A sensitive CCD image system for detection of chemiluminescent reactions

A new ultra-sensitive light detection system is described for the detection of chemiluminescence reactions. The system consists of a CCD camera equipped with a two-stage image intensifier coupled to an image analysis system with real-time filtering capabilities. The sensitivity was determined with the enhanced chemiluminescence system using horseradish peroxidase as label. Additional possibilities for further increase in sensitivity and the potential use in immuoassays and other applications are discussed.     

Chemiluminescence-based detection and comparison of protein amounts adsorbed on differently modified silica surfaces

The biological consequences of protein adsorption on biomaterial surfaces are considered to be of utmost importance for their biocompatibility. A new method based on amino group-labeling coupled to a chemiluminescence reaction for direct determination of proteins adsorbed on material surfaces was employed. This method was used to explore the effects of surface chemistry and surface roughness on protein adsorption in a silicon oxide model system. Corundum sandblasting was applied to silicon wafers to create roughened surfaces while immobilization of fluorocarbon-, hydrocarbon-, and poly(ethylene glycol)-containing silanes produced surfaces of varying wettability. The adsorption behavior of two complex body fluids, human serum and saliva, and of two purified components, human serum albumin and fibronectin, was strongly influenced by the surface parameters. A general tendency to higher amounts of adsorbed protein was found on roughened surfaces and modification with poly(ethylene glycol) or with fluorocarbon moieties reduced protein adsorption. The values obtained with the new method could be confirmed by a colorimetric determination of protein amounts adsorbed on identically modified silica beads and were in accordance with those previously reported utilizing established methods for protein quantification. The presented method, which was methodically simple to perform and allowed the simultaneous measurement of a large number of samples, may be of future value for high-throughput surveying of the protein adsorption characteristics of biomaterials.     

Simultaneous measurement of superoxide generation and intracellular Ca2+ concentration reveals the effect of extracellular Ca2+ on rapid and transient contents of superoxide generation in differentiated THP-1 cells

We invented a simultaneous measuring instrument of fluorescence and chemiluminescence, realizing the analysis of chronological correlation between change in intracellular Ca2+ concentration ([Ca2+]i) and superoxide generation. A human monocytic cell line, THP-1, differentiated to be neutrophil-like cells generated superoxide with increase in intracellular Ca2+ concentration when stimulated with formyl-methionyl-leucyl-phenylalanine (fMLP) whereas PMA, phorbol ester-stimulated superoxide response occurred without change in [Ca2+]i. The cells treated with TMB-8, an intracellular Ca2+ antagonist, generated superoxide rapidly as well as transiently with transient [Ca2+]i elevation after stimulation with fMLP, whereas EGTA-treated cells generated superoxide slowly as well as persistently with transient [Ca2+]i elevation after the stimulation. These results suggest that the rapid and transient contents of superoxide generation are specific for Ca2+ influx from the extracellular domain. Verapamil, voltage-dependent Ca2+ channel blocker, dose-dependently inhibited fMLP-stimulated extracellular Ca2+ influx and superoxide generation without affecting PMA-stimulated superoxide generation. Other channel blockers tested, nifedipine and diltiazem, similarly inhibited these fMLP-stimulated responses. Numerical analysis of the values of the response curves elucidated that TMB-8 or the channel blocker reveals or eliminates the same contents of superoxide generation by the antagonism of intracellular Ca2+ release or extracellular Ca2+ influx, respectively. Taking these results together, the characteristic extracellular Ca2+ influx essential for superoxide generation was first revealed by the simultaneous measurement of superoxide generation and change in [Ca2+]i.     

Enhanced immunoassay sensitivity using chemiluminescent acridinium esters with increased light output

Chemiluminescent acridinium ester labels are widely used in clinical diagnostics especially in automated immunochemistry analyzers such as Siemens Healthcare Diagnostics’ ADVIA Centaur® systems. Although, chemiluminescence from acridinium compounds was discovered more than 50 years ago, details regarding the excitation process are still not well understood particularly in relation to acridinium structure and overall light output. Herein, we report an empirical study that correlates the presence of electron-donating methoxy groups at C-2 and/or C-7 in the acridinium ring with increased light output. We further demonstrate that these high light output labels can be combined with hydrophilic functional groups such as hexa(ethylene)glycol to generate unique acridinium esters that are stable and are useful in improving immunoassay sensitivity for both competitive and sandwich automated immunoassays.     

