Solid-phase electrochemical enzyme immunoassay with attomole detection limit by flow injection analysis

A sandwich electrochemical enzyme immunoassay with flow injection analysis for the model antigen mouse IgG has been developed with alkaline phosphatase as the enzyme label. The enzyme substrate, 4-aminophenyl phosphate and its enzymatic reaction product, 4-aminophenol have been studied by cyclic and hydrodynamic voltammetry. The determination of 4-aminophenol by flow injection analysis with electrochemical detection (FIAEC) has a linear range of 5.0 × 10⁻⁸ to 1.0 × 10⁻⁵ M, a detection limit of 2.4 × 10⁻⁸ M, and a sample throughput of 72 samples/h. The detection limit is set by a background capacitance response, which depends on the ionic strength difference between the sample and the mobile phase. The sandwich immunoassay has been characterized with respect to substrate concentration for the enzymatic reaction, detection limit, dynamic range and sources of error. Mouse IgG can be determined with a detection limit of 0.81 pg ml⁻¹ by a 30-min substrate incubation time and a six orders of magnitude linear dynamic range.     

A new spectrophotometric method for the toxicological diagnosis of cyanide poisoning

A spectrophotometric method for the determination of hydrogen cyanide in biological fluids based on the release of cyanide ion by the addition of a strong acid and its subsequent specific reaction with hydroxocobalamin to give cyanocobalamin is proposed. The release of cyanide ion is accelerated by aeration with a stream of an inert gas (nitrogen) that carries it into the hydroxocobalamin solution. Although the in vitro reaction develops to completion within 20 min, reproducible quantitation in biological media takes 45 min. The cyanocobalamin formed is quantitated by second-derivative visible spectrophotometry from the absorbance difference between 333 and 361 nm, the measured signal being proportional to the cyanide ion concentration in the sample.     

Underestimation of rat serum vancomycin concentrations measured by an enzyme‐multiplied immunoassay technique and the strategy for its avoidance

An enzyme‐multiplied immunoassay technique (EMIT) has been widely adopted for the measurement of serum concentrations of vancomycin (VCM) in clinical practice. Because of the growing demand for its application to fundamental pharmacokinetic studies, we examined whether VCM concentrations in rat serum were accurately measured by EMIT. It was found that measured values of known amounts of VCM spiked to rat serum were markedly underestimated with a large analytical variance. When ultrafiltrated rat serum was used as the sample matrix, interference was significantly improved, and the degree of underestimation was attenuated also by diluting samples with physiological saline. These results suggest that endogenous substances of a high molecular weight in rat serum interfere with the analysis of VCM concentrations by EMIT. However, measured values of rat serum VCM concentrations by EMIT were restored to theoretical levels by exposing samples to 70°C for 3–7 min. A likely explanation for the avoidance of interference is that an appropriate thermal force eliminated the immunological function of endogenous substances falsely recognizing VCM without affecting the VCM molecule itself. Regarding serum samples collected from rats that were administered VCM, values measured by EMIT following the heat‐treatment agreed well with those by the high performance liquid chromatography (HPLC) method. This is the first report showing interference by endogenous high‐molecular substances in the measurement of drug concentrations in rat serum using EMIT. Our findings will contribute to the appropriate use of VCM based on evidence provided by clinical‐oriented rat experiments requiring the measurement of serum VCM concentrations by EMIT. Copyright © 2013 John Wiley & Sons, Ltd.     

Receptors and enzymes for medical sensing of L-glutamate

Medical sensing systems using isolated or intact glutamate receptor (GluR) ion channels and glutamate oxidase (GluOx) are discussed for L-glutamate, one of the principal neurotransmitter in the central nervous systems of mammalian brain, and related agonists. The GluR-based sensing system used for the evaluation of signal transduction ability of GluR channels demonstrate that the agonist selectivity based on the signal transduction ability is not parallel to that of the binding assay. On the other hand, the appropriate design of the enzyme system, namely glutamate oxidase (GluOx), in combination with horseradish peroxidase (HRP), enables to real-time monitoring of L-glutamate in vivo and in vitro and also to visualize its release in submerged, acute mouse hippocampal slices.     

