Patent Grant
7842454
The present invention relates to a kit for preparing a cancer cell detection sample and a kit for cancer cell detection using the same.
Cancer is a primary cause for mortality of Japanese, and it is said that if early treatment by early diagnosis is possible, the mortality can be remarkably decreased.
Currently, diagnosis of cancer is performed by discriminating a normal cell and a cancer cell by a morphological test of a cell in a test sample collected from a subject with microscopic examination generally by a cell testing technician. However, since this diagnosis method is a visual test by a cell testing technician, the method is not suitable for handling of a large amount of test samples as in group medical examinations, and there is a problem that unevenness occurs in test results depending on the status of a test sample, and the sample cannot be analyzed quantitatively.
In addition, a method for diagnosing cancer by detecting a tumor-derived DNA in plasma and serum of a subject is also being studied. Specifically, cancer is diagnosed by concentrating cells contained in blood collected from a subject, and biochemically analyzing a DNA and the like related to a blood free cancer cell in the concentrated cells. This method can relatively safely treat a large amount of test samples, but there is a problem that a step of concentrating blood free cancer cells is difficult, and simplicity is lacked.
On the other hand, in recent years, an anti-cancer agent (US2006239967) and a cancer cell detection reagent (US2006067890) using an oncolytic virus which specifically grows in a cancer cell are reported. The oncolytic virus is a virus which specifically grows in a cancer cell and, by infecting a cancer cell with the virus, the cancer cell can be directly destroyed and killed and, by incorporating a gene of a target protein into a genome thereof, it becomes possible to simply detect a cancer cell.
However, since the oncolytic virus has infectivity also on a normal cell apart from simple detection of a cancer cell, a facility at a P2 level becomes necessary for the detection. Therefore, there is a problem that it is difficult to actually use the virus in group medical examinations or the like.
The scope of the present invention is defined solely by the appended claims, and is not affected to any degree by the statements within this summary.
In view of such circumstances, the present inventors intensively studied and, as a result, found out a kit for preparing a cancer cell detection sample used in detection of a cancer cell with a reagent containing a virus, particularly, an oncolytic virus, which is not accompanied by contamination due to diffusion of the virus to the outside, which resulted in completion of the present invention.
That is, the present invention provides:
(1) A kit for preparing a detection sample for detecting a cancer cell, comprising a test container having an opening for receiving a biological sample collected from a subject, and a reagent inclusion part for accommodating a reagent containing a virus, a seal part for sealing the reagent inclusion part of the test container, a cap for closing the opening, and an opener for breaking the seal part;
(2) The kit for preparing a detection sample according to (1), wherein the cap has a virus-impermeable breathable filter;
(3) The kit for preparing a detection sample according to (1), wherein the opener breaks the seal part accompanying with an action of closing the opening by the cap;
(4) The kit for preparing a detection sample according to (1), wherein the cap has the opener;
(5) The kit for preparing a detection sample according to (1), wherein the virus in an interior portion of the test container is isolated from the outside when the opener breaks the seal part;
(6) The kit for preparing a detection sample according to (1), wherein the cap and the opener are integrally configured, the opener has a leading edge for breaking the seal part, and a middle part for connecting the cap and the leading edge, and the middle part is configured so as to be inserted into the opening in the state where it is adhered to the opening;
(7) The kit for preparing a detection sample according to (1), wherein a genome of the virus comprises a promoter of a carcinogenic gene and a gene encoding a target protein;
(8) The kit for preparing a detection sample according to (1), wherein the virus is an oncolytic virus;
(9) The kit for preparing a detection sample according to (1), wherein the virus is d12.CALP, d12.CALP delta RR, Telomelysin, Telomelysin-RGD, AxE1AdB-UPRT, AdSLP1.E1AdB, AxE1CAUP, AdE3-IAI.3B, Ad-MKAdMK, AdCEAp/Rep, AdAFPep/Rep, MMP-sub II SeV/delta M, or CD-MMP-sub II-SeV/delta M;
(10) The kit for preparing a detection sample according to (1), wherein the sample is collected from blood, sputum or uterus;
(11) A method for diagnosing cancer, comprising using the kit for preparing a detection sample as defined in (1);
(12) The diagnosis method according to (11), wherein the cancer is blood cancer, lung cancer or uterine cervical cancer;
(13) A kit for detecting a cancer cell, comprising a kit for preparing a detection sample for detecting a cancer cell comprising a test container having an opening for receiving a biological sample collected from a subject, and a reagent inclusion part for accommodating a reagent containing a virus, a seal part for sealing the reagent inclusion part of the test container, a cap for closing the opening, and an opener for breaking the seal part, and a slide glass comprising an insertion part for inserting the test container, a second opener for breaking a bottom of the test container by an action of inserting the test container into the insertion part, and a holding part for guiding a reaction solution of the biological sample and the reagent from the test container in which a bottom is broken with the second opener, and observably holding the reaction solution from the outside;
(14) The kit for cancer cell detection according to (13), wherein the cap has a virus-impermeable breathable filter;
(15) The kit for cancer cell detection according to (13), wherein the opener breaks the seal part accompanying with an action of closing the opening with the cap;
(16) The kit for cancer cell detection according to (13), wherein a virus in the interior portion is isolated from the outside when the second opener breaks the bottom of the test container;
(17) The kit for cancer cell detection according to (13), wherein a bottom of the test container has a second seal part which is broken with the second opener;
(18) A method for diagnosing cancer, comprising using the kit according to claim 13 for detection;
(19) The diagnosis method according to (18), wherein the cancer is blood cancer, lung cancer or uterine cervical cancer;
(20) A method for preparing a sample for detecting a cancer cell comprising steps of, adding a sample collected from a subject to a test container having an opening, and a reagent inclusion part for accommodating a reagent containing a virus in the state where it is sealed with a seal part, closing the opening with a cap, and breaking the seal part with an opener.
