The present invention relates to a masking member, a light measuring method, a light measuring kit and a light measuring container.
Conventionally, measurements of fluorescence and light emission have been used for various purposes including screening or the like of the chemical library in drug discovery. For such a measurement, firstly, a measurement object such as incubated cell or the like is disposed on the bottom of a transparent container such as petri dish or Bayer bottle or the like. In a case of fluorescence measurement as an example, a buffer (buffer solution, external solution) including a dye is filled additionally. If nothing is done, fluorescence of fluorescent dye not absorbed by cells or the like acts as a background light, and it becomes difficult to identify and measure fluorescence from a measurement object, and therefore, washing (buffer replacement, washout) is attempted repeatedly by sucking and removing a buffer containing such excess fluorescent dye and by adding another buffer free from fluorescent dye. Following this, a test compound to be subjected to screening is supplied and a screening takes place while irradiation of exciting light is made from the bottom composed of a transparent member of the container to a measurement object and measurement of fluorescence originated from the measurement object is carried out.
Note that, in the above-mentioned fluorescence measurement, there exist test compounds to be subjected to screening that emit fluorescence by themselves (self-fluorescent compound). Some predict that self-fluorescent compounds may reach as much as 70 to 80% of these compounds. Here, when a test is carried out by supplying self-fluorescent compound for the measurement object, it is difficult to determine whether fluorescence level measured is attributable to a measurement object incorporating fluorescent dye of cells or the like or to self-fluorescent compounds in the buffer (artifact). As, a result, there arises a problem that pseudopositive grows significantly. In this case, after a test compound having self-fluorescence is supplied for the measurement object, an additional step of washing with buffer free from such compound is necessary, while such washing is time-consuming, and especially in the high-throughput screening of compounds, the washing causes reduction in detection accuracy and also in throughput. Further, addition of washing step may not only hinder real-time measurement of effects of a test compound on the measurement object, but also invite detachment of the measurement object from the bottom of the container.
In order to overcome the drawbacks relating to background fluorescence (nonspecific fluorescence) due to excessive fluorescent materials in the buffer, for example, as a first fluorescence measurement method, such a method has been used practically that black pigment or dyes of various types are mixed into the buffer to reduce effects resulting from excessive fluorescent dyes without involving washing step (see, for example, FIG. 2 of Patent Document 1). As a second fluorescence measurement method, such a method has been proposed that buffers to which are added polymer latex beads, inorganic particles or the like are laminated on a measurement object to form an optical separation layer between the measurement object and the buffer, and background fluorescence resulting from the buffer above this separation layer is shielded (see, for example, FIG. 6 of Patent Document 1).
Patent Document 1: Japanese Patent No. 3452068
The present inventors made an intensive study of the conventional light measuring method for measuring fluorescence or light emission and found the following problems. Namely, in the first fluorescence measuring method by which black pigment or the like are mixed into the buffer, black pigment or the like themselves influence functions of cells, which are the measurement object, and in some cases, various reactions are hindered and therefore the first method is not able to cope with entire measurement system. Further in some cases, effects of a test compound on the measurement object can not be measured accurately due to adsorption of the test compound to pigments. Moreover, in a case of optical quenching in which pigment is involved, absorption band is different depending on wavelength, and therefore, optimum pigment should be selected depending on types of fluorescent dye and measuring system.
On the other hand, in the second fluorescence measuring method, in which a buffer to which are added polymer latex beads or the like is laminated on the measurement object to form an optical separation layer between the measurement object and the buffer, particles of polymer latex beads or the like make direct contact with cells, which are the measurement object, and it is probable that cells are badly affected (effects due to physical contact). Moreover, reagent or a test compound which needs to be subjected to screening is further dispensed, after a separation layer is formed by polymer latex beads or the like, craters will be caused to the separation layer due to delivery pressure of a pipette or the like, and thickness of the separation layer becomes uneven. When this is the case, thinner portion and thicker portion are caused to the separation layer and as a result, there is a possibility that high-resolution detection of fluorescence attributable to cells or the like becomes impossible.
