动物源食品中残留克伦特罗检测抗体的制备与应用

采用重氮化法将盐酸克伦特罗 (CL)与牛血清白蛋白 (BSA)交联 ,用紫外扫描和凝胶电泳对交联物鉴定后免疫新西兰纯毛白兔 ,制备抗血清。经鉴定 ,抗血清中含有针对CL的抗体 ,效价为 3.2× 10 5;以此抗体为基础 ,设计了间接酶联免疫法 ,该法的检出限为 0 .0 96ng/mL ,对加于尿液、血液和组织中CL的回收率为 80 %～ 12 0 % ;通过重复性、灵敏度、准确性等指标对该法进行了评估 ,并与高效液相层析 (HPLC)及国外进口试剂盒进行了比较 ,表明该法的各项参数均能满足实际检测的需要。     

Evaluating the sensitivity,stability, and cross-reactivity of commercial fentanyl immunoassay test strips

Illicit fentanyl has flooded the United States' drug market, increasing the risk of overdose and poisonings throughout the general population and accidental exposure among law enforcement officers confiscating the increasing number of seizures. Fentanyl test strips (FTS) are used to obtain presumptive information about the presence of fentanyl in a suspected sample. However, their adoption by law enforcement personnel and seized-drug analysts has been limited because most products are advertised for urine testing, not for assays using water solutions. This study presents an evaluation of four commercial FTS: Rapid Response from BTNX, Inc.; T-Dip Fentanyl (FTY) Urine Dip Cards obtained from Amazon.com ; Premier BioDip FYL10 from Premier Biotech Inc.; and MobileDetect Fentanyl strips from DetectaChem, Inc. Performance characteristics curves were used to compare the products' sensitivity, showing that all can reliably detect fentanyl in aqueous solutions at concentrations below 1 μg/mL, with some of the tests able to reliably detect the drug at 200 ng/mL. A stability study demonstrates the performance of all four FTS brands was only slightly affected after 30 days of storage at two extreme environmental conditions. Fentanyl-related substances are also evaluated using the Rapid Response FTS, which showed high cross-reactivity with para-fluorofentanyl and acetylfentanyl, but lower with ortho-chlorofentanyl, carfentanil, and 4-ANPP. Users should be aware that FTS may give false-negative results even when potentially dangerous levels of carfentanil are present. When testing other common drugs, adulterants, and diluents frequently encountered in seized tablets, concentration-dependent results were obtained and multiple instances of false positives were recorded.     

Utility of immunoassay in drug screening in skeletal tissues: sampling considerations in detection of ketamine exposure in femoral bone and bone marrow following acute administration using ELISA

Detection of ketamine exposure in skeletal tissues by automated enzyme-linked immunosorbent assay (ELISA) and gas chromatography with electron capture detection (GC-ECD) is described. Rats (n = 18) received 0, 15, 30, or 75 mg/kg ketamine hydrochloride acutely (i.p.), and were euthanized within 15 min or 1 h. Ketamine was extracted from ground femoral bone by methanolic incubation followed by liquid-liquid extraction (LLE), while marrow was homogenized in alkaline solution, and then underwent LLE. Extracts were analyzed by ELISA, and subsequently by GC-ECD following derivatization with trifluoroacetic acid anhydride. The effect of tissue type (i.e., diaphyseal bone vs. epiphyseal bone vs. bone marrow) on the immunoassay response was examined through determination of binary classification test sensitivity (S) and measurement of the relative decrease in absorbance (%DA, drug-positive tissues vs. drug-free controls) in each tissue type. The %DA varied significantly between different tissues examined under a given dose condition, and generally decreased in the order marrow > epiphyseal bone > diaphyseal bone, at all dose levels examined. Measured S values for marrow, epiphyseal bone, and diaphyseal bone were 100%, 77%, and 23%, respectively (75 mg/kg dose). These results suggest that the type of skeletal tissue sampled and position sampled within a given bone (diaphyses vs. epiphyses) are important parameters in drug screening of skeletal tissues.     

