WO2008024052A1

WO2008024052A1 - A method and a kit for determination of an enzyme activity involved in metabolic production of a deoxynucleoside triphosphate and use thereof

Info

Publication number: WO2008024052A1
Application number: PCT/SE2007/000749
Authority: WO
Inventors: Simon Gronowitz
Original assignee: Rönnerbol International Ab
Priority date: 2006-08-24
Filing date: 2007-08-24
Publication date: 2008-02-28

Abstract

A method and assay kit for determination and quantification of an enzyme activity, involved in a metabolic pathway leading to a deoxynucleoside triphosphate and derivates thereof, in a sample, are disclosed. The method comprises contacting, in a container with single stranded polynucleotide as a primer or template attached to a solid surface, a basic reaction mixture comprising a single stranded polynucleotide acting as a template and/or primer; a deoxynucleoside, precursor or analog thereof as an enzyme substrate; a phosphate donor; and an enzyme source for further processing of an enzymatically produced precursor product to the final nucleoside triphosphate, said source being devoid of the enzyme activity to be determined and quantified, and a DNA polymerase. The resulting mixture is incubated, and the amount of deoxynucleoside incorporated into the solid surface-attached primer and/or template is determined, indicating the enzyme activity present in the sample.The method and kit are useful in diagnosing, monitoring and/or prognosis of cell-proliferation disorders or diseases, such as cancer or certain viral infections, and in screening of compounds affecting enzymatic pathways.

Description

A method and a kit for determination of an enzyme activity involved in metabolic production of a deoxynucleoside triphosphate and use thereof.

The present invention relates to a method and kit for determination of an enzyme activity involved in metabolic production of a deoxynucleoside triphosphate. The determined enzyme activity is selected from the different enzyme activities involved. The metabolic pathway involves inter alia synthesis of nucleoside bases, nucleosides and nucleotides (See e.g. Figure 1 and Table 1 ). The method of the invention involves detection and quantification of a non-modified, modified or labeled deoxynucleoside incorporated into a newly synthesized DNA on a solid phase. The method of the invention makes use of the fact that an enzyme activity in the metabolic pathway for a specific precursor substance can be complemented by an added exogenous enzyme activity. Furthermore, the method of the invention makes use of the fact that labeled derivatives of non-modified deoxynucleotide precursor product(s) can be processed and used in the syntheses of modified DNA by DNA-polymerase or terminal transferase.

The invention is useful in the diagnosing, monitoring and prognosis of cell- proliferation disorders or diseases, such as cancer and certain infectious diseases, and in screening of compounds, e.g. new drug candidates, affecting an enzyme activity included in the enzymatic pathways involved in the metabolic production of deoxynucleoside triphosphates and thereby enabling therapeutic and useful treatment of the mentioned diseases. Background

A method for the determination of thymidine kinase activity has been claimed in the co-pending International Patent Application PCT/ SE 2006/ 000246, subsequently published as WO 2006/091158. However, this PCT application is concerned with determination of thymidine kinase activity only, whereas the present invention is concerned with determination of other enzyme activities involved in metabolic production of a deoxynucleoside triphosphates.

The EP 1395675 B1 describes a method for measuring DNA-dependent DNA polymerization in a biological sample by contacting a primer with a single stranded short specific sequence, which is unable to base pair internally, bound to a solid phase with a reaction mixture containing a single stranded deoxynucleotide template, that has a part of the sequence complementary to the primer, and the four deoxynucleoside triphosphates, one of which is modified so that it is specifically recognized by a labeled antibody; and adding a biological sample comprising the DNA polymerase to the mixture; allowing the polymerase reaction to proceed; incubating the immobilized reaction product with the labeled antibody; detecting the amount of bound labeled antibody; and measuring the amount of incorporated modified deoxynucleoside triphosphate, as a measure of the DNA polymerisation.

Description of the invention The present invention provides a sensitive procedure for detection of an enzyme activity (see e.g. Figures 2 and 3) involved in the synthesis of nucleoside bases, nucleosides and nucleotides and derivates of normal substrate substances that all can be used for syntheses of non-modified, modified or labeled DNA by DNA-polymerase or terminal transferase. The normal metabolic pathway that includes several enzymatic steps is followed so that an enzymatically produced precursor product is directly processed further via one or several enzymatic steps in the pathway, finally leading to a desired or selected, non- modified, modified or labeled deoxynucleoside triphosphate, which is continuously removed from the reaction solution through polymerization by a DNA polymerase incorporating the desired or selected, non-modified, modified or labeled deoxynucleoside into a DNA strand attached to a solid phase. The DNA-polymerase elongates a polynucleotide primer which is immobilized on a solid surface, or indirectly immobilized on the solid surface by binding to a complementary polynucleotide-chain, which in this case is bound to the solid surface. The enzyme activity level is directly determined and quantified by measurement of the amount of incorporated processed deoxynucleoside or by incorporation of another nucleoside triphosphate tracer that will only be incorporated if the desired, processed deoxynucleoside is first incorporated (see Figure 2), by e.g. using standard non-radioactive ELISA procedure with a specific antibody directed against the desired or selected, non-modified or modified deoxynucleoside, or via another labeled tracer only incorporated if the desired product is incorporated (see Figure 2). The whole reaction may take place in just one container without need for transfer of samples for secondary measurements. The later procedure is illustrated in figure 2 disclosing generalized template structure for measurement of all 4 deoxynucleoside kinases.

Thus, the invention is directed to a method for determination and quantification of an enzyme activity, involved in a metabolic pathway ultimately leading to a deoxynucleoside triphosphate and derivates thereof, in a sample, comprising the steps of contacting, in a container with single stranded polynucleotide as a primer or template attached to a solid surface, a basic reaction mixture, in a buffer, comprising the following components: optionally a non-attached single stranded polynucleotide acting as a template and/or primer, a non-modified, modified or labeled deoxynucleoside, precursor or analog thereof or a mixture thereof as an enzyme substrate, a phosphate donor and other co- factors, and an enzyme source for further processing of an enzymatically produced precursor product to the corresponding final nucleoside triphosphate, said source being devoid of the enzyme activity to be determined and quantified, the enzyme activity to be determined and quantified being present in the sample, and a DNA polymerase, and optionally another deoxynucleoside triphosphate which functions as a tracer indicating the amount of the enzyme activity to be determined, incubating the resulting mixture, optionally washing the incubated mixture to remove non-attached reaction components, determining the amount of the non-modified, modified or labeled deoxynucleoside that has ., been incorporated or the tracer that indicates such incorporation, or has been prevented from incorporation, into the solid surface-attached primer and/or template, and determining the enzyme activity present in the sample from the determined amount of incorporated non-modified, modified or labeled deoxynucleoside or from the reduction of incorporation of said deoxynucleoside. The last mentioned alternative is illustrated in figure 3.

