重组人抗TNFα单抗制品中CHO宿主细胞蛋白质残留夹心ELISA检测方法的建立及验证

目的建立检测重组人抗TNFα单抗制品中CHO宿主细胞蛋白质(host cell protein,HCP)残留的夹心ELISA法,并进行验证。方法采用二维电泳-Western blot(2D-Western blot)法筛选兔抗CHO HCP多克隆抗体(产品号:AB000103-A、AB000103-C)及羊抗CHO HCP多克隆抗体(批号:3G-0016-PA)中识别重组人抗TNFα单抗特异性CHO HCP覆盖率最高的一种作为检测抗体,建立检测样品中残余HCP的夹心ELISA法,并验证方法的线性、基质干扰、稀释线性、灵敏度、精密度及准确性。结果以覆盖率达57%的AB000103-C号兔抗CHO HCP多克隆抗体作为检测抗体;样品基质干扰HCP检测,样品需稀释6倍;HCP标准品浓度在3. 33～810 ng/mL范围与A450值曲线拟合良好,R2=1,试验内及试验间精密性验证的CV均小于12%,检测限及定量限分别为2. 4和20 ng/mL;20、150及300 ng/mL的加标样品回收率分别为99. 5%、97. 1%及100. 3%。结论成功建立了重组人抗TNFα单抗HCP残留的夹心ELISA检测方法,该方法灵敏度高,准确性、精密性及线性良好,适用于重组人抗TNFα单抗制品的残留HCP检测。     

玉米赤霉烯酮BSA-ELISA检测试剂盒的制备

目的制备玉米赤霉烯酮(ZEN)生物素-链霉抗生物素ELISA(BSA-ELISA)检测试剂盒。方法应用BSA-ELISA法制备ZEN检测试剂盒,并对试剂盒进行特异性、灵敏度、精密性、准确性及稳定性检测。结果该试剂盒对玉米赤霉烯酮特异性较好;检出范围为0.0125～192.0000ng/ml;灵敏度达0.0125ng/ml;试验内变异系数为1.8%～3.1%,试验间变异系数为4.5%～8.8%;检测人工污染的香肠样品中ZEN的平均回收率为(92.00±2.71)%,检测人工污染的生理盐水样品中ZEN的平均回收率为(85.02±4.33)%;试剂盒在4℃可保存4个月,-20℃可保存6个月。结论已成功制备了特异性强、灵敏度高、精密性好、准确性高的ZENBSA-ELISA检测试剂盒。     

基于光谱自编码微球的多样品酶联免疫检测混合筛选

将苯乙烯及其同系物共聚合成具有不同特征光谱的光谱自编码微球,应用于酶联免疫检测(ELISA),AP酶标抗体筛选灵敏度达体积稀释比1∶160000。将20种编码微球与20个样品对映包被后混合,进行多样品ELISA混合筛选,分析阳性信号最强微球的光谱编码,得到了阳性反应样品的编号。成功开发了一种多样品的ELISA混合筛选技术。     

ELISA法与胶体金试纸条检测HBsAg差异分析

目的探究胶体金试纸条与ELISA法检测HBsAg临床应用价值。方法分别用胶体金试纸条与ELISA法检测360份标本中的HBsAg,并对阳性率进行比较,然后将强阳性的10份标本作倍比稀释,观察2种检测方法灵敏度的差异。结果2种方法的阳性率之间无显著差异,ELISA法灵敏度较胶体金试纸条灵敏度高。结论2种方法皆是检测HBsAg的良好方法,胶体金试纸条法操作简便、快速适用于急诊及健康人群的筛查,ELISA法适用于常规检测。     

检测M、N血型抗原的双位点一步ELISA方法的建立

目的　建立一种特异性强、灵敏度高的检测MN抗原的新方法。方法　应用抗M、抗N及抗血型糖蛋白A单克隆抗体 ,建立双位点一步ELISA法。结果　检测M、N抗原的最适pH值分别为 6 0及 7 5。适宜的反应温度为 2 0 30℃。利用表面活性剂l o n octyl β D glucopyranoside(OBG)浸泡血痕 ,可明显提高ELISA法检测陈旧血痕中N抗原的灵敏度 ,检测微量液体血和新鲜血痕的灵敏度分别为 0 .0 3μg血液和 0 .0 1～0 .0 5 μg干血。 结论　该法特异性强、灵敏度高 ,为法医血清学MN分型检验提供了一种新方法     

