Abstract: Proteins are provided having luciferase activity with greater heat stability than wildtype luciferases by replacing the glutamate equivalent to that at position 354 of Photinus pyralis luciferase or 356 of Luciola luciferases with an alternative amino acid, particularly lysine. DNA, vectors and cells that encode for and express the proteins are also provided as are test kits and reagents for carrying out luminescence assays using the proteins of the invention. Preferred proteins have a second replaced amino acid at a position equivalent to position 215 of Photinus pyralis luciferase or 217 of Luciola luciferases.

Abstract: A method and apparatus for monitoring a gaseous environment for the presence of cellular material capable of providing a measure of presence and/or numbers of cellular microorganisms, such as bacterial cells, in a large volume of air such as in a warehouse or production facility or in an open air location where bacterial presence is suspected. The method and apparatus are particularly suited for determining the likelihood of pathogenic material being present in an environment by batch or on-line measurement of cell numbers. On-line measurement provides continuous monitoring of an environment for presence of pathogens. The device includes a continuous flow luminometer preferably fed by a cyclone or high velocity virtual impactor and luminescence reagents which detect the amount of ATP or adenylate kinase present in a sample of air.

Abstract: Luciferase is conjugated to a chemical entity, particularly to a specific binding agent such as an antibody, antigen or a nucleic acid, and more particularly an antibody, by (a) mixing the luciferase with one or more of D-luciferin, magnesium ions and adenosine triphosphate and (b) performing a covalent coupling reaction between the luciferase and the binding reagent using a covalent coupling reagent where the amount of D-luciferin, magnesium ions and/or adenosine triphosphate is sufficient to protect the luciferase activity against inhibition by the covalent coupling reagent. Preferably, step (a) is carried out by mixing the luciferase with its substrates in solution and preferably both magnesium and adenosine triphosphate are present as magnesium adenosine triphosphate (Mg.sup.2+ ATP), optionally together with D-luciferin. Also disclosed is a labeled chemical entity comprising a chemical entity conjugated to active luciferase as formed by the method.

Abstract: A method is provided for determining the presence and/or amount of a microorganism and/or its intracellular material present in a sample performed by: (a) exposing the sample to a specific binding agent that has been immobilized upon a solid substrate, the specific binding agent being capable of binding to the microorganism or its intracellular material such that it becomes associated with the solid substrate, (b) exposing the solid substrate to an agent capable of making adenylate kinase associated with the microorganism and/or its intracellular material accessible to solutions applied to the substrate, (c) applying a solution containing adenosine diphosphate (ADP) to the substrate under conditions whereby adenosine triphosphate (ATP) may be produced by any adenylate kinase present, and (d) measuring the amount of adenosine triphosphate (ATP) and relating that to the presence and/or amount of microorganism or intracellular contents.

Abstract: A method and apparatus for monitoring a gaseous environment for the presence of cellular material capable of providing a measure of presence and/or numbers of cellular microorganisms, such as bacterial cells, in a large volume of air such as in a warehouse or production facility or in an open air location where bacterial presence is suspected. The method and apparatus are particularly suited for determining the likelihood of pathogenic material being present in an environment by batch or on-line measurement of cell numbers. On-line measurement provides continuous monitoring of an environment for presence of pathogens. The device includes a continuous flow luminometer preferably fed by a cyclone or high velocity virtual impactor and lytic and luminescence reagents which detect the amount of ATP or adenylate kinase present in a sample of air.

Abstract: A method for detecting the presence and/or mount of microorganisms is described by adding adenosine diphosphate (ADP) to a sample suspected of containing microorganisms and/or their intracellular material, determining the amount of adenosine triphosphate (ATP) generated by adenylate kinase present, for example using a bioluminescent assay involving luciferase and luciferin, and relating the results to the presence and/or amount of microorganism. Test kits and apparatus for use in the method are also described.