抗人膀胱癌抗独特型抗体佐剂疫苗的动物实验

目的：研究抗独特型抗体佐剂疫苗（Ab2 SAF)对膀胱癌的免疫治疗作用。为临床实验提供动物实验依据，方法：用抗人膀胱癌Ab SAF免疫BALB／C小鼠为实验组；对照组用生理盐水，免疫3次后，于未次免疫后1周，将新鲜人膀胱移行细胞癌组织移植于小鼠肾包膜下，分别于移植后第2，4，6，8和10天处死小鼠，取血清进行抗抗独特型抗体（Ab3)分析，移植瘤行组织学检查，观察宿主淋巴细胞浸润情况及瘤细胞可见了率，结果：实验组小鼠肾包膜下人膀胱癌细胞很快受到排斥，在移植后第6天，淋巴细胞浸润即达高峰，而对照组在第10天才达高峰，瘤细胞可见率，在移植后第6天即明显降低，而对照组呈逐渐下降，实验组Ab3为阳性，而对照组为假阳性或阴性，结论：Ab2作为抗原免疫小鼠后，能够排斥瘤细胞的生长。     

双功能螯合剂BAT与鼠IgG1 Fab''''片段的点特异性交联方法的研究

目的报道一种用双功能螯合剂BAT与鼠单克隆抗体Fab’片段行点特异性交联的新方法。方法通过位于Fab’铰链区、远离抗原结合位点的活性巯基(-SH)直接与BAT相交联。用简单的两步法制备B43单抗的Fab’片段。首先单抗用胃蛋白酶处理，产生稳定的F(ab’)2片段，然后F(ab’)3经半胱氨酸还原后产生Fab’片段，最后Fab’片段直接与BAT交联。结果每个Fab’分子平均含1．8个-SH基团，74％的Fab’片段可与BAT生成交联物，平均每个Fab’分子携带1．28个BAT。经检测，F(ab’)2、Fab’及Fab，BAT均保持着良好的免疫活性。结论该交联法简单、高效，为双功能螯合剂BAT与鼠单抗Fab’片段的交联提供了一个新途径。     

单克隆抗体阿霉素偶联物导向治疗膀胱癌的临床研究

抗人膀胱癌单克隆抗体ＨＢＴＡ通过葡聚糖与阿霉素联交成化学免疫偶联物对７例膀胱癌病人进行免疫导向治疗。病人接受ＨＢＴＡ抗体蛋白量为１５ｍｇ～５０ｍｇ，平均４０ｍｇ。治疗后７例病人的肿瘤组织经免疫组化检测均呈阳性的抗体定位，血清膀胱肿瘤相关抗原显著降低（Ｐ＜０．０１），电镜观察到治疗后肿瘤细胞超微结构有坏死性改变。结果表明以鼠抗人膀胱癌单克隆抗体与化疗药物偶联制备的生物导弹能安全地用于临床，并能特异性地在靶部位发挥肿瘤细胞杀伤作用，为膀胱癌的治疗提供了一条新途径。     

抗人膀胱癌单克隆抗体BDI-1的扩增和鉴定

目的 制备纯化的抗人膀胱癌单克隆抗体BDI-1。方法 给BALB／C小鼠腹腔注射BDI-1杂交瘤细胞，收集的腹水过Protein G-agaros亲和层析柱即得到人膀胱癌BDI-1单克隆抗体。采用酶联免疫吸附试验（ELISA）法、间接免疫荧光法、SDS-聚丙烯酰胺凝胶电泳法和点印迹法鉴定单克隆抗体。结果 腹水可以得到1．0～1．5g／L的高效价（10^6）的纯化抗体。结论 该方法可以扩增高效价的单克隆抗体。     

抗人肾癌G250单克隆抗体的制备及其功能鉴定

目的 大量制备、纯化抗人G250单克隆抗体(G250-MeAb),并鉴定其与G250合成多肽及细胞天然抗原的结合特异性.方法 将抗人G250杂交瘤细胞接种BALB/C小鼠腹腔,大量制备G250-McAb.采用正辛酸-硫酸铵沉淀法获得粗制抗体,然后经蛋白A亲和层析柱纯化抗体.采用荧光激活细胞分类术及免疫组织化学法鉴定G250-McAb与抗原结合特异性.结果 成功制备抗人G250-MeAb,腹水经纯化后,获得了高纯度抗人G250单克隆抗体,蛋白浓度达4.78 g/L.荧光激活细胞分类术及免疫组织化学结果 显示G250-McAb与G250表达阳性的Ketr-3细胞特异性结合,而不与G250表达阴性的ACHN细胞结合.结论 成功制备并纯化G250-McAb,为进一步开展G250-McAb介导的肾癌诊断、治疗奠定了基础.     

