酶联免疫吸附法和薄层色谱法联合分析黄曲霉毒素B1的研究

目的 研究酶联免疫吸附法和薄层色谱法的特点，评价联合两种方法检测黄曲霉素B1的可行性。方法 应用酶联免疫吸附法进行初步筛选试验和最终定量分析，以薄层色谱法进行确证试验。结果 黄曲霉毒素B1含量为1．0～20．00μg／kg范围内，标准曲线r值为-0．993～-0．999，CV为0．4～2．6％，回收率92．0～105．0％，当酶联免疫吸附法测定结果为10.0～45．0μg／kg范围内，应用薄层色谱法进行确证试验，符合率达100％。常见的其它霉菌毒素干扰试验表明，该方法特异性反应好，无干扰现象。结论 酶联免疫吸附法是目前国际测定黄曲霉毒素B1的先进方法，灵敏度高，干扰少，测定步骤简便、快速、操作安全。薄层色谱法中的确证试验是利用三氟醋酸和黄曲霉毒素B1的特异性反应所生产的衍生物，确证黄曲霉毒素B1。结合国际先进酶联免疫吸附法和传统方法的优点，科学地实现了快速、可靠、准确测定黄曲霉毒素B1的目的。     

应用ELISA检测艰难梭状杆菌毒素A和毒素B的抗体

艰难梭状杆菌已被证明是实验性动物和病人患抗生素相关性腹泻(Antbioticassociated diarrhea)和结肠炎的病原。认为疾病是由毒素间接引起。目前明确艰难梭菌产生二种毒素:A和B。它们是用阴离子交换色谱法分离出的抗原性不同的大分子量蛋白。作者实验室研究推论毒素B有强的细胞毒,在通常组织培养试验中显示出较大的活性反应。在检测实验动物肠道疾病时毒素A看来更具有活性,提示疾病的临床表现过程中毒素A起主要的作用。毒素抗体可以通过细胞毒性的中和试验检测,但组织培养需要专门的设备从而     

院内感染艰难梭菌的毒素检测及核糖体分型研究

目的 了解艰难梭菌临床分离株的毒力情况,并运用核糖体分型技术进行流行病学研究.方法 采集住院腹泻患者的未成形粪便标本,无水乙醇处理后接种CDMN选择培养基行艰难梭菌分离培养,并通过革兰染色、耐氧试验和凝集试验行菌落鉴定;提取菌株DNA,选择特异性引物用PCR法扩增毒素基因tcdA和tcdB;核糖体分型针对细菌基因组16S～23S rDNA间区序列进行扩增,并根据电泳带型的多态性实现分型.结果 共计分离得44株艰难梭菌,3种毒素型分别为A+B+型、A-B+型和A-B-型,分别占 57％ 、34％和9％;18种核糖体型中以R8 型和R4 型为主,分别占 20％和18％.结论 本研究临床分离的艰难梭菌菌株中,毒素型以A+B+型为主,核糖体分型存在相对优势的型别;无证据提示存在院内艰难梭菌感染的暴发流行.     

人工污染标本及肉毒中毒标本的检测

应用酶标SPA—ELISA夹心法对7种18份食物标本作了肉毒毒素的检测。其定性试验的结果与反向被动血凝试验、小鼠试验及污染所用菌型完全相符。对A型肉毒毒素的最低检出量,除1份标本外,均高于Notermans等的报道(50～100 LD50/ml);而对B型肉毒毒素的最低检出量则明显低子Kozaki等的报道(400LD/ml)。应用亲和层析交叉吸收法纯化的A、B型肉毒抗毒素分别作为包被抗体,消除了A、B型间的交叉现象,提高了试验的特异性。本法在快速及稳定性上也较良好。     

黄曲霉毒素B_1加合物检测方法研究进展

黄曲霉毒素B1污染是我国肝癌发生的主要危险因素之一,检测黄曲霉毒素B1暴露的生物标志物来研究黄曲霉毒素B1和肝癌之间的关系已成为趋势。随着多学科的交叉发展,现在已经具备多种试验方法检测黄曲霉毒素B1生物标志物,其中的一些检测方法具有高敏感度和精确度,对研究黄曲霉毒素B1早期低暴露具有重要价值。     

