日本血吸虫单克隆抗独特型抗体NP30的建株及初步鉴定

用感染8条日本血吸虫尾蚴,时间长达一年半的BALB/c小鼠脾细胞与SP2/0骨髓瘤细胞融合,用兔抗日本血吸虫肠相关抗原和兔抗可溶性虫卵抗原免疫血清与细胞培养上清进行ELISA筛选阳性克隆,最终建立了分泌单克隆抗独特型抗体细胞株NP30。作者用一系列试验证明,NP-30是日本血吸虫肠相关抗原的内影象抗独特型抗体,有替代日本血吸虫虫源性抗原用于血清学诊断的可能性。     

日本血吸虫童虫保护性单克隆抗体的建株和鉴定

本研究获8株抗日本血吸虫(中国大陆株)童虫的单克隆抗体,从中确定一株具有体内保护性功能的单克隆抗体(编号为N15D9),其保护率范围为14-39％。经间接免疫荧光法和ELISA试验检测,N15D9能与尾蚴、3h龄机械转化童虫和5d龄肺童虫的表膜抗原反应。经3h龄机械童虫可溶性抗原的免疫印迹法分析,N15D9能识别132、96和10kDa抗原分子。     

抗日本血吸虫噬菌体抗体库的筛选和阳性克隆的鉴定

目的　研制抗日本血吸虫循环抗原SjMAg的单链抗体。　方法　用日本血吸虫成虫代谢抗原SjMAg包板,对已构建的抗日本血吸虫噬菌体抗体库进行3轮富集,然后用ELISA法从富集的次级抗体库中筛选阳性克隆,最后借助ELISA、SDSPAGE和Western印迹等方法,根据阳性克隆表达产物与其它4种吸虫抗原反应的情况,进行特异性鉴定。　结果　从随机挑取的72个克隆中筛选到阳性克隆6个,经交叉筛选和鉴定最终获得特异性抗日本血吸虫阳性克隆2个。　结论　用固相富集和ELISA筛选法可以从抗体库中有效地筛选到抗日本血吸虫循环抗原的阳性克隆,获得的抗体克隆可用于抗日本血吸虫循环抗原单链抗体的制备。     

抗日本血吸虫重组信号蛋白14-3-3单克隆抗体的制备和鉴定

目的制备抗日本血吸虫重组信号蛋白14-3-3单克隆抗体,并鉴定抗体性质。方法以重组表达纯化的日本血吸虫信号蛋白14-3-3为抗原免疫BALB/c小鼠,采用杂交瘤技术制备单克隆抗体,采用酶联免疫吸附试验及免疫印迹法对获得的单克隆抗体进行类和亚类、效价、浓度、纯度、亲和常数、特异性和检测灵敏度的测定与鉴定。结果共获得6株针对日本血吸虫信号蛋白14-3-3的单克隆抗体,分别为5G9、3F1、3F7、5C6、5D1和1G6,抗体类型分别为IgG1、IgG1、IgG2a、IgG2b、IgG1和IgG1。对其中的单克隆抗体5G9分析显示,制备的腹水抗体效价为1∶1.28×105,亲和层析纯化后的浓度为5.3 mg/ml,纯度95%,亲和常数为5.1×107mol/L。免疫印迹试验结果表明,该单克隆抗体可与体外重组表达纯化的日本血吸虫14-3-3蛋白特异性结合,同时也能与日本血吸虫可溶性虫卵抗原、成虫排泄分泌抗原和成虫抗原特异性反应,而与大肠埃希菌、健康人血清和华支睾吸虫抗原均无明显交叉反应。HRP标记的单克隆抗体5G9检测14-3-3蛋白的灵敏度为31.25 ng/ml。结论本研究获得了6株能稳定分泌抗日本血吸虫14-3-3蛋白的单克隆抗体杂交瘤细胞株,为建立新的日本血吸虫活动性感染检测方法奠定了良好的基础。     

斑点-ELISA和独特型/抗独特型抑制试验检测日本血吸虫病患者循环表膜抗原

将血吸虫成虫表膜机原的单克隆抗体8SE4联接辣根过氧化物酶制成结合物,用于直接法斑点-ELISA检测循环抗原。结果48例血吸虫病患者的阳性率为81.3％(39例),24例正常人未出现假阳性反应。检测华支睾吸虫及并殖吸虫病患者各18例,均无交叉反应。本结果证实日本血吸虫病患者血清中有循环表膜抗原存在。直接法斑点-ELISA方法简便,具有较高的敏感性和特异性,可作为诊断日本血吸虫病的补充方法。本文还报告了独特型/抗独特型(8SE_4/抗8SE_4)抑制试验检测循环表膜抗原的初步结果。     

