基于荧光淬灭克百威流式快速检测方法的建立及表征

目的制备满足流式分析要求的聚苯乙烯微球,进行量子点荧光编码,建立克百威流式快速检测方法,并对荧光编码微球性能进行表征。方法采用无皂乳液聚合法和分散聚合法制备聚苯乙烯微球,通过溶剂挥发法对其进行量子点荧光编码,借助扫描电镜、能谱分析和红外分析对微球进行考察,利用流式细胞仪检测并记录微球的平均荧光强度。结果在0.2~1.0 mg·L-1克百威水溶液中,随着克百威质量浓度的增加,量子点荧光编码聚苯乙烯(polystyrene labeled with quantum dots,QDs@PS)微球的荧光值逐渐下降。结论基于荧光淬灭原理,初步建立了一种水体中克百威残留量的快速、高通量检测方法。     

流式微球分析技术联合检测血清MMP-9、MPO CD40L和t-PA的方法学建立及评价

目的 建立流式微球分析技术联合检测人血清中基质金属蛋白酶-9 (MMP-9)、髓过氧化物酶(MPO)、CD40配体(CD40L)、血浆纤溶酶原激活物(t-PA)的方法并对其进行系统性评价.方法 分别将四种选择好一定浓度的捕获抗体(MMP-9、MPO、CD40L和t-PA鼠抗人单克隆抗体)包被在四种已激活的不同荧光强度编码的羧基化聚苯乙烯微球上,用棋盘法对试验条件进行优化选择,并应用CLSI的有关规则进行方法学评价.结果 反应体系中四种鼠抗人单克隆抗体最佳加入量为10 μg,最佳生物素标记抗体浓度为1∶4000倍稀释,最佳反应时间为2h,亲和素最适孵育时间为1h.MMP-9线性范围是0.20～333.33 ng/mL,批内变异系数为4.60％～8.80％,批间变异系数为8.80％～9.60％,准确度相对偏倚为2.80％～3.18％,回收率是94.8～102.50％,灵敏度为0.20 ng/mL;MPO线性范围是0.26～111.11ng/mL,批内变异系数为5.90％～7.70％,批间变异系数为9.10％～11.40％,准确度相对偏倚为1.44％～4.12％,回收率是97.8～103.2％,灵敏度为0.26 ng/mL;CD40L线性范围是0.32～111.11g/mL,批内变异系数为5.40％～6.50％,批间变异系数为8.90％～12.40％,准确度相对偏倚为2.72％～5.95％,回收率是97.20～105.30％,灵敏度为0.32 ng/mL;t-PA线性范围是1.21～111.11 ng/mL,批内变异系数为2.20％～2.90％,批间变异系数为6.30％～12.20％,准确度相对偏倚为1.23％～4.60％,回收率是97.20～101.30％,灵敏度为1.21 ng/mL.高浓度的甘油三酯、胆固醇和胆红素对四种因子有一定的干扰率,低浓度的甘油三酯、胆固醇和胆红素对三种因子干扰较小.分别与ELISA方法比较无显著差异.结论 自建的MMP-9、MPO、CD40L和t-PA1流式微球联合检测技术,可拓展流式细胞分析技术,值得临床推广使用.     

测定从生物降解微球中抽提的破伤风类毒素的方法(英)

用聚丙交酯／乙交酯共聚物制成的微球是单剂破伤风类毒素（TT）菌苗的一种新释放系统。由于微球中包被的TT含量很低，所以为准确测定微球中抗原TT的含量，作者筛选了一种既能识别中和位点又能与TT发生交叉反应的单克隆抗体（McAb）TT010作为捕获抗体，建立了一种敏感度为0.001絮状反应单位（Lf）／ml的夹心 ELISA法。     

