华支睾吸虫病免疫诊断研究进展

华支睾吸虫病是华支睾吸虫寄生于人体肝胆管内引起,又称肝吸虫病,该病的诊断主要依靠检获虫卵,但因虫卵小或感染度轻、排卵少而易于漏诊,免疫诊断可弥补不足。随着高新技术的发展和应用,显著提高了免疫诊断的敏感性、特异性和诊断效果,其在临床辅助诊断和疫区流行病学调查中的作用越来越突出。现将近年来有关本病免疫诊断的研究进展综述如下。１　特异性抗体的检测１１　检测用特异性抗原的选择　检测抗体的特异性主要取决于所用抗原的特异性,华支睾吸虫诊断抗原的研究,大多限于粗抗原或初步纯化抗原［１］。１１１　华支睾吸…     

华支睾吸虫成虫14～33 ku抗原诊断价值的研究

目的探讨纯化华支睾吸虫成虫14~33 ku抗原在华支睾吸虫病免疫诊断中的应用价值。方法使用SDS-PAGE和电洗提法,从可溶性华支睾吸虫成虫抗原中分离纯化14~33 ku抗原,用此抗原经ELISA方法检测华支睾吸虫病、卫氏并殖吸虫病、日本血吸虫病、姜片虫病患者血清及健康人血清特异性IgG抗体,并与粗制可溶性成虫抗原和可溶性成虫脱脂抗原ELISA结果相比较。结果检测63例华支睾吸虫病患者血清,纯化华支睾吸虫成虫14~33 ku抗原ELISA阳性率为76.2%,可溶性成虫抗原和可溶性成虫脱脂抗原ELISA阳性率均为100%;检测25例卫氏并殖吸虫病、85例日本血吸虫病、27例姜片虫病患者血清,14~33 ku抗原的ELISA交叉反应阳性率分别为8.0%、3.5%、0,而可溶性成虫抗原分别为80.0%、62.4%、14.8%,可溶性成虫脱脂抗原分别为64.0%、55.3%、7.4%;检测127例健康人血清,14~33 ku抗原的ELISA假阳性率为0,可溶性成虫抗原和可溶性成虫脱脂抗原的假阳性率分别为5.5%、3.1%。结论纯化华支睾吸虫成虫14~33 ku抗原用于华支睾吸虫病免疫诊断的特异性优于粗抗原,但敏感性较低。     

免疫金电转移印斑技术检测华支睾吸虫病人血清抗体

我们应用电转移印斑结合免疫金技术,进行了华支睾吸虫病人血清中抗体的研究,以寻找一个敏感、特异的华支睾吸虫病免疫诊断方法。一、材料和方法 (一)抗原的制备自人工感染华支睾吸虫囊蚴的猫肝中取华支睾吸虫成虫100条,以生理盐水洗涤数次.超声粉碎100w,10min,4℃冰箱冷浸72h,5000r/min离心15min,取上清液。 (二)实验血清1.华支睾吸虫病人血清:采自山东金乡县及宁阳县,经粪便检查,华支睾吸虫虫卵阳性     

亲和层析法纯化华支睾吸虫抗原的研究

目的探索华支睾吸虫特异性抗原的纯化方法。方法从华支睾吸虫病流行区的淡水鱼中分离华支睾吸虫囊蚴,接种动物,收集成虫,制备匀浆液(粗抗原),借助偶联华支睾吸虫病人血清抗体的Sepharose4B亲和层析柱,提取特异性抗原,用浓度梯度聚丙烯酰胺凝胶电泳(CG-PAGE)和免疫印迹方法鉴定纯化抗原的纯度和特异性。结果亲和层析法纯化抗原经CG-PAGE显示5条蛋白带,免疫印迹试验鉴定均有抗原性,强阳性带蛋白分子质量单位为31ku,各区带蛋白与并殖吸虫、血吸虫血清抗体无交叉反应。粗抗原有16条蛋白带。结论经亲和层析法纯化的华支睾吸虫抗原特异性高。亲合层析是获取华支睾吸虫诊断用抗原的较佳方法。     

两种方法纯化的抗体筛选华支睾吸虫模拟抗原表位的比较

目的比较用不同方法纯化的IgG从噬菌体随机12肽库中筛选出的华支睾吸虫模拟抗原表位。方法用饱和硫酸铵沉淀法和饱和硫酸铵沉淀法加层析法(两步法)纯化提纯的华支睾吸虫病患者血清IgG筛选华支睾吸虫模拟抗原表位,扩增随机挑取噬菌斑并进行序列分析和免疫学鉴定。结果硫酸铵沉淀法纯化IgG筛选的20个噬菌体克隆,6个有不同的DNA插入序列,Western blot显示6个克隆均可与华支睾吸虫患者血清反应,斑点免疫金银染色法有3个克隆可被华支睾吸虫患者血清识别。两步法纯化IgG筛选的20个噬菌体克隆,9个有DNA插入序列,其中有2个序列相同;Western blot和斑点免疫金银染色法显示9个克隆均可与华支睾吸虫患者血清反应。结论两种方法纯化的IgG均筛选到了可与华支睾吸虫患者血清结合的模拟抗原表位,用两步法纯化IgG筛选的模拟抗原表位更具有潜在的诊断价值。     

