聚丁二炔纳米粒快速检测日本血吸虫**☆◆

背景：采用PCR、ELISA、免疫荧光球法及基因芯片技术进行日本血吸虫的快速检测,需要数小时才能得到结果,并且对日本血吸虫有量的要求,还需配备专门人员、一定的仪器设备,故非常不适用于现场快速检验。 目的：制备聚丁二炔纳米粒,并对日本血吸虫进行免疫检测。 方法：采用超声分散法制备聚丁二炔纳米粒,并利用其检测日本血吸虫。 结果与结论：制备的聚丁二炔纳米粒粒径均匀,分布范围窄,结构稳定。取血吸虫感染兔血清稀释液滴入聚丁二炔免疫纳米粒稀释液中,溶液颜色由蓝变红,颜色变化约20 s时,体系有70%~80%发生了颜色变化,在约2 min体系颜色变化即基本完成。吸收光谱、透射电镜、激光粒径分布均显示,加入血吸虫感染兔血清后聚丁二炔免疫纳米粒变化很大。表明利用聚丁二炔免疫纳米粒比色检测日本血吸虫操作简便、快速。 关键词：聚丁二炔;纳米粒;PCDA;日本血吸虫;生物材料与纳米技术 doi:10.3969/j.issn.1673-8225.2012.08.007     

产超广谱β-内酰胺酶大肠埃希氏菌临床分离株药敏分析

目的了解郑州地区临床分离大肠埃希氏菌产超广谱β-内酰胺酶情况和耐药特征。方法双纸片法筛选及确证实验检测超广谱β-内酰胺酶(ESBLs),琼脂二倍稀释法对大肠埃希氏菌进行MIC检测。结果产ESBLs大肠埃希氏菌检出率为57.4%,产ESBLs菌株多数呈多重耐药,仅亚胺培南的耐药率在10%以下。结论本地区临床分离大肠埃希氏菌具有较高的产ESBLs流行率,对于产ESBLs大肠埃希氏菌应以亚胺培南为首选药物。     

化学发光磁酶免疫法检测O157:H7大肠埃希菌

目的：建立灵敏稳定检测O157：H7大肠埃希菌的化学发光磁酶免疫法.方法：利用AMPPD-ALP化学发光体系,通过磁珠酶联免疫法对O157：H7大肠埃希菌进行检测.结果：其检测灵敏度达到850个,线性范围为1 000～50 000个,批间变异小于15%,批内变异小于20%,与人工计数培养检测相比相关系数达0.9807.结论：化学发光磁酶免疫分析法操作简便,检测精密度和灵敏度高,是一种很有发展前景的可靠方法.     

人博卡病毒VP2蛋白单克隆抗体的制备

目的表达纯化人博卡病毒（human Bocavirus,HBoV）VP2蛋白,采用杂交瘤方法制备抗HBoVVP2蛋白的单克隆抗体。方法应用原核表达载体pET-30a和大肠埃希菌表达VP2蛋白,并用固定化金属亲和层析,纯化后的蛋白作为抗原免疫BALB／c小鼠,利用杂交瘤技术和间接ELASA方法筛选阳性的杂交瘤细胞,并对单克隆抗体进行分型检测和滴度测定。结果表达并纯化获得了重组HBoVVP2蛋白,利用杂交瘤技术得到单克隆IgG抗体,抗体效价达到1：4×10^5。结论利用HBoVVP2蛋白免疫制备了单克隆抗体,并具有较高的效价。本研究为快速诊断和研究HBoV打下基础。     

