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.     

器官移植患者红细胞膜流动性与环孢素A药代动力学的动态分析

目的 探讨肝、肾移植术后,患者红细胞膜流动性与环孢素A（CsA)药代动力学相关性。方法 采用荧光偏振免疫分析法（FPLA）测定CsA全血药物浓度,DPH荧光探针法测定红细胞膜流动性。结果 肾移植术后患者红细胞膜流动性的动态改变与血中环孢素A药物浓度的变化没有相关性,但满一年的患者红细胞膜流动性与体内CsA谷浓度呈负相关。肝移植术后患者红细胞膜流动性的动态改变与血中CsA药物浓度的变化呈负相关。结论 器官移植术后患者长期服用CsA,导致细胞膜流动性降低,是肝肾损害和术后并发症的病理学基础。建议器官移植术后监测患者红细胞膜流动性的变化,合理使用免疫抑制剂。     

Glycogen detection by in vivo 13C NMR: a comparison of proton decoupling and polarization transfer.

The performance of gated proton decoupling and polarization transfer with respect to glycogen detection by 13C NMR was investigated. Experiments were performed on a 1.5-T whole-body scanner using a 13C surface coil in combination with a proton head coil. Spectra were acquired from a glycogen phantom and from the lower leg of a healthy volunteer using proton decoupling and the polarization transfer method SINEPT. The signal strength of the C1 resonance of glycogen was determined and compared to a reference spectrum acquired without any form of sensitivity enhancement. In the phantom experiment both decoupling and SINEPT produced a signal gain of 3.5. Under in vivo conditions, the signal gain was approximately 2.5 for both techniques. We conclude that decoupling and polarization transfer are equivalently useful techniques for glycogen detection.     

Volume localized shift selective 13C spectroscopy using pulsed rotating frame transfer sequences with windows (PRAWN)

Some novel techniques for volume localized, chemical shift selective ¹³C spectroscopy are described in this work. These techniques are based on rotating frame J cross polarization and are reported for both direct and indirect modes of ¹³C detection. The performance of two selective mixing sequences, viz., pulsed rotating frame transfer sequences with windows (PRAWN) and PRAWN‐π has been studied systematically with different liquid and gel phantoms. Two different front‐end modules are used for volume localization, viz., point resolved spectroscopy (PRESS) and localized distortionless enhancement by polarization transfer (LODEPT). It is shown experimentally that both the selective J cross polarization sequences can operate efficiently with very low radiofrequency duty cycle; further, they have considerable tolerance to Hartmann‐Hahn mismatch. A simple theoretical analysis is also presented to understand J cross‐polarization dynamics at low RF field amplitudes. Finally, the performance of LODEPT‐PRAWN‐π is demonstrated for the selective detection of saturated fat in pigeon egg in indirect detection mode. Magn Reson Med, 2011. © 2011 Wiley Periodicals, Inc.     

Proton-enhanced 13C imaging/spectroscopy by polarization transfer

Carbon-13 magnetic resonance imaging/spectroscopy (CMRI/S) was performed using polarization transfer techniques where sensitivity of the carbon signal was enhanced by transferring the proton spin order to the carbon nuclei. The experimental feasibility of using polarization transfer techniques at 2.0 T was demonstrated with a phantom and an intact chicken egg. The potential clinical applications of CMRI/S with polarization transfer include the assessment of prostate cancer. Preliminary results using human prostate specimens are presented.     

Methods for the detection of salmonellae in food

Presented are results of modern immunological and culture medium techniques for the salmonella isolation from chicken trunks compared with those obtained by standard technique (ST). By using fluorescent antibody technique (FAT), enriched serum techniques (ES) and immune stain-reaction (ISR) a larger number of positive samples was detected (31%-41%) in comparison with standard culture medium techniques (ST) (25%). Statistically significant differences were obtained by FAT. By both Rappaport-Vassiliadis (RV) and modified semi-solid Rappaport-Vassiliadis (MSRV) culture medium techniques about thrice higher percentage of salmonella positive findings were obtained than in comparison with the standard techniques. Compared to RV, the MSRV culture media technique reduces the salmonella isolation period for 48.     