亲和层析法纯化华支睾吸虫抗原的研究

目的 探索华支睾吸虫特异性抗原的纯化方法。方法从华支睾吸虫病流行区的淡水鱼中分离华支睾吸虫囊蚴，接种动物，收集成虫，制备匀浆液（粗抗原），借助偶联华支睾吸虫病人血清抗体的Sepharose4B亲和层析柱，提取特异性抗原，用浓度梯度聚丙烯酰胺凝胶电泳（CG-PAGE）和免疫印迹方法鉴定纯化抗原的纯度和特异性。结果亲和层析法纯化抗原经CGPAGE显示5条蛋白带，免疫印迹试验鉴定均有抗原性，强阳性带蛋白分子质量单位为31ku，各区带蛋白与并殖吸虫、血吸虫血清抗体无交叉反应。粗抗原有16条蛋白带。结论经亲和层析法纯化的华支睾吸虫抗原特异性高。亲合层析是获取华支睾吸虫诊断用抗原的较佳方法。     

不同抗原的免疫学方法诊断华支睾吸虫病的实验观察

近来有人将纯化抗原应用于华支睾吸虫病的诊断,取得了较满意的结果。我们进一步应用硫酸铵分段沉淀华支睾吸虫不同抗原,进行酶联免疫吸附试验及皮内试验,以评价其应用阶值,报道如如下。一、材料与方法(一)华支睾吸虫抗原1.粗提抗原:自人工感染华支睾吸虫囊蚴的猫肝脏中取华支睾吸虫成虫200余条。以生理盐水洗     

抗华支睾吸虫代谢抗原单克隆抗体的制备及鉴定研究

目的建立分泌抗华支睾吸虫代谢抗原的单克隆抗体杂交瘤细胞株。方法用华支睾吸虫成虫代谢抗原免疫BALB/c小鼠，取其脾细胞与小鼠骨髓瘤细胞SP2／0融合，筛选分泌高滴度单克隆抗体的杂交瘤细胞株，测定单抗免疫球蛋白亚类和单抗效价，检测单抗与日本血吸虫全虫可溶性抗原、卫氏并殖吸虫成虫抗原和猪囊尾蚴抗原的交叉反应。结果获得3株分泌高滴度抗华支睾吸虫代谢抗原的杂交瘤细胞株，其分泌的抗体与日本血吸虫、卫氏并殖吸虫和猪囊尾蚴抗原均不发生交叉反应，3株单抗均属IgG。结论制备的抗华支睾吸虫代谢抗原的杂交瘤细胞株能分泌高滴度和高特异性的单抗，为制备免疫诊断试剂盒奠定了基础。     

华支睾吸虫特异性富甘氨酸2a类抗原基因的克隆、表达和免疫学诊断价值评价

目的采取免疫学方法筛选华支睾吸虫成虫cDNA表达文库,寻找新的抗原基因。方法使用华支睾吸虫病人混合血清以及华支睾吸虫排泄分泌抗原的免疫小鼠血清,分别免疫学筛选华支睾吸虫成虫λ ZAP cDNA表达文库;将阳性噬菌体克隆、测序以及核苷酸序列比对分析;将目的基因的编码区克隆至原核表达质粒pET28a(+)中,采用制备不带N端融合标签的天然蛋白的策略表达融合蛋白,使用组氨酸标签亲和纯化柱（Ni NTA树脂）纯化融合蛋白;采用间接ELISA法,检测血清中的特异性抗体,共检测35例华支睾吸虫虫卵粪检阳性血清,36例正常人血清,15例日本血吸虫、15例卫氏并殖吸虫和13例猪囊尾蚴病人血清,评价重组蛋白的免疫学诊断价值。结果发现华支睾吸虫特异性富甘氨酸2a（GRA2a）类抗原基因家族,将其中的Cs4抗原基因植入重组表达质粒pET28a(+)的NcoI位点,成功实现融合蛋白的表达,并进一步纯化得到可溶性重组蛋白。采用间接ELISA法检测血清中的特异性抗体,该ELISA法的敏感性为80.0%,特异性为97.2%,总符合率为88.7%;日本血吸虫、卫氏并殖吸虫和猪囊尾蚴病人血清的假阳性率分别为6.7%、6.7%和7.7%。经核苷酸序列同源性比较,华支睾吸虫GRA2a类抗原是迄今为止尚未进行功能研究的华支睾吸虫特异性抗原。结论发现华支睾吸虫GRA2a类抗原基因家族;成功构建重组表达质粒,并表达、纯化出可溶性重组蛋白;该抗原具有较高的免疫学诊断价值。     

