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Fluorescence Immunoassay (fluorescence + immunoassay)
Selected AbstractsCompetitive Enzymatic Fluorescence Immunoassay for Human IgG by Using a Temperature-Sensitive Phase Separating Polymer with Regulated Phase Transition TemperatureCHINESE JOURNAL OF CHEMISTRY, Issue 4 2008Peng LIN Abstract A new enzymatic fluorescence immunoassay for human IgG was developed using a temperature-sensitive polymer, poly(N -isopropylacrylamide-co-acrylamide) [P(NIP-AA)], as a carrier. The lower critical solution temperature of the P(NIP-AA) containing molar fraction of 8% for AA was 37 °C. In a competitive immunoassay, immobilized IgG and the standard IgG (or sample) competed for binding to a horseradish peroxidase labeled antibody at 33 °C in homogeneous format. After changing the temperature to separate the polymer-immune complex, the complex precipitate was re-dissolved and determined by coupling with the fluorescence reaction of hydrogen peroxide and p -hydroxyphenylacetic acid. The calibration graph for human IgG was linear over the range of 100,1000 ng/mL with a detection limit of 2.0 ng/mL. The method is rapid, sensitive and simple. The immune reaction efficiency was improved. In addition, the sensitivity of this method was close to that using traditional microtitration plates as carriers. However, the assay was much faster (the assay time decreased from 100,120 to 30 min). The method has been applied to the determination of the human IgG levels in human sera with satisfactory results. [source] Assessment of urinary total testosterone production by a highly sensitive time-resolved fluorescence immunoassay ,JOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 6 2008Shihua Bao Abstract Radioimmunoassay (RIA) that involves purification of the analyte by organic solvent extraction is widely used. Although the extraction RIA is reliable when properly validated, it is time consuming and radioactive, we measure urinary total testosterone with a highly sensitive rapid and accurate time-resolved fluorescence immunoassay (TRFIA) method. High affinity antitestosterone antibody and Eu3+ labeled Donkey antisheep IgG as tracers were used. The assay was evaluated for specificity, sensitivity, analytical recovery, precision and dilution linearity by the TRFIA method on urine samples. A satisfactory standard curve for testosterone TRFIA has been developed with good sensitivity (5.1,pmol/L). The validity of the assay for urinarytotal testosterone was confirmed by thegood correlation between the results obtained by TRFIA (X) and those RIA (Y) (Y=0.075+0.971X, R=0.992). Specificity, analytical recovery, precision and dilution linearity studies were determined and all found to be satisfactory. Male urinary total testosterone excretion ranged from 64.00 to 374.11,nmol/24,hr, which was about four times more than the range for women urinary testosterone excretion (14.16,100.65,nmol/24,hr), which suggests that a direct, reliable, easy to automate, highly sensitive and specific TRFIA type assay for the measurement of total testosterone in urine samples has been developed. J. Clin. Lab. Anal. 22:403,408, 2008. © 2008 Wiley-Liss, Inc. [source] Risk factors for periodontitis in HIV+ patientsJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2004Tamer Alpagot Objective:, The purpose of this study was to identify risk factors for periodontitis associated with human immunodeficiency virus (HIV) infection. Methods:, A total of 152 HIV+ patients were recruited from the CARE clinic at the University of the Pacific School of Dentistry. Clinical measurements (gingival index, plaque index, bleeding index, probing depth, and attachment loss), gingival crevicular fluid (GCF) and subgingival plaque samples were taken from eight sites of each patient at baseline and 6-month visits. GCF neutrophil elastase was determined by measurement of p -nitroanalide resulting from hydrolysis of an elastase-specific peptide. GCF ,-glucuronidase was determined by release of 4-methylumbelliferone from hydrolysis of a specific substrate. A bacterial concentration fluorescence immunoassay was used to detect periodontopathic bacteria in subgingival plaque samples. Results:, Viral load, age, smoking pack-years, Fusobacterium nucleatum, Prevotella intermedia, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, neutrophil elastase, and ,-glucuronidase were significantly correlated with clinical measurements (0.0001 < p < 0.05). Significantly higher levels of elastase, ,-glucuronidase, F. nucleatum, P. intermedia, and A. actinomycetemcomitans were found at progressing sites than in non-progressing sites (0.001 < p < 0.05). Conclusions:, These data indicate that age, smoking pack-years, viral load, F. nucleatum, P. intermedia, A. actinomycetemcomitans, elastase, and ,-glucuronidase are risk factors for periodontitis in HIV+ patients. [source] Immunological Detection of in Vitro Formed Phosphatidylethanol,An Alcohol Biomarker,With Monoclonal AntibodiesALCOHOLISM, Issue 6 2008Antti E. Nissinen Background:, Phosphatidylethanol (PEth) is a promising new marker for detecting long-term alcohol abuse with excellent sensitivity and specificity. Current methods are based on the high performance liquid chromatography,mass spectrometric method and therefore require high levels of expertise and expensive instrumentation. This study was designed to generate PEth-specific monoclonal antibodies for PEth immunoassay development. Methods:, C57/BL6 mice were immunized with PEth in 3 different carriers, mouse serum albumin, mouse high-density lipoproteins, and human low-density lipoprotein (LDL). Mouse splenocytes were fused with a mouse myeloma cell line using the hybridoma technique. Mouse IgM-producing cell lines were selected by limiting dilutions. Binding characteristics of the anti-PEth antibodies were studied using luminometric immunoassays and sequence analysis of the variable region mRNA sequences of the antibodies. Produced antibodies were purified by chromatographic methods. PEth was detected with these antibodies in fluorescence immunoassay and flow cytometric analysis. Results:, We generated monoclonal cell lines (2B1 and 2E9) that produce IgM antibodies binding specifically to PEth but not to structurally or chemically similar phospholipids, such as phosphatidylcholine, phosphatidic acid, and cardiolipin. We