氟苯尼考残留的酶联免疫检测方法的研究

对氟苯尼考进行结构改造制备半抗原及抗原,免疫家兔得到多克隆抗体。经测定,该抗体对氟苯尼考和氟苯尼考胺均有反应,且灵敏度较高,效价达到1∶480 000;在此基础上通过对各实验条件参数的优化建立了间接竞争酶联免疫检测方法,该方法检测限为0.5μg/kg,以20,50μg/kg进行空白样品添加实验,回收率为82.5%～96.0%,批间变异系数在4.1%～15.2%,可初步用于动物组织样本中氟苯尼考残留的检测.     

一种基于人工免疫的本体匹配算法

针对目前本体匹配算法存在运行效率低和匹配准确度不高等问题,提出一种基于人工免疫的动态本体匹配算法,用来快速地从现有本体中筛选出用户所需的子本体。该算法根据用户行为信息构建抗原本体模型,利用情景匹配确定其领域上下文环境,然后通过结构匹配获得匹配度最高的本体,最后对本体执行语义匹配得到最终需要的子本体。实验表明,该算法提高了本体匹配的准确度和效率。     

Ionic liquid and nanogold-modified immunosensing interface for electrochemical immunoassay of hepatitis B surface antigen in human serum

A mediator-free electrochemical immunoassay protocol based on a disposable immunosensor for the detection of hepatitis B surface antigen (HBsAg) in human serum was developed. To fabricate such an immunosensor, a layer of sol–gel composite film containing room temperature ionic liquid and chitosan was initially formed on a glassy carbon electrode. Nanogold particles were then adsorbed onto the membrane via the amine groups of chitosan molecules, and then horseradish peroxidase (HRP)-labeled hepatitis B surface antibodies (HRP-anti-HBs) were immobilized onto the nanogold surface. With a non-competitive immunoassay format, the antibody–antigen complex could be formed by a simple one-step immunoreaction between the immobilized HRP-anti-HBs and HBsAg in sample solution. The formed immunocomplex inhibited partly the active center of the HRP, which decreased the immobilized HRP toward the reduction of H₂O₂. The performance and factors influencing the performance of the immunosensor were evaluated. Under optimal conditions, the current change obtained from the carried HRP relative to H₂O₂ system was proportional to HBsAg concentration in the range of 1.5–400 ng/mL with a detection limit of 0.5 ng/mL (at 3δ). The reproducibility, selectivity, and stability of the proposed immunosensor were acceptable. Moreover, the proposed immunosensors were used to analyze HBsAg in human serum specimens. Analytical results of clinical samples suggested that the developed immunosensor has a promising alternative approach for detecting HBsAg in the clinical diagnosis.     

Proteomics-based identification of HSP60 as a tumor-associated antigen in colorectal cancer

Patients with cancer frequently develop autoantibodies. The identification of tumor autoantigens may have utility in early cancer diagnosis and immunotherapy. In this study, we used serological proteomics analysis (SERPA) to identify tumor proteins that elicit humoral response in colorectal cancer (CRC). The CRC cell line HCT116 was used as a source of proteins for 2-DE and subsequent Western blot analysis in which individual serum from patients with CRC was analyzed for autoantibodies. An autoantibody against HSP60 identified by MS was detected in 13 out of 25 patients with CRC and 1 out of 15 healthy subjects. In addition, the HSP60 expressions in tumor tissues collected from 40 patients with CRC were assessed by immunohistochemistry, and serum specimens from 100 patients with cancer and 30 healthy controls were screened for antibody titer to HSP60 by ELISA. The results showed that expressions of HSP60 in tumor tissue and serum antibody titer to HSP60 were significantly higher in patients with CRC than in healthy subjects. Thus, we conclude that the SERPA is an excellent assay for the identification of tumor-associated antigens and tumor markers. The detection of HSP60 may have clinical utility in CRC screening, diagnosis, and immunotherapy.     

