抗华支睾吸虫单克隆抗体的制备和鉴定

目的：建立分泌抗华支睾吸虫成虫的单克隆抗体杂交瘤细胞株并进行鉴定。方法：用华支睾吸虫成虫可溶性抗原免疫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虫卵抗原、卫氏并殖吸虫成虫抗原和猪囊尾蚴抗原的交叉反应，用ＩＦＡＴ进行单抗识别的抗原定位。结果：获得５株分泌高滴度抗华支睾吸虫成虫单抗的杂交瘤细胞株，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；５株单抗均属ＩｇＭ；单抗所识别的抗原定位于华支睾吸虫肠管壁。结论：制备的抗华支睾吸虫成虫的杂交瘤细胞株能分泌高滴度和高特异性的单抗。     

金标特异抗原单克隆抗体斑点免疫金染色用于检测脑囊虫…

应用金标记猪囊尾蚴囊液抗原为探针，以抗猪囊尾蚴抗原的ＭｃＡｂ为联桥，建立了非常规Ｄｏｔ－ＩＧＳ用于检测脑囊虫病患者ＣＡｇ的方法，用４Ｇ２ＭｃＡｂ检测８４例脑囊虫病人，１３例检出ＣＡｇ，阳性率为９２．８６％。检测其他疾病患者３６例，均为阴性。用４Ｇ２与１Ｆ１１株ＭｃＡｂ可分别检出最小抗原量为１ｎｇ／ｍｌ和０．２５ｎｇ／ｍｌ。本法敏感、特异、操作简单，可用于脑囊虫病的诊断。     

检测血吸虫循环抗原的多价单克隆抗体诊断试剂盒的研制和应用

用杂交瘤技术生产抗血吸虫肠相关循环阳极抗原（ＣＡＡ）单克隆抗体与血吸虫卵糖蛋白单克隆抗体经混合后标记辣根过氧化物酶（ＨＲＰ），制成Ｄｏｔ－ＥＬＩＳＡ诊断试剂盒，检测轻、中、重不同疫区粪检阳性的血吸虫病人血清中循环抗原，阳性率分别为８４．３％、８７．２％和９１．５％。累计阳性率为８９．２％（６１９／６９４）。对健康人、肝吸虫、肺吸虫病人血清均未出现阳性反应，显示有较高的敏感性和特异性。ＥＰＧ＞８０的病人循环抗原检出率高干ＥＰＧ／８０的病人。说明循环抗原检出率与感染度有关。血吸虫病人经吡喹酮治疗后半年和１年循环抗原转阴率分别为７１．４％和８８．６％。结果表明．该试剂盒可用于确诊病人和考核疗效。试剂盒质量稳定，操作简便快速，整个试验可在１小时内完成，且不需特殊仪器，适于现场大规模查病应用。     

单克隆抗体74D_(11)、SJA_(111)检测慢性日本血吸虫病循环抗原的研究

本研究用双抗夹心ＥＬＩＳＡ法检测慢性日本血吸虫病人血清循环抗原，观察血吸虫感染家兔血清循环抗原的动态变化。该方法采用兔抗ＡＷＡ－ＩｇＧ作包被抗体，抗日本血吸虫成虫和虫卵单抗ＳＪＡ１１１和７４Ｄ１１组合作Ⅱ抗，１７２例经粪孵毛蚴确诊的血吸虫病人中，１３６例循环抗原阳性（７９．１％），其中轻度（毛蚴数＜５只／２０克粪）、中度（６－２０）、重度（大于２０）感染者循环抗原阳性率分别为７３．４％、８７．７％、９２．８％，检测６５例健康献血员，２０例肺吸虫病人，１２例华支睾吸虫病人，１０倒包虫病人，交叉反应率依次为１．５％、５％、０％、０％，４２例经吡喹酮治疗后的血吸虫病人用该方法复查，治后３月和６月随访粪孵毛蚴阴转者中，循环抗原阴转率分别为７８．０％和９２．１％，考核疗效明显优于检测循环抗体。用单抗ＳＪＡ１１１检测感染家兔循环抗原动态变化，循环抗原于感染后３—４周可检出，７周达高峰，以后逐渐消退，而循环抗体（ＩｇＧ）推迟１周出现，８－９周达高峰。实验结果表明，血吸虫病血清ＣＡｇ阳性率与感染度有关，可在早期检出，检测方法具有较高敏感性和特异性，可用于早期诊断和疗效考核。     

