抗独特型卵巢癌疫苗的研究

根据免疫网络学说,抗独特型抗体中的Ab2β具有模拟原始抗原的作用,同时也是重要的免疫调节因子.抗独特型抗体能打破免疫耐受,作为肿瘤疫苗具有良好的应用前景.卵巢癌相关抗原OC166-9在大多数上皮性卵巢癌有表达,我中心经过系列研究陆续制备了鼠源的模拟OC166-9的抗独特型抗体6B11、6B11单链抗体(6B11scFv)、6B11scFv和人粒细胞集落刺激因子(hGM-CSF)的融合蛋白(6B11GM).鼠源抗体反复应用于人体可产生人抗鼠抗体,为了实现人源化、增加6B11scFv的免疫原性、提高分子质量、降低鼠源性,我中心又制备了抗独特型微抗体(6B11VLVHhc).同时为了验证上述抗体的免疫作用,我们构建了人淋巴细胞免疫重建的荷人卵巢癌的严重联合免疫缺陷小鼠模型.上述系列研究为6B11卵巢癌抗独特型疫苗用于临床积累了大量实验依据.     

移植前诱导抗独特型抗体对小鼠皮肤排斥反应的抑制作用

目的　探讨抗独特型抗体诱导对异品系小鼠皮肤移植排斥反应的影响.方法　以C57BL/6小鼠脾细胞免疫BALB/c小鼠制备抗同种异品系抗体(Ab1).将Ab1与KLH交联后,免疫BALB/c小鼠诱导产生抗独特型多克隆抗体(Ab2),并以之为受体,观察Ab2对小鼠皮肤移植排斥反应的影响.结果　Ab1交联KLH加弗氏佐剂免疫可有效地诱导抗独特型抗体(Ab2)产生.与对照组相比较,Ab2诱导组小鼠移植物存活时间明显延长.结论　移植前在受体体内诱导产生以移植物抗原为模拟抗原的抗独特型抗体,可对移植排斥反应产生有效的抑制作用移植前诱导抗独特型抗体对小鼠皮肤排斥…     

卵巢癌抗抗独特型单克隆抗体3D12的制备及初步研究

目的:制备卵巢癌抗抗独特型单克隆抗体并进行初步鉴定,为深入研究卵巢癌疫苗的主动免疫机制奠定基础.方法:以卵巢癌抗独特型单克隆抗体6B11作为免疫原,采用杂交瘤技术制备抗抗独特型单克隆抗体3D12.经酶联免疫吸附实验(ELISA)、免疫组织化学、流式细胞学、免疫印迹杂交等检测其特异性结合抗原的性质;竞争抑制实验鉴定其特异性结合的抗原表位.结果:获得1株稳定分泌3D12的杂交瘤细胞,所分泌的3D12能够特异性结合6B11及卵巢癌初始抗原OC166-9,并能竞争抑制COC166-9(抗OC166-9的单克隆抗体Ab1)与OC166-9的结合.免疫印迹杂交显示,3D12与COC166-9免疫沉淀的蛋白的相对分子质量相同.该抗体为IgM,亲和力约为8.34×10⁷ L/mol.结论:卵巢癌抗抗独特型单克隆抗体3D12属Ab1样Ab3,间接证实6B11为抗原内影像型抗独特型抗体.     

Monoclonal anti-idiotype antibody bearing the internal image of nasopharyngeal carcinoma associated antigen

目的　制备和鉴定具有鼻咽癌相关抗原内影像的抗独特型单克隆抗体 (Ab2 )。方法　用鼻咽癌单抗 (Ab1)免疫小鼠 ,免疫鼠脾细胞与骨髓瘤细胞SP2 / 0融合获得杂交瘤细胞系。用SandwichELISA和结合抑制试验筛选阳性克隆。为进一步证实Ab2是否具有鼻咽癌相关抗原的内影像 ,用Ab2免疫小鼠获得抗血清 ,以ELISA和竞争抑制试验检测Ab3。用迟发性变态反应和T细胞增殖试验检测Ab2诱导的细胞免疫反应。结果　选用两株抗鼻咽癌单抗FC2、HNL5 (Ab1)为免疫原 ,制备两株抗相应Ab1独特型的抗独特型抗体 2H4和5D3(Ab2 )。 2H4、5D3能分别与FC2、HNL5特异性结合 ,并能抑制相应Ab1与靶细胞HNE2的反应 ,诱导产生能与相应Ab1竞争结合靶抗原的抗抗独特型抗体 (Ab3)。并能诱发特异性迟发型超敏反应 (DTH)和细胞增殖反应。结论　抗独特型抗体 2H4、5D3具有鼻咽癌相关抗原的内影像 (Ab2 β) ,能模拟鼻咽癌相关抗原诱导体液和细胞免疫反应。     

