Patent Application
20070292882
A method for detecting a cancer cell which is one embodiment of the present invention is a method for detecting a cancer cell in a biological sample containing a plurality of cells. This method comprises a first quantitation step of quantitating an mRNA transcribed from a predetermined tumor marker gene, a second quantitation step of quantitating a polypeptide translated from this gene, and a detection step of detecting a cancer cell in the biological sample based on the result of quantitation of the mRNA and the result of quantitation of the polypeptide.
As used herein, a cancer is a tumor which has became malignant, and has the same meaning as a malignant tumor. The cancer includes carcinoma, sarcoma, and a cancer derived from a hematopoietic organ. As the carcinoma, an epithelial cell-derived cancer such as a breast cancer, a stomach cancer, a colon cancer, a prostate cancer, a cervical cancer, and a cancer of uterine body is exemplified. As the sarcoma, osteosarcoma and soft-tissue sarcoma are exemplified. As the cancer derived from a hematopoietic organ, leukemia and malignant lymphoma are exemplified.
The biological sample is not particularly limited, as far as it is a sample containing a plurality of cells collected from an animal such as human. Examples include excretion such as urine and feces, blood, and a tissue collected by a surgical procedure such as biopsy and isolation operation. Particularly, a tissue collected by biopsy is preferable.
For example, when the method is practiced using a tissue, whether this tissue has been transformed into a cancer or not can be determined. In addition, when primary focus is recognized in another tissue, whether a cancer cell has been metastasized from the primary focus to this tissue or not can be determined.
This determination result can be used as one of indices upon determination of therapeutic policy and a dissection region of a tissue. For example, collection of a lymph node tissue in the vicinity of a tumor from a breast cancer patient by biopsy, and determination of the presence or absence of metastasis of a cancer to a lymph node by the above method assist determination of whether a lymph node should be isolated, or to what extent a lymph node should be dissected.
In the first quantitation step, an mRNA transcribed from a tumor marker gene contained in the biological sample is quantitated. The tumor marker gene is a gene such that an expression amount in a cancer cell, and an expression amount in a normal cell are significantly different, as described above. The tumor marker gene is different depending on a biological sample used, and a kind of a cancer. Examples of the tumor marker gene include genes encoding CK (cytokeratin) such as CK18, CK19 and CK20, CEA (carcinoembryonic antigen), MUC1, MMG (mammaglobin), PSA (prostate specific antigen), CA15-3, EpCAM (epithelial cellular adhesion molecule), and the like.
Upon quantitation of an mRNA, it is preferable to prepare a sample for detection from the biological sample, and quantitate an mRNA contained in this sample for detection. For example, a biological sample and a buffer are mixed, cells in the buffer are chemically and/or physically treated to transfer an RNA in cells to a solution, and the solution containing the RNA can be used as a sample for detection.
It is preferable that a buffer is strongly acidic in order to suppress degradation of an RNA. A range of a pH is preferably 2.5 to 5.0, more preferably 3.0 to 4.0. In order to keep a pH in this range, a known buffer can be used.
In addition, it is preferable that the buffer contains a surfactant. A cell membrane and a nuclear membrane are damaged with the surfactant. Therefore, it becomes easy for a nucleic acid in cells to transfer into a solution through this damage. The kind of the surfactant is not particularly limited as far as it has such action. A preferable surfactant is a nonionic surfactant. A polyoxyethylene nonionic surfactant is more preferable. Particularly, a polyoxyethylene nonionic surfactant represented by the general formula:
R1-R2—(CH2CH2O)n—H
(wherein R1 is an alkyl group, an alkenyl group, an alkynyl group, or an isooctyl group having 10 to 22 carbon atoms; R2 is —O— or —(C6H6)—O—; n is an integer of 8 to 120) is suitable.
Specific examples of the polyoxyethylene nonionic surfactant include polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, polyoxyethylene myristyl ether, polyoxyethylene stearyl ether, polyoxyethylene nonyl phenyl ether, and polyoxyethylene isooctyl phenyl ether.
More specifically, Brij35(polyoxyethylene(35)lauryl ether) and the like are suitable. A concentration of the surfactant in the buffer is preferably 0.1 to 6% (v/v), more preferably 1 to 5% (v/v).
