Luminescent Proteins in Binding Assays

Roda, Aldo; Guardigli, Massimo; Michelini, Elisa; Mirasoli, Mara; Pasini, Patrizia

doi:10.1002/9783527610754.fa20

Cited by 3 publications

(4 citation statements)

References 92 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…fluorescence) stem from its higher sensitivity (attomolar detection) and simplicity as the light generated obviates the need for an excitation light source. [1][2][3] Assays that employ this technology have been developed to aid in the diagnosis of infectious diseases, [4][5][6][7] oncology, [8] cardiovascular disease, [9] and therapeutic drug monitoring. [10,11] Chemiluminescent acridinium labelled antibodies have been successfully commercialized on high-throughput, automated in vitro diagnostic analyzers.…”

Section: Introductionmentioning

confidence: 99%

A rainbow of acridinium chemiluminescence

et al. 2021

View full text Add to dashboard Cite

Multicolor chemiluminescent acridinium derivatives were synthesized by attaching various common fluorophores to the N 10 -acridinium position through a piperazine linker. Triggering of each acridinium derivative using alkaline hydrogen peroxide resulted in a chemiluminescence spectrum dominated by a strong emission (>95%) from the attached fluorophore. The highly quenched emission from the triggered acridinium, acting as a donor, points to a highly efficient intramolecular energy transfer in acridinium-based chemiluminophore-fluorophore tandems. A variable, and in many cases minimal, spectral overlap between the donor emission and the acceptor absorption may indicate that in such tandems the energy transfer follows the Dexter electron exchange mechanism. Moreover, fluorophores affixed through the acridinium 9-position produce a typical acridinium emission profile, demonstrating the need for close distances and favorable intramolecular orientation of the donor and acceptor moieties for the energy transfer to occur. A family of red-shifted chemiluminescent labels, all sharing a uniform triggering method, will find immediate application in multicolor ligand-receptor assays. Along with the multiplexing capabilities, the red-shifted chemiluminescent detection offers a higher tolerance to green-colored biological interferences and will therefore benefit many screening and diagnostic clinical tests.

show abstract

Section: Introductionmentioning

confidence: 99%

A rainbow of acridinium chemiluminescence

et al. 2021

View full text Add to dashboard Cite

show abstract

“…In comparison with luciferases from other sources, firefly luciferases have several advantages as labels in biospecific analysis: (1) the high quantum yield of bioluminescent reaction (1) provides a high sensitivity of detection; (2) highly active thermostable luciferase mutants with different spectral parameters are available; and (3) the luciferase preparation and extraction procedure is simple and yields required quantities of the protein (2,3). Luciferase fusion proteins that comprise the components forming high-affinity complexes with a target under study are of particular interest.…”

Section: Introductionmentioning

confidence: 99%

“…(a) The effect of probe immobilization methods on bioluminescence signals. The probe was immobilized on a Pluronic-modified surface (1) and on nonmodified surface(2). In method (2), the plate was treated with the bovine serum albumin (BSA)-casein mixture after probe immobilization.…”

mentioning

confidence: 99%

A Novel Streptavidin–luciferase Fusion Protein: Preparation, Properties and Application in Hybridization Analysis of DNA

Smirnova

Rubtsova

Grigorenko

et al. 2016

Photochem & Photobiology

View full text Add to dashboard Cite

A streptavidin-luciferase fusion protein comprising the thermostable mutant form of firefly luciferase Luciola mingrelica and minimal core streptavidin was constructed. The streptavidin-luciferase fusion was mainly produced in a tetrameric form with high luciferase and biotin-binding activities. It was shown that fusion has the same K values for ATP and luciferin and the bioluminescence spectra as initial luciferase. The linear dependence of the bioluminescence signal on the content of the fusion was observed within the range of 10 -10 mol per well. Successful application of obtained fusion in a biospecific bioluminescence assay based on biotin-streptavidin interactions was demonstrated by the example of a specific DNA hybridization analysis. A DNA hybridization analysis for Escherichia coli cells identification was developed using unique for these cells gadB fragment encoding glutamate decarboxylase. The amplified biotinylated GadB fragments were hybridized with the immobilized oligonucleotide probes; then, the biotin in the DNA duplexes was detected using the streptavidin-luciferase fusion protein. To reach the high sensitivity of the assay, we optimized the conditions of the assay. It was shown that the use of Pluronic for plate modification resulted in a significant reduction in the DNA detection limit which finally was 0.4 ng per well.

show abstract

“…Bioluminescence resonance energy transfer (BRET) is a phenomenon of nonradiative transfer of energy from a donor (luciferase) to an acceptor. Photoproteins, dyes or quantum dots can act as acceptors of bioluminescence energy (1,2). BRET is observed when both entities are in close proximity (≤10 nm) to each other.…”

Section: Introductionmentioning

confidence: 99%

The Bioluminescence Resonance Energy Transfer from Firefly Luciferase to a Synthetic Dye and its Application for the Rapid Homogeneous Immunoassay of Progesterone

Smirnova

Samsonova

Ugarova

2015

Photochem & Photobiology

View full text Add to dashboard Cite

The sensitive BRET system for the homogeneous immunoassay of a low-molecular weight antigen was developed using progesterone as an example. Two thermostable mutants of the Luciola mingrelica firefly luciferase (Luc)-the "red" mutant with λmax.em = 590 nm (RedLuc) and the "green" mutant with λmax.em = 550 nm (GreenLuc)-were tested as the donors. The water-soluble Alexa Fluor 610× (AF) dye was selected as the acceptor because its two absorption maxima, located at 550 and 610 nm, are close to the bioluminescence maxima of the GreenLuc and RedLuc, respectively. The methods for the synthesis of the luciferase-progesterone (Luc-Pg) conjugate and the conjugate of the dye and the polyclonal antiprogesterone antibody (AF-Ab) were developed. Both conjugates retained their functional properties, had high antigen-antibody binding activity, and demonstrated a high BRET signal. The homogeneous immunoassay system based on the BRET from the firefly luciferase to the synthetic dye was established to assay progesterone as a model antigen. Optimization of the assay conditions, the composition of the reaction mixture, and the concentrations of the donor and the acceptor made it possible to reach the minimum detectable progesterone concentration of 0.5 ng mL(-1) .

show abstract

Luminescent Proteins in Binding Assays

Cited by 3 publications

References 92 publications

A rainbow of acridinium chemiluminescence

A rainbow of acridinium chemiluminescence

A Novel Streptavidin–luciferase Fusion Protein: Preparation, Properties and Application in Hybridization Analysis of DNA

The Bioluminescence Resonance Energy Transfer from Firefly Luciferase to a Synthetic Dye and its Application for the Rapid Homogeneous Immunoassay of Progesterone

Contact Info

Product

Resources

About