氯霉素-BSA和氯霉素-OVA偶联物的制备与鉴定

目的：制备氯霉素（CAP）-牛血清闩蛋白（BSA）及氯霉素（CAP）-卵清白蛋白（OVA）的偶联物并进行鉴定。方法：分别采用混合酸酐法和重氮化法制备CAP—BSA及CAP—OVA的偶联物，前者是将CAP先与琥珀酸酐反应生成CAP-半琥珀酸酯（CAP—HS），再进行混合酸酐反应。将反应产物CAP—HS与BSA结合，合成CAP—HS—BSA偶联物；后者是将CAP分子中的苯环上的硝基还原为氯基后，进行重氮化处理，然后分别与BSA和OVA连接，合成CAP—BSA和CAP—OVA复合物。结果：紫外图谱分析表明，CAP—BSA、CAP—OVA和CAP—HS—BSA的紫外最大吸收峰分别是262nm、213nm和275nm；计算得CAP与载体蛋白BSA、OVA及HS的偶联比分别为5：1、12：1和22：1。结论：成功的制备CAP—BSA及CAP—OVA的偶联物，为免疫动物制备抗CAP的抗体奠定了基础。     

25-羟基维生素D3人工完全抗原的合成与鉴定

目的:合成25-羟基维生素D3人工完全抗原,并制备抗25-羟基维生素D3的特异性抗体。方法:将25-羟基维生素D3进行化学修饰加入羧基活性基团,合成具有半抗原结构特征的25-羟基维生素D3-半琥珀酸酯。采用碳二亚胺法,将25-羟基维生素D3-半琥珀酸酯,分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联,合成人工完全抗原25-羟基维生素D3-半琥珀酸酯-BSA和25-羟基维生素D3-半琥珀酸酯-OVA。通过紫外吸收光谱,SDS-PAGE和MALDI-TOF进行偶联鉴定。用25-羟基维生素D3-半琥珀酸酯-BSA免疫小鼠,获得抗25-羟基维生素D3抗体免疫血清。结果:25-羟基维生素D3-半琥珀酸酯与BSA的偶联比为(12±0.16)∶1,免疫小鼠后获得高效价(效价为6.25×10-4)的抗体,且标准品浓度在37.5～600 ng/mL范围具有显著的竞争性线性关系,检测的灵敏度为37.5 ng/mL。结论:成功合成了25-羟基维生素D3人工完全抗原,制备出25-羟基维生素D3的抗体且其线性关系显著,灵敏度较高,为进一步研制检测25-羟基维生素D3的试剂盒奠定了基础。     

甲氰菊酯人工抗原的合成和鉴定

目的 合成与鉴定甲氰菊酯的人工抗原.方法 通过水解、酰化、酯化等反应合成了甲氰菊酯的半抗原2,2,3,3.四甲基环丙烷羧酸-a-(N-丁酸基)-甲酰氨-3-苯氧基苄酯(Ⅳ)和2,2,3,3-四甲基环丙烷羧酸-a-羧基-3-苯氧基苄酯(Ⅲ).通过碳二亚胺法将半抗原Ⅳ与牛血清蛋白(BSA)偶联制备免疫抗原,通过混合酸酐法将半抗原Ⅲ与卵清蛋白(OVA)偶联制备包被抗原.结果 用质谱和核磁共振对Ⅲ和Ⅳ进行结构表征,合成产物为目标物.紫外光谱法鉴定结果表明,免疫抗原和包被抗原都发生了有效偶联,偶联比38:1和15:1,免疫抗原通过免疫新西兰大白兔得到的抗体效价为5.12×105.结论成功的合成了甲氰菊酯的人工抗原,为其免疫方法的建立奠定了基础.     

四环素人工抗原的合成与鉴定

目的:对牛血清白蛋白进行羧基化和四环素的衍生化,制备更高偶联比的四环素(TC)-牛血清白蛋白(BSA)偶联物并进行鉴定.方法:四环素通过两步衍生化后,形成一种活泼的重氮化中间产物,与羧基化的BSA上的表位羧基反应,合成TC-BSA偶联物.结果:HPLC-MASS图谱鉴定表明成功的制备了四环素衍生物,红外及紫外扫描图谱表明,偶联成功,计算得偶联比分别为7:1和11:1. 结论:BSA经过羧基化后的偶联效果明显好于未经羧基化的BSA,并成功制备了TC-BSA人工抗原.     