Nono蛋白的原核表达、纯化和多克隆抗体的制备

目的表达和纯化带多聚组氨酸(6×His)标签的Nono(non-POU-domain-containing,octamer-bindingprotein)融合蛋白并制备抗Nono多克隆抗体。方法构建pET-28a( )-Nono重组表达质粒,转入Rosetta(DE3)大肠埃希菌,以IPTG诱导6×His-Nono融合蛋白表达,经镍离子金属螯合树脂纯化后,用纯化出的蛋白免疫BALB/C小鼠制备多克隆抗体,并用ELISA检测多克隆抗体的效价,Western印迹检测多克隆抗体的特异性。结果在大肠埃希菌中诱导出高水平表达的His-Nono融合蛋白,经亲和树脂纯化后免疫小鼠,获得了高特异性的抗Nono抗血清。结论成功构建pET-28a( )-Nono原核表达质粒,表达并纯化出高纯度的目标蛋白,制备出高滴度、高特异性的多克隆抗体。     

化学发光磁酶免疫法检测O157∶H7大肠埃希菌

目的建立灵敏稳定检测O157∶H7大肠埃希菌的化学发光磁酶免疫法。方法利用AMPPD-ALP化学发光体系,通过磁珠酶联免疫法对O157∶H7大肠埃希菌进行检测。结果其检测灵敏度达到850个,线性范围为1000~50000个,批间变异小于15%,批内变异小于20%,与人工计数培养检测相比相关系数达0.9807。结论化学发光磁酶免疫分析法操作简便,检测精密度和灵敏度高,是一种很有发展前景的可靠方法。     

柯萨奇病毒B组5型非结构蛋白3C表达纯化及多克隆抗体的制备

目的 表达和纯化柯萨奇病毒(CVB5)非结构蛋白3C,免疫雄性大鼠制备多克隆抗体.方法 通过酶切质粒获取3C基因序列,构建到原核表达系统大肠埃希菌中诱导表达重组的3C蛋白,使用镍柱亲和层析法纯化重组蛋白,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和考马斯亮蓝染色法(BSA)对重组蛋白的纯度与浓度进行分析,重组的3C蛋白作为抗原,免疫雄性SPF级大鼠获得多抗血清,用ELISA测定抗体效价并Western印迹检测抗体特异性,用该抗体检测病毒在细胞内的复制.结果 构建了重组表达载体命名为3C-pET-28a,并在大肠埃希菌BL21(DE3)中诱导表达重组3C蛋白.ELISA测定制备的多抗血清效价为1:500 000,Western印迹检测在EV71、CVA16中都有交叉反应,并确定病毒感染细胞后24 h开始复制.结论 实验成功地利用原核表达系统表达柯萨奇病毒非结构蛋白3C,为进一步研究柯萨奇病毒的感染机理以及疫苗制备提供了基础.     

携带抗人表皮生长因子受体2与阿霉素的靶向药物载体构建及其抗肿瘤效应

目的 构建携带抗人表皮生长因子受体2(HER2)与阿霉素的靶向药物载体,并探讨具体外抗肿瘤效应.方法 以复乳法制备包载阿霉素的纳米粒,检测其外观形态、粒径分布、Zeta电位和体外释药情况.以耦联剂将阿霉素纳米粒与人源化抗HER2抗体Trastuzumab(Herceptin)耦联成免疫纳米粒,ELISA法检测其免疫活性,噻唑蓝法(MTT)检测其对高表达HER2的肿瘤细胞SKBR3的抗肿瘤效应.结果 构建的阿霉素纳米粒呈球形或类球形,平均粒径为( 198.2±12.4) nm,Zeta电位为-41mV,包封率为68.6％,体外96h药物释放量达50％.ELISA检测显示免疫纳米粒对肿瘤细胞SKBR3及SKOV3具有免疫活性,而对MCF-7几乎无免疫活性反应.SKBR3的细胞存活率比较显示,免疫纳米粒对细胞的抑制作用分别明显优于阿霉素纳米粒和阿霉素(均P＜0.05).结论 免疫纳米粒具有免疫性和靶向性,可有效抑制高表达特异抗原HER2肿瘤细胞的生长.     