利用表面等离子共振技术检测牛奶中庆大霉素残留方法的建立及验证

目的 建立一种基于表面等离子共振(SPR)技术快速测定牛奶中庆大霉素残留的方法。方法 采用表面SPR技术并利用抗体抗原反应的高特异性特点,通过将庆大霉素-BSA偶联蛋白固定于S系列CM5芯片表面,利用竞争性免疫原理,建立了牛奶中庆大霉素残留的方法并对该方法的准确度、重复性、专属性、检测范围等参数进行考察。结果 本实验室建立的牛奶中庆大霉素残留定量方法准确度高(平均回收率为102.60%,RSD值为9.58%)、重复性好(庆大霉素对照品3.2、12.8、51.2ng/mL高中低三个浓度分析重复性所得RSD分别为4.56%、4.83%、3.84%),中间精密度高(RSD值为4.65%),庆大霉素在0.8～102.4ng/mL的范围内,仪器响应值(Ru)与庆大霉素浓度呈现良好的回归关系。结论 本研究建立的方法可用于牛奶中庆大霉素残留的检测。     

免疫层析法测定动物源性食品中磺胺嘧啶残留

建立了动物源性食品中磺胺嘧啶残留的免疫层析快速测定方法。用胶体金标记磺胺嘧啶单克隆抗体作为显色剂，磺胺嘧啶竞争物包被于硝酸纤维素层析膜上作为捕获试剂，采用竞争反应模式制成胶体金免疫层析试纸。该免疫层析试纸可以在15min内完成对磺胺嘧啶残留的半定量检测。读条系统判定灵敏度为5ng／ml，肉眼判定检测限灵敏度为10ng／ml，与其它磺胺类药物无交叉反应。该方法在不同动物源性食品（鸡肉、鸡蛋、鸡肝、猪肉、猪肝、牛奶、蜂蜜）中添加磺胺嘧啶的回收率在68．1％-118．8％范围内。动物试验样品检测结果表明，该方法与ELISA及HPLC具有好的符合率。该方法灵敏度高，检测准确、简便、快速，有良好的开发应用前景。     

应用ELISA测定睫状神经营养因子中卡那霉素残留量

目的:建立ELISA检测卡那霉素残留量的方法并进行方法学验证,用于重组技术产品的质量控制。方法:采用间接竞争ELISA方法,样本中残留的卡那霉素将和微孔板上预包被的偶联抗原竞争抗卡那霉素抗体,加入酶标二抗后,用3,3,5,5-四甲基联苯胺(TMB)底物显色,样本吸光值与残留卡那霉素的含量成负相关,用酶标仪进行检测并用SOFT MAX分析软件进行分析。结果:ELISA方法测定卡那霉素残留量在0.5~40.5 ng.mL-1范围内线性良好,且符合四参数方程式:y=(A-D)/[1+(X/C)B]+D,r2≥0.99;板内变异小于15%,板间变异小于20%,实验重复性好,加标回收率在80%~120%之间;该方法检测与庆大霉素、盐酸四环素、链霉素和氨苄西林药物未显示交叉反应。应用该法对1批睫状神经营养因子(CNTF)原液的卡那霉素残留量进行测定,结果表明,每人用剂量CNTF(100μg)卡那霉素残留量小于0.5 ng。结论:该方法具有灵敏度高、操作简便、检测迅速等特点,可用于重组技术产品中卡那霉素残留量的检定。     

多聚赖氨酸预处理ELISA(PL-ELISA)检测抗DNA抗体

抗DNA抗体的检查对于活动期特别是合并肾炎的SLE的诊断有重要意义,目前常用检测方法有放免法和以短膜虫为抗原片的间接荧光抗体法,都由于试剂供应和需专门仪器尚未普遍推广。用DNA直接包被聚苯乙烯反应板作ELISA测定,由于DNA难于吸附,迄今未被广泛接受。我们用多聚赖氨酸预处理反应板,包被DNA后用间接ELISA法检测抗DNA抗体,取得较满意结果,现报告如下。材料与方法一、包被用DNA DNA(上海牛奶公司综合厂)     

QuEChERS-SPE-UPLC-MS/MS法快速测定水产品中氯霉素

水产品养殖中违法违规使用禁用药物氯霉素,可导致水产品中氯霉素残留,对人体健康造成危害.本文建立了一种QuEChERS-固相萃取-液质联用法(QuEChERS-SPE-UPLC-MS/MS)测定水产品中氯霉素的方法.经过方法学考察,氯霉素在0.2893～11.57 ng/ml范围内线性关系良好,r2为0.9995,检测限...     