show here that these anti-PEth antibodies can be used to detect PEth in a fluorescent PEth assay and FACS analysis of human red blood cell samples spiked with PEth. Conclusions:, The present study shows that PEth-specific monoclonal antibodies can be generated using traditional hybridoma technique. Immunogenicity of PEth was enhanced using human LDL as an immunization carrier. The generated monoclonal anti-PEth antibodies, 2B1 and 2E9 bind to PEth in fluid phase and in biological membranes. [source] Prevalence of Escherichia coli O157:H7 in industrial minced beefLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2002C. Vernozy-Rozand Aims: ,The lack of baseline data on the prevalence of Escherichia coli O157:H7 in retail minced beef in France prompted this survey of industrial minced beef production. Methods and Results: ,An automated enzyme-linked fluorescence immunoassay (ELFA), the VIDAS E. coli O157 method, was used to detect E. coli O157 in industrial minced beef samples. Confirmation of samples positive according to the ELFA was performed using an automated immunoconcentration (ICE) system, VIDAS ICE, which allows the selective capture and release of target organisms. The ICE was followed by culture on cefixime tellurite sorbitol MacConkey agar and a chromogenic medium, O157:H7 ID. Of the 3450 minced beef samples tested, 175 samples were positive with the ELFA method and, of these, four were confirmed by the ICE method. They were identified as sorbitol-negative, O157-positive, H7-positive, mobile, verotoxin-producing E. coli . Conclusions: ,The prevalence of E. coli O157:H7 in industrial French minced beef was 0·12%, consistent with many other reports. Significance and Impact of the Study: ,The low infective dose of E. coli O157:H7 presents a major threat. The main means of combating this organism are thermal destruction and good food hygiene covering activities on-farm, in the abattoir and in minced beef industries. [source] Simultaneous detection of two verotoxin genes using dual-label time-resolved fluorescence immunoassay with duplex PCRLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 2 2002Kazuyuki Watanabe Abstract Verotoxin (VT) produced by several Escherichia coli serotypes causes haemorrhagic colitis and has been associated with haemolytic uraemic syndrome in humans. Two types of verotoxin are known. Conventional diagnosis of verotoxin-producing Escherichia coli (VTEC) is conducted after isolation of bacteria from clinical specimens, followed by serological determination and identification of VTs. This method is complicated and time-consuming. Recently, rapid, direct immunological methods for identification of VTEC, i.e. immunochromatography and latex agglutination, have been developed. However, these techniques continue to suffer from limited sensitivity and a lack of specificity. These difficulties arise from the fact that the antibody used in these procedures reacts exclusively with the O157 antigen; moreover, VTEC strains with non-O157 antigens, such as O26, O103 and O111 antigens, exist. These VTEC groups did not react with anti-O157 antibody. Consequently, it is necessary to diagnose the VT gene in these bacteria. Therefore, we have designed a sensitive and specific method for the detection of two VT genes simultaneously, utilizing duplex PCR with time-resolved fluorescence immunoassay (TRFIA). Copyright © 2002 John Wiley & Sons, Ltd. [source] Competitive Enzymatic Fluorescence Immunoassay for Human IgG by Using a Temperature-Sensitive Phase Separating Polymer with Regulated Phase Transition TemperatureCHINESE JOURNAL OF CHEMISTRY, Issue 4 2008Peng LIN Abstract A new enzymatic fluorescence immunoassay for human IgG was developed using a temperature-sensitive polymer, poly(N -isopropylacrylamide-co-acrylamide) [P(NIP-AA)], as a carrier. The lower critical solution temperature of the P(NIP-AA) containing molar fraction of 8% for AA was 37 °C. In a competitive immunoassay, immobilized IgG and the standard IgG (or sample) competed for binding to a horseradish peroxidase labeled antibody at 33 °C in homogeneous format. After changing the temperature to separate the polymer-immune complex, the complex precipitate was re-dissolved and determined by coupling with the fluorescence reaction of hydrogen peroxide and p -hydroxyphenylacetic acid. The calibration graph for human IgG was linear over the range of 100,1000 ng/mL with a detection limit of 2.0 ng/mL. The method is rapid, sensitive and simple. The immune reaction efficiency was improved. In addition, the sensitivity of this method was close to that using traditional microtitration plates as carriers. However, the assay was much faster (the assay time decreased from 100,120 to 30 min). The method has been applied to the determination of the human IgG levels in human sera with satisfactory results. [source] High-throughput single molecule screening of DNA and proteinsTHE CHEMICAL RECORD, Issue 2 2001Edward S. Yeung Abstract We report a novel imaging technology for real time comprehensive analysis of molecular alterations in cells and tissues appropriate for automation and adaptation to high-throughput applications. With these techniques it should eventually be possible to perform simultaneous analysis of the entire contents of individual biological cells with a sensitivity and selectivity sufficient to determine the presence or absence of a single copy of a targeted analyte (e.g., DNA region, RNA region, protein), and to do so at a relatively low cost. The technology is suitable for DNA and RNA through sizing or through fluorescent hybridization probes, and for proteins and small molecules through fluorescence immunoassays. This combination of the lowest possible detection limit and the broadest applicability to biomolecules represents the final frontier in bioanalysis. The general scheme is based on novel concepts for single molecule detection (SMD) and characterization recently demonstrated in our laboratory. Since minimal manipulation is involved, it should be possible to screen large numbers of cells in a short time to facilitate practical applications. This opens up the possibility of finding single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction or other biological amplification. © 2001 John Wiley & Sons, Inc. and The Japan Chemical Journal Forum Chem Rec 1:123,139, 2001 [source] | |||||||||||||