According to the present invention, since a reagent containing a virus is mixed and reacted with a sample without being diffused to the outside, and it becomes possible to observe the sample after the reaction, a kit for preparing a cancer cell detection sample which is highly safe, simple, and high in precision, and can detect a cancer cell quantitatively, and a kit for cancer cell detection using the same, as well as a method for diagnosing cancer using those kits, and a method for preparing a sample for cancer cell detection are provided.
When the kit and the method of the present invention are used, since it is not necessary to provide a large scale test facility at a P2 level, a group medical examination of cancer which is simple and high in precision becomes possible.
a) shows a top view of the slide glass for microscopic examination shown in
b) shows a cross-sectional view (b) of the slide glass for microscopic examination shown in
First, a construction of the kit for preparing a cancer cell detection sample relating to the present embodiment will be explained.
Then, a motion when a cancer cell detection sample is prepared using the kit for preparing a cancer cell detection sample relating to the present embodiment will be explained. First, a user injects a sample into the test container 3. A cross-sectional view of the test container 3 into which a sample is injected is shown in
Then, the user closes the opening of the test container 3 with the cap 1.
The reaction solution prepared by the test container 3 as described above is supplied to a slide glass 11 explained later, and is tested with microscopic examination. A construction of the slide glass 11 will be explained below.
a) shows a plane view of the slide glass 11, and
At a part held by the first slide glass 17 and the second slide glass 18 of the slide glass 11, a holding part 13 for holding a reaction solution to be subjected to microscopic examination is provided. The holding part 13 of the slide glass 11 is constructed of a microscopic examination part 14 for microscopically examining a reaction solution, and a reaction sample passageway part 15 for guiding a reaction solution flown into the insertion part 12 to the microscopic examination part 14. The microscopic examination part 14 is formed as a flat space so that a supplied reaction solution can be observed by microscopic examination.
Then, a motion of use of the kit for cancer cell test relating to an embodiment of the present invention will be explained. First, a user injects a sample, and inserts the test container 3 in the state where an opening is closed with the cap 1, into the insertion part 12 of the slide glass 11. Thereby, the second aluminum sheet is broken with the second opener 16, and a reaction solution accommodated in the test container 3 is supplied to the insertion part. Thereby, as shown in an arrow of
An example of specific embodiments has been explained, but the present invention is not limited to these embodiments, and a variety of variations are possible.
The sample in the present invention is not particularly limited as far as it contains a cell derived from a living body collected from a subject, but examples include samples from blood, sputum and uterus. Particularly, samples of blood, sputum and uterus are preferable.
The virus used in the present invention is not particularly limited as far as it has the ability to infect a cell in a sample, but an oncolytic virus is exemplified, and specifically d12.CALP, d12.CALP delta RR, Telomelysin, Telomelysin-RGD, AxE1AdB-UPRT, AdSLP1.E1AdB, AxE1CAUP, AdE3-IAI.3B, Ad-MKAdMK, AdCEAp/Rep, AdAFPep/Rep, MMP-sub II SeV/delta M, or CD-MMP-sub II-SeV/delta M are preferable.
The seal part and the second seal part in the present invention are not particularly limited as far as a reagent containing a virus is not flown to the outside, and the virus is not diffused to the outside, but examples include an aluminum sheet.