抗独特型抗体替代马来丝虫感染期幼虫表面抗原

应用D1E5单克隆抗体(AB1,为小鼠IgM McAb,能识别马来丝虫感染期幼虫表面抗原决定簇)发现有一期特异性表面抗原仅存在于马来丝虫第二期后期和第三期幼虫。感染动物后2～3天内该抗原即从虫体表面丧失。它可能与丝虫传播有关和/或可以作为免疫攻击的靶抗原,然而分离和鉴定这种抗原的工作均未成功,因此制备含有D1E5独特型“内影像”的兔抗独特型抗体来替代该抗原。将AB1免疫兔产生抗独特型抗体(AB2),继用经典竞争试验检测AB2抗独特型特异性,结果表明AB2能特异性抑制AB1与马来丝虫幼虫表面结合,而不能抑制犬恶     

日本血吸虫单克隆抗抗独特型抗体NP48单特异性双链抗体的构建表达与初步鉴定

目的构建和表达日本血吸虫单克隆抗抗独特型抗体NP48单特异性双链抗体，并初步鉴定表达产物活性。方法应用SOE法扩增双链抗体基因VH-linker—VL（Diabody，简称D）。将D重组入原核表达载体pBAD／g Ⅲ。表达质粒转化E、coli TOP10，左旋阿拉伯糖诱导表达。对D的表达产物进行分离纯化，采用Dot—ELISA法检测纯化蛋白与NP30、可溶性虫卵抗原（SEA）、可溶性成虫抗原（SWAP）的结合活性。结果测序证实双链抗体基因D构建成功；构建了D的原核表达系统，诱导表达产物主要以包涵体形式存在，分子量约为27kD；经纯化、变性复性后用Dot—ELISA法鉴定结果表明，D表达的纯化蛋白可与NP30特异性结合。结论所构建、表达的NP48单特异性双链抗体具有亲本单抗的部分特性。     

抗rSjCTPI单克隆抗体检测血吸虫循环抗原的研究

为探讨抗重组血吸虫磷酸丙糖异构酶（rSjCTPI）的单克隆抗体检测血吸虫循环酶抗原在血吸虫病免疫诊断和疗效考核中的价值。制备日本血吸虫rSjCTPI表达蛋白,以此重组蛋白免疫小鼠,筛选出高效价的抗rSjCTPI单抗,建立以纯化的抗血吸虫可溶性虫卵抗原（SEA）的多克隆抗体（IgG）为捕捉系统,以酶标的抗rSjCTPI单抗为检测系统的酶联免疫吸附试验（P-M-ELISA）法检测血吸虫病人等血清。并对该法的敏感性、特异性、交叉反应以及疗效考核与常规的SEA-ELISA比较。结果获得了一株抗rSjCTPI的单克隆抗体,其抗体同型为IgG1。P-M-ELISA检测急性、慢性血吸虫病人、健康人以及华枝睾吸虫病人、肺吸虫病人血清的阳性率分别为100%、91.7%、0、0和0。而SEA-ELTSA为100%、98.3%、6.69%、10%和20%。P-M-ELISA法对30例同批血吸虫病人治疗前的阳性率和治愈后4个月的阴转率分别为100%和30%;而SEA-ELTSA分别为100%和10%。结果表明,应用抗rSjCTPI单抗检测血吸虫病人循环抗原的P-M-ELISA法具有较高的敏感性和特异性,并有一定的疗效考核价值。     

一组抗日本血吸虫卵抗原并表达株间交叉反应独特型的单克隆抗体的特性

为检测抗日本血吸虫卵可溶性抗原(SEA)并表达株间交叉反应独特型的单克隆抗体的特性,作者采用菲律宾株日本血吸虫尾蚴感染的C57BL/6鼠及纯化的日本血吸虫卵可溶性抗原免疫的BALB/c鼠的脾细胞与P_(?)NSI脊髓瘤细胞融合杂交生产单克隆抗体,并用免疫吸附和印渍技术进行鉴     