Comparison of performance of genetics 4N6 FLOQSwabs™ with or without surfactant to rayon swabs

The collection of traces is the first step in the process of forensic genetics analysis. Currently, several different techniques are used (eg. gauze). Nevertheless, swabbing appears to be the most common of these. In a second step, the sampling devices should allow the use of preliminary tests in combination with an immunological confirmatory test (e.g. Hexagon Obti or Hemdirect). Our previous study shows that sampling with Genetics 4N6FLOQswabs™ coated with surfactant reduces by a factor of at least 100 the detection threshold of blood using two immunological tests. The aim of this work was to compare the ability to recover blood trace and the compatibility with immunological confirmatory test of various Genetics 4N6FLOQswabs™ nylon flocked swabs with or without surfactant.The results obtain in this study show that Genetics 4N6FLOQswabs™ not coated with surfactant and Human DNA free FLOQswabs™ were suitable for the used in combination with immunological blood detection tests. Nevertheless, the Genetics 4N6FLOQswabs™ not surfactant coated give a better blood trace recovery.     

低剂量辐射诱导免疫适应性反应的实验研究

目的　研究低剂量辐射能否诱导免疫适应性应。方法　３ＨＴｄＲ 掺入法检测淋巴细胞增殖率;ＣＴＬＬ依赖株检测ＩＬ２ 活性;单克隆抗体免疫荧光标记,流式细胞术（ＦＣＭ） 检测淋巴细胞亚组及膜受体表达;ＰＩ标记,ＦＣＭ 检测细胞周期及细胞凋亡;双抗体夹心间接ＥＬＩＳＡ 法测定可溶性白介素２ 受体;Ｆｕｒａ２ 负载,双波长荧光测定法检测细胞内［Ｃａ２＋ ］ｉ;传代培养细胞测定细胞倍增时间。结果　低剂量辐射预照射可明显减轻其后大剂量照射对诸多淋巴细胞功能的抑制作用。结论　低剂量Ｘ 射线可诱导多参数免疫适应性反应。     

In vivo imaging of chromogranin A-positive endocrine tumours by three-step monoclonal antibody targeting

The detection of chromogranins (Cg) by immunohistochemistry and serology represents a new in vitro diagnostic tool for endocrine tumours. We have recently reported on the feasibility of targeting chromogranin A (CgA) for in vivo detection of pituitary adenomas by immunoscintigraphy (ISG). The scintigraphic procedure, based on an anti-CgA monoclonal antibody and on the avidin-biotin three-step method (Cg-3S-ISG), was evaluated on a group of 29 consecutive patients with known or suspected endocrine tumours other than pituitary adenomas, i.e. medullary thyroid carcinoma, carcinoid, insulinoma and parathormone- or ACTH-producing tumours. Primary tumours (10) and recurrences (16) were visualised in 26 patients, whereas conventional imaging techniques (planar radiography, computerised tomography, magnetic resonance imaging and ultrasonography) failed to detect the tumour sites in ten of the same (Cg-3S-ISG-positive) patients. Therefore, these preliminary results indicate that Cg-3S-ISG, the first immunological method able to detect endocrine tumours in vivo, has a higher diagnostic accuracy than conventional imaging techniques (93.1% compared with 65.5%).     

Increase in SNR for 31P MR spectroscopy by combining polarization transfer with a direct detection sequence

The sensitivity of (31)P MRS can be increased using higher magnetic fields, but also by using (1)H to (31)P polarization transfer techniques where the sensitivity is determined by the polarization of the proton spins and thus the signal-to-noise per unit time is unaffected by the slow T(1) relaxation properties of the (31)P spins. This implies that (31)P spins can be manipulated during the T(1) relaxation of the (1)H spins without affecting the signal-to-noise of the (1)H to (31)P polarization transferred spins. It is shown here that by combining (1)H to (31)P polarization transfer with a direct (31)P detection sequence in one repetition time, one can gain more signal-to-noise per unit of time as compared to a polarization transfer sequence alone. Proof of principle was demonstrated by phantom measurements and additionally the method was applied to the human calf muscle and to the human breast in vivo at 7 T.     

In vivo detection of a pH-sensitive nitroxide in the rat stomach by low-field ESR-based techniques.

A study was made of the in vivo detectability of a pH-sensitive, imidazolidine spin probe, and the efficacy of low-frequency electron spin resonance (ESR)-based techniques for pH measurement in vitro and in vivo in rats. The techniques used were longitudinally-detected ESR (LODESR) and field-cycled dynamic nuclear polarization (FC-DNP) for in vitro and in vivo measurements, and radiofrequency (RF)- and X-band ESR for comparisons in vitro. The spin probe was hexamethyl imidazolidine (HMI) with a pK of 4.6. All techniques detected HMI. Detection by FC-DNP implies coupling between the free radical and solvent water spins. Separations between the three spectral lines of the nitroxide radical, relative to measurement frequency, were consistent with theory. The overall spectrum width from unprotonated HMI (pH > pK) was greater than that from protonated agent (pH < pK). This was observed in vitro and in vivo. Longer-term studies showed that HMI is detectable and has the same spectral width (i.e., is at the same pH) up to 2 hr after gavage into the stomach, although the magnitude of the signal decreases rapidly during the first hour. These findings demonstrate the suitability of LODESR and FC-DNP for monitoring HMI and measuring pH in vivo. These techniques would be useful for monitoring disease and drug pharmacology in the living system.     

