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Magnetic Bead‑Based Sandwich immunoassay for Viral Pathogen Detection by Employing Gold Nanoparticle as Carrier

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Journal of Analysis and Testing 2017年第4期1卷 298-305页

作者： Lei Zhan Wen Bi Wu Chun Mei Li Cheng Zhi Huang Key Laboratory on Luminescence and Real‑Time Analytical Chemistry(Ministry of Education) College of Pharmaceutical ScienceSouthwest UniversityChongqing 400716China Chongqing Key Laboratory of Biomedical Analysis(Chongqing Science&Technology Commission) College of Chemistry and Chemical EngineeringSouthwest UniversityChongqing 400715China

enzyme-linked immunosorbent assay(ELISA)provides a convenient way for the detection of viral ***,conventional ELISA performed on mirowell plates suffers from poor sensitivity,laborious coating and complicated blocking ***,we designed a sensitive colorimetric immunoassay by taking advantages of the enrichment and isolation ability of magnetic beads(MBs)and the high loading capacity of gold nanoparticles(AuNPs)for detecting respiratory syncytial virus(RSV)as a pathogen *** was selectively captured and preconcentrated from samples with antibodies functionalized MBs,followed by binding with antibodies labeled AuNPs,which carrying a large amount of alkaline phosphatase(ALP)molecules for colorimetric signal amplification by catalyzing the dephosphorylation of non-colored pNPP to generate colored product *** optimizing the experimental conditions based on the principle of low nonspecific signal,low cost,and high sensitivity,the analytical sensitivity of the developed immunoassay can be improved to 0.27 pg/mL,which is over sevenfold higher than that of commercially available RSV ELISA kits(2 pg/mL).In addition,the total assay time was less than 2.5 h without any pretreatment,which is much more rapid than other reported ***,the proposed immunoassay holds great promise for the fabrication of rapid,sensitive,and economic method for the viral pathogen detection.

关键词： Magnetic bead Gold nanoparticles enzyme immunoassay Signal amplification Respiratory syncytial virus

Comparing techniques for measuring corticosterone in tadpoles

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Current Zoology 2015年第5期61卷 835-845页

作者： Pablo BURRACO Rosa ARRIBAS Saurabh S. KULKARNI Daniel R. BUCHHOLZ Ivan GOMEZ-MESTRE Department of Wetland Ecology Estacion Biologica de Dofiana (EBD-CSIC) C/Americo Vespucio s/n 41092 Sevilla Spain Department of Pediatrics Yale School of Medicine Yale University New Haven CT 0651 USA Department of Biological Sciences University of Cincinnati Cincinnati OH 45221 USA

Glucocorticoids play a key role in mediating stress responses in vertebrates. Corticosterone （CORT） is the main glucocorticoid produced in amphibians, birds, and reptiles, and regulates several metabolic functions. The most common methods for quantifying CORT are competitive binding immunoassays： radioimmunoassay （RIA） and enzyme immunoassay （EIA）. RIA has been broadly used since the 1980＇s but it requires radioactivity. Commercial EIA kits permit quantifying hormone levels without radioactivity although the requirement for a larger sample volume may be a strong limitation for measurements involving larval amphibians. Here we used Xenopus laevis tadpoles to compare the performance of three commonly used procedures for determination of CORT： RIA on a chloroform extract of whole-body homogenate, EIA on plasma, and EIA on supematant of whole-body homogenate. We treated tadpoles with exogenous CORT at 0, 25, 50, and 100 nM. RIA could distinguish between 0 and 25 nM, and EIA on plasma between 0 and 50 nM, whereas whole-body homogenate EIA only detected significant differences between 0 and 100 riM. Each procedure presents advantages and disadvantages regarding sensitivity, the use of radioactivity, sample size, handling time, and economic cost. RIA is preferred when studying small-bodied animals from which blood samples cannot be obtained. When CORT level differences are intermediate and blood sampling is possible, EIA on plasma is a good non-radioactive alternative. EIA on whole-body homogenates may be useful to assess qualitative changes in CORT levels when considerable differences are expected. Finally, we discuss our findings in the context of previous studies on CORT in amphibians [Current Zoology 61 （5）： 835-845, 2015].

