Clinical applications of luminescent assays for enzymes and enzyme labels

Bronstein, Irena; Kricka, Larry J.

doi:10.1002/jcla.1860030511

Cited by 48 publications

(13 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In a dilution series of biotinylated PCR products with known DNA contents, we could detect as little as 4 pg of DNA (10 amol) using ssDNA probes. The detection limit of our ECL hybridization assay was comparable to that reported from the use of other peroxidase-and ECL-based detection systems, ranging from about 1 amol (22) to 100 amol (3,30); furthermore, the detection limit was comparable to that achieved by the use of radiolabelled oligonucleotides (30).…”

Section: Discussionsupporting

confidence: 78%

“…Either a second round of PCR (i.e., nested PCR) is necessary (13,17), or a more sensitive detection of the PCR product is required, involving hybridization with labelled DNA probes (8, 10, 19-21, 31, 33). Enhanced chemiluminescence (ECL) following hybridization with a peroxidase-coupled DNA probe was shown to be almost as sensitive as hybridization with 32 P-labelled probes (30), allowing detection of 25 to 100 amol of DNA (3,30). Because of its sensitivity and ease of detection (3,7,22), ECL is superior to most nonradioactive detection and labelling methods.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Quantitative assay of PCR-amplified hepatitis B virus DNA using a peroxidase-labelled DNA probe and enhanced chemiluminescence

et al. 1996

View full text Add to dashboard Cite

We have developed a sensitive and quantitative assay for hepatitis B virus (HBV) DNA in serum or plasma in which PCR and then microtiter hybridization analysis are used. Assay of HBV DNA in serum or plasma is important for demonstrating viral replication, indicating and monitoring antiviral therapy, determining the infectivities of virus carriers, and ensuring the safety of blood products. Under optimum conditions PCR can amplify one HBV DNA molecule to 10 8 copies, but detection of this amount of DNA still requires hybridization with labelled probes or a nested PCR. We labelled one strand of the PCR product with a biotinylated primer. The double-stranded amplicon was incubated in streptavidin-coated microplate wells. The nonlabelled strand was removed after denaturation of the double-stranded DNA with alkali, and the bound strand was hybridized with a peroxidase-coupled single-stranded probe. The amount of bound peroxidase was measured in a luminometer. Four picograms of amplicon was detectable in this system, whereas conventional ethidium bromide staining requires a 1,000 times higher amplicon concentration. The performance of the new assay was compared with those of nested PCR and a PCR system that uses a digoxigenin label, hybridization to a solid-phase adsorbed probe, and colorimetric detection. The chemiluminescence assay was found to be almost as sensitive as nested PCR and approximately five times more sensitive than the colorimetric test.

show abstract

Section: Discussionsupporting

confidence: 78%

mentioning

confidence: 99%

Quantitative assay of PCR-amplified hepatitis B virus DNA using a peroxidase-labelled DNA probe and enhanced chemiluminescence

et al. 1996

View full text Add to dashboard Cite

show abstract

“…The sensitivity of alkaline phosphatase-dioxetane chemiluminescence detection has been shown to be superior to other nonisotopic systems based on colorimetric detection in membrane-based hybridization assays (Bronstein and Kricka, 1989;Bronstein and Voyta, 1989;Bronstein et al, 1989c;Musiani et al, 1991aMusiani et al, ,1992. Finally, alkaline phosphatase-dioxetane detection has been demonstrated to be two to five times more sensitive than enhanced luminol chemiluminescent detection (described subsequently) in a solution hybridization assay system Urdea et al, 1990).…”

Section: A Dioxetanesmentioning

confidence: 99%

Detection Methods Using Chemiluminescence

Bronstein¹,

Olesen²

1995

Molecular Methods for Virus Detection

Self Cite

View full text Add to dashboard Cite

“…tion of the reaction and the measurement of the light emission. Also, the detection limit for peroxidase (25 attomoles) is significantly lower than the detection limits for this enzyme using conventional assay techniques (Bronstein and Kricka, 1989;Thorpe and Kricka, 1986).…”

Section: Introductionmentioning

confidence: 99%

Chemiluminescent assay of enzymes using proenhancers and pro‐anti‐enhancers

Kricka

Schmerfeld-Pruss

Edwards³

1991

J. Biolumin. Chemilumin.

Self Cite

View full text Add to dashboard Cite

Enhanced chemiluminescent assays for hydrolase enzymes have been developed using proenhancer and pro-anti-enhancer substrates. Alkaline phosphatase is measured using disodium para-iodophenyl phosphate (proenhancer) which is converted to para-iodophenol and this in turn enhances the light emission from the horseradish peroxidase catalysed chemiluminescent oxidation of luminol by peroxide. An alternative strategy uses para-nitrophenyl phosphate which is converted by alkaline phosphatase to para-nitrophenol which inhibits the enhanced chemiluminescent reaction. The detection limit for the enzyme using the proenhancer and pro-anti-enhancer assays was 100 attomoles and 1 picomole, respectively. The proenhancer strategy was effective in assays for beta-D-galactosidase, beta-D-glucosidase and aryl sulfatase. A limited comparison of the proenhancer and a conventional colorimetric assay for an alkaline phosphatase label in an enzyme immunoassay for alpha-fetoprotein showed good agreement.

show abstract

Clinical applications of luminescent assays for enzymes and enzyme labels

Cited by 48 publications

References 33 publications

Quantitative assay of PCR-amplified hepatitis B virus DNA using a peroxidase-labelled DNA probe and enhanced chemiluminescence

Quantitative assay of PCR-amplified hepatitis B virus DNA using a peroxidase-labelled DNA probe and enhanced chemiluminescence

Detection Methods Using Chemiluminescence

Chemiluminescent assay of enzymes using proenhancers and pro‐anti‐enhancers

Contact Info

Product

Resources

About