氯霉素全抗原结合比的定量检测研究

 目的 探讨简便、快速定量检测氯霉素（CA）全抗原结合比的方法。 方法 以牛血清白蛋白（BSA）为载体蛋白，采用混合酸酐法将之与氯霉素琥珀酸酯（CA-HS）偶联，合成 CA-BSA全抗原。应用紫外分光光度法对 CA-BSA全抗原进行定性鉴定；分别采用自行改进的三硝基苯磺酸（TNBS）法和紫外分光光度法对 CA-BSA 全抗原结合比进行定量检测。 结果 紫外分光光度法检测显示 CA-BSA 的最大吸收峰高于相同蛋白浓度的 BSA 最大吸收峰，表明 CA-BSA 全抗原偶联成功。TNBS 法测得的 CA-BSA 全抗原结合比为 26，紫外分光光度法测得的 CA-BSA 全抗原结合比为 23，两种方法的检测结果基本一致。 结论 TNBS 法和紫外分光光度法用于氯霉素全抗原结合比的定量检测均具有快速、灵敏、方便和可靠的特点。TNBS 法对半抗原在紫外区无吸收的全抗原结合比测定也可以胜任，因此适用范围更为广泛。     

抗土霉素单克隆抗体的制备及其特性分析

目的:制备抗土霉素(OTC)的单克隆抗体(mAb),对其特异性和竞争ELISA的实用性进行分析。方法:通过碳二亚胺法和羰基二咪唑法分别将OTC与载体蛋白(BSA、OVA)偶联制备免疫原和检测抗原,用杂交瘤技术制备mAb,鉴定mAb的特异性并用于竞争ELISA效果分析。结果:获得1株能稳定分泌抗OTC的mAb杂交瘤细胞株,mAb腹水的ELISA效价为3×104,土霉素竞争ELISA检测下限为1mg/L。结论:抗OTC的mAb具有抗原结合特异性,可用于OTC竞争ELISA免疫学检测。     

5-氟尿嘧啶多克隆抗体的制备和鉴定

目的制备5-氟尿嘧啶(5-FU)免疫原并制备其多克隆抗体。方法采用化学合成法对5-FU进行修饰制备5-氟尿嘧啶-1-基乙酸半抗原(5-FUAA),并经碳酰二亚胺盐法将其分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)连接以制备免疫原。采用5-FU与BSA结合的免疫原(5-FUAA-BSA)免疫BALB/c小鼠制备多克隆抗体,5-FU与OVA结合物(5-FUAA-OVA)包被酶标板经间接ELISA检测抗血清效价,并采用ELISA与Western blot法检测抗血清的特异性。结果成功合成半抗原5-FUAA,并分别制备5-FUAA-BSA和5-FUAA-OVA完全抗原。将5-FUAA-BSA免疫BALB/c小鼠制备的免疫血清效价达1∶1 280 000,且该多克隆抗体能特异地结合5-FU半抗原。结论成功制备了5-FU的多克隆抗体。     

百草枯人工抗原的合成及抗体的制备

目的: 制备百草枯人工抗原和抗血清, 为建立酶联免疫吸附法提供技术储备.方法: 以4, 4'-联吡啶和碘甲烷为起始原料, 于暗处通氮气保护下合成了百草枯半抗原; 混合酸酐法偶联大分子蛋白载体牛血清白蛋白(BSA)和卵清蛋白(OVA)制备免疫抗原和包被抗原; 免疫新西兰大白兔, 制备多抗.结果: 合成的半抗原经HPLC-MS、 1HNMR和IR鉴定, 初步确定合成成功; 百草枯半抗原与BSA和OVA的结合比分别为19∶ 1和14∶ 1; 抗血清经间接ELISA法检测, 效价达1∶ 2.56×104, 经饱和硫酸铵沉淀法纯化后抗体的效价达1∶ 5.12×104.结论: 合成的百草枯人工抗原具有较好的免疫原性, 为百草枯酶联免疫检测(ELISA)试剂盒的研制提供了基础.     

