New detection method for ribonuclease 2 (RNase 2) using immunoblotting with specific antibody

A ribonuclease (RNase) was isolated from the urine of a 35-year-old male and purified to electrophoretic homogeneity. The enzyme was tentatively designated RNase 2. A rabbit antibody produced by injection of the purified RNase 2 was able to distinguish RNase 2 from another type of RNase coexisting in body fluids. With this antibody it was possible to detect RNase 2 isozymes in human serum and urine without difficulty using isoelectric focusing or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting. Both RNase 2 in serum and urine seemed to exist in multiple forms with regard to their molecular masses and pI values. This technique may prove to be useful in genetic and forensic studies of RNase polymorphism.     

Dual examinations for identification of urine as being of human origin and for DNA-typing from small stains of human urine

Concurrent methods for identification of urine as being of human origin, and for DNA-typing from small stains of human urine were examined. A urine stain was extracted with phosphate-buffered saline (PBS), and the extract was filtered using a Centricon-100 device. The filtrate was subjected to electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS) for identification of human urine and a DNA-typing sample was obtained by dialfiltration of the residue using a DNA purification kit. After the purified residue was treated with an AmpflSTR Profiler PCR amplification kit, the DNA-types were analyzed by capillary electrophoresis using a Genetic Analyzer. It was possible to identify a urine stain as being of human origin, and complete DNA profiles could be successfully obtained from a urine stain which had been created by 50 microL of female urine. Serial analyses of urine stains found at a crime scene provide effective information for forensic investigation. This method is recommended for stain identification and for DNA-typing from a urine stain.     

A sandwich enzyme immunoassay for human pancreatic elastase III

To develop a method for the determination of pancreas injuries using a pancreas-specific antigen as a marker, human elastase III was purified from the pancreas by chromatographic methods. A rabbit anti-human elastase III antibody was prepared, and this antibody was confirmed using immunoblotting to react only with elastase III among proteins from the pancreas. A sensitive sandwich enzyme immunoassay for human elastase III was developed. The detection limit for human elastase III was 0.3 pg (10 amol) per assay. Proteins extracted from the pancreas showed the strongest response, whereas reactions of the other organs were less than the detection limit. These results suggest that a sandwich enzyme immunoassay for human elastase III is useful for the determination of pancreas injury.     

MMP-11多克隆抗体的制备及其法医学意义

目的制备兔抗人基质金属蛋白酶-11(MMP-11)多克隆抗体,建立用MMP-11抗体检测月经血的可行方法,探讨其法医学意义。方法将用基因工程制备的人MMP-11融合蛋白免疫新西兰白兔,饱和硫酸铵法进行抗体纯化。运用蛋白印迹法检测月经血痕、外周血痕、阴道液斑、精液斑、唾液斑和尿液斑,盲测验证该方法的可靠性。结果高效价的兔抗人MMP-11多克隆抗体检测月经血MMP-11蛋白的阳性率为90.48%(93/105),而外周血痕、精液斑、唾液斑、尿液斑和阴道液斑均未检出MMP-11。结论用自制的抗MMP-11多克隆抗体所建立的蛋白印迹法检测月经血中的MMP-11特异性好,灵敏有效,可用于月经血及外周血的鉴别。     

Analysis of LSD in human body fluids and hair samples applying ImmunElute columns

Immunoaffinity extraction units (LSD ImmunElute) are commercially available for the analysis of lysergic acid diethylamide (LSD) in urine. The ImmunElute resin contains immobilized monoclonal antibodies to LSD. We applied the ImmunElute procedure to serum and also to human hair samples. For hair analysis the samples were first extracted with methanol under sonication. The extracts were then purified using the ImmunElute resin. LSD analysis was carried out with HPLC and fluorescence detection. The immunoaffinity extraction provides highly purified extracts for chromatographic analysis. The limit of detection (signal-to-noise ratio = 3) has been determined to be < 50 pg regardless of which sample material was used. The procedure was applied to authentic hair samples from drug abusers (n = 11). One of these samples tested positive with an amount of 110 pg LSD in 112 mg extracted hair corresponding to a concentration of 1 pg/mg.     