GC-MS测定纺织品中有机磷农残含量检测方法研究

本实验在模拟人体环境下,利用人工汗液对生态纺织品中的有机磷农药残留进行提取,用GC/MS联用进行测定,采用外标法定量。实验过程中用21种农药混标,确定实验条件后(萃取时间25 min、萃取温度为室温、吸附时间3.5 min、Na2SO4以及pH为7),进行方法重现性、线性范围和检测限试验。发现重现性好,RSD值在1.56%~24.18%之间;线性良好,其中乙拌磷线性范围10~350 ppb,乙基溴硫磷50~500 ppb,丙溴磷20~500 ppb,另外还有烯虫磷、速灭磷、马拉硫磷、喹硫磷、毒虫畏5种有机磷的线性范围是0.1~350 ppb,二嗪磷、倍硫磷2种线性范围0.1~200 ppb,甲基对硫磷、乙基对硫磷2种0.1~500 ppb,敌敌畏、三唑磷2种100~350 ppb,检测限均低于20μg/L。对实际样品(100%棉织物)进行了加标回收实验,平行测量3次的相对平均偏差在0.75%~13.92%之间,回收率在84.96%~110.91%之间,这说明SPME-GC-MSD法用于检测纺织品中有机磷农药残留,有很好的重现性和精密度,可进行纺织品中有机磷农药残留的定量检测分析。     

GC-MS测定纺织品中有机磷农残含量检测方法研究

本实验在模拟人体环境下,利用人工汗液对生态纺织品中的有机磷农药残留进行提取,用GC/MS联用进行测定,采用外标法定量。实验过程中用21种农药混标,确定实验条件后(萃取时间25 min、萃取温度为室温、吸附时间3.5 min、Na2SO4以及pH为7),进行方法重现性、线性范围和检测限试验。发现重现性好,RSD值在1.56%~24.18%之间;线性良好,其中乙拌磷线性范围10~350 ppb,乙基溴硫磷50~500 ppb,丙溴磷20~500 ppb,另外还有烯虫磷、速灭磷、马拉硫磷、喹硫磷、毒虫畏5种有机磷的线性范围是0.1~350 ppb,二嗪磷、倍硫磷2种线性范围0.1~200 ppb,甲基对硫磷、乙基对硫磷2种0.1~500 ppb,敌敌畏、三唑磷2种100~350 ppb,检测限均低于20μg/L。对实际样品(100%棉织物)进行了加标回收实验,平行测量3次的相对平均偏差在0.75%~13.92%之间,回收率在84.96%~110.91%之间,这说明SPME-GC-MSD法用于检测纺织品中有机磷农药残留,有很好的重现性和精密度,可进行纺织品中有机磷农药残留的定量检测分析。     

EV71抗原BA-ELISA检测方法的建立及应用

目的建立EV71抗原生物素-亲和素酶联免疫(BA-ELISA)检测方法,并进行验证及初步应用。方法以抗EV71抗体为包被抗体,利用生物素-亲和素系统建立双抗体夹心ELISA法,并对其灵敏度、特异性、准确性及精密性进行验证。采用该方法检测组织培养及临床样品中EV71抗原的含量。结果所建立的BA-ELISA法在EV71蛋白含量625～156.25ng/ml之间线性关系最佳,R2达0.9976,灵敏度为78.125～156.25ng;与包括CoxA16在内的42种肠道病毒及相关病毒均无交叉反应;检测10份EV71阳性样品和10份阴性样品,结果符合率均为100%;检测EV71原液和高、中浓度样品的变异系数分别为3.09%、6.69%和6.43%;可检出约103～104CCID50的活病毒及手足口病患者的大便悬液和部分咽拭子悬液中的病毒。结论用生物素标记EV71抗体与亲和素系统建立的双抗体夹心ELISA法具有较高的特异性、灵敏度、准确性和精密性,为临床检验及抗原分析提供了参考。     

