可卡因及其代谢物苯甲酰爱康宁胶体金检测试剂盒的研制

目的根据胶体金免疫层析的原理,建立一种可以在现场快速检测可卡因及其代谢物苯甲酰爱康宁的方法。方法将胶体金标记的抗可卡因单克隆抗体均匀浸涂在玻璃纤维膜上,用点膜仪将可卡因的完全抗原以及羊抗鼠多抗在硝酸纤维素膜上划线,分别作为可卡因的检测区（T）和质控区（C）。并且对样品垫用0.2%Tween 20和0.5%PVA封闭处理。样本（尿样、唾液、血样等）中游离的可卡因及其代谢物同包被的完全抗原分别免疫竞争结合胶体金标记的抗可卡因单抗。肉眼观察试剂盒的质控区和检测区上有无紫红色带来判读结果。结果可卡因胶体金检测试剂盒对65种毒品、药物的特异性测试中仅识别可卡因及其代谢物。对人体尿样中的可卡因和苯甲酰爱康宁检测阈值均为300ng/mL。与GC/MS对照试验,其准确性分别为98.9%和98.6%。在常温下能稳定保存2年。结论该可卡因检测试剂盒在5min内可以检测出样本中的可卡因及其代谢物成分。     

氯胺酮胶体金标记单克隆抗体免疫层析检测板的研制

目的建立一种简便快速检测氯胺酮的方法。方法将20nm胶体金颗粒标记的抗氯胺酮单克隆抗体,均匀浸在吸水玻璃纤维上,用点膜机将氯胺酮-BSA和纯化后的羊抗鼠IgG多克隆抗体在硝酸纤维薄膜分别划1mm宽的检测线(T线)和质控线(C线),然后依次将吸水玻璃纤维、硝酸纤维薄膜和吸水滤纸粘贴于白色的塑料片上,切成0.5 cm×10 cm大小,制成氯胺酮胶体金标记单克隆抗体免疫层析检测条,并组装成检测板,并检测其特异性和灵敏度。结果氯胺酮胶体金标记单克隆抗体免疫层析检测板在对46种药(毒)物的测试中,仅识别氯胺酮及其主要代谢产物,其准确性为97.6%,检测氯胺酮的阈值为1000ng/m l。结论氯胺酮胶体金标记单克隆抗体免疫层析检测板在5m in内可检出样品中的氯胺酮及其代谢物。     

抗丁丙诺啡单克隆抗体的制备

目的建立抗丁丙诺啡单克隆抗体的杂交瘤细胞株，制备高特异性的丁丙诺啡单克隆抗体，并对其免疫学特性进行鉴定。方法在丁丙诺啡的分子上连接活性羧基基团，通过缩合反应将丁丙诺啡半抗原连接于血蓝蛋白（KLH）和小牛血清白蛋白（BSA），形成完全抗原。以完全抗原免疫Balb／c小鼠，通过细胞融合，筛选等杂交瘤技术，建立稳定的分泌抗丁丙诺啡单克隆抗体的杂交瘤细胞株。通过腹腔注射杂交瘤细胞，诱导小鼠产生含有单抗的腹水。用辛酸-硫酸铵加亲和层析法纯化抗丁丙诺啡单克隆抗体。采用酶联免疫反应和胶体金膜层析实验测定丁丙诺啡单抗的特异性以及免疫反应动力学参数。结果共获得3株分泌抗丁丙诺啡单克隆抗体的杂交瘤细胞株，分别命名为7E6，6G4和3C2。7E6，6G4抗体灵敏度为10．0ng／ml，3C2抗体灵敏度为20．0ng／ml。7E6，6CA和3C2抗体的亲和常数分别为3．6×10^-9 mol／L，4．3×10^-9 mol／L和6．3×10^-9 mol／L。特异性测试结果表明7E6和6G4抗体与40种药物、毒品无任何交叉反应，而3C2抗体与吗啡有交叉反应。结论杂交瘤细胞株7E6和6G4产生的抗丁丙诺啡单克隆抗体具有很高的特异性和灵敏度。     

