Patent Application
20090203056
The present invention relates to a kit for assaying cancer or other diseases.
Cancer is highly lethal. One factor to improve the cancer survival rate is early detection. However, current early detection assays and effective treatments for cancer have many limitations.
The incidence and development of cancer are highly correlated to irregular expression of enzymes. During cancerization of cells, the activities of enzymes and their isozymes related to cell proliferation are up-regulated. Arylsulphatase (ARS) and its isozymes that can hydrolyze arylsulphates are related to detoxification in organism. It is generally agreed that the increase of serum ARS activity is related to inflammation that is referable to distinguish tumor or non-tumor. The determination of the ARS concentration in serum can provide very useful information for clinical diagnosis, treatment and prognosis of the disease.
Some studies showed that arylsulphatase (ARS) and its isozymes are hydrolases that can catalyze the hydrolysis of arylsulphate, which have been proved to be more active in serum of patients with lung cancer than in normal human serum and their activities are reduced by treatment. The increase of activity of lysosome enzymes including ARS can be used for effective diagnosis and therapeutic effect monitoring (Wozniak, A., et al, Neoplasma, 2002, 49(1): p. 10-5). Moreover, the activities of ARSA and ARSB in different histological types of human primary and secondary tumors increase significantly in almost all the primary lung cancers (Gasa, S., et al, Cancer Res, 1980, 40(10): p. 3804-9). The ARS activities in breast cancer patients are 2-5 times of those in healthy women. The activity is reduced to normal level after surgery or chemotherapy. The activity of enzyme is correlated to the size of tumor and its metastasis (Rozwodowska, M., et al, Pol Merkuriusz Lek, 2004, 17(99): p. 252-4). It is also found that the activity of sulphatase in breast cancer tissue is roughly double of that in normal tissue (Chetrite, G. S., et al., Anticancer Res, 2005, 25(4): p. 2827-30). Moreover, it is found that ARS activity in stomach cancer, rectum cancer, breast cancer and skin cancer is increased by using a non-specific substrate (Dzialoszynski, L. M., et al, Clin. Chim. Acta, 1966, 14(4): p. 450-3).
4-methyl umbelliferone sulfate (4-MUS) can be used as a suitable substrate for examining the activity of ARS (Uchimura, K., et al, Methods In Enzymology, 2006, 416: p. 243). Furthermore, 4-MUS has better Km, Vmax, sensitivity and stability than other substrates, and its hydrolysate 4-methyl umbelliferone (4-MU) has higher fluorescence in alkaline solution, which is convenient for detecting. The present techniques all examine either ARS A or ARS B, but the present invention is to examine both ARS A and B enzyme activity together in one assay because 4-MUS cannot distinguish A/B (Sherman, W. R., et al, Biochem J., 1967, 102: p. 905).
According to the biochemical reaction of 4-MUS catalyzed by ARS enzymes, based on the principle of resultant 4-MU in reaction solution reflecting enzyme activity, we examine the fluorescence intensity of 4-MU by 365 nm excitation and at wavelength of 445-450 nm, and draw standard curve based on 4-MU standard product with the known concentration so as to quantificationally examine the ARS activity in serum/urine samples. The initial screening, therapeutic effect monitoring and recurrence inspecting are carried out accordingly.
The present invention relates to a kit for assaying cancer, including 4-MUS and 4-MU. Preferably, the concentration of 4-MUS in the kit is 1-10 mM, the concentration of 4-MU is 100-5000 nM. The buffer in the kit is sodium acetate, with preferable concentration in the range of 1-10000 mM, pH value between 1 and 14, more preferable concentration in the range of 10-1000 mM, pH value between 2 and 14. The stop solution in the kit is mixed solution of sodium carbonate and sodium bicarbonate or mixed solution of sodium carbonate and glycin, with preferable concentration in the range of 1-50000 mM, pH value between 1 and 14, more preferable concentration in the range of 10-5000 mM, pH value between 7 and 12. The precipitator in the kit is lead acetate, with preferable concentration in the range of 1%-90% (w/v), more preferable concentration in the range of 10%-40% (w/v).