抗甘草酸二铵抗血清的制备

目的通过人工抗原的构建,制备抗甘草酸二铵（DG）的特异性抗血清。方法采用碳二亚胺法将DG与牛血清蛋白（BSA）和鸡卵清蛋白（OVA）共价偶联,分别合成免疫抗原DG-BSA和包被抗原DG-OVA。用合成的人工抗原免疫家兔,制得抗甘草酸二铵的特异性抗血清,并通过酶联免疫方法（ELISA）对抗血清进行鉴定。结果制备了抗甘草酸二铵的多克隆抗体,获知理想的包被抗原的浓度为200ng／mL,抗血清的工作浓度为1：10000,雌、雄家兔抗血清的效价分别为1：51200和1：25600。结论采用化学反应构建人工抗原的方法,可以制备出针对甘草酸二铵的特异性抗血清,从而为甘草酸二铵的鉴定及体内代谢的免疫学分析研究奠定了基础。     

己烯雌酚免疫原的合成及其抗血清制备

目的: 制备人工合成的己烯雌酚 (DES)的多克隆抗体。方法: 采用碳二亚胺法将DES与牛血清白蛋白 (BSA)交联, 经紫外扫描估算其交联比为 22, SDS- PAGE实验初步表明DES与BSA发生交联。用双向免疫琼脂扩散实验检测经DES -HS- BSA多次免疫新西兰白兔的抗血清。结果: 合成的DES- HS- BSA复合物, 具有免疫原性, 并产生了针对小分子DES的抗体。结论: DES免疫原的合成是成功的, 为其试剂盒的研制提供了条件。     

杜氏肌营养不良患者及其基因携带者红细胞膜蛋白颗粒定量分析

目的　研究 Duchenne型肌营养不良症 (Duchenne muscular dystrophy,DMD)患者及其基因携带者红细胞膜蛋白颗粒的变化情况 ,探讨 DMD的发病机理以及红细胞冷冻断裂技术的诊断价值。方法　采用冷冻断裂法把固定好的红细胞块制成复型膜样品 ,在电镜下观察拍照 ,计算其红细胞膜劈裂面中面向细胞质的一面 (extracellular face,EF)和红细胞膜劈裂面中面向细胞外隙的一面 (protoplasmic face,PF)单位面积内蛋白颗粒数 ,并进行统计学对比分析。结果　 DMD患者及 DMD携带者红细胞膜 EF面和PF面蛋白颗粒数与正常对照组相比均明显减少 (P<0 .0 0 1)。结论　红细胞冷冻断裂电镜技术可作为诊断DMD的辅助检查手段 ,也可作为检测 DMD基因携带者的一种方法。本研究结果为 DMD的全面性膜缺陷理论提供了可靠的证据。     

载氧药物的冷冻干燥和冻干制品的保存稳定性

对以聚合胎盘血红蛋白为有效成分的载氧药物进行冷冻干燥.采用蔗糖作保护剂,减少冷冻干燥过程中高铁血红蛋白(Methemoglobin,MetHb)的增加.测定冷冻干燥前后的MetHb含量、UV光谱、十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)、高效液相色谱法(HPLC)等指标.结果表明:冷冻干燥中,当蔗糖/蛋白质的重量比≥0.5时,MetHb增加得到有效控制, UV光谱中血红蛋白(Hb)的特征吸收峰、SDS-PAG、HPLC无显著改变.冻干制品分别在室温或冰箱中保存3个月,考察其稳定性.结果表明:保存过程中,MetHb的增加与蔗糖/蛋白质的重量比、保存温度有关,试验组C(蔗糖/蛋白质的重量比为1.0)在室温或冰箱中保存3个月,MetHb含量、UV特征吸收峰、SDS-PAGE、 HPLC等指标均无显著改变.     

