Expression Hybridization Assays Combining cDNAs from Firefly and <i>Renilla</i> Luciferases as Labels for Simultaneous Determination of Two Target Sequences

Laios, Eleftheria; Obeid, Pierre J.; Ioannou, Penelope C.; Christopoulos, Theodore K.

doi:10.1021/ac0004198

Cited by 13 publications

(10 citation statements)

References 21 publications

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“…Multi‐enzyme systems containing labels synthesizing ATP which is detected using firefly luciferase and hybridization analysis using expressible DNA fragment encoding firefly luciferase as a label represent valid alternative for detection of DNA with the help of firefly luciferase. The DNA detection limit in these systems is usually lower , although the assay includes the step of signal amplification (synthesis of ATP or luciferase molecules ). Moreover, the duration of luminescence detection increases significantly .…”

Section: Luciferase Fusions With Dna or Rna‐binding Proteins And Theimentioning

confidence: 99%

Firefly Luciferase‐based Fusion Proteins and their Applications in Bioanalysis

Smirnova

Ugarova

2016

Photochem & Photobiology

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Firefly luciferase is widely used in molecular biology and bioanalytical systems as a reporter molecule due to the high quantum yield of the bioluminescence, availability of stable mutant forms of the enzyme with prescribed spectral characteristics and abundance of bacterial expression systems suitable for production of recombinant proteins in limitless quantities. In this review, we described fusion proteins of luciferase with biotin-binding domain and streptavidin, with proteins A and G, antibodies, with DNA-and RNA-binding proteins, as well as fusion proteins designed for BRET systems. The firefly luciferase-based fusion proteins are represented as an effective tool for the development of different bioanalytical systems such as (1) systems in which luciferase is attached to the surface of the target and the bioluminescence signal is detected from the specific complexes formed; (2) BRET-based systems, in which the specific interaction induces changes in the bioluminescence spectrum; and (3) systems that use modified or split luciferases, in which the luciferase activity changes under the action of the analyte. All these systems have wide application in biochemical analysis of physiologically important compounds, for the detection of pathogenic bacteria and viruses, for evaluation of proteinprotein interactions, assaying of metabolites involved in cell communication and cell signaling.Abbreviations: Ab, antibody; b, biotin; bccp, biotin carboxyl carrier protein domain of acetyl-CoA carboxylase from E. coli; HRP, horseradish peroxidase; IA, immunoassay; KPBT, Klebsiella pneumoniae oxaloacetate decarboxylase; Luc, firefly luciferase; PG, pepsinogen; PSA, prostate-specific antigen; SA, streptavidin.

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Section: Luciferase Fusions With Dna or Rna‐binding Proteins And Theimentioning

confidence: 99%

Firefly Luciferase‐based Fusion Proteins and their Applications in Bioanalysis

Smirnova

Ugarova

2016

Photochem & Photobiology

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“…Two DNAs encoding firefly luciferase (FLuc) and Renilla luciferase (RLuc) were used as labels for the development of a microtiter well-based expression hybridization assay that allowed simultaneous determination of two target DNA sequences in the same well. 32 The RLuc DNA was chosen because RLuc, like FLuc, is a monomeric protein, it requires no posttranslational modification and its activity can be readily measured in the transcription/translation reaction without prior purification. The constructed FLuc Biotinylated (B) target DNA is captured on streptavidin (SA)-coated wells and denatured by NaOH.…”

Section: Aq1mentioning

confidence: 99%

“…42 Few reports describe the combination of two labels in a single hybridization assay with applications to the quantification of PCR products. 4,32 These assays are based on the combination of two chemiluminescent labels (detection of two targets in the same sample and in one microtiter well).…”

Section: (D T T T T T T T T…) (D a A A A A A A A A…)mentioning

confidence: 99%

Gene Assays Based on Bio(Chemi)luminescence

Laios

Ioannou²,

Christopoulos

2010

Chemiluminescence and Bioluminescence

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One of the first reports describing the use of recombinant aequorin as a reporter molecule in a bioluminometric nucleic acid hybridization assay was in 1996. 1 Microtiter wells were coated with anti-digoxigenin antibody. The target DNA was hybridized simultaneously with an immobilized digoxigenin-labeled capture probe and a biotinylated detection probe. The hybrids were determined using an aequorin-streptavidin conjugate followed by the measurement of luminescence in the presence of excess Ca 21 (Figure 9.1). The linearity of the assay was in the range 0.1-200 pM (5 amol well À1 to 10 fmol well À1) and the S/B ratio at 0.1 pM (5 amol well À1) was 5.3. A configuration in which the aequorinstreptavidin conjugate was replaced by a preformed complex of biotinylated aequorin to streptavidin resulted in equivalent signals and detectability. An advantage of using the preformed complex is that it was conveniently prepared by simply mixing the two components (streptavidin and biotinylated aequorin), thus providing a practical alternative to the use of aequorin-streptavidin conjugates prepared by covalent crosslinking techniques. The aequorin-biotin and the aequorin-streptavidin conjugates in this study were obtained commercially (the same group later reported the construction of a plasmid suitable for bacterial expression of in vivo-biotinylated aequorin, facilitating further the development of highly sensitive hybridization assays, 2 as well as a method for

