Multispot, multianalyte, immunoassay

Consideration of the basic principles of immunoassay design reveals that highly sensitive assays can, in principle, be developed using amounts of "sensor" antibody far smaller than are currently conventional in this field. Furthermore, when using such amounts, the fractional occupancy of antibody binding sites by analyte is independent of both sample volume and antibody concentration. Labelling of both the sensor-antibody and a developing antibody (designed to recognize either occupied or unoccupied sensor-antibody binding sites) permits the development of "ratiometric" immunoassays relying on measurement of the ratio of signals emitted by the two labelled antibodies. Furthermore, the sensor-antibody can be located within a "microspot" a few microns 2 in area. By labelling both sensor and developing antibodies with fluorescent labels, and scanning the microspot using a highly focussed laser beam, microspot immunoassays at least comparable in sensitivity with conventional "macroscopic" immunoassays are made possible. This in turn permits the development of immunoassay "arrays" capable in principle of measuring very large numbers of different substances within small samples (such as a drop of blood). The general principles and theory underlying these concepts are discussed, and preliminary experimental data using currently available instrumentation reported.     

应用蛋白微阵列同时检测人血清抗ToRCH抗体

目的：建立一种基于多元蛋白微阵列的免疫检测方法，用于同时检测人血清中识别弓形虫（TOXO）、风疹病毒（RV）、巨细胞病毒（CMV）、单纯疱疹Ⅰ型、Ⅱ型病毒（HSV-1、HSV-2）的特异性抗体。方法：将TOXO、RV、CMV、HSV-1和HSV-2抗原喷印到活化的玻片表面，抗原与活化基团共价结合后，制成蛋白微阵列。固定在玻片表面的抗原与病人血清中的特异性抗体反应、结合后，加入荧光标记的二抗，然后用高分辨率的激光共聚焦芯片扫描系统对蛋白微阵列进行扫描成像。所获得的图像用自行开发的软件进行分析，并自动判定并生成待测样本的定性结果。结果：使用此套蛋白微阵列系统检测抗TORCH抗体参考品，并与其它多种检测方法进行比较，各检测项目的符合率分别为92％～100％、92％～100％、96％、84％～96％、76％-96％。结论：多元蛋白微阵列法特异性强，灵敏度、准确度、精密度较高，具有应用和推广价值。     

Simultaneous detection of platelet and lymphocyte antibodies by slide enzyme immunoassay

The feasibility of using an enzyme immunoassay (EIA) to detect simultaneously the antibodies bound to platelets and lymphocytes on glass slides was investigated. A peroxidase-antiperoxidase (PAP) immune complex method was used in which lymphocytes and platelets were attached to glass slides by poly-L-lysine and the preparations were stored at -20 degrees C for subsequent assays. Test sera were incubated with the cells. Reagents linking the platelet and/or leukocyte antibody to the PAP enzyme marker were added, followed by a staining step to localize the antigen-antibody complex. Any brown staining of the perimeter of the cells and in the cytoplasm was considered indicative of a positive reaction. A total of 146 sera were assayed; 36 were from Roswell Park Memorial Institute (RPMI) plasmapheresis donors, 75 were from RPMI oncology patients, and 35 were from other laboratories. The PAP method agreed with complement lysis inhibition assay (CLIA) by 97 percent in detecting antibodies capable of reacting with platelets while concordance with complement dependent lymphocytotoxicity (CDL) was 81 percent. With further investigation, this method could be adapted as a screening procedure in the clinical laboratory. It is easy, inexpensive, and could be performed in a few hours.     

Simultaneous detection of antibodies to six nonhuman-primate viruses by multiplex microbead immunoassay

To maintain healthy nonhuman primates for use in biomedical research, animals are routinely screened for several infectious agents at most facilities. Commonly, monkey serum samples are tested by conventional immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) or Western blotting, for antibodies to specific infectious agents. For testing for antibodies against multiple agents in each sample, conventional immunoassays are laborious and time-consuming. More efficient immunoassays are needed. Accordingly, we have developed a novel multiplex serodiagnostic system based on individually identifiable, fluorescent microbead sets, where each bead set is coated with antigens from a purified preparation of a specific virus. The coated bead sets are mixed to enable the detection of antibodies to multiple viruses in one serum or plasma sample. These viruses include four agents that are routinely tested for maintenance of specific-pathogen-free monkeys, namely, simian immunodeficiency virus, simian type D retrovirus, simian T-cell lymphotropic virus, and herpes B virus, as well as simian foamy virus and rhesus cytomegalovirus, both of which are commonly found in nonhuman primates. This multiplex microbead immunoassay (MMIA) enabled the simultaneous detection of antibodies to all six viruses in single serum samples as small as 1 microliter. The results obtained by MMIA analysis correlated with results of conventional ELISAs, which detect antibodies to single agents. Thus, this multiplex microbead detection system is an efficient diagnostic modality for serosurveillance of nonhuman primates.     