Application of a modified version of Habeeb's trinitrophenylation method for the characterization of hapten-protein conjugates in a reversed micellar medium

We describe here a protocol for determining the epitope density of hapten-carrier conjugates at the nanomolar level. Conjugates of bovine serum albumin (BSA) and hen egg albumin (OVA) were prepared with two model haptens: 4-acetyl benzoic acid (ABA) as a chromophoric carboxylic hapten and cholesterol hemisuccinate (Chol HS) as a carboxylic derivative of a nonchromophoric hydroxylated hapten. Small-scale but valuable haptenization of carriers was achieved (approximately 4.3 nmol with an input molar ratio of hapten to carrier within the range from 50:1 to 100:1) that proved yet compatible with immunogenicity and antibody detection. We used a modified version of the amide bond-generating mixed anhydride method of Erlanger performed in a reversed micellar medium. Microsample aliquots of the coupling reaction (carriers plus activated haptens) or control experiment (carriers plus nonactivated haptens) mixtures were directly subjected to trinitrophenylation in the reversed micellar medium. The results for carrier substitution were strongly correlated (r2=0.94; n=12) with those obtained by other methods such as UV analysis (for ABA) and chromatographic determination (for Chol HS). This method was found directly applicable to other hapten-carrier conjugates coupled by the same procedure, provided that the haptens do not absorb at about 335 and 420 nm (absorption peaks of trinitrophenyl groups). With this simple, rapid, reproducible and low-cost analysis method, the separation of uncoupled hapten and conjugate is unnecessary. This facilitates the optimization of reaction conditions. Finally, using this procedure, kinetic studies of conjugate formation can be carried out in a very simple manner.     

2006-2007年广东省登革病毒Ⅰ型分离株E基因序列的分子进化

目的 揭示广东省2006-2007年各地登革病毒流行株的遗传关系,探讨其可能来源.方法 收集广东省2006年5个流行区、2007年4个流行区登革毒株和近几年广东省分离的登革病毒流行毒株,设计覆盖登革Ⅰ型病毒E(包膜蛋白)基冈的3对扩增片段瓦相嵌套的引物,应用逆转录-聚合酶链反应(RT-PCR)进行扩增,扩增产物纯化后直接进行序列测定,所得序列经拼接成E基因全长序列后,同时与GenBank上扶得的登革Ⅰ型病毒E基因序列一起,用MEGA 4.1软件进行分析.结果 2006年广州流行株来源于越南;阳江、南海流行株来源于阳江本地;汕头、潮州流行株来源于新加坡.2007年流行株都来源于新加坡.结论 广东省2006年发生的登革热疫情来源多样,而2007年疫情来自同一源头,近几年广东省流行的登革病毒主要来源于东南亚等国,但在广东省局部地区已形成地方性流行.     

Genotypes and phylogenetic characterization of hepatitis B and delta viruses in Egypt

Hepatitis B virus (HBV) and hepatitis D virus (HDV) sequences among HBV carriers from Egypt have not been evaluated sufficiently. The genotypes of HBV isolated from 105 serum samples from Egyptian carriers were determined. Four complete genomes and 11 entire preS1/S2/S genes were sequenced and evaluated. All serum samples were classified into HBV genotype D using serologic and genetic methods. The length of four complete nucleotide sequences was 3,182 bp. In all 15 samples, the common 33 nucleotides (11 amino acids) deletions in the preS1 region specific for HBV genotype D were observed. In the phylogenetic analysis based on the complete nucleotide sequences, all samples were clustered with the HBV isolates reported from previously Western and Mediterranean countries with nucleotide homology ranging from 96.0-98.0%. Of 75 HBsAg positive samples, anti-HDV was found in 15 (20%), and HDV RNA was detected in 9 of 15 (60%). The proportion of the patients with liver disease was higher in HBV carriers of anti-HDV positive with HDV RNA than in HBV carriers of anti-HDV positive without HDV RNA (P < 0.05). In the phylogenetic analysis based on the sequences in nucleotide position 853-1267 of HDV, nine samples were classified into HDV genotype I with the nucleotide homology ranging from 88.3-92.1% (mean; 90.5%) and clustered with HDV strains reported previously from Ethiopia, Somalia, Egypt, and Lebanon. These results indicate that HBV genotype D and HDV genotype I are most prevalent in Egypt, and HDV co-infection in HBV carriers is related to severity of liver disease.     

