生物样品中蓖麻毒素的双夹心酶联免疫定量检测方法及应用

目的建立定量检测生物样品中蓖麻毒素的双夹心ELISA方法并研究毒素在大鼠体内的分布。方法利用抗蓖麻毒素的单克隆抗体（MAb）3D74和辣根过氧化物酶（HRP）标记的小鼠抗蓖麻毒素单克隆抗体4C13,建立了定量检测血清和组织样品中蓖麻毒素的双夹心ELISA方法,并对方法学进行了必要的验证。结果建立的双抗夹心ELlSA方法检测纽织中蓖麻毒素的浓度范围为1．25～320-g／ml,最低定量限为2．5ngy／ml,日内和日间精密度分另Ij小于3．5％和9％。将该方法应用于蓖麻毒素组织分布的定量测定,结果显示,大鼠静脉染毒后毒素可以在心、肝、脾、肺、肾、肠和脂肪等组织分布,以脾脏的分布为最高,其次为肝脏,染毒后0．5和2h脾与血清的比值分别为3．9和7．2。在脑和肌肉中未检出毒素。结论建立的双夹心ELISA方法简便、灵敏,可以用于定量检测血清和组织等生物样品中的毒素含量,为蓖麻毒素的毒物代谢动力学研究及毒素中毒的快速检测提供了技术支撑。     

基于镧系高敏荧光的生物毒素现场快速检测方法的研究

目的 利用时间分辨荧光技术建立一种可快速、准确地对蓖麻毒素、A型肉毒毒素及产气荚膜梭菌ε毒素进行定量检测的方法.方法 首先合成了一种可见光激发的镧系荧光微球,通过比较不同粒径的铕离子荧光微球,筛选出最佳粒径的微球偶联标记效价最高的捕获抗体,分别与相应检测抗体平行组合,确定最优的配伍组合条件建立检测方法.其次,对该法的敏感性、精密性、定量能力和特异性进行评价.结果 所建立的时间分辨荧光免疫层析法(TRFIC)可在20 min内完成对3种生物毒素的定性/定量检测.3种毒素的最低检出限均可达0.5 ng/ml,定量范围为0.5～50 ng/ml,3种毒素间及与黄曲霉毒素B1、T-2毒素均无交叉反应,批内重复性和批间重复性变异系数均小于15％.结论 该研究建立了一种基于可见光激发的TRFIC,在生物毒素的检测中获得了较高的最低检测限,与配套的小型便携式检测仪器一起使用,具有检测快速、可远程传输结果的优势,未来在生物反恐、生物武器核查等领域具有较高的应用价值和应用前景.     

HPLC法测定放射增敏剂苯并三嗪衍生物YABQ含量及有关物质

目的：建立测定放射增敏剂YABQ含量及有关物质的高效液相色谱检测方法。方法采用Diamonsil C18（250 mm ×4．6 mm,5μm）色谱柱;流动相为甲醇－0．1％甲酸（22∶78）等度洗脱;流速1．0 ml／min;检测波长266 nm;柱温30℃;进样体积10μl。结果运用上述色谱方法进行检测,YABQ在7～84μg／ml的范围内线性关系良好（r＝0．9992）;该化合物检测限响应值为8 ng（信噪比S／N≥3）,定量限响应值为24 ng（信噪比S／N≥10）;经破坏性试验,各色谱峰间分离度良好（＞1．5）,说明该色谱法满足试验要求。结论经方法学验证及破坏性试验,该法精密度高,专属性良好,可用于YABQ及其有关物质的质量控制。     

HPLC法测定甲氧氯普胺鼻腔喷雾剂的含量和有关物质

目的：建立测定甲氧氯普胺（ MCP）鼻腔喷雾剂含量和有关物质的HPLC检测方法。方法采用Agilent TC－C18柱（250 mm ×4．6 mm,5μm）,流动相为乙腈－磷酸盐缓冲液（取0．05 mol／L磷酸二氢钾溶液,加三乙胺5 ml,用磷酸调至pH 4．0）（19∶81）,检测波长为275 nm,柱温30℃,流速为1．0 ml／min。结果与结论在建立的色谱条件下,MCP与有关物质峰基线分离,MCP浓度在10～200μg／ml 范围内线性关系良好,r＝0．9999,回收率为100．3％～101．6％, RSD为0．68％（n＝9）,MCP检出限0．3 ng。该方法准确、可靠,重复性较好,可作为MCP鼻腔喷雾剂的质量控制方法。     