卵巢癌6B11抗独特型微抗体诱导抗肿瘤免疫应答的体外实验

目的:研究6B11抗独特型微抗体在体外抗肿瘤免疫情况,探讨其作为卵巢癌疫苗的可能性.方法:分离人外周血单个核细胞,用6B11抗独特型微抗体刺激并培养.采用3HTdR参入法、51Cr细胞毒实验分别观察淋巴细胞增殖情况和细胞毒作用,ELISA方法测定用6B11微抗体刺激后培养上清液中IFN-γ水平的变化,流式细胞术检测微抗体刺激后淋巴细胞表型的改变.结果:6B11抗独特型微抗体可以刺激人外周血淋巴细胞的增殖,最佳刺激剂量为20 mg/L;并对OC166-9表达阳性的卵巢癌细胞系有细胞毒作用;用6B11微抗体刺激后培养上清液中IFN-γ水平升高;淋巴细胞的表型表现为,疫苗刺激后CD3 的细胞明显增高, CD4 的细胞也增加, CD8 的细胞增加不明显.CD4 /CD8 的比率明显增高,差异有统计学意义.结论:6B11抗独特型微抗体在体外可诱导机体产生特异体液免疫和细胞免疫反应,为抗独特型微抗体疫苗的临床应用提供了一定的实验依据.     

湖南省2000年医药卫生科研进展

一、基础医学研究 1.鼻咽癌抗独特型抗体的制备及主动免疫机制研究:中南大学湘雅医学院研究人员根据Jerne免疫网络理论,首先用小鼠杂交瘤单克隆抗体技术,以鼻咽癌细胞株为免疫原,制备了一系列抗鼻咽癌的单抗(Ab1)。再用与钥孔(虫戚)血兰素的交联物抗体为免疫原和小鼠杂交瘤技术制备了作用与Ab1的V区的抗独特型抗体(Ab2),并证实其中某些Ab2具有鼻咽癌相关抗原的内影像(Ab2β),以此抗体(Ab2)免疫小鼠,诱导与鼻咽癌细胞发生特     

具有鼻咽癌相关抗原内影像的抗独特型抗体研究

用抗人鼻咽癌单抗FC2 ,HNL5 (Ab1)分别免疫Balb/C小鼠 ,制备了两株抗独特型抗体 2H4和 5D3(Ab2 )。结果显示 :2H4和 5D3分别与FC2和HNL5特异性结合 ,抑制Ab1与靶细胞的反应 ,诱导产生能与相应Ab1竞争结合靶抗原的抗抗独特型抗体 (Ab3) ;并能诱发特异性迟发型超敏反应 (DTH)和细胞增殖反应。表明 2H4和 5D3具有鼻咽癌相关抗原的内影像 (Ab2 β) ,能模拟鼻咽癌相关抗原诱导体液和细胞免疫反应。     

具有鼻咽癌相关抗原内影像的抗独特型抗体研究

用抗人鼻咽癌单抗FC2,HNL5(Ab1)分别免疫Balb／C小鼠,制备了两株抗独特型抗体2H4和5D3(Ab2)。结果显示：2H4和5D3分别与FC2和HNL5特异性结合,抑制Ab1与靶细胞的反应,诱导产生能与相应Ab1竞争结合靶抗原的抗抗独特型抗体(Ab3);并能诱发特异性迟发型超敏反应(DTH)和细胞增殖反应。表明2H4和5D3具有鼻咽癌相关抗原的内影像(Ab2β),能模拟鼻咽癌相关抗原诱导体液和细胞免疫反应。     

抗独特型抗体对小鼠移植低免疫反应状态的诱导作用

目的：探讨抗独特型抗体对小鼠移植耐受的诱导作用。方法：以C57BL/6小鼠脾细胞免疫BALB/c小鼠制备抗同种异品系抗体（Abl），Abl交联KLH后，免疫BALB/c小鼠诱导产生抗独特型多克隆抗体（Ab2），以此为移植受体，观察Ab2对小鼠心脏移植耐受的诱导作用。结果：Abl交联KLH和弗氏佐剂免疫可有效地诱导Ab2，与对照组相比，Ab2诱导组的小鼠移植物存活时间明显延长。结论：移植前在受体体内诱导产生以移植物抗原为模拟抗原的Ab2，可以对小鼠特异性低免疫反应状态起到有效的诱导作用。     