关键词： Corticosterone Radioimmunoassay enzyme immunoassay Glucocorticoids Amphibians Stress

Hormonal control of seasonal color change in female spiny-footed lizards:an observational and experimental approach

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Current Zoology 2019年第6期65卷 633-642页

作者： Belen FRESNILLO Josabel BELLIURE Diego GIL Jose J.CUERVO Department of Life Sciences Ecology SectionUniversity of AlcalaMadridSpain Department of Functional and Evolutionary Ecology Estacion Experimental de Zonas Aridas(CSIC)Carretera de Sacramento s/nAlmerfaSpain Department of Evolutionary Ecology Museo Nacional de Ciencias Naturales(CSIC)MadridSpain

Breeding coloration of females often signals aspects of their reproductive status,suggesting a link between color and sex steroid *** this study,we examined the relationships between 2 sex steroid hormones(progesterone and p-estradiol)and reproductive coloration in female spinyfooted lizards Acanthodactylus *** first explored natural variation in female plasma hormone levels and coloration during their reproductive cycle,p-estradiol was negatively related to brightness and positively related to red saturation,whereas progesterone was not significantly related to *** identifying key relationships,plasma hormone concentrations were manipulated by creating 3 experimental female groups(p-estradiol-treated,progesterone-treated,and control),and the effects on coloration were monitored,p-estradiol-treated females,in which there was a rise in both p-estradiol and progesterone levels,lost their red coloration earlier than females in the other 2 experimental groups,whereas progesterone treatment had no significant effect on female *** results suggest that high levels of either p-estradiol alone or(3-estradiol together with progesterone trigger the loss of red coloration in female spiny-footed lizards,and that progesterone alone does not affect *** hypothesize that changes in female breeding color might be regulated by(3-estradiol in species in which conspicuous coloration is displayed before ovulation,and by progesterone in species in which this color is displayed during gravidity.

关键词： color signaling enzyme immunoassay hormone-induced color change sex-specific coloration spectrophotometry transdermal hormone application

Accuracy of Helicobacter pylori stool antigen for the detection of Helicobacter py/ori infection in dyspeptic patients

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World Journal of Gastroenterology 2005年第3期11卷 386-388页

作者： Ari Fahrial Syam Abdul Aziz Rani Murdani Abdullah C. Manan D. Makmun M. Simadibrata D. Djojoningrat Daldiyono Tadashi Sato Division of Gastroenterology First Department of Medicine Yamanashi Medical University

AIM: The premier platinum Helicobacter pylon (Hpylori) stool antigen (HpSA) test is an enzyme immunoassay (EIA) that detects an Hpyloriantigen present in human stools. However, at present there is no uniformity about the cut off level required to consider the test as positive or negative. So we need the cut off level for our local population. The aim of this study was to evaluate the HpSA for the detection of H pylori infection in dyspeptic patients and to determine the sensitivity, specificity of the HpSA test in the diagnosis of H pylori infection, as compared to other standardized diagnostic techniques. METHODS: Sixty-three dyspeptic patients were selected from patients who came to the Division of Gastrointestinal Clinic in Cipto Mangunkusumo Hospital, Jakarta, Indonesia. H pylori infection was confirmed in all patients by histology and rapid urease test (CLO test). Positive results for H pylori were based on positive results from both rapid urea test and microscopic detection of H pylori. Stool specimens were analyzed for H pylori antigen using HpSA immunoassay. RESULTS: A total 63 patients consisted of 31 (49.2%) males and 32 (50.8%) females ranging in ages between 16 and 73 years with a mean age of 42.4±15 years. The mean age of men was 43.2±15.7 years and women was 41.6±14.4 years. Endoscopic findings in this study included gastric cancer 1.6%, peptic ulcer 4.8%, duodenal ulcer 7.9%, esophagitis 6.3%, gastritis 77.7%, and gastroduodenitis 4.8%. According to the predefined study criteria, 6 (9.5%) of 63 patients were positive for H pylori. In the diagnosis of infection, the area under the receiver operating characteristic (ROC) curve for the HpSA test was 0.722 (95% CI, 0.518-0.927). Using a cut-off value of 0.274 instead of 0.16 (as recommended by the manufacturer) the sensitivity and the specificity were 66.7% and 78.9% respectively. CONCLUSION: The HpSA stool test, using a cut-off value of 0.274, may be useful for the primary diagnosis of H pylori infection