黄曲霉毒素B1快速检测方法的研究进展

黄曲霉毒素B1是黄曲霉毒素系列中毒性、危害性、污染性最大的一种,且分布广,对世界范围内的大多数食品原料及成品均有不同程度的污染,严重威胁人类健康。目前,国内外对于黄曲霉毒素B1的检测要求越来越高,新发展起来的快速分析技术也逐渐应用于黄曲霉毒素B1的检测,除了常规的液相色谱法、薄层层析法、胶体金法、酶联免疫吸附法得到更充分的研究和应用外,时间分辨荧光免疫法等新兴免疫学快速检测方法以及具有创新意义的ELISA-TLC组合分析法也已初步建立,这些为实现快速筛查、现场快检,提高检测效率、降低检测成本提供了很好的思路;同时也为更好地维护人民群众饮食安全提供重要支撑。本文就黄曲霉毒素B1的快速检测方法做一综述,以期为进一步提高对黄曲霉毒素B1的监控水平和效率提供参考。     

葡萄球菌肠毒素的纯化及免疫血清的制备

用国际标准菌株生产并纯化葡萄球菌肠毒素A,B,C2,D,经聚丙烯酰胺凝胶电泳(SDS-PAGE)图谱分析,表明所提纯的毒素与国外商品纯化毒素一致。用本室纯化的毒素免疫绵羊和家兔,制备免疫血清,并经Western印迹法试验证实葡萄球菌肠毒素各型的抗体与参比抗原专一结合。     

用HPLC法测定银杏种子中的银杏毒素和银杏毒素-5’-葡糖苷

银杏种子中含有某种有毒成分，能引起惊厥和丧失意识，有研究显示，这种有毒成分为银杏毒素。还有人发现该种子中存在银杏毒素-5＇-吡喃葡糖苷。由于尚未得到银杏-5L葡糖苷纯品，所以至今未对该化合物进行直接测定。作者开发了1个伴有荧光检测的HPLC法，同时测定银杏毒素、银杏毒素-5＇-葡糖苷和维生素B6。     

吉林地区某高校学生维生素B1、B2、C营养状况调查

目的 了解大学生维生素B1、B2和维生素C营养状况,为大学生合理膳食提供依据.方法 膳食调查采用称量法和询问法,维生素B1、B2和维生素C缺乏采用4h尿负荷试验及体征检查.结果 大学生维生素B1、B2和维生素C摄入量分别是1.1 mg、0.7 mg和86.0 mg.4h尿负荷试验维生素B1缺乏49.4％,正常46.8％,充裕为3.8％;维生素B2缺乏55.8％,正常42.9％,充裕1.3％;维生素C缺乏46.8％,正常48.1％,充裕6.4％.维生素缺乏体征检查,维生素B1没有检出,维生素B2检出率是27.2％,维生素C为17.6％.结论 大学生维生素B1、B2和维生素C营养状况不理想,应进一步调整膳食结构,增加食物的种类或补充维生素制剂.     

葡萄球菌肠毒素的纯化及免疫血清的制备

用国际标准菌生产并纯化葡萄球菌肠毒素A、B、C2、D，经聚丙烯酰胺凝胶电泳（SDS－PAGE）图谱分析，表明所提纯的毒素与国外商品纯化毒素一致。用本室纯化的毒素免疫绵羊和家兔，制备免疫血清，并经Western印迹法试验证实葡萄球菌肠毒素各型的抗体与参比抗原专一结合。     

Studies on shiga-like toxin produced by enterohemorrhagic Escherichia coli: purification and characterization of the toxin and development of methods for identifying the toxin

A simple purification method using DEAE cellulose column chromatography and immunoaffinity column chromatography was developed for purifying Shiga-like toxin produced by Escherichia coli O157:H7. About 0.75 mg of purified toxin was obtained from 5 liters of culture (62% recovery). The purified toxin was demonstrated to be immunologically, biologically and structurally indistinguishable from Shiga toxin. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detection of Shiga-like toxin. In the ELISA assay, Shigella dysenteriae type 1, Escherichia coli O157:H7 and some strains of Escherichia coli isolated from traveller's diarrhea were positive. Shiga toxin-resistant Vero cells were isolated by treatment of the cells with nitrosoguanidine. Immunofluorescence studies showed that the mutant Vero cells had lost toxin binding capacity. Samples of S. dysenteriae type 1 and E. coli O157:H7 showed cytotoxicity to the parent cells, but not to the mutant cells. Samples of other organisms showed either no cytotoxicity or cytotoxicity to both cell lines. The results suggested that (1) the presence of a receptor for Shiga-like toxin on Vero cells is essential for expression of cytotoxicity of the toxin, (2) the mutant Vero cells could be used to identify Shiga-like toxin producing organisms.     