Ethanol involvement in putrescine and histamine oxidation by guinea pig liver

The in vitro influence of ethanol and acetaldehyde on diamine oxidase catalyzed reaction was measured with guinea pig liver and intestine. Acetaldehyde, having no influence on diamine oxidase activity, diminished the formation of gamma-aminobutyric acid or imidazole-4-acetic acid when putrescine or histamine were used, respectively, as enzyme substrate. During ethanol ingestion by guinea pig the enhancement of hepatic diamine oxidase activity was observed after 10 days of treatment with subsequent decrease to the control value on the 18th day. The metabolic products of putrescine in diamine oxidase catalyzed reaction by livers of alcohol animals show similar increase and the same time course as enzymic activity. The per cent distribution of particular products in diamine oxidase reaction was not affected by ethanol administration.     

Mechanisms of oxygen activation by nitrofurantoin and relevance to its toxicity

Purified ferredoxin-(cytochrome c)-NADP+ oxidoreductase and xanthine oxidase were found to catalyse the reduction of nitrofurantoin to the free radical. Under aerobic conditions, the nitrofurantoin radical underwent autoxidation to regenerate the parent compound with the concomitant production of superoxide and eventually hydrogen peroxide. The nitrofurantoin radical was also shown to react with hydrogen peroxide to generate a highly reactive species which was capable of oxidising methionine to ethylene. This active oxygen radical appeared to be identical with the crypto-OH . radical, previously proposed as being formed from the analogous reaction of the methyl viologen radical with hydrogen peroxide [R.J. Youngman and E.F. Elstner, FEBS Lett. 129, 265 (1981)]. Catalase inhibited nitrofurantoin-dependent ethylene formation in both enzyme systems, whereas superoxide dismutase was only inhibitory in the xanthine oxidase mediated reaction. Although the primary function of the respective enzyme systems is to generate the nitrofurantoin radical, the xanthine oxidase reaction is markedly more complex than that of ferredoxin-(cytochrome c)-NADP+ oxidoreductase. The differences between the two enzyme reactions appear to be due to the endogenous autoxidation of xanthine oxidase. The aerobic activation of nitrofurantoin by xanthine oxidase involved the superoxide anion as an intermediate, whereas the nitrofuran was directly reduced by ferredoxin-(cytochrome c)-NADP+ oxidoreductase without a requirement for active oxygen species.     

Interaction of drugs with a model membrane protein: Effect of dibucaine on cytochrome oxidase proteoliposomes

Cytochrome oxidase is a mitochondrial trans-membrane protein which catalyzes the vectorial transfer of electrons from cytochrome c to molecular oxygen. When the oxidase was incorporated into liposomes composed of saturated phospholipids, enzymatic activity was reduced as compared to the activity of either the isolated enzyme or the enzyme incorporated into soy bean phospholipid (asolectin) liposomes. This reduced activity probably resulted from partial replacement of retained oxidase boundary lipid with exogenously added lipid and an unfavourable orientation of a portion of the oxidase molecules for reaction with externally added substrate. On the other hand, substrate binding at the low affinity site was enhanced by incorporation of the oxidase into vesicles composed of either saturated phospho-lipids or asolectin. At pH 7.4 the local anesthetic dibucaine behaved as an uncompetitive inhibitor of the enzyme, while at pH 6.0 the inhibition pattern became mixed in type. Dibucaine had similar effects on both the isolated and incorporated enzyme except that, in general, the anesthetic caused less inhibition of the incorporated oxidase. It is postulated that positively charged anesthetic molecules act predominantly by competing with substrate for binding while non-charged anesthetic molecules interact with the oxidase boundary lipid to form non-productive complexes.     

Effects of oxygen on the antagonism of cyanide intoxication: cytochrome oxidase, in vitro

Since oxygen was reported to be an effective cyanide antagonist in vivo, particularly in the presence of the classic antidotal combination of sodium nitrite and sodium thiosulfate, in vitro studies were initiated in an attempt to investigate the mechanism of oxygen-mediated cyanide antagonism. The effect of oxygen on cyanide-inhibited cytochrome oxidase with and without cyanide antagonist(s) was investigated in a purified membraneous enzyme system prepared from rat liver mitochondria. Cyanide produced a concentration dependent inhibition of cytochrome oxidase, and 100% oxygen did not alter the inhibition produced by KCN either in the presence or absence of sodium thiosulfate. However, the addition of sodium thiosulfate and rhodanese to the assay reactivated the cyanide-inhibited cytochrome oxidase. Kinetic analysis indicated rhodanese competes with cytochrome oxidase for cyanide, and oxygen had no effect on this coupled reaction. In conclusion, the in vivo antidotal properties of oxygen cannot be attributed to oxygen-mediated reactivation of cyanide-inhibited cytochrome oxidase or an oxygen-mediated acceleration of rhodanese detoxification.     