抗核抗体独特型多肽对慢性移植物抗宿主病狼疮样肾炎小鼠的作用及其机制探讨

狼疮肾炎（LN）是育龄妇女的好发病之一，LN的治疗仍是临床的一大难题。目前国际上已有人尝试用主动免疫治疗LN。抗核抗体独特型多肽具有SNF1小鼠抗-dsDNA抗体的独特型，其结构与此抗体重链可变区的一组多肽片段相似。为探讨独特型多肽这种保护作用的机制，我们在以往实验的基础上进一步观察了慢性移植物抗宿主病狼疮样肾炎小鼠体重、尿蛋白定量、肾脏组织学变化和血清IL-6水平的变化㈣。     

抗肝癌血管内皮细胞的单克隆抗体的制备和鉴定

目的　研究制备抗肝癌区血管内皮细胞的单克隆抗体,并进行初步的鉴定。方法　单克隆抗体制备：将制备好的抗原、免疫动物,行ＰＥＧ 细胞融合、免疫组织化学和ＥＬＩＳＡ抗体筛选,进行细胞克隆。抗体的生产及纯化;组织学鉴定分析：亲和层析蛋白分离、ＳＤＳＰＡＧＥ 电泳、免疫组织化学分析;抗体的体外细胞毒作用试验选用补体介导的细胞毒实验ＭＴＴ法。结果　以经肝癌细胞系培养上清诱导扩增的人胎脐静脉血管内皮细胞为抗原,制备并获得了抗肝癌区血管内皮细胞的单克隆抗体;组织学研究表明,抗体在肝癌组织的血管内皮细胞中有特异性,在体外对诱导扩增的血管内皮细胞有补体介导的细胞毒作用。结论　制备并获得抗肝癌区血管内皮细胞的单克隆抗体;抗体在体外具有细胞毒作用。     

抗人类癌抗原单克隆抗体的制备与肝组织中的鉴定

目的运用杂交瘤技术制备人类癌抗原(Human carcinoma antigen,HCA)的IgG单克隆抗体。方法用现有的HCA-IgM抗体HAE3分别从PC3、PCaT及LCaT总蛋白中分离纯化得到三组糖蛋白复合物组分作为抗原分别免疫Balb/c小鼠,获得鼠抗人HCA抗体.并对这些抗体进行ELISA检测效价、western-blot以及免疫组化等分析,筛选合适的抗体进行下一部分研究。结果共制备出62株高亲和力、高特异性并且针对不同抗原结合位点的鼠抗人HCA单克隆抗体。通过在肝组织上的鉴定,发现这些抗体无论效价、western-blot以及免疫组化都适合进行进一步研究。结论我们利用天然HCA抗原制备了高亲和力,高特异性并且针对不同抗原结合位点的单克隆抗体,为我们进一步研究其结构及功能提供了有力的特异的工具。     

抗内毒素类脂A单链抗体基因的克隆、表达和初步鉴定

为探索Ｇ－杆菌脓毒症的治疗问题，应用基因工程技术从本所制备的分泌抗内毒素单克隆抗体的杂交瘤细胞中提取ｍＲＮＡ，然后反转录成ｃＤＮＡ第一链，用特异性轻、重链引物扩增反应产物，获得重链（３４０ｂｐ）和轻链（３２０ｂｐ）基因片段，经纯化后将轻链和重链基因用一多肽连接物连接，构成单链抗体基因片段。此单链抗体基因经再次ＰＣＲ扩增后克隆入ＰＣＡＮＴＡＢ５噬菌粒中再转化于感受态的ＴＧ１大肠杆菌中，通过噬菌体抗体库技术筛选重组噬菌体，结果在ＴＧ１细菌中表达了９Ｇ６单链抗体基因。ＥＬＩＳＡ阻断试验初步证明了该单链抗体能阻断其亲本单克隆抗体对内毒素的结合。提示：抗内毒素单链抗体可为脓毒症的治疗提供一条途径。     

大鼠抗小鼠组织相容性抗原抗体的制备与纯化

为进一步了解异种移植间免疫排斥反应的抗体特性，采用NIH小鼠牌细胞免疫SD大鼠，制备大鼠抗小鼠组织相容性抗原抗血清，经辛酸沉淀、羟基磷灰石层析技术纯化抗体。结果：制备的大鼠抗小鼠组织相容性抗原抗血清具有较高效价（＞1：640），辛酸、羟基磷灰石二步法纯化的抗体具有纯度好、收获率高、能保留免疫原性、条件温和等特点。表明此法是制备各种抗组织相容性抗原抗体的有效方法。     