种球溶胀法制备分子印迹聚合物并对其吸附性能进行研究

目的采用种球溶胀法制备毒死蜱、阿特拉津和克百威的分子印迹聚合物并对其吸附性能进行研究。方法采用分散聚合法制备微米级聚苯乙烯单分散微球,以聚苯乙烯微球为种球,采用种球溶胀法制备毒死蜱、阿特拉津和克百威3种分子印迹聚合物微球,用扫描电子显微镜观察微球的形貌,恒温振荡实验考察微球的亲和性和选择性。结果吸附实验证明制备的印迹微球具有高亲和性和高选择性,可用于检测果蔬中的毒死蜱、阿特拉津和克百威。果蔬中印迹微球的吸附时间为2 h,吸附率在71.84%⁸7.2%内,相对标准偏差在2.34%87.2%内,相对标准偏差在2.34%⁴.52%内。结论种球溶胀法制备的分子印迹聚合物可以用于果蔬中农药残留的快速检测。     

对用于包被冠状动脉支架的抗CD34单克隆抗体的生物学指标的质量控制研究

目的:对鼠源抗CD34单克隆抗体的结合活性以及包被在冠状动脉支架上的抗体量和牢固度等技术指标进行评价。方法:采用流式细胞术测定抗CD34单克隆抗体结合KG-1 a细胞的活性;酶联免疫显色方法测定支架上抗体包被量;体外模拟冲刷方法测定支架上抗体牢固度;采用显微镜计数方法测定包被抗CD34抗体支架体外捕获CD34(+)细胞的能力。结果:在抗CD34 M cAb浓度为1μg.mL-1时,细胞平均阳性率为96.34%;支架上抗体含量为50.97 ng.cm-2,而且体外模拟冲刷后支架上抗体含量无显著变化;包被抗CD34单抗的冠状动脉支架捕获细胞数为4万个.cm-2。结论:该方法可用于体外检测抗CD34单克隆抗体包被冠状动脉支架的生物学参数。     

Novel 3‐Substituted Ocotillol‐Type Triterpenoid Derivatives as Antibacterial Candidates

Plant‐derived triterpenoid saponins are involved in the plant defense system by targeting bacterial membranes. A series of ocotillol‐type triterpenoid derivatives were synthesized starting from PPD, one of the main components of Panax ginseng and their antibacterial activity against several representative bacteria were evaluated. Compounds 5 and 11 exhibited excellent antibacterial activity with MIC values of 1 μg/mL against Staphylococcus aureus and 8 μg/mL and 4 μg/mL against Bacillus subtilis, respectively. Furthermore, when compounds 5 and 11 were combined with two commercial antibiotics kanamycin and chloramphenicol, they showed strong synergistic activity at sub‐MIC levels against S. aureus USA300 and B. subtilis 168. Moreover, chloramphenicol turned from a bacteriostatic to a bactericidal agent when combined with compound 11 against B. subtilis 168.     

