Fluorescence polarization immunoassay for the detection of drugs of abuse in human whole blood

Fluorescence polarization immunoassay (FPIA) is a technique which has been known for a number of years. Since the development of the fundamental principles of fluorescence polarization by Perrin in a series of papers beginning in 1926, immunological techniques using labelled reactants have gained an extraordinary importance in the field of medical research and in routine diagnosis. As one of the non-radioactive immunological techniques, FPIA has found broad application in clinical and forensic toxicology. The authors report a new method to quickly screen autopsy, police and hospital blood samples for opiates, benzodiazepines, benzoylecgonine, barbiturates and methadone after Extrelut extraction utilising the FPIA methodology.     

Immunchemisches Screening von Serumproben: Vergleich der Cannabinoid- und Opiat-Assays Mahsan MTP und Abbott FPIA mit Kontrolle durch Gaschromatographie-Massenspektrometrie

By the update of §24?a of the German road traffic act driving under the influence of drugs of abuse can now be fined even in ¶absence of an actual driving impairment. ¶The only requisite is the detection of certain drugs/metabolites in a blood sample. Therefore for pretesting there is a need for fast and reliable immunological screening methods in blood/serum. The new Mahsan microtiterplate enzyme immunoassay (MTP-EIA) is claimed to be sensitive and independent of the matrix. For an evaluation we carried out a comparison of the cannabinoid and opiate assays from Mahsan (MTP-EIA) and the routinely used Abbott fluorescence polarization immunoassay (FPIA) with a batch of routine serum samples. For the MTP-EIAs we used the cut-off recommendations of the supplier (cannabinoids 2 ng/mL, opiates 10 ng/mL). For the Abbott FPIAs the serum samples had to be pretreated (deproteinization with acetone) and appropriate cut-offs were defined by our investigations (cannabinoids 20 ng/mL, opiates 30 ng/mL). Out of 136 samples screened for cannabinoids 42% were positive with GC-MS and 18% from 123 samples were positive for opiates. The Abbott cannabinoid FPIA gave false negative results in 23% of GC-MS positive cases and 9% false negatives for opiates while Mahsan MTP-EIA gave no false negatives. Each of the two cannabinoid assays gave false positive results in 13% of the cases and for the MTP opiate assay 2% of the positives could not be confirmed with GC-MS. As a result of our study the cut-off values for the MTP-EIAs have proved to be suitable for a distinction between potential positives and true negatives.     

Development of a psilocin immunoassay for serum and blood samples

After the immunisation of rabbits with a psilocin-specific immunogen, polyclonal antisera were obtained. With these antisera a competitive, heterogeneous radioimmunoassay for the detection of psilocin was developed. As tracer a derivative of psilocin was synthesised, which contained a tritiated CH₃ group. The antisera showed a specific reaction with psilocin. The cross-reactivity of structurally related endogenous substances like serotonin, tryptophan and tyrosine was below 0.01%. Also common drugs of abuse (⁹-tetrahydrocannabinol, cocaine, morphine, amphetamine) showed negligible cross-reactivity (0.01–2%). Only tricyclic neuroleptics with a (dimethylamino)ethyl side-chain showed some cross-reactivity (20%). Spiked serum and blood samples were analysed with this new immunoassay and the results obtained were compared with the values measured with a validated GC-MS method.     