甲型肝炎病毒荧光定量RT-PCR检测方法的建立及在贝类检测中的应用

目的建立甲型肝炎病毒(hepatitis A,HAV)荧光定量逆转录PCR(qRT-PCR)检测方法,用于贝类中HAV的检测。方法选择HAV高度保守的5’UTR区设计引物及探针,以HAV质粒为标准品,HAV活病毒为样本,建立HAV qRT-PCR检测方法;验证该方法的特异性、灵敏度、重复性、准确性及中间精密度;采用建立的方法检测HAV活病毒和贝类样本,并与我国贝类HAV检测国标方法(GB/T22287-2008法)进行比较。结果通过14对引物探针分别对HAV质粒(101~108拷贝/μL)检测结果的比较,选择灵敏度最优的Pan1引物,建立了HAV qRT-PCR检测方法(PAN法)。PAN法对CVA16、CVB3、EVA71、PV等11种RNA病毒均无阳性扩增;灵敏度达10 TCID50/mL;检测2.699~5.699 LgTCID50/mL各浓度HAV活病毒的回收率为82.9%~127.0%,单人和双人检测的变异系数分别为3.0%~7.0%和2.6%~8.4%。PAN法检测HAV活病毒的灵敏度(10 TCID50/mL)优于GB/T22287-2008法;两种方法对20份贝类样本的检测结果均为阴性,符合率为100%。结论建立的PAN法具有良好的特异性、灵敏度、准确性、重复性和中间精密度,可用于贝类样本中HAV的核酸定量快速检测。     

ELISA及电化学法检测藠头中氨基甲酸酯类农药残留

建立了克百威ELISA检测技术,配合电化学法对200份出口菖头样品进行氨基甲酸酯类农药残留检测.ELISA法对克百威的最低检测限达10-7g/L,检出10例,检出率5%;电化学法检测,对乙酰胆碱酯酶抑制率超过60%的样品有7例,检出率3.5%.对比分析检测结果得出2种方法检测结果基本吻合.     

Hybrid Plasticizers Enhance Specificity and Sensitivity of an Electrochemical-Based Sensor for Cadmium Detection

In addition to their use as an additive to improve physical properties of solvent polymeric membranes, plasticizers have a considerable impact on the specificity and sensitivity of membrane-modified electrochemical sensors. In this work, we aim at the hybridization of two different plasticizers using the electropolymerization technique in the development of a cadmium(II)-selective electrochemical sensor based on screen-printed gold electrode along with cyclic voltammetric measurement. At this point, we first screen for the primary plasticizer yielding the highest signal using cyclic voltammetry followed by pairing it with the secondary plasticizers giving rise to the most sensitive current response. The results show that the hybridization of DOS and TOTM with 3:1 weight ratio (~137.7-μm-thick membrane) renders a signal that is >26% higher than that from the sensor plasticized by DOS per se in water. The solution of 0.1 mM hydrochloric acid (pH 4) is the optimal supporting electrolyte. In addition, hybrid plasticizers have adequate redox capacity to induce cadmium(II) transfer from bulk solution to the membrane/water interfaces. Conversion of voltammetric signals to semi-integral currents results in linearity with cadmium(II) concentration, indicating the irreversible cadmium(II) transfer to the membrane. The DOS:TOTM hybrid sensor also exhibits high sensitivity, with a limit of detection (LOD) and limit of quantitation (LOQ) of 95 ppb and 288 ppb, respectively, as well as greater specificity towards cadmium(II) than that obtained from the single plasticizer sensor. Furthermore, recovery rates of spiked cadmium(II) in water samples were higher than 97% using the hybrid plasticizer sensor. Unprecedentedly, our work reports that the hybridization of plasticizers serves as ion-to-electron transducer that can improve the sensor performance in cadmium(II) detection.     