The virus-impermeable breathable filter used in the present invention is not particularly limited as far as it dose not diffuse a virus in a reaction solution to the outside, and has breathability, but examples include a breathable anti-virus filter and a virus-adsorbable filter. Specifically, a charcoal filter is preferable.
Isolation in the present invention is not particularly limited as far as it is the state where a virus in not diffused to the outside, and a position with breathability rather than the complete closed state is preferable because a cell in a sample and a virus in a reagent can be reacted sufficiently.
The kit for preparing a cancer cell detection sample in the present invention is not particularly limited in its use as far as it is a test by which a cancer cell can be confirmed, and examples include a microscopic examination test and a test with a flow cytometer.
A variety of characteristics shown in the aforementioned embodiments can be combined mutually. When a plurality of characteristics are included in one embodiment, one or a plurality of characteristics among them can be appropriately extracted, and they can be adopted alone or in combination, in the kit for preparing a cancer cell detection sample, and the kit for cancer cell detection using the same, of the present invention.
The present invention will be specifically explained below using Examples, by referring to results of a method for detecting cancer which actually uses the kit for cancer cell detection of the present invention.
(Preparation of Sample)
A sputum collected from a healthy person was recovered into 1.5 ml of a cell culturing solution (Dulbecco's Modified Eagle Medium; DMEM), and pre-culturing was performed under the condition of 37° C. and 5% CO2 for 60 minutes. The pre-cultured cell culturing solution was diluted with DMEM to 1.0×106 cells/ml, and to this was added a solution of an A431 cell (purchased from ATCC) which is a human lung cancer cell strain to 1000 cells/300 μl, and this was used as a lung cancer sample.
Separately, as a control, a cell culturing solution (1.0×106 cells/ml) before addition of an A431 cell solution, and an A431 cell solution which had been diluted with DMEM to 1000 cells 300 μl were prepared.
(Preparation of Reagent)
A reagent containing OBP-301 which is an oncolytic virus encoding a green fluorescent protein (GFP) was prepared by a known method described in CANCER RESEARCH 64, 6259-6265, Sep. 1, 2004. The prepared reagent was sealed into the reagent inclusion part 6 of the aforementioned test container 3.
(Virus Infection on Sample)
Each 250 μl of the control or the cancer sample was injected into the test container 3 in which the reagent containing OBP-301 was sealed into the reagent inclusion part 6 of the aforementioned kit for preparing a cancer cell detection sample. Thereafter, the cap 1 was inserted until the jaw part 1a was abutted against an upper end of the test container 3, and culturing was performed under the condition of 37° C. and 5% CO2 for 24 hours, thereby, the sample was infected with a virus.
(Observation of Sample)
The test container 3 after virus infection was inserted into the insertion part 12 of the aforementioned slide glass 11, and a reaction solution held by the microscopic examination part 14 was observed with a fluorescent microscope, and the results are shown in Table 1 and
Sputum indicates a cell culturing solution (1.0×106 cells/ml) before addition of an A431 cell, Sputum/A431 indicates a lung cancer sample, and A431 indicates an A431 cell solution which has been diluted with DMEM to 1000 cells/300 μl.
(Preparation of Sample)
Using a sterilized swab, an oral cavity mucosa cell was recovered into 10 ml of a cell culturing solution (Dulbecco's Modified Eagle Medium; DMEM), and this was pre-cultured under the condition of 37° C. and 5% CO2 for 60 minutes. The pre-cultured cell culturing solution was diluted with DMEM to 1.0×106 cells/ml, and to this was added a Hela cell solution (purchased from ATCC) which is a human lung cancer cell strain to 1000 cells/300 μl, and this was used as a uterine cervical sample.
Separately, as a control, a cell culturing solution (1.0×106 cells/ml) before addition of a Hela cell solution, and a Hela cell solution which had been diluted with DMEM to 1000 cells/300 μl were prepared.
According to the same procedure as that of Example 1 except for the aforementioned procedure, the sample was observed. The results are shown in Table 2 and
Oral indicates a cell culturing solution (1.0×106 cells/ml) before addition of a Hela cell, Oral/Hela indicates a uterine cervical sample, and Hela indicates a Hela cell solution which has been diluted with DMEM to 1000 cells/300 μl.
As apparent from Examples, a cancer cell can be actually detected using the kit for preparing a cancer cell detection sample of the present invention, and the kit for cancer cell detection using the same.
The foregoing detailed description and examples 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 |
|---|---|---|---|
| 2006-150753 | May 2006 | JP | national |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20060067890 | Fujiwara et al. | Mar 2006 | A1 |
| 20060239967 | Fujiwara et al. | Oct 2006 | A1 |
| Number | Date | Country | |
|---|---|---|---|
| 20070287149 A1 | Dec 2007 | US |
