Thymidine Kinase Assay

Field of the Invention

The present invention relates to an alternative method for the determination of thymidine kinase (TK) activity in a biological sample. The invention is useful for the diagnosis, monitoring and/or prognosis of diseases wherein levels of thymidine kinase activity are altered from normal levels. In particular, the invention can be used in the diagnosis of cancer, monitoring and/or prognosis of cancer and in the screening of compounds and drug candidates affecting the enzymatic pathway involved in the formation of thymidine monophosphate and 5-bromodeoxyuridine monophosphate.

Background of the Invention Two cellular genes encode thymidine kinase (TK). Thymidine kinase 1 is a serum enzyme present in human or animal bodily fluids as well as in tissue/cell extracts. Thymidine kinase 2 participates in mitochondrial DNA synthesis and has a different amino acid sequence, localisation in the cell, and expression profile from thymidine kinase 1. Whilst TK2 has been connected to mitochondrial diseases, it is not involved in cell proliferation.

Thymidine kinase 1 (TK1 ) is a cellular enzyme involved in DNA synthesis. TK1 catalyses the transformation of thymidine to thymidine monophosphate (TMP) in the presence of adenosine triphosphate (ATP). In general terms TK1 transports a gamma phosphate group from a nucleoside triphosphate to a 5'-hydroxyl group of thymidine or deoxyuridine. The origin of the substrate for TK1 can be exogenous or endogenous. Suitable substrates include, thymidine, phosphorylate derivatives of deoxyuridine or modified thymidine wherein modifications

are present at the 5' position of the pyrimidine ring or the 3' position of the ribose.

Thymidine kinase 1 has been determined to be present in the cytosol of dividing cells, but not in resting cells and, as such, an increased concentration of TK1 correlates with the proliferative activity of a tumour. As a general rule, in healthy individuals, TK1 levels can be less than 5 U/L, whilst in neoplastic diseases, and in some viral infections, TK levels can be more than 80 U/L.

Thymidine kinase 1 has been described as an oncofetal enzyme. As it is a cellular enzyme, its levels increase most significantly when a tumour cell has direct contact with a biological fluid, for example, in cases of haematological malignant diseases, such as epicardium carcinomatosis and central nervous system tumours. An assay of TK1 within the cerebrospinal fluid can be used for the identification of a tumour(s) affecting the central nervous system. Elevated levels of TK1 activity, from increased enzyme activity and/ or increased amounts of TK enzyme, in serum can also be found in the acute stages of malignant diseases of the haematopoetic system, such as bronchoendogenic carcinoma, in breast cancer, in colorectal, prostate, testicular and bladder carcinoma. In non tumour diseases, elevated TK1 levels are most frequently found in viral infections, psoriasis, tuberculosis pleuritis, and in pulmonary eosinophilic infiltrate in sarcoidosis.

The TK1 activity of chronic and acute leukaemia cells is higher than in other neoplasias and serum levels of TK1 can reach even several tens of U/L. Repeated examination of leukemic infiltrates into the central nervous system can be used in therapy control at the intrathecal cytostatics application.

In Hodgkin's and non-Hodgkin's lymphoma, the level of TK1 is typically markedly increased and correlates with the stage, the course and the prognosis of the disease.

TK1 has been determined to be the best single biomarker for the determination of the clinical stage of lung small cell carcinoma. In colorectal carcinoma, an elevated TK1 level indicates disease generalisation, i.e. progression of the disease. In breast carcinoma, elevated TK1 levels are observed in both the localised type and the generalised disease. TK1 determination can serve as a useful addition marker in prostate, testicular, and bladder carcinoma.

Furthermore, TK1 estimation can also be useful in the evaluation of tumour response to treatment. In most cases, a dramatic decrease in TK1 levels appear within 48 hours after the start of therapy. TK1 activity in cancer patients has been proven to be one of the best markers for diagnosis of cancer disease and estimation of the effectiveness of a respective drug treatment.

There are several available methods presently used to determine TK levels as described by Krungkari J. et al. 1989 (High performance liquid chromatographic assay for thymidylate synthesis from the human malaria parasite, Plasmodium falciparum). Journal of chromatography Biomedical Application 487, 51 -60; D ^' Cruz et al. 2001 (Thymidine kinase-independent intracellular delivery of bioactive nucleotides by aryl phosphate derivatives of bromo-methoxy zidovudine (compounds WHI-05 and WHI-07) in normal human female genital tract epithelial cells and sperm). Biology of Reproduction 64 (1 ), 51-59; Ronnerbol Holding. 2006 (A method and kit for determination of thymidine kinase and use thereof). WO 2006/091158

A1 ; Oehrvik et ai. 2004 (Method for determination of thymidine kinase 1 activity and the use thereof). EP1385005A1 ; and Gronowitz et al. (1987) Method of determining DTK isoenzyme activity and the use thereof) US 4,637977.