关键词： Helicobacter pylori infection Dyspepsia HpSA enzyme immunoassay

Current knowledge on the laboratory diagnosis of Clostridium difficile infection

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World Journal of Gastroenterology 2017年第9期23卷 1552-1567页

作者： Adrián Martínez-Meléndez Adrián Camacho-Ortiz Rayo Morfin-Otero Héctor Jesús Maldonado-Garza Licet Villarreal-Trevino Elvira Garza-González Departamento de Microbiología e Inmunología Facultad de Ciencias BiológicasUniversidad Autónoma de Nuevo León.Pedro de Alba S/NCiudad UniversitariaSan Nicolás de los Garza Servicio de Infectología Hospital Universitario“Dr.JoséEleuterio González”Universidad Autónoma de Nuevo León.Av.Francisco I.Madero Pte.S/NCol.Mitras Centro.Monterrey Hospital Civil de Guadalajara “Fray Antonio Alcalde”e Instituto de Patología Infecciosa y ExperimentalCentro Universitario de Ciencias de la SaludUniversidadde Guadalajara.Sierra Mojada 950Col.Independencia Servicio de Gastroenterología Hospital Universitario“Dr.JoséEleuterio González”Universidad Autónoma de Nuevo León.Av.Francisco I.Madero Pte.S/NCol.Mitras Centro.Monterrey

Clostridium difficile (C. difficile) is a spore-forming, toxin-producing, gram-positive anaerobic bacterium that is the principal etiologic agent of antibiotic-associated diarrhea. Infection with C. difficile (CDI) is characterized by diarrhea in clinical syndromes that vary from selflimited to mild or severe. Since its initial recognition as the causative agent of pseudomembranous colitis, C. difficile has spread around the world. CDI is one of the most common healthcare-associated infections and a significant cause of morbidity and mortality among older adult hospitalized patients. Due to extensive antibiotic usage, the number of CDIs has increased. Diagnosis of CDI is often difficult and has a substantial impact on the management of patients with the disease, mainly with regards to antibiotic management. The diagnosis of CDI is primarily based on the clinical signs and symptoms and is only confirmed by laboratory testing. Despite the high burden of CDI and the increasing interest in the disease, episodes of CDI are often misdiagnosed. The reasons for misdiagnosis are the lack of clinical suspicion or the use of inappropriate tests. The proper diagnosis of CDI reduces transmission, prevents inadequate or unnecessary treatments, and assures best antibiotic treatment. We review the options for the laboratory diagnosis of CDI within the settings of the most accepted guidelines for CDI diagnosis, treatment, and prevention of CDI.

关键词： Clostridium difficile Toxigenic culture Nucleic acid amplification tests enzyme immunoassay Diagnosis Glutamate dehydrogenase

The M Protein of SARS-CoV: Basic Structural and Immunological Properties

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Genomics, Proteomics & Bioinformatics 2003年第2期1卷 118-130页

作者： JunWang SiqiLiu ChangqingZeng JianWang HuanmingYang YongwuHu JieWen LinTang HaijunZhang XiaoweiZhang YahLi JingWang YujunHan GuoqingLi JianpingShi XiangjunTian FengJiang XiaoqianZhao BeijingGenomicsInstitute ChineseAcademyofSciencesBeijing101300China CollegeofLifeSciences PekingUniversityBeijing100871China JamesD.WatsonInstituteofGenomeSciences ZhijiangCampusZhejiangUniversityandHangzhouGenomicsInstituteHangzhou310008China