In addition, when quantitation of an mRNA is performed by nucleic acid amplification described later, it is preferable that the buffer further contains dimethyl sulfoxide (DMSO). Although a substance inhibiting an enzymatic reaction in nucleic acid amplification (inhibitory substance) is contained in the biological sample in some cases, influence of this inhibitory substance can be effectively reduced by the action of DMSO. In addition, DMSO also has the effect of suppressing reduction in the activity of a nucleic acid amplification enzyme. A concentration of DMSO in the buffer is preferably 1 to 50% (v/v), more preferably 5 to 30% (v/v), most preferably 1 to 25% (v/v).
Upon preparation of the sample for detection, it is preferable that the biological sample is subjected to physical treatment such as homogenization. Thereby, a cell membrane and a nuclear membrane of cells in the biological sample are physically ground, and it becomes easy for a nucleic acid in cells to transfer into a solution. Homogenization may be performed manually with a pestle, or may be performed using a commercially available electric-powered homogenizer. A cell piece floating in a homogenate can be precipitated by centrifuging the resulting homogenate for a few seconds to a few minutes. Thereby, a supernatant containing an RNA and the like can be used as the sample for detection.
Quantitation of an mRNA can be performed by a known method using nucleic acid amplification, a DNA chip or the like. When a nucleic acid amplification method is used, an RT-PCR (Reverse Transcription PCR) method and an RT-LAMP (Reverse Transcription LAMP: for LAMP method, see U.S. Pat. No. 6,410,278) method comprising a reverse transcription reaction are suitably used before the nucleic acid amplification method. Particularly, as a quantitative nucleic acid amplification method, known methods such as an SYBR Green method and a TaqMan (registered trademark of Roche Diagnostics) method (see Linda G. Lee, 1993, Nucleic Acids Research, vol. 21, p3761-3766 etc.) can be used.
As the DNA chip which can be used in quantitating an mRNA, a substrate on which a polynucleotide of a DNA capable of hybridizing with a cDNA of the tumor marker gene and/or a fragment thereof is immobilized, can be used. Detection of an RNA using the DNA chip can be performed by a known method which is generally used.
For example, detection of an RNA using the DNA chip can be performed as follows. First, a reverse transcription reaction is performed utilizing a polyA sequence present on a 3′ end of an mRNA in a sample for detection. Upon the reverse transcription reaction, for example, by using a nucleotide labeled with a fluorescent substance such as Cy3 and Cy5, a fluorescently labeled cDNA is synthesized. This is contacted with the substrate on which a polynucleotide is immobilized. Thereby, this polynucleotide and the labeled cDNA form a double strand. After formation of the double strand, an mRNA can be quantitated by measuring fluorescence of the cDNA.
A quantitated value of an mRNA, measured by the above method, may be a substance amount of an mRNA, an mRNA mass, or a copy number per unit volume. Alternatively, it may be a time or a cycle number (when PCR is used) until a fluorescent intensity or a turbidity of a reaction solution reaches a predetermined value.
In the second quantitation step, a polypeptide translated from the tumor marker gene is quantitated. The polypeptide may be a protein translated from the tumor marker gene, or a fragment thereof.
Upon quantitation of the polypeptide, it is preferable that a sample for detection is prepared from the biological sample, and a polypeptide contained in this sample for detection is quantitated. This sample for detection may be a sample for detecting a polypeptide different from the sample for detection used upon quantitation of an mRNA. Preferably, the same sample as the sample for detection, prepared upon quantitation of an mRNA, is used. Thereby, labor for preparing two kinds of samples, a sample for quantitating an mRNA and a sample for quantitating a polypeptide, can be omitted. Therefore, the sample can be prepared more easily and in a short time.
Conventionally, upon quantitation of an mRNA, treatment such as extraction and purification was repeated on a biological sample using an RNA extraction kit or the like. Thereby, a sample substantially containing only an RNA was prepared, and this was used as a sample for detection. When this treatment is performed, a polypeptide in the biological sample and a piece of ground cells are discarded. Therefore, in order to quantitate a polypeptide from the same biological sample, it was necessary to prepare again a sample for detection for quantitating a polypeptide.
However, a sample for detecting an mRNA, which was prepared using a buffer having the aforementioned pH and containing the aforementioned surfactant, contains a polypeptide of a tumor marker gene. For this reason, quantitation of an mRNA and quantitation of a polypeptide can be performed using the same sample.
