An analysis of molecular diagnostic patents published in the first nine months of 1999 shows that roughly two-thirds of documents from the top three companies (Innogenetics, Abbott Laboratories and Akzo Nobel) are focused on immunoassay technologies. However, this focus is shifting toward nucleic acid based approaches. Interestingly, companies with a strong emphasis on nucleic acid based approaches also own their own proprietary detection techniques, threatening Roche's polymerase chain reaction (PCR) as the dominant technology. There is some evidence that the mainstream pharmaceutical companies are becoming active in nucleic acid-based technologies for infection targets, using these techniques to develop new drugs.

Roche occupies the number one position in this survey of patent documents published in the first nine months of 1999 that are concerned with the underlying, or enabling, technologies. There is a strong emphasis toward nucleic acid based approaches, and the list of top companies includes those with their own proprietary amplification methods. These technologically active companies are gaining a competitive edge over those that are still focussed on the older immunoassay technologies, or those that have no underlying technology R&D of their own.

Pharmacogenomic advances mean that simple prescribing of drugs will no longer be enough. Instead the genetic profile of the patient, which will determine his or her response to the available drugs, will need to be assessed. For example, a certain drug may have its most beneficial effect on patients with a particular genetic profile. The effect may be less potent on other patients, and the drug might have adverse side effects on yet further patients. By identifying the patient group that is most receptive to treatment with this drug, problems with low efficacy and side effects are circumvented. This could mean that some drugs chat fail in present day clinical trials may find utility in the future for a small group of patients only. This targeted treatment will thus lead to more appropriate and more economic patient treatment. It is inevitable that the trend in drug administration will be toward combined Rx/Dx solutions, and in vitro diagnostics will become even more integrated into the disease management process. In addition, pharmaceutical companies are embracing pharmacogenomic and other molecular approaches to develop assays for use in high-throughput screening systems, so that the high numbers of hits are efficiently and economically converted into a rather smaller list of lead compounds. This internal market is estimated co be worth between US$1.5 and US$2 bn. Thus, molecular diagnostics has a dual role for the pharmaceutical industry. On the one hand there is a market for pharmacogenomic diagnostics for internal use in pharmaceutical companies, and on the other there will be the demand for combined Rx/Dx solutions. In this review, patent documents published between January and September 1999 by EPO, WIPO and US PTO and Japanese patents published in January and February 1999 have been analysed. The analysis has utilised the Diagnostics Insight database, which characterises documents according to whether they are concerned with infection, disease, analyte or enabling technology patents. Documents are further classified into nucleic acid (NA) based approaches (either using gene probes or amplification techniques) and immunoassay-based approaches.

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 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 P1as 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 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.

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.

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 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.

Summary  Dissociation of FAD from D-aminoacid oxidase occurred most rapidly at pH 6.0 in the presence of 1 M KBr. Diafiltration of 0.6 g of enzyme under these conditions yielded apoenzyme containing 1.3% of residual holoenzyme activity, which was subsequently reduced to less than 0.01% by chromatography on Blue Sepharose and ion exchange, giving material containing <1 ppb of contaminating phosphatase and nucleotidase.

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.

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.

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.

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.

Production of 7,8-epoxy-1-octene from 1,7-octadiene by non-growing Pseudomonas putida PpG6 in a two-liquid phase reaction has been characterized. The measured activity of the bacteria depended on the ratio of the organic and aqueous phases and the degree of agitation, which suggested that mass transfer across the liquid-liquid interface was a controlling factor. At very high aqueous phase cell concentrations the reaction rate became further limited by the increased viscosity of the cell suspension. Bacteria lost their activity in a stirred tank reactor after 14 h at a stirrer speed of 1750 rev min−1and after only 4 h at 2500 rev min−1. Faster volumetric epoxide production rates were obtained than have been reported for growing cells in a two-liquid phase fermentation.

The disruption of Pseudomonas putida cells capable of n-alkane assimilation was investigated by enzymic lysis and mechanical disruption in a high pressure-homogeniser, with a view to the isolation of alkane hydroxylase activity. Examination of the conditions for enzymic lysis showed that disruption with lysozyme/EDTA could be replaced effectively with lysozyme alone in phosphate buffer, pH 8.0 (I=0.05). This allowed inclusion of DNase during the lysis procedure for high bacterial concentrations and gave improved cell disruption. Mechanical disruption resulted in the solubilisation of alkane hydroxylase activity. In contrast enzymic lysis allowed the isolation of an insoluble fraction containing alkane hydroxylase activity, and although some solubilisation of the enzyme system did occur much of the activity was retained in the insoluble fraction. This fraction also contained a high level of n-alkane or diethoxymethane inducible, NAD-independent alcohol dehydrogenase activity.

The properties of spinach leaf sucrose-phosphate synthetase (EC 2.4.1.14) and cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11) have been studied. These two enzymes have been considered to be important in the control of sucrose synthesis. Sucrose-phosphate synthetase from leaf tissue has not been studied in detail previously and we report a technique for purifying this enzyme 50-fold by chromatography on AH-Sepharose 4B. This method frees the enzyme from contaminants which interfere with assay procedures with little or no loss of activity. The partially purified enzyme has a Km for UDP-glucose of 7.1 mm and for fructose 6-phosphate of 0.8 mm. Fructose 1,6-bisphosphate, inorganic phosphate and UDP are strong inhibitors. The inhibition patterns of these suggest that the enzyme operates either by an ordered bi-bi or a Theorell-Chance mechanism. Partially purified cytosolic fructose-1,6-bisphosphatase is not only inhibited by AMP as previously reported, but is also inhibited by fructose 6-phosphate and UDP. From our observations, we conclude that sucrose biosynthesis is indeed controlled through these two enzymes and it appears that the rate of sucrose synthesis is largely dependent upon the supply of triose phosphate and ATP from the chloroplast.

A continuous spectrophotometric assay for sucrose phosphate synthetase is described. In this assay, the production of UDP is coupled to NADH oxidation by the enzymes nucleoside-5′-diphosphate kinase, pyruvate kinase, and lactate dehydrogenase. The assay could not be used with crude extracts, but was found suitable for use with partially purified sucrose phosphate synthetase from the leaves of spinach, wheat, and maize. It has obvious advantages for kinetic studies.