Abstract: A kit or solution for use in extension of an oligonucleotide primer having a first single-stranded region on a template molecule having a second single-stranded region homologous to the first single-stranded region, comprising a first agent able to cause extension of the first single-stranded region of the primer on the second single-stranded region of the template in a reaction mixture, and a second agent able to reduce the amount of pyrophosphate in the reaction mixture below the amount produced during the extension in the absence of the second agent.

Abstract: N-alkylacridan carboxylic acid derivative compounds (I) are used to generate chemiluminescence by the action of a peroxidase enzyme and an oxidant. The compounds I are useful in assays of all types.

Abstract: Methods for detecting nucleic acids in a sample, using naphthol derivative phosphates, are provided. Nucleic acids present in a sample are contacted with phosphatase, producing a modified phosphatase. A naphthol derivative phosphate is contacted with the modified phosphatase to produce a reaction product. Any reaction product formed is detected by irradiating it with an excited light and detecting a fluorescence emitted from the reaction product. Naphthol derivative phosphates useful in these methods are also provided. Methods for production of naphthol derivative phosphates are also provided.

Abstract: A quantitative assay for 1,5-anhydroglucitol in a specimen is described. This assay comprises treating a specimen with a phosphorylating enzyme in the presence of 5 to 500 mM adenosine-5'-triphosphate in a pH range of 7.2 to 8.5, to selectively eliminate sugars other than 1,5-anhydroglucitol, and reacting the product of this reaction, without removing the residual ATP, with pyranose oxidase in the above pH range. This assay is quite rapid and suitable for performance using an automated device.

Abstract: A method and apparatus for performing an enzyme spot test without traditional instrumentation is disclosed. The method utilizes various reagents to allow the visual determination of the presence of an enzyme in a sample above a predetermined critical level. The enzyme reacts with the reagents producing a product according to a reaction which is timed by a chemical fuse. At the end of the reaction, a colored by-product is visible, the color intensity indicating that the enzyme occurs in the sample in quantities above the critical level.

Abstract: The present invention provides a non-mouse pluripotential embryonic stem cell which can:(a) be maintained on feeder layers for at least 20 passages; and(b) give rise to embryoid bodies and multiple differentiated cell phenotypes in monolayer culture. The invention further provides a method of making a pluripotential embryonic stem cell comprising administering a growth enhancing amount of basic fibroblast growth factor, leukemia inhibitory factor, membrane associated steel factor, and soluble steel factor to primordial germ cells under cell growth conditions, thereby making a pluripotential embryonic stem cell.

Abstract: A first solution containing Tris-HCl, MgCl.sub.2, a homopolymer of 2-acrylamide-2-methylpropanesulfonate (AMPS) and agarose is applied to a filter having alkaline phosphatase to be detected. A second solution containing Tris-HCl, MgCl.sub.2, homopolymer of AMPS, 5-bromo-4-chloroindoxyl phosphate, nitroblue tetrazolium and agarose is applied onto a film formed by the first solution.

Abstract: The present invention makes available assays and reagents for identifying anti-proliferative agents, such as mitotic and meiotic inhibitors, especially inhibitors of cdc25 phosphatase. The present assay provides a simple and rapid screening test which relies on scoring for positive cellular proliferation as indicative of anti-mitotic or anti-meiotic activity, and comprises contacting a candidate agent with a cell which has an impaired cell-cycle checkpoint and measuring the level of proliferation in the presence and absence of the agent. The checkpoint impairment is such that it either causes premature progression of the cell through at least a portion of a cell-cycle or inhibition of normal progression of the cell through at least a portion of a cell-cycle, but can be off-set by the action of an agent which inhibits at least one regulatory protein of the cell-cycle in a manner which counter-balances the effect of the impairment.

Abstract: A rapid method of determining the efficacy of a sterilization cycle, and an indicator adapted to perform such method, comprising subjecting to the sterilization cycle a source of active enzyme having activity which correlates with the viability of a microorganism commonly used to monitor sterilization, and incubating the enzyme source, following the completion of the sterilization cycle, with an effective amount of a substrate system capable of reacting with any residual active enzyme to produce a detectable enzyme-modified product.

