFP)不同抗原决定簇的单克隆抗体分别与纤维素及酶结合,建立酶联免疫吸附测定(ELISA)法。应用这种方法测定18例人血清标本,结果与放射酶免疫测定比较,二者趋于一致。此外,本文对单克隆抗体的ELISA与抗血清的RIA进行了比较与讨论。     

非标记抗体技术在流行性出血热病毒抗原研究中的应用

将非标记抗体技术应用于流行性血热病毒抗原研究，对感染细胞内病毒抗原及经免疫转印到硝酸纤维膜上的病毒多肽抗原进行了检测，并分别与间接免疫荧光和间接酶标抗体方法进行了比较。结果表明，非标记抗体技术敏感性高，背景浅，可用于流行性出血热病毒抗原检测与分析。用非标记抗体技术及间接免疫荧光法对流行性出血热单克隆抗体培养上清液进行筛选，两种方法阳性检出率相同（７．２９％，７／９６），但前者滴度（１：１６～２５６     

口服滋养细胞抗原诱导免疫耐受的实验性研究

目的 :探讨免疫性流产口服耐受疗法的可行性。方法 :采用抗人滋养细胞单克隆抗体纯化滋养细胞抗原 ,通过口服该抗原诱导小鼠产生免疫耐受 ,利用 EL ISA法检测血清中抗滋养细胞 Ig G抗体水平。结果 :口服不同剂量滋养细胞特异性抗原 ,4次免疫后小鼠血清中抗滋养细胞 Ig G抗体水平均明显低于对照组小鼠血清中抗体水平 ,其差异具有显著性 (P<0 .0 5 ) ,而且小鼠血清中抗滋养细胞 Ig G抗体水平随着抗原剂量的增加呈下降趋势 ;小鼠血清中抗滋养细胞Ig G抗体水平随着口服耐受诱导时间的增加而降低 ,但差异无显著性 (P>0 .0 5 )。结论 :口服纯化的滋养细胞抗原可特异性抑制血清中抗滋养细胞 Ig G抗体的产生 ,这种疗法可能是一种简单、有效、特异性预防或治疗免疫性流产的方法     

抗华支睾吸虫单克隆抗体杂交瘤细胞株的建立及其初步应用

本文用华支睾吸虫成虫可溶性抗原免疫的BALB/c小鼠与SP_2/o骨髓瘤细胞融合,经筛选和克隆化后获得4株抗华支睾吸虫单克隆抗体杂交瘤细胞。该4株荜克隆抗体与华支睾吸虫成虫抗原作ELISA,结果均为阳性。经交叉试验(ELISA法)证明与日本血吸虫成虫抗原,虫卵抗原和卫氏并殖吸虫成虫抗原均呈阴性反应。4株单克隆抗体免疫球蛋白亚类鉴定除3F_7株为IgG_1外,其余3株均属IgM。初步试用3F_7株单克隆抗体作ELISA双抗体法检测人体华支睾吸虫循环抗原。     

自身免疫性梅尼埃病的体液与细胞转移免疫示踪研究

目的：探究能否经体液和（或）细胞转移造成被动免疫性自身免疫性梅尼埃病（AIMD）,并了解针对内耳组织的特异性抗体和淋巴细胞到达内耳的途径,部位及其对内耳组织和生理功能的影响。方法：对AIMD模型动物的免疫球蛋白（IgG)行荧光标记,脾细胞（主要为T淋巴细胞）行同位素标记,继而进行被动免疫（同时在内淋巴囊周围局部注射内耳抗原）。观察内耳听觉和前庭功能变化,不同部位组织中荧光聚集反应和同位素含量改变。结果：特异性抗体（IgG)首先到达血管纹和纳淋巴囊部位,特异性淋巴细胞则首先出现在内淋巴囊局部,内耳听觉和前庭功能障碍发生与内耳组织中抗体（即荧光反应强度）及淋巴细胞含量（即CPM值）呈明显相关性。结论：体液和细胞转移免疫均可诱发出被动免疫性AIMD;内耳组织抗原的特异性抗体和淋巴细胞可经过体循环到达内耳局部,抗体主要经血管纹和内淋巴囊部位造成内耳病变（包括膜迷路积水）,而特异性淋巴细胞则主要通过内淋巴囊局部造成内耳免疫炎性病理变化。     

SPA结合HSV免疫血清交叉吸收抗原后对抗体分型的初步探讨

以HSV-1(F株)或HSV-2(G株)免疫血清结合SPA,然后用于交叉吸收HSV-2或HSV-1抗原,经吸收的抗原作ELISA,目的是检测HSV型特异性抗体。在未经吸收的抗原反应中,八份兔抗血清的同型反应与交叉反应差别不大,其抗体效价比值在2倍以内,而经交叉吸收的抗原反应中,抗体以同型反应为主,其抗体效价比值都在4倍以上,最高达16倍。经多次交叉吸收抗原后,抗体的交叉反应进一步降低,但其同型反应也有所降低。     