The histopathology of drugs of abuse

The use of drugs for recreational purposes is widespread. The drugs used can be divided into groups including stimulants (cocaine, amphetamines, etc.), opiates and opioids (heroin, oxycodone, methadone, fentanyl, etc.), sedatives (benzodiazepines and related substances) and miscellaneous drugs, including ketamine and cannabis (marijuana). These drugs can have profound effects on all organ systems in the body. The method of administration, whether by injection or inhalation, can cause localized and systemic effects, including the transmission of infection and granulomata at the site of injection and in the lungs. Suppurative abscesses from injection can result in systemic amyloidosis. Stimulants have profound effects on the cardiovascular and cerebrovascular systems, with enlarged hearts with fibrosis seen microscopically and cerebral infarction and haemorrhage. Crack cocaine use is also associated with changes in the pulmonary system, including carbon pigmented intra-alveolar macrophages, emphysema and pulmonary arterial changes. Cannabis use is associated with brown pigmented macrophages in the lung as well as changes in the respiratory tract epithelium. Opiates/opioids are associated with inhalational pneumonitis and hypoxic brain damage due to their respiratory depressant effects. Heroin use has been associated with focal segmental glomerulonephritis (heroin-associated nephropathy: HAN). 3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) use is associated with changes in the cardiovascular system. Its use can lead to hyperpyrexia, which results in systemic changes. Ketamine abuse has been associated with cystitis. Drugs of abuse may affect testicular function. In analysing the effects of drugs at autopsy a systematic approach to sampling of histology is required.     

Investigating drugs of abuse at autopsy

Post-mortem toxicology is an important diagnostic part of the investigative process, not least when drugs of abuse are suspected, some of which require special attention. This review summarises key information regarding current common drugs of abuse, including amphetamines, benzodiazepines, cannabis, cocaine, ketamine, pregabalin as well as new psychoactive substances (NPS). The importance of sufficient and appropriate information (such as case circumstances and drug history) in addition to scene evidence and other considerations (including analytical and sampling factors) are presented that are relevant to the interpretation of results and the pathological process.     

Investigating drugs of abuse at autopsy

Post-mortem toxicology is an important diagnostic part of the investigative process, not least when drugs of abuse are suspected, some of which require special attention. This updated article summarizes key information regarding current common drugs of abuse, including amphetamines, benzodiazepines, cannabis, cocaine, ketamine, pregabalin as well as new psychoactive substances (NPS). There have also been recent trends in substances used in suicide (e.g. gases and chemicals). The importance of sufficient and appropriate information (such as case circumstances and drug history) in addition to scene evidence and other considerations (including analytical and sampling factors) are presented that are relevant to the interpretation of results and the pathological process.     

Preclinical assessment of abuse liability of drugs

Studies that are used in preclinical assessment of the liability of a drug to become an abuse problem are reviewed. These studies examine the capacity of a drug to produce physiological dependence or to function as a reinforcer. Studies that examine physiological dependence by assessing whether a drug reverses signs of withdrawal from a standard drug are rapid, reliable and inexpensive methods for determining if a drug produces dependence of a type similar to the standard. However, these techniques will not determine if the drug produces a unique type of dependence. Studies that examine whether a drug functions as a reinforcer have been predictive of whether a drug will be abused in human populations. Attempts to rank order drugs with respect to their efficacy as reinforcers, however, are not predictive of measures of extent of abuse in human populations. Since abuse of drugs in human populations is a function of societal variables in addition to pharmacological factors, it is unlikely that preclinical assessments will ever yield more than qualitative information on abuse liability of drugs.     

Hypnotic drugs: Use and abuse

Hypnotic drugs have a number of pharmacologic properties which contribute to their safety and efficacy as well as to their abuse potential. Benzodiazepines are among the safest of all depressant compounds and do not lose their effectiveness as quickly as do nonbenzodiazepine drugs. They differ in their half-lives and duration of action. Short- and intermediate-acting benzodiazepines can cause rebound insomnia and rebound anxiety in certain patients but can aid in daytime alertness in others. Long-acting benzodiazepines can impair daytime performance and cause daytime sleepiness but can be useful in minimizing withdrawal effects. Selection of a proper hypnotic should depend on the clinical situation and the needs of the patient. Principles for proper use are discussed.     