抗猪囊尾蚴单抗,双特异单抗建株及检测血清循环…

检测猪囊尾蚴病人的血清循环抗原。     

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。     

抗弓形虫McAb的建立及应用研究

应用杂交瘤技术,建立了５株分泌抗弓形虫单克隆抗体的杂交瘤细胞。其中１Ｅ９和４Ｃ１０２株分泌的ＭｃＡｂ效价达１：１２８００。该２株单克隆抗体均属ＩｇＣ１亚类,其识别的弓形虫抗原分别为Ｐ２２、Ｐ２８。１Ｅ９和４Ｃ１０ＭｃＡｂ混合后与兔抗弓形虫抗体联合,进行双抗体夹心ＥＬＩＳＡ测定弓形虫的敏感性分别为１２．５和２５个虫体／ｍｌ。检测１４６份有不良孕产史妇女血清,１１份弓形虫循环抗原（ＣＡｇ）阳性（７．５     

单克隆抗体检测囊虫病人循环抗原及其在疗效考核中的应用

用单克隆抗体致敏绵羊红细胞作反向间接血凝试验，检测７７例囊虫病人血清循环抗原，阳性率为６４．９％；３０例正常人血清的假阳性率为０；２２例华支睾吸虫病人、２４例包虫病人和１７例弓形虫病人的交叉反应率分别为４．６％、４．２％和１１．８％。结果表明，该法较为敏感、特异，且稳定可靠、简单易行，尤适于在基层医疗单位推广应用。检测经治疗１￣１０个疗程囊虫病人血清中循环抗原和抗体，发现随着疗程的增加循环抗原的消     

单克隆抗体检测囊虫病人循环抗原及其在疗效考核中的应用

用单克隆抗体致敏绵羊红细胞作反向间接血凝试验，检测７７例囊虫病人血清循环抗原，阳性率为６４．９％；３０例正常人血清的假阳性率为０；２２例华支睾吸虫病人、２４例包虫病人和１７例弓形虫病人的交叉反应率分别为４．６％、４．２％和１１．８％。结果表明，该法较为敏感、特异，且稳定可靠、简单易行，尤适于在基层医疗单位推广应用。检测经治疗１～１０个疗程囊虫病人血清中循环抗原和抗体，发现随着疗程的增加循环抗原的消失明显快于抗体，且大部分患者的循环抗原和其临床症状、体征同时消失。表明循环抗原的检测对囊虫病的诊断和疗效考核有一定的实用价值。     

单克隆抗体74D11,SJA111检测慢性日本血吸虫病循环抗原的研究

本研究用双抗夹心ＥＬＩＳＡ法检测慢性日本血吸虫病人血清循环抗原，观察血吸虫感染家兔血清循环抗原的动态变化。该方法采用兔抗ＡＷＡ－ＩｇＧ作包被抗体，抗日本血吸虫成虫和血卵单抗ＳＪＡ１１１和７４Ｄ１１组合作Ⅱ抗，１７２例经粪孵毛蚴确诊的血吸虫病人中，１３６例循环抗原阳性（７９．１％），其中轻度（毛蚴数〈５只／２０克粪）、中度（－２０）、重度（大于２０）感染者循环抗原阳性率分别为７３．４％、８７．７％、     

Pentameric complex of viral glycoprotein H is the primary target for potent neutralization by a human cytomegalovirus vaccine