Summary of Invention

Due to difficulties with the above methods these methods have not been widely used in clinical practice. At present, a radioenzymatic assay is the most common commercially available assay used to measure TK activity in patient samples. However, this radioenzymatic assay is disadvantageous due to the use of a radioactive isotope. In addition, the radioenzymatic assay is time consuming and cannot be easily automated. Immunological detection methods which have been tried have indicated that such methods are not best suited for clinical practice due to the difficulties with the stability of components used in such assays.

Accordingly, the present invention provides a method to determine thymidine kinase enzyme activity in a sample comprising the steps of;

providing a reaction mixture comprising a sample to be tested, a phosphate donor, and at least one thymidine analogue, selected from the group comprising 5'Bromodeoxyuridine (BrdU), lododeoxyuridine (IdU), 5' fluorodeoxyuridine, 3' fluoro3'deoxythymidine, and phenyldeoxythymidine,

incubating the reaction mixture such that any thymidine kinase in the sample can utilse the thymidine analogue and the phosphate donor,

measuring the level of at least one of the thymidine analogue and a monophosphate of the thymidine analogue present in the incubated reaction mixture, and

correlating the level of at least one of the monophosphate and the thymidine analogue present in the incubated reaction mixture with the activity of thymidine kinase.

In embodiments of the method, there is provided a step of separating the thymidine analogue and any monophosphate of the thymidine analogue present in the incubated reaction mixture from each other prior to the step of measuring. In such methods, the step of separating can be performed by a chromatographic method. Preferably, the chromatographic method is a reversed-phase chromatography.

In embodiments of the methods of the present invention the phosphate donor can be ATP or an analogue of ATP.

In particular embodiments, the method of separating comprises the steps of:

providing the incubated reaction mixture to a support under a condition wherein said support binds to the thymidine analogue and a monophosphate of the thymidine analogue with different affinities under the same condition,

providing a condition wherein the affinity of the support to one of the thymidine analogue and the monophosphate of the thymidine analogue is reduced such that a first elutant comprising the thymidine analogue or the monophosphate thymidine analogue is eluted,

optionally, providing a condition wherein the affinity of the support to the thymidine analogue or the monophosphate of the thymidine analogue is

reduced such that a second elutant comprising the thymidine analogue or the monophosphate thymidine analogue is eluted,

measuring the level of at least one of the eluted thymidine analogue and the monophosphate thymidine analogue present in the elutants,

correlating the level of at least one of the measured eluted thymidine analogue and monophosphate thymidine analogue present in the elutants with the activity of thymidine kinase.

In embodiments, the activity of the thymidine kinase is determined from measuring monophosphate thymidine analogues.

In embodiments the support can be a reverse phase matrix.

In the method of the present invention, the support does not require to be provided with a nucleic acid complex or an antibody which can bind to the thymidine analogue to allow separation of the thymidine analogue from the monophosphate of the thymidine analogue.

The assay may be used to measure the activity of TK1 and/or TK2.

Typically, the measurement of TK2 activity is of little practical significance.

In particular embodiments the thymidine analogue used in the method can be the thymidine analogue 5-bromodeoxyuridine which is converted to 5'- Bromodeoxyuridine monophosphate in the presence of thymidine kinase 1 enzyme in the sample to be tested. This is particularly advantageous, as 5-bromodeoxyuridine has a significantly higher UV absorbance than other known TK substrates.

The assay of the present invention has a number of advantages including the assay is less expensive to perform than conventional assays, the assay is relatively simple to perform, the assay does not depend on the use of expensive antibodies or isotopes, and the assay has a comparable sensitivity to the conventional radioenzymatic assay used in clinical practice.