抗MUC-1抗体通过独特型免疫网络抗肿瘤生长

目的 探讨应用单克隆抗体于小鼠肿瘤模型，通过诱发产生抗独特型免疫应答而获取的抗肿瘤作用。方法 用经微球包裹识别MUC-1抗原的小鼠单克隆抗体CS20免疫小鼠，观察其诱导的体液及细胞免疫反应，并通过小鼠乳腺癌模型，验证CS20的抗肿瘤作用。结果 CS20治疗组可激活小鼠体内的抗独特型免疫网，产生高水平的Ab2和Ab3，且肿瘤的生长速度明显减慢，甚至消失。结论 抗体CS20可以诱导抗独特型抗体以及抗一抗独特型抗体的产生，增强体液免疫应答。CS20抗体对荷MUC-1阳性乳腺癌小鼠有一定治疗作用，为肿瘤免疫治疗提供一条可能途径。     

卵巢癌抗独特性单链抗体6B11基因在大肠杆菌中的高效表达

目的 制备在大肠杆菌中高效表达６Ｂ１１卵巢癌抗独特型单链抗体。方法 采用聚合酶链反应（ＰＣＲ）克隆技术,将６Ｂ１１ｓｃＦｖ基因克隆到原核高效表达载体ｐｋＰＬ－３ａ上,重组子转化大肠杆菌ｐｏｐ２１３６,温度诱导表达,复性蛋白经二乙氨乙基琼脂糖快离子交换层析纯化,十二烷基硫酸钠－聚丙烯酰胺凝腕鉴定蛋白纯度,直接法和竞争抑制法ＥＬＩＳＡ检测其活性,结果 ６Ｂ１１ｓｃＦｖ以包涵体形式表达获高效表达,包涵体     

Humoral immune responses induced by anti-idiotypic antibody fusion protein of 6B11scFv/hGM-CSF in BALB/c mice

Background We have previously developed and characterized a monoclonal anti-idiotype antibody, designated 6B 11, which mimics an ovarian carcinoma associated antigen OC166-9 and whose corresponding monoclonal antibody is COC166-9 （Abl）. In this study, we evaluate the humoral immune responses induced by the fusion protein 6B11 single-chain variable fragment （scFv）/human granulocyte macrophage colony-stimulating factor （hGM-CSF） and 6B 1 lscFv in BALB/c mice. Methods The fusion protein 6B 11 scFv/hGM-CSF was constructed by fusing a recombinant single-chain variable fragment of 6B11scFv to GM-CSE BALB/c mice were administrated by 6B11scFv/hGM-CSF and 6B11scFv, respectively. Results The fusion protein 6B11scFv/hGM-CSF retained binding to the anti-mouse F（ab）2＇ and was also biologically active as measured by proliferation of human GM-CSF dependent cell TF1 in vitro. After immunization with the 6B11scFv/hGM-CSF and 6BllScFv, BALB/c mice showed significantly enhanced Ab3 antibody responses to 6B11 scFv/hGM-CSF compared with the 6B11 scFv alone. The level of Ab3 was the highest after the first week and maintained for five weeks after the last immunization. Another booster was given when the Ab3 titer descended, and it would reach to the high level in a week. Conclusion The fusion protein 6B11scFv/hGM-CSF can induce humoral immunity against ovarian carcinoma in vivo. We also provide the theoretical foundation for the application of the fusion protein 6B11 scFv/hGM-CSF for active immunotherapy of ovarian cancer.     

卵巢癌抗独特型单链抗体6B11基因在大肠杆菌中的高效表达

目的制备在大肠杆菌中高效表达６Ｂ１１卵巢癌抗独特型单链抗体。方法采用聚合酶链反应（ＰＣＲ）克隆技术,将６Ｂ１１ｓｃＦｖ基因克隆到原核高效表达载体ｐＫＰＬ３ａ上,重组子转化大肠杆菌ｐｏｐ２１３６,温度诱导表达;复性蛋白经二乙氨乙基琼脂糖快速离子交换层析纯化,十二烷基硫酸钠聚丙烯酰胺凝胶鉴定蛋白纯度;直接法和竞争抑制法ＥＬＩＳＡ检测其活性。结果６Ｂ１１ｓｃＦｖ以包涵体形式表达获高效表达;包涵体经溶解复性纯化,纯度达９５％以上;表达蛋白能与卵巢癌单抗ＣＯＣ１６６－９特异结合并能有效抑制卵巢癌抗原ＯＣ１６６－９与卵巢癌单抗ＣＯＣ１６６－９的特异结合。结论６Ｂ１１ｓｃＦｖ基因可在原核细胞中高效表达,表达产物经复性纯化后具有较好的免疫活性。     