Leishmanial Excreted Factor: Protein-Bound and Free Forms from Promastigote Cultures of Leishmania tropica and Leishmania donovani

Leishmania spp. growing in culture produce an immunologically active substance called excreted factor (EF), which precipitates antibodies raised against intact cells and has been implicated as the conditioning agent for parasite infection of host macrophages. An improved method for isolation of the material is described, based on Sephadex column chromatography of growth medium which had been boiled at pH 5.0. This procedure allows the detection of differences among the EF molecules of different species, and it overcomes previous shortcomings through the monitoring of immunological activity throughout. Analysis of the products of this procedure revealed that EFs from Leishmania tropica and Leishmania donovani share a common carrier protein, identified as rabbit serum albumin, and are chemically quite similar. Growth medium from L. tropica boiled at acidic pH contains primarily an EF-albumin complex of 75,000 molecular weight. Treated growth medium from L. donovani, on the other hand, contains both the albumin complex and a smaller molecule (less than 27,000 molecular weight) that is not associated with rabbit protein. This material accounts for nearly 20% of the EF of one L. donovani strain, but constitutes only a minute fraction of L. tropica EF. Treatment of the EF-albumin complex with trichloroacetic acid separates the molecule into two major subunits, one having a molecular weight of about 61,000 (without anti-Leishmania activity) and the other having a molecular weight of about 18,000 (with no anti-rabbit activity). The protein-free EF of L. tropica differs from that released by trichloroacetic acid extraction in that it is capable of precipitating antisera of nonhomologous serotypes, whereas the albumin complex and the trichloroacetic acid-treated EF fragment are not. EFs from both species display pH-dependent affinity for certain lectins.     

Anthrax edema toxin impairs clearance in mice

The anthrax edema toxin (ET) of Bacillus anthracis is composed of the receptor-binding component protective antigen (PA) and of the adenylyl cyclase catalytic moiety, edema factor (EF). Uptake of ET into cells raises intracellular concentrations of the secondary messenger cyclic AMP, thereby impairing or activating host cell functions. We report here on a new consequence of ET action in vivo. We show that in mouse models of toxemia and infection, serum PA concentrations were significantly higher in the presence of enzymatically active EF. These higher concentrations were not caused by ET-induced inhibition of PA endocytosis; on the contrary, ET induced increased PA binding and uptake of the PA oligomer in vitro and in vivo through upregulation of the PA receptors TEM8 and CMG2 in both myeloid and nonmyeloid cells. ET effects on protein clearance from circulation appeared to be global and were not limited to PA. ET also impaired the clearance of ovalbumin, green fluorescent protein, and EF itself, as well as the small molecule biotin when these molecules were coinjected with the toxin. Effects on injected protein levels were not a result of general increase in protein concentrations due to fluid loss. Functional markers for liver and kidney were altered in response to ET. Concomitantly, ET caused phosphorylation and activation of the aquaporin-2 water channel present in the principal cells of the collecting ducts of the kidneys that are responsible for fluid homeostasis. Our data suggest that in vivo, ET alters circulatory protein and small molecule pharmacokinetics by an as-yet-undefined mechanism, thereby potentially allowing a prolonged circulation of anthrax virulence factors such as EF during infection.     

Immunological specificity of chloramine-T-induced IgE antibodies in serum from a sensitized worker

A procedure for the preparation of chloramine-T (CT) conjugates used to assay IgE antibodies was developed using response surface methodology and serum from a subject occupationally exposed to the substance. The conjugates, synthesized by reacting CT with human serum albumin (HSA) and other protein carriers, were used as antigens in a radio-allergosorbent test (RAST). Human serum albumin was found to be a suitable carrier, although other protein carriers also gave specific IgE-binding of a similar extent. The CT-HSA conjugates used in the RAST were characterized by high performance liquid chromatography, electrophoresis, immunodiffusion and ammonium sulphate precipitation. However, no strong correlation was seen between the ability of the conjugates to bind IgE and their physical or immuno-chemical properties. The hapten and carrier specificity of CT-induced IgE antibodies in the subject's serum were studied by direct RAST and RAST inhibition. No existence of new antigenic determinants related to the carrier could be demonstrated. Although HSA as a carrier was altered immunochemically by CT, the IgE antibodies were found to be specific to hapten only. Chloramine-T-specific IgG antibodies could not be demonstrated in the subject's serum.     