超高效液相色谱-串联质谱法同时测定尿液中16种抗生素

目的 建立同时分析尿液中16种抗生素的超高效液相色谱-串联质谱(UPLC-MS、MS)方法.方法 以哌拉西林为内标.尿样中的目标化合物经Oasis HLB固相萃取柱富集、纯化,利用ZORBAX SB-C18色谱柱,以0.1%的甲酸溶液-乙腈为流动相经梯度洗脱分离,采用多反应监测(MRM)模式进行分析.结果各化合物的最低...     

Validity testing of commercial urine cocaine metabolite assays: III. Evaluation of an enzyme-linked immunosorbent assay (ELISA) for detection of cocaine and cocaine metabolite

A validity assessment study was performed on the Genetic Diagnostic Enzyme Immunoassay test kit, a new enzyme-linked immunosorbent assay (GDC ELISA) for detection of cocaine and cocaine metabolite in urine. A set of 290 urine specimens, comprised of clinical cocaine urines collected from 5 male subjects who had received single doses of intravenous cocaine, drug-free urines spiked with cocaine, cocaine metabolites, cocaine isomers, and other drugs of abuse, were assayed by GDC ELISA. The results were compared with results by gas chromatography/mass spectrometry (GC/MS) assay for benzoylecgonine. Concordance was high between the GDC ELISA assay and GC/MS and with results reported earlier for other commercial assays. Detection times and specificity of the GDC ELISA antibody were most similar to those of the Abuscreen radioimmunoassay for cocaine metabolite. Overall, the assay produced no false negative or false positive results and appeared to be a reliable screening test for detection of cocaine and benzoylecgonine in human urine.     

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。     

胶体金标记检测大麻单克隆抗体免疫试剂盒研制

目的 建立准确、快速、简便的检测尿液中大麻的胶体金免疫层析技术(ICT)。方法 采用柠檬酸三钠还原法制备胶体金颗粒,标记抗主要代谢物四氢大麻酚-9-羧酸,THC与GC/MS检测方法相比,用ICT法的216份尿样,其检测限为50 ng/ml,灵敏度为96.67％,准确性为98.61％。结论 ICT法检测尿液中THC,其特异性强,对确定大麻的存在具有广泛的应用价值。     

Analysis of dimethylamphetamine and its metabolites in human urine by liquid chromatography-electrospray ionization-mass spectrometry with direct sample injection

An analytical method to identify and determine dimethylamphetamine (DMA) and its metabolites in human urine was developed with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) involving the direct injection of a urine sample. The urine samples were directly injected by using a gel permeation column, whose stationary phase was polyvinyl alcohol with a small amount of a carboxyl group, so DMA and its metabolites were analyzed rapidly and simply without pretreatment such as extraction, concentration and derivatization. DMA and its metabolites were identified in drug-free human urine spiked with 1 microg of DMA, dimethylamphetamine N-oxide (DMANO) and methamphetamine (MA), and 3 microg of amphetamine (AM) in 1 ml of urine under the full-scan mode. Under the selected ion monitoring (SIM) mode, the limits of detection (signal-to-noise ratio=5) for DMA, DMANO, MA and AM were 20, 20, 20 and 60 ng in 1 ml of urine, respectively. This method was applied to the identification and determination of DMA and its metabolites in urine samples of 10 DMA abusers. The concentrations of DMANO were higher than those of unchanged DMA in all urine samples; thus, DMANO is considered to be a useful metabolite as an indicator to prove DMA intake.     