抗广州管圆线虫成虫单克隆抗体的研制及初步应用

目的制备抗广州管圆线虫成虫单克隆抗体并研究其初步应用。方法用广州管圆线虫成虫可溶性抗原免疫BALB/c小鼠,经融合,筛选分泌高滴度、高特异性单克隆抗体的杂交瘤细胞株。使用所得的单克隆抗体以Western blotting检测广州管圆线虫病患者血清,并建立双抗体夹心ELISA法检测感染大鼠血清。结果获得3株分泌高滴度抗广州管圆线虫成虫可溶性抗原的单克隆抗体杂交瘤细胞株(2A2、3F1、4H2),其分泌的抗体与日本血吸虫、卫氏并殖吸虫、猪囊尾蚴、旋毛虫抗原均不发生交叉反应。3株单克隆抗体均可识别广州管圆线虫成虫可溶性抗原蛋白Mr 15 000,以及患者血清中两种循环抗原Mr24 000和Mr15 000。双抗体夹心ELISA检测阳性率为76.5%。结论制备的抗广州管圆线虫成虫可溶性抗原的杂交瘤细胞株能分泌高滴度、高特异性的单克隆抗体,且在循环抗原的检测方面有应用前景。     

Production and characterization of human monoclonal anti-idiotype antibodies to anti-dsDNA antibodies

Anti-dsDNA autoantibodies are the hallmark of systemic lupus erythematosus (SLE) and frequently correlate with disease activity. In this study we report the isolation and characterization of human anti-Id monoclonal antibody fragments as single-chain Fv fragments (scFv) against anti-dsDNA antibody. The anti-Id monoclonal antibodies, specific for anti-dsDNA antibodies, have been cloned from phage display antibody scFv libraries derived from a patient with SLE. The V gene repertoires were derived from the RNA obtained from the B cells of an SLE patient with anti-Ro/SSA and anti-La/SSB antibodies. Affinity-purified anti-dsDNA antibodies were used for selection of bacterial clones producing specific scFv antibody fragments against anti-dsDNA antibodies and little reactivity with normal IgG and other IgG antibodies by ELISA. The anti-Id antibody recognizes a public idiotope that is broadly cross-reactive with polyclonal and monoclonal anti-dsDNA antibodies. This binding was largely inhibited by dsDNA antigen. The anti-Id antibody inhibited anti-dsDNA binding to dsDNA antigen in immunoassays and in the Crithidia luciliae assay. The anti-Id scFv antibody fragments derived from human genes could modulate the pathogenicity of anti-dsDNA autoantibodies and may have therapeutic implications in SLE. They may also be used as probes in studies of the structure of the idiotype.     

Monoclonal anti-idiotypic antibody mimics the CD4 receptor and binds human immunodeficiency virus.

A monoclonal anti-idiotypic (anti-Id) antibody, HF1.7, was generated against anti-Leu-3a, a mouse monoclonal antibody (mAb) specific for the CD4 molecule on human helper/inducer T lymphocytes. The anti-Id nature of HF1.7 was demonstrated by the following properties. (i) It reacted in a solid-phase immunoassay with anti-Leu-3a and not with a panel of irrelevant mouse mAbs. (ii) It partially inhibited the binding of anti-Leu-3a to CD4+ T cells. (iii) It detected a common idiotype present on various anti-CD4 mAbs. Because the CD4 molecule represents the receptor site for human immunodeficiency virus (HIV), the etiologic viral agent of acquired immunodeficiency syndrome, we examined the ability of the anti-mAb HF1.7 to mimic CD4 and bind HIV. This anti-Id mAb reacted with HIV antigens in commercial HIV ELISAs and recognized HIV-infected human T cells but not uninfected cells when analyzed by flow cytofluorometry. Attesting further to the HIV specificity, the anti-Id mAb reacted with a recombinant gp160 peptide and a molecule of Mr 110,000-120,000 in immunoblot analysis of HIV-infected cell lysates. The anti-Id mAb also partially neutralized HIV infection of human T cells in vitro. These results strongly suggest that this anti-Id mAb mimics the CD4 antigenic determinants involved in binding to HIV.     

Characterization of a family of monoclonal antibodies which bind Schistosoma japonicum egg antigens and express an interstrain cross-reactive idiotype