mPEG-CS纳米粒介导livin shRNA基因纳米复合物的制备及转染效率

背景：作为非病毒基因转染载体,由可降解的高聚物形成的纳米载体目前被广泛由于基因转染,因为他们具有良好的缓释性,靶向性和生物相容性。 目的：制备mPEG-CS纳米粒,探讨mPEG-CS作为Livin shRNA基因转染载体的可行性。 方法：通过离子交联法制备mPEG-CS纳米粒,利用聚乙二醇对壳聚糖进行改性,通过静电吸附法制备载livin shRNA的基因纳米复合物。Zeta-size分析仪和透射电镜检测空白纳米粒和载livin shRNA的基因纳米复合物的形态、粒径和zeta电位,测定基因纳米复合物的包封率,凝胶电泳阻滞实验和DNase I酶消化实验验证纳米粒对基因的保护作用。利用最佳条件下制备的基因纳米复合物,转染大肠癌HT-29细胞,考察转染效率。 结果及结论：成功制备出约60 nm的mPEG-CS纳米粒,当纳米粒与基因体积比为3∶1时,得到的基因纳米复合物形较规则,粒径100 nm左右;其包封率为(94.32±0.35)%。凝胶电泳阻滞实验表明纳米粒能够紧密结合DNA,对基因具有良好的基因保护作用。该基因纳米复合物转染大肠癌细胞的转染效率高,持续作用时间长。mPEG-CS纳米粒作为基因转染载体,对基因具有保护作用,能够将livin shRNA重组质粒高效转染入大肠癌细胞,能够在大肠癌细胞内长时间表达,克服了RNA干扰在基因治疗肿瘤中基因作用时间较短的缺点。     

人星状病毒非结构蛋白nsP1a／1表达纯化及多克隆抗体的制备

目的：表达纯化人星状体病毒（ human astrovirus, HAstV）非结构蛋白nsP1a／1,免疫动物制备多克隆抗体。方法利用PCR技术扩增nsP1a／1基因序列,构建到大肠埃希菌原核表达系统中表达重组nsP1a／1蛋白,使用镍柱亲和层析法对重组蛋白进行纯化,十二烷基磺酸钠?聚丙烯酰胺凝胶电泳（ SDS?PAGE）和二噻啉甲酸（BCA）实验对重组蛋白的纯度与浓度进行分析,以重组的nsP1a／1蛋白为抗原,免疫雄性SPF级SD 大鼠获得多抗血清,用 ELISA 测定抗体效价、 Western 印迹检测抗体特异性。结果nsP1a／1?pET28a原核表达载体构建成功,将其转化至大肠埃希菌BL21（DE3）细菌中诱导表达了重组蛋白,免疫大鼠获得的多抗血清几何平均效价达到1∶406374。结论本实验成功地运用原核表达系统表达并鉴定了人星状体病毒非结构蛋白nsP1a／1,为进一步研究人星状病毒的复制及病毒感染的临床诊断奠定基础。     

Detection of Escherichia coli O157:H7 using chicken immunoglobulin Y

A sandwich ELISA technique was examined to detect Escherichia coli O157:H7 using chicken anti-E. coli O157:H7 IgY as the capture-antibody and an anti-E. coli O157 mouse mAb conjugated with biotin as the detection antibody. The anti-E. coli O157:H7 IgY was harvested from eggs laid by hens (23 weeks of age, Single Comb White Leghorn) immunized with formalin-killed E. coli O157:H7. The IgY was purified by water dilution methods and gel chromatography on Sephacryl S-300 followed by ammonium sulfate precipitation. The sensitivity (CFU/ml) of sandwich ELISA for the E. coli O157:H7 was repeatedly examined with 10 replicates of each sample and a standard curve was plotted. The sandwich ELISA can detect as low as 40CFU/ml of E. coli O157:H7. The data suggest that chicken IgY-based sandwich ELISA provides a reliable, inexpensive and sensitive assay for the detection of the food-borne pathogen E. coli O157:H7.     

