检测禽流感病毒的压电免疫芯片两种抗体固定方法的研究

目的探讨构建检测禽流感病毒压电免疫芯片的2种抗体固定方法。方法以AT切型、基频10 MHz的石英晶体为材料,分别采用聚乙烯亚胺-戊二醛交联法（简称戊二醛法）、葡萄球菌A蛋白（SPA）法将禽流感病毒H5亚型单克隆抗体固定在石英晶体的金膜电极表面,构建检测禽流感病毒压电免疫芯片。结果戊二醛法构建的压电免疫芯片检测H5抗原的线性范围为稀释度1∶2~1∶40,Y=75.814-13.748X,r2=0.978 9,与H9抗原不存在交叉反应,与鸡胚分离法的符合率达到91.7%。SPA法若先固定抗体,则无法检测到抗原;如先进行抗原抗体反应再与SPA结合,可以达到定性检测的效果。结论戊二醛法适合于构建禽流感病毒压电免疫芯片,其检测线性满足检测要求;SPA法在检测抗原时存在局限性,尚不能直接检测病毒抗原。     

压电免疫芯片检测禽流感病毒H9亚型抗原的方法研究

目的 为用压电免疫芯片检测禽流感病毒抗原寻找一种可行的抗体固定方法.方法 以AT切型、基频10 mHz的石英晶体为材料,分别采用聚乙烯亚胺-戊二醛交联法、葡萄球菌A蛋白法将禽流感病毒H9亚型单克隆抗体固定在石英晶体的金膜电极表面构建禽流感压电免疫芯片,用于检测禽流感病毒H9抗原.结果 ①聚乙烯亚胺-戊二醛交联法构建的压电免疫芯片检测禽流感病毒H9抗原的线性范围为稀释度1∶10～1∶160,Y=-8.868X+73.514,r2=0.966 6,与H5抗原不存在交叉反应,与鸡胚分离法的符合率达到91.7%.②葡萄球菌A蛋白法若先固定抗体,则无法检测到抗原;如先进行抗原抗体反应再与葡萄球菌A蛋白结合,可以达到定性检测的效果.结论 聚乙烯亚胺-戊二醛交联法适合于构建禽流感病毒H9亚型抗原压电免疫芯片,其检测特异度、重复性和线性均满足检测要求;葡萄球菌A蛋白法在检测抗原时存在局限性,尚不能直接检测抗原.     

基于压电石英晶体传感器质量效应研制的肌钙蛋白Ⅰ检测压电免疫芯片

以AT切型、基频13MHz的石英晶体为材料,设计四通道芯片微阵列。采用双功能基团交联剂Sulfo-LC-SPDP将肌钙蛋白I单克隆抗体巯基化,固定于石英晶体的金膜电极表面制备生物敏感膜,构建一种新型、快速的检测肌钙蛋白I的压电免疫芯片。结果显示该芯片用于检测肌钙蛋白I的线性范围为1～20μg/L,与酶联荧光分析法具有较好的相关性。其灵敏度高、特异性好,可用于定性检测肌钙蛋白I,具有一定的临床应用价值。     

基于压电石英晶体传感器质量效应研制的肌钙蛋白I检测压电免疫芯片

以AT切犁、基频13 MHz的石英晶体为材料,设计阴通道芯片微阵列.采用双功能基团交联剂Sulfo-LC-SPDP将肌钙蛋白I单克隆抗体巯基化,固定于石英晶体的金膜电极表面制备生物敏感膜,构建一种新型、快速的检测肌钙蛋白I的压电免疫芯片.结果显示该芯片用于检测肌钙蛋白I的线性范围为1～20 μ g/L,与酶联荧光分析法具有较好的相关性.其灵敏度高、特异性好,可用于定性检测肌钙蛋白I,具有一定的临床应用价值.     