直肠指检和经直肠超声检查对血清前列腺特异抗原≤4ng／ml的前列腺癌的诊断价值

目的探讨直肠指检（digitalrectalexamination,DRE）和经直肠超声检查（transrectalultrasonography,TRUS）对血清前列腺特异抗原（prostatespecificantigen,PSA）≤4ng／ml的前列腺癌的诊断价值。方法对112例血清PSA≤4ng／ml而DRE或（和）TRUS异常的男性患者进行了TRUS引导的前列腺系统10针＋可疑区穿刺活检。根据血清PSA范围将病例分成0～0．9ng／ml,1．0～1．9ng／ml,2．0—2．9ng／ml和3．0～4．0ng／ml4组。结果112例病例中有14例被诊断为前列腺癌,检出率为12．5％。4组中：检出前列腺癌病例分别为0、2、5、7例。血清PSA在0—1．9ng／ml和2．0—4．0ng／ml范围：DRE异常患者中前列腺癌检出率分别为5％和21．1％（P〈0．05）;TRUS异常患者中前列腺癌检出率分别为2．4％和28．6％（P〈0．05）。在14例前列腺癌患者中有4例仅被TRUS发现,并且这4例PSA值均在2．0～4．0ng／ml范围内。结论血清PSA〈2rig／ml时,除非DRE高度异常,患前列腺癌风险低,不需行前列腺穿刺活检。血清PSA在2．0—4．0ng／ml,应行TRUS检查,以提高前列腺癌检出敏感性。     

高效液相色谱法测定盐酸右哌甲酯缓释微丸的有关物质及含量

目的：应用高效液相色谱技术建立盐酸右哌甲酯缓释微丸有关物质检查和含量测定的方法。方法采用Agilent ZORBAX SB-C18色谱柱（250 mm ×4．6 mm,5μm）;磷酸二氢钾溶液[25 mmol／L,三乙胺0．2％（v／v）,磷酸调至pH 3．5]－甲醇（65∶35）为流动相,检测波长209 nm,流速1．0 ml／min,柱温40℃。结果在选定的色谱条件下,盐酸右哌甲酯与相关杂质分离良好,制剂辅料不干扰盐酸右哌甲酯的测定;检测限为81．12 ng／ml（S／N＝3）,在质量浓度2～30μg／ml范围内,盐酸右哌甲酯与峰面积线性关系良好,相关系数r为0．9995;平均回收率为100．83％,溶液在12 h内保持稳定（ RSD为0．10％）。结论该方法简便、准确、专属性强,可用于盐酸右哌甲酯缓释微丸有关物质及其含量的测定。     

孕母血清游离β绒毛膜促性腺激素时间分辨荧光免疫试剂盒的研制与评价

目的研制一种用于产前筛查孕母血清游离β绒毛膜促性腺激素（freehCGβ）的时间分辨荧光免疫（TRFIA）试剂盒。方法采用2株freehCG13单克隆抗体（McAb）,一株用于固相包被（3μg／ml）,另一株用于标记铕（Eu3＋）制备Eu3＋-freehCG13MeAb,以β-萘甲酰三氟丙酮为主要成分的增强液,应用双抗体夹心法建立freehCGBTRFIA法,并对其进行性能评价。采用线性回归分析及配对t检验进行统计学分析。结果该试剂盒的剂量一反应曲线线性相关系数（r）达0．9990;分析内、分析间变异系数（CV）均小于10．00％;标准品各点实测值与标示值的偏差（DIFF）均小于5．00％;灵敏度不高于0．20ng／ml;以国家标准品为对照,标准品效价比在0．9130～1．100间;与人促甲状腺激素（hTSH）、人黄体生成素（hLH）、人卵泡刺激素（hFSH）和人绒毛膜促性腺激素（hCG）交叉反应低;线性范围为2．00—200ng／ml;与同类国外试剂比对,线性回归方程为y=x＋0．0565,r=0．9771,检测结果间差异无统计学意义（t=0．1839,P〉0．05）;国内外同类仪器比对检测,线性回归方程为y=0．9894x＋0．0957,r=0．9995,检测结果间差异无统计学意义（t=-0．9669,P〉0．05）;室间质量评价实测值与靶值偏倚（-11．93％～7．01％）符合标准;干扰实验表明乙二胺四乙酸钾（EDTA．K2）≥0．54mmol／L、柠檬酸钠≥1．06mmol／L、草酸钾（K2C204H20）≥2．12mmol／L、氟化钠（NaF）≥29．76mmo]／L时,自制试剂盒检测结果明显偏低（干扰程度均〉10％）,肝素≤150U／L、血红蛋白≤12g／L、三酰甘油≤21．54mmol／L和胆红素≤818txmol／L对该试剂盒检测结果无明显干扰（干扰程度均〈10％）;温度升高会使样本检测结果偏高。结论该试剂盒具有灵敏度高、特异性强、准确度好、线性范围宽等优点,与国外的同类产品性能相仿,能满足临床需要。     

