3- 1,2 dioxetane phosphate

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... 

Luminescent substrate buffer 50 mM Tris-Cl, pH 7.5 (appendix2a HRP), or dioxetane phosphate substrate buffer (see recipe AP)... 

Dioxetane phosphate substrate buffer 1 mM MgCl2 0.1 M diethanolamine 0.02% (w/v) sodium azide (optional)... 

Dioxetane phosphate visualization solution Prepare 0.1 mg/ml AMPPD or CSPD (Applied Biosystems) or 0.1 mg/ml Lumigen-PPD (Lumigen) substrate in dioxetane phosphate substrate buffer (see recipe). Prepare just before use. Lumi-Phos 530 (Boehringer Mannheim or Lumigen) is a ready-to-use solution and can be applied directly to the membrane. 

Signal antibodies have most often been radiolabeled (for immunoradiometric assays [IRMA]) with ( 25j) 7,62,i%,344,4i4 labeled with a chemiluminescent (for immunochemilummo-metric assays [ICMA]) compound, such as acridinium ester, or an enzyme (enzyme-linked immunosorbent assay [ELISA] or enzyme immunoassay [EIA]), such as ALP, converting a substrate (1,2-dioxetane phosphate) to a chemiluminescent product. 

As of 2004, the College of American Pathologists Interlaboratory Survey listed five manufacturers as providing first generation noncompetitive immunoassays for intact PTH. Most of these commercially available methods are on fully automated immunoassay analyzers using chemiluminescence detection including ALP with 1,2-dioxetane phosphate, acridinium ester, or electrochemiluminescence (ruthenium chelate). Signal antibody is less commonly radiolabeled with L... 

Automated noncompetitive chemiluminescent (ICMA) methods have recently been reported using acridinium. ester or ALP with 1,2-dioxetane phosphate. 

E2. Edwards, B., Sparks, A., Voyta, J. C., Strong, R., Murphy, O., and Bronstein, 1., Naphthyl dioxetane phosphate Synthesis of novel substrates for enzymatic chemiluminescent assays. J. Org. Chem. 55, 6225-6229 (1990). 

Replacement of this colorimetric reaction with a chemiluminescent process provides for better sensitivity as well as easier reusability of membranes. The development of compounds, such as adamantyl 1,2 dioxetane phosphate (AMPPD) (1) provides an easy alternative to previous detection schemes as this compound is also destabilized by alkaline phosphatase, resulting in the production of light, which will expose a standard X-ray film. Probes produced by exactly the same methods as for NBT detection are perfectly usable in this process. The membranes with the bound alkaline phosphatase are soaked in a... 

Chemiluminescent substrate Admantyl 1,2-dioxetane phosphate (AMPPD) from Tropix, Bedford, MA. The stock solution is at a concentration of 10 mg/mL. 

In analogy to FIA, CLIAs have also been developed using chemiluminogenic substrates. Dioxetane derivatives such as adamantyl-l,2-dioxetane phosphate are widely used substrates, which exhibit chemiluminescence upon cleavage by alkaline phosphatase. 

FIGURE 5.2 Dioxetane chemiluminescence reaction pathway. A, AMPPD adamantyl 1,2-dioxetane phosphate) when X = H or CSPD when X = Cl B, dioxetane anion C, adamantanone D, methyl-oxybenzoate anion and E, light emission at 477 nm. 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

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