Goldmag-based enzyme-linked immunosorbent assay for determination of α-lactalbumin in milk

In this work, a goldmag-based enzyme-linked immunosorbent assay was developed for determination of α-lactalbumin (α-LA) in milk. The magnetic nanoparticle functionalized with polyetherimide was synthesized by one-pot method and coated with two layers of gold nanoparticles on the surface to synthesize goldmag nanoparticles. Anti-α-LA monoclonal antibody, prepared by hybridoma cell lines via cell fusion, was then bound to this goldmag nanoparticle to develop a capture nanoprobe. The results showed that this developed immunoassay had a good linear range of 2.33–127.1?ng/mL with IC₅₀ of 17.2?ng/mL. Besides, recovery rates for α-LA in four commercial milks were from 86.7% to 109.8%. The coefficients of variation in intra-assay and inter-assay were 3.9–6.8% and 5.5–9.8%, separately, which could meet the requirements to quantify α-LA content in milk. This goldmag-based immunoassay might have considerable potentials in the detection of food allergies.     

Fluorescent nanoparticle-based indirect immunofluorescence microscopy for detection of Mycobacterium tuberculosis

A method of fluorescent nanoparticle-based indirect immunofluorescence microscopy (FNP-IIFM) was developed for the rapid detection of Mycobacterium tuberculosis. An anti-Mycobacterium tuberculosis antibody was used as primary antibody to recognize Mycobacterium tuberculosis, and then an antibody binding protein (Protein A) labeled with Tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate (RuBpy)-doped silica nanoparticles was used to generate fluorescent signal for microscopic examination. Prior to the detection, Protein A was immobilized on RuBpy-doped silica nanoparticles with a coverage of approximately 5.1 x 10(2) molecules/nanoparticle. With this method, Mycobacterium tuberculosis in bacterial mixture as well as in spiked sputum was detected. The use of the fluorescent nanoparticles reveals amplified signal intensity and higher photostability than the direct use of conventional fluorescent dye as label. Our preliminary studies have demonstrated the potential application of the FNP-IIFM method for rapid detection of Mycobacterium tuberculosis in clinical samples.     

Use of monoclonal antibodies to probe subunit- and polymer-specific epitopes of 987P fimbriae of Escherichia coli.

The relationship between the structure and biological function of 987P fimbriae of a strain of enterotoxigenic Escherichia coli (O9:K103:H-) from piglets was investigated. A set of four monoclonal antibodies was prepared from the spleen cells of mice immunized with isolated 987P fimbriae. Antibodies E11, D5, and C3, but not G10, reacted in enzyme-linked immunosorbent assays with 987P fimbriae-bearing E. coli. Electron microscopy showed that E11 and D5 reacted in a discrete periodic pattern forming a spiral motif along the length of the fimbriae. The results of enzyme-linked immunosorbent assays were in agreement with these results; antibodies E11 and D5 reacted at a high dilution (1:12,000) with native fimbriae on the surface of E. coli, whereas antibody C3 reacted at an intermediate dilution (1:3,000) and G10 failed to react at all (less than 1:250). In contrast, C3 and G10 reacted at a dilution of 1:3,276,000 with the fimbrial subunits derived by treating the isolated fimbriae with 6 M guanidine hydrochloride, whereas E11 and D5 reacted with the subunits at much lower dilutions of 1:800 and 1:6,400, respectively. Moreover, fimbriae reassembled from the subunits regained reactivity with antibodies D5 and E11, indicating that these antibodies are directed against quaternary conformational epitopes. Only the three antibodies (D5, E11, and C3) that recognized epitopes accessible on intact fimbriae were able to efficiently block the adhesion of 987P fimbriated E. coli to piglet enterocytes. These results indicate that certain epitopes of 987P fimbriae are dependent on quaternary structural conformation, whereas others are present on monomeric subunits; some of the latter appear to remain accessible on fully assembled fimbriae.     

Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate for one-step immunodetection in molecular hybridization.