An ultrasensitive chemiluminescence immunoassay of chloramphenicol based on gold nanoparticles and magnetic beads

A competitive, direct, chemiluminescent immunoassay based on a magnetic beads (MBs) separation and gold nanoparticles (AuNPs) labelling technique to detect chloramphenicol (CAP) has been developed. Horseradish peroxidase (HRP)‐labelled anti‐CAP monoclonal antibody conjugated with AuNPs and antigen‐immobilized MBs were prepared. After optimization parameters of immunocomplex MBs, the IC₅₀ values of chemiluminescence magnetic nanoparticles immunoassay (CL‐MBs‐nano‐immunoassay) were 0.017 µg L^?1 for extract method I and 0.17 µg L^?1 for extract method II. The immunoassay with two extract methods was applied to detect CAP in milk. Comparison of these two extract methods showed that extract method I was advantageous in better sensitivity, in which the sensitivity was 10 times compared to that of extract method II, while extract method II was superior in simple operation, suitable for high throughout screen. The recoveries were 86.7–98.0% (extract method I) and 80.0–103.0% (extract method II), and the coefficients of variation (CVs) were all <15%. The satisfactory recovery with both extract methods and high correlation with traditional ELISA kit in milk system confirmed that the immunomagnetic assay based on AuNPs exhibited promising potential in rapid field screening for trace CAP analysis. Copyright © 2013 John Wiley & Sons, Ltd.     

Assessment of antibacterial drug residues in milk for consumption in Kosovo

The objective of this study was to assess the occurrence of drug residues in the raw milk collected from individual farms and milk collection points during 2009–2010 in six different major regions of Kosovo (Prishtinë, Gjilan, Mitrovicë, Pejë, Gjakovë, Prizren). In the present study, a total of 1734 raw milk samples were collected, and qualitatively screened with two different tests, the Delvotest SP assay and an enzyme-linked receptor-binding assay (SNAP). Overall, 106 (6.11%) out of 1734 samples examined with Delvotest SP contained possible drug residues (5.12% and 7.51% of samples from 2009 and 2010, respectively). All suspect samples were further analyzed by three distinct enzyme-linked receptor-binding assays specific for β-lactams (new β-lactam test), tetracyclines (SNAP tetracycline test), and sulfonamides (SNAP sulfamethazine test). Only the new SNAP β-lactam test detected residues in 40 out of 52 samples in 2009 and 54 out of 54 suspect samples in 2010. A confirmatory method based on liquid chromatography-tandem mass spectrometry was used to confirm the presence of β-lactam drug residues in samples detected by the enzyme-linked receptor-binding assay. Amoxicillin, penicillin G, and cloxacillin were the most frequently detected residues and were in a concentration range between 2.1 μg/kg and 1973 μg/kg. Seventeen of the positive samples exceeded the maximum residue levels for one or more β-lactam drug. The highest number of positive milk samples came from the Pejë Region (58.8%) and Gjakovë Region (23.5%), and the lowest number of positive samples originated from Gjilan (5.88%), with no positive samples detected in two regions, Mitrovicë and Prizren.     

Morning glory resin glycosides as modulators of antibiotic activity in multidrug-resistant gram-negative bacteria

Twenty-six microbiologically inactive (MIC >?512?μg/mL) convolvulaceous resin glycosides ( 1- 26) were tested for resistance modulatory activity in vitro against Escherichia coli Rosetta-gami and two nosocomial pathogens, Salmonella typhi and Shigella flexneri. These compounds exerted a potentiation effect of the clinically useful antibiotics tetracycline, kanamycin, and chloramphenicol against the tested gram-negative bacteria by increasing antibiotic susceptibility up to 32-fold at concentrations of 25?μg/mL. Therefore, the oligosaccharides from the morning glory family (Convolvulaceae) represent metabolites that reverse microbial resistance mechanisms, favoring an increase in the strength and effectiveness of current antibiotics that are not effective in the treatment of refractive infections caused by multidrug-resistant strains.     