The present invention has been developed to overcome above-mentioned problems and the object of the present invention is to provide masking member for light measurement, light measuring method, light measuring kit and light measuring container by which effects attributable to fluorescence or light emission resulting from fluorescent dye in the buffer and the test compound are removed surely without giving any bad influence on the measurement of fluorescence or light emission resulting from the measurement object, and by which a need for washing of fluorescent dye in the buffer and test compound or the like is eliminated.
The masking member according to the present invention is a measuring member used for measuring fluorescence or light emission, originated from a measurement object placed in the liquid in the container, through the bottom of the container, and comprises a light shielding part and an outer frame part. The light shielding part has liquid permeability and also has light shielding effect to shield a background light traveling from the liquid, located on the opposite side of the container bottom across the measurement object, toward the bottom of the container. The outer frame part positions the light shielding part, while supporting the same, on the opposite side of the container bottom across the measurement object.
In accordance with the above-mentioned composition, in the case of fluorescence measurement, the masking member shields an exciting light irradiated through the container bottom, with respect to liquid containing fluorescent dye and test compound or the like located at higher portion than the measurement object in the liquids accommodated in the container, and therefore, materials above the measurement object are not excited. Thus, generation of background light is suppressed, thereby improving accuracy of detection of fluorescence specific to the measurement object. Even when excessive fluorescent dye or test compound located at higher portion than the measurement object is excited by a part of the exciting light passing through the masking member, background light from the liquid containing them to the container bottom can be shielded, and thereby separation of fluorescence originated from the measurement object from background light is possible. Further, at light emission measurement, the masking member shields background light from the liquid containing light emission reagent or the like located at higher portion than the measurement object to the container bottom, and thereby separation of light emission originated from the measurement object from background light becomes possible. In the meantime, the masking member can allow transmission of liquids and allow fluorescent dye in the liquid, test compound and light emitting reagent or the like to react with the measurement object. In other words, the light shielding part having liquid permeability is disposed at higher portion than the measurement object due to the function of the outer frame part while being immersed in the liquid in the container.
In this configuration, the masking member can shield surely fluorescence of fluorescent dye and the test compound other than the measurement object while the masking member itself does not contact with the measurement object such as cells or the like, and therefore, high-sensitivity fluorescence measurement becomes possible excluding effects of fluorescence originated from fluorescent dye in the buffer and self-fluorescence of the test compound without giving any bad influence to the measurement object such as cells. In this case, it is possible to cope with any measurement regardless of types of the fluorescent dyes (fluorescence wavelength) and measuring system. In a case that reagent is dispensed after the light shielding part in the masking member is inserted in the container, since the light shielding part itself has liquid permeability, the reagent is transmitted through the light shielding part and reaches the measurement object. Therefore, light shielding effect of the light shielding member itself is maintained uniform, which enables high-resolution detection of fluorescence originated from the measurement object. Further, in light emission measurement, same as the fluorescence measurement, since the masking member itself does not make contact with the measurement object and light emission originated from surplus light emission regent or the like, which is not concerned with the measurement object, can be surely shielded, high-sensitivity light emission measurement excluding effects of light emission from light emission reagent in the buffer becomes possible without giving bad influences to the measurement object. In this case, it is possible to cope with any measurement regardless of types of the light emission reagents and measuring system.
Further, since it is possible to shield fluorescence or light emission from excessive fluorescent dye, test compound or light emission reagent in the liquid by merely inserting a light shielding part of the masking member in the container where the measurement object is disposed, before or after the liquid containing fluorescent dye, test compound, light emission reagent or the like is added, there is no need for washing of the fluorescent dye, test compound or light emission reagent contained in the liquid, thereby enabling to improve throughput in the fluorescence or light emission measurement.