华支睾吸虫cDNA文库的构建

目的　构建华支睾吸虫cDNA文库。方法　采集华支睾吸虫阳性鱼 ,分离囊蚴 ,感染实验动物兔 ,解剖兔收集华支睾吸虫成虫虫体。应用“一步法”提取华支睾吸虫总RNA ;经过mRNA纯化、cDNA合成 ,以PcDNA3(Amp +)质粒为载体构建文库。挑取 2 0个克隆进行扩增 ,选择 9个克隆进行DNA序列测定。序列结果与GenBank中相关基因进行比对。结果　获得含有 9 7× 10 5个重组子库容量的华支睾吸虫cDNA文库。其中PC6号克隆基因序列与华支睾吸虫半胱氨酸蛋白酶序列具有 93%的同源性。结论　已构建成华支睾吸虫cDNA文库。     

日本血吸虫病诊断抗原研究进展

血吸虫病免疫诊断技术的发展为血吸虫病控制做出了重要贡献。血吸虫抗原检测特异性抗体是目前血吸虫病免疫诊断的常用方法,所使用的抗原可分为粗制虫源抗原、纯化虫源抗原和重组抗原。研制高敏感性、高特异性和具有早期诊断价值的抗原是免疫诊断研究的重点。本文对近年日本血吸虫病诊断抗原研究进展进行了综述。     

The specificity of the humoral immune response to soluble mycobacterial antigens in tuberculosis

A high proportion of patient with tuberculosis have been shown to produce significant levels of antibodies to antigens unique to Mycobacterium tuberculosis, antigens restricted to the slowly growing mycobacteria and antigens common to all species in the genus. About 20% of patients with this disease do not have significantly elevated levels of antibodies to any of these groups of antigens. Consequently, the availability of highly purified soluble antigens specific for M. tuberculosis would not permit more cases of tuberculosis to be diagnosed serologically.     

Biochemical characterization of alien H-2 antigens expressed on a methylcholanthrene-induced tumor.

A methylcholanthrene-induced tumor of BALB/c (H-2d) origin had been shown to express H-2 antigens normally associated with the H-2k haplotype. The molecules expressing the alien antigens have been partially purified and shown to be distinct from molecules expressing normal H-2d antigens. Like normal H-2 antigens, the alien antigens have a molecular weight of 48,000, are glycoproteins, and are noncovalently bound to beta 2-microglobulin. The alien antigens differ from normal H-2 antigens in their susceptibility to papain digestion, behavior during gel filtration, and overall stability. Possible mechanisms accounting for the expression of the alien antigen are discussed.     

Cell-free synthesis of mouse H-2 histocompatibility antigens.

The mRNAs coding for the histocompatibility (H-2) antigens of the mouse have been identified by cell-free translation of poly(A)-containing RNA obtained from the livers of mice (strain A/J), followed by immunoprecipitation of the cell-free products by using an antiserum directed against purified H-2a. Unlike the 47,000- and 46,000-Mr H-2 glycoproteins synthesized in splenic lymphocytes, the cell-free translation products have Mrs of 45,000 and 44,500, representing the unglycosylated forms of these antigens. The cell-free products are shown to be related to the H-2 antigens by competition immunoprecipitation with purified H-2a and by two-dimensional tryptic peptide mapping. The H-2 mRNAs which sediment at 17 S are found associated predominantly with membrane-bound polysomes and are actively translated in the liver where as many as 16 ribosomes are associated with each molecule of H-2 mRNA. The implications of these studies for molecular cloning and for an understanding of the organization and expression of the genes encoding these H-2 antigens are discussed.     

Human antibodies reactive with purified envelope antigens of primate type C tumor viruses.

Human sera from healthy individuals have been shown to react with viral antigens in preparations of detergent-disrupted C type viruses from monkeys. In this report, it is demonstrated that (populations of) these human antibodies react with highly purified envelope glycoproteins of the simian sarcoma virus-simian sarcoma-associated virus complex and the Friend leukemia virus complex. Several immunological parameters influencing human antibody binding to C type tumor virus antigens have been characterized. These parameters indicate that human antibodies tend to bind to what is probably a subset of the antigenic determinants on the virus envelope antigens and that different human sera recognize the same antigenic determinants on the virus envelope antigens tested. The possible origin of these antibodies is discussed.     