SMCY性别特异融合抗原的克隆表达及其抗体制备

目的利用蛋白检测的快速性优势,研究不同性别在SMCY抗原氨基酸序列的差异,筛选出特异性氨基酸序列,并克隆表达性别特异融合抗原,制备相应抗体,建立一种快速鉴别法医物证性别的方法。方法通过对人SMCY和SMCX进行序列分析,发现了三段差异片段,采用搭桥PCR方法获得差异片段全长基因,连接入p ET-28a载体进行原核表达,用Ni柱纯化后的性别特异融合抗原免疫制备多克隆抗体,用ELISA法和western blot检测SMCY多抗与抗原的反应特异性,制作胶体金试纸条检测样本。结果筛选出具有性别特异性的氨基酸序列,获得SMCY性别特异融合抗原,成功制备出多克隆抗体及胶体金试纸条。结论获得SMCY性别特异融合抗原具有很好的抗原活性,制备的多克隆抗体可以与抗原特异性地结合,用于性别鉴定。     

抗人血红蛋白胶体金检测试剂条的研制

目的制备法医学检验所用的确定人血的免疫胶体金层析试剂条。方法选取抗人血红蛋白单克隆细胞株,制备其小鼠腹水,从腹水中纯化出单克隆抗体。制备胶体金并用一纯化的单克隆抗体包被,制成免疫胶体金。取玻璃纤维以免疫胶体金浸泡,烘干。在一硝酸纤维素膜上两个不同位置分别点加另一抗人血红蛋白抗体和羊抗鼠IgG。搭建试剂条并检测其灵敏度和特异性。结果制成的免疫胶体金试剂条可对稀释至20万倍的人血红蛋白溶液显示阳性,对法医学检验常见8种动物的血溶液显示阴性。结论所制备抗人血红蛋白胶体金试剂条可以应用于法医学检验。     

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。     

Compared interest between hair analysis and urinalysis in doping controls. Results for amphetamines, corticosteroids and anabolic steroids in racing cyclists

In France during a famous bicycle race, the newspapers documented the degree in which doping seemed to be supervised in some teams by managers and doctors. Use of anabolic steroids and other substances was officially banned in the mid-seventies by sports authorities. This policy has been enforced through urine testing before competition. It is well known, however, that a latency period is all that is necessary to defeat these tests. Nevertheless, hair analysis could be a promising tool when testing for periods that are not accessible to urinalysis any more. We have developed different sensitive methods for testing hair for amphetamines, anabolic steroids and their esters and corticosteroids. For amphetamines, 50 mg of hair were digested with 1 M NaOH, extracted with ethyl acetate, derivatized with TFA and analyzed by gas chromatography positive chemical-ionization mass spectrometry. For corticosteroids, 50 mg of powdered hair were treated with methanol in an ultrasonic bath and subsequently purified using a C18 solid phase extraction column. Analysis was realized by high performance liquid chromatography coupled to electrospray-ionization tandem mass spectrometry. For anabolic steroids and their esters, 100 mg of powdered hair were treated with methanol in an ultrasonic bath for extraction of esters, then alkaline digested with 1 M NaOH for an optimum recovery of other drugs. The two liquid preparations were subsequently extracted with ethyl acetate, pooled, then finally highly purified using a twin solid phase extraction on aminopropyl and silica cartridges. Residue was derivatized with MSTFA prior to injection. Analysis was conducted by gas chromatography coupled to a triple quadrupole mass spectrometer. Thirty cyclists were sampled and tested both in hair and in urine. Amphetamine was detected 10 times in hair (out of 19 analyses) compared to 6 times in urine (out of 30 analyses). Corticosteroids were detected 5 times in hair (methylprednisolone 1 case, triamcinolone acetonide 3 cases and hydrocortisone acetate 1 case) in hair (out of 12 analyses) compared to 12 times (triamcinolone acetonide 10 cases and betamethasone 2 cases) in urine (out of 30 analyses). Anabolic steroids were detected twice (nandrolone 1 case, and testosterone undecanoate 1 case) in hair (out of 25 analyses) compared to none in urine (out of 30 analyses).     