The optionally present non-attached single stranded polynucleotide acting as a template and/or primer in the basic reaction mixture may not be present in case a terminal transferase is used, only a solid phase polynucleotide chain is required in that case.

The expression "devoid of enzyme activity" is in the present specification intended to mean that there is so low amount of enzyme activity, if at all present, that it will not interfere with the performance of the method of the invention.

Furthermore, it should be understood that the determined enzyme activity is proportional to the amount of incorporated non-modified, modified or labeled nucleoside or the degree of prevention of such incorporation.

The enzyme activity to be determined may be selected from the enzyme activities exemplified in Figure 1 and Table 1 , such as phosphoribosyl transferases, adenylphosphotransferases, deoxycytidine kinase, deoxyadenosine kinase, deoxyguanosine kinase, thymidylate kinase, adenylate kinase, guanylate kinase, nucleoside diphosphate kinase, nucleoside monophosphate kinase, deaminases, cytidine/deoxycytidine deaminase, thymidylate synthetase, thymidylate synthetase, uridine phosphorylase, thymidine phosphorylase, deoxyuracilphosphatase, nucleoside phosphotransferase, ribonucleoside- diphosphate reductases, phosphotransferases, oxireductases, methyltransferases, pentosyltransferases, phosphoric monoester hydrolases, and hydrolases. However, as mentioned, a method for the determination of thymidine kinase activity has already been claimed in the co-pending International Patent Application PCT/ SE 2006/ 000246.

In one embodiment of the invention the incubated mixture is washed, the amount of incorporated non-modified or modified deoxynucleoside being the processed product or an added nucleoside tracer only incorporated if said processed product is first incorporated (see figure 2), is determined by contacting the solid surface-attached primer and/or template with a labeled affinity molecule with affinity for the incorporated non- modified, modified or labeled deoxynucleoside and determining the amount of non-modified or modified deoxynucleoside that has been incorporated by determining the amount of attached labeled affinity molecule with the aid of the label.

In another embodiment, especially for drug screen purposes, a radioactive non- modified, modified or labeled substrate is used, removing the need of labeled affinity molecule, as the amount of deoxynucleoside is determined on the basis of radioactivity retained after wash of the solid surface. Before cell division, the DNA carrying the genetic information has to duplicate by a DNA-polymerase. This requires among other necessary components the presence of large amounts of substrates, i.e. the presence of all four deoxyribonuclotides, i.e. Thymidine Tri-Phosphate (TTP), deoxyCytidine Tri-Phosphate (dCTP), deoxyGuanonsine Tri-Phosphate (dGTP) and deoxyAdenosine Tri-Phosphate (dATP), required for complete DNA-synthesis. In an embodiment of the method of the invention the extract holding the enzyme source for further processing of an enzymatically produced precursor product to the corresponding final nucleoside triphosphate is selected from the group consisting of a mammalian cell line extract, a fungal cell line extract, a bacterial cell line extract, either the cell being devoid of the enzyme to be measured or holding that enzyme as a temperature sensitive mutant, or said enzyme should be sensitive to an inhibitor non-efficient on the enzyme to be measured, said enzymes being optionally produced by recombinant technology. Thus, under certain circumstances such enzyme source may be a combination of relevant recombinant enzymes.

In another embodiment of the method of the invention, when directing the method to drug screens, the enzyme source for further processing of an enzymatically produced precursor product may be an extract from a virus infected mammalian cell, or an extract from relevant disease generating bacteria, amoeba, rickettsiae, Plasmodium etc holding both the enzyme to be analyzed as well as the enzyme source for further processing of an enzymatically produced precursor product to the corresponding final nucleoside triphosphate, and optionally also including the polymerase necessary for the DNA synthesis, said enzymes being optionally produced by recombinant technology.

The presently preferred source of enzyme for further processing of an enzymatically produced precursor product to the corresponding final nucleoside triphosphate is a yeast cell extract, such as an extract of Saccharomyces cerevisiae. In another embodiment the incorporated modified nucleotide is derived from a correspondingly non-modified, modified or labeled deoxynucleoside or precursor thereof. In yet another embodiment, the modified enzyme substrate is selected from the group consisting of Bromo-, lodo-, Fluoro- and Vinyldeoxy- etc- modified nucleoside/nucleotide or from the group of nucleosides/nucleotide with modified sugars, and it is e.g. BromodeoxyUridine. In a presently preferred embodiment the solid surface is a plastic surface, e.g. a plastic microtiter plate. However, the solid surface may be of another material onto which affinity molecules can be attached, such as plastic beads, magnetic beads, and agarose or silica surfaces as beads or coated wells.

In a further embodiment the solid surface-attached single stranded polynucleotide is attached via its 5'-end.

The biological sample analyzed by the method of the invention may be selected from the group consisting of blood, serum, plasma, Cerebral Spinal Fluid (CSF), pleural fluid, ascites, tissues, cell supematants, cells and extracts thereof. Tissue and cell samples refer to cytosolic or nuclear extract samples. The invention is also directed to an assay kit for determination of an enzyme activity in a sample comprising, in several separate containers, a solid surface-attached single stranded polynucleotide as a primer and/or template, optionally a non-attached single stranded polynucleotide as a template and/or primer, a non-modified, modified or labeled deoxy nucleoside/nucleotide or precursor as an enzyme substrate, a phosphate donor and other co-factors, a nucleotide polymerizing enzyme, and an enzyme source, devoid of the enzyme to be quantified, for further processing of an enzymatically produced precursor product to the corresponding final nucleoside triphosphate.

In one embodiment, the kit comprises reference enzyme(s) for standardization of the assay. In a further embodiment of the assay kit of the invention, the kit additionally comprises one or several microtiter plates containing, in one or several wells, surface- attached single stranded polynucleotide.

In another embodiment of the kit of the invention, the assay kit additionally comprises a labeled affinity molecule with affinity for a non-modified, modified or labeled deoxynucleoside to be detected, e.g. an enzyme labeled affinity-conjugate, such as an antibody labeled with alkaline phosphatase or with horseradish peroxidase.