Beta-aminopropionitrile treatment can accelerate recovery of mice after spinal cord injury.

Modulations of the extracellular matrix and scar formation following central nervous system (CNS) injuries are considered prohibitive for axon regeneration, thus restricting functional recovery. Recent findings indicating that lysyl oxidase, an extracellular matrix-forming enzyme, appears in a time-dependent manner at brain injury sites have suggested that inhibition of this enzyme may be conducive for regeneration and functional recovery. Here, we report that after unilateral spinal cord transection in adult mice, daily treatment (for 20 days) with the lysyl oxidase inhibitor beta-aminopropionitrile (100 mg/kg intraperitoneal) resulted in accelerated and more complete functional recovery. The mode of functional recovery, however, indicates that axonal regeneration of long descending tracts did not occur.     

聚丁二炔生物传感器变色免疫法检测乳腺癌MCF-7细胞

目的 合成能特异性识别肿瘤细胞的聚丁二炔生物传感器。方法 超声乳化-共价修饰法制备聚丁二炔/磷脂(polydiacetylene/Phospholipid,PDA/PC)纳米囊泡,通过共价修饰法将鼠抗人细胞角蛋白抗体CK19共价修饰固定在PDA/PC纳米囊泡表面,制备可变色的PDA/PC生物传感器,用于检测肿瘤细胞。透射电镜负染技术,激光散射粒径测定仪、紫外-可见分光光度计扫描,计算比色响应(colorimetric response,CR)等对合成的PC/PDA生物传感器进行表征,以乳腺癌细胞株MCF-7为模型,模拟考察PDA/PC生物传感器识别鉴定外周血循环肿瘤细胞(circulating tumorcells,CTCs)可行性。结果 TEM检查结果显示制备的PDA/PC生物传感器呈球形或类球形、粒径均匀,平均粒径约为(223.4±23.6)nm;激光散射粒径分析仪显示生物传感器的强均粒径和多分散系数为298.4 nm和0.184;特异性抗原加入后,纳米粒发生团聚,颜色出现由蓝至红的变化,且CR值随着抗原浓度增加而增加,与高表达的乳腺癌细胞MCF-7发生特异性结合,充分混匀后,溶液颜色产生由蓝至红的变化。结论 成功合成由单克隆抗体抗CK19修饰的PDA/PC生物传感器,通过免疫化学显色技术,可快速、有效、灵敏的检测溶液中微量乳腺癌MCF-7细胞,为PDA/PC生物传感器技术在CTC识别上的应用奠定基础。     

基于“碘-罗丹明B”缔合物荧光探针测定β-内酰胺酶活性

碘(I3-)对罗丹明B(RB)具有荧光猝灭作用，可使RB的荧光信号强度减弱甚至消失，而β-内酰胺酶作用下的青霉素水解产物青霉噻唑酸可将I3-还原为I-，使体系荧光信号再现，据此建立了以碘-罗丹明B缔合物为荧光探针测定药剂中青霉素含量的新方法。在激发波长360nm，发射波长580nm条件下进行了β-内酰胺酶活性的荧光测定。结果表明，在0~2.4U/mL范围内，β-内酰胺酶活性与其荧光强度变化值呈良好的线性关系，检出限为0.002U/mL。方法的加标回收率为96.67%~103.3%，相对标准偏差 (n=6)2.0%~4.5%。该方法简便快速、准确可靠、灵敏度高，成功用于自制酶液酶活性测定。     

A homogeneous immunological detection system for soman using the in vitro protection of acetylcholinesterase by a monoclonal antibody

In this study a monoclonal antibody (MAb) based soman detection system was investigated. Since the MAb F71D7 recognizes the pinacolyl group of soman, non-toxic soman analogues are also detected when using an indirect competitive ELISA. This can lead to falsely positive results. The toxic effect of soman is, however, independent of the pinacolyl group. In the described homogeneous enzyme immunoassay (EIA), the inhibitory effect of soman on acetylcholinesterase (AChE) was combined with its specific binding to the MAb F71D7 in order to minimize false positive results and enhance the specificity of the detection system. In this rapid EIA no incubation or washing steps are necessary, so only time for pipetting and reaction have to be considered. Soman could be detected in concentrations of 1.6–25 nM using the EIA. This corresponds to 8 pg soman per 25 l sample and means that compared to other ELISA systems, besides enhanced specificity, the limit of detection could be improved by 3 orders of magnitude.     

