Excessive production of reactive oxygen metabolites by inflamed colon: analysis by chemiluminescence probe.

Reactive oxygen metabolites (ROMs) are involved in inflammatory diseases and are postulated to contribute to tissue injury in colitis. To determine whether excessive ROMs are generated by inflamed colonic mucosa and to identify possible sources and type of ROMs, mucosal ROMs were estimated in rats and humans using a chemiluminescence probe. Colitis was induced in rats by intracolonic injection of acetic acid or intraperitoneal injection of mitomycin C. Intact, inflamed colon in rats produced more ultraweak chemiluminescence than normal colon. Inflamed mucosal scrapings from both rat models produced significantly more luminol-enhanced chemiluminescence. Addition of catalase, an H2O2 scavenger, or azide, a myeloperoxidase inhibitor, into the media significantly decreased chemiluminescence from inflamed mucosal scrapings. Indomethacin, an antioxidant cyclo-oxygenase inhibitor, also decreased chemiluminescence, but MK-866, a 5-lipoxygenase inhibitor, had no effect. Colonic biopsy specimens obtained during colonoscopy from patients with ulcerative colitis also produced more catalase-inhibitable chemiluminescence than normal colonic mucosa. These data indicate that excessive ROMs are produced by inflamed colonic mucosa in both humans and rats, which may contribute to tissue injury.     

Determination of oxygen radical production in spondyloarthropathies by whole blood chemiluminescence.

Oxygen derived free radicals are considered to play an important part in the development of inflammation. A whole blood chemiluminescence assay was used to study N-formyl-methionyl-leucyl-phenylalanine induced oxygen radical production in subjects with ankylosing spondylitis or previous yersinia arthritis. In luminol enhanced chemiluminescence, the subjects with previous yersinia arthritis showed significantly increased initial activation (at one minute), whereas the subjects with ankylosing spondylitis showed decreased responses at both the initial activation and at peak activation (at two to three minutes). This finding gives credence to the view that, in terms of oxygen radical production, the pathogenesis of yersinia arthritis is different from that of ankylosing spondylitis.     

化学发光酶免疫分析法测定血清三碘甲状腺原氨酸的建立与研究

目的建立化学发光酶免疫分析法测定血清三碘甲腺原氨酸(T3)及临床甲状腺疾病病情的监测和预后。方法化学发光酶免疫分析法(CLEIA)采用辣根过氧化酶(HRP)-鲁米诺竞争法。结果该方法的灵敏度为0.19nmol/L,在0.52~10.28nmol/L的范围内线性良好。CLEIA测定血清T3的批内与日间变异系数分别为4.96%和4.935%。在血红蛋白浓度<2.4g/L、总胆红素浓度<342μmol/L、甘油三酯<9.9mmol/L时的干扰率无临床意义。与放射免疫分析法有较好的相关性(r=0.9264)。结论CLEIA法测定血清T3能及时有效地满足甲状腺疾病患者的临床需要,有助于进一步对检测试剂盒的研制。     

化学发光免疫分析检测丙肝病毒抗体在血液筛查中的应用评价

目的:评价化学发光免疫分析(CLIA)测定丙肝病毒(HCV)抗体的方法学性能在血液筛查中的应用。方法:以辣根过氧化物酶(HRP)为酶标记物,以鲁米诺为发光底物,采用双抗原夹心法检测血样中的抗-HCV。建立抗-HCV化学发光酶免疫分析方法,与常规使用的抗-HCV酶联免疫分析方法(ELISA)同时检测20例发光值(RLU)大于14 000的抗-HCV血清阳性标本,再用荧光定量RT-PCR检测;每天用卫生部临床检验中心指定生产的HCV的质控血清对CLIA进行检测,建立自己实验室的该项目临界值浓度,连续6个月对HCV质控血清检测,计算S/CO值,统计分析精密度、CV;用EQA的质控物检测HCV抗体,进行标本符合率计算。结果:CLIA方法的灵敏度为0.02ng/mL,阳性符合率为100%,ELISA的阳性符合率为75.2%,荧光定量RT-PCR检测的阳性符合率为100%。结论:HCV抗体CLIA检测抗-HCV试验方法的敏感性、准确性、稳定性较好,能满足临床输血早期筛查,以排除假阳性,杜绝漏检的风险。     