HPLC-CAD法测定硫酸卡那霉素及注射液含量及有关物质

目的建立测定硫酸卡那霉素原料及其注射液中有关物质与卡那霉素含量的高效液相色谱-电喷雾检测器(HPLCCAD)方法。方法采用Waters HSS T3色谱柱(4.6mm×250mm,5μm),以0.2 mol/L三氟乙酸水溶液为流动相,流速0.3m L/min,柱温30℃,电喷雾检测器的喷雾器温度为35℃。结果新建方法对卡那霉素与硫酸盐、有关物质分离良好,精密度、重复性、回收率均满足分析要求。卡那霉素浓度与峰面积线性关系良好(R2>0.99),检出限可达到6.1ng。采用该方法对单硫酸卡那霉素、硫酸卡那霉素以及硫酸卡那霉素注射液进行了测定,检出的有关物质个数及含量均高于现行标准的结果。结论新建方法灵敏度更高、分离度更好,能检出更多杂质,可以满足硫酸卡那霉素原料及注射液有关物质及含量测定分析要求。     

Effect of digoxin fab antibody on the measurement of total and free digitoxin by fluorescence polarization and a new chemiluminescent immunoassay

Digoxin fab antibody (Digibind; Burroughs Wellcome, Research Triangle Park, NC, USA) is used in the treatment of digoxin overdose. The effect of digibind on the measurement of total and free digoxin has been extensively studied. However, the effect of digibind on digitoxin measurements has not been studied thoroughly. The authors studied the effect of digibind on the measurement of total and free digitoxin in vitro using the fluorescence polarization immunoassay and a new chemiluminescent immunoassay. We also studied the capability of digibind to bind digitoxigenin, the major aglycon metabolite of digitoxin. Digibind neutralized both digitoxin and digitoxigenin in vitro, as evidenced by significant reductions in free digitoxin and digitoxigenin (measured as digitoxin equivalent) concentrations. Digibind caused negative interference in the measurement of total digitoxin concentrations by both fluorescence polarization and chemiluminescent assays. However, the magnitude of negative interference was significantly higher with the chemiluminescent assay. For example, in a serum pool supplemented with 80 ng/mL of digitoxin, the concentrations of total and free digitoxin measured by the fluorescence polarization immunoassay were 82.1 ng/mL and 3.3 ng/mL respectively. In the presence of 5 microg/mL of Digibind, the corresponding total and free digitoxin concentrations were 73.9 ng/mL and none detected, respectively. In another serum pool supplemented with 70 ng/mL of digitoxin, the concentrations of total and free digitoxin as measured by the chemiluminescent assay were 69.1 ng/mL and 3.8 ng/mL, respectively. In the presence of 5 microg/mL of Digibind, the corresponding total and free digitoxin concentrations were 29.0 ng/mL and none detected, respectively. Because this effect may also occur in vivo, the progress of Digibind therapy in treating a patient with digitoxin overdose may be monitored by measuring the free digitoxin concentrations.     

The use of hair as a long‐term indicator of low‐dose β2 agonist treatments in cattle: Implications for growth‐promoting purposes monitoring

The objective of this study was to assess the feasibility of using hair as a long‐term indicator of cocktail (low‐dose β₂ agonists) treatments in cattle. Six male Simmental cattle were treated with a mixture of low‐dose clenbuterol, ractopamine, and salbutamol at dosages of 5.3, 223.3, and 50.0 μg/kg, respectively. The trial lasted for 112 days and included 28 days of treatment and 84 days of withdrawal. Plasma and urine samples taken during the treatment period contained the highest residues, with maximum concentrations of clenbuterol, ractopamine, and salbutamol in plasma of 1.49 ng/mL (Day 21), 43.78 (Day 14) ng/mL, and 8.07 ng/mL (Day 7), respectively, and in urine of 62.40 ng/mL (Day 28), 3995.77 ng/mL (Day 28), and 503.72 ng/mL (Day 1), respectively. On day 42 of withdrawal, the residues of all three β₂ agonists in plasma were below the limit of quantification (LOQ; 0.3 ng/mL for clenbuterol, and 0.5 ng/mL for ractopamine and salbutamol), and in urine samples were below or near the LOQ (the highest being ractopamine at 1.10 ng/mL). The highest concentrations of clenbuterol, ractopamine, and salbutamol in hair were 88.36, 1351.92, and 100.58 ng/g, respectively, on day 14 of withdrawal; and the residues were long‐lasting, with 7.64, 28.55, and 8.77 ng/g, respectively, on day 84 of withdrawal. The results of this study demonstrate that hair could be utilized as a long‐term indicator of the use of a combination of low‐dose β₂ agonists in cattle, which could have implications for growth‐promoting purposes monitoring.     