Enzyme immunoassay of bradykinin using β-d-galactosidase as a labeling enzyme

An enzyme immunoassay of bradykinin, using β-d-galactosidase from Escherichia coli as a labeling enzyme, is described. Bradykinin, conjugated to the enzyme with a hetero bifunctional type of coupling agent, N-(m-maleimidobenzoyloxy)succinimide, was prepared as a labeled antigen, Antisera against bradykinin were obtained from male rabbits immunized with bradykinin linked to albumins (ovalbumin or bovine serum albumin) with toluene 2,4-diisocyanate or l-ethyl-3-(3-dimethylaminopropyl) carbodiimide. These antisera were tested for their abilities to bind the labeled antigen and for their sensitivities. The antigen-antibody reaction was performed in an ice bath for 18 hr; this was incubated for another 4 hr, after addition of anti-rabbit IgG antiserum from goat (double antibody method) to separate the bound antigen from free antigen; the enzyme activity in the precipitate was measured with a fluorogenic substrate. Some of the antisera showed good sensitivity when assayed by this method, the sensitivity having been comparable to that of radioimmunoassays of bradykinin. With this method, 30 pg/tube (0.2 ml) of bradykinin could be measured, and the standard curve was obtained in the range of 30 pg to 10 ng of bradykinin. The kininogen level in human plasma was determined by conversion of kininogen to bradykinin by trypsin after heating plasma at 60°. Kininogen levels obtained from six human subjects were in good agreement with those obtained by bioassay.     

Enhancement of rat brain cytosolic monoamine oxidase activity by clorgyline. Comparison with (-)-deprenyl and MDL 72145

The presence of unsedimentable forms of monoamine oxidase (EC 1.4.3.4) in liver and brain homogenates has prompted fresh studies on the effects of inhibitors on this cytosolic monoamine oxidase. Clorglycine is a specific monoamine oxidase A inhibitor and (-)-deprenyl and MDL 72145 are specific monoamine oxidase B inhibitors. We investigated the effects of (-)-deprenyl, MDL 72145 and clorgyline on the purified enzyme from mitochondria and cytosolic monoamine oxidase along with high speed cytosol and 1% Triton X-100 treated mitochondrial preparations. Clorgyline enhanced the activity of the purified enzyme several-fold. (-)-Deprenyl and MDL 72145 also enhanced and inhibited the activity of cytosolic monoamine oxidase in a concentration-dependent manner.     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Rapid and simple quantitation of methamphetamine by using a homogeneous time-resolved fluoroimmunoassay based on fluorescence resonance energy transfer from europium to Cy5

A rapid and simple homogeneous time-resolved fluoroimmunoassay based on fluorescence resonance energy transfer from europium (Eu) to cyanine dye (Cy5) has been developed for the quantitation of methamphetamine. In this assay, Eu chelate was labeled to a conjugate of methamphetamine and bovine serum albumin (MA-BSA), as an energy donor, and Cy5 was labeled to anti-MA as an energy acceptor. The close proximity between the two labels in the immunocomplex permits energy transfer from the excited Eu(3+) donor. Therefore, by measuring the sensitized emission of Cy5 with the time-resolved assay, immunocomplex of MA-BSA and anti-MA can be measured in the homogeneous solution without separation steps within 30 min. By a competitive immunoassay, MA could be assayed in the range 0.1-1,000 ng/mL. The intra-assay variations were 5.4-14.8% at 5 different concentrations. When urine or serum samples were examined, the quenching of Eu fluorescence was observed, but the acceptor-to-donor ratio constantly depended upon the dilution of samples. Twenty urine samples were assayed, and the data showed a good correlation to those obtained by gas chromatography (r = 0.94). The homogeneous assay using Eu-Cy5 energy transfer is time-saving without any washing procedures and is suitable for screening drugs that are commonly abused.     