Urinary tract infection in infants and children: an update with special regard to the changing role of reflux

The aim of this study was to present current theories of pathogenesis and prognosis in urinary tract infection (UTI) and renal scarring during infancy and childhood, with special regard to new insights concerning the role of vesico-ureteral reflux (VUR). For a long time VUR and UTI were considered the only risk factors for renal scarring in childhood. Now a wider spectrum of contributing conditions is commonly accepted, which all may pose different clinical consequences and require different imaging approaches. Particularly bilateral renal scarring causes long-term sequalae; therefore, renal involvement in UTI with potential scarring has become the clinical and imaging focus, and proper diagnosis of UTI as the key factor for further management has become even more important. The VUR still remains one issue on a list of important factors such as treatment onset and response, bacterial virulence, immunological factors, genetic disposition, anatomical variants, and lower urinary tract dysfunction. Recent advances in knowledge leading to changed patho-physiological concepts, and new imaging techniques, may consecutively impact the presently established standard imaging algorithms. New, advanced imaging techniques offer improved and accelerated comprehensive imaging of the paediatric urinary tract. At present, this is complimentary to the established gold standard techniques. Strong research efforts have to be made before suggesting significant changes of current imaging concepts; however, based on recent technical advances and new insight on the natural history of paediatric urological diseases, potential changes of established imaging algorithms need to be discussed and evaluated.     

Urinary tract infection in infants and children: an update with special regard to the changing role of reflux

The aim of this study was to present current theories of pathogenesis and prognosis in urinary tract infection (UTI) and renal scarring during infancy and childhood, with special regard to new insights concerning the role of vesico-ureteral reflux (VUR). For a long time VUR and UTI were considered the only risk factors for renal scarring in childhood. Now a wider spectrum of contributing conditions is commonly accepted, which all may pose different clinical consequences and require different imaging approaches. Particularly bilateral renal scarring causes long-term sequalae; therefore, renal involvement in UTI with potential scarring has become the clinical and imaging focus, and proper diagnosis of UTI as the key factor for further management has become even more important. The VUR still remains one issue on a list of important factors such as treatment onset and response, bacterial virulence, immunological factors, genetic disposition, anatomical variants, and lower urinary tract dysfunction. Recent advances in knowledge leading to changed patho-physiological concepts, and new imaging techniques, may consecutively impact the presently established standard imaging algorithms. New, advanced imaging techniques offer improved and accelerated comprehensive imaging of the paediatric urinary tract. At present, this is complimentary to the established gold standard techniques. Strong research efforts have to be made before suggesting significant changes of current imaging concepts; however, based on recent technical advances and new insight on the natural history of paediatric urological diseases, potential changes of established imaging algorithms need to be discussed and evaluated.     

Sensitivity enhancement in whole-body natural abundance 13C spectroscopy using 13C/1H double-resonance techniques at 4 tesla.

In vivo 13C spectroscopy experiments were performed using a whole-body MR system at a static field of 4 T. The main goal of the investigations was to evaluate the sensitivity increase achievable by means of 13C/1H double-resonance techniques at 4 T. Spectra from subcutaneous fat as well as muscle glycogen from the lower leg were acquired using frequency selective proton decoupling and the polarization transfer method SINEPT. With respect to measurements on subcutaneous fat, polarization transfer turned out to be more efficient than selective decoupling. About a fourfold enhancement in spectral peak intensity for the C = C line doublet of the unsaturated fatty acid chain was obtained. Combining polarization transfer with decoupling yielded a factor of 6 in signal amplitude. In contrast to that, the signal enhancement observed in measurements on the glycogen C-1 resonance was only around twofold. The lower efficiency is explained by fast T2 relaxation of the proton transition. A T2 value of about 3 ms was derived from the experimental data. Acquisition times as low as 3 min were realized for normal level glycogen in human calf muscle, enabling a time resolution adequate for dynamic studies on muscle glycogen depletion. Aspects of RF power absorption in tissue and the generally higher efficiency make polarization transfer methods preferable to selective decoupling in whole-body 13C spectroscopy at 4 T.     