Sequence of oxygen binding by hemoglobin

A nitroxide spin-label probe was attached directly to a propionic acid group of heme in either the alpha or the beta chain of hemoglobin. The electron paramagnetic resonance (EPR) spectrum of the spin label is altered by the spin-state change of the heme iron to which the spin label is attached. These hybrid hemoglobins showed normal optical and functional properties, indicating that the attachment of the spin label did not perturb the function of hemoglobin. Upon deoxygenation of alpha-heme-spin-labeled hemoglobin, EPR signals changed proportionally with oxygen saturation (determined by measuring absorption spectra). This result indicates that there is no binding preference between the alpha and beta chains of hemoglobin. However, the cross plot for the fraction of the EPR changes vs. the fraction of oxygen saturation deviated significantly from the diagonal straight line in response to the addition of 2,3-diphosphoglycerate and inositol hexaphosphate. The deviation indicated that the EPR change precedes the optical change at low oxygen tension. This result implies that, in the presence of organic phosphate, oxygen binds preferentially to the alpha subunit of deoxyhemoglobin. This conclusion was supported by the result obtained with beta-heme-spin-labeled hemoglobin: the direction of the deviation for beta-heme-spin-labeled hemoglobin in the presence of diphosphoglycerate and inositol hexaphosphate was opposite to that obtained for alpha-heme-spin-labeled hemoglobin. However, the curve deviated even in the absence of organic phosphate. This deviation for beta-heme-spin-labeled hemoglobin can be explained by the intersubunit interaction of hemoglobin. From these results, it was concluded that, in the absence of organic phosphate, oxygen combines with the alpha and beta chains with equal probability whereas, in the presence of organic phosphate, oxygen binds preferentially to the alpha chains of hemoglobin.     

Quantitation of reversible binding by particle counting: hapten-antibody interaction as a model system.

With a view toward developing a general method for measuring intrinsic equilibrium constants for the reversible interactions between two ligands, we used an antibody-hapten model system [2,4-dinitrophenyl (DNP) hapten and anti-DNP antibody] to explore an approach based on particle counting of uniform polystyrene spheres to which the hapten is coupled covalently. This approach was made possible by an optical pulse particle size analyzer that accurately counts individual sphere clusters and quantitates with high precision specific aggregation of spheres crosslinked by antibody. The reduction in crosslinking that results from competition for antibody binding sites between a soluble DNP ligand and immobilized DNP groups on the spheres provides the basis for measuring the intrinsic equilibrium constant for the soluble ligand-antibody interaction. The binding constants measured in this way for several DNP ligands and an anti-DNP antibody (2A1) agreed with the values obtained by conventional methods. The range of intrinsic equilibrium constants that can be determined by particle counting is likely to be exceptionally wide and a value as low as 10(3) liters/mol has been measured. And since all soluble antigens, regardless of their mass, acquire the same ability to scatter light as a result of their immobilization on the much larger uniform spheres (0.36 microns), the approach described here should be applicable to virtually any molecularly dispersed antigen and its monoclonal antibody.     

磁微粒酶联免疫吸附法检测人体尿液中日本血吸虫抗体

目的应用磁微粒酶联免疫吸附法（MPAIA）检测日本血吸虫病患者尿液中日本血吸虫特异性抗体,探索血吸虫病新的无创性免疫诊断方法。方法采用MPAIA对158例日本血吸虫病患者和100例健康人的尿液进行检测,并同步进行血清检测,评价其检测效果。结果 MPAIA尿液检测加样量确定为10μl未特殊处理原尿,尿液与血清阳性检出率分别为48.10%（76/158）和88.61%（140/158）,两者差异有统计学意义（χ2=60.24,P〈0.05）,尿液及血清检测特异度均为100%。结论 MPAIA可用于检测日本血吸虫病患者尿液中抗日本血吸虫抗体,但还须对影响其阳性检出率的多种因素进行系统研究,以提高其敏感性。     

Myocardial oxygen usage: its measurement regulation and meaning.

The general status of the problems of coronary perfusion, myocardial work, and their measurement have been reviewed; a new method of oximetry outlined; data obtained with it and by the polarographic method by Weiss presented, compared and analyzed; and their import to man noted.     

Performance of protein induced by vitamin K absence or antagonist-II assessed by chemiluminescence enzyme immunoassay for hepatocellular carcinoma detection: a meta-analysis

Objectives: In the setting of surveillance for hepatocellular carcinoma (HCC) detection, the use of serum biomarkers in addition to ultrasonography (US) is still a matter of debate. Hence, we performed a meta-analysis to evaluate the diagnostic accuracy of protein induced by vitamin K absence or antagonist-II (PIVKA-II) and alpha-fetoprotein (AFP) alone or in combination for HCC detection in patients at risk of tumor development.

Materials and methods: We performed a systematic search in PubMed and Scopus database for original articles published in English from 2011 to 2017, investigating the accuracy of PIVKA-II alone or in combination with AFP (reported as area under the curve [AUC]) for HCC detection among patients at risk of tumor development. Furthermore, we focused on studies in which serum PIVKA-II was assessed by highly sensitive chemiluminescence immunoassay (CLEIA).

Results: A total of 11 studies (873 patients with HCC and 1244 patients with advanced liver disease/cirrhosis) were included in the meta-analysis. The weighted summary AUC (sAUC) of PIVKA-II and AFP for the discrimination between patients with HCC and those without was 0.791 (0.746–0.837) and 0.767 (0.732–0.803), respectively. The combination of PIVKA-II?+?AFP results in a sAUC of 0.859 (0.837–0.882). The performance for HCC detection of PIVKA-II?+?AFP was significantly superior to each biomarker used alone (ΔsAUC?=?0.068, p?=?.032 and ΔsAUC?=?0.092, p?Conclusion: In clinical practice, the use of PIVKA-II?+?AFP in addition to US examination may improve the effectiveness of surveillance among patients at risk for HCC development.     