Abstract: Methods and compositions are provided for the detection of analytes. The method employs a fluorescence production layer which comprises a fluorescent polymerized polyunsaturated lipid layer in association with a ligand which is a member of a specific binding pair, where the ligand is competitive with the analyte for the complementary binding member or is a complementary binding member. By providing for a fluorescence modulation reagent which binds to the fluorescence production layer in proportion to the amount of analyte in the sample, by measuring the resulting fluorescence after carrying out the assay methodology, the amount of analyte can be determined quantitatively.

Abstract: A method is described for diagnosing rheumatoid arthritis by providing a recombinant IgM-specific rheumatoid arthritis-associated antigen and detecting antibodies against the antigen in patient sera. Preliminary steps of making a cDNA library from polyadenylated RNA purified from human cells, selecting recombinants that express the antigen, recloning cDNA containing the antigen gene in a high level expression vector, expressing the antigen in transformed cells, and purifying the antigen are also described.

Abstract: The invention refers to a method for determining the relative amounts of all cholesterol-containing lipoproteins in body fluids comprising electrophoretically separating the lipoproteins of an aliquot of body fluid on a thin layer carrier matrix, incubating the carrier matrix, containing the separated lipoproteins with cholesterol esterase and cholesterol dehydrogenase, forming a provable complex, and determining the relative amounts of the different lipoprotein classes2. The new method makes it possible to simultaneously determine HDL-, LDL-, VLDL- and LP (X)-cholesterol in body fluids with a high accuracy even at small concentrations. The thin layer matrices obtained electrophoretically, are very easy to handle and to record.

Abstract: Method for assaying nucleic acids and proteins employing no radiosotope labeling, which is suitable for data storage and allows to carry out the detection efficiently and high sensitivity. The sample selectable from the group of nucleic acids, proteins, and other chemical compounds is adhered on a nylon membrane filter to be bound to a phosphatase. Then the phosphatase is reacted with an anthracene derivative phosphate followed by irradiating the reaction product with ultraviolet light to detect a fluorescence emitted therefrom.

Abstract: The invention provides a method for supplying an enzyme substrate to a membrane-based, enzyme-linked reaction, comprising providing an open pore, high liquid retention capacity material impregnated with a predetermined amount of a substrate for the enzyme; and contacting the material with a membrane containing the enzyme-linked reaction under conditions which permit diffusion of the enzyme substrate to sites on the membrane containing the enzyme linked reaction.

Abstract: The present invention relates to chromogenic compounds which are represented by the general Formula (I): ##STR1## wherein R.sub.1 is a sugar group, ester group, hydrocarbyl group, phosphate group, sulfate group or a salt thereof, with the proviso that R.sub.1 is other than .beta.-D-glucuronic acid or .beta.-D-galactopyranoside, R.sub.2 is H or hydrocarbyl group containing 1 to about 5 carbon atoms, X is Cl or H, and Y is Cl or H. The present invention further relates to a method for quantitatively identifying and differentiating a first biological material having enzyme specificity for a first chromogenic compound as represented by Formula (I) above, and a second biological material having enzyme specificity for a second chromogenic compound.

Abstract: The present invention discloses a rapid assay method for quantitatively determining the complete isoenzyme profile of a biological fluid and a multiple-assay reagent system for carrying out the method. In one preferred embodiment, a multiple-assay reagent system is disclosed for quantifying the complete isoenzyme profile of lactate dehydrogenase in blood serum, based on two measured parameters and three performed assays.

Abstract: The subject invention provides a method for analyzing the metabolic activity in cells by improving the retention of a detectable reporter molecule only in intact cells where a particular enzyme is present. In particular, improved retention results from a two part process involving conjugation of haloalkyl-substituted derivatives of a reporter molecule with intracellular cysteine-containing peptides while unblocking the reporter molecule. The method for analyzing metabolic activity of cells involves the use of a substrate having the formXR-REPORTER-BLOCKwherein -BLOCK is a group selected to be removable by action of a specific analyte, to give REPORTER spectral properties different from those of the substrate,-REPORTER- is a molecule that, when no longer bound to BLOCK by a BLOCK-REPORTER bond, has spectral properities different from those of the substrate, andXR-- is a haloalkyl moiety that can covalently react with an intracellular thiol (Z--S--H) to form a thioether conjugate (Z--S--R--).

Abstract: A method of evaluating the immunosuppressive activity of a compound including contacting the compound with calcineurin and determining the ability of the compound to bind to the calcineurin. The ability to bind to the calcineurin is positively correlated to the immunosuppressive activity of the compound.