The present invention can be used for screening, therapeutic effect monitoring and recurrence inspecting:
Screening in normal people; diagnosis in patients with symptoms and distinguishing benign tumors and malign tumors; determining patient's conditions and evaluating treatment plans; monitoring the curative effects of tumors; predicting recurrence and prognosis.
The tumors include: lung cancer, stomach cancer, ovary cancer, pancreas cancer, breast cancer, liver cancer, oesophagus cancer, intestines cancer, nasopharynx cancer, kidney cancer, bladder cancer, lymph cancer, cholecyst cancer, ampullae cancer, penis cancer, testicle cancer, throat cancer, hypothyroid cancer, prostate cancer, palace cancer, colorectal cancer, bile duct cancer, parenchyma sarcoma, spermatogonium tumor, Ewing's sarcoma, leukaemia, osteosarcoma, encephaloma and malignant lymphoma. More preferably are lung cancer, stomach cancer, ovary cancer, pancreas cancer, breast cancer, liver cancer, oesophagus cancer, kidney cancer, lymph cancer, colorectal cancer, leukaemia and malignant lymphoma.
The advantages of the present invention (compared with ELISA):
Because the reaction between enzymes and substrates is highly effective and consistent, the determination of enzyme activity is better than antigen-antibody reaction technique, i.e. without false positive results. At the same time, the determination of enzyme activity can better reflect the biological functions of the enzyme in tumorigenesis, i.e. the experiment results are more authentic. And the product to be examined has special excitation and detection wavelength, making the whole experiment operable.
(1) Determination of Reaction Parameters
1 Reaction Parameters
1.1 Impact of anticoagulant agent on the enzyme activity (
From the
*EDTA did not seem to affect the ARS activity (
*sodium heparin (
To ensure activities of the enzyme, we choose serum samples without anticoagulant agent treatment as experiment samples.
1.2 Impact of preservatives on the enzyme activity (
From
*under preservative condition at room temperature, the activity of the serum ARS enzyme is dramatically reduced every day, with about 10% reduction each day compared to the previous day
*at 4° C., the activity of the serum ARS enzyme is insignificantly changed with time
*by freezing-thawing for 10 times, the activity is reduced by about 20%
For not affecting the activity of enzyme, we choose the temperature of −20° C.
1.3 Reaction Parameters determined by Basic Enzyme Experiments:
*OD-[P] is linear
*[P]-time is linear in early phase
*initial reaction speed v-[E] is linear (if [S]>>[E]0)
1.3.1 Determination of OD-[4-MU] standard curve: in order to ensure the fluorescence reading is linear with 4-MU concentrations,
1.3.2 Determination of [4-MU]-time curve:
We selected 3 serum samples to determine the optimal reaction time, the result shows (
In order to ensure values of [4-MU] readings from experimental measurements are within linear range and are obvious different from background (comparative) value, the reaction time is set as 24 h. Therefore, all our experiments using serum samples described below used 24 hours as the reaction time if not specified elsewhere.
1.3.3 [4-MU]-time curve of serum samples at different diluting conditions (
In order to ensure the dilution ratio of serum samples is within linear range, the ratio of 1:8 is selected, then 20 μL of which is added to the reaction system.
2 Determination of reaction parameters: the serum is diluted to ⅛, take 20 μl for enzymatic reaction, fluorescence reading is measured after 24 h, and at the same time, OD-[4-MU] standard curve is determined, finally (ODsample-ODcontrol(water)) is converted into [4-MU]
(2) Experimental Steps:
1 Serum sample examination
Take 5 mL blood into a vacuum cuvette with separation gel and without anticoagulant agent, standing for about 30 min at temperature, then centrifugate it with horizontal centrifugal machine for 10 min (3000 r/min). The supernatant serum is sucked/poured out to a 1.5 mL EP tube/or other type of unused tubule, cool it at −20° C. before use.
