Irena Bronstein

Genomic Southern analysis with alkaline-phosphatase-conjugated oligonucleotide probes and the chemiluminescent substrate AMPPD

We have used a chemiluminescent detection method to improve both the sensitivity and the speed of detection of human genes with oligonucleotide probes. A direct chemiluminescent substrate (AMPPD) was used in combination with an alkaline-phosphatase-labeled oligonucleotide probe to detect the human tissue of plasminogen activator gene by Southern blot analysis. X-ray exposures obtained after 4 h were comparable to those obtained after 7 days with a 32P-labeled oligomer. After 16 h, the signal was 12 times greater than the 32P signal. The detection of the single-copy tissue plasminogen activator gene in 0.25 micrograms of human genomic DNA (76,000 molecules) was achieved. The improved sensitivity obtained by chemiluminescent detection should increase the usefulness of oligonucleotide probes in the direct Southern analysis of human genetic disorders.

[13] Novel methods for chemiluminescent detection of reporter enzymes

Chemiluminescent reporter gene assays provide highly sensitive, quantitative detection in simple, rapid assay formats for detection of reporter enzymes that are widely employed in gene expression studies. Chemiluminescent detection methodologies typically provide up to 100-1000x higher sensitivities than may be achieved with fluorescent or colorimetric enzyme substrates. The variety of chemiluminescent 1,2-dioxetane substrates available enable assay versatility, allowing optimization of assay formats with the available instrumentation, and are ideal for use in gene expression assays performed in both biomedical and pharmaceutical research. In addition, 1,2,-dioxetane chemistries can be multiplexed with luciferase detection reagents for dual detection of multiple enzymes in a single sample. These assays are amenable to automation with a broad range of instrumentation for high throughput compound screening.

Comparison of 5-Hydroxy-2, 3-Dihydrophthalazine-1, 4-Dione and Luminol as Co-Substrates for Detection of Horseradish Peroxidase in Enhanced Chemiluminescent Reactions

The utility of 5-hydroxy-2, 3-dihydrophthalazine-1, 4-dione (HDP) as a co-substrate for the chemiluminescent detection of horseradish peroxidase was assessed. Several substituted aryl boronic acid derivatives (4-phenyl, 4-iodo) acted as potent enhancers of the peroxidase catalyzed reaction. Addition of chelating agents (EDTA) and surfactants (Tween-20 and [poly (vinylbenzyl)tributylphosphonium chloride-poly (vinylbenzyl) trioctylphosphonium chloride copolymer]) modulated background light emission and the intensity and duration of the signal from both HDP and luminol. However, HDP was found to be inferior to luminol in the peroxidase assay. Comparative studies revealed that at 500 amol of peroxidase the S/B was ten-fold higher using a commercial luminol-based signal reagent as compared with an HDP-EDTA-Tween-20 reagent (S/B t = 0 min 21.8 vs 1.7, S/B t = 10 min 17.8 vs 2.0).

Chemiluminescent immunodetection protocols with 1,2-dioxetane substrates

Chemiluminescent 1,2-dioxetane enzyme substrates provide a highly sensitive and versatile detection method for immunoblots and other membrane-based detections. 1,2-Dioxetane substrates, coupled with either alkaline phosphatase or beta-galactosidase enzyme labels, generate glow light emission kinetics, with a signal duration that is significantly longer than most enhanced luminol/horseradish peroxidase chemiluminescent detection systems. The long-lived, high-intensity light signal is ideal for imaging using a variety of formats, including X-ray film, photographic film, chemiluminescence phosphor imaging screens, and the rapidly expanding selection of camera imaging systems.

Detection of DNA in Southern Blots with Chemiluminescence

The chemiluminescent detection methods described in this chapter have been successfully applied to the detection of plasmid DNA and genomic DNA in Southern and sequencing protocols. The high sensitivity and the simplicity of AMPPD are instrumental in making the chemiluminescent detection of DNA successful in hybridization assays. This detection technique has also been used to detect DNA in dot blots and in situ hybridization experiments as well as proteins in enzyme-linked immunosorbent assays (ELISAs) and Western blots.