压电石英晶体免疫传感器在前列腺特异抗原检测的实验研究

目的构建一种检测前列腺特异抗原(PSA)的新型压电石英晶体免疫传感器.方法采用AT切型、基频10 MHz的金膜电极石英晶体用金属夹具和乳胶套圈固定形成检测池,抗人PSA单克隆抗体采用巯基化方法固定在金膜电极表面制成抗体识别膜,构成压电PSA免疫传感器,同时对56例临床血清标本进行检测,并与化学发光免疫分析法进行比较.结果构建的压电PSA免疫传感器对PSA的响应特性良好,其线性检测范围为1.25～50 ng/ml,批内、批间变异系数(CV)分别小于4.0%和6.0%,与化学发光免疫分析法具有良好的相关性(r=0.95,P>0.05),癌胚抗原(CEA)、甲种胎儿球蛋白(AFP)、人绒毛膜促性腺激素(hCG)等对PSA的检测基本无干扰;传感器可再生后重复使用5次.结论压电PSA免疫传感器具有灵敏度高、特异性好、不需标记、操作简单、省时、能实时在线检测和重复使用等优点,是一种检测PSA的新方法,适用于临床检测,有推广价值.     

四通道骨唾液蛋白压电免疫传感器阵列的研制

背景:骨唾液蛋白是细胞外基质中的一种高度磷酸化和糖基化的分泌性蛋白,其血清表达水平的检测在评估骨代谢水平方面具有重要的临床检测价值.目的:以AT切型、10Hz双面镀金的石英晶体为换能器,拟制作一种4通道压电蛋白芯片阵列,用于检验临床血清中骨唾液蛋白的表达水平.设计、时间及地点:观察实验,于2008-12/2009-07在解放军广州军区广州总医院医学实验科生物传感器实验室完成.材料:实验所用压电免疫传感器包括包被过的石英晶体、振荡器和频率计等.骨质疏松症患者血清(n=10)、正常人血清(n=9),参考标准血清由解放军广州军区广州总医院检验科、血液中心提供.小牛血清、胎牛血清为Hydone公司产品.方法:石英晶体的厚度切片模式采用AT切割,10MHz,大小为8.00mmx8.00mmx0.15mm,在石英晶片的双面采用真空镀膜的方法形成金电极,该阵列由弹簧支架以及4个石英晶体组成.采用双功能试剂Sulfo-LC-SPDP将鼠抗人骨唾液蛋白单克隆抗体巯基化,固定于石英晶体的金电极表面,通过免疫反应检测血清中骨唾液蛋白的表达.主要观察指标:不同抗体浓度与抗体固定量之间的关系,临床患者血清与正常人血清之间骨唾液蛋白表达的差异.结果:①随着抗体浓度的增加,石英晶体频率响应值也随之增大.实验以80mg/L为抗体固定浓度,检测稀释度为1:4的参考标准血清,结果显示传感器阵列检测的最佳pH值条件在7.4左右.对稀释度分别为1:9, 1:8,1:7,1:6,1:5,1:4,1:3,1:2的参考标准血清进行检测,结果显示在血清稀释度1:9-1:2的范围内,构建的骨唾液蛋白传感器阵列对血清检测的响应呈较好的线性关系,线性方程为△F =521.54c-46.548,相关系数为0.9703.②骨质疏松症患者血清中骨唾液蛋白的表达水平明显高于正常人血清(1=5.702,P<0.05).结论:实验研制的骨唾液蛋白压电免疫传感器阵列具有结构简单、稳定性好、灵敏度高、特异性好等优点,可用于骨唾液蛋白的定量检测.     

梅毒快速检测压电免疫传感阵列的研制

目的研究压电免疫传感器阵列用于梅毒快速检测的可行性。方法采用吸附-交联法将梅毒基因工程重组抗原固定在镀金石英晶体表面,以5%聚乙二醇6000作为反应加速剂,运用以高效空气过滤器、空气干燥器和流量控制电路组成的洁净空气条件下的动态检测技术,检测90份临床血液样本,并与ELISA法检测结果比较。结果压电免疫传感阵列快速检测与ELISA检测比较,梅毒抗体的阳性、阴性血清的检出符合率分别为90%(27/30)和100%(60/60),压电免疫传感阵列在4℃空气密封袋中至少可保存3周性能不变。结论免疫传感阵列检测梅毒螺旋体抗体,具有灵敏度高、特异性好,检测速度快等特点,为野外作业下的献血者血液快速筛查分析提供了一种新的选择。     