血清降钙素原对危重病人细菌感染的诊断价值

目的 评价血清降钙素原（PCT）对危重病人感染诊断和鉴别诊断的临床价值。方法 53例危重病人按细菌感染、非细菌感染分为两组，采用微量双夹心免疫发光法测定血清PCT水平。结果 细菌感染组PCT水平明显高于非细菌感染组（χ^2=10．05，P〈0．05）。随血清PCT浓度升高，其对危重病人感染诊断的敏感度降低、特异度增高；以1．5ng／ml为阳性诊断标准时，Youden指数和诊断符合率分别为0．65和83．0％，明显高于以1ng／ml和2ng／ml为阳性诊断标准时。结论 血清PCT检测对危重病人细菌感染的早期诊断、鉴别诊断及指导临床治疗具有重要意义。     

HPLC-MS 测定大鼠体内白杨素及其药代动力学简

目的建立HPLC—MS的方法测定大鼠血浆中白杨素含量。方法以柚皮素为内标；采用WatersXteraC-18色谱柱（150mm×4．6mm，5μm）分离，柱温设定为30oC；梯度洗脱：A相为乙腈，B相为0．05％甲酸；流速为0．8ml·min，使用分流器将0．2ml·min。的洗脱液注入离子源；选择离子监测检测（SIM）：白杨素m／z255[M＋H]＋，内标柚皮素m／z273[M＋H]＋。结果本研究所采用的方法检测限为1．5ng·ml-1，定量限为4．0ng·ml-1，且精密度、稳定性及回收率满足测定要求，并成功用于白杨素在大鼠体内的药动学评价。结论本研究建立了快速、准确的HPLC—MS法用于测定大鼠血浆中的白杨素含量，并成功拟合其药动学参数，白杨素在大鼠体内的药动学特性符合二室开放模型含量。     

血清降钙素原水平对发热患者的诊断价值研究

目的：探讨血清降钙素原（PCT）水平对发热患者的诊断价值。方法：选择我院住院发热且PCT表达阳性300例,观察各种疾病所占比例及PCT水平,为临床鉴别诊断及治疗提供一定线索。结果：发热且PCT表达阳性300例中,败血症64例（21．3％）,腹膜炎60例（20．0％）,混合感染56例（18．7％）,局部感染组52例（17．3％）,肺部感染25例（8．3％）,肝衰竭25例（8．3％）,不明原因发热18例（6．0%）;感染性疾病所占比例非常显著高于非感染性疾病（P〈0．01）。败血症PCT表达水平为（18．69±1．11）ng／ml,局部感染为（8．45±0．66）ng／ml,混合感染为（4．98±1．03）ng／ml,腹膜炎为（3．51±0．71）ng／rnl,肺部感染为（2．71±0．85）ng／ml,肝衰竭为（2．13±0．62）ng／ml,不明原因发热为（1．24±0．62）ng／ml;感染性疾病PCT表达水平显著或非常显著高于非感染性疾病（P〈0．05,P〈0．01）。结论：及时检测发热患者PCT表达水平,可为临床诊断及治疗提供一定参考。     

Detection of proteinous toxins using the Bio-Threat Alert system, part 4. Differences in detectability according to manufactural lots and according to toxin subtypes