The masking member according to the present invention can be applied to a microplate that has a plurality of wells, each of which accommodates a liquid containing the measurement object. In this case, the masking member comprises a plurality of light shielding parts prepared corresponding to each of the wells and a supporting structure for supporting these light shielding parts in a predetermined position in each of corresponding wells. Each of light shielding parts has liquid permeability and also has light shielding effect for shielding a background light from the liquid, located on the opposite side of the corresponding well bottom across the measurement object accommodated in the corresponding well, to the bottom of the well. The supporting structure comprises a sheet-shaped part covering an upper plane of the microplate and a plurality of outer frame parts each prepared corresponding to one of the wells. Note that each of outer frame parts in the supporting structure positions the light shielding part in the state supporting the same on the opposite side of the corresponding well bottom across the measurement object accommodated in the corresponding well.
In above-mentioned composition, even a case where the objects to be measured are put into a plurality of wells on the microplate and a number of fluorescence or light emission measurements are carried out at one time, light shielding of each of wells can be performed collectively, thereby enabling to further improve throughput of fluorescence or light emission measurements.
The light measuring method according to the present invention comprises: a first step of inserting a measurement object in a container; a second step of adding a liquid containing fluorescent dye, test compound or light emission reagent to the container; a third step of shielding a background light from the liquid, located on the opposite side of the container bottom across the measurement object, toward the bottom of the container by disposing a masking member having above-mentioned structure oppositely to the container; and a fourth step of measuring from the bottom of the container fluorescence or light emission originated from the measurement object. In this case, the third step prepares a masking member that comprises a light shielding part having light shielding effect and liquid permeability, and disposes the masking member so as to sandwich the measurement object between the light shielding part and the bottom of the container. On the other hand, the light measuring method according to the present invention may comprise; a first step of inserting a measurement object in a container; a second step of disposing the masking member having above-mentioned structure with regard to the container; a third step of adding a liquid containing fluorescent dye, test compound or light emission reagent in the container; and a fourth step of measuring from the bottom of the container fluorescence or light emission originated from the measurement object. In this case, in the second step, when the masking member is disposed with regard to the container, a light shielding part is positioned in a predetermined place in the container. Further, measurement of fluorescence or light emission in the fourth step is carried out while a background light from the liquid, located on the opposite side of the container bottom across the measurement object, toward the bottom of the container is being shielded by the masking member.
In above-mentioned composition, for fluorescence measurement, the masking member shields an exciting light irradiated via the container bottom, for liquid containing fluorescent dye and test compound or the like located in the higher portion than the measurement object out of the whole liquid accommodated in the container, and therefore materials above the measurement object are not excited. Thus, generation of background light can be suppressed, thereby improving accuracy of detection of fluorescence specific to the measurement object. Even when a part of exciting light is passed through the masking member and excessive fluorescent dye or test compound located higher than the measurement object is excited, a background light from the liquid located higher than the measurement object toward the bottom of the container is shielded by the masking member, and separation between fluorescence originated from the measurement object and the background light becomes possible. Further, for light emission measurement, the masking member shields a background light from the liquid containing light emission reagent or the like located higher than the measurement object to the bottom of the container, separation between fluorescence originated from the measurement object and the background light becomes possible. Besides, since constituents of fluorescent dye or the like in the liquid can transmit the light shielding part in the masking member, reaction with the measurement object is possible.
In this composition, high-sensitivity fluorescence measurements becomes possible excluding effects of fluorescence originated from fluorescent dye and self-fluorescence of the test compound in the liquid in the container. Further, for light emission measurement, same as the fluorescence measurement, high-sensitivity light emission measurement excluding effects of light emission from light emission reagent or the like in the liquid becomes possible. Further, it is possible to cope with various measurements regardless of types of fluorescent dye, test compounds or light emission reagents. Furthermore, even when reagent is dispensed after the masking member is disposed, uniformity of light shielding is maintained. In addition, since it is possible to shield fluorescence originated from excessive fluorescent dye in the liquid, self-fluorescence of test compound or light emission originated from light emission reagent, by merely inserting a light shielding part of the masking member in the container before or after fluorescent dye, test compound or light emission reagent are added, there is no need for washing of the fluorescent dye, test compound or light emission reagent contained in the liquid accommodated in the container, thereby improving throughput in the fluorescence measurement or light emission measurement.