Modern screening strategies in analytical toxicology with special regard to new benzodiazepines

Summary Screening procedures for the detection of toxicologically relevant substances have become of ever-increasing importance due to the rapid development of new substances. Identification methods must be simple, sensitive, and practicable. This article describes standardized chromatographical (corrected R _f ^c values, retention indices) and immunological methods (enzyme-multiplied immunoassay technique, fluorescent polarization immunoassay) with special regard to the screening of some newer benzodiazepines, a class of substances that is still expanding. Some of these new compounds may be integrated in well-known screening procedures (via aminobenzophenones and detection by the Bratton-Marshall reagent); others require special concepts for detection. The problems are indicated and discussed, including the use of high-pressure-liquid chromatography and mass spectrometry; recommendations are given.In memoriam Johann Bösche     

单克隆抗体研究新进展

单克隆抗体技术是现代生命科学研究的重要工具，在基因和蛋白质的结构和功能研究方面有着不可或缺的作用。本文全面综述了单克隆抗体研究最新进展。通过构建真核表达载体，采用DNA免疫、细胞免疫、消减免疫和多位点重复免疫等现代分子免疫学方法，快速制备高亲合力的单克隆抗体，改进常规方法制备单抗费时费力且亲合力低的弱点，对后基因时代蛋白质组学等基础研究领域的研究具有巨大的推动作用；同时在生物芯片、临床医学和疾病的诊断与治疗以及空间生命科学等应用领域有着广阔的应用前景。     

Measurements of the persistent singlet state of N2O in blood and other solvents--potential as a magnetic tracer

The development of hyperpolarized tracers has been limited by short nuclear polarization lifetimes. The dominant relaxation mechanism for many hyperpolarized agents in solution arises from intramolecular nuclear dipole-dipole coupling modulated by molecular motion. It has been previously demonstrated that nuclear spin relaxation due to this mechanism can be removed by storing the nuclear polarization in long-lived, singlet-like states. In the case of N(2)O, storing the polarization of the nitrogen nuclei has been shown to substantially increase the polarization lifetime. The feasibility of utilizing N(2)O as a tracer is investigated by measuring the singlet-state lifetime of the N(2)O when dissolved in a variety of solvents including whole blood. Comparison of the singlet lifetime to longitudinal relaxation and between protonated and deuterated solvents is consistent with the dominance of spin-rotation relaxation, except in the case of blood.     

替硝唑对氨茶碱在兔体内药物动力学的影响

采用荧光偏振免疫分析法(PFIA)测定了单用氨茶碱和氨茶碱与替硝唑合用时免体内血清茶碱浓度,观察了替硝唑对氨茶碱药物动力学的影响。结果显示,替硝唑在与氨茶碱合并用药时,对茶碱在体内的药物动力学参数除K_(21)略升高外均无显著影响,表明替硝唑对氨茶碱的体内过程无影响。     

Molecular Imaging of Biological Gene Delivery Vehicles for Targeted Cancer Therapy: Beyond Viral Vectors

Cancer persists as one of the most devastating diseases in the world. Problems including metastasis and tumor resistance to chemotherapy and radiotherapy have seriously limited the therapeutic effects of present clinical treatments. To overcome these limitations, cancer gene therapy has been developed over the last two decades for a broad spectrum of applications, from gene replacement and knockdown to vaccination, each with different requirements for gene delivery. So far, a number of genes and delivery vectors have been investigated, and significant progress has been made with several gene therapy modalities in clinical trials. Viral vectors and synthetic liposomes have emerged as the vehicles of choice for many applications. However, both have limitations and risks that restrict gene therapy applications, including the complexity of production, limited packaging capacity, and unfavorable immunological features. While continuing to improve these vectors, it is important to investigate other options, particularly nonviral biological agents such as bacteria, bacteriophages, and bacteria-like particles. Recently, many molecular imaging techniques for safe, repeated, and high-resolution in vivo imaging of gene expression have been employed to assess vector-mediated gene expression in living subjects. In this review, molecular imaging techniques for monitoring biological gene delivery vehicles are described, and the specific use of these methods at different steps is illustrated. Linking molecular imaging to gene therapy will eventually help to develop novel gene delivery vehicles for preclinical study and support the development of future human applications.     

Parahydrogen-induced polarization in imaging: subsecond (13)C angiography.

High nuclear spin polarization of (13)C was reached in organic molecules. Enhancements of up to 10(4), compared to thermal polarization at 1.5 T, were achieved using the parahydrogen-induced polarization technique in combination with a field cycling method. While parahydrogen has no net polarization, it has a high spin order, which is retained when hydrogen is incorporated into another molecule by a chemical reaction. By subjecting this molecule to a sudden change of the external magnetic field, the spin order is transferred into net polarization. A (13)C angiogram of an animal was generated in less than a second. Magn Reson Med 46:1-5, 2001.