建立微量空斑减少方法测定人血清H7N9禽流感病毒中和抗体水平

目的 建立微量空斑减少的方法测定人血清中H7N9禽流感病毒中和抗体水平。方法 确定H7N9禽流感病毒感染MDCK细胞后形成空斑数目,进行病毒定量,系列稀释待测抗体,加入已定量病毒,计算病毒被抗体中和后空斑减少数目,确定中和抗体的滴度。实验中以H7N9病例血清测定该方法的灵敏度,以其他亚型流感病毒抗体阳性人群血清评估微量空斑减少方法的特异性,同时将该方法与微量中和实验进行比较分析。结果 微量空斑减少方法不仅能够检测H7N9禽流感病例中和抗体水平,而且该方法检测季节性流感病毒抗体、H5N1和H9N2禽流感病毒抗体与H7N9禽流感病毒抗体之间均无交叉反应,与微量中和实验检测结果相一致。结论 建立的微量空斑减少方法具有较好的灵敏度和特异性,能够用于人血清H7N9禽流感病毒中和抗体的检测。     

中国职业暴露人群感染H9N2禽流感病毒血清学调查

目的 评估我国职业暴露人群感染H9N2禽流感病毒的风险因素. 方法 利用2009-2011年间在我国22个省(直辖市、自治区)采集的职业暴露人群血清13 715份,筛选了在我国主要流行的G9系毒株A/Chicken/AK4/Anhui/2011作为检测抗原,使用血凝抑制方法初筛和微量中和方法复核两种血清抗体检测方法,在我国大陆地区开展系统的H9N2禽流感病毒血清学研究. 结果 H9N2禽流感病毒在中国职业暴露人群的中和抗体血清阳性率为0.41%(63份阳性血清标本).所有家禽职业暴露人群均有阳性血清标本检出,活禽市场暴露人群感染H9N2禽流感病毒风险显著高于其他职业暴露人群. 结论 活禽市场是人感染禽流感病毒的重要暴露风险因素.加强H9N2禽流感病毒在我国职业暴露人群的监测,对于有效评估和防控禽流感对人类的危害具有重要的意义.     

用于RhD抗原流式细胞检测的固定透化方法评价

目的寻找具有回收率高且适于RhD抗原荧光检测的红细胞固定透化方法。方法分别采用4%甲醛、4%多聚甲醛、2%戊二醛对红细胞进行固定,同时采用4%甲醛-Triton X-100、4%多聚甲醛-Triton X-100、2%戊二醛-Triton X-100对红细胞进行透化固定,未处理红细胞作为对照。冰上固定20 min,洗涤后计算红细胞回收率。在各组红细胞中加入1∶128的RhD Ig G单克隆抗体,同时设组内空白对照,37℃孵育30 min。PBS洗涤3次后加入1∶200的FITC荧光抗体,37℃孵育30 min,PBS洗涤3次后流式细胞仪检测。结果 2%戊二醛及2%戊二醛-Triton X-100 2组的回收率分别为72.83%与72.80%,高于对照组(58.32%),甲醛及多聚甲醛2组的回收率在4.00%左右,远低于对照组。流式检测结果显示,不同固定剂的荧光本底均高于未处理红细胞。与对照组相比甲醛与多聚甲醛会减弱RhD抗原荧光强度,而戊二醛不影响RhD荧光强度,戊二醛-Triton X-100可增强RhD抗原检测的灵敏度。结论2%戊二醛-Triton X-100固定透化法不仅可得到较高的红细胞回收率,且RhD荧光检测的灵敏度也得到提升,是荧光检测RhD抗原较理想的固定透化方法。     

Determination of creatine kinase isoenzyme MB activity in serum using immunological inhibition of creatine kinase M subunit activity. Activity kinetics and diagnostic significance in myocardial infarction.

This is a new method for the determination of creatine kinase isoenzyme MB activity in serum. The method uses direct activity measurement of creatine kinase B subunit activity after blocking of CK-M subunit activity by inhibiting antibodies. The test takes no longer than 15 min. The method yields an intra-serial C.V. of 2.0-12.9%, and a C.V. from day to day of 5.5%. The detection limit is 3.4 U/l creatine kinase MB. In the 95 cases with proven myocardial infarction several types of creatine kinase MB activity kinetics could be determined. The percentage of creatine kinase MB of peak CK-total is 6-25%, with a mean of 11.1%. The amount of creatine kinase MB with respect to total CK activity after reinfarction is higher than the amount after initial infarction.     