Chemiluminescent reporter gene assays with 1,2-dioxetane enzyme substrates

1,2-Dioxetane chemiluminescent substrates provide highly sensitive, quantitative detection with simple, rapid assay formats for the detection of reporter enzymes that are widely used in gene expression studies. Chemiluminescent detection methodologies typically provide up to 100-1000x higher sensitivities than can be achieved with the corresponding fluorescent or colorimetric enzyme substrates. The varieties of 1,2-dioxetane substrates available provides assay versatility, allowing optimization of assay formats with the available instrumentation, and are ideal for use in gene expression assays performed in both biomedical and pharmaceutical research. These assays are amenable to automation with a broad range of instrumentation for high throughput compound screening.

7 – Detection Methods Using Chemiluminescence

This chapter describes the use of various chemiluminescence methodologies for the detection of viruses in DNA hybridization assays. Chemiluminescent processes constitute a very special class of chemical reactions, in which products are produced in electronically excited states that are very short-lived and rapidly decay with concomitant emission of light. Chemiluminescent reactions do not produce very high intensity light signals because of many efficient quenching processes that compete with the radiative decay of the excited states. 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. A wide spectrum of instruments is currently available for recording and quantifying chemiluminescent signal intensities. These instruments, known as luminometers, use a light detector that consists of a photomultiplier tube in photon counting mode, positioned close to the light source to maximize photon collection efficiency. In situ hybridization with chemiluminescence detection has been used to detect immunodeficiency (HIV)-infected cells and human papillomavirus (HPV) type 16 in a cervical carcinoma cell line. Chemiluminescence detection technologies combined with DNA hybridization methods provide rapid, sensitive, nonradioactive, automatable assay formats for the clinical diagnosis of infectious agents, as well as for research use.

Immunoassay protocol for quantitation of protein kinase activities.

Quantitation of at least two orders of magnitude of kinase enzyme concentration is achieved with detection of less than 0.1 U/well of src kinase activity (Fig. 3). A comparison between a sequential protocol, in which biotinylated peptide substance is captured prior to incubation with the kinase enzyme, and a simultaneous protocol, in which peptide capture and the kinase reaction proceed concurrently, demonstrates that the simpler simultaneous protocol provides similar detection sensitivity. these have also been demonstrated with 0.1 microM peptide substrate in a protein kinase A assay.5 Quantitation of protein kinase activity with chemiluminescent detection has been demonstrated with several different protein kinases, including both tyrosine and serine/threonine kinases.5 An immunoassay format provides high sensitivity and can be performed under conditions that most closely mimic physiological substrate and ATP concentrations with chemiluminescent detection. This assay format is also automated easily for use in high-throughput screening.

Chemiluminescence:Properties of 1,2-Dioxetane Chemiluminescence

The first synthesis of a dioxetane was described in 1969 by Kopecky and Mumford, and the first thermally stable dioxetane, adamantylidene-adamantyl-1,2-dioxetane, was described in 1972 by Wierynga et al. The resulting product decomposed chemiluminescently only when heated above 165°C. McCapra and coworkers (1977) reported on the synthesis of an un-symmetrically substituted adamantyl 1,2-dioxetane (9-(2-adamantylidene)-N-methylacridan-1,2-dioxetane) which, when heated generated chemiluminiscence exclusively from the excited singlet of N-methylacridone. It has been shown that the decomposition of this unsymmetrically substituted 1,2- dioxetane leads to the formation of the excited state carbonyl-based product with the lowest singlet energy state. McCapra also proposed that dioxetanes which are substituted with electron donating groups decompose via charged intermediaties in an electron transfer process (McCapra et al., 1977). In 1983, Adam and coworkers (Adam et al., 1983) synthesized and studied the stability of several unsymmetrically substituted adamantyl 1,2-dioxetanes and concluded that the stabilization mechanism is complex and depends on several factors, such as conformational isomerism (McCapra, 1977). More recently a theroretical investigation on the decomposition modes of 1,2-dioxetanes has been reported (Reguero et al., 1991).

Quantitative polymerase chain reaction and solid-phase capture nucleic acid detection.

The combination of PCR amplification and chemiluminescent detection of PCR products provides a highly sensitive system for the quantitation of DNA and RNA. The broad dynamic range of the chemiluminescent detection assay simplifies the selection of cycling and concentration parameters critical to harnessing the quantitative aspects of PCR amplification. Detection of 200 amol of PCR product is attained using the described procedures. The tube or microplate format of the assay avoids many of the limitations associated with other methods of PCR quantitation involving gel electrophoresis. This detection methodology can be applied to a variety of quantitative nucleic acid assays, including viral load and gene expression analysis.