检测丁丙诺啡的胶体金标记单克隆抗体免疫试剂盒研制

目的采用免疫竞争法原理,建立一种简便快速检测丁丙诺啡的方法。方法将20nm胶体金颗粒标记的抗丁丙诺啡单克隆抗体,均匀浸在吸水玻璃纤维上,将丁丙诺啡-BSA和纯化后的羊抗鼠IgG多克隆抗体在硝酸纤维薄膜分别划1mm宽的检测线(T线)和质控线(C线),制成丁丙诺啡胶体金标记单克隆抗体免疫层析检测条,并组装成检测板。结果以GC/MS为标准对照,100例样品检测结果显示,本检测板的准确率为99.0%。丁丙诺啡胶体金标记单克隆抗体免疫层析检测板在对45种药(毒)物的测试中,仅识别丁丙诺啡及其主要代谢产物。结论丁丙诺啡胶体金标记单克隆抗体免疫层析检测板在5min内可检出样品中的丁丙诺啡及其代谢物,检测丁丙诺啡的阈值为100ng/mL。     

胶体金标记苯丙胺单克隆抗体免疫试剂盒研究

目的建立一种准确、快速、简便的检测尿液中苯丙胺(AMP)的胶体金免疫层析技术。方法采用柠檬酸三钠还原法制备胶体金颗粒,标记抗AMP单抗,将AMP—BSA抗原固相于硝酸纤维素膜上,制备胶体金免疫层析测试条。通过尿液、血液和唾液中的可能存在的苯丙胺成分与测试条上的苯丙胺-BSA完全抗原竞争结合有限的单抗结合位点,来判定检测结果。结果用ICT法和GC/MS检测217份尿样,本法检测阈值为1000ng/mL,特异性为99.17%,准确性为99.54%。结论ICT法检测尿液中的AMP特异性强,灵敏度高、简便快速、无需特殊仪器设备,具有广泛应用价值。     

抗体芯片竞争抑制法检测尿液中吗啡含量

目的建立抗体芯片竞争抑制法检测尿液中吗啡含量的方法。方法将吗啡单克隆抗体固定在用琼脂糖包被的芯片上,与含有吗啡的尿液检材和Cy3荧光标记-吗啡-BSA复合物进行竞争抑制反应,共聚焦扫描仪采集反应图像并进行分析。结果吗啡单克隆抗体及Cy3-吗啡-BSA的最佳浓度是31.25μg/m l、12.50μg/m l,检测线性范围0.01~10ng/m。回收率在91.2%~109.2%之间,尿液检测限为0.02ng/m l。甲基苯丙胺、安非他明与吗啡抗体之间无交叉反应,与可待因有一定的交叉反应。结论抗体芯片竞争抑制法检测尿液中的吗啡含量具有灵敏度高,特异性好、操作简单、高通量等优点,可用于法医毒物检测、戒毒效果监测。     

检验人精浆特异蛋白P30免疫胶体金试剂条的研制

目的制备用于检验人精浆特异蛋白P30-主要是来自法医学案件的免疫胶体金层析试剂条.方法选取针对不同抗原决定簇的抗P30单克隆抗体细胞株,并制备其小鼠腹水,分离纯化单克隆抗体.制备胶体金并以纯化抗体包被,制成免疫胶体金,以免疫胶体金浸泡玻璃纤维.选取适宜的硝酸纤维素膜并于其上不同位置以未金标的另一株P30单克隆抗体和羊抗鼠IgG包被.搭建试剂条并检测其灵敏度和特异性.结果所制成的试剂条灵敏度至少可达4ng/ml;对6人份混合的人精液物质在稀释20万倍后仍出阳性结果,且无非特异性反应.结论检验人精浆特异蛋白P30的免疫胶体金试剂条可对嫌疑人精物质做出排查,有利于法医物证检验.     