In activity determination, take 50μl serum into 350 μl 100 mM sodium acetate buffer (pH 5.4).
20 μl described diluted serum is added to 180 μl reaction system (containing 100 mM sodium acetate, pH 5.4, 8 mM 4-MUS).
It is placed in a thermal container (37° C.) for 24 h, then the reaction is stopped by adding 1 ml stop solution (500 mM sodium carbonate/sodium bicarbonate buffer, pH 10.7), finally fluorescence of each sample is determination (365 nm excitation, recording 445-450 nm emitting light value).
Standard sample 4-MU (pure product) is used for OD-[4-MU] standard curve, and serum reaction readings are converted according to this standard curve to yield [4-MU] for each reaction.
Based on the described [4-MU], the activity of the serum ARS or its isozymes is determined as the amount of 4-MU produced by 1 L serum protein in 1 minute.
2 Urine Sample Examination
Take about 3 mL morning urine sample into sterilized EP tube. In case of with impurity, it is centrifugated for 10 min (3000 r/min) to remove impurity and stored at −20° C.
In activity determination, take 40 μL urine sample with micropipettor into reaction tube, add 40 μL precipitator (30% lead acetate solution), mix well, let the samples stand at 4° C. for 5 min. It is then centrifugated for 5 min at room temperature (3000 r/min).
The supernatant is taken away with micropipettor. Add 40 μL substrate preparation (containing 100 mM sodium acetate, pH 5.4, 8 mM 4-MUS) to the tube, mix well, leave no coagulum, cultivate for at 37° C. 30-40 min, the reaction time is recorded as T. Thereafter, the reaction tube is quickly placed on ice, added by 200 μL stop solution (500 mM sodium carbonate/sodium bicarbonate buffer, pH 10.7), mix well. The reaction tube is centrifugated for 1 min at room temperature(6000-8000 r/min).
Take 200 μL post-reaction mixed urine sample and zero control solution (100 mM sodium acetate buffer, pH 5.4) for fluorescence determination (365 nm excitation, recording 445-450 nm emitting light value).
Standard sample 4-MU (pure product) is used for OD-[4-MU] standard curve, and urine reaction readings are converted according to this standard curve to yield [4-MU] for each reaction.
According to the above [4-MU], the activity of the urine ARS or its isozymes is determined as the amount of 4-MU produced by 1 L urine protein in 1 minute.
(3) Data Processing I:
Measure the standard sample in buffers as described above (containing sodium acetate and stop solution component in same concentration), i.e. OD of 4-MU (0.003 mM), recorded as ODs
Determine the OD of control reaction (reaction of 20 μl sodium acetate buffer to replace serum/reaction of 40 μl sodium acetate buffer to replace urine), which is recorded as ODO
(ODn−OD0)/[4−MU]n=ODs/[4-MU]s
[4-MU]n=(ODn−D0)*0.003 mM/ODs
It is possible to calculate the amount of product 4-MU produced from reaction of sample n, i.e. amount of substrate 4-MUS joining the reaction.
It is possible to obtain the activity of sample n, i.e. molar number of substrate that can be converted by 1 L enzymatic protein in Imin.
The equation is [4-MU]n/(L*24*60)
The unit is converted into nmol/min/L
Data Processing II:
Measure the standard sample in buffers as described above (containing sodium acetate and stop solution component in same concentration), i.e. OD of 4-MU (0.003 mM), recorded as ODs
Determine the OD of control reaction (reaction of 20 μl sodium acetate buffer to replace serum/reaction of 40 μl sodium acetate buffer to replace urine), which is recorded as OD0
(ODn−OD0)/[4-MU]n=ODs/[4-MU]s
[4-MU]n=(ODn−OD0)*0.003 mM/ODs
It is possible to calculate the amount of product 4-MU produced from reaction of sample n, i.e. amount of substrate 4-MUS joining the reaction.