We studied structural and immunological properties of the SARS-CoV M (membrane) protein, based on comparative analyses of sequence features, phylogenetic investigation, and experimental results. The M protein is predicted to contain a triple-spanning transmembrane (TM) region, a single N-glycosylation site near its N-terminus that is in the exterior of the virion, and a long C-terminal region in the interior. The M protein harbors a higher substitution rate (0.6% correlated to its size) among viral open reading frames (ORFs) from published data. The four substitutions detected in the M protein, which cause non-synonymous changes, can be classified into three types. One of them results in changes of pI (isoelectric point) and charge, affecting antigenicity. The second changes hydrophobicity of the TM region, and the third one relates to hydrophilicity of the interior structure. Phylogenetic tree building based on the variations of the M protein appears to support the non-human origin of SARS-CoV. To investigate its immunogenicity, we synthesized eight oligopeptides covering 69.2% of the entire ORF and screened them by using ELISA (enzyme-linked immunosorbent assay) with sera from SARS patients. The results confirmed our predictions on antigenic sites.

关键词： SARS-CoV the M protein enzyme immunoassay antigenicity

Level of serum neuron-specific enolase and brain damage in children with febrile seizures

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Neural Regeneration Research 2006年第5期1卷 472-474页

作者： Lang Chen Qiaobin Chen Fang Yang Zhi Lin Xinfu Lin Ying Huanc Xin Zheng Yu Lin Department of PediatncsFujian Provincial Hospital Fuzhou 350001 Fujian Province China Department of Laboratory Fujian Provincial Hospital Fuzhou 350001 Fujian Province China

BACKGROUND ： Febrile seizure （FS） has good prognosis in the majority of cases. But there is an ongoing debate on the relationship between complicated febrile seizure （CFS） and later development of temporal lobe epilepsy （TLE） due to hippocampal sclerosis.OBJECTIVE： To evaluate the level of serum neuron-specific enolase （S-NSE） of children with simple febrile seizure （SFS） and complicated febrile seizure and compare with children with non-FS respiratory tract infection. DESIGN： Contrast observation.SETTING ： Department of Pediatrics and Department of Laboratory, Fujian Provincial Hospita. PARTICIPANTS： Forty-nine patients who were admitted to Department of Pediatrics of Fujian Provincial Hospital from June 2002 to September 2003 with FS were enrolled in this study. There were 28 boys and 21 girls aged from 5 to 72 months. All children were divided into 2 groups based on frequency and duration. Thirty-two children whose FS occurred within 24 hours （lasting shorter than single and twice durations and also shorter than 10 minutes） were regarded as SFS group; meanwhile, 17 children whose FS occurred within 24 hours （lasting longer than single and twice durations and also longer than 10 minutes） were regarded as CFS group. Another 23 patients who were admitted to our hospital with respiratory tract infection in the same period, without the history and positive symptoms/features of neurological dysfunction, were enrolled as control group. There were 13 boys and 10 girls aged from 5 months to 86 months. All parents were told the facts. METHODS： 2 mL venous blood was collected from FS children within 24 hours after the seizures. Meanwhile, 2 mL venous blood was also collected from children in the control group. Level of S-NSE was measured with enzyme immunoassay procedure, and differences among groups were compared with ttest. MAIN OUTCOME MEASURES： Level of S-NSE in each group RESULTS： A total of 49 children in FS group and 23 ones in control group were involved

关键词： febrile seizure respiratory tract infection venous blood temporal lobe epilepsy enzyme immunoassay brain damage

Changes of cerebral blood flow in rats with acute cerebral ischemia and the effect of nitric oxide donor S-nitroso-N-acetyl-penicillamine

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Neural Regeneration Research 2006年第5期1卷 393-396页

作者： Feng Gao Zhiqiang Yi Guijun Lin Department of Neurosurgery Aerospace Central HospitalPeking University Beijing 100049 China

