Substrates chemiluminescence

Alkaline phosphatase-labeled probes are synthesized so that 18 bases are complementary to sequences on the arms of the bDNA. Three hybridization sites are located on each branch for a total binding capacity of 45 labeled probes per bDNA molecule. The alkaline phosphatase catalyzes the dephosphorylation of chemiluminescent substrate, dioxetane (Lumi-Phos Plus, Lumigen, Detroit, MI). The intensity of the light emission is measured with a plate luminometer as relative luminescent units. 

Light emission from the chemiluminescent substrate is directly proportional to the amount of the target nucleic acid in the sample, and the results are recorded as relative luminescence units (RLUs). All samples, standards, and controls are run in duplicate, and the mean RLU is used in data analysis. The percent coefficient of variation (%CV) for duplicate RLU for controls and samples must be within the recommended limit for that assay for the results to be valid. For example, negative samples must have a CV of <30% and positive samples <20% in the HCV assay. 

In the Hybrid-Capture assay (Digene), a full-length RNA probe is hybridized to denatured HBV DNA in solution and the hybrids are captured on the surface of a tube coated with anti DNA RNA hybrid antibody. The bound hybrids are reacted with antihybrid antibody labeled with alkaline phosphatase. A chemiluminescent substrate is converted to a luminescent compound by the bound alkaline phosphatase. Light emission is measured in a luminometer and the concentration of HBV DNA, in pg/ml, is determined from a standard curve. The concentrations of the standards are determined spectrometrically (A260nm/A280nm). 

A CL ISH assay for the detection of human papillomavirus (HPV) DNA was developed, in which the hybridization reaction was performed using either digoxigenin-, biotin-, or fluorescein-labeled probes [64], The hybrids were visualized using AP as the enzyme label and a highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. This assay was applied to biopsy specimens from different pathologies associated with HPV, which had previously proved positive for HPV DNA by polymerase chain reaction (PCR). The analytical sensitivity was assessed using samples of HeLa and CaSki cell lines, whose content in HPV DNA is known (10-50 copies of HPV 18 DNA in HeLa cells and 400-600 copies... 

AMPPD is the best chemiluminescent substrate for detecting an ALP-labeled probe [2, 3], The enhanced sensitivity of the chemiluminescence based on the reaction of AMPPD with ALP depends on the enzymatic reaction time (Fig. 2), because the slow kinetics of the signal decay result in the accumulation... 

In the method shown in Figure 9A, a biotin-labeled cDNA probe is first immobilized to a polyvinylchloride microtiter plate well that is coated with bio-tinylated-bovine serum albumin [33], The target DNA is hybridized in the liquid-phase with a digoxigenin-labeled probe, so that the biotin-labeled probe can capture a marker enzyme. An antibody-conjugated enzyme is then added, followed by a chemiluminescent substrate. 

Briefly, the membrane is incubated in the appropriate buffer for the chemiluminescence substrate then placed on a glass plate and a thin layer of substrate solution is pipetted onto the membrane to completely cover the surface and allowed to incubate for the required time (usually... 

An elegant approach is to capture the target DNA or RNA with specific oligonucleotides on to a microwell plate. Synthetic branched DNA bearing multiple alkaline phosphatase-labeled probes hybridizes to the target. A chemiluminescent substrate is added to produce signal. This branched DNA assay has been used in infectious disease detection (W3). 

Schaap, A. P., Akhavan, H., and Romano, L. J., Chemiluminescent substrates for alkaline phosphatase Application to ultrasensitive enzyme-linked immunoassays and DNA probes. Clin. Chem. (Winston-Salem, N.C.) 35, 1863-1864 (1989). 

Applied Biosystems Ltd. CDP-Star <6 CSPD Chemiluminescent Substrates for Alkaline Phosphatase , 2003. www.appliedbiosystems.com/products/productde-tail.cfm procLid = 114. 

Super Signal West Pico Chemiluminescence Substrate (ECL Perbio, Bonn, Germany). 

Horseradish peroxidase (HRP)-coupled secondary anti-IgG antibodies (species depending on primary antibody) enhanced chemiluminescence substrate... 

A modification of these coloring systems has recently been developed that leads to more sensitive detection. Chemiluminescent substrates have been designed that are converted by the enzymes to products that generate a light signal that can be captured on photographic film. This increases the level of sensitivity about 1000-fold over standard color detection methods. 

Working enhanced chemiluminescence substrate solution (see Note 9)—either a. /Modophenol-enhanced substrate luminol 1.25 mM, p-iodophenol 4 pM,... 

Add 150 pL of enhanced chemiluminescent substrate to each well in the same order and preferably with the same timing as used by the plate reader. Allow at least 2 min for the light output to stabilize before reading the plate. 

Since high-affimty recombinant antibodies are becoming easier to produce, it is important to be able to measure affinities with rigorous and reliable, yet user-friendly methods. Competition immunoassays (45,46) meet these criteria, but radioactive antibody labeling or use of fluorogenic or chemiluminescent substrates is needed for affinities >109 owing to the limits of sensitivity in... 

Sandhu, G.S., Eckloff, B.W.. and Kline, B.C. 1991. Chemiluminescent substrates increase sensitivity of antigen detection in Western blots. Bio-Techniques 11 14-16. 

This problem can also be lessened by using stronger fluorochromes and chemiluminescent substrates for use in ELISAs, immunofluorescence-based staining, and immunoblotting. 

Matsumoto, M., Watanabe, N., Kasuga, N.C., Hamada, F. andTadokoro, K. (1997) Synthesis of 5-alkyl-l-aryl-4,4-dimethyl-2,6,7-trioxabicyclo[3.2.0]heptanes as a chemiluminescent substrate with remarkable thermal stability. Tetrahedron Letters, 38 (16), 2863-2866. 

SuperSignal West Pico chemiluminescent substrate for Western blotting (Pierce Catalog 34080)... 

SuperSignal West Pico Chemiluminescent Substrate— Pierce catalog 34080. Prepare the working solution immediately before use by mixing equal volumes of the SuperSignal luminol/enhancer solution and the stable peroxide solution. Each group will require 5 to 10 ml, depending on the size of the membrane. 

Fig. 24.13 Diagnosis using DNA hybridization with biotin-labelled probes. The DNA from the pathogen to be identified is first purified and bound to a membrane. The membrane is then incubated with a diagnostic biotinylated probe. The biotin from the probe will be recognized by streptavidin which has several biotin recognition sites. Hence, subsequent incubation with biotin-labelled alkaline phosphatase results in the recognition of the bound streptavidin. As several molecules of biotin-labelled alkaline phosphatase will bind to a single streptavidin molecule, incubation of the membranes with chemiluminescence substrate for this enzyme will lead to an amplified reaction and the generation of light-emitting products.

A number of nonisotopic immunoassays for estradiol have been developed and adapted for use on fuHy automated immunoassay systems. All are heterogeneous assays (separation step needed), but most are direct assays and do not require prehminary extraction. Most procedures offer the convenience of solid-phase separation methods. For routine clinical applications, the greatest experience is with enzyme immunoassays. Most commercial enzyme immunoassays use horseradish peroxidase or alkaline phosphatase to label estradiol antigens enzyme activity is determined using a variety of photometric,fluorescent,or chemiluminescent substrates. ... 

Design and synthesis of dioxetanes as highly efficient chemiluminescent substrates 03YGK595. 

Chemiluminescent substrates have also been developed for lucifer-ase (Neufeld et al., 1965), GPDase (Haggerty et al., 1978), catalase (Neufeld et al., 1965) and BGase (Puget et al., 1977). 

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