In Vitro Studies on Sterically Stabilized Liposomes (SL) as Enzyme Carriers in Organophosphorus (OP) Antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Highly Sensitive and Specific Detection of Staphylococcal Enterotoxins SEA,SEG, SEH,and SEI by Immunoassay

Staphylococcal food poisoning (SFP) is one of the most common foodborne diseases worldwide, resulting from the ingestion of staphylococcal enterotoxins (SEs), primarily SE type A (SEA), which is produced in food by enterotoxigenic strains of staphylococci, mainly S. aureus. Since newly identified SEs have been shown to have emetic properties and the genes encoding them have been found in food involved in poisoning outbreaks, it is necessary to have reliable tools to prove the presence of the toxins themselves, to clarify the role played by these non-classical SEs, and to precisely document SFP outbreaks. We have produced and characterized monoclonal antibodies directed specifically against SE type G, H or I (SEG, SEH or SEI respectively) or SEA. With these antibodies, we have developed, for each of these four targets, highly sensitive, specific, and reliable 3-h sandwich enzyme immunoassays that we evaluated for their suitability for SE detection in different matrices (bacterial cultures of S. aureus, contaminated food, human samples) for different purposes (strain characterization, food safety, biological threat detection, diagnosis). We also initiated and described for the first time the development of monoplex and quintuplex (SEA, SE type B (SEB), SEG, SEH, and SEI) lateral flow immunoassays for these new staphylococcal enterotoxins. The detection limits in buffer were under 10 pg/mL (0.4 pM) by enzyme immunoassays and at least 300 pg/mL (11 pM) by immunochromatography for all target toxins with no cross-reactivity observed. Spiking studies and/or bacterial supernatant analysis demonstrated the applicability of the developed methods, which could become reliable detection tools for the routine investigation of SEG, SEH, and SEI.     

Effect of a plant histaminase on asthmalike reaction induced by inhaled antigen in sensitized guinea pig

This study evaluates the effects of a copper amine oxidase (histaminase) purified from the pea seedling as a free or immobilized enzyme on asthmalike reactions to inhaled antigen in actively sensitized guinea pig in vivo. Male albino guinea pigs, sensitized with ovalbumin, were challenged with the antigen given by aerosol; free histaminase or CNBr-Sepharose immobilized histaminase was given intraperitoneally (20 microg, 3 or 24 h before antigen challenge) or by aerosol (4 microg, 30 min before or during ovalbumin aerosol). The following parameters were examined: latency time for the onset of respiratory abnormalities, cough severity score, and occurrence and duration of dyspnea. We also evaluated lung histopathology, mast cell degranulation, and lung myeloperoxidase and malonydialdehyde levels. Histaminase significantly reduced the severity of cough and the occurrence of dyspnea and delayed the onset of respiratory abnormalities. Both enzymes prevented bronchial constriction, pulmonary air space inflation, leukocyte infiltration (evaluated as myeloperoxidase activity), and lipoperoxidation of cell membranes (evaluated as malonyldialdehyde production). No relevant differences in pharmacological potency were noted between free or immobilized enzyme. This study provides evidence that histaminase counteracts acute allergic asthmalike reaction in actively sensitized guinea pigs, raising the possibility of new therapeutic strategies for allergic asthma in humans.     

Elevation of molybdenum hydroxylase levels in rabbit liver after ingestion of phthalazine or its hydroxylated metabolite

Oral administration of phthalazine (50 mg/kg/day) or 1-hydroxyphthalazine (10 mg/kg/day) to female rabbits caused an increase in the specific activity of the hepatic molybdenum hydroxylases aldehyde oxidase and xanthine oxidase, whereas no effect on microsomal cytochrome P-450 activity was observed. The rise in the specific activity of purified aldehyde oxidase fractions was accompanied by a similar increase in molybdenum content. A significant lowering of the Km value for phthalazine was demonstrated with enzyme from treated rabbits whereas Km values for structurally similar substrates such as isoquinoline were unchanged from control values. Iso-electric focusing of DEAE-cellulose fractions showed the presence of an additional band of activity indicating that genuine induction of aldehyde oxidase had occurred in rabbits treated with phthalazine or 1-hydroxyphthalazine.