Abstract: A chromogenic .beta.-galactosidase substrate producing an insoluble precipitate of a first color when reacted upon by .beta.-galactosidase and a chromogenic .beta.-glucuronidase substrate producing an insoluble precipitate of a second, contrasting color when reacted upon by .beta.-glucuronidase are combined in test medium for quantitatively identifying and differentiating general coliforms and E. coli. The .beta.-galactosidase substrate 5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside which produces an indigo blue precipitate when reacted upon by .beta.-galactosidase may be used with one of the novel compounds 6-chloroindolyl-.beta.-D-glucuronide, 4,6-dichloroindolyl-.beta.-D-glucuronide, 6,7-dichloroindolyl-.beta.-D-glucuronide, and 4,6,7-trichloroindolyl-.beta.-D-glucuronide, which produce mauve or magenta precipitates when reacted upon by .beta.-glucuronidase. The .beta.-glucuronidase substrate 5-bromo-4-chloro-3-indolyl-.beta.

Abstract: A non-radioactive method of detecting a ligand and antiligand complex labelled with alkaline phosphatase or a tracer having alkaline phosphatase conjugated thereto comprises reacting the complex with bromo-chloro-indolyl phosphate (BCIP), phenazine methosulfate (PMS) and dimethylthiazol diphenyl tetrazolium (MTT) and allowing the reaction to proceed to produce a colored formazan or a color change indicative of the presence of the labelled complex. A solution or composition of bromo-chloro-indolyl phosphate (BCIP), phenazine methosulfate (PMS) and dimethylthiazol diphenyl tetrazolium (MTT), as well as a test kit including the same, is also provided for carrying out the chromogenic method of detection.

Abstract: The substrate, 3,4-dinitrophenylphosphate, has been found to be useful in an assay of acid phosphatase in body fluids.

Abstract: Enzymatically cleavable chemiluminescent 1,2-dioxetane compounds capable of producing light energy when decomposed, substantially stable at room temperature before a bond by which an enzymatically cleavable labile substituent thereof is intentionally cleaved, are disclosed. These compounds can be represented by the formula: ##STR1## wherein: X and X.sup.1 each represent, individually, hydrogen, a hydroxyl group, a halo substituent, an unsubstituted lower alkyl group, a hydroxy (lower) alkyl group, a halo (lower) alkyl group, a phenyl group, a halophenyl group, an alkoxyphenyl group, a hydroxyalkoxy group, a cyano group or an amide group, with at least one of X and X.sup.1 being other than hydrogen; andR.sub.1 and R.sub.2, individually or together, represent an organic substituent that does not interfere with the production of light when the dioxetane compound is enzymatically cleaved and that satisfies the valence of the dioxetane compound's 4-carbon atom, with the provisos that if R.sub.1 and R.sub.

Abstract: A method of measuring adenylate cyclase (AC) in a sample of physiological material which does not employ radioactive reagents is provided, comprising:(a) providing a physiological sample of physiological material comprising cAMP produced by endogenous AC, and other endogenous adenine nucleotides selected from the group consisting of ATP, AMP, ADP and mixtures thereof;(b) combining the sample with effective amounts of apyrase, 5'-nucleotidase and adenosine deaminase, so as to enzymatically eliminate the other endogenous adenine nucleotides in the sample;(c) enzymatically converting the cAMP into AMP; and(d) measuring the amount of AMP, the amount providing a measure of the amount of cAMP and AC in the sample.

Abstract: An enhanced chemiluminescent assay, in which a dihydrophthalazinedione such as luminol, a peroxidase such as HRP and an oxidant such as H.sub.2 O.sub.2 are co-reacted in the presence of an enhancer such as p-iodophenol, is modified. The enhancer is generated by enzyme-catalysed reaction of a pro-enhancer, e.g. p-iodophenol phosphate is cleaved by alkaline phosphatase, enabling this enzyme to be assayed instead of peroxidase. Alternatively, the enhancer is added, an anti-enhancer such as p-nitrophenol is generated by enzymatic reaction of a pro-anti-enhancer such as p-nitrophenol phosphate and the reduction in luminescent emission is measured.