An evaluation and survey of enzyme immunoassay and fluorometric enzyme immunoassay tests for TSH and HCG

Assays for human choriogonadotropin (HCG) and thyroid stimulating hormone (TSH) have typically been performed using radioisotopic procedures. Two newer, nonradioisotopic approaches, enzyme immunoassay (EIA) and fluorometric enzyme immunoassay (FEI), were evaluated for the determination of HCG and TSH. Two TSH EIA assays (Abbott, Hybritech) and one FEI assay (Dade) were compared with each other and an immunoradiometric (IRMA) assay. The FEI assay performed better than the EIA assays with a sensitivity of 0.3 microIU/ml, within-run closing volumes (CVs) of less than 4.5%, and a carryover of 0.7%. All of the assays correlated well with each other and the IRMA assay. Three HCG EIA assays (Abbott, Hybritech, Roche) and one FEI assay (Dade) were evaluated. Again, the FEI assay performed better than the EIA assays with a sensitivity of 1.3 mIU/ml, within-run CVs of 4.0% or less, and a carryover of 1.7%. The new generation of nonradioisotopic assays offers performance comparable to radioisotopic assays coupled with various degrees of automation. A clinical laboratory may not only take advantage of these methods but can choose an assay system that is well suited to its particular requirements.     

False-negative results for bromazepam use observed by the EMIT II Plus benzodiazepine assays

During our extensive routine analyses of drugs of abuse and other drugs such as benzodiazepines using both immunoassays and gas chromatography-mass spectrometry (GC-MS), we have noticed that the EMIT II benzodiazepine assays for urine samples sometimes gave false-negative results for bromazepam use or abuse. The negative benzodiazepine immunoassays and positive GC-MS results for bromazepam in urine could be explained by the absence (or below the detection limit) of 3-hydroxybromazepam (3HB) and the presence of 2-amino-3-hydroxy-5-bromobenzoylpyridine (AHBBP), which are the two major metabolites of bromazepam. 3HB cross-reacted with the antibody contained in the EMIT II, but AHBBP did not, due to their structural characteristics. To avoid such false-negative results for bromazepam, AHBBP screening by GC-MS is recommended. The false-negative results observed for bromazepam are probably not limited to the EMIT II kit; the same phenomena may be also true for other commercially available kits that are based on immunochemical reactions.     

标记免疫分析技术及其进展

主要介绍了放射免疫分析、酶免疫分析、发光免疫分析及荧光免疫分析的现状及近来发展的新技术,并对标记免疫分析技术的发展趋势及发展前景作了评价。     

Ultrastructural alterations and environmental exposure influence the opiate concentrations in hair of drug addicts

Hair samples were taken at autopsy from the head of 1 male and 1 female subject both known as drug abusers. Some of the strands were bleached by in-vitro cosmetic treatment. The bleached hair as well as the original hair samples were partly exposed to water or soil prior to further investigations and drug monitoring. The exposure times were 4 weeks or 6 months for water and 6 months for soil. The hair fibers were examined by transmission electron microscope (TEM) and by scanning electron microscope (SEM) investigations. The electron microscope studies confirmed that all experimental conditions had produced morphological alterations in the hair fibers. After exposure to water or to soil for 6 months as well as after storage of the clipped bleached hair in tap water at room temperature for 4 weeks, drug monitoring of formerly positive hair samples gave negative results. After storage of natural hair in soil or in water for 4 weeks the opiate levels had dramatically decreased. The samples were screened by fluorescence polarization immunoassay after enzymatic digestion. The results were confirmed by GC/MS.     

化学发光免疫分析

化学发光免疫分析技术基于放射免疫分析的基本原理,其特点是以化学发光物质为示踪物,灵敏度高,快速,简便,主要试剂包括氧化剂、发光剂和催化剂等,测试中不使用有害的试剂,因此成为非放射性免疫分析法中最有前途的方法。     

Zum Suchtmittelnachweis in Haaren. VII. Untersuchungen bei Methadonpatienten unter konstanter Wirkstoffeinnahme

Hair samples and perspiration collected from long term drug addicts (n = 18) under constant methadone treatment were investigated. Hair segments representing the individual hair growth of 4 weeks were analyzed for methadone, opiates and cocaine by GC/MS. The results obtained from the transdermal collection devices confirmed the presence of drug substances in variable amounts on the skin surface, methadone being the main analyte. The perspiration test period lasted for 4 days. For six persons illicit drugs similar to those in the corresponding hair samples were found. Overall, a poor relationship (r = 0,602) was found between the methadone dose under steady state conditions and the methadone level in hair. The results are discussed in the light of pharmacokinetic aspects, drug uptake by perspiration and forensic interpretation.     