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“…Laios et al 104 discovered that firefly and Renilla luciferase cDNAs could be transcribed and translated simultaneously and independently over a broad range of concentrations in the same expression mixture. Based on this fact, they developed an expression hybridization assay that combines firefly-luciferase DNA with Renilla-luciferase DNA as labels for the simultaneous determination of two target DNA sequences.…”

Section: ·2 Substrate Zone-resolved Techniquementioning

confidence: 99%

Chemiluminescence Platforms in Immunoassay and DNA Analyses

Fan

Cao

et al. 2009

ANAL. SCI.

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IntroductionCL is the production of electromagnetic radiation by a chemical reaction. The CL intensity is directly proportional to the concentration of a limiting reactant involved in the CL reaction. CL has been exploited for its use in a wide range of applications due to its high sensitivity, wide calibration range, and suitability for miniaturization. Protein and DNA analyses have recently attracted much attention. Many different types of techniques, such as electrochemical, optical, radiochemical, and piezoelectronic methods have been applied for protein and DNA analyses. To provide an overview of CL detection assays for DNAs and proteins, we have summarized their essential techniques. Monoplex and multiplex assays are described, and their current reports are intended to provide an overview of the CL techniques currently employed in numerous laboratories. Monoplex AssayA monoplex assay aims to measure the presence of a single analyte in any given sample, in which nanoparticle, DNA enzyme, horseradish peroxidase (HRP), alkaline phosphatase (AP) and acridinium ester are commonly used as labels. 2·1 Nanoparticle-based CL techniquesNanoparticles offer excellent prospects for DNA detection and immunoassay, owing to their many attractive properties. Compared to existing labels, nanoparticles are more stable and cheaper, and easier to be purified. In addition, they indicate faster binding kinetics with high sensitivity and a high reaction rate for many types of monoplex assays, ranging from immunoassays to DNA detection. 2·1·1 Gold nanoparticlesColloidal gold labels are ideal in biotechnological systems for several reasons. Firstly, they can be readily prepared in a wide range of sizes, from approximately 2 nm to above 100 nm. Secondly, the biochemical activity of the labeled biomolecules can be retained when colloidal gold is coupled to the biomolecules and, thirdly, colloidal gold labels can be easily visualized as dense structures within biological entities using transmission electron microscopy. As a biological tag, colloidal gold has been employed in several CL detection systems. 2·1·1·1 Gold nanoparticle-based CL immunoassay. The Au 3+ -luminol reaction, in which 10 -9 M Au 3+ ions can be sensitively detected, is a classic CL reaction. Thousands of Au atoms are contained within the gold nanoparticles; for example, 2.3 ¥ 10 5 Au atoms are theoretically contained in a 20-nm spherical gold particle and, consequently, picomolar detection limits can be attained by employing oxidative gold metal dissolution in acidic solutions. Our group 1,2 reported a sensitive CL immunoassay based on a colloidal gold label after oxidative gold metal dissolution (using 0.01 M HCl-0.5 M NaCl-0.5 mM Br2), using a simple and sensitive luminol-CL reaction (Fig. 1). Reviews Chemiluminescence Platforms in Immunoassay and DNA AnalysesAiping FAN,* Zhijuan CAO,* Huan LI,* Masaaki KAI,** and Jianzhong LU* † *School of Pharmacy, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China ** Faculty of Pharmaceutical Sciences, Graduate Scho...

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Expression Hybridization Assays Combining cDNAs from Firefly and Renilla Luciferases as Labels for Simultaneous Determination of Two Target Sequences

Cited by 13 publications

References 21 publications

Firefly Luciferase‐based Fusion Proteins and their Applications in Bioanalysis

Firefly Luciferase‐based Fusion Proteins and their Applications in Bioanalysis

Gene Assays Based on Bio(Chemi)luminescence

Chemiluminescence Platforms in Immunoassay and DNA Analyses

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