In particular embodiments the support to which the thymidine analogue of the reaction mixture is bound can be a reverse phase column, more particularly a reverse phase HPLC column. In particular embodiments the reverse phase HPLC column can be a C-18 reverse phase column

In particular embodiments, the method used to measure the thymidine analogue and/or the monophosphate of the thymidine analogue is UV absorbtion. Preferably, the range of wavelengths used to measure the level of at least one of the thymidine analogue and the monophosphate of the thymidine analogue can be 260 nm to 300 nm. In preferred embodiments the range of wavelengths can be 275 nm to 295 nm. In particularly preferred embodiments the wavelength can be 280 nm. In alternative embodiments, mass spectrometry may be used to measure the amount of a thymidine analogue and/or monophosphate thymidine analogue present, for example, in a first and/or second elutant.

In embodiments the level of a thymidine analogue or the level of monophosphate of the thymidine analogue can be the concentration of the a thymidine analogue or the monophosphate of the thymidine analogue.

In embodiments the reaction mixture can be incubated in the range 3O ⁰ C to 40 ⁰ C for a time of between 1 hour to 5 hours. In particular embodiments the reaction mixture can be incubated at 37 ⁰ C for three and

a half hours before the concentration of thymidine analogue and / or monophosphate of the thymidine analogue is determined or measured.

In particular embodiments the enzymatic reaction within the reaction mixture can be stopped by incubating the reaction mixture for ten minutes at around 9O ⁰ C. Advantageously, whilst this incubation at 9O ⁰ C abolishes the thymidine kinase activity, it does not affect the recovery of the thymidine analogue from the sample.

In particular embodiments the support is a chromatography column and the conditions are provided using standard techniques. In particular embodiments conditions provided to the support are changed using HPLC. In such embodiments acetonitrile and TFA (Trifluoroacetic acid) may be provided to the support such that at least one of the thymidine analogue and a monophosphate of the thymidine analogue bind to the support. In particularly preferred embodiments the support is a C-18 column 3OθA 5μm (Phenomenex).

In particular embodiments the biological sample can be selected from the group consisting of blood, serum, plasma, cerebral spinal fluid (CSF), pleural ascites, tissues, cells and extracts thereof. In one embodiment the sample is an extract from eukaryotic cells. In preferred embodiments the sample is an extract from human cells. In one embodiment a sample includes serum and/or plasma samples or a tissue sample or cell sample.

In one embodiment of the invention there is provided the use of the method for determining TK1 activity in a human or animal sample for diagnosing diseases involving elevated levels of thymidine kinase 1 activity.

According to a second aspect of the present invention there is provided the use of the method according to the first aspect of the invention in the diagnosis of cell proliferation disorders or other diseases in mammals, especially humans.

According to a third aspect of the present invention there is provided the use of the method of the first aspect of the present invention to monitor progression of cell proliferation disorders or other diseases in mammals, especially humans, wherein the method includes the steps of

measuring the level of at least one of the thymidine analogue and a monophosphate of the thymidine analogue present in a patient sample taken at a first time point, and

measuring the level of at least one of a thymidine analogue and a monophosphate of the thymidine analogue present in a second or further sample obtained from the patient at a second or further time point, and

comparing the levels of a thymidine analogue and a monophosphate of the thymidine analogue at the first and subsequent time points.

In an embodiment, the method of the present invention is used for the diagnosis of cancer or tumours. In another embodiment the method of the present invention is used for monitoring the progression of a cancer or tumour. In the present invention a tumour or neoplasm refers to a tissue comprised of cells that grow in an abnormal way. In particular, a cancer can refer to a tumour or neoplasm which is malignant, for example haematological cancer, gastrointestinal cancer, breast cancer and prostate cancer.

Additionally or alternatively, there is provided the method of the present invention for use in determining thymidine kinase activity, particularly TK1 activity in a human or animal sample to identify a subgroup of patients wherein the patients of the subgroup are at high risk of disease progression, wherein those patients identified as having an increased TK1 activity level compared to healthy patients analysed by the method are considered to be at risk.

In particular embodiments such patients are considered to be at risk of developing haematological malignant diseases, such as epicardium carcinomatosis and central nervous system tumours, malignant diseases of the haematopoetic system such as bronchoendogenic carcinoma, other cancers such as breast cancer, colorectal, prostate, testicular or bladder carcinoma and/or of non tumour diseases such as viral infections, psoriasis, tuberculosis pleuritis, pulmonary eosinophilic infiltrate and sarcoidosis.

In particular embodiments, the method of diagnosis refers to a determination of the nature of a disease or ailment.