The measuring kit according to the present invention comprises, for the sake of measurement of fluorescence or light emission originated from a measurement object disposed in a liquid in a container from the bottom of the container, a container for accommodating the measurement object together with the liquid, and one or more masking members each having above-mentioned structure that comprises a light shielding part and an outer frame part. Further, the light measuring kit according to the present invention may be equipped with, as a container, a microplate having one or more wells, each of which is to accommodate a measurement object, and in this case, the light measuring kit has one or more masking members each having a light shielding part and an outer frame part as mentioned above. Further, in the light measuring kit according to the present invention, as a container, when there is provided a microplate having a plurality of wells, each of which is to accommodate a measurement object, a masking member applied to the microplate may have such a structure having a plurality of light shielding parts and supporting structure supporting these light shielding parts. In this case, supporting structure in the masking member is composed of a sheet-shaped part covering upper plane of the microplate and a plurality of outer frame parts prepared corresponding to each of wells, and sheet-shaped part and these outer frame parts function as a positioning means for disposing each light shielding part to a predetermined location in corresponding well of the microplate.
In accordance with the above-mentioned composition, in the container or each of wells of the microplate prepared as the container, in case of fluorescence measurement, the masking member shields an exciting light irradiated via the bottom of the container for the liquid containing fluorescent dye or test compounds or the like located higher than the measurement object out of the whole liquids accommodated in the container, and therefore, materials above the measurement object are not excited. Thus, generation of background light is suppressed, thereby improving accuracy of detection of fluorescence specific to the measurement object. Even when a part of exciting light is passed through the masking member, and excessive fluorescent dye or test compound located higher than the measurement object is excited, a background light from the liquid containing fluorescent dye or the like located higher than the measurement object to the bottom of the container is shielded by the masking member, and separation between fluorescence originated from the measurement object and the background light becomes possible. Further, in case of light emission measurement, the masking member shields a background light from the liquid containing light emission reagent or the like located higher than the measurement object to the bottom of the container, separation between light emission originated from the measurement object and the background light becomes possible. In the meantime, the light shielding part in the masking member can allow transmission of liquids, and enables fluorescent dye and various compounds or the like in the liquid to react with the measurement object.
In this configuration, by simply using the light measuring kit, it becomes possible to perform high-sensitivity fluorescence measurements and high-sensitivity light emission measurements in a simple manner without giving influences to the measurement object. Various kinds of fluorescent dyes and measuring apparatuses or the like can be selected to be applied, and high-accuracy fluorescence measurements or high-sensitivity light emission measurements are made possible even at the time of dispensing of the reagent. Besides, there is no need for washing of fluorescent dyes and various reagents of the liquid accommodated in the container, thereby resulting in high-throughput fluorescence measurement or light emission measurement.
Note that, in the light measuring kit, when the masking member applied to the microplate comprises masking member having a plurality of light shielding parts and supporting structure supporting the same, various kinds of objects to be measured can be accommodated in a plurality of wells of the microplate, and by inserting each of corresponding light shielding parts of the masking member in a part of or the whole of well inside, background lights from inside the well can be shielded collectively, thereby ensuring various fluorescence measurements or light emission measurements efficiently.
In addition, the light measuring container according to the present invention is used to perform measurement of fluorescence or light emission of a measurement object through the bottom of the container, while the measurement object is accommodated inside together with the liquid. This light measuring container is used together with the masking member having light shielding effect and liquid permeability for shielding background light from the liquid located at higher portion of the measurement object toward the bottom. In particular, the light measuring container is provided with a positioning means for positioning a masking member to the inner wall thereof.