Dissociable correlates of two classes of retrieval processing in prefrontal cortex

Although substantial evidence suggests that the prefrontal cortex (PFC) implements processes that are critical for accurate episodic memory judgments, the specific roles of different PFC subregions remain unclear. Here, we used event-related functional magnetic resonance imaging to distinguish between prefrontal activity related to operations that (1) influence processing of retrieval cues based on current task demands, or (2) are involved in monitoring the outputs of retrieval. Fourteen participants studied auditory words spoken by a male or female speaker and completed memory tests in which the stimuli were unstudied foil words and studied words spoken by either the same speaker at study, or the alternate speaker. On "general" test trials, participants were to determine whether each word was studied, regardless of the voice of the speaker, whereas on "specific" test trials, participants were to additionally distinguish between studied words that were spoken in the same voice or a different voice at study. Thus, on specific test trials, participants were explicitly required to attend to voice information in order to evaluate each test item. Anterior (right BA 10), dorsolateral prefrontal (right BA 46), and inferior frontal (bilateral BA 47/12) regions were more active during specific than during general trials. Activation in anterior and dorsolateral PFC was enhanced during specific test trials even in response to unstudied items, suggesting that activation in these regions was related to the differential processing of retrieval cues in the two tasks. In contrast, differences between specific and general test trials in inferior frontal regions (bilateral BA 47/12) were seen only for studied items, suggesting a role for these regions in post-retrieval monitoring processes. Results from this study are consistent with the idea that different PFC subregions implement distinct, but complementary processes that collectively support accurate episodic memory judgments.     

俯卧位通气对高海拔地区肺复张治疗无效急性呼吸窘迫综合征患者氧合的影响

目的 探讨俯卧位通气对高海拔地区肺复张术(RM)治疗无效急性呼吸窘迫综合征(ARDS)患者的治疗作用.方法 从海拔2260m的地区医院筛选RM治疗无效的41例ARDS患者[平均氧合指数( PaO2/FiO2)较RM前升高＜20％视为RM无效],依不同病因分为肺内源性ARDS组(ARDSp组)和肺外源性ARDS组(ARDSexp组),每组再按信封法随机分为俯卧位组和仰卧位组,即ARDSp俯卧位组(11例)、ARDSp仰卧位组(9例)、ARDSexp俯卧位组(10例)、ARDSexp仰卧位组(11例).在通气前及通气1、2、3、4h监测动脉血氧分压( PaO2)、PaO2/FiO2、静态顺应性(Cst)、气道阻力(Raw)的变化.结果 通气lh时,ARDSexp俯卧位组PaO2/FiO2( mm Hg,l mm Hg=0.133 kPa)即较通气前显著升高(157.4±40.6比129.3±48.7,P＜0.05),并随通气时间延长呈持续增高趋势,4h达峰值(219.1 ±41.1);且ARDSexp俯卧位组通气3h内PaO2/FiO2较其他3组显著增高,另3组间则差异无统计学意义.ARDSp俯卧位组、ARDSexp俯卧位组通气4h时PaO2/FiO2均较相应仰卧位组显著增高(208.8±39.7比127.4±47.1,219.1±41.1比124.9±50.8,均P＜0.05).4组通气前后Cst无显著改变,各组间差异也无统计学意义.ARDSp俯卧位组通气4h时Raw(cmH2O·L-1·s-1)较通气前显著降低(6.8±1.7比10.7±1.8,P＜0.05),且明显低于其他3组;其他3组各时间点Raw组内及组间比较差异均无统计学意义.结论 俯卧位通气作为ARDS机械通气重要策略之一,可以改善RM无效高原ARDS患者的氧合,为抢救患者赢得宝贵的时间.     

Digital imaging among medical facilities

The Department of Veterans Affairs (VA) in the USA operates a network of 172 medical centres which all utilize a hospital information system (HIS) which has been developed and is currently maintained by the VA. During the past several years, an image management and communication module has been developed, installed and clinically utilized at the Washington DC and Maryland VA Medical Centres. This image management and communication system, referred to as the decentralized hospital computer program (DHCP) imaging system, is fully integrated with a commercial picture archiving and communication system (PACS). The system is utilized to capture, archive, and display all images generated within the hospital including radiology, nuclear medicine, pathology, endoscopy, bronchoscopy, and dermatology, intraoperative photographs, ECG data, and a limited number of paper documents. The ultimate goal of the project is to have all patient text and image data available at any clinical workstation to any authorized user anywhere within the network of medical centres. Clinical requirements for an imaging workstation include ease of use, rapid and reliable access to the complete set of patient information, and images which are of acceptable quality to meet the requirements of the user and the subspecialty. Patient confidentiality and data security must be safeguarded at all times. Integration of the images with the remainder of the patient's database was found to be critical to the success of the project. The experience at the Washington and Maryland facilities suggests that an imaging system that is successfully integrated with a hospital information system can provide substantial clinical and economic benefits both within and among medical centres. Clinical acceptance and utilization of the system has been excellent, particularly in diagnostic radiology where DHCP Imaging has been interfaced to a commercial PAC system. Based upon this initial experience, the VA has begun to deploy the system throughout its large network of medical centres.     