Also in an embodiment of the assay kit of the invention the enzyme source for further processing of an enzymatically produced precursor product to the corresponding final nucleoside triphosphate essentially devoid of the enzyme activity sought for, is selected from the group consisting of a mammalian cell line extract, a fungal cell line extract, a bacterial cell line extract, a combination of purified enzymes of biological or recombinant nature necessary for converting the enzyme product into nucleoside triphosphate. It should be understood that the enzyme source for further processing of an enzymatically produced precursor product may be from different sources in the above combinations. However, the presently preferred source of enzyme for further processing of an enzymatically produced precursor product to the corresponding final nucleoside triphosphate is a yeast cell extract, such as an extract of Saccharomyces cerevisiae.

The invention is further directed to the use of the method and/or the assay kit of the invention in the diagnosing, monitoring and/or prognosis of cell-proliferation disorders or diseases, such as cancer or certain viral infections, in mammals, especially humans.

The invention is additionally directed to the use of the method and/or the assay kit of the invention in the screening of compounds, e.g. new drug candidates, affecting enzymatic pathways, which may obstruct the formation of deoxynuclosides/tides phosphates and the DNA synthesis.

The two last mentioned aspects of the invention may be formulated in an alternative way, namely as a method for use in the diagnosing, monitoring and prognosis of cell-proliferation disorders or diseases, such as cancer or certain viral infections, in mammals, especially humans comprising the method according to the invention and/or the assay kit according to the invention, and a method for use in the screening of compounds, e.g. new drug candidates, affecting enzymatic pathways which may obstruct the formation of deoxynucleosides/tides or interfere with the synthesis of nucleic acids comprising the method according to the invention and/or the assay kit according to the invention.

An important feature of the method of the invention is that the determination of an enzyme activity in a body fluid sample is achieved by utilizing an enzyme source, such as a cell extract devoid of such enzyme activity, for further processing of an enzymatically produced precursor product to the corresponding final nucleoside triphosphate. This in combination with a presently preferred solid phase immunodetection of processed enzyme product or complementary tracer (see figure 2) incorporated in newly synthesized DNA overcomes both the problem with enzyme feed-back inhibition and the problems connected with use of radioactive methods most commonly used for quantification of enzymes associated with nucleotide metabolism. For the phosphorylation steps to occur in the method of the invention there is an absolute requirement for a phosphate donor to be present in the basic reaction mixture in a buffer. As phosphate donors several nucleoside triphosphates can be used, especially ATP. Preferably the choice is a nucleotide that will not become incorporated into the growing chain of the primer/template system on the solid surface. It is at present particularly preferred to use a ribo nucleotide triphosphate as phosphate donor. In one embodiment of the invention there is provided a method for the determination enzyme activity in a biological sample by contacting, in a buffer, the sample with a Basic Reaction Mixture, containing a single stranded polynucleotide attached to a solid surface as a template and/or primer, optionally a non-attached single stranded polynucleotide as a template and/or primer, and an enzyme source for further processing of an enzymatically produced precursor product to the corresponding final nucleoside triphosphate, such as a cell extract, devoid of the enzyme activity to be measured so that the biological sample provides the only source for the enzyme to be measured. The enzyme activity in the biological sample is then determined by determination of the amount of non- modified, modified or labeled nucleotide, or added tracer nucleotide, that has been incorporated/or prevented to be incorporated by polymerase(s) into the newly synthesized double stranded nucleic acid attached to the solid surface, e.g. polymerase- directed solid phase incorporation of e.g Brd modified base or nucleoside/tide that is subsequently detected by using an enzyme-antibody conjugate against incorporated Brd nucleotide. In one embodiment the extract, devoid of the enzyme activity to be measured is derived from an extract of eukaryotic cells. In a presently even more preferred embodiment the extract is derived from extracts of mammalian cells. In another embodiment the extract, devoid of the enzyme activity, to be measured, is derived from purified mammalian cell extract e.g. using, but not limited to, affinity purification. In yet another embodiment the extract, devoid of enzyme activity to be measured is constructed by combining relevant recombinant enzymes.

In a presently particularly preferred embodiment the cell extract, devoid of the enzyme activity to be measured, is derived from baker's yeast, Saccharomyces cerevisiae. The present invention reveals that an enzyme activity, which is present in a biological sample, can be detected and determined by using an enzyme source for further processing of an enzymatically produced precursor product to the corresponding final nucleoside triphosphate devoid of that enzyme activity, combined with a polymerase giving direct incorporation or prevention of a modified nucleotide or natural substrate into a solid phase attached template/primer. The determination of the amount of incorporated non-modified, modified or labeled nucleoside can be made in a number of different ways, such as by detection of the modification of the nucleotide, e.g. Br in BrdUMP or by use of autoimmune antibody directed to natural incorporated deoxynucleotide (anti-DNA antibody). The invention is illustrated by use of an immunological method, namely Enzyme Linked lmmuno Sorbent Assay (ELISA). Thus, an enzyme-labeled affinity conjugate is used for the determination of the incorporated modified or non-modified nucleoside, which is related to the level of enzyme activity in the sample. Examples of affinity molecules include antibodies, such as monoclonal antibodies or antigen-binding fragments thereof.

In a presently preferred embodiment the polymerase, used for the incorporation of modified or non-modified nucleoside triphosphate into the solid surface primer template setup, is RNA directed DNA polymerase (RNA template dependant DNA polymerase = Reverse Transcriptase) (RT) and the solid surface attached nucleic acid template is poly-rA (polyriboadenylic acid). In another equally preferred embodiment the polymerase is a DNA directed DNA polymerase and the solid surface attached nucleic acid template is an poly-dA and the primer is a shorter complementary oligo-dT. In a further embodiment the polymerase is any polymerase, including Terminal Transferase known in prior art and the solid phase attached template is a variable DNA containing 2 or more of the 4 deoxynucleosides, with and without a homopolymeric region, with bases occurring sequentially or not in the single part of the template after end of primer, and the shorter primer is complimentary either to a variable region or the homopolymeric part of the template, or only a single strand is present. Depending on how the nucleic acid is attached to the surface, either the template or primer is attached to the surface. A provision is that the primer has to be attached to the solid surface, via its the 5'-end.

A number of native and modified DNA directed DNA polymerases are known in the art; these are exemplified by, but not limited to, eukaryotic DNA polymerase alpha from various sources, Escherichia coli DNA polymerase I, modified Klenow fragment from Escherichia coli DNA polymerase I₁ Phage T4 polymerase native or modified, Taq polymerase from Thermophilus aquaticus native or modified, Vent polymerase from Thermococcus litoralis and Pfu polymerase from Pneumococcus furioυsus.

Several native and modified RNA-directed DNA-polymerases, (Reverse Transcriptase = RT) (EC: 2.7.7.49) are known in the art; these are exemplified by, but not limited to, alfalfa Mosaic Virus RT AMV-RT), Moloney Murine Leukemia Virus RT (M-MuLV- RT), Simian Immunodeficiency Virus (SIV RT) and Human Immunodeficiency Virus (HIV), which are typical examples of this type of polymerases.