单克隆抗体在ELISA法检测B型肉毒毒素中的应用

于ELISA—双夹心法系统中采用B型肉毒毒素单克隆抗体作为包被抗体,可检出B型肉毒类毒素4—31ng/ml,与A型交叉最低浓度为4—8μg/ml,特异性比值为250—1,000;可检出B型肉毒毒素16ng/ml(4LD_(50)/ml) A型大于32μg/ml(大于301LD_(50)/ml),特异性比值大于2,000;检出污染食物标本中的B型肉毒毒素最低浓度为0.67—7.8LD_(50)/ml,与A型间未见交叉现象。其敏感度接近RPHA,特异性明显优于RPHA。     

HUMAN SERUM IgE ASSAY BY REVERSED PASSIVE HEMAGGLUTINATION TEST

A reversed passive hemagglutination test (RPHA) has been established tol determine human serum IgE levels Sheep erythrocytes stabilized by glut.araldehyde are coated with purified IgG fraction of ho,rse anti-human. IgE serum with chromic chloride and tannic acid. Va.rious fa.ctors we're determined, including method of stabiliza.tion, coating pH, purification and heat-treatment of purified horsei anti-human IgE serum and chromic chloride and tannic acid conclecntrat.ion yielding the best results. RPHA sensitivity was compared with that of the ra- dioa.ctive single radial diffusion test (RSRD) using a working standard coded '751' which was calibrated with the Internatioinal Ref'eirence Standard 68/341, WHO. It was found that '751' IgE level is 2,100 units/ml by RSRD a.nd the corresponding RPHA reiproducible tAiter is usual- Iy around l:160. RPHA sensitivity is about 13.3 units/ml. The test s.pecificity was confirmecl by hemagglutination inhibition test (HAl). 196 serum samples were tested by both methods and showed definit.e correlation. If attention is paid to the conditions mentioned, RPHA can be used as a sensitive semiquantitative method for screening serum IgE levels in numerous samples.     

乙型肝炎病毒消毒指标探讨

本文选用四种微生物际本,在进行物理与化学的消毒处理后,作反相间接血凝试验、DNA-P 试验及电镜检查,通过与常规培养结果的比较,阐明其与微生物活性之间的相关及其作为消毒效果评价指标的意义。实验结果表明,微生物抗原性破坏甚为困难,且与微生物本身的耐力不相关,而微生物体内聚合酶的存在与消失与微生物本身的存亡亦无同步相关,均不宜作为消毒效果的评价指标。在致死剂量的消毒处理后,电镜下可见细菌胞壁破坏,原浆外流,病毒颗粒减少与结构模糊,可望作为消毒效果评价的有效手段。     

模式抗原破伤风毒素C蛋白的克隆、表达条件优化及免疫原性初步分析

目的 获得高纯度、高免疫原性的破伤风毒素C片段(TTC)蛋白.方法 以PCR从破伤风杆菌质粒DNA中扩增TTC基因片段,将其插入载体pET43.1a( )并导入大肠杆菌BL21(DE3)plysS中表达.用Ni2 NTA琼脂糖柱纯化融合蛋白,经凝血酶酶切,再用Ni2 -NTA琼脂糖柱分离目的 蛋白.SDS-PAGE和免疫印迹分析目的 蛋白的相对分子质量和抗原性,动物免疫实验鉴定其免疫原性.结果 获得1373 bp的TrC基因片段,构建成重组表达载体pET43.1a( )-TTC并在大肠杆菌中表达.TTC-Nus融合蛋白的表达量占菌体总蛋白的22％,酶切后TTC蛋白可与载体蛋白有效分离,获得纯度为95.5％的TTC蛋白,该蛋白可被破伤风抗毒素识别;将TTC蛋白免疫小鼠后,免疫血清可与破伤风毒素特异性结合,三次免疫后其抗体效价可达1:25 600.结论 所获TTC蛋白纯度高,免疫原性好,为研究体内外抗原提呈、免疫应答提供了良好的模式抗原.     

