Employment of a phenoxy-substituted acridinium ester as a long-lived chemiluminescent indicator of glucose oxidase activity and its application in an alkaline phosphatase amplification cascade immunoassay.

This paper describes the employment of a novel phenoxy-substituted acridinium ester (di-ortho-bromophenyl-AE) as a chemiluminescent endpoint indicator for ligand binding assays. The reactivity of this compound is such that it is capable of generating a high-intensity chemiluminescent signal at neutral pH. Under these conditions, when present in excess, it has been used as an indicator of hydrogen peroxide generation by the action of glucose oxidase (GOx, EC 1.1.3.4) on glucose substrate. The resulting chemiluminescent signal is a long-lived glow. The magnitude of the chemiluminescent signal is directly proportional to the quantity of GOx present and has been used to measure GOx with a sensitivity of 1.8 x 10(-16) mol. In addition, this ability to monitor GOx activity has been utilized in an alkaline phosphatase (ALP, EC 3.1.3.1) amplification cascade assay. Here ALP catalyzes the formation of FAD from a prosthetogenic substrate FADP. FAD, a cofactor for a number of oxidase enzymes, then converts inactive apo-GOx to holo-GOx, the activity of which is monitored by the chemiluminescent endpoint and facilitates detection of ALP over the range 10(-15) to 4.1 x 10(-19) mol. The clinical utility of this system has been demonstrated by application to the assay of human thyrotrophin (TSH, sensitivity 0.005 mU/liter).

A system for the quantitation of DNA using a microtiter plate-based hybridization and enzyme amplification technology.

A quantitative hybridization technique for the detection of plasmid DNA by the action of a nuclease enzyme is described. The process utilizes the specific capture and detection of a sandwich hybridization, in a microtiter plate, that occurs in a single step. The detector probe is labeled with nuclease P1. The pH-dependent specificity of this enzyme for 3'-dinucleotides is used to generate a measurable signal by activating apo-glucose oxidase, which triggers an enzyme amplification cascade in the same microtiter plate. The sensitivity of the assay system is demonstrated in an assay of a mutated form of the human pancreatic ribonuclease gene inserted into the plasmid pUC 18. The system was able to detect 35 amol of target DNA in an assay composed of a 60-min hybridization and 20 min of signal generation. This use of nuclease P1 as the enzyme label and apo-glucose oxidase as the trigger for the amplification cascade results in an assay that is more sensitive than previously described enzyme amplification systems using colorimetric detection.

An amplified chemiluminescent assay for the detection of alkaline phosphatase.

An amplification assay for the measurement of alkaline phosphatase has been combined with a luminescent end point using the luminol-peroxidase system to produce the first enzyme-amplified chemiluminescent assay based on the principle of prosthetogenesis. This assay is both quantitative and extremely sensitive. When the assay was used to detect alkaline phosphatase in solution, the detection limit was 0.4 amol in a 5-min assay. The interassay variance ranged from 4 to 20% and 7 to 19% across the dynamic range of the assay for a chemiluminescent assay and an enhanced chemiluminescent assay, respectively, employing two different preparations of luminol.

Purification and semienzymic synthesis of flavin adenine dinucleotide-3'-phosphate.

Nonradioactive immunoassays incorporating an element of amplification in their detection system require the use of components that are highly purified. Flavin adenine dinucleotide-3'-phosphate (FADP) is the primary substrate used in such an amplification assay. For incorporation into a simple, single-pot assay system, the concentration of contaminating flavin adenine dinucleotide (a prosthetic group for the enzyme D-aminoacid oxidase used in the amplification cascade assay) in this primary substrate must be minimized to achieve maximum sensitivity. Production of the substrate to a high degree of purity has been achieved using apo-glucose oxidase to specifically remove contaminating flavin adenine dinucleotide from solution and hydrolysis of a cyclic intermediate as a final production protocol by ribonuclease T2 to give the product in high yield. The use of continuous ultrafiltration reactors at each stage is described and compared to a final production step utilizing immobilized ribonuclease T2. These reactors allow large volumes of material to be handled and assist in the scale-up of these processes. The suitability of each protocol is assessed for the commercial production of FADP.

Identification by site-directed mutagenesis of amino acids in the B2 subsite of bovine pancreatic ribonuclease A.