Quantitation of the polypeptide can be performed by a known method, being not limiting. For example, an analysis method using a protein chip or the like, an immunoblot method such as a dot blot method and a Western blot method, radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescent immunoassay (FIA), chemical emission immunoassay (CLIA), counting immunoassay (CIA: see Sysmex Journal Vol. 20 No. 1, 77-86 (1997)) can be used.
Among them, when the Western blot method is used, the kind of a polypeptide contained in a sample for detection can be separated based on a molecular weight. For this reason, an objective polypeptide can be quantitated more specifically.
It is preferable that a quantitated value of a polypeptide is calculated based on a calibration curve which was produced in advance. The calibration curve can be produced by measurement under the same condition as that of measurement of a polypeptide of a sample for detection, using a sample containing a known amount of a protein.
Based on the quantitation result of an mRNA and the quantitation result of a polypeptide measured as described above, a cancer cell in the biological sample is detected. In this step, whether a cancer cell is contained in the biological sample or not is predicted. A cancer cell may be detected based on a combination of both quantitation results. Alternatively, determination may be first performed based on the quantitation result of one of an mRNA and a polypeptide, and the result of the determination may be confirmed using the other quantitation result. The determination result obtained based on both quantitation results of an mRNA and a polypeptide has higher precision and higher reliability than those of a determination result using either one of quantitation results.
A cancer cell in a biological sample may be detected based on the comparison result obtained by comparing a quantitated value of an mRNA with a corresponding first threshold, and the comparison result obtained by comparing a quantitated value of a polypeptide with a corresponding second threshold. For example, in the case of a tumor marker gene in which an expression amount is greater in a cancer cell than in a normal cell, when a quantitated value of an mRNA is not smaller than a first threshold, and a quantitated value of a polypeptide is not smaller than a second threshold, it can be predicted that a cancer cell is contained in a biological sample. In addition, when a quantitated value of an mRNA is not smaller than a first threshold, or a quantitated value of a polypeptide is not smaller than a second threshold, it may be predicted that a cancer cell is contained in a biological sample.
The first threshold and the second threshold are values which are conveniently set depending on a kind of a cancer and that of a tumor marker. These thresholds can be set at a value being not larger than an amount of a tumor marker contained in a biological sample, for which the presence of a cancer cell has been confirmed (positive sample), and higher than an amount of a tumor marker contained in a biological sample, for which the absence of a cancer cell has been confirmed (negative sample). Particularly, it is preferable that a value obtained by measuring tumor marker amounts of a plurality of positive samples and tumor marker amounts of a plurality of negative samples in advance, by which a positive sample and a negative sample can be discriminated at a highest probability, is set as a threshold.
Alternatively, an mRNA transcribed from a tumor marker gene contained in a sample for detection prepared from the buffer is qualitatively detected, a polypeptide translated from the tumor marker gene contained in this sample for detection is qualitatively detected and, based on these detection results, a cancer cell in a biological sample may be detected. In this case, for example, when it is determined that an mRNA is present and a polypeptide is present in the sample for detection, it is determined that a cancer cell is contained in a biological sample.
A method for qualitatively detecting an mRNA is not particularly limited, but a known method can be used. For example, detection can be performed by conducting agarose gel electrophoresis or the like using a reaction solution from nucleic acid amplification by the aforementioned RT-PCR or RT-LAMP. In addition, a method for qualitatively detecting a polypeptide is also not particularly limited, but the aforementioned known immunoassay and the like can be used.
Another embodiment of the present invention is a cancer cell detection apparatus for implementing the aforementioned method. Based on the drawings, this apparatus will be explained below.
The nucleic acid quantitation part 100 of the apparatus 1 will be explained using
The nucleic acid quantitation part 100, as shown in
In addition, the dispensing mechanism part 10, as shown in
In addition, as shown in
In addition, in the present embodiment, a sample container 22 accommodating an extract (sample for detection), which is prepared by solubilizing a pre-excised biological tissue with the aforementioned treatment (homogenization, filtration etc.) is set in the sample container setting pores 21c , 21e, 21g and 21i on a front left side. Furthermore, a sample container 23 accommodating a diluted sample obtained by 10-fold diluting the sample is set in the sample container setting pores 21d, 21f, 21h and 21j on a front right side.