Human cytomegalovirus (HCMV) can cause serious morbidity/mortality in transplant patients, and congenital HCMV infection can lead to birth defects. Developing an effective HCMV vaccine is a high medical priority. One of the challenges to the efforts has been our limited understanding of the viral antigens important for protective antibodies. Receptor-mediated viral entry to endothelial/epithelial cells requires a glycoprotein H (gH) complex comprising five viral proteins (gH, gL, UL128, UL130, and UL131). This gH complex is notably missing from HCMV laboratory strains as well as HCMV vaccines previously evaluated in the clinic. To support a unique vaccine concept based on the pentameric gH complex, we established a panel of 45 monoclonal antibodies (mAbs) from a rabbit immunized with an experimental vaccine virus in which the expression of the pentameric gH complex was restored. Over one-half (25 of 45) of the mAbs have neutralizing activity. Interestingly, affinity for an antibody to bind virions was not correlated with its ability to neutralize the virus. Genetic analysis of the 45 mAbs based on their heavy- and light-chain sequences identified at least 26 B-cell linage groups characterized by distinct binding or neutralizing properties. Moreover, neutralizing antibodies possessed longer complementarity-determining region 3 for both heavy and light chains than those with no neutralizing activity. Importantly, potent neutralizing mAbs reacted to the pentameric gH complex but not to gB. Thus, the pentameric gH complex is the primary target for antiviral antibodies by vaccination.Human cytomegalovirus (HCMV) is an important pathogen in transplant patients (1–5), and its infection can lead to invasive end-organ diseases, such as pneumonitis and hepatitis, as well as vascular pathology contributing to graft failure (4, 6, 7). HCMV is also the most common cause of in utero viral infections in North America and Europe, affecting 0.5–2% of newborns annually (8–10). Congenital HCMV infection can lead to symptomatic diseases at birth and also cause developmental disabilities in children (10, 11). Maternal seropositivity before conception protects against congenital transmission (12, 13), and both maternal humoral and cellular immunity are likely to contribute to the protection (14–16). Antibodies in particular are important for preventing congenital infection, serving as the first line of defense against maternal infection. It may also play a role in preventing transmission to the fetus, supported by the results of a small, nonrandomized study in pregnant women with primary HCMV infection, in which the passive immunity of monthly infusions of HCMV hyperimmune human IgG (HCMV-HIG) (200 mg/kg maternal weight) was ∼60% effective in protecting against congenital HCMV infection (17, 18). These studies suggest that it is feasible to develop a vaccine for preventing congenital HCMV infection and its sequelae. However, despite the fact that the Institute of Medicine has identified development of an effective vaccine for prevention of congenital HCMV as a top priority since 1999 (19), progress toward this goal has only been incremental (8, 20, 21). One of the hurdles to the efforts is our limited understanding of component of natural immunity associated with protection against HCMV infection.HCMV is a large, complex virus, with a genome capable of encoding >150 proteins (22–26). Because of the strict species specificity, options of animal models for HCMV research are limited (27). Thus, the functions of most HCMV antigens in viral infection in vivo and their roles as targets for host immunity are poorly understood. Furthermore, culture systems of single cell types have limitations for studying HCMV pathogenesis. Immunohistochemistry studies showed that HCMV can infect varieties of cells in vivo, including endothelial, epithelial cells, fibroblasts, and leukocytes (28–36). Many HCMV end-organ diseases, such as pneumonitis and gastroenteritis, are due to infection of the epithelial/endothelial cells in the affected organ (35–39). However, common laboratory strains, such as AD169 and Towne, were culture-adapted in fibroblast cells, with genomic mutations (22, 24, 40) and, more importantly, have lost their tropism to endothelial and epithelial cells, in contrast to pathogenic clinical isolates (32, 33, 41, 42).Loss of viral tropism to endothelial and epithelial cells was mapped to various mutations in the viral UL131-128 locus, and these mutations abrogated the expression of the pentameric glycoprotein H (gH) complex, composed of gH, gL, UL128, UL130, and UL131 proteins, a determinant for viral tropism to endothelial and epithelial cells (42–44). Because the pentameric gH complex is missing in common laboratory strains (42, 43), its importance in viral tropism, viral pathogenesis, and vaccine design was not fully appreciated until recently (42, 45). With this understanding, it is not surprising that Towne virus and recombinant glycoprotein B (gB) vaccines, although with ∼50% efficacy against primary infection in the clinic (46–49), induced poor neutralizing titers against viral infection of epithelial cells, in contrast to immune sera from HCMV-seropositive donors (50, 51). Thus, missing the pentameric gH complex is likely a deficiency in antigen composition for both vaccines (50). Studies of monoclonal antibodies (mAbs) isolated from HCMV-seropositive donors or polyclonal IgG enriched for antigen specificity supported the hypothesis that the pentameric gH complex, not gB, appears to be important for neutralizing activity in human subjects with natural infection (52).We recently described an experimental vaccine virus in which expression of the pentameric gH complex was restored (53). Unlike the parental AD169 virus and the recombinant gB vaccine, this virus can elicit high levels of neutralizing antibodies in rabbits and rhesus macaques (53). To support clinical development of this vaccine centered its concept on the pentameric gH complex, we established a comprehensive panel of 45 mAbs from a single rabbit that received vaccination. Of the 45 mAbs, 25 had neutralizing activity against viral entry in epithelial cells, including 11 elite neutralizers with ≥10-fold greater potency than HCMV-HIG. Biochemical analysis demonstrated that all elite neutralizers preferentially bound to the virus expressing the pentameric gH complex, and the majority of elite neutralizers (8 of 11) specifically recognized a recombinant form of the pentameric gH complex. Interestingly, binding affinity for intact virions was not correlated with neutralizing activity. Moreover, genetic analysis of the 45 mAbs based on their heavy- and light-chain sequences identified at least 26 B-cell linage groups characterized by distinct binding or neutralizing properties. In addition, neutralizing antibodies had longer complementarity-determining region 3 (CDR3) for both heavy and light chains than those of antibodies with no neutralizing activity. These data establish the importance of the pentameric gH complex as the primary target for potent neutralizing antibodies by vaccination, and support development of an experimental HCMV vaccine featuring the pentameric gH complex.     