In a series of experiments, we have tested the usefulness and limitations of the Guardian Bio-Threat Alert (BTA) system for detection of staphylococcal enterotoxin B (SEB), botulinum toxins (BTXs) A and B, and ricin. In this report, the BTA system has been further evaluated for toxin subtypes and the detection ability of manufactural lots of the BTA strips. The SEB strips failed to detect staphylococcal enterotoxin A, C, and D; the BTX strips generally failed to detect BTXs C, D, E, and F, but one lot showed positive results for BTXs C and D with very low sample values. Differences were observed in sample values at 1 μg/ml for all main toxins according to the different manufactural strip lots: 3.9-fold difference for SEB, 6.3-fold difference for BTX A, 10.9-fold difference for BTX B, and 6.4-fold difference for ricin. The ricin strips showed high cross reactivity toward RCA120. The BioWarfare Agent Detection Devices system showed much lower sensitivity than the BTA system for BTX and ricin (detection limit: about 10 μg/ml).     

胶体金免疫层析技术快速定量检测相思子毒素

目的：建立胶体金免疫层析技术快速定量检测相思子毒素。方法：利用胶体金标记和双抗体夹心免疫层析技术,建立相思子毒素的快速检测方法,评价其特异性和敏感性,并拟合检测曲线进行定量检测。在面粉、饼干、奶粉、苹果汁、牛奶、果冻等食品样品中添加相思子毒素模拟污染样品,评价该方法对固体、半固体、液体等食品样品的检测能力。结果与结论：该法可在15min内完成定性和半定量检测,灵敏度为30ng/ml,线性范围30～600ng/ml、回收率80%～110%,变异系数小于15%。所建立的检测相思子毒素的胶体金免疫层析方法,能快速、灵敏、特异、准确地检测样品中的相思子毒素,并可实现定量,适用于现场快速检测。     

A型肉毒毒素胶体金免疫层析法检测试纸条的研制

目的：研制一种敏感、特异、快速并适用于非专业人员的胶体金免疫层析法(immuno-chromatographic assay,ICA)试纸技术，用于检测食物、患者呕吐物或粪便以及血清和土壤中的肉毒毒素(botulinum toxin)。方法：采用柠檬酸盐还原法制备胶体金颗粒、标记A型肉毒毒素的多克隆抗体，制成免疫层析检测试纸条。待测样中的A型肉毒毒素与试纸条上金标记抗体结合后沿着硝酸纤维膜移动，并与膜上的抗体结合形成肉眼可视红色带。结果：肉毒毒素ICA试纸条可以特异性检测A、B和E型肉毒毒素。检测中国兰州生物制品研究所注射用A型肉毒毒素标准品，灵敏度可达2U／ml。与金黄色葡萄球菌肠毒素等抗原无交叉反应。结论：A型肉毒毒素ICA试纸条具有特异性强、灵敏度高、简便快速的特点，适用于专业和非专业人士，实用性较强。     

石头鱼毒素胶体金检测试纸条的研制

目的：研制石头鱼毒素（neoverrucotoxin,stonefish toxin,neoVTX）的胶体金检测试纸条,建立一种该毒素的快速检测方法。方法采用柠檬酸三钠还原法制备胶体金溶液,以neoVTX马血清抗体用于胶体金标记,并将该抗体固定于硝酸纤维素膜作为捕获抗体,利用双抗体夹心法原理制备胶体金检测试纸条。结果制备的检测试纸条对该毒素检测灵敏度为50 ng／ml,该试纸与牛血清白蛋白、卵清蛋白及水母毒素等蛋白无反应,具有较好的特异性。结论成功研制了石头鱼毒素胶体金检测试纸,该试纸条灵敏度高,特异性好,可用于该毒素的快速检测。     

Comparative evaluation of various solid phases for the development of coated tube assays for the estimation of progesterone in human serum,bovine serum and bovine milk

Immobilization of progesterone antibody using three polystyrene surfaces and two progesterone radiotracers for use in the development of a coated tube assay for the evaluation of progesterone levels in human serum, bovine serum and bovine milk was studied. The selection of the solid phase and the tracers were based on the maximum binding, non-specific binding, sensitivity and percentage recovery. Amongst the polystyrene tubes studied, streptavidin coated tubes showed the acceptable assay features such as low non-specific binding (0.5–1.0%), adequate sensitivity (0.13–0.16 ng/ml) and recovery (85–115%) for all the three sample matrices, human serum, bovine serum and bovine milk.     