Genistein abrogates pre-hemolytic and oxidative stress damage induced by 2,2'-Azobis (Amidinopropane)

The pre-hemolytic mechanism induced by free radicals initiated from water-soluble 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) and its reversal by genistein was investigated in human erythrocytes. The time course of K+ efflux compared to the occurrence of hemolysis suggests that AAPH-induced hemolysis occurs indirectly via pore formation and band 3 oxidation as expected. However, genistein inhibited hemolysis, LDH release and membrane protein oxidation but not K+ efflux. This indicated that erythrocyte protein oxidation possibly in the hydrophobic core plays a significant role in the membrane pre-hemolytic damage. Chemiluminescence (CL) analysis carried out in non-lysed erythrocytes treated with AAPH showed a dramatic increase in CL indicating both reduced levels of antioxidants and increased membrane lipid peroxide. The V0 value was also increased up to 6 times, denoting a high degree of membrane peroxidation very early in erythrocyte membrane damage. The whole process was inhibited by genistein in a dose-dependent manner. These results indicate that the genistein inhibited both hemolysis and pre-hemolytic damage and also hindered membrane lipid peroxide formation and protein oxidation. In addition, it is suggested that pre-hemolytic damage is mediated mainly by the oxidation of both phospholipid and protein located in the deeper hydrophobic region of the membrane.     

Oxidation of tetrahydrobiopterin by peroxynitrite or oxoferryl species occurs by a radical pathway

The molecular mechanisms of tetrahydrobiopterin (BH₄) oxidation by peroxynitrite (ONOO^-) was studied using ultra-weak chemiluminescence, electron paramagnetic resonance (EPR) and UV-visible diodearray spectrophotometry, and compared to BH₄ oxidation by oxoferryl species produced by the myoglobin/hydrogen peroxide (Mb/H₂O₂) system. The oxidation of BH₄ by ONOO^- produced a weak chemiluminescence, which was altered by addition of 50 mM of the spin trap α-(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN). EPR spin trapping demonstrated that the reaction occurred at least in part by a radical pathway. A mixture of two spectra composed by an intense six-line spectrum and a fleeting weak nine-line one was observed when using ONOO^-. Mb/H₂O₂ produced a short-living light emission that was suppressed by the addition of BH₄. Simultaneous addition of POBN, BH₄ and Mb/H₂O₂ produced the same six-line EPR spectrum, with a signal intensity depending on BH₄ concentration. Spectrophotometric studies confirmed the rapid disappearance of the characteristic peak of ONOO^- (302 nm) as well as substantial modifications of the initial BH₄ spectrum with both oxidant systems. These data demonstrated that BH₄ oxidation, either by ONOO^- or by Mb/H₂O₂, occurred with the production of activated species and by radical pathways.     

Determination of ferulic acid by flow injection chemiluminescence analysis based on enhancement of the N‐bromobutanimide–eosin–CrCl3 system in alkaline solution

A simple and sensitive flow injection chemiluminescence method has been developed for the determination of ferulic acid (FA) based on the significant enhancement effect of FA on the CL signal of the N‐bromobutanimide (NBS)–eosin–CrCl₃ system in alkaline solution. Under optimum conditions, the enhanced CL intensity is linearly related to the concentration of FA in its pharmaceutical preparations and human plasma samples. The corresponding linear regression equations were established over the 4.0 × 10^–10–1.0 × 10^–7 g/mL for FA tablets and 2.0 × 10^–10–1.0 × 10^–7 g/mL for plasma samples. The limit of detection for FA tablets and limit of quantification for plasma samples were 2.8 × 10^–10 g/mL (3 σ) and 3.04 × 10^–10 g/mL (10 σ), respectively. A complete analysis could be performed within 40 s, including washing and sampling, giving a throughput of ≈90/h. The proposed method was successfully applied to the determination of FA in pharmaceutical preparations and human plasma samples with satisfactory results. The recoveries of pharmaceutical preparations and human plasma samples at three different concentrations were 97.8–102.6% and 96.7–104.0%, respectively. Furthermore, the possible mechanism of CL reactions was also discussed briefly. Copyright © 2013 John Wiley & Sons, Ltd.     

广州地区人腺病毒5型血清流行病学初步调查

目的评估广州地区人群中人腺病毒5型(HAdV-5)先存中和抗体的流行情况。方法采用以β-半乳糖苷酶(LacZ)为报告基因,结合CMV启动子的人重组腺病毒5型,使用化学发光法,检测了209份免疫功能正常的成人血清样本中HAdV-5的中和抗体阳性率情况。结果 HAdV-5中和抗体的阳性率为82.3%(172/209)。HAdV-5中和抗体在<1/20(低滴度)、1/40-1/160(中滴度)、1/320-1/1280(高滴度)、>1/1280(超高滴度)时均能检测到,而且随着滴度的增加,阳性率逐渐升高。在20～40岁时HAdV-5中和抗体阳性率最高,在<20岁时HAdV-5阳性率最低。结论广州地区人群中针对HAdV-5的先存中和抗体阳性率高,应用基于该种DNA病毒作为载体进行基因疫苗及基因治疗的研究时,具有一定的局限性。