Determine the protein amount of sample n: detected with bovine serum albumin (BSA) as control, ultraviolet spectrophotometer at λ=280 nm to obtain the protein concentration of sample n that is recorded as Mn, from which the protein amount of sample n can be calculated as Mgn.
It is possible to obtain the specific activity of sample n, i.e. molar number of substrate that can be converted by 1 mg enzymatic protein in 1 min.
As formula: [4-MU]n/(Mgn*24*60)
The unit is converted into nmol/min/mg
Data Processing III:
The fluorescence values of 4-MU standard samples B1, B2, B3 and B4 are used as ordinate(y), the corresponding concentration values of standard solution (in nM) as abscissa (x), draw the standard curve through the origin: y=kx
Calculate net fluorescence value of the samples Ai′=Ai−A0, which is substituted into the above equation as y to obtain concentration of 4-MU in post-reaction solution, i.e. [4-MU]i, in nM
Enzymatic activities of samples are calculated as below:
the total volume of post-reaction solution, 40 uL is initial urine volume)
the total volume of post-reaction solution, 0.4 μL is initial serum volume, i.e. add 4 μL 1/10 diluting serum)
Value unit can be converted into U/L, in which 1U=1 μmol·min−1
(4) Instruments for the Experiments:
Horizontal centrifugal machine: Heraeus Labofuge 400R Instrument for examination: it can detect fluorescence signal at 365 nm of excitation wavelength and 445-450 nm of detecting wavelength
Electro-thermostatical incubator: Tianjin Zhonghuan Experimental Circuit Ltd.
Vortex shaker: IKA MS2 Minishaker Pipetman: Gilson 1 mL, 200 μl, 20 μl
(5) Experimental Data:
1. Measurement of the ARS activity in each serum sample from normal human (15 cases) and patients with cancer (7 cases of lung cancer, 1 case of stomach cancer, 3 cases of ovary cancer, 1 case of pancreas cancer, 1 case of breast cancer) is repeated for 3 times. Refer to
2. Average of the ARS activity in serum samples from normal human and patients with cancer
T-test is completed for normal human serum (15 samples in total) and serum from patients with cancer (13 samples in total) to obtain p=0.005415<0.01.
3. Refer to
4. Determination of standard:
According to the correlation of sensitivity-specificity, we choose cutoff values with relative high sensitivity and specificity as standards.
5. Measurement of the ARS activity in each urine sample from normal human (54 cases) and patients with cancer (12 cases of lung cancer, 10 cases of stomach cancer, 13 cases of colorectal cancers 9 cases of oesophagus cancer, 5 cases of kidney cancer and 5 cases of breast cancer) is repeated for 3 times. Refer to
As the Figure shows, the activities of arylsulphatase in urine samples from patients with cancer are dramatically higher than that in normal human, indicating that urine ARS activity can be used as a marker for identifying tumor from normal samples.
6. The activities of arylsulphatase in urine samples from patients with cancer(57 cases of oesophagus cancer, 83 cases of stomach cancer, 79 cases of colorectal cancer, 51 cases of kidney cancer, 63 cases of bladder, 211 cases of lung cancer, 76 cases of liver cancer, 107 cases of breast cancer, 91 cases of ovary cancer, 28 cases of hematopoietic tumor) are examined, the sensitivities in different cancer types (true positive cases/(true positive cases+false positive cases)) are shown in
Among many urine samples from patients with different types of cancers examined, the average sensitivity of the arylsulphatase activities is not less than 80%, indicating that abnormal increases of the ARS activities in urine samples are common in patients with cancer.
7. Measurement of the ARS activity in each urine sample from normal human (29 cases) and cancer patients after treatments (7 cases of lung cancer, 9 cases of oesophagus cancer, 9 cases of liver cancer, 2 cases of kidney cancer, 3 cases of ovary cancer, 1 case of hypopharynx cancer and 2 cases of hematopoietic tumor) is repeated for 3 times. Refer to
We observed that in cancer patients after therapeutic treatments the urine ARS activity was dramatically reduced to the level close to that of normal controls (