Myocardial elastography at both high temporal and spatial resolution for the detection of infarcts

Myocardial elastography is a novel method for noninvasively assessing regional myocardial function, with the advantages of high spatial and temporal resolution and high signal-to-noise ratio (SNR). In this paper, in-vivo experiments were performed in anesthetized normal and infarcted mice (one day after left anterior descending coronary artery [LAD] ligation) using a high-resolution (30 MHz) ultrasound system (Vevo 770, VisualSonics Inc., Toronto, ON, Canada). Radiofrequency (RF) signals of the left ventricle (LV) in longitudinal (long-axis) view and the associated electrocardiogram (ECG) were simultaneously acquired. Using a retrospective ECG gating technique, 2-D full field-of-view RF frames were acquired at an extremely high frame rate (8 kHz) that resulted in high-quality incremental displacement and strain estimation of the myocardium. The incremental results were further accumulated to obtain the cumulative displacements and strains. Two-dimensional and M-mode displacement images and strain images (elastograms), as well as displacement and strain profiles as a function of time, were compared between normal and infarcted mice. Incremental results clearly depicted cardiac events including LV contraction, LV relaxation and isovolumetric phases in both normal and infarcted mice, and also evidently indicated reduced motion and deformation in the infarcted myocardium. The elastograms indicated that the infarcted regions underwent thinning during systole rather than thickening, as in the normal case. The cumulative elastograms were found to have higher elastographic SNR (SNR(e)) than the incremental elastograms (e.g., 10.6 vs. 4.7 in a normal myocardium, and 6.0 vs. 2.4 in an infarcted myocardium). Finally, preliminary statistical results from nine normal (m = 9) and seven infarcted (n = 7) mice indicated the capability of the cumulative strain in differentiating infracted from normal myocardia. In conclusion, myocardial elastography could provide regional strain information at simultaneously high temporal (>/=0.125 ms) and spatial ( approximately 55 microm) resolution as well as high precision ( approximately 0.05 microm displacement). This technique was thus capable of accurately characterizing normal myocardial function throughout an entire cardiac cycle, at the same high resolution, and detecting and localizing myocardial infarction in vivo.     

手转胎头术失败的原因与分娩结局临床分析

目的 探讨手转胎头术失败的原因与分娩结局.方法 选择2008年1月至2010年12月于我院住院分娩的持续性枕横位、枕后位产妇198例,根据行手转胎头术后结果分为成功组126例、失败组72例.比较两组分娩结局,对比分析失败原因.结果 失败组胎儿体质量≥3500 g的发生率[76.4％(55/72)]明显高于成功组[31.7％(40/126)],差异有统计学意义(x2=30.177,P=0.001)、失败组宫缩乏力发生率[58.3％(42/72)]高于成功组[38.1％ (48/126)],差异有统计学意义(x2=7.569,P=0.006)、失败组骨盆临界或轻度狭窄发生率[38.9％ (28/72)]高于成功组[23.8％(30/126)],差异有统计学意义(x2 =5.030,P=0.002)、失败组手转胎头时机不当(宫口开大＜6 cm、胎头位于坐骨棘上及宫口开大8～10 cm、胎头位于坐骨棘下≥2 cm)发生率[61.1％(44/72)]高于成功组[38.9％(49/126)],差异有统计学意义(x2=9.084,P=0.003).失败组母儿并发症(产后出血、产褥病率、胎儿窘迫、新生儿窒息)发生率高于成功组(x2 =9.586,P=0.002、x2=9.334,P=0.002、x2=5.910,P=0.015、x2=5.240,P=0.022)、失败组剖宫产发生率[72.2％(52/72)]明显高于成功组[34.1 ％(43/126),x2=26.641,P=0.001)].结论 手转胎头术能使难产变顺产,降低剖宫产率,减少母儿并发症,但须积极预防、处理导致手转胎头术失败的原因,对矫正失败后继续矫正及试产应慎重.