Rapid Spectrographic Determination of Beryllium in Biological Samples

A direct spectrochemical method for the determination of beryllium in biological samples is described. Charred samples are mixed with dilute HCl and used directly for excitation by the rotating-disc spark technique. Chemical separation of Be is eliminated, simplifying the estimation and increasing the speed at which the analysis can be performed. The limit of detection is 1 ppb of Be in urine samples, 20 ppb in lung and other tissues. The coefficient of variation ranges from 15% to 25%. This precision is sufficient when the determination is intended for rapid diagnostic purposes.     

Glassy carbon electrodes coated with poly(allylamine hydrochloride), PAH: Characterization studies and application to ion-exchange voltammetry of trace lead(II) at combined PAH/mercury film electrodes

Glassy carbon electrodes coated with adsorbed single layers of the cationic polyelectrolyte poly(allylamine hydrochloride), PAH, were produced by solvent evaporation for subsequent development of PAH modified thin mercury film electrodes (PAH/TMFE). The present work describes the preparation, incorporation features towards lead(II) species, as well as the morphological characterization of PAH coatings of different molar loadings, prepared from polyelectrolyte solutions of different composition. The present PAH films revealed interesting morphologic features, related to the process of solvent evaporation and to the specific structural properties of the PAH polyelectrolyte in the assembling medium.The novel PAH-thin mercury film electrodes, developed for the determination of trace lead(II) as its anionic forms in chloride medium, were found to be stable, sensitive and reproducible. Concentrations in the ppb concentration region could be easily assessed using 20 s accumulation time (detection limit 1.2 ppb, i.e. 6 nM, 3σ) with low relative standard deviations (<2.5%). The sensitivity of the SWASV determination of lead(II) increased 20% compared to the uncoated TMFE. The linearity range reached at least two orders of magnitude. Additionally, the PAH-coated mercury film electrodes presented an improved resistance to fouling by common surfactants.     

重组细胞中鼠细小病毒检测方法的建立及初步应用

目的建立生物技术产品用重组细胞中鼠细小病毒(Murine minute virus,MMV)污染的检测方法,并进行验证及初步应用。方法以NB324K细胞为指示细胞,建立MMV感染性检测方法,并以不同CCID50的MMV分别感染NB324K细胞,验证方法的灵敏度;通过设计针对编码非结构蛋白1(Non-structural protein 1,NS1)保守序列的引物和探针,建立用于重组细胞中MMV检测的实时荧光定量PCR,并对其线性、特异性、精密性、灵敏度、最低检测限及试验可行性和干扰性进行验证;将NB324K细胞感染试验与荧光定量PCR法相结合,建立NB324K细胞感染-PCR法,并通过对大量样本的检测,分析荧光定量PCR法和NB324K细胞感染-PCR法的可行性。结果 NB324K细胞感染试验的灵敏度为0.2 CCID50;荧光定量PCR检测方法最佳线性范围为1×108～1×104拷贝/μl,R2达0.99以上,特异性良好,与其他种属的细小病毒均无交叉反应,试验内和试验间Ct值的变异系数(CV)均小于5%,试验内病毒定量拷贝数的CV在20%～30%之间,试验间病毒定量拷贝数的CV在20%～50%之间,灵敏度为5×103拷贝/μl,最低感染性病毒颗粒检测限为2 CCID50。该方法能够用于细胞样品中MMV的检测,部分样品基质对病毒的检测具有一定的干扰性。NB324K细胞感染-PCR法的灵敏度为0.02 CCID50,检测时间可缩短为96 h,与荧光定量PCR法分别对47份样品进行检测,结果均为阴性。结论成功建立了MMV荧光定量PCR检测方法和NB324K细胞感染-PCR法,可应用于重组细胞中MMV污染的检测,为进一步提高生产用重组细胞的安全性奠定了基础。     

Immuno-strip biosensor system to detect enrofloxacin residues

The immuno-strip biosensor was developed by combination of immuno-chromatography assay and sandwich enzyme linked immunosorbent assay (ELISA) techniques to detect enrofloxacin, a widely used broad-spectrum antibiotic. A proposed analytical system, consisted of immuno-reaction step and enzyme reaction step, has several advantages such as a very cheap manufacturing cost, a short analysis time (approximately 20 min), an easy-to-use, a high sensitivity, and a qualitatively confirmation by bare eyes. Enzyme reaction step in a proposed analytical tool could enhance the signal of immunosensor, resulting in the high sensitivity. A proposed system was successfully applied to detect enrofloxacin in a range of 100–10000 ppb. The lower detection limit was 100 ppb, which is the maximum residue limit of enrofloxacin in animal product.     