In particular embodiments therapeutic monitoring refers to the monitoring of the efficacy of a treatment with a known active compound or of a candidate drug compound. In particular embodiments the known active compound or candidate drug compound can be an anti-cancer agent. The efficacy of treatment of such compounds can be determined by measuring a decrease in activity or expression of TK1 in samples taken over a time period.

In one embodiment there is provided a method for determining the efficacy of treatment to inhibit thymidine kinase comprising the steps:

providing a reaction mixture comprising a sample to be tested, a phosphate donor, and at least one thymidine analogue, selected from the group comprising 5'bromodeoxyuridine (BrdU), lododeoxyuridine (Idu), 5' fluorodeoxyuridine, 3' fluoro3'deoxythymidine, and phenyldeoxythymidine in the presence of a test agent,

incubating the reaction mixture such that any thymidine kinase in the sample can utilise the thymidine analogue and the phosphate donor,

measuring the level of the thymidine analogue and/or monophosphate of the thymidine analogue present in the incubated reaction mixture in the presence of the test agent,

providing a reaction mixture comprising a sample to be tested, a phosphate donor, and at least one thymidine analogue, selected from the group comprising 5'bromodeoxyuridine (BrdU), lododeoxyuridine (Idu), 5' fluorodeoxyuridine, 3' fluoro3'deoxythymidine, and phenyldeoxythymidine in the absence of a test agent,

incubating the reaction mixture such that any thymidine kinase in the sample can utilise the thymidine analogue and the phosphate donor, and

measuring the level of the thymidine analogue and/or monophosphate of the thymidine analogue present in the incubated reaction mixture in the absence of the test agent,

wherein a decrease in the level of thymidine analogue or an increase in the level of monophosphate of the thymidine analogue is indicative that the treatment does not inhibit thymidine kinase.

In particular embodiments a quantitative measurement of the amount of TK1 can be determined.

According to a fourth aspect of the present invention there is provided a method to screen for a compound, for example new drug candidate, affecting enzymatic pathways which obstruct the formation of thymidine phosphates or interfere with nucleic acid synthesis comprising the steps:

providing reaction mixtures comprising a sample to be tested, a phosphate donor, and at least one thymidine analogue, selected from the group comprising 5'bromodeoxyuridine (BrdU), lododeoxyuridine (Idu), 5' fluorodeoxyuridine, 3' fluoro3'deoxythymidine, and phenyldeoxythymidine in the presence and absence of a test agent,

incubating the reaction mixture such that any thymidine kinase in the sample can utilise the thymidine analogue and the phosphate donor,

measuring the level of at least one of thymidine analogue and monophosphate of the thymidine analogue present in the incubated reaction mixture in the presence and absence of the test agent,

wherein when the level of thymidine analogue is unchanged or reduced or the monophosphate of the thymidine analogue is unchanged or increased in the presence of the test agent, the test agent does not obstruct the formation of thymidine phosphates or interfere with nucleic acid synthesis.

In embodiments, the method of the present invention can further comprise the step of:

correlating the level of the analogue present with the activity of thymidine kinase, wherein when the activity of thymidine kinase in the reaction mixture including the test agent is reduced, it is indicative that the test agent is a modulator of enzymatic pathways which obstruct the formation of thymidine phosphates and / or a modulator of nucleic acid synthesis.

In embodiments the methods of the present invention comprise a step of separating the thymidine analogue and the monophosphate of the thymidine analogue from each other.

In such embodiments the step of separating can include:

providing a reaction mixture, wherein the test agent is present, to a support wherein said support binds to the thymidine analogue and a monophosphate of the thymidine analogue with different affinities under the same condition such that the thymidine analogue and a monophosphate of the thymidine analogue of the reaction mixture, wherein the test agent is present, can be separated from each other,

providing a condition wherein the affinity of the support to one of the thymidine analogue and the monophosphate of the thymidine analogue is reduced such that a first elutant comprising the thymidine analogue or the monophosphate thymidine analogue is eluted,

providing a condition wherein the affinity of the support to the thymidine analogue or the monophosphate thymidine analogue is reduced such that a second elutant comprising the thymidine analogue or the monophosphate thymidine analogue is eluted,

measuring the level of at least one of the eluted thymidine analogue and monophosphate thymidine analogue present in the first and second elutant of the reaction mixture, wherein the test agent is present,

providing a reaction mixture wherein the test agent is absent to a support wherein said support binds to the thymidine analogue and a monophosphate of the thymidine analogue with different affinities under the same condition such that the thymidine analogue and a monophosphate of the thymidine analogue of the reaction mixture, wherein the test agent is absent, can be separated from each other,