Immunchemische und gaschromatographisch-massenspektrometrische Befunde im Blut bei Verdacht auf Drogenkonsum – 1. Mitteilung: Opiate, Kokain, Cannabis und Amphetamine

The extension of § 24 a of the road traffic regulations is expected to lead to more frequent investigations of blood for cannabinoids, opiates, cocaine (or metabolites) and amphetamines. Nearly 200 blood (serum) samples were analysed by the inhomogenous MTP-immunoassay for cannabinoids, opiates and amphetamines and 140 for cocaine consumption. In about half the specimens a gas chromatographic-mass spectrometric analysis followed. In generally immunassay results above 10 ng/ml for opiates, cannabinoids, or amphetamines and above 25 ng/ml for benzoylecgonine resulted in additional GC-MS analyses. Moreover cases with corresponding statements in the anamnesis were analysed gas-chromatographically otherwise only random tests were performed. Cannabinoids were detected in 47.4% of the samples, opiates in 31.1%, cocaine in 25.7% and amphetamines in 13.2%. The MTP-immunoassay has proved to be a good screening method to differentiate between probably positive and negative cases. In view of the threshold values above an offence is considered to have been proven, the results of our investigations result in Cut off values of 30 ng/ml with opiates and cannabinoids, 50 ng/ml with benzoylecgonine and 10 ng/ml with amphetamines. The tested assays have proven to be practicable and neglibable susceptible to interference. However it is recommendable to make a 3-point calibration before each series of samples.     

Zum Suchtmittelnachweis in Haaren

Hair samples collected from drug addicts (n=25) were analysed for opiates and cocaine by GC/MS. A decrease in drug concentration was observed even after a single bleaching or permanent wave application. The stability of the drug molecules in hair seemed to be different and cocaine was less affected than morphine or 6-acetylmorphine. When drug concentrations were low, the drug content often declined below the detection limit after cosmetic treatment. In hair samples which showed a drug content above 2 ng/ mg hair the qualitative detection of drugs was regularly successful. However, a distinct rule could not be established mainly due to influences of the individual hair matrix.     

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     

Development of an immunoassay for fluvoxamine detection using a recombinant single-chain variable fragment antibody

In forensic toxicology, immunoassays for drug screening are widely used because of the simple test procedures and instantaneous outcome of results. However, commercial immunoassay products are available for only a limited number of drugs. Preparation of antidrug antibodies is a crucial, but time-consuming in creating an immunoassay system. In this study, we focused on the application of a single-chain variable fragment (scFv) antibody for drug screening and developed a fluvoxamine (FLV) detection system for indirect competitive enzyme-linked immunosorbent assay (icELISA) using the scFv against FLV. To clarify the influence of domain order on the scFv binding activities, we prepared two kinds of scFv with different domain orders (HL, V_H-linker-V_L, and LH, V_L-linker-V_H) and examined their kinetic parameters against FLV. The scFvs showed sufficient FLV binding activities (K _D = 3.8 and 7.6 nM), and the HL scFv was slightly more favorable for FLV binding than the LH scFv. The developed icELISA using the HL scFv could detect FLV in the range of 10–200 ng/mL, and the scFv has no cross-reactivity below 100 μM except for chlorpromazine and imipramine. We also quantified the plasma FLV concentrations in forensic autopsy cases, and the results showed that this method could be applied effectively for FLV quantification without the need for extraction steps. Although recombinant antibodies against small molecule drugs for immunoassays have not yet been commonly used, we can predict that they could be a powerful tool to screen drugs in the near future, because of their advantages.     