超高效液相色谱法（UPLC）同时筛选检测吗啡等7种常见毒品

目的建立快速筛选检测中毒者血液、尿液中吗啡、甲基苯丙胺、苯丙胺、麻黄碱、3，4-亚甲基双氧甲基苯丙胺（MDMA）、3，4-亚甲基双氧苯丙胺（MDA）、氯胺酮并定量分析的方法；方法采用超高效液相色谱（UP—LC）-二极管阵列检测器（PAD）；结果峰面积和质量浓度的线性关系良好，分离效果好、速度快、灵敏度提高；结论该方法与传统的HPLC相比能够更好满足实际办案中吗啡、甲基苯丙胺、苯丙胺、麻黄碱、MDMA、MDA、氯胺酮等中毒者血液、尿液的筛选检测并定量分析。     

特异性可卡因核酸适体测试条的研制

目的筛选识别可卡因的特异性核酸适体,制备可卡因核酸适体测试条（盒）。方法通过SELEX技术筛选可卡因核酸适体,制备胶体金标记可卡因完全抗原（COC-BSA）,制作可卡因层析测试条,并对方法特异性、灵敏度、准确性等性能指标进行考察和验证。结果甲基苯丙胺等64种药品和毒品采用本文研制的可卡因测试条检测,结果均为阴性,无交叉反应;检测可卡因最低浓度在5ng/mL,比胶体金单克隆抗体方法检测高5倍;将测试条密封45℃烘烤1个月后,检测结果与未烘烤过的测试条检测结果一致;与GC/MS检测结果对照,准确性为100%。结论本文研制的可卡因核酸适体检测试条,性能稳定,特异性良好,灵敏性和准确性均可达到实际检测的要求,在毒品、毒物的检测中具有广阔的应用前景。     

抗人Tf及抗人Hb试剂盒的比较

目的研究比较抗人Tf(转铁蛋白)和抗人Hb(血红蛋白)试剂盒的实验方法。方法采用胶体金标记单克隆抗体结合免疫层析技术,对不同稀释度的人血、动物血进行检测,并对保存时间、溶解度等影响因素进行研究。结果抗人Tf和抗人Hb试剂盒同样具有简单、快速、灵敏、稳定、特异性好的优点,但抗人Tf试剂盒能检测出陈旧及难以溶解血痕。结论抗人Tf试剂盒适用于法医物证的血痕种属检验。     

定量检测毒品的蛋白芯片的研发

目的利用蛋白芯片技术建立一种快速、高通量的毒品定量检测方法。方法采用硝酸纤维膜作为蛋白芯片的基片,将多种毒品和蛋白偶联物抗原包被在芯片基片上后封闭,不同毒品的单克隆抗体与之结合,再用荧光染料Cy5标记的二抗孵育,样本中如果含有毒品成分,将会与包被的毒品偶联物竞争性地与毒品的抗体相结合,导致最终的荧光信号发生改变,荧光信号的变化与样品中的毒品的浓度相关。通过荧光芯片检测仪CCD可以进行定量分析,最终判断样本中毒品的含量。结果对506例样本进行定性定量检测,与GC-MS仪器比较后总相关性在88%以上,芯片的检测灵敏度比胶体金方法提高10倍,其检测的特异性达到99%。结论蛋白芯片是一种有效、准确的高通量检测毒品含量的方法。     

A screening method for urinary methamphetamine--latex agglutination inhibition reaction test

Methamphetamine in urine samples from abusers was detected by the latex agglutination inhibition reaction test with latex-antibody (Latex-Ab) and latex-methamphetamine (Latex-MA) reagents. Anti-methamphetamine antibody was produced in rabbits by immunization with bovine serum albumin (BSA)-methamphetamine conjugate. Latex particles were coated with antibodies or with rabbit serum albumin (RSA)-methamphetamine conjugate to obtain Latex-Ab and Latex-MA reagents, respectively. The results are read at 4-5 min after mixing the latex reagents. The sensitivity of this method for methamphetamine was 0.4 micrograms/ml urine. Methamphetamine analogs (methylephedrine, amphetamine, phentermine, methoxyphenamine, ephedrine, beta-phenylethylamine, OH-methamphetamine, OH-amphetamine, and OH-ephedrine) all cross-react in varying degrees, while glucosiurea and albuminurea give false positive results in the tests. Though attention must be paid to these effects this simple and rapid test is suitable for the mass screening of urine samples.     