Abstract: A device for colorimetric assay of at least one component in a liquid sample, which device comprises a support, a reagent layer formed on a part of one surface of the support and a sample-receiving layer which covers at least a part of the reagent layer and at least a part of the support surface, with which the timing of application of a sample on the device is automatically detected with accuracy.

Abstract: A method of identifying compounds or molecules which alter (enhance or inhibit) stimulation of kinase activity of pre-MPF and, thus, alter (enhance or inhibit) activation of MPF and entry into mitosis. The present method thus makes it possible to identify compounds or molecules which can be administered to regulate the cell cycle; such compounds are also the subject of this invention.

Abstract: A method of detecting hydrolase activity using as substrates the compounds of the formula ##STR1## wherein each of R.sup.1, R.sup.2 and R.sup.3 is C.sub.1 -C.sub.4 alkyl, or phenyl optionally substituted in the meta- or para- position by C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 alkoxy or mono- or di-(C.sub.1 -C.sub.6)-alkylamino, or optionally substituted by an O-X group in which X is a glycosyl, phosphate or acyl moiety of a natural substrate of the corresponding glycosidase, phosphatase or esterase.

Abstract: The invention is directed to new drug hapten analogues comprising:(A) an active ester group;(B) a drug hapten nucleus selected from a hydantoin nucleus or a barbiturate nucleus and(C) a linking chain linking the 3-position of the drug hapten nucleus to the active ester group.

Abstract: A method for immunoassay for a ligand is performed on a porous membrane with alkaline phosphatase as the label. The assay protocol includes a wash step with an organic acid to deactivate endogenous alkaline phosphatase. The invention includes a kit of materials for performing the assay.

Abstract: A process is disclosed for the enhancement of the chemiluminescence of enzyme-triggered 1,2-dioxetanes detectable within a sample. The process comprises first providing a solid support having sample disposed thereon and suitably treated with a solution including enzyme-triggered 1,2-dioxetanes. The solid support is next dried and optionally heated either simultaneously with drying or thereafter. The steps of drying and/or heating are conducted either prior to or simultaneously with detection.

Abstract: A method and compounds useful for this method are described for enzyme-amplified signal detection in analytical assays requiring extremely high detection sensitivity which uses a substrate capable of being transformed by an enzyme from a compound which does not form a luminescent lanthanide chelate into a product which forms a luminescent lanthanide chelate. The method in which a substrate not capable of forming a highly luminescent lanthanide chelate is enzymatically altered to produce a product which forms a highly luminescent lanthanide chelate and hence is particularly useful in time-resolved luminescence analysis as required in many different heterogeneous or homogeneous assay formats is described herein.

Abstract: Novel assay methods employing compounds which are chemically or enzymatically cleavable and which give rise to an intermediate which further decomposes by an intramolecular anchimeric displacement reaction which releases a signal producing species are disclosed. Also disclosed are probe hybridization assays employing the compounds of the invention employing thermostable enzymes which are not denatured by the hybridization conditions. Such signal producing species may include chemiluminescent dioxetanes and other colored products.

Abstract: Alkaline phosphatase to which is covalently bound a carbohydrate of the general formula ##STR1## wherein n is 0, 1, 2 or 3, A is an acyl radical containing 2 to 5 carbon atoms, R.sup.1 and R.sup.2 are each hydrogen atoms or hydroxyl groups, R.sup.3 is --COOH or --CH.sub.2 OH, R.sup.4 is a hydroxyl group or a --CHOH--CHOH--CH.sub.2 OH or --NH--CO--CH.sub.2 --CH(NH.sub.2)--COOH radical or a complex containing the carbohydrate, is used as a standard for the determination of human alkaline phosphate. Preferred carbohydrates are ovomucoid, chitobiose, chitotriose or N-acetylglucosamine-N-acetylneuraminic acid.

Abstract: The present invention provides a process for the determination of phospholipase A in body fluids, wherein the body fluid is incubated with a mixture containing a substrate having a fatty acid residue for the phospholipase and at least two emulsifiers of different chemical classes, namely, at least one neutral emulsifier and a bile acid salt, and the amount of the free fatty acids formed by the cleavage of the substrate is determined in known manner.