In addition, a container 24 accommodating a positive control for confirming that a nucleic acid to be amplified is normally amplified is mounted in the sample container setting pore 21a. Furthermore, a container 25 accommodating a negative control for confirming that a nucleic acid not to be amplified is not normally amplified is set in the sample container setting pore 21b.
An enzyme reagent container 26 accommodating a nucleic acid amplification enzyme reagent for amplifying a cDNA (hereinafter, also referred to as CK19cDNA) corresponding to an mRNA of CK19 (hereinafter, also referred to as CK19 mRNA), and a primer reagent container 27 accommodating a reagent comprising a primer (hereinafter, referred to as primer reagent), which can be hybridized with CK19cDNA are set in the enzyme reagent container setting pore 21k and the primer reagent container setting pore 211, respectively.
In addition, each reaction detection block 50a of the reaction detection part 50, as shown in
In addition, the turbidity detection part 52 is constructed of an LED light source part 52a consisting of blue LED having a wavelength of 465 nm attached to a substrate 55a arranged on one side face side of the reaction part 51, and a photodiode light receiving part 52b attached to a substrate 55b arranged on the other side face side of the reaction part 51. In each reaction detection block 50a, two sets of the turbidity detection part 52 are arranged, one set consisting of one LED light source part 52a and one photodiode light receiving part 52b.
In addition, the detection cell 54 has two cell parts 54a for accommodating a sample, and a lid part 54b for covering the two cell parts 54a.
In addition, the transferring part 60, as shown in
Next, referring to
First, as shown in
When motion of the nucleic acid quantitation part 100 is started, first, after the arm part 11 of the dispensing mechanism part 10 is moved from an initial position to the chip setting part 30 with the transferring part 60 shown in
After suction of the primer reagent, after two syringe parts 12 are moved upwardly, the arm part 11 of the dispensing mechanism part 10 is moved to above the reaction detection block 50a situated at the cell setting position 1 which is an innermost side (front inner side of the apparatus), with the transferring part 60. Then, at the reaction detection block 50a on the innermost side, by moving two syringe parts 12 downwardly, two pipette chips 31 fitted into two syringe parts 12 are inserted into two cell parts 54a of the detection cell 54, respectively. Then, using the syringe parts 12, primer reagents are discharged into two cell parts 54a, respectively.
After discharge of the primer reagents, after the two syringe parts 12 are moved upwardly, the arm part 11 of the dispensing mechanism part 10 is moved towards above the chip discarding part 40 in an X axis direction with the transferring part 60. Then, at the chip discarding part 40, the pipette chip 31 is discarded. Specifically, by moving two syringe parts 12 downwardly, pipette chips 31 are inserted into two chip discarding pores 40a (see
Then, by a similar motion, an enzyme reagent is discharged to the cell part 54a from an enzyme reagent container 26 and, further, by a similar motion, samples are discharged to the cell part 54a from a sample container 22 and a sample container 23.
Then, after the primer reagent, the enzyme reagent and the sample are discharged into the cell part 54a, motion of closing a lid of a lid part 54b of the detection cell 54 is performed. After completion of this lid closing motion, by elevating a liquid temperature in the detection cell 54 from about 20° C. to about 65° C., a cDNA corresponding to a CK19 mRNA is amplified by an RT-LAMP reaction. Then, clouding due to magnesium pyrophosphate produced accompanied with amplification is detected by nephelometry. Specifically, using an LED light source part 52a and a photodiode light receiving part 52b shown in
Turbidity data of the sample is sent to the personal computer 102 from the nucleic acid quantitation part 100 in real time. CPU102d of the personal computer 102 calculates an mRNA copy number per unit volume from turbidity data of the sample, and compares this with a predetermined threshold.
Then, based on
The protein quantitation part 101, as shown in
The dispensing part 210, as shown in
The reagent setting part 220 is provided for mounting a reagent bin 203 accommodating a buffer, a latex reagent and a specimen diluting solution. Thereupon, reagents (buffer, latex reagent, specimen diluting solution) in the reagent bin 203 are maintained at a predetermined temperature (not higher than, 15° C.). In addition, in the reagent setting part 220, a buffer container setting part 221, a latex reagent container setting part 220 and a specimen diluting solution container setting part 223 are provided in an order from an inner side of the apparatus.