The length of the template is preferably 30 to 300 nucleotides long and the primer is preferably 10 to 30 nucleotides long. Methods for covalently attaching proteins to the solid surface of e.g. polystyrene plastic 96-well micro well plates are well known in the art. Binding to the polystyrene surface can be facilitated by the use of Imidazole, which is hereby included as an example, but not limiting, of this technology. Use of 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDCC) activated surface to couple 5'-amine modified oligonucleotide primers is well known in the art using photo coupling. Many different methods for employing technologies for the measurement of bio- molecules present in a sample, using Enzyme Linked lmmuno Sorbent Assay (ELISA) technology are well known in the art. In a presently preferred embodiment a monoclonal antibody directed against the hapten BrdU, is conjugated to the enzyme Alkaline Phosphatase, (AP) (Alkaline Phosphatase: Orthophosphoric-monoesterphosphohydrolase (alkaline optimum), EC 3.1.3.1.). However, any other enzyme known in the prior art to be useful for ELISA procedure can be used in the present invention, such as an enzyme conjugated to the anti-BrdU antibody, e.g. horseradish peroxidase. (Hydrogen peroxide oxidoreductase EC 1.11.1.7) (HRP). In another approach, any chromatophore, fluorophore, luminophore that is known to the prior art may be conjugated to the tracer antibody, e.g. the anti-BrdU antibody.

Several suitable substrates for either AP or HRP are well known in the art. Cleavage of the substrate para-Nitro Phenyl Phosphate (pNPP) by the AP turns the substrate colour from colourless to yellow. The absorbance can be read at 405 nm wavelengths using a spectrophotometer. Chemiluminescent AP substrates include, but are not limited to, derivatives of adamantyl 1 ,2-dioxetane (e.g. AMPPD, CSPD, CDP, and CDP- Star substrates all marketed by Tropix (Bedford, MA, US)), which emit a steady state glowing light at 477 nm upon de-phosphorylation by AP and the subsequent decomposition of the remaining molecule. Other examples of popular immunodetection chemiluminescent substrates for AP are lsoluminol (Diasorin S.p.a.) and Acridine ester (Bayer diagnostics). An example of, but not a limitation to, fluorescent substrates for AP is 4-Methylumbelliferyl Phosphate (4-MUP). After dephosphorylation of MUP, the remaining Methylumbelliferyl (MU) is excited at 370 nm wavelength. The emitted fluorescence is detected at 430 nm using a fluorometer. The invention will now be further illustrated by the following description of a novel technique for the detection of deoxycytidine kinase activity, deoxyadenosine kinase activity and deoxyguanosine kinase activity as well as a novel technique for the determination and quantification of thymidylate synthetase activity, and by the drawings.

Detection of deoxycvtidine kinase activity, deoxyadenosine kinase activity and deoxyguanosine kinase activity

Background

Deoxycytidine kinase, deoxyadenosine kinase and deoxyguanosine kinase are enzymes expressed in some species but not all. These enzymes salvages their substrates, i.e deoxycytidine, deoxyadenosine, and deoxyguanosine, into the DNA-synthesis by converting their substrates into monophosphates, which then are phosphorylated to the corresponding triphosphates before use for enzymatic DNA-polymerization. Conventionally mentioned kinases have been measured by incubating a radioactive substrate (³H- or ^Relabeled) together with enzyme and cofactors, whereupon the phosphorylated product is bound to a solid phase most commonly used is a DEAE-paper, whereupon the substrate is washed away,. The paper is then dried and put in scintillation fluid and the radioactivity bound is measured. The amount of radioactivity found is directly proportional to the kinase activity found. The described technique has several drawbacks like being sensitive to product feedback, low sensitivity due to long half-life of isotopes, low specific radiolabel of substrate, and problems with handling of radioactivity with reference to safety and regulations. Thus, in the following a novel technique automatically solving the separation between substrate and product, also useful with both non-radioactive and radioactive detection for the above mentioned kinase activities, is described. General description

A sample containing any or several of the following kinases, deoxycytidine kinase, deoxyadenosine kinase and deoxyguanosine kinase, is incubated, with a reaction mixture holding relevant co-factors, e.g. phosphate donor, metal-ion, and an extract or mixture devoid of the kinase activity to be measured but holding all enzymes involved in converting the kinase product, i.e. the deoxynucleoside-monophosphate to the corresponding deoxynucleoside-triphosphate, and a DNA-polymerase or terminal transferase, and a solid phase to which a relevant primer or template is bound and in the soluble phase the primer relevant to the immobilized template, or the template relevant to the immobilized primer, and a tracer molecule if not a radioactive kinase substrate is used.

After incubation the solid phase is washed and processed, and the processed kinase product that has been incorporated into the solid phase bound DNA is determined either by the amount of incorporated radioactivity or by the amount of incorporated nonradioactive tracer molecule which is quantified with conventional ELISA technique.

Example 1 For non-radioactive deoxycytidine kinase activity determination the assay outlined according to the invention may be as follows (see also figure 2 A: ):

The deoxycytidine kinase holding sample to be analyzed may be a recombinant enzyme, a cell extract or a supernatant from a culture of mammalian cells, a bacteria or a culture of any parasite like amoebas Plasmodium etc. The sample may also be a clinical specimen like serum, ascites fluid, spinal fluid, pleural effusion or an extract from a cell fraction collected anywhere in e.g a human being. The reaction mixture holds the substrate deoxycytidine, a metal ion e.g. Mg²⁺ and an phosphate donor e.g. ATP and additional components essential for the deoxycytidine kinase activity, a processing extract from a yeast devoid of deoxynucleoside kinase and preferentially from Saccaromyces cervisae, a tracer molecule BrdUTP, a DNA-polymerase e.g. Klenow fragment or a RT enzyme, and a template which in the 3'-end has a sequence base pairing with the solid phase bound primer and a template only holding outside the primer-binding region dG bases followed by several dA bases in the far 5'-end.

The sample and reaction mixture is incubated, in the presence of the solid- phase bound primer complementary to the 3' end of the template present, for desired time whereupon the reaction mixture is washed away.

Thereafter the amount of incorporated BrdUMP is determined using anti- BrdUMP antibody conjugate with alkaline phosphatase using standard Elisa technique with coloro- or fluorometric reading. (Illustrated in figure 2)

Determination and quantification of thvmidylate synthetase activity. Background.