Detection of fumonisin b1 and ochratoxin a in grain products using microsphere-based fluid array immunoassays

Grain products are a staple of diets worldwide and therefore, the ability to accurately and efficiently detect foodborne contaminants such as mycotoxins is of importance to everyone. Here we describe an indirect competitive fluid array fluoroimmunoassay to quantify the mycotoxins, fumonisin B1 and ochratoxin A. Both toxins were immobilized to the surface of microspheres using a variety of intermediate molecules and binding of biotinylated "tracer" antibody tracers determined through flow cytometry using streptavidin-phycoerythrin conjugates and the Luminex100 flow cytometer. Competitive assays were developed where the binding of biotinylated monoclonal antibodies to fumonisin B and ochratoxin A was competitively inhibited by different concentrations of those toxins in solution. Concentrations of fumonisin giving 50% inhibition were 300 pg/mL in buffer, 100 ng/g in spiked oats, and 1 μg/g in spiked cornmeal; analogous concentrations for ochratoxin A were 30 ng/mL in buffer, 30 ng/g in spiked oats, and 10 ng/g in spiked corn. The future challenge will be to expand the number of mycotoxins tested both individually and in multiplexed format using this platform.     

Buprenorphine and norbuprenorphine concentrations in human breast milk samples determined by liquid chromatography-tandem mass spectrometry

Buprenorphine (BUP) is considered to be safe during pregnancy. However, the extent of BUP transfer into breast milk has not been investigated thoroughly. Because the drug concentration in the milk is 1 of the determinants in the assessment of the exposure risk, a rapid and sensitive LC-MS/MS method has been developed and evaluated to measure BUP and norbuprenorphine (norBUP) concentrations in milk. A solid-phase and 2 liquid-liquid extraction procedures have been compared. The lower limits of detection and quantification were 0.05 ng/mL and 0.18 ng/mL for BUP and 0.05 ng/mL and 0.20 ng/mL for norBUP, respectively, using a sample volume of 0.5 mL milk. BUP and norBUP concentrations determined from 10 random breast milk samples collected over 4 successive days from a lactating woman during buprenorphine maintenance therapy ranged from 1.0 to 14.7 and 0.6 to 6.3 ng/mL, respectively. Drug exposure of the infant may be considered to be low. Further investigations may seek to extend these preliminary findings to evaluate an infant's level of BUP exposure through breast milk.     

血清CA125、CEA与卵巢癌的相关性分析

目的检测血清中糖类抗原CA125和癌胚抗原（CEA）两种肿瘤标志物的浓度,分析CA125和CEA联合检测在卵巢癌诊断中的临床价值。方法采用全自动化学发光免疫分析法测定23例卵巢癌、37例良性卵巢肿瘤和50例健康体检者外周血清中CA125和CEA的水平,采用t检验进行数据统计分析。结果卵巢癌患者组中CA125和CEA的水平及阳性率分别是（278.43±182.27）U/mL与73.92%、（8.49±8.37）ng/mL与17.4%,明显高于良性卵巢肿瘤组的（23.75±13.59）U/mL与18.92%、（3.43±1.48）ng/mL与0.0%及健康对照组的（12.46±7.65）U/mL与0.0%、（2.21±1.03）ng/mL与0.0%,且差异均有统计学意义（P〈0.05）。结论血清CA125与卵巢癌的相关性较理想,CEA与卵巢癌的相关性较差;血清CA125是卵巢癌筛查比较可靠、理想的肿瘤标志物,有较好的诊断价值;而CEA在卵巢癌中也有一定的参考价值,但其阳性率较低。因此CA125和CEA联合检测对卵巢癌的诊断无明显临床价值。