Using phage-display technology, a recombinant single-chain Fv antibody fragment (scFv) was rapidly generated from the K16-16 hybridoma secreting mouse monoclonal antibody (MAb) that binds to acetylaminofluorene-labeled DNA (AAF-DNA). The selected A4 phage-scFv specifically bound to AAF-DNA. The anti-AAF scFv gene was then recloned into a fusion vector for the production of a hybrid protein comprising the antibody fragment fused to a potent bacterial alkaline phosphatase variant (PhoAv). The anti-AAF scFv-PhoAv hybrid protein was bifunctional and possessed both antigen binding capacity and PhoA activity. The recombinant conjugate was directly used, without further purification, for one-step immunodetection in dot-blot hybridization. The detection limit was identical and the test was quicker than the conventional two-step procedure with the purified anti-AAF MAb revealed with a secondary enzyme-labeled antibody. To assess the value of this new reagent for the immunodetection of genomic nucleic acids, genomic DNAs of Campylobacter jejuni and Campylobacter coli were then one-step immunodetected with non-purified recombinant scFv-PhoAv conjugate in a Southern-blot hybridization experiment. The present study shows that the genetic fusion with PhoAv provides a new tool for immunodetection which presents easier and quicker production and use with the same sensitivity and specificity as classical reagents. The recombinant anti-AAF scFv-PhoAv conjugate is a promising alternative reagent for applications involving the immunodetection of specific DNA or RNA sequences, such as the detection and characterization of microorganisms.     

Production and characterization of a monoclonal antibody specific for enterohemorrhagic Escherichia coli of serotypes O157:H7 and O26:H11

A monoclonal antibody (MAb 4E8C12) specific for Escherichia coli O157:H7 and O26:H11 was produced by immunizing BALB/c mice with a rough strain of E. coli O157:H7. The antibody reacted strongly by a direct enzyme-linked immunosorbent assay with each of 36 strains of E. coli O157:H7. No cross-reactivity was observed with strains of Salmonella spp., Yersinia enterocolitica, Shigella dysenteriae, Proteus spp., Escherichia hermanii, Klebsiella pneumoniae, Campylobacter jejuni, Serratia marcescens, Citrobacter spp., Enterobacter cloacae, Hafnia alvei, Aeromonas hydrophila, and all except five strains of E. coli other than serotype O157:H7 (including strains of serotype O157 but not H7). The E. coli strains (all of serotype O26:H11) that reacted with the antibody were enterohemorrhagic E. coli (EHEC) that were isolated from patients with hemolytic uremic syndrome or hemorrhagic colitis and produced verotoxin similar to that of E. coli O157:H7. MAb 4E8C12 belongs to the subclass immunoglobulin G2a and has a kappa light chain. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins of E. coli of different serotypes followed by Western immunoblot analysis revealed that MAb 4E8C12 reacted specifically with two proteins of EHEC strains of serotypes O157:H7 and O26:H11 with apparent molecular weights of 5,000 to 6,000. These proteins appeared to be markers specific for EHEC strains of serotypes O157:H7 and O26:H11. This MAb, because of its specificity, may be a useful reagent of an immunoassay for the rapid detection of these types of EHEC isolates in clinical and food specimens.     

Monoclonal antibodies against Escherichia coli heat-stable toxin (STa) and their use in a diagnostic ST ganglioside GM1-enzyme-linked immunosorbent assay.