抗人膀胱癌/抗VEGF双功能基因抗体对膀胱癌生物学行为的影响

目的:探讨抗人膀胱癌/抗VEGF双功能基因抗体对人膀胱癌生长及转移的影响。方法:通过构建人膀胱癌裸鼠皮下移植瘤模型并注射双功能抗体,观察肿瘤生长及盆腔淋巴结转移情况,同时采用免疫组化法检测肿瘤微血管密度值及凋亡的肿瘤细胞指数。结果:肿瘤大小:双抗组为(19.50±4.51)mm2,对照组为(57.62±8.31)mm2,两组比较P<0.01;肿瘤微血管密度双抗组为(2351±207)个,对照组(4356±548)个,两组相比P<0.05;凋亡指数双抗组为19.25,对照组为9.31,两者比较P<0.05;双抗组无一只发生盆腔转移,而对照组为75.0%,两组比较P<0.05。以上各项组间差异均有统计学意义。结论:抗人膀胱癌/抗VEGF双功能基因抗体对人膀胱癌具有良好的靶向性,能够通过抑制肿瘤微血管形成和加速肿瘤细胞凋亡,遏制实验性人膀胱癌的生长转移,为该抗体用于临床膀胱癌的治疗提供了一定的实验基础。     

人精子抗原基因表达产物HSG_2Ag的纯化和鉴定

本研究运用离子交换层析法分离纯化人精子抗原基因表达克隆HSG2的溶原蛋白,并用ELISA法鉴定其中的表达产物(HSG_2Ag)。结果显示:(1)经离子交换层析法分离HSG_2溶原蛋白为12个主要蛋白峰;(2)用抗人精子抗体和纯化的人SIT阳性血清经ELISA法识别,在HSG_2溶原蛋白中,第3峰为人精子抗原基因表达克隆HSG_2的表达产物(HSG_2Ag)。结果提示,用上述方法分离和纯化的HSG_2Ag纯化度高,特异性高,适合应用于抗精子抗体检测试剂盒的制备,并为进一步研制精子抗原免疫避孕疫苗提供候选成分。     

Detection of onco-fetal bladder antigen in urine of patients with transitional cell carcinoma

Studies of antigens associated with transitional cell carcinoma were extended by using murine IgM monoclonal antibody E7, developed earlier by this laboratory. These antibodies react preferentially with human bladder tumors and transitional cell carcinoma (TCC) cell line 647V. We now report that monoclonal antibody E7 detected the presence of antigen in midgestational and third trimester amniotic fluids, and in urine of patients with advanced transitional cell carcinoma. Western blot analysis showed that the antigen present in amniotic fluids consists of a sharp band with molecular weight greater than 200 kdaltons. A similar molecular weight pattern was seen with the solubilized membrane of 647V. A sensitive and convenient sandwich ELISA was developed and the urine of patients with bladder cancer was assayed for the presence of the E7 antigen. Antigen was detected in the urine of patients with advanced transitional cell carcinoma but not in the urine of normal adults or in urine from patients with prostate cancer, renal cell carcinoma, or benign prostate hyperplasia. An inhibition enzyme immunoassay was developed with monomeric forms of the E7 antibody and confirmed the presence of antigen in the urine of patients with TCC. We conclude that the E7 antigen is an onco-fetal antigen expressed in patients with transitional cell carcinoma of the bladder.     

UNME/K1: an IgG2a monoclonal antibody specific to cytokeratin of human urinary bladder squamous cell carcinoma

Summary The main distinctive feature of carcinoma in schistosomal bladder is keratinized squamous cell carcinoma. Keratins/cytokeratins constitute a multigeneic family of structurally related polypeptide markers for the malignant state of epithelial cells. A monoclonal antibody (UNME/K1) regognizing keratins associated with squamous cell carcinoma of the human urinary bladder was generated at the Urology and Nephrology Center, Mansoura, Egypt (UNME), by fusion of spleenocytes from a BALB/c mouse immunized with a keratin extract (K1) of human squamous cell carcinoma and P3X63Ag8/U1 syngeneic myeloma cells. UNME/K1 was purified by a protein-A affinity column and was of the IgG2a type, as determined by immunoelectrophoresis and gel diffusion techniques. When tested against keratins of different types of urinary bladder tumors using enzym linked immunosorbent assay (ELISA), UNME/K1 reacted only with the high molecular weight keratin of squamous cell carcinoma and showed selectivity towards specific histopathological grades of tumors.     