Thymidylate synthetase is an enzyme present in most, if not all, species. The enzyme is essential in the de novo pathway for production of thymidine nucleotides and catalyses the reaction converting deoxyuridine-monophosphate to thymidine-monophosphate by transfer of a methyl group from tetrahydrofolate to the 5 position in the deoxyuridine pyrimidine ring. This reaction is addressed both by anticancer- and infectious disease therapies. Conventionally thymidylate synthetase activity has been measured either by spectrophotometric technology or by analyzing the release of ³H from 3H-labeled deoxyuridine-monophosphate which occurs in connection with transfer of the methyl group to the deoxyuridine-monophosphate molecule. The mentioned techniques have several drawbacks like low sensitivity, low specific radiolabel of substrate, and problems with handling of radioactivity with reference to safety and regulations. Thus, in the following, a novel technique automatically solving the separation between substrate and product also useful with both non-radioactive and radioactive detection for thymidylate synthetase is described.

General description

A sample containing thymidylate synthetase is incubated, with a reaction mixture holding relevant co-factors, methyl donor, metal-ion, and an extract or mixture devoid of thymdylate synthetase activity to be measured, but holding all enzymes necessary for converting the kinase product, i.e. the thymidine-monophosphate to its corresponding deoxynucleoside-triphosphate, and a DNA-polymerase or terminal transferase, and a tracer substrate competing with the thymidylate synthetase product, and a solid phase to which a relevant primer or template is bound, and in the soluble phase the primer relevant to the immobilized template, or the template relevant to the immobilized primer.

After incubation, the solid phase is washed and the amount of processed thymidylate synthetase product incorporated is determined on the basis of reduced incorporation of the tracer substrate. Said tracer being quantified immunologically with conventional Elisa technique. Alternatively the thymidylate synthetase substrate is radioactively labeled anywhere except in the 5-position of the pyrmidine ring, and the amount of incorporated product is then directly determined on the basis of radioactivity incorporated into the solid phase.

Example 2

For non-radioactive thymidylate synthetase activity determination the assay outlined, according to the invention, may be as follows (see also figure 3):

The thymidylate synthetase activity holding sample to be analyzed may be a recombinant enzyme, a cell extract, or a supernatant from a culture of mammalian cells, a bacteria or a culture of any parasite like amoebas Plasmodium etc. The sample may also be a clinical specimen like serum ascites fluid spinal fluid, pleural effusion or an extract from a cell fraction collected anywhere in e.g. a human being.

The reaction mixture holds the substrate deoxyuridine-monophosphate a metal ion e.g. Mg²⁺ and a methyl donor e.g. tetrahydrofolate and additional components essential for the thymidylate synthetase activity, a processing extract from a yeast devoid of thymidylate synthetase activity or optionally holding a temperature sensitive mutant of thymidylate synthetase and preferentially from Saccaromyces cervisae, a tracer molecule either BrdUMP or BrdUTP, a DNA-polymerase e.g. a SIV-RT enzyme, and a homopolymeric template preferentially prA base pairing with the solid phase bound odT primer. Alternatively the prA template may be bound to the solid phase, then the odT primer is present in the reaction solution.

The sample and reaction mixture is incubated, in the presence of the solid- phase bound primer, for desired time, whereupon the reaction mixture is washed away. Thereafter the amount of incorporated BrdUMP is determined using anti-BrdUMP antibody conjugate with alkaline phosphatase using standard Elisa technique with coloro- or fluorometric detection. The thymidylate synthetase activity measured is directly proportional to the reduction in incorporated BrdUMP in the solid phase, (see figure 3). Abbreviations

4-MUP 4-Methyl Umbelliferyl Phosphate AMPPD Adamantyl 1 ,2-dioxetane aryl phosphate

Alkaline Phosphatase: EC 3.1.3.1 ; Orthophosphoric-monoester

AP phosphohydrolase (alkaline optimum)

ATP Adenosine 5'-Tri-Phosphate

AZT 3'-azido-3'-deoxythymidine

BrdU BromodeoxyUridine

BrdUMP BromodeoxyUridine Mono-Phosphate

BrdUTP BromodeoxyUridine Tri-Phosphate

CEM cells Cells from a cell line derived from a childhood acute lymphoblastic leukaemia

CSF Cerebral Spinal Fluid

DEAE Di-Ethyl Amino Ethyl

DTT Dithiothreitol

EDCC 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide

EDTA Ethylenediamineteraacetic acid

EGTA Ethylene glycol-bis[beta-aminoethyl ether]-N,N,N^'N'-tetraacetic acid

ELISA Enzyme Linked lmmuno Sorbent Assay

FdU Fluorodeoxy Uridine

HIV Human Immunodeficiency Virus

HRP Hydrogen peroxide oxidoreductase EC 1.11.1.7

IdU lododeoxy Uridine

NdK Nucleoside di-phosphate kinases: EC 2.7.4.6 pNPP para-Nitro Phenyl Phosphate

PSA Prostate Specific Antigen

RT Reverse Transcriptase; RN A-directed DNA-polymerase: EC:2.7.7.49

SIV RT Simian immunodeficiency virus reverse trancriptase

TK Thymidine kinase: E. C. 2.7.1.21 ; ATP: thymidine - 5'-phosphotransferase

TMPK Thymidylate kinase: EC 2.7.4.9; ATP:dTMP phosphotransferase

TMP deoxyThymidine Mono-Phosphate

TTP deoxyThymidine Tri-Phosphate dUTP 2'-deoxyUridine 5'-Tri-Phosphate Materials and Methods

Primer/ Template used for illustration of invention experiments A and B.

The primer sequence is 18 bases δ'-GTC-CCT-GTT-CCG-GCG-CCA-S' (SEQ ID NO: 12 in EP 1395675 B1) and linked at the 5' end to a primary amine by a C6 spacer arm.

The template (CTGA)₆-A₁₂ (SEQ ID NO: 10 in EP 1395675 B1 ) construct contains three parts with different functions. From the 5' end: A (A)₁₂ polymer used to amplify the BrdU signal, a variable part (CTGA)_δ to obtain a polymerase reaction that is dependent on the four deoxynucleoside triphosphates and a sequence complementary to the primer.