日本血吸虫抗独特型单克隆抗体的建株及特性测定

BALB/C小鼠感染10条日本血吸虫尾蚴,半年后用一株在小鼠体内、外具有杀伤血吸虫作用的IgM单抗ssj14进行加强免疫,3d后取其脾细胞和骨髓瘤细胞(SP2/0)融合,筛选到10株单克降抗独特型抗体(抗-Id).选择其中6株作进一步特征测定,ELISA抑制法表明它们能不同程度地抑制单抗ssj14识别可溶性虫卵抗原(SEA),抑制率与抗-Id的浓度呈正比,而这些抗-Id都不与SEA直接起反应.双向琼扩试验测定表明,6株抗-Id中2株为IgG:亚型(C_8和D_5),3株为IgG_3亚型(B_3、E_3和C_(10) ),1株为非IgM和IgG亚型(E_4).小鼠免疫试验结果显示,两株抗-Id(D_5和E_4)诱导了较高的减虫率(44.67%,(P<0.05)、47.28%(P<0.01))和减雌率(46.99%(P<0.01)、54.26%(P<0.01)),2株抗-Id(C_3和E_4)诱导了较高的减卵率(56.68%和61.13%);ELISA检测结果显示,小鼠用抗-Id免疫后均不同程度地产生了针对抗-Id和抗SEA及抗可溶性雄虫抗原(m-SWAP)的抗体,说明抗-Id模拟了单抗ssj14的靶抗原的部分结构,诱导了免疫鼠特异性的免疫应答.     

Leu 7+, Leu 11a- acute T-lymphoblastic leukemia having low K cell activity and no NK cell activity

The phenotypic and functional features of the leukemic blasts from a child with T-acute lymphoblastic leukemia (T-ALL) were studied. The leukemic cells lacked the usual markers of T-cell lineage (T3-, T11-, E-sheep-) although they displayed some T-lymphocyte markers (T6+, T8+, T9+, T10+) and were T gamma-. Furthermore, these cells had a strong reaction with anti-Leu 7 but were negative to anti-Leu 11a antibody and exhibited low K cell activity, no NK activity, and showed virtually no response to PHA. These leukemic cells probably represented the leukemic counterpart of the Leu 7+, Leu 11a- subset that has been demonstrated in the peripheral blood of normal individuals.     

卡氏肺孢子虫病实验与临床研究 Ⅲ．抗卡氏肺孢子虫单克隆抗体杂交瘤细胞株的建株及鉴定

从感染肺孢子虫肺炎大鼠模型中分离纯化肺孢子虫包囊并免疫ＢＡＬＢ／ｃ小鼠，采用杂交瘤技术，获得１株能长期稳定分泌抗人源性肺孢子虫单克隆抗体杂交瘤细胞株４Ｄ７。其染色体众数为８２，分泌ＩｇＧ１亚型抗体。该单抗可识别５４ｋＤａ肺孢子虫包囊蛋白区带，并能准确识别肺孢子虫包囊，其灵敏性与特异性可达１００％（１１／１１）；并与正常人体组织、肺炎双球菌、白色念珠菌和新型隐球菌无交叉反应。     