Abstract: Enzymatic immunoassay for estimating the concentration of antigen or antibody comprising bringing an antigen-antibody complex, which is labelled by an enzyme and is present heterogeneously in a solution, into contact with a substrate where pH of the solution is so adjusted as to be suitable for the enzyme to be activated and also for measuring the fluorescence of the substrate, measuring the time-variation of fluorescent intensity of the substrate produced by the enzyme reaction, and estimating the concentration of the antigen or the antibody from the slope of the substantially linear portion, on a characteristic curve representing variation of the fluorescence intensity with the time.Fluorescence is measured while magnetic beads with antigen-antibody complex attached are oscillated at a specific frequency.

Abstract: Vitamin D derivatives corresponding to the following formula I ##STR1## in which R.sub.1 denotes a substituted alkyl group having 1 to 15 carbon atoms, in particular the side chains of vitamin D.sub.2 (C.sup.20 to C.sup.28) or D.sub.3 (C.sup.20 to C.sup.

Abstract: Novel methods and compositions for detecting a member of a ligand pair on solid supports having intrinsic fluorescence are disclosed. A target member of a ligand pair is contacted with a capture member of a ligand pair, wherein the capture member is immobilized on a solid support having intrinsic fluorescence, and the contacted pair is in association with a colorimetric reporter, then the solid support is irradiated the solid support, and the resultant fluorescence is determined. Preferably, methods include hybridization assays of nucleic acid sequences on such solid supports. The extent of detecting a member of a ligand pair, preferably a nucleic acid sequence, is determined using a method or technique described throughout this document as fluorescent quenching. Methods and compositions pertaining to solid supports having their intrinsic or natural fluorescence quenched or masked are also described herein. The present invention has utility in detection assays for a member of a ligand pair.

Abstract: Phosphoric acid derivatives represented by formula (I): ##STR1## wherein X is a halogen and R is --(CH.sub.2).sub.n CH.sub.3 (n=0 to 3), or salts thereof are stable to non-enzymatic hydrolysis and are capable of specifically reacting with acid phosphatase. Therefore, the activity of acid phosphatase in the sample can be determined extremely accurately by reacting said compound with a sample containing acid phosphatase and quantitatively determining the reaction product by colorimetry.

Abstract: A method for purifying an aqueous intrinsic factor solution which contains R-protein is disclosed. The method involves adding to the intrinsic factor solution an amount of colloidal silica to disperse lipid emulsion, an amount of cobinamide sufficient to bind substantially all of the R-protein in the solution and an amount of an intrinsic factor affinity resin sufficient to bind the intrinsic factor in the solution, washing the bound cobinamide and the R-protein from the resin, eluting the intrinsic factor from the resin, and dialyzing the eluted intrinsic factor. The purified intrinsic factor possesses less than 0.004 percent cross reactivity with cobinamides, and at least 95 percent of the proteins in the purified material can bind cobalamins. A conjugate of microparticles and the purified intrinsic factor is also disclosed, as is a kit for conducting an assay for cobalamins which includes a conjugate of microparticles and purified intrinsic factor.

Abstract: A method of detecting, identifying, and enumerating microbes in biological and non-biological samples includes the steps of combining the sample with a triggerable chemiluminescent compound specifically susceptible to the initiation of chemiluminescent decomposition by at least one microbial enzyme in the sample and detecting and integrating light emission over an extended period of time as an indication of the presence, identification, or enumeration of the microbes in the sample.

Abstract: A unitary sterility indicator and a method for its use, the indicator comprising an outer container having liquid impermeable and gas non-absorptive walls, and having a gas-transmissive, bacteria impermeable opening therein; contained within the outer container, a detectable amount of a source of active enzyme and/or another microorganism commonly used to monitor sterilization; a sealed, openable gas and liquid impermeable inner container containing an aqueous medium and/or a nutrient growth medium, disposed in the outer container; an enzyme substrate system capable of reacting with active enzyme to produce a detectable enzyme-modified product and/or a detector material sensitive to microorganism growth, contained in one of the containers; and means contained within the outer container for restricting the area in which enzyme-modified product and/or growing microorganisms are contained, after the inner container is opened, to an area which is less than the volume of aqueous solution contained in the inner con

Abstract: An optically active 1,3-butanediol can be produced by either (1) treating a mixture of 1,3-butanediol enantiomers with a microorganism, which has been optionally treated, capable of asymmetrically assimilating said mixture, or (2) preparing a microorganism, which has been optionally treated, capable of asymmetrically reducing 4-hydroxy-2-butanone, and collecting optically active 1,3-butanediol.

