人体结肠癌细胞单克隆抗体的纯化及其在血清诊断上的初步应用

目的：为了进一步证明抗人体结肠癌细胞膜表面抗原（ＬＥＡ）的单克隆抗体（ＮＤ－１）有很高的特异性及其应用价值。方法：应用单克隆抗体纯化系统（ＭＡＰＳ－１００）对分泌ＮＤ－１抗体的杂交瘤细胞株（ＩＣ２）产生的小鼠腹水进行纯化，对纯化后的收集液用不同方法进行ＮＤ－１成分的检测、效价、浓度、纯度测定及亚型鉴定，还利用ＮＤ－１阳性收集液对１０例结肠腺癌患者和８例正常人的血清进行抗原成分的检测。结果：结肠癌患者血清中ＬＥＡ阳性率为６０％，正常人均为阴性。结论：ＮＤ－１对人结肠癌患者血清具有较好的特异性，是具有应用价值的新型血清标志物。     

Studies on immunodiagnosis of dracunculiasis. II. Search for circulating antigens

Sera from individuals living in a dracunculiasis endemic area of northern Ghana were examined for circulating Dracunculus medinensis antigens by applying protocols previously developed for detection of circulating antigens in other helminth infections. Antisera from rabbits immunised with homogenized first stage D. medinensis larvae were used for antigen capture and detection in three different forms, namely non-treated, biotinylated and horse-radish peroxidase (HRP)-labelled. Three different preparations of human sera were examined, namely non-treated, pre-treated with polyethylene glycol/ethylenediaminetetra-acetic acid (PEG/EDTA) for analysis of precipitated immune complexes, and pre-treated with trichloroacetic acid (TCA) for analysis of isolated glycoproteins. In both SDS-PAGE/Western blotting and ELISA, significant reactivity was observed between non-treated and treated rabbit-antisera on the one hand and non-treated and treated human sera on the other. However, no significant response differences were observed between sera obtained from individuals with dracunculiasis and non-endemic controls. The reasons are analysed and possible explanations presented. The study provided no evidence that D. medinensis-specific circulating antigens, detectable by relatively simple means, occur in infected individuals.     

Monoclonal antibodies against Opisthorchis viverrini antigens

Summary Monoclonal antibodies (MoAb) were produced against somatic antigens of adult human liver fluke Opisthorchis viverrini. Earlier studies attached diagnostic potential to an 89–90 kD antigen present in both somatic extracts and in vitro culture supernatants as well as to the abundant 16–17 kD tegumental protein doublet. Mice made excellent immune responses to low dose somatic extract adsorbed onto nitrocellulose or to the 80–95 kD region of SDS gel Western blots. The antigen specificities of hybridomas reactive with somatic antigen by ELISA were determined by radioimmunoprecipitation or immunoblotting. Six MoAb reacted with the desired 16 kD tegumental protein. A 90 kD somatic protein was identified by 9 clones. By indirect immunofluorescence, monoclonals reactive with the 16 kD polypeptide identified the outermost surface of the tegument. The 90 kD antigen was associated with all major muscle systems, most strikingly the crossed subtegumental layers, oral and ventral suckers, pharynx and a thin layer surrounding caeca. The biochemical identity of this muscle-associated antigen is unknown, but it is clearly distinct from the previously identified species-specific 89 kD exoantigen. The 16 kD tegumental protein shares epitopes with a number of related flukes. However, 2 MoAb which react with this protein show no crossreaction.     

并殖吸虫病的流行病学、临床学及血清学诊断的研究

目的 :研究并殖吸虫病在河南省的流行病学特点、临床特征及其血清学诊断方法。方法 :对我们诊治的 5 0例并殖吸虫病患者进行病例分析 ;在南阳地区进行本病的流行病学调查 ;应用斯氏狸殖吸虫成虫冰冻切片抗原间接荧光抗体试验 (IFAT)对本病患者血清进行抗并殖吸虫抗体的检测。结果 :本病在河南省主要分布于西部和西南部的山区及丘陵地带 ,在 5 0例患者中儿童占 6 2 % ,男性多于女性 ,夏季为本病的高发季节 ,主要因生食或半生食溪蟹和饮生溪水而感染。胸肺型、皮下包块型、脑型、腹型及心包型患者分别占本病的 5 0 %、32 %、2 2 %、18%及 12 %。嗜酸性粒细胞 (Eos)增多者占 72 %。痰、粪检查均未发现虫卵 ,皮下包块活检后病理检查主要表现为嗜酸性肉芽肿和隧道样改变。南阳地区居民并殖吸虫的感染率为 16 .3% ( 4 2 /2 5 7) ,感染率随年龄增长而下降 ;当地河南华溪蟹的囊蚴感染率为 40 % ( 4 8/12 0 ) ,实验感染大鼠后获得斯氏狸殖吸虫成虫和虫卵。应用IFAT对 5 0例本病患者血清检测时抗体阳性率为 10 0 % ,2 0例丝虫病、30例包虫病、9例结核病及 2 3例健康人血清均为阴性反应 ,抗原片在 -2 0℃可保存 5年而不丧失抗原活性。结论 :河南省的并殖吸虫病主要因斯氏狸殖吸虫所致 ,患者在临床上主要表现为幼虫?     

Serologic diagnosis of human infections with lymphocytic choriomeningitis virus: comparative evaluation of seven methods.