单克隆抗独特型抗体NP30用于日本血吸虫病血清学诊断的研究

本研究用单克隆抗独特型抗体NP30与日本血吸虫肠相关抗原(GAA)和可溶性虫卵抗原(SEA)检测了702份不同病期及正常人群中的血清抗体,结果显示,在急性感染时,NP30抗体的检出率为98％,与GAA(94％)和SEA(98％)的无差别。在慢性感染时NP30抗体的检出率为87％,与GAA(86％)的无差别,但低于SEA(98％)的。在正常人群中,上述3种的抗体假阳性率均为3％左右,无差别。NP30的抗体滴度几何均数在急性血吸虫感染时高于GAA的,低于SEA的,在慢性感染时低于后两者,提示NP30的抗体在血吸虫感染期间出现比较早,消退较快。上述结果提示,NP30可以替代虫源性抗原,用于日本血吸虫病诊断。     

抗人胰腺癌单克隆抗体的制备及其特性的研究

本实验采用常规细菌融合方法，在PEG作用下，成功地制备了五株能稳定分泌抗人胰腺癌单克隆抗体的杂交瘤。以间接型ELISA方法和ABC技术进行抗体检测，有限稀释法进行克隆。用羟基磷灰石法纯化单克隆抗体（MAb)，所得MAbB6的免疫球蛋白类型为IgG1,它在培养上清及小鼠腹水中的效价分别为1：16和1：2600。MAbB6具有较好的特异性，其所针对的抗原可能为一种胰腺癌细胞胸膜抗原，是一种肿瘤相关性抗原，羟基磷灰石方法是一种比较理想的单克隆抗体提纯方法。     

执着 严谨 求实 创新——访我国著名血液学专家阮长耿院士

阮长耿院士心系振兴我国医学事业的宏伟目标,潜心学术、严谨求实、勇于创新,发现并从事世界上第一株被国际公认的抗血小板膜糖蛋白的单克隆抗体研究。创建了国内第一个血栓与止血研究室,并制备了一系列抗血小板膜糖蛋白及凝血成分的抗体,在血液学研究中取得多项重大突破。他的奋斗历程和科研态度对我国科研工作者,特别对年轻科研工作者是一种重要启迪。     

Transient response of pure red cell aplasia to anti-thymocyte globulin in a patient with T-cell chronic lymphocytic leukemia

Pure red cell aplasia (PRCA) is an unusual complication of chronic lymphoproliferative disorders. A patient with T-cell chronic lymphocytic leukemia (T-CLL) had severe anemia and neutropenia. Initial in vitro studies demonstrated no evidence of T-cell suppression of erythropoiesis. Sequential bone marrow examinations demonstrated progressive red cell aplasia. In vitro studies showed that the T-cells from the patient suppressed allogeneic but not autologous BFU-E. Treatment with antithymocyte globulin (ATG) reduced circulating leukemic cells and produced a definite but transient improvement in erythropoiesis.     

全人源肝癌噬菌体单链抗体基因的克隆及活性分析

从噬菌体单链抗体库中筛选克隆全人源肝癌抗体基因并进行活性鉴定.PCR鉴定阳性重组菌中人肝癌ScFv的插入率,以肝癌细胞SMMC-7721为抗原对所建抗体库进行4轮"吸附-洗脱-扩增"的亲和筛选.将筛选后的ScFv采用ELISA法鉴定其与人肝癌细胞的结合活性.ScFv基因插入率为70%.在亲和筛选过程中,肝癌噬菌体单链抗体得到富集,收获率逐轮提高,第4轮为第一轮的381倍.利用噬菌体抗体库技术筛选出了肝癌噬菌体单链抗体,且筛选后的抗体片段与人肝癌细胞有特异性的结合活性.     

抗人EGFR/抗CD3双功能抗体的制备、检测及对胃癌细胞毒性作用的实验研究

目的探讨抗EGFR/抗CD3双功能抗体体外对胃癌细胞的杀伤能力,为临床应用该抗体治疗胃癌打下实验基础。方法采取化学偶联法合成抗EGFR/抗CD3双功能抗体并使用间接细胞免疫荧光法检测该抗体的功能。通过细胞结合率检测及MTT杀伤实验检测其对胃癌细胞结合及杀伤的能力并与单纯的EGFR单抗和CD3单抗比较。结果抗EGFR/抗CD3双功能抗体联合效应细胞与胃癌细胞株SGC7901结合率显著高于两组单抗对照组(P<0.05);MTT细胞杀伤实验结果提示:抗EGFR/抗CD3双功能抗体组对胃癌细胞株SGC7901杀伤率显著高两组单抗对照组(P<0.05)。结论初步的体外实验显示由化学偶联法合成的抗EG-FR/抗CD3双功能抗体可能对胃癌有一定的治疗作用,具有进一步研究的价值。