Production of primer coated microtiter plates

1-ethyl-3-(3-dimetylamino-propyl)carboimide hydrochloride (final concentration 10 mg / ml) was added to a 100 mM 1-Methylimidazole buffer (pH 7.0) and the mixture was used to dilute the primer construct to a final concentration of 1 μg /ml. 100 μl of the primer solution was aliquoted to each well of a microtiter plate consisting of Nalge Nunc

NucleoLink® transparent strips (Cat no 248259). The plates were incubated 6-8 hours at 37 ⁰C, washed thoroughly in 2M NaOH with 2 mM Ethylenediaminetetraacetic acid (EDTA) and soaked in three 5 L vials with water. Residual fluid in the wells was removed by tapping the plates upside down on absorbing cloth or paper. The plates were allowed to dry for 30 min at room temperature and finally frozen for storage at - 20 ⁰C.

Production of yeast extract

Yeast extract containing complementing/rescuing kinase activity was prepared in a cold room facility (8⁰C) from bakers yeast (Jastbolaget, Rotebro, Sweden). 80 g yeast was first washed with 800 ml pre-cooled water for approximately 30 min in a Nalgene centrifuge bottle, then the yeast slurry was centrifuged for 5 min at 1500 rpm. The supernatant was discarded and the pellet was resuspended with 40 ml cool 3x lysis buffer (see Table 2) and transferred to a BeadBeater bottle (BSP) containing 200 ml Zirconium beads (1.0 mm in diameter; Biospec Products, Inc., Bartlesville PO Box 788, OK 74005, USA). The bottle was filled completely with 1x lysis buffer and the lid was added. The BeadBeater bottle was placed on the Beadbeater (BSP), the space between the bottle and lid was filled with crushed ice. The yeast was beadbeaten and all liquid was then transferred to a 500 ml beaker, and thereafter to 50 ml Sorvall centrifuge tubes. The liquid was centrifuged at 30 000 g for 20 min using a pre-cooled Sorvall RC-5B centrifuge. The supernatant, i.e. the yeast extract was aliquoted and stored at -70⁰C until use. For the illustrated experiments the yeast extract was further purified just before use in order to remove nucleoside triphophates. This was done by incubating the extract, diluted a 20-fold, together with Fractoge.l EMD TMAE Hicap gel on a shaker board at 4°C for 30 minutes. The supernatant was then collected and used in the assay mixture giving the final concentration indicated.

Reaction mixture for control of dCMP incorporation by the yeast: (N-(2-

Hydroxyethylpiperazine-N'-(2-ethanesulfonic acid) (Hepes) 20.0 mM pH 7.6, Sodiumazide 0.01 %, MgCl2 7 mM, EGTA 0.007 mM, spermine 1 mM, trehalose 1.5 mM, glutathione 0.25 mM, DTT 6.3 mM, ATP 1.5 mM, template 33 ng/100 μl, SIV 1.8 ng/100 μl, yeast extract 1.33%, Triton-X 100 0.85 %(v/v), dATP 0.1 μM, dGTP 0.1 μM, BrdUTP 0.1 μM.

Reaction mixture for control of BrdUMP incorporation by the yeast: (N-(2-

Hydroxyethylpiperazine-N'-(2-ethanesulfonic acid) (Hepes) 20.0 mM pH 7.6, Sodiumazide 0.01 %, MgCl2 7 mM, EGTA 0.007 mM, spermine 1 mM, trehalose 1.5 mM, glutathione 0.25 mM, DTT 6.3 mM, ATP 1.5 mM, template 33 ng/100 μl, SIV 1.8 ng/100 μl, yeast extract 1.33%, Triton-X 100 0.85 %(v/v), dATP 0.1 μM, dGTP 0.1 μM, dCTP 0.1 μM.

Deoxyguanosine kinase Reaction mixture: (N-(2-Hydroxyethylpiperazine-N'-(2- ethanesulfonic acid) (Hepes) 20.0 mM pH 7.6, Sodiumazide 0.01 %, MgCl2 7 mM, EGTA 0.007 mM, spermine 1 mM, trehalose 1.5 mM, glutathione 0.25 mM, DTT 6.3 mM, ATP 1.5 mM, template 33 ng/100 μl, SIV 1.8 ng/100 μl, yeastextract 1.33%, octophenoxy- polyethoxyethanol (Triton-X 100) 0.85 %(v/v), dATP 0.1 μM, dCTP 0.1 μM, BrdUTP 0.1 μM, dG 0.1 mM

Deoxycytidine kinase Reaction mixture: (N-(2-Hydroxyethylpiperazine-N'-(2-ethanesulfonic acid) (Hepes) 20.0 mM pH 7.6, Sodiumazide 0.01 %, MgCl2 7 mM, EGTA 0.007 mM, spermine 1 mM, trehalose 1.5 mM, glutathione 0.25 mM, DTT 6.3 mM, ATP 1.5 mM, template 33 ng/100 μl, SIV 1.8 ng/100 μl, yeast extract 1.33%, Triton-X 100 0.85 %(v/v), dATP 0.1 μM, dGTP 0.1 μM, BrdUTP 0.1 μM, dC 0,1mM.

Deoxythymidine kinase Reaction mixture: (N-(2-Hydroxyethylpiperazine-N'-(2-ethanesulfonic acid) (Hepes) 20.0 mM pH 7.6, Sodiumazide 0.01 %, MgCl2 7 mM, EGTA 0.007 mM, spermine 1 mM, trehalose 1.5 mM, glutathione 0.25 mM, DTT 6.3 mM, ATP 1.5 mM, template 33 ng/100 μl, SIV 1.8 ng/100 μl, yeast extract 1.33%, Triton-X 100 0.85 %(v/v), dATP 0.1 μM, dGTP 0.1 μM, dCTP 0.1 μM, BrdU 0.2 mM. Preparation of cell extracts. Extracts were prepared by spinning down cells from spinner cultures of CEM wt and CEM TK negative cells with approximately 70% of the cells in dividing stage, using a cooled centrifuge. The cells were then resuspended to 100 million cells per ml using cold PBS. The cells were then freeze-thawed for three cycles and the material was aliquoted and frozen at -20 ⁰C until further use.

Protocol for illustrative experiments and deoxynucleoside kinase activity assays.

For the illustration of the capacity of the bakers yeast extract together with SIV, to phosphorylate deoxynucleoside monophosphates and to immobilise the final triphosphate using a generalised primer/template system given above, reaction mixtures devoid of deoxynucleotide in question was used as given above. The monophosphates, were added at various concentrations as indicated in Fig 4 illustrates the results using dCMP and for reference BrdUMP.

For the illustration of the deoxynucleoside kinase activity assays the same generalized template structure was used with a matching primer sequence that was covalently bound to the wells of a 96 well microtiter plate (see materials). The reaction mixture depended on kinase activity to be measured (see materials). For example, for measurement of deoxyguanosine kinase activity the dGTP was substituted with dG, while for deoxycytidinekinase activity the dCTP was substituted with dC, while for the control thymidine kinase activity the BrdUTP was substituted with BrdU. The amount of bromodeoxyuridine monophosphate (BrdUMP) finally incorporated into DNA due to the kinase reaction in presence of yeast extract and excess reverse transcriptase was detected with an Ap conjugated anti-BrdU monoclonal antibody. An Ap substrate, pNNP was used for colorimetric product detection.