Differential in vitro immune responses to biliary tract antigens in primary biliary cirrhosis and chronic active hepatitis

ABSTRACT— Cellular immune reactions against normal biliary tract antigens have been investigated in 21 patients with primary biliary cirrhosis (PBC) and 18 with chronic active hepatitis (CAH) using the leucocyte migration inhibition test with partially purified antigens from normal human gall-bladder bile. Four antigen fractions, containing (either separately or together) three previously-described biliary antigens, were employed: (1) Antigen I; (2) Canalicular antigen; (3) Canalicular and Ductular antigens; (4) All three antigens. Seventeen (81%) of the PBC patients showed migration inhibition with all four fractions. Five (28%) of the CAH patients showed inhibition with three fractions but none exhibited sensitization to the fraction containing only the antigen derived from the bile canalicular portion of the hepatocyte membrane. In experiments with purified lymphocyte sub-populations from PBC patients, leucocyte migration inhibitory factor production was shown to be a function of T-lymphocytes. The antigenic selectivity with respect to the responses in CAH patients suggests that sensitization to biliary tract antigens is probably not a secondary phenomenon resulting from »unmasking« of antigens after bile duct damage has occurred but may be more directly related to the disease process.     

The detection of antibodies to the trophozoite antigens of Lamblia by an immunoenzyme method]

Whether purified antigens of Lamblia intestinalis trophozoites can be used to detect these antibodies by immunoassay. The drugs of immunodominant Lamblia antigens were prepared by anion-exchange chromatography of solubilized trophozoite components and they are mainly presented by proteins having molecular weights of 70, 56, and 49 kD. Immunoassay using these antigens revealed antibodies to Lamblia trophozoite antigens in sera of 87.6% of patients with lambliasis (its diagnosis was established on the basis of microscopic data on the duodenal content) and only in 16.2% of clinically healthy blood donors. Twenty six sera from patients with trichomoniasis having high levels of antibodies to trichomonad antigens were studied to evaluate the specificity of this method for detection of antibodies. It has been found that the proportion of subjects in this group who have also antibodies to Lamblia antigens does not greatly differ from that of healthy blood donors (19.2 and 16.2, respectively).     

细粒棘球蚴囊液抗原纯化方法的比较评价

对于绵羊肝脏细粒棘球蚴囊液用常规粗制法,通过抗绵羊全血清抗体免疫吸附柱的亲和层析法,Oriol氏纯化法,Burstein氏纯化法,以及将纯化抗原再经酚提取法制备的六种抗原,以SDS-PAGE,免疫电泳,ELISA,IHA等方法进行了抗原得率、纯度、组份、免疫活性和特异性等方面的比较。结果表明:亲和层析抗原得率最高(l6.25％～22.64％),Oriol氏法纯化抗原最低(3.35％)。与囊液粗抗原相比,纯化抗原中Burstein抗原组份最多。经酚提取的纯化抗原组份最少。各种抗原组份数目显然相差明显,但在免疫电泳中均出现弧5沉淀线。酚提取法不能达到将抗原5和抗原B分离的目的。在ELISA和IHA中,Burstein法纯化抗原的活性最高。作者分析了亲和层析抗原活性低于Burstein和Oriol两种抗原的原因可能是亲和层析抗原缺少68kDa和36.5kDa两种宿主组份。因而认为这两个组份可能同时具有寄生虫抗原的性质。在ELISA试验中,各种纯化抗原均与囊虫病人血清产生部分交叉反应,但比粗抗原低。在IHA试验中,纯化抗原均不与囊虫病人血清产生交叉反应。作者推荐由于Burstein法纯化抗原得率高,提纯程序较简单,抗原活性高而且在IHA中没有与囊虫病人血清的交叉反应,因而可作为常规诊断抗原使用。     

Identification of the immunogenically active components of the Sm and RNP antigens

The spectrum of cellular targets in the autoimmune diseases is both large and varied and includes among the nuclear components the so-called Sm and RNP antigens associated with systemic lupus erythematosus. The use of immunoaffinity chromatography with dual specificity for the Sm and RNP antigens has allowed for their substantial purification from rabbit thymus in parallel and in quantity. In lieu of a functional assay, the use of a counterimmunoelectrophoresis assay provided a sensitive and rapid means of monitoring the distribution of the two antigens during purification and ensured the isolation of complexes containing the components required for antigenicity. The resulting purified complex consisted of nine polypeptides having molecular weights of approximately 9000 to 44,000 and two small RNAs of similar size. However, limited proteolysis of the isolated complex suggested that most of these polypeptides were not actually required for antigenic activity. Unlike Sm in crude thymus extracts, purified Sm was RNase sensitive. Thus, one of the major diagnostic criteria used to distinguish Sm and RNP antigens in crude extracts was shown to be invalid for purified material, suggesting that both antigens from rabbit thymus are actually ribonucleoprotein complexes.