The aim of this work was to compare different procedures for the serodiagnosis of human infections with the lymphocytic choriomeningitis (LCM) virus and to single out those which are both reliable and practicable. By the use of 46 sera from as many persons who had undergone infection with this virus either some time in the past or very recently and of 26 control sera, seven methods were evaluated. For making a rapid diagnosis soon after infection, determination of antibody by the immunofluorescence procedure appears to be the method of choice. Of equal reliability, though less easy to perform, is demonstration of sensitizing antibody by a plaque reduction assay, this procedure having the additional advantage of detecting antibody many years after infection, probably for life. For the demonstration of neutralizing antibody, three of four methods which were analyzed in this study gave the expected information, two employing mice, one employing cell cultures for the determination of residual infectivity. Neutralizing antibody was found to appear relatively late after infection and cannot, therefore, be recommended for the demonstration of seroconversion early in covalescence. This antibody, however, persists for many years, presumably lifelong, and is well suited to detect infections with this virus if they have occurred in the more distant past. Based on previous observations by ourselves and others, as well as on the work reported here, the complement fixation test appears to be of little value for the serological diagnosis of infection with LCM virus.     

日本血吸虫表皮膜蛋白的研究 Ⅰ.细胞免疫和体液免疫反应观察

本文报道以血吸虫表皮膜蛋白作抗原,采用淋巴细胞转化试验和ELISA方法对感染血吸虫的家免作纵向免疫反应观察,发现感染后25天淋巴细胞转化反应达高峰,40天后逐渐下降,而IgG抗体于感染后25天开始升高并维持在一个高水平,用ELIsA方法检测10例血吸虫病人均呈阳性反应,对5例正常人及35例其他寄生虫感染者血清均为阴性反应,提出表皮膜蛋白有可能用于血吸虫病的免疫诊断。     

血清丙肝病毒RNA二种提取方法的比较

２４例抗－ＨＣＶ阳性的血清标本，同时以硫氰胍半字符热酚变性方法和蛋白酶ＫＩ肖化法裂解提取丙肝病毒ＲＮＡ（ＨＣＶＲＮＡ），并应用反转录半字符多聚酶链反应（ＲＴ－ＰＣＲ）和套式多聚酶链反应（Ｎｅｓｔ－ＰＣＲ）检测ＨＣＶＲＮＡ。结果表明，硫氰胍半字符热酚法明显优于蛋白酶Ｋ消化法。前者处理标本的阳性检出率为５８．３％，后者处理标本的阳性检出率为３３．３％（Ｐ＜０．０５）；前者操作步骤简便，在试剂的保存等方面优于后者，故前者更适于临床ＨＣＶＲＮＡ的检测     

日本血吸虫病PVF抗原片环卵沉淀试验规范化研究

为解决日本血吸虫病环卵沉淀试验(COPT)的稳定性和可比性,对改良的PVF-COPT中所涉及的各种因素进行了系统地比较观察。在一系列实验研究的基础上,对PVF-COPT操作方法规范化提出以下建议:PVF-COPT在反应板内进行,每孔放入一块PVF抗原片,作抗原的虫卵数量应控制在100个左右。抗原片可按要求大批量预制,置干燥器内,在室温能保存抗原性达7个月,从而防止因作抗原的虫卵数量过多而导致结果的不一致。被测血清为75μl按3∶1混入0.4%NaN_3防腐。孵育温度在30～37℃孵育48h观察结果。当环沉率≥1%,反应强度≥1+即可作为阳性反应,以免抗体水平低的患者漏诊。     

梅毒血清学诊断用抗原Gpd蛋白的重组表达及鉴定

目的 利用基因工程技术重组表达梅毒螺旋体Gpd蛋白,探讨其在梅毒血清学诊断中的应用.方法 PCR扩增Gpd基因序列,构建表达载体pET-28b-Gpd,将表达载体转化感受态细胞E.coli BL21(DE3)并以IPTG诱导蛋白表达,经镍柱亲和层析纯化后,利用质谱和免疫印迹法鉴定重组蛋白.以重组蛋白建立间接ELISA法,并检测20份TPPA阳性和20份TPPA阴性血清去评价其在梅毒血清学诊断中的应用.结果 PCR扩增出约1.1 kb的Gpd片段,成功构建重组质粒pET-28b-Gpd.表达的重组蛋白的相对分子质量约41×103,主要以包涵体形式存在,占菌体总蛋白的40%.质谱和免疫印迹分析证实重组蛋白为Gpd蛋白,并能够与梅毒患者血清发生免疫学反应.间接ELISA法测定TPPA阳性血清和TPPA阴性血清的符合率分别为95%(19/20)和100%(20/20).结论 重组Gpd蛋白能够与梅毒患者血清发生特异性的免疫学反应,是一种可潜在应用于梅毒血清学诊断的新抗原.     

Application of Dirofilaria immitis immunoreactive proteins in serodiagnosis

Dirofilariasis is a zoonotic global vector‐borne disease caused by Dirofilaria immitis. The present study focuses on the somatic and excretory/secretory (E/S) proteins released from adult D. immitis. We aimed to fractionate and identify adult D. immitis immunoreactive proteins. Somatic and E/S extracts were immunoblotted to identify the immunoreactive proteins. In the current study, we used matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐TOF/MS) to characterize the immunogenic proteins. Additionally, we used fast protein liquid chromatography (FPLC) to fractionate and evaluate the immunogenicity of the D. immitis secretome. The most immunoreactive proteins were between 10 and 48 kDa. Six proteins including polyprotein antigen, P22u, pepsin inhibitor Dit33, neutrophil chemotactic factor (DiNCF) precursor, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and heat‐shock protein 70 (HSP70) were found in both somatic and E/S extracts. Eluting the FPLC column with NaCl resolved two peaks in which the immunoreactivities of the purified proteins were conserved. Characterization of these proteins could provide a novel perspective for understanding the pathogenesis and diagnosing of this disease.