In accordance with this composition, the masking member is disposed by the positioning means at the predetermined position above the measurement object. In case of fluorescence measurement, the masking member thus disposed shields an exciting light irradiated via the bottom of the container, for the liquid containing fluorescent dye, test compound or the like located at higher than the measurement object, out of the whole liquid accommodated in the container, and therefore, materials above the measurement object are not excited. That is, generation of background light is suppressed, thereby improving detection accuracy of fluorescence specific to the measurement object. Even when a part of exciting light passes through the masking member and excessive fluorescent dye or test compound located higher than the measurement object is excited, a background light from the liquid containing fluorescent dye or the like located higher than the measurement object to the bottom of the container is shielded by the masking member, and separation between fluorescence originated from the measurement object and the background light becomes possible. Further, in case of light emission measurement, the masking member is able to shield a background light from the liquid containing light emission reagent or the like located higher than the measurement object to the bottom of the container, separation between light emission originated from the measurement object and background light becomes possible, and high-sensitivity light emission measurement becomes possible without influencing the measurement object. Various kinds of fluorescent dyes and measuring apparatuses or the like can be selected to be applied, and high-accuracy fluorescence measurements or high-sensitivity light emission measurements are possible even at the time of dispensing of the reagent. Besides, there is no need for washing of fluorescent dyes or the like contained in the liquid accommodated in the container, thereby resulting in high-throughput fluorescence measurement or light emission measurement. Besides, even when no means is provided to the masking member itself for positioning in the container, it is possible to dispose the light shielding part to a position where sufficient light shielding is achieved without making contact with the measurement object.
The light measuring container according to the present invention may be a microplate equipped with a plurality of wells, each of which accommodates liquid containing the measurement object. Also in this case, a means for positioning the masking member is provided to the inner wall of each well.
In the light measuring container of microplate type as mentioned above, a plurality of fluorescence measurements or light emission measurements become possible at one time while various kinds of objects to be measured are placed in a plurality of wells. Since a means for positioning the masking member is provided at the inner wall of each well, each corresponding masking member is inserted and shielding of a background light from the liquid accommodated in each of wells becomes possible. With these configurations, high-sensitivity fluorescence measurement or high-sensitivity light emission measurement is possible excluding effects of fluorescence from excessive fluorescent dye in the liquid, self-fluorescence of test compound or light emission from light emission reagent.
The present invention will be more fully understood from the detailed description given hereinbelow and the accompanying drawings, which are given by way of illustration only and are not to be considered as limiting the present invention.
Further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art from this detailed description.
In the masking member, light measuring method, light measuring kit and light measuring container according to the present invention, high-sensitivity fluorescence measurement or high-sensitivity light emission measurement becomes possible, excluding influences of fluorescence from excessive fluorescent dye in the buffer being accommodated in the container together with the measurement object, self-fluorescence of test compound or light emission from light emission reagent, without giving bad effects to the measurement object such as cells. Besides, it is possible to cope with any measurement regardless of types of fluorescent dyes (fluorescence wavelength) and measuring system. In addition, even a case where reagent is dispensed after a masking member is mounted to the container, light shielding effect in the masking member is maintained uniform ensuring high-resolution detection of fluorescence or light emission originated from measurement object. Further, washing step after various reagents are added is unnecessary, thereby improving throughput in fluorescence or light emission measurements.
1
a, 1b, 1c . . . light shielding part; 2, 2′ . . . mesh; 3, 3′ . . . mesh hole; 4 . . . diagonal hole; 10, 20 . . . outer frame part; 11 . . . rim-shaped part; 21 . . . sheet-shaped part; 100, 100′, 200 . . . masking member; 400 . . . container; 500 . . . microplate; 550, 750 . . . well; 600, 700 . . . light measuring container; 630, 730 . . . positioning means; 800, 810, 820 . . . light measuring kit; B . . . liquid (buffer) containing fluorescent dye, test compound or light emission reagent; S . . . measurement object; SS . . . sample solution; l . . . background light from buffer; L . . . fluorescence from measurement object.