The reaction part 240 is provided for reacting a sample accommodated in a cuvette 201a in two reaction plates 201, and various reagents (buffer, latex reagent containing latex particle obtained by sensitizing anti-CK19 antibody, specimen diluting solution). Specifically, by stirring and mixing a sample dispensed from the dispensing part 210, and various reagents (buffer, latex reagent, specimen diluting solution) and, at the same time, maintaining the stirred and mixed sample and various reagents at a predetermined temperature, a reaction of aggregating the latex reagent is promoted. That is, at this reaction part 240, as shown in
When a CK19 protein is present in a sample, as shown in a right figure of
The measurement dilution dispensing part 250, as shown in
The sample receiving part 260 is provided for receiving the prepared sample and the measurement diluting solution in the cuvette 201a of the reaction plate 201 of the reaction part 240. In addition, a particle suspension (prepared sample and measurement diluting solution) received in the sample receiving part 260 is guided to a sheath flow cell 274 (see
The optical detection part 270, as shown in
The reaction plate tray 280 can accommodate a maximum of four unused reaction plates 201 (see
The washing part 300a is provided for washing the specimen·latex pipette part 211 of the dispensing part 210. In addition, the washing part 300b is provided for washing the measurement dilution pipette part 251 of the measurement dilution dispensing part 250.
The CPU 102d of the personal computer 102 acquires scattered light information sent from the photodiode 275 and, based on this, calculates an amount of the CK19 protein in a sample. The CPU 102d compares the calculated CK19 protein quantitated value with a predetermined corresponding threshold.
A process flow of the CPU 102d will be explained based on
The nucleic acid quantitation part 100 and the protein quantitation part 101 maybe separate devices (i.e. nucleic acid quantitation device and protein quantitation device).
In addition, since the nucleic acid quantitation part 100 and the protein quantitation part 101 use the same sample, a conveying device for conveying this sample to each quantitation part may be provided.
A result of a method for detecting a cancer which actually uses the kit for detecting a cancer cell of the present invention will be specifically explained below by way of Example.
Using 31 lymph nodes excised from a breast cancer patient, samples for measurement were prepared. Among them, by microscopy of a hematoxylin-eosin stained tissue section, metastasis of a cancer cell was recognized in 22 lymph nodes, and metastasis of a cancer cell was not recognized in 9 lymph nodes.
Using 22 lymph nodes for which metastasis of a cancer cell was histologically recognized (positive lymph node), and 9 lymph nodes for which metastasis of a cancer cell was not histologically recognized (negative lymph node), a sample for measurement was prepared as follows.
First, 200 μL of a buffer (containing 200 mM glycine-HCl 5% Brij35(polyoxyethylene(35)lauryl ether, manufactured by SIGMA CORPORATION) and 20% DMSO (Wako Pure Chemical Industries, Ltd.)) of a pH of 3.4 was added to each lymph node (about 50 to 600 mg/node), and this was homogenized with a blender. The resulting homogenate was centrifuged at 10,000×g and room temperature for 1 minute, and 50 to 200 μL of a supernatant was collected, which was used as a sample for detection.
Samples for detection from positive lymph nodes and negative lymph nodes obtained as described above were set on a nucleic acid amplification apparatus (GD-100, manufactured by Sysmex Corporation), and a CK19cDNA was amplified by an RT-LAMP reaction (as a reagent for measurement, a reagent for nucleic acid amplification, a cytokeratin reagent (manufactured by Sysmex Corporation) was set in GD-100). By measuring a turbidity of the reaction solution in real time, an mRNA of CK19 (copy number) was quantitated.