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.     

Validation of a high performance liquid chromatographic method for alpha amanitin determination in urine

The objective of the present study was to develop and validate a liquid chromatographic method with electrochemical detection to measure alpha amanitin concentrations in urine after sample pretreatment with double mechanism (reversed phase/cation exchange) solid-phase extraction cartridges. The urine samples (10 ml) were purified and concentrated to 1 ml with elimination of matrix contaminants. The extracts were then separated by isocratic reversed-phase chromatography using a C18 column (4.6 mm×25 cm) with a mobile phase composed of 0.005 M phosphate buffer (pH 7.2) and acetonitrile (90:10). Coulometric detection was performed by applying an oxidation potential of +500 mV to a porous graphite electrode in a low-volume analytical cell. The limit of quantitation was 10 ng/ml with a signal-to-noise ratio=25. The linearity studied on spiked urine was satisfactory (r=0.9966) from 10 ng/ml to 200 ng/ml. The average extraction recovery of alpha amanitin was 78%, determined using spiked urine samples ranging from 10–300 ng/ml. The intra-assay precision was checked at 10, 50 and 100 ng/ml levels (n=10) in spiked urine samples, with resulting coefficients of variation of 3.6%, 2% and 1.5%, respectively.     

A sandwich enzyme immunoassay for pulmonary surfactant protein D and measurement of its blood levels in drowning victims

A sensitive sandwich enzyme immunoassay for human pulmonary surfactant protein D (SP-D) was developed and used to examine the blood SP-D levels of drowning victims. Human SP-D was purified from amniotic fluid by chromatographic methods, and an antibody against human SP-D was prepared. A polystyrene ball coated with anti-SP-D IgG was incubated with purified human SP-D, and then with anti-SP-D Fab'-peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as the hydrogen donor. The detection limit of human SP-D was 5.2 pg per assay tube. Examination of cross-reactions of this sandwich enzyme immunoassay with proteins from other human organs showed it to be highly specific for lung, and Northern blot analysis detected specific SP-D mRNA expression only in lung. The SP-D concentration of normal human serum was 6.4+/-2.7 (mean+/-S.D.) ng ml(-1) (n=20). The recovery rates of 0.52 ng and 5.2 ng SP-D added to 5 microl normal human serum were 93.6+/-2.7% and 93.6+/-6.1%, respectively. Blood SP-D levels of victims from the saltwater drowning group (n=14) revealed higher concentrations (105.8+/-53.7 ng ml(-1)), while freshwater drowning victims (n=12) were estimated to be 74.1+/-43.9 ng ml(-1). The SP-D levels of 15 subjects who died of hemorrhage (n=5), heart failure (n=8), traumatic shock (n=1), and electrocution (n=1) were lower (22.0+/-8.5 ng ml(-1)), and those of asphyxia victims (n=10) were slightly higher (36.2+/-17.1 ng ml(-1)) than those of other causes of death, except for drowning. These results suggest that in drowning victims, SP-D flowed into the systemic circulation by physiological and physical mechanisms, and the differences of blood SP-D levels between saltwater drowning and freshwater drowning victims are presumed to be influenced by the type of agony and/or the length of survival time in water.     

Use of the "SMITEST" PSA card to identify the presence of prostate-specific antigen in semen and male urine

To determine whether the prostate-specific antigen (PSA) could be identified in semen using the "SMITEST" PSA immunochromatographic membrane test card, we examined semen and other body fluids, including urine. Although PSA activity was detected in semen with high sensitivity using the "SMITEST" PSA card, it was also detected in adult male urine. However, the lower detectable limit in the urine was 1000-fold lower than that in semen. The concentration of PSA in adult male urine was found to be 800 ng/ml using the card. PSA activity usually can be detected in urine of individuals over 14 years old and it has been detected in urine from children as young as 11 years old. Therefore, the appearance of PSA in urine may occur anytime between the age of 12 and 14 years. To determine the stability of PSA activity in urine, dried samples of urine on filter paper were kept at room temperature for up to 3 years. Although the immunoreactive line showing PSA activity became weak after storage, it was still detectable, but faint, after 3 years. In addition, PSA activity was not detected in male serum or saliva and in the urine from human females, male cats or male dogs using the PSA card. We conclude that the PSA card is useful for identification of PSA in both semen and adult male urine.     