Abstract: Antigens extracted from one or more serotypes of a Bacteroides microorganism are rapidly and sensitively detected when directly bound to a water-insoluble substrate. The bound antigens are detected by forming an immunological complex on the substrate when the antigen reacts with the appropriate antibody. The antibody-antigen complex can be detected directly or by means of a second antibody which is directed to the Bacteroides antibody. The entire assay can be carried out at room temperature in less than about 15 minutes.

Abstract: An improved chromatographic device and method for quantitating the activity of an enzyme utilizes a bibulous test strip as the stationary phase, an aqueous solution as the mobile phase, and a chromophore as the indicator. The enzyme activity is quantitated by transforming a substantially depletable amount of a chromogenic substrate from soluble chromogen to insoluble chromophore as the mobile phase advances, whereupon the insoluble chromophore becomes immobilized on the test strip to produce a column of color. The length and/or the intensity of the column of color are related to the activity of the enzyme. The present invention is useful for the direct determination of enzyme-analytes and for the indirect determination of analytes which can be coupled to an enzyme.

Abstract: Chromogenic merocyanine enzyme substrate compounds of the general formula: ##STR1## where Y is an enzymatically-cleavable group such as a radical of a sugar, carboxylic acid, amino acid, peptide, phosphoric acid, or sulfuric acid; A and B represent residues that complete 5- or 6-membered ring systems; R.sup.1 is substituted or unsubstituted alkyl; R.sup.2 and R.sup.3, independently, are hydrogen or lower alkyl; m, n, and p, which can be different, are integers from 0 through 3 provided that m+n+p must be at least 2; and X is an appropriate counterion (anion).

Abstract: An assay for Chlamydia includes contacting Chlamydia organisms in a liquid with a solid support having an antispecies Fe antibody immobilized thereon and an anti-Chlamydia capture antibody. After binding of Chlamydia antigen to the capture antibody and binding of the capture antibody to the antispecies antibody on the support, a tracer including a label conjugated to a signal antibody is added. After binding of the signal antibody to the antigen, the presence of Chlamydia organisms in the liquid is detected by a signal associated with the label thereby bound to the support. The invention includes a kit of materials for performing an assay according to the method of the invention.

Abstract: A rapid, competitive enzyme linked immunosorbent assay (cELISA) for the determination of Bluetongue virus antibodies in serum is described. This method utilizes either a biotinylated monoclonal antibody to Bluetongue virus and streptavadin-enzyme in conjunction with synthetic substrate, or an enzyme-conjugated monoclonal to detect antibodies specific for Bluetongue virus.

Abstract: A method and kit for typing platelets in whole blood for human leucocyte antigens (HLA). A panel of HLA antigens is made on a microtiter plate. Each well is contacted with serum to be typed and the amount of reaction is followed with anti-human (Fab '2) alkaline phosphatase and disodium p-phenylphosphate.

Abstract: The present invention relates to a method for quantitatively assaying the presence of DSP toxins such as okadaic acid and dinophysistoxin-1 in marine samples. The method comprises the steps of preparing a marine extract, fractionating the prepared marine extract and selecting the extract fraction containing the toxin to be assayed. Once the desired extract fraction has been selected, a labelled substrate for protein phosphatase and at least one protein phosphatase are added to the extract in an assay. The amount of toxin present is quantitatively measured by the ability of the extract fraction to inhibit catalysis, mainly dephosphorylation, of the labelled substrate by protein phosphatases, such as phosphatase-1 (PP1) or phosphatase-2A (PP2A). Preferably, the method of the present invention is used to assay the presence of okadaic acid in marine organisms such as mussels, oysters, scallops, phytoplankton and the like.

Abstract: A new method of rapidly analyzing plural substances in the presence of biological catalyzers is disclosed. The method is practiced by way of the steps of injecting both pH buffer solution and specimen into a reaction cell, successively adding a plurality of enzymes to induce up-take reaction of dissolved oxygen, causing the plurality of substances to be subjected to selective oxidation in the stepwise manner, obtaining a stepdown curve of dissolved oxygen by automatically recording the oxidative process by means of a dissolved oxygen sensor, qualitatively determining each of the substances with reference to the kind of added enzymes and the order of their addition and quantitatively determining the same with reference to the extent of decrease in dissolved oxygen. Typically, oxidation of the substances is carried out by way of two or three or further more steps.