Identification of nasopharyngeal carcinoma antigens that induce humoral immune response by proteomic analysis

A proteomics-based approach has been used to identify proteins that commonly elicit a humoral immune response in nasopharyngeal carcinoma (NPC). Sera from 19 newly diagnosed NPC patients and 19 healthy individuals were analyzed for IgG autoantibodies against NPC proteins resolved by 2-DE. Protein spots that exhibited selective reactivity with sera from NPC patients were identified by MS. Among nine identified proteins, cytokeratin 19 (CK19), Erb3 binding protein (EBP1), and Rho GDP dissociation inhibitor-beta (Rho-GDI-2) induced autoantibodies in more than 36.8% of NPC patients but not in healthy individuals. Furthermore, Western blot analysis and immunohistochemical staining were performed to determine the expression and localization of CK19, EBP1, and Rho-GDI-2 in NPC and normal nasopharyngeal mucosal tissues. Up-regulated CK19 and EBP1, but not Rho-GDI-2, were observed in NPC vs. normal tissue. Subcellular localization of the three proteins in NPC tissue was same as that in the normal tissue. Thus, overexpression of CK19 and EBP1 may be one of the mechanisms for their autoantibody development in NPC. To validate the findings of a proteomic analysis, occurrence of autoantibodies against these three proteins was detected by immunoprecipitation and Western blot analysis in additional 30 NPC patients, 23 other types of cancer patients and 20 healthy individuals. Results showed that frequency of autoantibodies against CK19, EBP1 and Rho-GDI-2 in NPC patients was significantly higher than that in other types of cancer patients and healthy individuals. We conclude that CK19, EBP1 and Rho-GDI-2 may have utility in NPC screening and diagnosis.     

人工免疫系统中人工抗原提呈细胞的设计

反向选择模型是人工免疫系统中的经典模型,但存在训练代价高、覆盖率低的问题.危险理论模型是人工免疫系统中新引进的模型,在降低训练代价,提高覆盖率上具有优势.建立人工免疫危险理论模型必须解决的关键问题之一是:在保持人工免疫系统自适应特征的前提下,设计人工抗原提呈细胞.本文从抗原提呈细胞的生物学原理出发,设计了人工免疫系统中的人工抗原提呈细胞,给出单个人工抗原提呈细胞的结构、细胞上Toll样受体的结构,以及人工抗原提呈细胞群体的结构;设计了人工抗原提呈细胞群体的生命周期.初步的实验结果证明所设计的人工抗原提呈细胞具有自适应发现恶意软件的能力.     

基于小生境技术的多样性抗体生成算法

新的入侵方法以及网络计算环境的不断变化,使得入侵越来越难以防范.因此应用人体免疫机制构建下一代入侵检测系统成为一个新的研究热点.本文采用一种新的抗原－抗体编码方法,并在此基础上,提出一种基于共享函数的小生境遗传算法,用来产生多样性的抗体基因,最后给出多样性评价函数以验证算法的有效性.实验结果表明:该算法能够使抗体在演化过程中保持较好的多样性.     

基于聚硫堇的一次性O型口蹄疫抗原酶免疫传感器的研制

将O型口蹄疫酶标抗体掺杂于有机-无机溶胶凝胶中,并修饰于聚硫菫的丝网印刷碳电极表面,从而制备一次性口蹄疫病毒抗原(FMDV-Ag)酶免疫传感器。试验中采用直接免疫分析法检测口蹄疫病毒抗原。根据免疫反应前后还原峰电流下降的百分率K值的大小实现对抗原的检测。在优化条件下,免疫传感器检测抗原的线性范围为77～388ng/mL,最低检出限为32ng/mL。免疫电极具有较好的特异性、重现性(RSD=5.6%)、稳定性(10d后电流响应为初始值的89.5%)和准确性(与AGP符合率为95%)。因此,该免疫传感器有望用于O型口蹄疫抗原的快速检测。     

木瓜蛋白酶水解对豆乳中抗原蛋白含量和亚基构成的影响

采用木瓜蛋白酶水解传统生浆工艺制备的豆乳,用双抗体夹心酶联免疫吸附法（ELISA）测定酶解残余物中抗原蛋白含量,用SDS-PAGE分析其蛋白分子亚基组成,并通过正交实验优化了木瓜蛋白酶的水解条件。结果表明,当保持豆乳自然p H（6.5⁶.8）、酶浓度3500 U/m L、水解温度53℃、水解时间15 min时,β-伴球蛋白的α’、α亚基被完全水解,大豆球蛋白的酸性多肽链基本消失,碱性多肽链含量明显减少,同时产生了大量14 k Da以下组分,此时β-伴球蛋白残留率为56.6%。      

Preclinical Assessment of the Combination of PSMA-Targeting Radionuclide Therapy with PARP Inhibitors for Prostate Cancer Treatment