In addition to hydrolysing RNA, bovine pancreatic ribonuclease splits esters of pyrimidine nucleoside 3'-phosphates, including dinucleotides. For a series of 3':5'-linked dinucleotides of general structure CpN, where N is a 5' linked nucleoside, kcat for the release of N varies enormously with the precise structure of N. Structural studies have been interpreted to indicate that the group N interacts with a subsite, B2, on the enzyme that comprises Gln69, Asn71 and Glu111. We report studies by site-directed mutagenesis that indicate that Gln69 is not involved in productive interactions with any of the dinucleotide substrates and that Asn71 is an important component of subsite B2 for all dinucleotide substrates tested. Glu111 appears to be functionally involved in catalysis for dinucleotide substrates solely when N is guanosine.

A model for prosthetogenic enzyme amplification assays.

A mathematical model describing the behaviour of a new class of prosthetogenic enzyme amplification assays is described. The predictions of the model are favourably compared with an enzyme amplification assay for alkaline phosphatase. The model is used to kinetically characterise and optimise the enzyme amplification assay.

Amplified assay of alkaline phosphatase using flavin-adenine dinucleotide phosphate as substrate.

A simple to use, robust, quantitative, and extremely sensitive colorimetric assay for alkaline phosphatase (EC 3.1.3.1), designed to be used as a detection system in diagnostic assays employing antibodies or gene probes, is described. This technology is based on the novel principle of prosthetogenesis, according to which a purpose-designed substrate (a prosthetogen) for a primary analyte-linked enzyme label is hydrolyzed to produce a prosthetic group for a detector enzyme system. The prosthetogen employed here is a derivative of FAD which is phosphorylated at the 3'-position of the ribose ring (FADP), the label enzyme is alkaline phosphatase, and the detector is a D-amino-acid oxidase/horseradish peroxidase-coupled system. Essentially each turnover of every molecule of alkaline phosphatase produces a molecule of D-amino-acid oxidase for detection. Thus enormous amplification of the initial signal is achieved in short time periods because of the relatively high turnover number of alkaline phosphatase for FADP. The system can be formatted as a stable, preformed, freeze-dried preparation containing all analytical components, which is reconstituted simply by addition of buffer solution. This methodology can quantitate less than 0.1 amol of alkaline phosphatase in 30 min at 25 degrees C using microtiter plates.

Amplified luminometric assays of alkaline phosphatase using riboflavin phosphates.

An assay for alkaline phosphatase is described which is based on the hydrolysis of riboflavin phosphates (5'FMN or 4'FMN) to produce riboflavin. This is converted to 5'FMN using riboflavin kinase, and then assayed using the bacterial bioluminescent system from Vibrio harveyi or V. fischeri. The most sensitive assay is obtained using 4'FMN, which can measure less than 20 amol after a 1-hour incubation.

Amplified colorimetric assay of alkaline phosphatase using riboflavin 4'-phosphate: a simple method for measuring riboflavin and riboflavin 5'-phosphate.

Alkaline phosphatase hydrolyzes riboflavin 4'-phosphate to produce riboflavin. This is converted to riboflavin 5'-phosphate, using riboflavin kinase, which reconstitutes apoglycolate oxidase to give hologlycolate oxidase. This enzyme catalyzes the oxidation of glycolate with simultaneous production of hydrogen peroxide which is detected via the formation of a colored product through the action of peroxidase. The system allows the detection of 4 amol after a 2-h incubation.

Sensitive, colorimetric enzyme amplification cascade for determination of alkaline phosphatase and application of the method to an immunoassay of thyrotropin.

A highly sensitive flavin adenine dinucleotide-3'-phosphate (FADP)-based enzyme amplification cascade has been developed for determining alkaline phosphatase (ALP; EC 3.1.3.1). The cascade detects ALP via the dephosphorylation of the novel substrate FADP to produce the cofactor FAD, which binds stoichiometrically to inactive apo D-amino acid oxidase (D-AAO). The resulting active holo D-AAO oxidizes D-proline to produce hydrogen peroxide, which is quantified by the horseradish peroxidase-mediated conversion of 3,5-dichloro-2-hydroxybenzenesulfonic acid and 4-aminoantipyrine to a colored product. The FADP-based enzyme amplification cascade has been used in a novel releasable linker immunoassay (RELIA) to quantify thyrotropin (TSH). In the assay, TSH is first captured onto antibody-coated chromium dioxide particles. After formation of an antibody-TSH sandwich with a dethiobiotinylated second antibody, the complex is reacted with a streptavidin-ALP conjugate. Biotin is then used to release the conjugate into solution, and ALP is quantified in an automated version of the FADP-based amplification cascade on the aca discrete clinical analyzer (Du Pont). The sensitivity of the colorimetric RELIA assay for TSH (less than 0.1 milli-int. unit/L) is comparable with that of fluorometric assays. This technology provides a way to adapt to the aca high-sensitivity immunoassays for a wide range of analytes via colorimetric detection.