应用荷人卵巢癌腹水瘤模型研究免疫脂质体的导向治疗

用卵巢癌单抗脂质体阿霉素交联物对16只荷人卵巢癌裸鼠腹水瘤模型进行导向治疗。腹水瘤模型更接近实际,符合临床。结果表明,导向治疗组的疗效明显高于对照组、阿霉素组及正常IgG脂质体组。     

Nanoparticles as a vaccine adjuvant of anti-idiotypic antibody against schistosomiasis

Background The development of new adjuvants for human use has been the focus of attention. This study’s aim is to explore the possibility of using nanoparticle Ca nanoparticles (CA) as a vaccine adjuvant of anti-idiotypic antibody NP30 against schistosomiasis and its protective mechanisms. Methods Nanoparticle CA-NP30 conjugate (CA-NP30) was fabricated. BALB/c mice were immunized actively with CA-NP30 to evaluate its effects of protective immunity on mice. The serum levels of specific IgG, IgG1 and IgG2a antibodies against NP30 and the concentrations of IFN-γ and IL-4 in supernatant of splenocytes were determined via ELISA. Results Nanoparticle CA could enhance significantly the protective immunity of NP30 against infection of Schistosoma japonicum and the worm reduction rose from 36.0% (NP30 alone) to 52.6%. The serum levels of specific IgG, IgG1 and IgG2a antibodies against NP30 increased remarkably, as compared with those of the group immunized with NP30 alone. The concentration of IFN-γ in supernatant of splenocyte was drastically elevated ［the groups immunized with CA-NP30 and NP30 alone were (493.80±400.74) pg/ml and (39.03±39.58) pg/ml, respectively］, but the concentration of IL-4 showed no significant difference from that of NP30 alone ［(27.94±9.84） pg/ml vs （27.28±14.44） pg/ml］. Conclusions Nanoparticle CA could act as a vaccine adjuvant of anti-idiotypic antibody NP30 against schistosomiasis. The mechanism could be that CA-NP30 enhances humoral and cellular immune responses in mice.     

Mucosal administration of alpha-fodrin inhibits experimental Sjögren's syndrome autoimmunity

OBJECTIVE: To explore the effect of mucosal administration of alpha-fodrin in inhibition of autoimmunity in Sj?gren's syndrome (SS). METHODS: Thirty-four 4-week-old NOD mice were randomly divided into 4 equal groups: to be immunized by nasal administration of alpha-fodrin 1 microg/dose and 10 microg/dose respectively every two days (experimental groups), and phophate-buffered saline (PBS) or glutathion2 S-tansferase4 (GST) (control groups). The weekly volume of water drinking was calculated. The salivary flow was maintained. Serum samples were obtained to detect the anti-SSA, anti-SSB, RF, ANA, anti- -fodrin and anti-type 3 muscarinic acetylcholie receptor polypeptide (M3RP) by immunofluorescence or ELISA. The cytokines of IFN-gamma and IL-10 were measured with ELISA. The salivary glands were examined by HE staining and immunohistochemical analysis. Flow cytometry was used to detect the proportion of Foxp3+ CD4+ CD25+ T cells. RESULTS: The titers of anti- -fodrin antibody and M3RP antibody of the mice immunized with-fodrin were lower than those of the 2 control groups (all P < 0.05), however, there was not significant differences between these two a-fodrin immunized groups. Five of the 8 mice in the GST group, 5 mice in the PBS group, 2 mice in the alpha-fodrin 1 microg/dose group, and 3 mice in the alpha-fodrin 10 microg/dose showed ANA positive. The serum IFN-gamma levels in the mice of alpha-fodrin 1 microg/dose and 10 microg/dose groups, PBS group, and GST group were (42 +/- 16), (37 +/- 15), (87 +/- 18), and (72 +/- 11) pg/ml respectively, those of the fodrin groups being significantly lower than those of the control groups (all P < 0.05). There were not significant differences in the level of serum IL-10 among these four groups. The numbers of Foxp3+ CD4 CD25+ regulatory T cells were higher in the fodrin groups than in the PBS and GST control groups (all P < 0.05). The lymphocytic infiltration and expression of alpha-fodrin were decreased in the alpha-fodrin administrated groups. The volume of water drinking of the alpha-fodrin 1 microg/dose group, alpha-fodrin 10 microg/dose group, PBS group, and GST group were (39.2 +/- 2.1), (40.4 +/- 2.5), (49.3 +/- 3.1), and (51.6 +/- 2.8) ml respectively. CONCLUSION: Mucosal administration of alpha-fodrin effectively inhibits the progression of experimental Sj?gren's syndrome autoimmunity.