Seven monoclonal antibodies (MAbs) against heat-stable enterotoxin (ST) from a human Escherichia coli isolate were prepared and evaluated for their usefulness in an ST immunodetection assay, the ST ganglioside GM1-enzyme-linked immunosorbent assay (ELISA). This assay is based on the ability of STa, as present in, for example, culture filtrates from ST-producing E. coli, to inhibit specific anti-ST antibody from binding to solid-phase-bound ST ganglioside (GM1-bound ST-cholera B subunit). Four of the MAbs were of immunoglobulin G1 (IgG1), one was of IgG2b, and two were of IgM isotype. All the IgG1 MAbs could be completely inhibited by addition of free ST; 0.2 to 0.4 ng of purified ST inhibited binding of these MAbs by 50%. The non-IgG1 MAbs were, in contrast, not inhibited by 200-fold-higher amounts of purified ST, probably because they were directed against linkage epitopes or were of low affinity or both. When the IgG1 MAbs were tested in the ST GM1-ELISA, ST could be detected in culture filtrates from stock human E. coli isolates with 100% sensitivity and specificity. ST in filtrates from fresh stool cultures was demonstrated with higher sensitivity with the MAbs ST GM1-ELISA than with the conventional infant mouse test. Both subtypes of STa, STaI and STaII, could be detected by the ST GM1-ELISA by using either IgG1 MAb in the immunodetection step, whereas infant-mouse-active ST from Yersinia enterocolitica failed to react.     

Development of a nanoparticle-labeled microfluidic immunoassay for detection of pathogenic microorganisms

The light-scattering properties of submicroscopic metal particles ranging from 40 to 120 nm in diameter have recently been investigated. These particles scatter incident white light to generate monochromatic light, which can be seen either by the naked eye or by dark-field microscopy. The nanoparticles are well suited for detection in microchannel-based immunoassays. The goal of the present study was to detect Helicobacter pylori- and Escherichia coli O157:H7-specific antigens with biotinylated polyclonal antibodies. Gold particles (diameter, 80 nm) functionalized with a secondary antibiotin antibody were then used as the readout. A dark-field stereomicroscope was used for particle visualization in poly(dimethylsiloxane) microchannels. A colorimetric quantification scheme was developed for the detection of the visual color changes resulting from immune reactions in the microchannels. The microchannel immunoassays reliably detected H. pylori and E. coli O157:H7 antigens in quantities on the order of 10 ng, which provides a sensitivity of detection comparable to those of conventional dot blot assays. In addition, the nanoparticles within the microchannels can be stored for at least 8 months without a loss of signal intensity. This strategy provides a means for the detection of nanoparticles in microchannels without the use of sophisticated equipment. In addition, the approach has the potential for use for further miniaturization of immunoassays and can be used for long-term archiving of immunoassays.     

Immunodetection of Canine Parvovirus (CPV) in clinical samples by polyclonal antisera against CPV-VP2 protein expressed in Esherichia coli as an antigen

The entire virion protein 2 (VP2) gene of Canine Parvovirus (CPV) was amplified by polymerase chain reaction (PCR) and engineered to be expressed by a bacterial expression vector pET-28a, under the control of the IPTG-inducible T7lac promoter. SDS-PAGE gel revealed that VP2 expressed as a 67kDa, and found mainly in the pellet of the bacterial lysates, suggesting that cytoplasmic expression is not preferred. The recombinant protein VP2 fused with His-tag was purified from Esherichia coli using Ni-NTA resin under denaturing conditions. SDS-PAGE analysis also showed the high expression of several lower molecular weight (LMW) bands. Western blot analysis showed that polyclonal antisera produced by rabbit against E. coli-VP2 protein reacted specifically with the purified VP2 protein as well as two other LMW bands. Some of the resulting LMW products failed to keep their antigenic site in the N-terminal region of the VP2. The degradation of recombinant VP2 protein in E. coli could be due to the action of host proteases. The immunodetection ability of the polyclonal antisera was compared with that of a commercial monoclonal antibody to test numerous clinical specimens by immuno-dot blot assays. There were distinctive differences in the degree of immunodetection ability of polyclonal antisera and monoclonal antibody to react with CPV antigens. The reaction time of polyclonal antisera was much faster in visual color appearance than that of monoclonal antibody during NBT/BCIP staining. The result from diagnostic PCR assay confirmed the presence of CPV in 44 out of 46 specimens collected, consistent with polyclonal antisera-positive result. Therefore, the polyclonal antisera can be used for CPV detection in the faeces of diarrhoeic dogs, which was found to be more rapid, sensitive, broad but less specific than the monoclonal antibody.