Use of on-site immunoassay devices to screen urine absorbed in disposable diapers for methamphetamine: a preliminary study with artificial urine

The aim of this preliminary study was to establish a simple, rapid method for recovering urine absorbed in disposable diapers in order to test for methamphetamine (MAP) using the Instant-View™ M-1 and Triage^? DOA on-site immunoassay devices. A 4-ml aliquot of drug-free artificial urine was absorbed into a disposable diaper (Pampers^?) that had been cut into 3 × 3 cm pieces. Further addition of 4 ml of saturated KCl solution to the piece of diaper led to the recovery of substantial amounts (c.a. 2 ml) of fluid sample within 3–5 min. After diluting recovered fluids two-fold with distilled water, both immunoassays showed all samples were negative for all drug classes. After absorption of artificial urine containing 500–5,000 ng/ml of MAP in similar-sized pieces of diaper using the identical processing method, positive results were observed with Instant-View™ M-1 for artificial urine containing 2,000 ng/ml or more of MAP and with Triage^? DOA for urine containing 4,000 ng/ml or more of MAP. Diapers dosed with artificial urine containing 500, 2,500, and 5,000 ng/ml of MAP were further examined by gas chromatography–mass spectrometry, with recoveries of MAP of 98.6 ± 36.7 % (n = 6), 115 ± 22.4 % (n = 6), and 102 ± 15.1 % (n = 6), respectively. Use of this new sample preparation method may be applicable for analyzing infant urine absorbed in disposable diapers. Additionally, the sensitivity of the method along with the availability of Instant-View™ M-1 screening suggests the potential usefulness of this technique in clinical settings.     

Specificity of immunoassays. I. Effect of plasma proteins on the specificity of steroid immunoassay.

The theory of the measurement of the specificity of antigen binding by antibodies is reviewed. The specificity of steroid immunoassay has been investigated using a new solid phase radioimmunoassay system. The effects of plasma proteins on the specificity of the immunoassays have been tested. The methods used and the conclusions reached have been tested with homogeneous antibodies in order to simplify the interpretation of the results obtained. A new criterium of antibody homogeneity is proposed. The importance of correcting all results accurately for "non-specific binding" is emphasised. A new method for plotting radioimmunoassay standard curves is presented.     

电化学发光免疫法与放射免疫法检测血清皮质醇的对比研究

目的采用电化学发光免疫（ECLIA）法与放射免疫（RIA）法测定血清皮质醇水平,对其结果进行对比分析。 方法采用简单随机抽样法抽取我院健康体检者、门诊和住院患者共188例血清样本,利用ECLIA法和RIA法同时进行检测并进行统计学分析。将质控血清进行两两对比分析,计算两种方法批内、批间差异系数（CV）,评价其精确性;将质控血清加入低、中、高（100.2、201.9、310.7 nmol/L）已知浓度的皮质醇血清中,测定回收率。对样本测定结果的相关性作回归性分析。 结果ECLIA法检测血清皮质醇表达水平的灵敏度和特异度均高于RIA法。ECLIA法检测的批内与批间CV均低于RIA法,但两种方法回收率相当。两种方法检测结果具有高度相关性（r=0.991,P < 0.01）。 结论在检测血清皮质醇水平上,两种方法各有优势,ECLIA法自动化程度高,RIA法技术成熟、价格低廉。随着技术的发展,ECLIA法要更具优势,但ECLIA法在检测皮质醇方面是否可以完全替代RIA法,还有待进一步的探讨。     