双蛋白载体A、C群脑膜炎球菌多糖结合物的免疫效果观察

目的 比较和评价在A、C群脑膜炎球菌多糖结合物中应用两种蛋白作为载体的免疫效果.方法 构建不耐热肠毒素B亚单位(LTB)的克隆载体和表达载体.确定rLTB主要为可溶性表达.应用一步阳离子交换层析对rLTB进行纯化,获得较纯的目的蛋白.利用GM1-ELISA和非变性SDS-PAGE的方法确定纯化后的rLTB能够形成具有生物学活性的五聚体.最后利用化学方法(ADH方法)将通过基因工程手段获得的重组LTB五聚体蛋白与C群脑膜炎球菌多糖(GCMP)耦联,获得多糖蛋白结合物GCMP-rLTB.以单一蛋白破伤风类毒素(TT)为载体的A+C群脑膜炎球菌多糖蛋白结合物(GAMP-TT和GCMP-TT)及同时应用两种蛋白载体的A+C群脑膜炎球菌多糖蛋白结合物(GAMP-TT和GCMP-rLTB)分别通过腹腔注射途径免疫小鼠.应用ELISA方法分别检测小鼠血清中IgG水平.结果 单独以TT为载体的A+C群脑膜炎球菌多糖蛋白结合物(GAMP-TT和GC-MP-TT)和同时以两种蛋白作为载体的A+C群脑膜炎球菌多糖蛋白结合物(GAMP-TT和GCMP-rLTB)通过注射途径免疫小鼠,后者产生的血清多糖特异性IgG水平显著高于前者.结论 双蛋白载体在改善A、C群脑膜炎球菌多糖结合物疫苗的免疫原性方面具有明显优势.

Abstract：

Objective To evaluate the immunogenicity of group A and C meningococcal polysaccharides conjugates using different proteins as carriers. Methods Heat-labile enterotoxin B subunit (LTB)pentamer form was expressed in E. coli. The target protein was identified and purified by cation-exchange chromatography. Then biological activity of rLTB was tested using GM1-ELISA. GCMP was conjugated to rLTB with the chemical method (ADH). Furthermore, the mice were immunized with GAMp-TT/GCMP-TT conjugates and GAMP-TT/GCMP-rLTB conjugates via peritoneal. Finally the anti-polysaccharide antibody was detected. Results The GAMP-TT/GCMP-rLTB conjugate elicits remarkably higher serum antibodies in mice than GAMP-TT/GCMP-TT conjugate. Conclusion These results indicated that polysaccharide conjugates using different proteins as carriers were superior to those using only one protein as carrier.     

EB病毒蛋白诱导IgG和IgM产生的作用

目的:检测热灭活或紫外线(UV)灭活的EB病毒(EBV)刺激培养的人脐带血B细胞产生IgG和IgM的效应。方法:常规分离人脐带血单个核细胞(UBMC),以L-亮氨酸甲酯去除法,去除单核细胞、NK细胞和细胞毒性T细胞,以2-氨乙基硫脲溴化物(AET)处理绵羊红细胞(SRBC)的花环形成分离法,去除T细胞以获得纯化的B细胞。分别用UV和热灭活的EBV刺激培养的B细胞后,用夹心ELISA法检测培养上清中IgG 和IgM的产生。结果:以UV灭活的EBV刺激培养B细胞后18 d起,IgG和IgM的产生具有意义(P<0.05);而热灭活的EBV刺激后,各时间点均无显著差异(P>0.05)。结论:UV灭活EBV具有诱导IgG和IgM产生的作用,但有明显的时效性,提示EBV诱导IgG和IgM产生,可能是通过病毒的蛋白成分实现的,为进一步验证EBV功能蛋白诱导天然自身抗体(NAA)产生中的作用奠定了基础。