Evaluation and applicability of Alere iCup DX 14 for rapid postmortem urine drug screening at autopsy

Performing point‐of‐care urine drug screen testing at autopsy by a forensic pathologist may provide an early indication of the presence of analytes of interest during autopsy. An evaluation for the screening of 14 classes of common drugs of abuse in postmortem urine by the point‐of‐care screening device, Alere iCup DX 14, is presented. One hundred ninety postmortem urine samples were screened with the iCup occurring at autopsy by the forensic pathologist. Positive and negative results obtained from the screening kit were evaluated against confirmatory test results obtained using routine forensic toxicology analyses that employed LC‐MS/MS and GC‐MS to detect a combination of over 85 common drugs of abuse and medications. Sensitivity for each respective iCup drug class ranged from 66% (buprenorphine) to 100% (methadone, tricyclic antidepressants). Specificity for each respective iCup drug class ranged from 89% (benzodiazepines) to 100% (amphetamines, barbiturates, buprenorphine, 3,4‐methylenedioxymethamphetamine, methadone). Positive predictive values ranged from 44% (benzodiazepines) to 100% (amphetamines, barbiturates, buprenorphine, methylenedioxymethamphetamine, methadone), while negative predictive values ranged from 96% (methamphetamine) to 100% (barbiturates, methadone, tricyclic antidepressants). A high false‐positive rate was yielded by the benzodiazepine class. The lack of fentanyl screening in the point‐of‐care device is a significant limitation considering its prolific prevalence in forensic casework. The results obtained in the study should be acknowledged when considering the use of the Alere iCup DX 14 in the context of postmortem casework to help indicate potential drug use contemporaneously with autopsy and when requiring such preliminary results prior to the release of a final forensic toxicology report.     

磺胺甲(口恶)唑的控温相变免疫分析

将已制备的抗磺胺甲唑(SMX)单克隆抗体(AbSMX)与温度敏感水凝胶(pNIPA)偶联形成水凝胶复合物(pNIPA-AbSMX),用异硫氰基荧光素(FITC)标记的抗原(FITC-AgSMX)和游离半抗原SMX竞争地与有限量的AbSMX和pNIPA-AbSMX反应,根据水凝胶相变温度以上免疫复合物沉淀的特性进行分离,结合抗原抗体免疫反应测定SMX,建立了一种检测SMX的控温相变免疫分析方法。结果表明,SMX浓度在0.1ng/mL~100μg/mL范围内有良好的线性关系,60%抑制率时灵敏度为0.03μg/mL。     

小分子半抗原抗体制备技术的研究进展

从半抗原的设计与合成、新型载体和佐剂的使用、基因工程抗体的应用等方面论述了小分子半抗原抗体制备技术的最新研究情况,旨在为制备一些采用常规方法不易获得的小分子半抗原抗体提供新的方法和思路。     

Detection of Acute Diazepam Exposure in Bone and Marrow: Influence of Tissue Type and the Dose‐Death Interval on Sensitivity of Detection by ELISA with Liquid Chromatography Tandem Mass Spectrometry Confirmation*

Abstract: Enzyme‐linked immunosorbent assay (ELISA) and liquid chromatography tandem mass spectrometry (LC/MS/MS) were used to detect diazepam exposure in skeletal tissues of rats (n = 15) given diazepam acutely (20 mg/kg, i.p.), and killed at various times postdose. Marrow, epiphyseal, and diaphyseal bone were isolated from extracted femora. Bone was cleaned, ground, and incubated in methanol. Marrow underwent ultrasonic homogenization. Extracts and homogenates were diluted in phosphate buffer, and then underwent solid‐phase extraction and ELISA. Relative sensitivity of detection was examined in terms of relative decrease in absorbance (ELISA) and binary classification sensitivity (ELISA and LC/MS/MS). Overall, the data showed differences in relative sensitivity of detection of diazepam exposure in different tissue types (marrow > epiphyseal bone > diaphyseal bone), which is suggestive of heterogenous distribution in these tissues, and a decreasing sensitivity with increasing dose‐death interval. Thus, the tissue type sampled and dose‐death interval may contribute to the probability of detection of diazepam exposure in skeletal tissues.     

Development of an Antibody Hapten‐Chip System for Detecting the Residues of Multiple Antibiotic Drugs*

Abstract: The abuse of antibiotic drugs during animal production remains a worldwide problem and the subsequent detection of the residues of various drugs present at low concentrations in complex biological matrices poses significant analytical challenges. The present study outlines a practical biochip assay system to identify antibiotic residues in different animal tissue extracts. The system uses a simple but efficient multiresidue sample extraction procedure to isolate the antibiotic residues which were then identified directly using high‐affinity monoclonal antibodies presented in a competitive immunoassay with conjugated antibiotic hapten‐chips. The hapten‐chip can analyze six samples each for eight antibiotics on a single chip within 3 h. The analytical results with both artificial positive standard samples and the incurred samples show that the antibody hapten‐chip system has a comparable accuracy and a similar sensitivity to a standard ultra performance liquid chromatography–mass spectrometry (MS)/MS assay. In conclusion, an effective analytical screening system based on antibody hapten‐chip was developed for detecting multiple antibiotic residues from multiple samples.     

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。     

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。