单克隆免疫金标试纸应用于血液中药物筛选的可靠性研究
发布时间：2019-05-20 07:58
【摘要】：目的:1.通过空白添加、动物实验,探索单克隆试纸对血液中苯二氮?类;巴比妥类;毒品类等药(毒)药物筛选的检测阈值及LC-MS/MS确认方法的研究;2.通过实际案例验证单克隆试纸用于血液中苯二氮?类;巴比妥类;毒品类等药(毒)物筛选的可靠性。方法:1.血液用单克隆免疫金标试纸检测前的处理方法的研究通过血液空白添加实验,分析了不同p H值缓冲液条件、不同离心转速以及不同温度下对血液前处理的影响,得到血液中不同药物的检测阈值。2.动物实验,建立血液筛选的检测阈值及检验方法的研究称重,按大鼠的LD50给动物灌胃,在动物血液药物浓度达到最大值时解剖取血液,按照第二部分的血液前处理,使用单克隆免疫金标试纸检测血液药物浓度,结果呈阳性,则减少1/2灌胃剂量,直至单克隆免疫金标试纸呈阴性。这时,阴性结果的前一次阳性灌胃剂量就是现有单克隆免疫金标试纸检测所能达到的最低血液药物浓度。取1m L的动物血液,加9m L乙酸乙酯,振荡,超声5min,离心,取上清液于蒸发皿中,60℃水浴挥干,加1m L的乙腈复溶,有机膜过滤,LC-MS/MS检测分析,得到血液药物浓度。3.通过实际案例验证单克隆免疫金标试纸应用于血液中药物筛选的可靠性研究实际案例的血液经过上述的前处理,使用单克隆免疫金标试纸检测和LC-MS/MS检测验证,得出结果。结果:1.使用单克隆免疫金标试纸对血液进行药(毒)物筛选时,有必要向血液中加入磷酸盐缓冲液,这样能得到较好的检测结果。另外,V(血液):V(磷酸盐缓冲液)=1:1进行等量添加,效果最好。2.使用Sigma 3-30K高速台式冷冻离心机在12000转、4℃条件下离心处理得到的检测限最低,检测效果最好,在条件允许时最好在筛选时选用高速冷冻离心机离心,能够获得最佳结果。但是这种离心机成本高、普及率低,因此,一般使用低速离心机也是可以的。3.磷酸盐缓冲液的p H值对各类药物检测阈值的影响不一致,其中对氯硝安定和氯胺酮的影响较大。4.实际案例。2012-2013年的38个实际案例的LC-MS/MS检验结果与用单克隆免疫金标试纸检测结果相比较,其中的89.47%结果一致。结论:1、用于尿液筛选的单克隆免疫金标试纸在应用于血液检材的筛选时差别不大。2、使用磷酸盐缓冲液稀释血液,用高速冷冻离心机离心,取上清液用于单克隆免疫金标试纸筛选血液能够获得较好的结果。3、根据空白实验结果,在使用单克隆免疫金标试纸筛选血液中苯二氮?类药物时,应注意分别使用p H6-8及p H11两种缓冲液分别稀释血液后进行筛选,以免漏掉氯硝安定这种需要在碱性条件下才能用单克隆免疫金标试纸较好筛选出的药物。4、使用单克隆免疫金标试纸筛选血液中巴比妥类药物时,使用p H6-8的缓冲液稀释血液可以满足检测需要。5、在使用单克隆免疫金标试纸筛选血液中的毒品时,吗啡类、苯丙胺类使用缓冲液p H6-8,均可满足检测要求,但氯胺酮筛选存在较大问题,在p H6-8时,阈值为4000ng/m L,在p H11时,阈值为2000ng/m L,均与试纸厂家说明书上标注的1000ng/m L相差较大。6、根据本课题研究,目前单克隆免疫金标试纸检测药(毒)物的阈值如表5-1,建议厂家对氯胺酮单克隆免疫金标试纸加以改进,以满足实际案件需要。
[Abstract]:Objective:1. Through the blank addition, animal experiment, explore the monoclonal test paper to the blood of the benzodiazepines? Class; barbiturates; drug-like drug (toxic) drug screening detection threshold and LC-MS/ MS validation method;2. Use a practical case to verify that the monoclonal test paper is used in the blood of benzodiazepines? Class; barbiturates; drug-like drugs (toxic) screening reliability. Method:1. The study of the treatment method before the detection of the monoclonal immunogold-labeled test paper for blood was carried out by a blank addition experiment, and the effects of different pH buffer conditions, different centrifugal rotation speeds and different temperatures on the pretreatment of the blood were analyzed, and the detection threshold of different drugs in the blood was obtained. an animal experiment is carried out to establish a test threshold for blood screening and a study weighing of the test method; the LD50 of the rat is given to the animal for the stomach, and the blood is dissected and taken according to the blood pre-treatment of the second part when the blood drug concentration of the animal reaches the maximum value; The concentration of blood drug was detected by using the monoclonal immunogold standard test paper, and the result was positive, and the dose of 1/2 was reduced until the monoclonal antibody test paper was negative. At this time, the first positive intragastric dose of the negative result is the minimum blood drug concentration that can be achieved by the prior monoclonal antibody test paper. 