To 20 μL of the sample for detection used in quantitation of the CK19 mRNA was added 10 μL of a 3×SDS treatment buffer (containing 150 mM Tris HCl (pH 6.8), 300 mM dithiothreitol, 6% sodium dodecylsulfate (SDS), 0.3% bromophenol blue, and 30% glycerol), and the mixture was warmed at 95° C. for 5 minutes to prepare a sample for electrophoresis. Using 15 μL of this sample for electrophoresis, SDS-PAGE was performed. A gel after electrophoresis was transferred to a PVDF membrane, this was immersed in a blocking buffer (containing 20 mM Tris (pH 7.6), 137 mM NaCl, 0.1% Tween20, and 5% skim milk), and shaken at room temperature for 1 hour, thereby, the membrane was subjected to blocking treatment. After the blocking treatment, the membrane was washed with TBS-T (20 mM Tris (pH 7.6), 137 mM NaCl, and 0.1% Tween20) for 2 minutes. Then, the membrane was immersed in a primary antibody solution (solution obtained by 1/500 diluting a primary antibody: Cytokeratin 19 (A53-B/A2):sc-6278 (manufactured by Santa Cruz Biotechnology, Inc., Lot:#L2403) with TBS-T), and allowed to stand at 4° C. overnight to perform an antibody reaction. After the reaction, this membrane was washed with TBS-T for 5 minutes four times, and the membrane was immersed in a solution of a secondary antibody which can bound to the primary antibody and is bound to horseradish peroxidase as a labeling enzyme (solution obtained by 1/2000 diluting a secondary antibody: ECL anti-mouse IgG HRP linked F(ab′2) fragment (manufactured by GE Health Care Biosciences) with TBS-T), and allowed to stand at room temperature for 30 minutes, to perform an antibody reaction. After the reaction, the membrane was washed with TBS-T for 5 minutes four times, and an enzyme reaction was performed with ECL-Advance Western Blotting Detection Kit (manufactured by GE Health Care Biosciences). A membrane image after the enzyme reaction was taken with LumiAnalyst (manufactured by Roche Diagnostics), and a fluorescent intensity of the membrane image was calculated with Quantityone (manufactured by Bio-Rad Laboratories). The fluorescent intensity measured as described above was fitted with a calibration curve to calculate an amount of the CK19 protein. The calibration curve was produced by accommodating a sample containing the CK19 protein having a known concentration in a well other than a well accommodating a sample for measurement with the same gel, and performing the aforementioned fluorescent intensity measurement.
The measured mRNA quantitated value (copy/μL) and protein quantitated value (ng/μL) were developed on a logarithmic axis to produce a graph. This graph is shown in
In addition, a first threshold corresponding to an mRNA quantitated value can be 5000 copy/μL, and a second threshold corresponding to a protein quantitated value can be 0.5 ng/μL. By comparing the mRNA quantitated value and the protein quantitated value with these thresholds, the presence or absence of cancer metastasis may be determined.
Using 63 lymph nodes excised from a colon cancer patient, samples for measurement were prepared. Among them, by microscopy of a hematoxylin-eosin stained tissue section, metastasis of a cancer cell was recognized in 35 lymph nodes, and metastasis of a cancer cell was not recognized in 28 lymph nodes.
Using 35 lymph nodes for which metastasis of a cancer cell was histologically recognized (positive lymph node), and 28 lymph nodes for which metastasis of a cancer cell was not histologically recognized (negative lymph node), a sample for measurement was prepared as in Example 1.
Using the above samples for detection, the CK19 mRNA and CK19 protein were quantitated as in Example 1.
The measured mRNA quantitated value (copy/μL) and protein quantitated value (ng/μL) were developed on a logarithmic axis to produce a graph. This graph is shown in
Using 30 lymph nodes excised from a stomach cancer patient, samples for measurement were prepared. Among them, by microscopy of a hematoxylin-eosin stained tissue section, metastasis of a cancer cell was recognized in 24 lymph nodes, and metastasis of a cancer cell was not recognized in 6 lymph nodes.
Using 24 lymph nodes for which metastasis of a cancer cell was histologically recognized (positive lymph node), and 6 lymph nodes for which metastasis of a cancer cell was not histologically recognized (negative lymph node), a sample for measurement was prepared as in Example 1.
Using the above samples for detection, the CK19 mRNA and CK19 protein were quantitated as in Example 1.
The measured mRNA quantitated value (copy/μL) and protein quantitated value (ng/μL) were developed on a logarithmic axis to produce a graph. This graph is shown in
Since the present Example is a detection method based on the mRNA quantitation result and the protein quantitation result, a detection result which has better precision and higher reliability than those of a detection result by a single method is obtained.
The foregoing detailed description and example have been provided by way of explanation and illustration, and are not intended to limit the scope of the appended claims. Many variations in the presently preferred embodiments will be obvious to one of ordinary skill in the art, and remain within the scope of the appended claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2006-168131 | Jun 2006 | JP | national |
