Simultaneous Analysis of Two-Site Gene Polymorphisms Using Time-Resolved Fluorescence Immunoassay

Watanabe, Kazuyuki; Oda, Miwa; Arakawa, Hidetoshi; Maeda, Masako

doi:10.2116/analsci.16.765

Cited by 4 publications

(2 citation statements)

References 15 publications

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“…Lanthanide labelling reagents, Assay buffer 2 and Enhancement solution 2 were purchased from Wallac. Avidin-coated microplates, washing solution and blocking solution were prepared as described elsewhere (9). Other reagents were of analytical grade.…”

Section: Reagentsmentioning

confidence: 99%

Simultaneous detection of two verotoxin genes using dual‐label time‐resolved fluorescence immunoassay with duplex PCR

Watanabe¹,

Arakawa²,

Maeda³

2002

Luminescence

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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).

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Section: Reagentsmentioning

confidence: 99%

Simultaneous detection of two verotoxin genes using dual‐label time‐resolved fluorescence immunoassay with duplex PCR

Watanabe¹,

Arakawa²,

Maeda³

2002

Luminescence

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“…Also, we have reported on the simultaneous determination of two or three analytes by using TR-FIA. [5][6][7] Recently, we established a highly sensitive bioluminescent assay of acetate kinase (AK) and pyruvate phosphate dikinase (PPDK) using firefly luciferase and luciferin as a detection system. We succeeded in detecting 8.6 × 10 -21 mol/assay for AK and 1.4 × 10 -20 mol/assay for PPDK, respectively.…”

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confidence: 99%

Highly Sensitive Simultaneous Bioluminescent Measurement of Acetate Kinase and Pyruvate Phosphate Dikinase Activities Using a Firefly Luciferase-Luciferin Reaction and Its Application to a Tandem Bioluminescent Enzyme Immunoassay

et al. 2003

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The immunoassay method has been improved by many researchers, and has became a mature method. Generally, only an antigen or an antibody in one assay medium is measured by the immunoassay. However, if we can determine many analytes by using an immunoassay in one assay medium, we can obtain many benefits, such as saving time, materials and labor. Trials of the simultaneous determination by immunoassay were carried out by time-resolved fluoroimmunoassay (TR-FIA). We can determine multiple substances with the simultaneous use of various rare earth ions e.g. Eu 3+ , Sm 3+ , Tb 3+ by TR-FIA. [1][2][3] Further, Xu et al. 4 reported on simultaneous quadruple TR-FIA. Antigens or antibodies were labeled with four kinds of rare earth ion (Eu 3+ , Sm 3+ , Tb 3+ and Dy 3+ ) chelate in the TR-FIA. Their method was very significant, but a particular enhancement solution was required for the assay of Tb 3+ and Dy 3+ . The method is less sensitive to Sm 3+ and Dy 3+ than the other ions. Also, we have reported on the simultaneous determination of two or three analytes by using TR-FIA. [5][6][7] Recently, we established a highly sensitive bioluminescent assay of acetate kinase (AK) and pyruvate phosphate dikinase (PPDK) using firefly luciferase and luciferin as a detection system. We succeeded in detecting 8.6 × 10 -21 mol/assay for AK and 1.4 × 10 -20 mol/assay for PPDK, respectively. These enzymes were applicable in bioluminescent an enzyme immunoassay (BL-EIA) for various biological samples. 8-13 AK and PPDK are enzymes that generate ATP, and react under similar conditions. We have now developed simultaneous bioluminescent assays using AK and PPDK. The principle of the proposed assay is shown in Fig. 1. In the first step, AK generates ATP from ADP and acetylphosphate, and ATP is determined by the firefly luciferase-luciferin reaction. In the second step, the bioluminescent emission from AK is quenched by the addition of glucose and ADP-dependent hexokinase (ADP-HK) 14 We have developed a simultaneous bioluminescent measurement of acetate kinase (AK) and pyruvate phosphate dikinase (PPDK) activities and its application to a tandem enzyme immunoassay. The principle of the proposed assay is as follows. In the first step, AK generates ATP from ADP and acetylphosphate, and the ATP is determined by the firefly luciferase-luciferin reaction. In the second step, the bioluminescent intensity from AK is eliminated by adding glucose and ADP-dependent hexokinase, which forms AMP from ADP. At the same time, the PPDK catalyzes the interconversion of AMP, diphosphate, and phosphoenolpyruvate to ATP, phosphate and pyruvate. The ATP formed by PPDK is also determined by the firefly luciferase-luciferin reaction. The detection limits (at blank + 3SD) of AK and PPDK were 1.03 × 10 -20 and 2.05 × 10 -20 mol per assay, respectively. The method was applicable to a bioluminescent enzyme immunoassay for the assay of insulin and C-peptide in the same sample.

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Highly sensitive and rapid tandem bioluminescent immunoassay using aequorin labeled Fab fragment and biotinylated firefly luciferase

Ito

Nishimura

Maeda

et al. 2007

Analytica Chimica Acta

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Simultaneous Analysis of Two-Site Gene Polymorphisms Using Time-Resolved Fluorescence Immunoassay

Cited by 4 publications

References 15 publications

Simultaneous detection of two verotoxin genes using dual‐label time‐resolved fluorescence immunoassay with duplex PCR

Simultaneous detection of two verotoxin genes using dual‐label time‐resolved fluorescence immunoassay with duplex PCR

Highly Sensitive Simultaneous Bioluminescent Measurement of Acetate Kinase and Pyruvate Phosphate Dikinase Activities Using a Firefly Luciferase-Luciferin Reaction and Its Application to a Tandem Bioluminescent Enzyme Immunoassay

Highly sensitive and rapid tandem bioluminescent immunoassay using aequorin labeled Fab fragment and biotinylated firefly luciferase

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