providing a condition wherein the affinity of the support to one of the thymidine analogue and the monophosphate of the thymidine analogue is reduced such that a first elutant comprising the thymidine analogue or the monophosphate thymidine analogue is eluted,

providing a condition wherein the affinity of the support to the thymidine analogue or the monophosphate thymidine analogue is reduced such that a second elutant comprising the thymidine analogue or the monophosphate thymidine analogue is eluted,

measuring the level of at least one of the eluted thymidine analogue and monophosphate thymidine analogue present in the first and second elutant of the reaction mixture, wherein the test agent is absent, and

correlating the level of at least one of eluted thymidine analogue and monophosphate thymidine analogue present in the elutants with the activity of thymidine kinase wherein when the activity of thymidine kinase in the reaction mixture including the test agent is reduced, it is indicative the test agent is a modulator of enzymatic pathways which obstruct the

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formation of thymidine phosphates and / or a modulator of nucleic acid synthesis.

According to a fifth aspect of the present invention there is provided an assay kit for the measurement of thymidine kinase wherein said kit comprises at least one thymidine analogue selected from 5'- bromodeoxyuridine (BrdU), lododeoxyuridine, 5'-fluorodeoxyurinine, 3' fluoro3'deoxythymidine and a phenyldeoxythymidine, a phosphate donor, and a support to which at least one of the thymidine analogue and a monophosphate of the thymidine analogue bind with different affinities under the same condition.

In an embodiment of the assay kit, the assay kit is provided for the in vitro assay of a sample wherein the result can be used for diagnosis and/or therapeutic monitoring of a disease in a human or animal.

In an embodiment of the assay kit, the thymidine analogue is 5'- bromodeoxyuridine. In a particularly preferred embodiment of the assay kit the derivative of thymidine and a monophosphate of the thymidine analogue can be detected by UV absorbance measurement. In particularly preferred embodiments the thymidine analogue and the monophosphate of the thymidine analogue can be detected by UV absorbance at a wavelength in the range 250 nm to 300 nm. In preferred embodiments detection by UV absorbance is performed at a wavelength in the range 265 nm to 290 nm.

In embodiments of the assay kit of the present invention the support can be a HPLC column. In preferred embodiments the HPLC column is a C-18 column. In embodiments the assay kit may further comprise TFA and acetonitrile or other suitable buffers for use with the support. In additional

embodiments of the assay kit, the assay kit can further comprise a reference Thimidine Kinase for standardisation of the assay.

In a particular embodiments of the present invention at least one elutant comprising one of thymidine analogue or monophosphate of the thymidine analogue eluted from the support is frozen substantially immediately following elution.

Preferred features and embodiments of each aspect of the invention are as for each of the other aspects mutatis mutandis unless context demands otherwise.

Embodiments of the present invention will now be described by way of example only with reference to the following figures in which:

Figure 1 shows the detection and quantification of 5-bromodeoxyuridine (BrdU) by HPLC;

Figure 2 illustrates the detection and quantification of lododeoxyuridine (IdU) by HPLC;

Figure 3 shows an HPLC based TK assay of the present invention using BrdU as a substrate;

Figure 4a shows an HPLC trace using purified human TK and 10 mM and 5-bromodeoxyuridine as a substrate;

Figure 4b shows an HPCL trace using purified human TK and 10 mM Thymidine;

Figure 5 illustrates optimisation and automation of an assay procedure of the invention wherein termination of the enzymatic reaction occurs A. Recovery after acetone precipitation B. Recovery after incubation at 90° ;

Figure 6 illustrates an optimisation and automation of the assay procedure wherein pre-incubation of thymidine kinase for 10 minutes at 9O ⁰ C fully abolishes enzyme activity;

Figure 7 illustrates the stability of the product (BrdU-PO ₄ )at 6 ⁰ C;

Figure 8 illustrates linear regression analysis of the thymidine kinase HPLC-based assay versus the thymidine kinase REA.

HPLC detection of TK activity was performed using the following method. 10 μl_ cytosol from exponentially growing HeLa cells was added to buffer containing 50 mM Tris pH 7.4, 10 mM ATP, 10 mM MgCI ₂ , 10 mM 5- bromodeoxyuridine and 1% BSA. The sample was incubated for 0, 1 , 2 and 3 h and immediately analysed by HPLC using C-18 reversed phase column. Analysis of the chromatograms revealed time-dependent depletion of ATP, accumulation of ADP as well as accumulation of a new compound, which elutes before the substrate. The appearance of this compound was dependent on the presence of ATP, substrate and enzyme, suggesting that this is a product of ATP-dependent enzymatic reaction.