A dot-blot-immunoassay for semen identification using a polyclonal antibody against semenogelin, a powerful seminal marker

Among various seminal plasma proteins, semenogelin (Sg), produced in the seminal vesicle, has been considered a candidate for demonstrating the presence of semen. Sg consists of two proteins, one 52 kDa (Sg-I) in size, and the other a mixture of 71 and 76 kDa proteins (Sg-II). Recombinant Sg-I and Sg-II proteins were obtained using a baculovirus system and then injected into a rabbit to produce the respective antibodies [Characterization of recombinant precursor proteins of the human seminal plasma sperm motility inhibitor synthesized in insect cells, Int. J. Mol. Med. 2 (1998) 693]. When liquefied seminal plasma was immunoblotted with the anti-Sg-I and Sg-II antibodies, the anti-Sg-II antibody identified a wider range of the polypeptides originating from Sg than did the anti-Sg-I antibody. A dot-blot-immunoassay using anti-Sg-II antibody revealed a clear immunoreactive spot even when the semen was diluted 6400-fold. However, this assay showed that the Sg antigen was undetectable in saliva, urine, vaginal secretions, sweat, nasal secretions and serum. To determine the stability of Sg antigenic activity, filter paper with dried semen stains were kept at 37, 4 and 22 degrees C for 1, 6 and 18 months, respectively, and the Sg antigenic activity was examined. The activity was detectable in an area not less than 0.5 cm x 0.5 cm under all of the above environmental conditions during each period. Finally, semen was mixed with saliva or blood at various volumetric ratios, and used as a source of dried stains. The Sg antigenic activity was detectable in the stains until the ratio of semen to saliva or blood reached 1:8. These results suggest that Sg may be useful as a marker for semen identification.     

A sandwich enzyme immunoassay for human muscle-specific beta-enolase and its application for the determination of skeletal muscle injury

A sensitive sandwich enzyme immunoassay for human beta-enolase was developed and used to examine beta-enolase in blood or bloodstains as a marker for the determination of skeletal muscle injury. Human beta-enolase was purified from human skeletal muscle, and then an antibody against it was prepared. Polystyrene balls coated with rabbit anti-human beta-enolase IgG were incubated with human beta-enolase and then with anti-human beta-enolase Fab'-peroxidase conjugate. Peroxidase activity bound to the polystyrene balls was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as a hydrogen donor. The detection limit for human beta-enolase was 2.6 pg (30 amol) per assay. The degree of cross-reaction of the sandwich enzyme immunoassay for other organs except for heart (1/10) was about 1/150 or less. Moreover, the localization of beta-enolase in various human tissues was examined by Northern blot analysis, and this confirmed that beta-enolase was expressed only in skeletal and cardiac muscle. Antigenic activity in bloodstains containing beta-enolase was recovered well after storage for 60 days at room temperature. The ratio of beta-enolase to total protein in bloodstains made from non-traumatic blood, nasal hemorrhage and menstrual blood, was within the normal range. In contrast, the ratio of beta-enolase in bloodstains from traumatic blood was obviously elevated (10-30 fold) in comparison with non-traumatic blood. Furthermore, the ratio of beta-enolase was proved to be higher in stains adhering to weapons that had passed through skeletal muscle, indicating that detection of beta-enolase in bloodstains could be used to distinguish crime weapons. These results suggest that beta-enolase is a useful marker for identification of skeletal muscle injury as well as for detecting the origin of bleeding.