Practically the same protocol was used for the yeast capacity experiment (Fig 4) and the kinase experiment (Fig 5) i.e.100 μl of reaction mixture was added to each well of the primer coated microtiter plates. The monophosphates used were diluted in tenfold steps using dil reag spe buffer (Table 3) and the reaction was initiated by transferring 10 μl monophosphate dilution to each well on the plate. Likewise the cell extract samples were diluted using tenfold steps and the reaction was also initiated by transferring 10 μl to the primerplate holding the reaction mixture. The plate was next sealed with plastic adhesive tape (Fasson S695, cat. no. SH 236269, Nunc Denmark) and incubated at 33 ⁰C overnight. The reaction was terminated by washing the plate with 1 mM borate buffer (pH 8.9) with dextransulfate 16 mg/L, 1 % (v/v), octophenoxypolyethoxyethanol (Triton X-100) and 0.26% ethanol. The plates were then washed by adding 4x200 μl wash buffer holding 0.2 M NaOH and 2 mM EDTA, after fourth addition the plates were soaked for 15 minutes at 33⁰C, thereafter the plates were thoroughly washed in buckets with distilled water. Next the plates were incubated for 90 minutes at 33°C with 100 μl Ap-conjugated anti-BrdU monoclonal antibody diluted to 4.8 μg/ml in 25 mM (bis[2-Hydroxyethyl]iminotris[hydroxyl- methyl]methane;2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl]-1 ,3-propanediol) (Bis-Tris) buffer (pH 7.2) with 50 mM NaCI, 37.5 mM (NH₄)₂SO₄, 1 mg/ ml Dextran sulphate, 1 % Triton X-100 and 25 mg/ml Sigma non-fat dried milk.

The plates were thereafter washed with 2.5 mM borate buffer (pH 8.9) with dextransulfate 40 mg/L, 0.8 % (v/v), Triton X-100 and 0.16 % ethanol to remove unbound labeled antibody. The alkaline phosphatase activity was determined using 4.1 mg/ml pNPP dissolved in a Tris-buffer (pH 9.75) holding 1 mM MgCI₂. Absorbance was read at 405 nm with a BioTek ELX 800 reader first within 5 minutes then after defined intervals.

The results using serially diluted deoxynucleoside monophosphates (Fig 4) shows that dCMP is well detected by the yeast extract and that BrdUMP can be detected by the yeast also using a variable DNA template contrary to the prA used in co-pending patent application PCT/ SE 2006/ 000246.

The results using serially diluted CEM cell extract with various kinase reaction mixtures are illustrated in Fig 5. As can be seen, approximately 100 cells were required for detection of deoxyguanosine kinase and deoxycytidine kinase, 500 cells were required for thymidine kinase using extracts from wild type CEM cells. Using TK1 negative CEM cell extract as sample, similar results were obtained for dG and dC kinase while TK was not detected at all.

In summary, the results show that the invention is useful for measurement of different enzymes e.g. kinases producing precursors of deoxynucleoside triphosphates. Obviously, as the invention is useful for deoxynucleoside kinases it is also useful for measuring enzymes producing the nucleosides such as deoxyribosytransferases if the reaction mixture is complemented with deoxynucleoside kinase. Measurement of enzymes modifying nucleotides making them available for DNA synthesis as riboreductase and thymidylate synthetase are also feasible provided the correct processing enzymes and co- factors are included in the reaction mixture and others excluded. Table 1.

Examples of enzymes which can be measured by use of the described invention

Table 2.

1X lysis buffer, pH 5.3

Ingredient

Hepes 50 m M

Potassium Acetate 50 m M

Glycerol 20% v/v

EDTA 1 mM

Complete protease inhibitor tablet (Roche Inc.) one tablet to 50 mL^~1

MilliQ™ water to the required volume

Table 3

DiI Reag Spe, pH 7.65

Ingredient Hepes 1O mM

MgCI2 3.75 mM

Sodium azide 0.01 %

EGTA 3.75 μM

BrdU 10 μM Spermine 0.5 mM

Triton X-100 0.85 %

Claims

1. A method for determination and quantification of an enzyme activity, involved in a metabolic pathway leading to a deoxynucleoside triphosphate and derivates thereof, in a sample, comprising the steps of contacting, in a container with single stranded polynucleotide as a primer or template attached to a solid surface, a basic reaction mixture, in a buffer, comprising the following components: optionally a non-attached single stranded polynucleotide acting as a template and/or primer, a non-modified, modified or labeled deoxynucleoside, precursor or analog thereof or a mixture thereof as an enzyme substrate, a phosphate donor and other co-factors, and an enzyme source for further processing of an enzymatically produced precursor product to the corresponding final nucleoside triphosphate, said source being devoid of the enzyme activity to be determined and quantified, the enzyme activity to be determined and quantified being present in the sample, and a DNA polymerase, and optionally another deoxynucleoside triphosphate which functions as a tracer indicating the amount of the enzyme activity to be determined, incubating the resulting mixture, optionally washing the incubated mixture to remove non-attached reaction components, determining the amount of the non-modified, modified or labeled deoxynucleoside that has been incorporated, or the tracer that indicates such incorporation, or has been prevented from incorporation, into the solid surface-attached primer and/or template, and determining the enzyme activity present in the sample from the determined amount of incorporated non-modified, modified or labeled deoxynucleoside or from the reduction of incorporation of said deoxynucleoside.

2. The method according to claim 1 , wherein the optionally present non- attached single stranded polynucleotide acting as a template and/or primer in the basic reaction mixture may be absent in case a terminal transferase is present in the mixture.

3. The method according to claim 1 or 2, wherein the enzyme activity to be determined is selected from the group consisting of nucleoside base modifying enzymes, nucleoside modifying enzymes and nucleotide modifying enzymes.

4. The method according to any one of claims 1 - 3, wherein the enzyme activity to be determined is selected from the group consisting of phosphoribosyl transferases, adenylphosphotransferases, deoxycytidine kinase, deoxyadenosine kinase, deoxyguanosine kinase, thymidylate kinase, adenylate kinase, guanylate kinase, nucleoside diphosphate kinase, nucleoside monophosphate kinase, deaminases, cytidine/deoxycytidine deaminase, thymidylate synthetase, thymidylate synthetase, uridine phosphorylase, thymidine phosphorylase, deoxyuracilphosphatase, nucleoside phosphotransferase, ribonucleosidediphosphate reductases, phosphotransferases, oxireductases, methyltransferases, pentosyltransferases, phosphoric monoester hydrolases, and hydrolases.