Simultaneous quantitation of diphtheria and tetanus antibodies by double antigen, time-resolved fluorescence immunoassay

A dual, double antigen, time-resolved fluorescence immunoassay (DELFIA) for the simultaneous detection and quantitation of diphtheria (D) and tetanus (T) antibodies in sera has been developed. In the double antigen format one arm of the antibody binds to antigen coated microtitre wells and the other arm binds to labelled antigen to provide a fluorescent signal. This assay was found to be functionally specific for IgG antibodies and showed a good correlation with established toxin neutralization assays. Furthermore, the double antigen set-up was species independent, permitting the direct use of existing international references of animal origin to measure protective antibody levels in humans in international units (IU/ml). The detection limit corresponded to 0.0003 IU/ml with Eu³⁺-labelled toxoids and to 0.0035 IU/ml using Sm³⁺-labelled toxoids. The assay was fast with a high capacity making it a suitable method for serological surveillance studies.     

On the abuse of marihuana and other drugs

The negative effect on motivation of marihuana and other psychedelic drugs is discussed. It is concluded that the problem is not whether the drugs should be legalized but if it is possible to enforce their prohibition.     

Microbial infections,immunomodulation, and drugs of abuse

The use of recreational drugs of abuse has generated serious health concerns. There is a long-recognized relationship between addictive drugs and increased levels of infections. Studies of the mechanisms of actions of these drugs became more urgent with the advent of AIDS and its correlation with abused substances. The nature and mechanisms of immunomodulation by marijuana, opiates, cocaine, nicotine, and alcohol are described in this review. Recent studies of the effects of opiates or marijuana on the immune system have demonstrated that they are receptor mediated, occurring both directly via specific receptors on immune cells and indirectly through similar receptors on cells of the nervous system. Findings are also discussed that demonstrate that cocaine and nicotine have similar immunomodulatory effects, which are also apparently receptor mediated. Finally, the nature and mechanisms of immunomodulation by alcohol are described. Although no specific alcohol receptors have been identified, it is widely recognized that alcohol enhances susceptibility to opportunistic microbes. The review covers recent studies of the effects of these drugs on immunity and on increased susceptibility to infectious diseases, including AIDS.     

Simultaneous detection of antibodies to mouse hepatitis virus recombinant structural proteins by a microsphere-based multiplex fluorescence immunoassay

We describe a new microsphere-based multiplex fluorescent immunoassay (MFI) using recombinant mouse hepatitis virus (MHV) proteins to detect antibodies to coronaviruses in mouse and rat sera. All the recombinant proteins, including nucleocapsid (N) and 3 subunits of spike protein, S1, S2, and Smid, showed positive reactivity in MFI with mouse antisera to 4 MHV strains (MHV-S, -A59, -JHM, and -Nu67) and rat antiserum to a strain of sialodacryoadenitis virus (SDAV-681). The MFI was evaluated for its diagnostic power, with panels of mouse sera classified as positive or negative for anti-MHV antibodies by enzyme-linked immunosorbent assay (ELISA) using MHV virion antigen and indirect fluorescent antibody assay. The reactivities of 236 naturally infected mouse sera were examined; 227 samples were positive by MFI using S2 antigen (96% sensitivity), and 208 samples were positive using N antigen (88% sensitivity). Based on the assessment by MFI using the S2 and N antigens, only 3 serum samples showed double-negative results, indicating a false-negative rate of 1.3%. In 126 uninfected mouse sera, including 34 ELISA false-positive sera, only 7 samples showed false-positive results by MFI using either the S2 or N antigen (94% specificity). Similarly, the S2 and N antigen-based MFI was 98% sensitive and 100% specific in detecting anticoronavirus antibodies in rat sera. Thus, this MFI-based serologic assay using the S2 and N antigens promises to be a reliable diagnostic method, representing a highly sensitive and specific alternative to traditional ELISA for detection of coronavirus infections in laboratory mouse and rat colonies.     

Free radical mediated release of histamine from rat mast cells induced by drugs of abuse

Inflammation Research -     

Self-reported use of psychotropic drugs and alcohol abuse in South-Verona

The data for this cross-sectional survey were obtained from a stratified probability sample of 510 residents in South-Verona during the period May-June 1980. The two-week prevalence of psychotropic drug consumption was found to be 13%. The prevalence was higher in women (18%) than in men (9%). The most commonly consumed category of psychotropic drugs were the benzodiazepines, which accounted for 70% of all the psychotropic drugs consumed by women and 85% of those consumed by men. Using a logistic analysis, a strong association between drug consumption and minor psychiatric morbidity, as indicated by the GHQ scores, was found for both sexes, a finding not significantly different from that found in the West London study by Murray et al. (1981). Alcohol abuse in men was less prevalent in the youngest age-group. Thirty-three (19%) of the male GHQ low scorers were alcohol abusers, compared with 12 (40%) of the high scorers. No significant association was found between psychotropic drug consumption and alcohol abuse in men.