Selective Extraction and Detection of Hg2+ in Aqueous Solution by using Rhodamine Dye-Modified Silica Gel

Novel silica gel-immobilized rhodamine (SGR) anchored by hydrazinium hydrate was synthesized. The selectivity and adsorption ability of the SGR for metal cations were investigated with fluorophotometry and atomic absorption spectrophotometery, respectively. The SGR exhibits high selectivity and adsorption capacity for Hg²⁺. The maximum static adsorption capacity of the SGR for Hg²⁺ was 25.8 μmol g^?1. The linear response range covers a concentration range of Hg²⁺ from 20 ppb to 2000 ppb and the detection limit is 10 ppb. The determination of Hg²⁺ in real water samples displays satisfactory results, and the SGR can be used repeatedly by treating with a solution of tetrabutylammonium hydroxide.     

Gas sensors based on thick films of semi-conducting single walled carbon nanotubes

A comparative study was made of sorted semi-conducting single walled carbon nanotube (SWCNT) films and unsorted SWCNT films for gas sensing applications. The transmission line method is used to monitor separately the SWCNTs film resistance and the contact resistance between electrodes and the SWCNTs, thus revealing that the sensing mechanism mainly relies on a modification of the tube conductivity during gas exposure. The fabricated sensors demonstrate a detection limit of 20 ppb NO₂ and 600 ppb NH₃ mainly attributed to experimental setup limitations. Moreover, semi-conducting nanotubes happened to be 2.5 times more sensitive to NH₃ than unsorted ones, thus proving that selectivity can be improved by sorting the SWCNTs. The temperature dependence of the sensor sensitivity was studied, and a good agreement was found between experimental results and the Langmuir adsorption model.     

Improvement of sensitivity in an interferometry by controlling pore size on the anodic aluminum oxide chip pore-widening technique

The pore size of an anodic aluminum oxide (AAO) chip, as well as uniform pore distribution, is one of the key parameters that should be adjusted, by choosing the appropriate etching conditions, in order to enhance the sensitivity of an interferometer. In this study, the pore size of AAO chips was optimized and characterized in order to lower the detection limit of prostate specific antigen (PSA) in an interferometric immunoassay system. After pore widening for 30–50 min, the AAO pore size was increased approximately 2-fold larger than that before pore widening. A large increase in effective optical density (ΔEOT) was obtained from the AAO chip fabricated by pore-widening technique, which thereby lowered the PSA detection limit. The present study results are not sufficiently validated to enable the immediate application to immunoassay for prostate cancer (PCa) screening, but they do demonstrate that controlling pore size can positively affect the sensitivity and lower the detection limit.     

Determination of the total iodide content in desalinated seawater permeate

An investigation was conducted to determine the iodide content of permeate collected from several operating facilities reliant upon synthetic membrane processes for seawater desalination. A possible, yet unintentional impact for communities that employ synthetic membrane processes for seawater desalination is the introduction of permeate streams containing iodide into their water supply, that then may result in the formation of iodinated disinfection by-products. To evaluate this potential, the iodide content of desalinated seawater permeate streams were measured using an analytical procedure based on the catalytic reduction of ceric sulfate by arsenious acid in a sulfuric acid solution. It was determined that iodide concentrations in permeate samples collected from seawater desalination facilities were less than the catalytic reduction method detection limit of 4.0 μg/L for membrane feed seawaters that ranged between 51.1 μg/L and 35.8 μg/L of total iodide. Results of this investigation indicated that synthetic membrane processes can remove greater than 89% of the total iodide from the feedwater of seawater based on an iodide detection limit of 4.0 μg/L.