Bacterial alkaline phosphatase assays

In a second procedure, poly(ADP-ribose) was first separated from the bulk of the nucleic acids and proteins by dihydroxyboryl-Sepharose affinity chromatography 147,190). The isolated polymer was treated with snake venom phosphodiesterase and bacterial alkaline phosphatase to yield the nucleoside 2, l"-ribosyladenosine from internal residues. This product was then treated with chloroacetaldehyde to produce the fluorescent derivative, l,iSr -ethenoribosyladenosine, which was then separated from other derivatized residues by reversed-phase high performance liquid chromatography picomole amounts were quantified by fluorescence detection. This procedure facilitates the accurate determination of minute quantities of endogenous poly(ADP-ribose) (102, 190). Niedergang et al. (147) have also utilized a fluorimetric assay for determination of the enzymatic digestion products of the polymer, ADP-ribose, or iso-ADP-ribose. 

Cambella and Antia [385] determined phosphonates in seawater by fractionation of the total phosphorus. The seawater sample was divided into two aliquots. The first was analysed for total phosphorus by the nitrate oxidation method capable of breaking down phosphonates, phosphate esters, nucleotides, and polyphosphates. The second aliquot was added to a suspension of bacterial (Escherichia coli) alkaline phosphatase enzyme, incubated for 2h at 37 °C and subjected to hot acid hydrolysis for 1 h. The resultant hot acid-enzyme sample was assayed for molybdate reactive phosphate which was estimated as the sum of enzyme hydrolysable phosphate and acid hydrolysable... 

Methods based on chemiluminescent and bioluminescent labels are another area of nonisotopic immunoassays that continue to undergo active research. Most common approaches in this category are the competitive binding chemiluminescence immunoassays and the immunochemiluminometric assays. Chemiluminescence and heterogenous chemiluminescence immunoassays have been the subject of excellent reviews (91, 92). Detection in chemiluminescence immunoassays is based on either the direct monitoring of conjugated labels, such as luminol or acridinium ester, or the enzyme-mediated formation of luminescent products. Preparation of various derivatives of acridinium esters has been reported (93, 94), whereas a variety of enzyme labels including firefly or bacterial luciferase (70), horseradish peroxidase (86, 98), and alkaline phosphatase are commercially available. 

Enzymes modified with carbohydrates (neoglycoenzymes) can be used in cytochemistry as described above or in biochemical detection of lectins in solid-phase assays to gain greater sensitivity in analysis. For example, bacterial 3-galactosidase modified with p-aminophenyl a-D-mannopyranoside via amide linkage was useful in determination of Con A immobilized on plastic microtiter plates, and lactose-modified P-galactosidase was effective in histochemical detection of galactoside-specific lectins [63]. Other enzymes frequently used for these applications are alkaline phosphatase and horse radish peroxidase. There are a number of colorimetric, fluorometric, and chemiluminescent substrates available for these enzymes. 

Immunoassay and nucleic acid probe assays Labels. recApoaequorin, firefly luciferase, marine bacterial luciferase, Vargula luciferase Detection reactions, alkaline phosphatase label (luciferin-O-phosphate/firefly luciferase), glucose 6-phosphate dehydrogenase label (marine bacterial luciferase/NADH FMN oxidoreductase reaction)... 

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