Mass spectrometry analysis of the HPLC fraction corresponding to the peak, demonstrated that this fraction contains a compound with a molecular weight of 384.96 Da, which is equivalent to the molecular weight of 5-bromodeoxyuridine monophosphate (Fig.3).

The experiment was repeated using purified human TK and 10 mM Thymidine or 5-bromodeoxyuridine as a substrate. The direct comparison of the two substrates demonstrated that the area under the peak corresponding to Thymidine monophosphate (Fig.4B) was significantly smaller in comparison to 5-bromodeoxyuridine monophosphate (Fig.4A) due to the lower sensitivity of UV detection of thymidine.

The sensitivity with which 5-bromodeoxyuridine can be measured using UV detection is advantageous.

Example 2: Termination of the enzymatic reaction

A procedure for termination of the assay, thus allowing several samples to be processed simultaneously was developed. The suitability of acetone precipitation and heat inactivation of TK as a means to terminate the reaction in the reaction mixture was explored (Fig.5). Acetone precipitation of the sample reduced the recovery of small molecules such as 5-bromodeoxyuridine, thus rendering this approach incompatible with the subsequent HPLC analysis (Fig.5A). In contrast, heat inactivation of the enzyme for 10 min at 90 ⁰ C did not affect the recovery of 5- bromodeoxyuridine from the sample and the following analysis by HPLC (Fig.δB).

In addition, pre-incubation of TK sample for 10 min at 90 ⁰ C, fully abolished the TK activity, demonstrating that this treatment is sufficient for complete inactivation of the enzyme (Fig.6).

Example 3: Stability of assay components

The assay method has been further optimised in terms of incubation temperature and duration. Results demonstrated that performing the enzymatic reaction at 37 ⁰ C for 3.5h gave optimal results. Stability of assay components is critical for the reproducibility of the assay. Of critical importance is the stability of the enzyme in human serum.

In order to determine enzyme stability, human serum was spiked with 80 U/L purified human TK and stored for 1 , 3 and 7 days at -20 ⁰ C and 4 ⁰ C. Samples were analysed for TK activity. No significant change was detected for the sample stored at -20 ⁰ C. Conversely, a substantial decrease of the activity of the enzyme has resulted from the storage at 4 ⁰ C.

As handling of patient's blood samples might require repeat freezing and thawing, the effect of such action on enzyme activity has been investigated. Serum samples were frozen quickly in liquid nitrogen, stored at -20 ⁰ C and thawed quickly by putting them in a water bath at 37 ⁰ C. Surprisingly, repeating this treatment up to 5 times did not significantly affect the activity of TK.

Having in mind the above results, it is suggested that patient serum samples should be frozen immediately after collection and analysed immediately after thawing. Several rounds of quick freezing and thawing (for aliquoting, etc.) are tolerated. The stability of the product (e.g. 5-bromodeoxyuridine monophosphate) in the reaction buffer has also been investigated. After the termination of the enzymatic reaction the samples were kept for 24, 48 and 72h at 6 ⁰ C. No significant change in the product concentration has been detected during this period (Fig.7). This result suggests that samples can be analysed up

to several days after termination of the assay, thus allowing for automation of the HPLC analysis, using a temperature-controlled autosampler.

Example 4: Validation of assay with samples from cancer patients

Sera from 5 healthy volunteers (samples 1 to 5) and 15 patients with breast cancer (samples 6 to 20) were tested in triplicate, in parallel with TK HPLC assay of the present invention and the TK REA (Table 1). All samples from the healthy volunteers except sample No4 showed low level 0 of TK activity below 5U/L. Surprisingly sample No4 showed slightly elevated level of TK activity, measured by both assays.

In addition the HPLC-based assay demonstrated higher reproducibility than the REA assay. The standard deviations in the first case were up to 5 10% while in the latter up to 20%.