A family of monoclonal antibodies (MoAb) was derived from the somatic cell fusion of P3NS1 myeloma cells and lymphoid cells from naturally infected mice of hyperimmunized mice. C57BL/6 mice were naturally infected with Schistosoma japonicum, and BALB/c mice were hyperimmunized with preparations of S. japonicum soluble egg antigens (SEA). The MoAbs which reflected the immune repertoire of naturally infected animals versus hyperimmune animals were characterized with regard to antibody isotype, antigen binding specificity, in-vitro immunosuppression of antigen-induced cell-mediated immune responses and the expression of SJ-CRIM, a major cross-reactive idiotype which appears on polyclonal anti-SEA antibodies generated during murine S. japonicum infection. The data indicate that for MoAbs of the IgG isotype which bound SEA by ELISA, the most immunosuppressive anti-SEA MoAbs identified expressed SJ-CRIM and were derived from naturally infected mice. All anti-SEA MoAbs expressing SJ-CRIM showed an identical banding pattern on immunoblot analysis which was abrogated by weak periodate treatment. The generation of expression of SJ-CRIM on MoAbs using differing methodologies across an allotype barrier indicates that the expression of SJ-CRIM is encoded by a germline gene. These data indicate an association between expression of this germline interstrain cross-reactive idiotype and immunosuppressive capacity. In addition, the immunoregulatory network which develops during immune S. japonicum infection is initiated by a carbohydrate epitope(s) found on various SEA. These data have profound implications in the use of the cross-reactive idiotype as a serodiagnostic tool in schistosomiasis.     

Production of anti-insulin monoclonal antibody and its application to immunoassay of insulin and immunohistochemistry

An unlimited supply of suitable antisera is wanted for immunoassays, analysis of antigenic determinants and precise localization of antigens in biological systems. Therefore, we produced a monoclonal antiporcine insulin antibody by the hybridoma technology and assessed it in comparison with polyclonal antibody. The spleen cells of BALB/c mice immunized against porcine insulin were hybridized with mouse myeloma cells (P3-X63-Ag8-U1). The monoclonal antibody thus generated was shown to have high binding capacity and specificity to porcine insulin in radioimmunoassay. It reacted with human insulin as well, but did not crossreact with other polypeptide hormones produced in the pancreatic islets such as glucagon, somatostatin and pancreatic polypeptide. In immunohistochemistry human and dog islets were stained by this monoclonal antibody. Rat islets were not stained, although they reacted with polyclonal anti-insulin antibody. The insulin of human serum samples measured using the monoclonal antibody was tightly correlated with that using the polyclonal antibody. These observations indicate that our hybridoma-derived monoclonal antibody is useful for immunoassay as well as localization of insulin.     

Cell-mediated and humoral immune responses in capuchin monkeys infected with Schistosoma japonicum or Schistosoma mansoni

Cell-mediated immune responses, assessed by lymphocyte clonal expansion in vitro, as well as humoral responses, assessed by an enzyme-linked immunosorbent assay (ELISA), were evaluated in capuchin monkeys during a 7-month infection with Schistosoma mansoni or with a Japanese or Philippine strain of Schistosoma japonicum. Although mounting a vigorous antibody response against parasite antigens, the S. mansoni-infected monkeys failed to show lymphocyte proliferation in response to stimulation with soluble adult worm antigen or soluble egg antigen derived from S. mansoni. Monkeys infected with S. japonicum responded to parasite antigens obtained from S. japonicum both by antibody production and lymphocyte blastogenesis. Monkeys infected with S. japonicum (Japanese strain) never developed detectable levels of circulating immune complexes (CIC). On the other hand high levels of CIC appeared at 7 months of infection in the monkeys infected with S. mansoni. The CIC levels exhibited negative correlations with intensity of infection. In studies of antigen species specificity, sera from S. mansoni-infected monkeys showed much higher IgG antibody titers to antigens derived from S. mansoni than to S. japonicum-derived antigens. On the other hand, monkeys infected with S. japonicum had comparable IgG antibody titers to antigens of both schistosome species.     

Analysis of the Heterogeneity of the Binding Site Specificities of Hyperimmune Human Anti-A and -A,B Sera: The Application of Competition Assays Using Murine Monoclonal Antibodies

Monoclonal antibodies PL41 and AL62 have previously been shown to recognize two distinct blood group A epitopes on the red cell surface. Competitive inhibition of the binding of 125I-PL41 and 125I-AL62 to group A1 red cells, by hyperimmune polyclonal human antibodies, has been employed to investigate the binding site specificities of 15 anti-A and 8 anti-A,B sera. Differences in the degree of inhibition of the binding of the two MABs by individual anti-A or -A,B samples indicate that polyclonal reagents are composed of varying proportions of up to 3 (or more) different antibody specificities, each recognizing a distinct epitope: PL41-like, AL62-like and a third (as yet undefined) category of antibody. In general, those anti-A sera with PL41-like specificities are superior agglutinators of A2B cells with weakly expressed A antigens; similarly, the specificity of potent anti-A,B, sera capable of strongly agglutinating Ax cells was likewise directed towards PL41-binding blood group A trisaccharide haptens.