鸡、鸭血清中抗HBs的证明

采用生物素-抗生物素蛋白 EIA 试验证明,32.1%(18/56)的鸡和43.1%(25/58)的鸭血清中存在有乙型肝炎病毒表面抗原的抗体。虽然该抗体在RPHA 法中无中和作用,但在 EIA 法中可被已知的 HBsAg 所阻断。     

抗葡萄球菌肠毒素B单克隆抗体的免疫学特性鉴定及其应用

作者以国产纯化葡萄球菌肠毒素B(SEB)免疫BALB/c小鼠制备出6株抗SEB单克隆抗体(McAb)杂交瘤细胞系(S_1,S_2,D_1,3D_6,B_4及E_7).对其分泌的抗SEB McAb的主要免疫学特性鉴定的结果表明,6株McAb具有相同的表位特异性或构象表位相关;它们的相对亲和力由高至低依次为:S_1>D_1>3D_6>B_4>S_2>E_7;对SEB刺激外周血单个核细胞(PBMC)增殖活性的中和抑制作用不一(抑制率65.1％-22.0％).在上述鉴定的基础上,应用D_1株McAb建立的BA-ELISA及反向被动血凝试验(RPHA),检测了SEB人工污染的4种食物标本,所获敏感性分别为:0.38-0.75ng/ml及9.4-15.6ng/ml.     

Quantification of circulating schistosome antigen by monoclonal antibody-based enzyme-linked immunosorbent assay.

A two-site enzyme-linked immunosorbent assay (2S-ELISA) was developed for the quantification of gut-associated schistosome circulating anodic proteoglycan (GASCAP) of Schistosoma japonicum using a monoclonal antibody (McAb M120), purified by one-step hydroxylapatite (HAP) chromatography, as both solid phase and peroxidase-conjugated reagent. In this system, a high detectable level of serum GASCAP concentration (0.25 microgram/L) could be achieved with the immunoaffinity purified GASCAP being used as the standard. The specificity of the assay was 97.2%, with minimized interference of rheumatoid factor. The intra-assay (4.01%) and interassay (6.09%) coefficients of variation and the mean analytical recovery (101.4%) for GASCAP demonstrated the precision and accuracy of the assay.

     

三种粪便隐血试验的比较研究

粪便隐血试验是检测消化道微量出血的重要手段。本文用三种隐血试验:血红蛋白定量试验(HQT)、反向间接血球凝集试验(RPHA)和联苯胺试验(BT)对不同消化道疾病患者的粪便进行检测。结果表明隐血的检出用HQT和RPHA较BT更为特异和敏感。     

Effects of chimeric molecule of recombinant Dsg3EC1-2 with toxin PE40 on T and B lymphocytes in Pemphigus Vulgaris

Objective:To observe the effects of the recombinant chimeric toxin Dsg3EC1-2PE40 on T and B lymphocytes isolated from Pemphigus Vulgaris (PV) patients to further study its biological therapeutic function for PV. Methods :Recombinant chimeric toxin Dsg3EC1-2PE40 was first identified, expressed and purified, and then its effects on T and B lymphocytes of PV patients in vitro were detected and quantified by ELISPOT assay and MTT assay. Results :The purity of the expressed protein Dsg3EC1-2PE40 was up to 80%. In ELISPOT assay, with Dsg3EC1-2PE40, the overall number of B cells that produce anti-Dsg3 antibodies among PV patients was only about 60% of the comparable number with Dsg3EC1-2. The proliferation of T cells of PV patients was inhibited markedly by Dsg3EC1-2PE40. There was significant difference between the different groups with Dsg3EC1-2PE40 and Dsg3EC1-2. Conclusion:The recombinant chimeric toxin Dsg3EC1-2PE40 decrease the number of B cells that produce anti-Dsg3 antibodies in PV patients and can inhibit or kill T cells of PV patients in vitro.