1 m L of animal blood,9 m L of ethyl acetate, shaking, ultrasonic for 5 min, centrifuging, collecting the supernatant in the evaporating dish, volatilizing at 60 & deg; C water bath, adding 1 ml of acetonitrile for reconstitution, filtering the organic membrane, and detecting the LC-MS/ MS to obtain the blood drug concentration. The actual case was used to verify the reliability of the application of the monoclonal immunogold standard test paper to the drug screening in the blood. The blood of the actual case was subjected to the above-mentioned pretreatment, and the test and the LC-MS/ MS detection and verification were performed using the monoclonal immune gold standard test paper and the results. Results:1. When using the monoclonal immunogold standard test paper to screen the blood, it is necessary to add a phosphate buffer to the blood, so that a better detection result can be obtained. In addition, V (blood): V (phosphate buffer) = 1:1 was added in the same amount, with the best effect. The lowest detection limit is obtained by using a Sigma 3-30K high-speed bench freezing centrifuge at the temperature of 12000 rpm and 4 鈩，
本文编号：2481485
[Abstract]:Objective:1. Through the blank addition, animal experiment, explore the monoclonal test paper to the blood of the benzodiazepines? Class; barbiturates; drug-like drug (toxic) drug screening detection threshold and LC-MS/ MS validation method;2. Use a practical case to verify that the monoclonal test paper is used in the blood of benzodiazepines? Class; barbiturates; drug-like drugs (toxic) screening reliability. Method:1. The study of the treatment method before the detection of the monoclonal immunogold-labeled test paper for blood was carried out by a blank addition experiment, and the effects of different pH buffer conditions, different centrifugal rotation speeds and different temperatures on the pretreatment of the blood were analyzed, and the detection threshold of different drugs in the blood was obtained. an animal experiment is carried out to establish a test threshold for blood screening and a study weighing of the test method; the LD50 of the rat is given to the animal for the stomach, and the blood is dissected and taken according to the blood pre-treatment of the second part when the blood drug concentration of the animal reaches the maximum value; The concentration of blood drug was detected by using the monoclonal immunogold standard test paper, and the result was positive, and the dose of 1/2 was reduced until the monoclonal antibody test paper was negative. At this time, the first positive intragastric dose of the negative result is the minimum blood drug concentration that can be achieved by the prior monoclonal antibody test paper. 1 m L of animal blood,9 m L of ethyl acetate, shaking, ultrasonic for 5 min, centrifuging, collecting the supernatant in the evaporating dish, volatilizing at 60 & deg; C water bath, adding 1 ml of acetonitrile for reconstitution, filtering the organic membrane, and detecting the LC-MS/ MS to obtain the blood drug concentration. The actual case was used to verify the reliability of the application of the monoclonal immunogold standard test paper to the drug screening in the blood. The blood of the actual case was subjected to the above-mentioned pretreatment, and the test and the LC-MS/ MS detection and verification were performed using the monoclonal immune gold standard test paper and the results. Results:1. When using the monoclonal immunogold standard test paper to screen the blood, it is necessary to add a phosphate buffer to the blood, so that a better detection result can be obtained. In addition, V (blood): V (phosphate buffer) = 1:1 was added in the same amount, with the best effect. The lowest detection limit is obtained by using a Sigma 3-30K high-speed bench freezing centrifuge at the temperature of 12000 rpm and 4 鈩，
本文编号：2481485
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