Estimation of time of death by quantification of melatonin in corpses

A method for the estimation of time of death (TOD), was evaluated by measuring the melatonin (MT) content of pineal bodies (PBs), sera and urine samples from 85 cadavers. A total of 44 cadavers were investigated in Sapporo (geographical coordinates N 43° 4, E 141° 21) and 41 in Tokyo (N 35° 39, E 139° 44). MT contents were measured by radioimmunoassay (RIA) in 75 PBs, 27 sera and 14 urine samples. Exponential differences of pineal MT content were recognized between peaks in nighttime and nadirs in daytime, ranging from 0.099 to 63.2 ng/PB. Circadian rhythms were also observed for the concentrations of MT in serum (11–205 pg/ml), and in urine (7.5–137.5 pg/ml). Consequently, criteria for the TOD estimation are proposed as follows. 1) Pineal MT contents — (1) 0–0.2 ng/PB: TOD 1100–1700 hours, (2) 0.2–0.3 ng/PB: TOD 0700–2000 hours, (3) 0.3–1 ng/PB: inconclusive, (4) 1–4 ng/PB: TOD 1600–1000 hours, (5) 4–8 ng/PB: TOD 2000–0800 hours, (6) over 8 ng/PB: TOD 2000–0500 hours, 2) Serum MT concentration — (1) 0–100 pg/ml: inconclusive, (2) over 100 pg/ml: TOD 2200–0100 hours, and 3) Urinary MT concentration — (1) 0–35 pg/ml: inconclusive, (2) over 35 pg/ml: TOD 1800–0600 hours. The range of the estimation can be limited by a combination of these 3 criteria. The present method can be combined with other methods for estimating the TOD to decrease the range.     

头低位—6°卧床中血,尿中Cu,Zn—SOD的酶免疫法测定

报道了用辣根过氧化物酶作标记，测定人血，尿Ｃｕ，Ｚｎ－ＳＯＤ含量的高灵敏度酶联免疫反应体系。该方法在０．０５－５ｎｇ范围内检测重复性好，灵敏度比以前报道的放射免疫法和栈联免疫法灵敏度高。批内实验和批是实验的变异系数分别为１．１７－７．９４％和１．９４－１０．７３％。血和尿Ｃｕ，Ｚｎ－ＳＯＤ的平均回收率分别为１０１．０－１１３．８％和９６．０－１１６．０％。用该方法测定人血、尿Ｃｕ－Ｚｎ－ＳＯＤ含量     

21天头低位(-6°)卧床对机体摄入营养状况和头发Zn、Ca、Fe、Cu含量的影响

目的探讨模拟失重对机体摄入营养状况和头发Ｚｎ、Ｃａ、Ｆｅ、Ｃｕ含量的影响。方法受试者－６°头低位卧床２１天,用称量法观察其营养素摄入量变化,记录卧床第１天、１１天和第２１天的体重、尿量和上臂围,同时收集受试者头发,用原子火焰吸收分光光度法测定头发Ｚｎ、Ｃａ、Ｆｅ、Ｃｕ元素的含量。结果膳食Ｚｎ、Ｃａ、Ｆｅ、Ｃｕ在模拟失重第２周降低,第３周后基本恢复正常。头发中的含量第１１天较第１天显著下降（Ｐ＜０．０１）,第２１天恢复原来水平。尿量增加,体重和上臂围无明显变化。结论模拟失重对机体摄入营养状况和头发Ｚｎ、Ｃａ、Ｆｅ、Ｃｕ含量有短期影响,经过适应期后可恢复到正常范围。     

Determination of dihydrocodeine in hair of opiate addicts by GC/MS

Summary After the examination of more than 300 hair samples of suspected heroin abusers, a large number of which proved positive, we can say that high concentrations of dihydrocodeine can be determined either in addition to, or in the place of, morphine and also frequently in combination with codeine. The opiates were extracted after dissolving the hair samples in NaOH and hydrolysis with HCI. The quantitative determination of dihydrocodeine was achieved by derivatisation with HFBA using GC/MS at m/u = 497. Dihydrocodeine is used in antitussive drugs. After the examination of individual hair samples, it was obvious that some heroin consumers had switched to dihydrocodeine. This may lead to the conclusion that dihydrocodeine itself is used either as an intoxicating drug or to reduce withdrawal symptoms. The increasing number of positive samples should be noted by the legal authorities.