Initial evaluation of the diagnostic performance of the new urinary bladder cancer antigen test as a tumor marker for transitional cell carcinoma of the bladder

PURPOSE: We evaluated the diagnostic performance of the new noninvasive bladder cancer test on voided urine samples from patients with transitional cell carcinoma compared to symptomatic and asymptomatic controls. MATERIALS AND METHODS: Urinary bladder cancer antigen was measured in urine from 86 patients with active transitional cell carcinoma of the bladder (group 1), 76 patients free of transitional cell carcinoma as confirmed by cystoscopy at followup (group 2), 25 patients with other benign urological diseases (group 3), 25 patients with other malignant pathological conditions (group 4) and 30 healthy subjects free of urological diseases (group 5). RESULTS: Mean urinary bladder cancer antigen concentrations were 104.84, 4.57, 11.79, 48.87 and 1.38 microg/l, for groups 1 to 5, respectively, which was statistically different (p = 0.00005) except for groups 1 and 4 (p = 0.187). Sensitivity was 87.0% (95% confidence interval 79.2 to 92.7) and specificity was 86.8% (77.1 to 93.5%), and both were optimized by receiver operating characteristics plot analysis at a threshold value of 9.74 microg/l using asymptomatic (group 2) compared to known cancer (group 1) cases. CONCLUSIONS: Urinary bladder cancer antigen might have a role as a potential tumor marker for diagnosing transitional cell carcinoma of the bladder.     

Antigen(s) on human transitional cell carcinoma detected by a xenogeneic antiserum

Summary In studies concerning human bladder cancer, antisera were raised in rabbits against human tumours, normal tissue, and cell lines derived from human tumours and were analysed by absorption and complement dependent microcytotoxicity tests. No significant selective cytotoxicity was discernible with any unabsorbed antisera. After absorption of A53, (an antiserum against the transitional cell carcinoma derived cell line T24) with peripheral blood cells and normal adult tissues, it was cytotoxic to two bladder cancer cell lines (T24 and J82) but not to four other cell lines. This activity was removed by absorption with each of two specimens of transitional cell carcinoma but not by normal bladder and by absorption with T24 or J82 but not with four other non-bladder cell lines. This functional specificity for transitional cell carcinomas could be due to a tumour associated antigen, a significant quantitative difference between tumour and normal cells, or an embryonic specificity reexpressed on the tumour. Further experiments are necessary to investigate these alternatives.Abbreviations used TAA tumour associated antigen(s) - NRS normal rabbit serum - MEM Eagle's minimal essential medium and additives - LuLiKi mixture of homogenates of human lung, liver and kidney - TCC transitional cell carcinoma - NBI normal bladder     

前列腺特异性膜抗原人源Fab抗体可变区基因的筛选与鉴定

目的:筛选、鉴定抗中国人前列腺特异性膜抗原(PSMA)的人源Fab抗体可变区的编码基因,为PSMA蛋白质生物学功能的研究及前列腺癌的基因治疗研究开辟新途径。方法:采用噬菌体表面展示技术,以PSMA原核表达重组子pET30 a(+)-PSMA诱导表达并纯化后的PSMA为固相抗原,从噬菌体Fab可变区抗体库中经过5轮“吸附-洗脱-扩增”筛选过程,获得抗原结合活性和特异性较强的PSMA人源Fab可变区抗体基因片段的阳性克隆,并对其进行免疫检测及序列测定。结果:筛选得到Fab抗体克隆的Fd段基因核苷酸序列为696 bp,编码由232个氨基酸残基组成的多肽,与人免疫球蛋白γ链恒定区的同源性为98%;轻链κ基因核苷酸序列为630 bp,编码由210个氨基酸残基组成的多肽,与κ链恒定区同源性为93%。结论:利用噬菌体抗体库技术,成功获得了PSMA人Fab抗体可变区编码基因克隆,该克隆抗体基因具有典型的免疫球蛋白轻链和重链可变区的结构特点,基因编码产物具有与PSMA反应的免疫学活性和特异性。     

抗人膀胱癌单链抗体的构建及表达的研究

目的 为膀胱癌的导向诊断和治疗提供免疫原性更低的单链抗体。方法 从1株分泌鼠抗人膀胱癌单克隆抗体的杂交瘤细胞株BDI-1中分离出总RNA。     

Cellular and humoral immunity after allogeneic renal transplantation in the rat. V. Appearance of anti-idiotypic antibody and its relationship to cellular immunity after treatment with donor spleen cells and alloantibody.

Enhancement of LBN F1 renal allograft survival in Lewis (L) rats is achieved by injecting the recipient i.v. with donor antigen (LBN F1 spleen cells) 1 day before transplantation and antidonor antibody (L anti-BN alloantiserum) at the time of transplantation. Treatment with this combination of antigen and antibody also induces the recipient to make L anti-(L anti-BN) anti-idiotypic antibody that reaches peak titers within 10 days. The degree of graft enhancement achieved was increased greatly by delaying transplantation until the peak of the anti-idiotypic antibody response 10 days after treatment with antigen and antibody. Two in vitro assays for cellular immunity (51Cr release and microcytotoxicity) failed to demonstrate antidonor activity in spleen cells from recipients for which transplantation had been delayed 10 days. The close correlation of enhancement, absence of cellular immunity in vitro, and the kinetics of the anti-idiotypic antibody response suggest that anti-idiotypic antibody may prevent either sensitization and generation of effector T lymphocytes or the destructive potential of sensitized cells.