Prostate specific membrane antigen targeted radionuclide therapy (PSMA-TRT) is a promising novel treatment for prostate cancer (PCa) patients. However, PSMA-TRT cannot be used for curative intent yet, thus additional research on how to improve the therapeutic efficacy is warranted. A potential way of achieving this, is combining TRT with poly ADP-ribosylation inhibitors (PARPi), which has shown promising results for TRT of neuroendocrine tumor cells. Currently, several clinical trials have been initiated for this combination for PCa, however so far, no evidence of synergism is available for PCa. Therefore, we evaluated the combination of PSMA-TRT with three classes of PARPi in preclinical PCa models. In vitro viability and survival assays were performed using PSMA-expressing PCa cell lines PC3-PIP and LNCaP to assess the effect of increasing concentrations of PARPi veliparib, olaparib or talazoparib in combination with PSMA-TRT compared to single PARPi treatment. Next, DNA damage analyses were performed by quantifying the number of DNA breaks by immunofluorescent stainings. Lastly, the potential of the combination treatments was studied in vivo in mice bearing PC3-PIP xenografts. Our results show that combining PSMA-TRT with PARPi did not synergistically affect the in vitro clonogenic survival or cell viability. DNA-damage analysis revealed only a significant increase in DNA breaks when combining PSMA-TRT with veliparib and not in the other combination treatments. Moreover, PSMA-TRT with PARPi treatment did not improve tumor control compared to PSMA-TRT monotherapy. Overall, the data presented do not support the assumption that combining PSMA-TRT with PARPi leads to a synergistic antitumor effect in PCa. These results underline that extensive preclinical research using various PCa models is imperative to validate the applicability of the combination strategy for PCa, as it is for other cancer types.     

诺氟沙星单克隆抗体的制备及初步应用

采用优化的碳二亚胺法制备诺氟沙星（NOR）人工抗原,紫外扫描和红外光谱图表明人工抗原偶联成功。采用细胞融合技术筛选抗NOR单克隆抗体（NORmAb）杂交瘤细胞株,体内诱生腹水法制备NORmAb,建立间接竞争ELISA（icELISA）标准曲线。结果表明,筛选的3株杂交瘤细胞分泌的抗体为IgG1亚型,具k轻链;建立的icELISA标准曲线在PBS中的的线性检测范围为0.04～43.6ng/mL,IC50值为0.62ng/mL,检测限（LOD）为0.02ng/mL;抗体与环丙沙星（87.2%）、培氟沙星（76.9%）、依诺沙星（66.8%）、恩诺沙星（48.6%）和洛美沙星（36.6%）有较高的交叉反应率;禽肉基质中NOR添加回收率满足检测要求。     

玉米赤霉烯酮多克隆抗体的制备及特性分析

合成玉米赤霉烯酮半抗原,应用液相色谱-质谱联用法进行鉴定,并采用活泼酯法将半抗原与载体蛋白OVA或BSA偶联,分别作为免疫原或包被原,并通过SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和紫外扫描法鉴定偶联效果,免疫3只BALB/c小鼠,制备玉米赤霉烯酮多克隆抗体。结果表明:抗血清效价最高达1∶32 000,以此多抗建立的玉米赤霉烯酮间接竞争标准曲线IC50为39.8ng/mL,IC10为0.71ng/mL,多抗与玉米赤霉烯酮类似物β-zearalenol、zear-alanone、α-zearalanol、β-zearalanol交叉反应率分别为4.80%,3.07%,0.96%,0.09%。说明试验成功制备了玉米赤霉烯酮人工抗原及高特异性多克隆抗体。     

高效液相色谱与质谱联用法分离与鉴定己烯雌酚合成抗原的研究

采用Lichrospher C-18柱，用甲醇和水为流动相，流速0．3ml／min，甲醇从60％-100％梯度洗脱25min，液质联用，电喷雾（ESI），对己烯雌酚及其衍生物己烯雌酚-单（双）-丁酸乙酯基-醚进行了分离和结构鉴定。以混合酸酐法将DES-MCPE与载体蛋白偶联形成完全抗原，并以紫外扫描分析了完全抗原的偶联比率。结果表明：在极性溶剂中，己烯雌酚和其衍生物存在着顺反异构转化现象，极性有机溶剂的溶剂化作用促使顺反异构体的转化。计算DES-MCPE-BSA偶联比率为10。     

苯甲酸人工抗原的合成与免疫效价的测定

通过苯甲酸的衍生物邻苯二甲酸酐(PA)的氨解法将苯甲酸(BA)偶联到载体蛋白上合成苯甲酸的人工抗原:免疫原苯甲酸-鸡卵清蛋白(BA-OVA)和包被原苯甲酸-牛血清白蛋白(BA-BSA).通过紫外光谱扫描、红外光谱扫描及SDS聚丙烯酰胺凝胶电泳的方法检测鉴定人工抗原,并用免疫原(BA-OVA)免疫BALB/c小鼠,检测抗体的生成.结果表明,苯甲酸人工抗原的合成成功.根据邻苯二甲酸酐、人工抗原和载体蛋白的紫外吸收值,计算出免疫原中苯甲酸(BA)与载体鸡卵清蛋白(OVA)的偶联比约为174:1.免疫小鼠血清中的抗体通过ELISA法检测为阳性(血清效价达到了1:3.2×10<'5>),表明合成的抗原已诱导出针对苯甲酸的特异性抗体生成,为进一步的苯甲酸单克隆抗体制各和免疫检测提供可靠的免疫原.