狼疮肾炎中新发现的抗肾小球系膜细胞抗体靶抗原的鉴定

目的 研究狼疮肾炎血清中新发现的抗肾小球系膜细胞抗体与抗DNA抗体的关系,及其靶抗原是否存在于细胞膜上。方法 DNA纤维素柱亲和层析分离狼疮肾炎患者血清中的抗DNA抗体,Western印迹法检测祛除抗DNA抗体的血清和亲和层析纯化的抗DNA抗体与系膜细胞靶抗原的结合。从体外培养的人系膜细胞系分离细胞膜并提取膜蛋白,以已知抗肾小球系膜细胞抗体阳性血清为探针应用Western印迹法鉴定膜蛋白中的靶抗原。结果系膜细胞中相对分子质量为74000、36000的蛋白可以被祛除抗DNA抗体的血清识别。而相对分子质量为63000、91000、125000的蛋白被亲和层析的抗DNA抗体识别。系膜细胞膜上至少相对分子质量为101000、91000、74000和31000的蛋白可以被狼疮肾炎患者的血清识别。结论部分抗肾小球系膜细胞抗体与抗DNA抗体无关。人系膜细胞系的膜蛋白中存在狼疮肾炎血清可识别的抗原物质,提示抗DNA抗体以外的直接针对肾小球系膜细胞的自身抗体在狼疮性肾炎的发病机制中可能起重要作用。     

Antibodies against major histocompatibility complex class I-related chain A in transplant recipients

Objective  To review the role of polymorphism of major histocompatibility complex class I-related chain A (MICA) gene and antibodies against MICA antigens in transplant immunology.

Data sources  The data used in this review were mainly from our own results and from the relevant English language literatures published from 1999 to 2010. Some data presented in this review are in press.

Study selection  Articles regarding MICA gene discovery and pioneering finding of antibodies against MICA antigen and allograft rejection were selected. This review chronicles the development of our understanding of the role that MICA antigens and antibodies may play in organ transplantation.

Results  Polymorphic glycoprotein MICA antigens were detected on freshly isolated human umbilical cord endothelial cells, but not on peripheral lymphocytes. Antibodies were found and typing of recipients and donors by sequencing the MICA alleles has established that de novo antibodies produced in kidney transplant recipients are directed at mismatched MICA epitopes and are associated with acute rejection and chronic transplant failure. The specificity of antibodies against the epitopes of MICA antigens were well characterized by donor MICA typing, single antigen array testing with antibody absorption and elution. Acute graft-versus-host disease was observed in stem-cell recipients who were mismatched for MICA.

Conclusions  Immunization against mismatched MICA epitopes encountered in donor organs after transplantation may result in antibodies against MICA alleles. Testing for MICA donor-specific antibodies (DSA) which are associated with early failure of kidney transplants may be helpful for identifying some of the targets of antibodies against antigens other than the human leukocyte antigen (HLA) and for improving transplantation outcome.

     

Rh血型系统抗CE复合抗原抗体的研究

目的鉴定Rh血型系统抗CE复合抗原的抗体,推测患者Rh血型基因型,制订患者的输血策略。方法对4例抗体筛选阳性且Rh表型均为DCcEe的患者的血清采用LISS/Coombs卡做抗体鉴定,根据阴性排除法的原则确定抗体的特异性。结果 4例患者血清中均存在Rh血型系统抗CE复合抗原抗体,分别为抗-RH6(f或ce)1例、抗-RH7(Ce)1例、抗-RH27(cE)2例。结论 RH表型一致的人由于基因型的不同,其红细胞上会存在或缺失某些CE复合抗原,缺失抗原的人经过输血或妊娠免疫,可能产生针对该复合抗原的抗体,而这种复合抗原抗体与单纯的C、c、E、e抗原均无反应性。     

Purification and partial characterization of a new group of gastric cancer associated antigens.

The corresponding antigens of MG series monoclonal antibodies (MG5, MG9, MGd1 and MGe1) against gastric cancer were purified and partially characterized. Each of these monoclonal antibodies was purified by passing through a DEAE-52 cellulose columns and covalently coupled with CNBr-activated sepharose 4B successively. By means of concanavalin A and antibody affinity chromatography, the corresponding antigens of MG series McAb were extracted from gastric cancer tissue respectively. Immunological and biochemical studies confirmed that the corresponding antigens of MG series McAb were a new group of gastric cancer associated neutral glycolipid and glycoprotein antigens.

     

Association of specific immune response to pork and beef insulin with certain HLA-DR antigens in type 1 diabetes

To test the association of HLA-DR antigens with high-responder and low-responder status to either beef or pork insulin, insulin antibodies in diabetic sera were separated into those with average low and those with average high affinity and their insulin-binding capacities for each insulin determined. Significantly less binding of pork insulin by the high affinity antibodies occurred in the group of patients with DR3 antigens compared with those with DR4 antigens (p less than 0.01) and DR3/4 antigens (p less than 0.01). The difference in the binding capacity of beef insulin by the high affinity antibodies between the groups with DR3 and DR4 antigens was less pronounced but still significant. The high-responder status of DR3/4 antigens to pork insulin suggests that the gene or genes associated with HLA-DR4, and responsible for a high response to pork insulin, are dominant to genes associated with HLA-DR3 and a low response. If extended to human insulin and different HLA-DR and HLA-B antigen patterns, these finding should help in the therapeutic selection of the appropriate insulin and thus reduce the induction of an anti-insulin response in patients with diabetes.     

Preliminary study of immunodiagnosis of trichinosis by ELISA using urea-soluble antigen of Trichinella spiralis

Urea-soluble antigens of Trichinella spiralis infected larva were preparedand the efficiency of the antigens in diagnosis of trichinosis by ELISA wasstudied.Specific IgG antibodies were detected by ELISA using urea-solubleantigens in 91.67% (22/24) of 24 serum samples taken from patients withtrichinosis and 91.67% (11/12) of 12 blood spots on filter paper.Specific IgMantibodies were seen in 95.24% (20/21) of 21 serum samples from patients withtrichinosis using the antigens.Sera of patients infected with Schistosoma japonicumhad some cross teactions to the antigens,but the sera of mice infected withSchistosoma japoni(?) Ancylostoma caninum larva and Plasmodium yoelti were allnegative in the test.