A recombinant chimeric antigen toward a standardized serodiagnosis of Taenia solium neurocysticercosis

Neurocysticercosis (NC) invokes formidable neurological problems worldwide. Previous proteomic analyses revealed most of the low‐molecular‐weight proteins might derive from two macromolecules of 120 kDa (consisting of 14–38 kDa subunits) and 150 kDa (7–15 kDa subunits) of Taenia solium metacestode (TsM) cyst fluid (CF). We characterized serological properties of these two proteins and established an immunopotent chimera. The 120 and 150 kDa proteins harbored 54–81 and 94–98% of the antibody‐binding activity of the crude CF with minimal antigenic cross‐reactivity to each other. The expression and immune recognition of the 150 kDa subunits were relatively constant, regardless of the different geographical origins of the CF collected, while those of the 120 kDa subunits varied by their origins (Asia vs. America). We cloned four representative proteins (one from the 120 kDa and three from the 150 kDa) that showed different epitope specificities, generated a chimera, and demonstrated that this regimen may bolster serodiagnostic reliability. Overall sensitivity and specificity, against sera from active‐/mixed‐stage NC and those from other infections, and healthy‐controls, were determined to be 97.5% (156/160 samples) and 97.8% (265/271 cases). Patient sera from adult taeniases, sparganosis, and fascioliasis showed weak cross‐reactions. Micro‐ELISA showed similar results. This chimera may prove useful in the construction of standardized platform for NC serodiagnosis.     

表达轮状病毒VP4抗原的重组腺病毒对新生小鼠的被动免疫保护作用

目的探讨表达轮状病毒VP4抗原的重组腺病毒(Ad5/VP4)对新生小鼠的被动免疫保护作用。方法在293细胞上扩增表达猴轮状病毒SA11株VP4抗原的重组腺病毒并纯化。将轮状病毒抗体阴性的小鼠交配后的雌性小鼠,分别通过肌肉注射、滴鼻和口服免疫纯化后的重组腺病毒,生产后第7天和第8天,用轮状病毒SA11(1·0×106PFU)口服攻击乳鼠2次,观察出现腹泻症状的乳鼠的比例,并用ELISA检测母鼠的血清以及乳鼠胃囊乳块中的轮状病毒特异性抗体。同时以含有部分E1区基因的腺病毒5型载体(Ad5)作为对照。结果经3种不同途径免疫母鼠后,在血清和乳汁中均能检测到轮状病毒VP4特异性抗体。经滴鼻免疫的母鼠喂养的乳鼠,经攻击后保护率为100%(11/11);经肌肉注射免疫的母鼠喂养的乳鼠,经攻击后保护率为30%(3/10);经口服免疫的母鼠喂养的乳鼠,经轮状病毒攻击后保护率为80%(8/10)。对照组的小鼠全部出现了腹泻症状。结论表达轮状病毒VP4抗原的重组腺病毒经滴鼻免疫母鼠后,能在乳汁中产生特异性的IgA和IgG抗体,并能保护新生小鼠对抗轮状病毒的攻击,保护率达100%。     

基于残余抗原学说的动态记忆风险识别模型

以小概率事件风险识别为研究对象,提出一个基于残余抗原学说的动态记忆风险识别模型DMRIM。DMRIM针对小概率事件风险的无规则等特点,将风险的强度和频度直观地、动态地映射为残余抗原的浓度,以残余抗原刺激免疫记忆、指导抗体进化、控制识别器的生命周期,突破了传统的记忆细胞生命周期,实现了识别器分布自制,提高了小概率事件的辨识能力。仿真实验表明,DMRIM充分体现免疫记忆的动态性,有效地识别小概率事件,其可行性在实际应用中得到了验证。     

口蹄疫病毒复合多表位DNA疫苗的设计及构建

目的构建口蹄疫病毒(FMDV)复合多表位基因工程疫苗表达盒OAAT及其DNA疫苗。方法以O型、A型FMDV结构蛋白VP1全基因和Asia1型FMDV两个基因拓扑型的结构蛋白VP1基因上的5个抗原表位基因作为主要免疫原基因,非结构蛋白3ABC上的2个Th2细胞表位基因及结构蛋白VP4上的1个Th2细胞表位基因作为辅助基因,构建表达盒。将构建好的表达盒OAAT克隆到真核表达载体pVAX1 PCMV启动子下游,构建三价口蹄疫核酸疫苗pVAX1-OAAT,并用Western blot和IFA方法检测目的蛋白在HeLa细胞中的表达。结果通过InsightⅡ软件同源建模和DNAStar5.0软件分析表明,所构建的FMDV复合多表位基因工程疫苗表达盒OAAT理论上符合设计要求,且在真核细胞获得了正确表达。结论已成功设计FMDV复合多表位基因工程疫苗表达盒OAAT,并构建了三价口蹄疫核酸疫苗pVAX1-OAAT。     

农药小分子半抗原合成的研究与应用

在农药残留免疫分析中,农药小分子半抗原的分子设计及合成是基础和关键。本文对近年来国内外关于农药小分子半抗原的设计及合成、合成的影响因素、半抗原与载体蛋白偶联方法的研究现状及应用作一综述。