多重生物检测技术研究进展

多重生物检测因其快速、耗费少、所需样品量少等优点,被广泛用于细菌、病毒、真菌、寄生虫及自身免疫相关抗体等抗原或抗体的快速筛查诊断。多重核酸检测的新技术包括巢式PCR(nPCR)、多重串联PCR、基于毛细管电泳的GeXP平台和luminex xMAP多重分析技术平台,多重免疫检测的新技术包括基于上转换发光、多线点、表面等离子共振技术的多重生物检测及基于蛋白芯片的阵列array-ELISA检测。本文综述了近年来多重生物检测新技术在呼吸道感染、腹泻、自身免疫性缺陷和肿瘤等疾病中的应用进展。     

Utility of disk solid-phase extraction for whole blood samples: analysis of some tetracyclic antidepressants by gas chromatography with nitrogen-phosphorus detection

Four tetracyclic antidepressants, maprotiline, mianserin, mirtazapine, and setiptiline, were extracted from human whole blood and plasma samples by disk solid-phase extraction with Empore C₁₈ cartridges. They were determined by gas chromatography (GC) with nitrogen-phosphorus detection. Recoveries of maprotiline, mianserin, mirtazapine, and setiptiline spiked into whole blood or plasma were more than 83%. Regression equations for the four drugs showed excellent linearity in the range of 25–1000 ng in 0.2 ml whole blood and in 0.5 ml plasma. The limits of detection for the drugs were 4.1–18.2 ng per 0.2 ml for whole blood and 3.4–13.5 ng per 0.5 ml for plasma. The limits of quantification for the four drugs were 25 ng in 0.2 ml whole blood and in 0.5 ml plasma. Intraday and interday coefficients of variation for the antidepressants in both human specimens were below 15%. The above data show that relatively hydrophobic and basic drugs in crude biological samples such as whole blood can be extracted by disk solid-phase extraction very efficiently and with good reproducibility, and suggest that the extraction method is also useful as pretreatments before more sophisticated detection methods such as GC-mass spectrometry (MS) and liquid chromatography-MS(-MS).     

On-site testing of saliva and sweat with Drugwipe and determination of concentrations of drugs of abuse in saliva, plasma and urine of suspected users

Potential drug users participated voluntarily in a Belgian study on the usefulness of the non-instrumental immunoassay Drugwipe (Securetec, Germany) for the screening of cocaine, opiates, amphetamine and cannabinoids in saliva and sweat. If one of the screening assays (urine, oral fluid, sweat) showed a positive result, blood and saliva were collected. The on-site Drugwipe results were correlated with the Drugwipe results for saliva in the laboratory and with the GC/MS results of the corresponding saliva, plasma and urine samples and pharmacological effects at the time of sampling. The Drugwipe assay proved to be sufficiently sensitive for the detection of recent cocaine (n = 6) and amphetamine (n = 15) abuse, whether the device was wiped on the tongue or on the surface of the body, or when a saliva sample was applied to the wiping part. In five of the six potential cocaine users, the saliva concentrations of cocaine exceeded 1000 ng/ml. In the amphetamine group, the saliva concentrations of amphetamine, MDMA or both were high (> 1000 ng/ml) in 13 subjects. For cocaine and amphetamine, the positive scores for Drugwipe matched the GC/MS results for the three body fluids. Recent heroin abuse (n = 5) could be demonstrated to some extent with Drugwipe on samples from the tongue but only the two subjects with the highest saliva concentrations of MAM (> 500 ng/ml) and morphine (> 500 ng/ml) were positive. If the legal cut-off value for driving under the influence of opiates in Belgium (20 ng/ml of free morphine in plasma) was taken into account, only three subjects would have been legally positive. For cannabinoids (n = 15), false negatives and even some false positives were observed. Saliva can be considered as a useful analytical matrix for the detection of drugs of abuse after recent abuse when analysed with GC/MS. Received: 26 January 1999 / Received in revised form: 19 May 1999 / Accepted: 17 June 1999