Table 1

Comparison of HPLC-based assay and REA detection of TK activity in serum samples 0

Patient

No TK activity HPLC assay U/L Average SD TK activity REA assay U/L Average SD

1 2.2 1.2 1 1.47 0.52 2.6 2.2 2.8 2.53 0.31

2 1.8 1 0.9 1.23 0.40 2.1 2 1.5 1.87 0.32

3 4.1 3.5 4 3.87 0.26 4.5 3.5 5.1 4.37 0.81

4 6.5 6.4 6.1 6.33 0.17 7.2 6.2 8.1 7.17 0.95

5 1.5 1.2 0.9 1.20 0.24 1.9 2.6 3.1 2.53 0.60

6 34.5 36.7 32.6 34.60 1.68 28.6 37.2 44 36.60 7.72

7 56.4 55.8 49.8 54.00 2.98 60.3 54.2 48.9 54.47 5.70

8 18.5 22.6 19.3 20.13 1.77 19.8 25 25.3 23.37 3.09

9 80.8 75.8 75 77.20 2.57 75 57.8 63.2 65.33 8.80

10 16.1 16.2 16.9 16.40 0.36 17 25 20.1 20.70 4.03

17.1 17.3 17.9 17.43 0.34 18.1 18.3 19 18.47 0.47

20 20.1 22 20.70 0.92 12.9 20 20.9 17.93 4.38

9 9.2 6 8.07 1.46 9.5 9.2 6.7 8.47 1.54

20.1 20.4 20.5 20.33 0.17 25 20.6 20.5 22.03 2.57

80.1 77.5 75 77.53 2.08 80 75.6 84 79.87 4.20

13 13.1 13.5 13.20 0.22 13 25 13.1 17.03 6.90

32.1 32.9 32 32.33 0.40 32 47 38.7 39.23 7.51

44.1 44.9 44.2 44.40 0.36 44 47 32 41.00 7.94

65.1 70 70.6 68.57 2.46 63 80 75.1 72.70 8.75

89.9 79 75.9 81.60 6.00 88.9 81 78.9 82.93 5.27

Linear regression analysis of the TK HPLC-based assay versus the TK REA was performed. Average results from the 20 samples were plotted on the same graph (TK REA results on the Y axis and TK HPLC assay on the X axis) (Figure 8). Results showed a strong correlation between the HPLC-based assay and the REA. The linear regression analysis yielded R=O.98 (theoretical maximum is 1), n=20.

Example 5: Protocol for HPLC-based TK assay

A kinase buffer containing 50 mM Tris pH 7.4, 10 mM ATP, 10 mM MgCI ₂ , and 1 % BSA was prepared. This buffer should not be vortexed.

Lyophilised thymidine kinase standards were reconstituted with the Thymidine kinase buffer. The volume to be used was marked on the vial label- solution of the thymidine kinase standard with the stated enzyme activity 80 U/L. The standards with respective concentrations values for the standard curve were prepared by further dilution. Six following values were typically used 80, 40, 20, 10, 5, 2.5 U/L. These solutions were not vortexed. Foaming of the enzyme solution, which may cause enzyme denaturation, should be avoided.

30 ml 5-bromodeoxyuridine in Thymidine kinase buffer was prepared and mixed gently. In addition HPLC buffers, Buffer A: 0.1% TFA in water and Buffer B: 0.1% in acetonitrile were prepared.

The following assay was prepared in triplicate.

25μl substrate solution, 50 μl Thymidine kinase standard or sample and 175 μl Thymidine kinase buffer were mixed.

This was incubated for 3.5 hours at 37°C in an incubator without shaking.

To stop the enzymatic reaction the reaction mixture was incubated for 10 minutes at 80-90 ⁰ C.

The reaction mixture was centrifuged for 10 min at 13,000 RPM on a micro centifuge and the supernatant was transferred in to HPLC vials.

The assay may be interrupted in this stage and the assay samples can be stored at 4°C for 48h.

The assay samples are HPLC analysed on a reverse phase C-18 column. Fractions were eluted at 1 ml/min with 10 minutes linear gradient from 0 to 100% acetonitrile and 0.1 % TFA. UV absorbance was detected at 280nm.

To identify the peak corresponding to the product positive and negative (no enzyme added) controls were overlaid and an additional peak in the former positive control was been detected. This peak appears approximately around the 5 ^th minute. The exact time can change depending on the factors such as column batch and age.

A Standard curve plotting area under the peak versus enzyme activity of the standards was prepared. The enzyme activity of the thymidine kinase in the unknown sample was read off the calibration curve. The calibration curve may be used to determine the activity of TK in samples assayed at the same time as the calibrators

Various modifications may be made to the invention herein described without departing from the scope thereof.