BACKGROUND ： Previous studies show that nitric oxide donor can increase cerebral blood flow and improve the function of neurons in cerebral ischemia, but the change does not happen in all the models of cerebral ischemia. OBJECTIVE ： To observe the effects of nitric oxide donor S-nitroso-N-acetyl-penicillamine （SNAP） on the cerebral blood flow, cyclic guanosine monophosphate （cGMP） content in cerebral cortex, infarct volume and blood pressure in acute ischemic rat brain.DESIGN ： A randomized and control animal experiment SEI-FING ： Department of Neurosurgery, Aerospace Central Hospital, Peking University.MATERIALS： Twenty-eight male Wistar rats of SPF grade, weighing 250-300 g, aged 10-12 weeks were randomly divided into control group （n =14） and SNAP-treated group （n =14）. SNAP （5 mg/bottle） was provided by Beijing Chemical Reagent Company. Laser Doppler Flowmeter （FLO C1; Omegawave Inc., Tokyo, Japan） and immunoassay kit （Amersham Pharmacia Biotech, UK） were applied. METHODS： ① Model establishment： In the control group, models of cerebral ischemia were induced by ligating right common, internal and external carotid arteries; In the SNAP-treated group, models of cerebral ischemia were induced by ligating right common and external carotid arteries, followed by occluding middle cerebral artery and ligating internal carotid artery. ②Administration： In the SNAP-treated group, SNAP （100 μg/kg） was intravenously infused within 2 minutes, whereas in the control group, phosphate buffered saline （PBS, 1 mL） was intravenously infused （0.5 mL per minute）. Six rats were used to measure the volume of cerebral infarction, and the other 8 rats were used to determine other indexes in each group respectively. ③ Determination of indexes： Regional cerebral blood flow （rCBF） was continuously measured by laser-Doppler flowmetry in the ischemic penumbra and contralateral cortex under the continuous monitoring of blood pressure, cGMP concentrations in brain tissue were determined

关键词： nitric oxide cerebral ischemia cerebral infarction cerebral blood flow blood pressure middle cerebral artery intravenous infusion carotid arteries internal carotid artery continuous monitoring enzyme immunoassay ischemic penumbra animal experiment measurement data Chemical Reagent cerebral cortex caudate nucleus Wistar rats

EXPRESSION OF X-GENE TRANSLATION PRODUCT IN HUMAN PRIMARY CHOLANGIOCARCINOMA AND ITS SURROUNDING NONTUMOROUS TISSUES

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Chinese Journal of Cancer Research 1993年第2期5卷 4-7页

作者：胡敏王文亮朱新生 Department of Pathology Fourth Military Medical University Xi' an 710032 Department of Pathology Fourth Military Medical University Xi' an 710032ntrahepatic cholangiocarcinoma is an uncommon type of primary hepatoma and its mechanism is still unknown. Twenty samples of the cancer and the tissues surrounding it were all taken from the Xi' an area and examined for the presence of X-gene product (HBxAg) by the avldin-biotin-peroxidase complex method. It was found that 17 cases (85%) showed positive reaction HBxAg was present in 15 cancer samples and 16 non-tumor tissue samples. This result suggests that there is a close relationship between human choiangiocarcinoma and HBV and the HBxAg might play an important role in the pathogenesis of cholanglocarcinoma.

Intrahepatic cholangiocarcinoma is an uncommon type of primary hepatoma and its mechanism is still unknown. Twenty samples of the cancer and the tissues surrounding it were all taken from the Xi' an area and examined for the presence of X-gene product (HBxAg) by the avldin-biotin-peroxidase complex method. It was found that 17 cases (85%) showed positive reaction, HBxAg was present in 15 cancer samples and 16 non-tumor tissue samples. This result suggests that there is a close relationship between human choiangiocarcinoma and HBV, and the HBxAg might play an important role in the pathogenesis of cholanglocarcinoma.

关键词： Bile duct neoplasms Hepatitis B virus X-gene enzyme immunoassay