Continuous measurement of oxygen consumption by pancreatic islets

The rate of oxygen consumption is an important measure of mitochondrial function in all aerobic cells. In pancreatic beta cells, it is linked to the transduction mechanism that mediates glucose-stimulated insulin secretion. However, measurement of oxygen consumption over long periods of time is technically difficult owing to the error resulting from baseline drift and the challenge of measuring small changes in oxygen tension. We have adapted an ultrastable oxygen sensor based on the detection of the decay of the phosphorescent emission from an oxygen-sensitive dye to a previously developed islet flow culture system. The drift of the sensor is approximately 0.3%/24 h, allowing for the continuous measurement of oxygen consumption by 300 islets (or about 6 x 10(5) cells) for hours or days. Rat islets placed in the perifusion chamber for 24 h were well maintained as reflected by membrane integrity, insulin secretion, and oxygen consumption. Both acute changes in oxygen consumption as induced by glucose and chronic changes as induced by sequential pulses of azide were resolved. The features of the flow culture system--aseptic conditions, fine temporal control of the composition of the media, and the collection of outflow fractions for measurement of insulin, and other products--facilitate a systematic approach to assessing metabolic and functional viability in responses to a variety of stimuli. Applications to the measurement of effects of hypoxia on insulin secretion, membrane integrity, and the redox state of cytochromes are demonstrated. The system has particular application to the field of human islet transplantation, where assessment and the study of islet viability have been hampered by a lack of experimental methods.     

The diagnosis of human hydatidosis by measurement of specific IgE antibody by enzyme immunoassay

The sensitivity and specificity of an enzyme immunosorbent assay (EIA) for measurement of anti-Echinococcus granulosus specific IgE antibody using microtiter strips was evaluated. The sensitivity was 92.2% for 90 patients with cysts in the liver or other body sites but excluding the patients with cysts in the lungs or bones. In this latter group (n = 26) a sensitivity of 61.5% was recorded. The specificity for control groups comprising 89 blood bank sera from Spain and 48 sera from atopic Swedes was 100%. Patients infected with other parasites (n = 78) were usually negative (81%). 11 of the 15 false positives were found in patients with total IgE greater than 5,000 kU/l. The microtiter EIA method employed can be considered as a very convenient method to be included in the range of immunodiagnostic tests for human hydatid disease.     

Energy-structure correlation in metalloporphyrins and the control of oxygen binding by hemoglobin.

The contribution of the porphyrin skeleton to the potential energy surface metalloporphyrins is calculated by the semiempirical method of quantum mechanical extension of the consistent force field to eta electron molecules. This calculation makes it possible to correlate the observed structure of metalloporphyrins with the strain energy of the porphyrin skeleton. It is found that the out-of-plane metal displacement in pentacoordinate heme systems is due to both the restricted size of the porphyrin hole and the "1-3" steric interaction between the axial ligand and the heme nitrogens. The main components of the active site of hemoglobin are simulated by a histidine-heme-oxygen system. The energy surface of this system provides a quantitative explanation for the control of ligand binding by hemoglobin. It is shown that the heme acts as a diaphragm, designed to provide simultaneous binding to the histidine and the sixth ligand under the steric requirements of the 1-3 interactions. The dependence of the hemoglobin potential surface on the distance between the proximal histidine and the heme plane is evaluated for the R and T states, using the calculated heme potential and the observed energy of heme-heme interaction.     

定量检测人甲状腺过氧化物酶抗体化学发光酶免疫分析法的临床应用

目的 应用人甲状腺过氧化物酶抗体(hTPOAb)的化学发光酶免疫分析(CLEIA)试剂盒,检测正常人及各类甲状腺疾病患者,探讨该试剂盒在临床应用中的价值.方法 用该试剂盒共测定333例样品血清中的hTPOAb,包括正常人133名,各类甲状腺疾病患者200例,其中桥本甲状腺炎(HT) 94例,Graves病68例,结节性甲状腺肿19例,亚急性甲状腺炎10例,单纯性甲状腺肿9例.结果 确立本试剂盒hTPOAb阳性切限值为3.22 IU/ml,hTPOAb浓度以中位数表示,分别为HT5.48 IU/ml,阳性率为91.50％;Graves病1.88 IU/ml,阳性率为29.40％;结节性甲状腺肿1.83 IU/ml,阳性率为10.50％,亚急性甲状腺炎2.54 IU/ml,阳性率为20.00％;单纯性甲状腺肿2.21 IU/ml,阳性率为0.HT患者血清hTPOAb阳性率高于其他患者(x2=67.22,60.13,35.49,49.89,P均＜0.01).结论 该定量检测hTPOAb的CLEIA试剂盒在HT中有很高的阳性率,可作为HT有效的诊断手段.