5. The method according to claim 1 , wherein the incubated mixture is washed, the amount of incorporated non-modified or modified deoxynucleoside being the processed product, or an added nucleoside tracer only incorporated if said processed product is first incorporated, is determined by contacting the solid surface-attached primer and/or template with a labeled affinity molecule with affinity for the incorporated non-modified, modified or labeled deoxynucleoside and determining the amount of non-modified or modified deoxynucleoside that has been incorporated by determining the amount of attached labeled affinity molecule with the aid of the label.

6. The method according to claim 1 , wherein a radioactive non-modified, modified or labeled substrate is used, and the amount of deoxynucleoside is determined on the basis of radioactivity retained after wash of the solid surface.

7. The method according to claim 1 , wherein the enzyme substrate in the basic reaction mixture comprises three out of all four deoxyribonuclotides: Thymidine Triphosphate (TTP), deoxyCytidine Tri-Phosphate (dCTP), deoxyGuanosine Tri-Phosphate (dGTP) and deoxyAdenosine Tri-Phosphate (dATP), required for complete DNA-synthesis.

8. The method according to claim 1 , wherein the enzyme source for further processing of an enzymatically produced precursor product to the corresponding final nucleoside triphosphate is an extract selected from the group consisting of a mammalian cell line extract, a fungal cell line extract, a bacterial cell line extract, either the cell being devoid of the enzyme to be measured or holding that enzyme as a temperature sensitive mutant, or said enzyme should be sensitive to an inhibitor non-efficient on the enzyme to be measured, said enzymes being optionally produced by recombinant technology.

9. The method according to claim 1 , wherein the enzyme source for further processing of an enzymatically produced precursor product is an extract from a virus infected mammalian cell, or an extract from relevant disease generating bacteria, amoeba, rickettsiae, or Plasmodium holding both the enzyme to be analyzed as well as the enzyme source for further processing of an enzymatically produced precursor product to the corresponding final nucleoside triphosphate, and optionally also including the polymerase necessary for the DNA synthesis, said enzymes being optionally produced by recombinant technology.

10. The method according to claim 8, wherein the source of enzyme for further processing of an enzymatically produced precursor product to the corresponding final nucleoside triphosphate is a yeast cell extract.

11. The method according to claim 10, wherein the yeast cell extract is an extract of Saccharomyces cerevisiae.

12. The method according to claim 1 , wherein the incorporated modified nucleotide is derived from a correspondingly non-modified, modified or labeled deoxynucleoside or precursor thereof.

13. The method according to claim 1 , wherein the modified enzyme substrate is selected from the group consisting of Bromodeoxy-, lododeoxy-, Fluorodeoxy- and Vinyldeoxy- modified nucleoside/nucleotide or from the group of nucleosides/nucleotides with modified sugars.

14. The method according to claim 13, wherein the modified enzyme substrate is BromodeoxyUridine.

15. The method according to claim 1 , wherein the solid surface is selected from a plastic surface, a plastic microtiter plate, plastic beads, magnetic beads, and agarose and silica surfaces as beads or coated wells.

16. The method according to claim 1 , wherein the solid surface-attached single stranded polynucleotide is attached via its 5'-end.

17. The method according to claim 1, wherein the sample in which an enzyme activity is to be determined and quantified is selected from the group consisting of blood, serum, plasma, Cerebral Spinal Fluid (CSF), pleural fluid, ascites, tissues, cell supematants, cells and extracts thereof, the tissue and cell samples being cytosolic or nuclear extract samples.

18. An assay kit for determination of an enzyme activity in a sample comprising, in several separate containers, a solid surface-attached single stranded polynucleotide as a primer and/or template, optionally a non-attached single stranded polynucleotide as a template and/or primer, a non-modified, modified or labeled deoxy nucleoside/nucleotide or precursor as an enzyme substrate, a phosphate donor and other co-factors, a nucleotide polymerizing enzyme, and an enzyme source, devoid of the enzyme to be quantified, for further processing of an enzymatically produced precursor product to the corresponding final nucleoside triphosphate.

19. The assay kit according to claim 18, comprising additionally reference enzyme(s) for standardization of the assay.

20. The assay kit according to claim 18, comprising additionally one or several microtiter plates containing, in one or several wells, surface-attached single stranded polynucleotide.

21. The assay kit according to claim 18, comprising additionally a labeled affinity molecule with affinity for a non-modified, modified or labeled deoxynucleoside to be detected.

22. The assay kit according to claim 21 , wherein the labeled affinity molecule is an enzyme labeled affinity-conjugate.

23. The assay kit according to claim 22, wherein the enzyme labeled affinity conjugate is an antibody labeled with alkaline phosphatase or with horseradish peroxidase.

24. The assay kit according to claim 18, wherein the enzyme source for further processing of an enzymatically produced precursor product to the corresponding final nucleoside triphosphate devoid of the enzyme to be quantified, is selected from the group consisting of a mammalian cell line extract, a fungal cell line extract, a bacterial cell line extract, and a combination of purified enzymes of biological or recombinant nature necessary for converting the enzyme product into nucleoside triphosphate.

25. The assay kit according to claim 24, wherein the enzyme source is a yeast cell extract, such as an extract of Saccharomyces cerevisiae.

26. Use of the method according to any one of claims 1 - 17 and/ or the assay kit according to any one of claims 18 - 24 in diagnosing, monitoring and/or prognosis of cell- proliferation disorders or diseases, such as cancer or certain viral infections, in mammals, especially humans.

27. Use of the method according to any one of claims 1 - 17 and/ or the assay kit according to any one of claims 18 - 24 in screening of compounds, e.g. new drug candidates, affecting enzymatic pathways, which obstruct the formation of deoxynuclosides/tides phosphates and nucleic acid synthesis.

28. A method for use in diagnosing, monitoring and prognosis of cell- proliferation disorders or diseases, such as cancer or certain viral infections, in mammals, especially humans comprising the method according to any one of claims 1 - 17 and/or the assay kit according to any one of claims 18 - 25.

29. A method for use in screening of compounds, e.g. new drug candidates, affecting enzymatic pathways which obstruct the formation of deoxynucleosides/tides or interfere with the synthesis of nucleic acids comprising the method according to any one of claims 1 - 17 and/or the assay kit according to any one of claims 18 - 25.