High prevalence of 6-acetylmorphine in morphine-positive oral fluid specimens

Identification of 6-acetylmorphine, a specific metabolite of heroin, is considered to be definitive evidence of heroin use. Although 6-acetylmorphine has been identified in oral fluid following controlled heroin administration, no prevalence data is available for oral fluid specimens collected in the workplace. We evaluated the prevalence of positive test results for 6-acetylmorphine in 77,218 oral fluid specimens collected over a 10-month period (January-October 2001) from private workplace testing programs. Specimens were analyzed by Intercept immunoassay (cutoff concentration=30 ng/ml) and confirmed by GC-MS-MS (cutoff concentrations=30 ng/ml for morphine and codeine, and 3 ng/ml for 6-acetylmorphine). Only morphine-positive oral fluid specimens were tested by GC-MS-MS for 6-acetylmorphine. A total of 48 confirmed positive morphine results were identified. An additional 107 specimens were confirmed for codeine only. Of the 48 morphine-positive specimens, 32 (66.7%) specimens were positive for 6-acetylmorphine. Mean concentrations (+/-S.E.M.) of morphine, 6-acetylmorphine and codeine in the 32 specimens were 755+/-201, 416+/-168 and 196+/-36 ng/ml, respectively. Concentrations of 6-acetylmorphine in oral fluid ranged from 3 to 4095 ng/ml. The mean ratio (+/-S.E.M.) of 6-acetylmorphine/morphine was 0.33+/-0.06. It is suggested that, based on controlled dose studies of heroin administration, ratios >1 of 6-acetylmorphine/morphine in oral fluid are consistent with heroin use within the last hour before specimen collection. The confirmation of 6-acetylmorphine in 66.7% of morphine-positive oral fluid specimens indicates that oral fluid testing for opioids may offer advantages over urine in workplace drug testing programs and in testing drugged drivers for recent heroin use.     

SPE/UPLC法检测血中吗啡、苯丙胺类及氯胺酮

目的建立SPE/UPLC方法在同一条件下同时检测血中吗啡、苯丙胺类及氯胺酮。方法采用SCX 3cc（60mg）固相萃取柱萃取血中吗啡、MA、MDMA、MDA及氯胺酮,用超高效液相色谱（UPLC）-二极管阵列检测器（PDA）检测,结合保留时间和紫外光谱进行定性、定量分析,对实验各环节进行优化,并进行实际案例检测。结果吗啡、MA、MDMA、MDA、氯胺酮的固相萃取提取回收率分别为81.4%±2.51%、88.2%±2.48%、91.8%±2.03%、93.8%±1.46%、74.8%±2.27%,峰面积和质量浓度的线性关系良好（r〉0.999）,线性范围分别为0.08～100μg/mL、0.4～100μg/mL、0.2～75μg/mL、0.3～75μg/mL、0.4～100μg/mL,检出限分别为30pg、200pg、80pg、100pg、200pg。结论本文所建方法适用于血中吗啡、苯丙胺类、氯胺酮常见毒品的筛选及定量分析。     

Rapid determination of ketamine in urine by liquid chromatography-tandem mass spectrometry for a high throughput laboratory

In recent years, the abuse of ketamine had gained popularity in rave parties in Hong Kong. The Urinalysis Unit of the Government Laboratory of Hong Kong faced a tremendous increase in workload for ketamine analysis. The number of tests performed rose from 10 in 1999 to 15,000 in 2002. As a fully validated immunoassay test for ketamine was not available in the market, most laboratories analyzed ketamine by chromatographic techniques after liquid-liquid extraction. However, these methods reported in the literature are not suitable for high throughput laboratories. Hence, a rapid screening/confirmation method for ketamine by liquid chromatography-tandem mass spectrometry (LC-MS-MS) with a detection limit of 5 ng/ml was developed. After automated solid-phase extraction (SPE), the urine extract was analyzed for ketamine by a 2.5-min chromatographic run, the estimated recovery was 89% and the precision was 11% R.S.D. at 20 ng/ml. With the aid of an in-house developed computer program, the results were presented in spreadsheet format for easy checking. The method has been applied to our laboratory for routine ketamine analysis and a maximum of 200 samples per day can be achieved.