In the following, each of embodiments of a masking member, a light measuring method, a light measuring kit and a light measuring apparatus according to the present invention will be explained in detail using
(First Example of Masking Member)
As shown in
As shown in
In the first specific example shown in
In the second specific example shown in
In the third specific example shown in
The light shielding part having the structure mentioned above has preferably hydrophobic nature and nonabsorbent nature same as the outer frame part. Specifically, as for materials, nylon, cellulose, cotton, wool or the like having higher hydrophilic nature are used for applications where a drug is dispensed after insertion of the masking member, or resin, glass, silicon member or the like subjected to hydrophilic processing by plasma processing, chemical processing, photocatalytic reaction or the like, are preferably used. In a case nonspecific adsorption of proteins or the like should be avoided, polyester, polystyrene, polypropylene, polyethylene, polycarbonate or the like are preferably used as the hydrophobic raw materials.
Next, referring to
In this state, an exciting light is irradiated from the transparent bottom of the container 400 and fluorescence L from the object S to be measured is measured from the bottom of the container 400. In this case, the exciting light irradiated from the bottom of the container 400 is shielded by the light shielding part 1 and does not reach the buffer located higher than the light shielding part 1, and is radiated selectively to the object S to be measured located lower than the light shielding part 1. Meanwhile, even when a part of exciting light is passed through the light shielding part 1, and excessive fluorescent dye or test compound existing in the buffer B is excited, a background light 1 (nonspecific fluorescence) from the buffer B due to fluorescence originated therefrom is shielded by the light shielding part 1. Accordingly, in the fluorescence measurement from the bottom of the container 400, a specific fluorescence L from the measurement object can be measured without being disturbed by the background light 1 from the buffer, and high-accuracy fluorescence measurement with less pseudopositive becomes possible.
In addition, in this case, since fluorescence from excessive fluorescent dye and test compound not absorbed by the object S to be measured in the buffer B is shielded by the light shielding part 1, replacement step (washing step) of the buffer required by the conventional method is unnecessary. As a result, with the washing step as mentioned, labor hour for washing is saved and a problem that a measurement object is detached from container bottom is not caused, thereby improving throughput of fluorescence measurement. Besides, dispensing of reagent or the like is possible even when the masking member 100 is provided, while the reagent dispensed reaches object S to be measured via light shielding part 1 having liquid permeability. On this occasion, the reagent and object S to be measured cause a predetermined reaction and fluorescence observation on the result can be carried out in real time.
Although the object S to be measured shown in the example of use shown in
In addition, although according to this example of use, the object S to be measured is caused to take in fluorescent dye to allow fluorescence labeling, if object S to be measured expresses fluorescent protein, for example, green fluorescent protein (GFP), cells may not necessarily be labeled by fluorescent dye. Even in this case, a background light originated from test compound having self-fluorescence can be shielded, and therefore, high-accuracy fluorescence measurement with less pseudo-positive becomes possible. Further, washing step is also unnecessary, thereby improving throughput of fluorescence measurement.
Although fluorescence labeled object S to be measured is used in this example of use, light emission measurement may be carried out using cells expressing gene of luminescent enzymes such as luciferases as a measurement object. In the light emission measurement, irradiation of an exciting light exciting fluorescent dye is unnecessary. In light emission measurement, since masking member can shield a background light from a liquid containing light emission reagent or the like located higher than the measurement object to the bottom of the container, separation between light emission originated from the measurement object and background light becomes possible, and high-accuracy and high-throughput light emission measurement becomes possible without giving influences to the measurement object.
(Second Embodiment of Masking Member)
The masking member relating to the second example differs from the masking member (
As shown in
As shown in the areas (b) and (c) of
In usage state of the masking member according to the second embodiment, as shown in the area (d) of
In the masking member 200 according to the second embodiment, same as above-mentioned first embodiment, fluorescent L from the object S to be measured in each well is fluorescence measured from transparent bottom of the well 550. In this case, an exciting light being irradiated from the bottom of the well 550 is shielded by the light shielding part 1 and therefore does not reach a buffer located higher than the light shielding part 1, and is radiated selectively to the object S to be measured located lower than the light shielding part 1. Meanwhile, even when a part of exciting light is passed through the light shielding part 1, and excessive fluorescent dye or test compound existing in the buffer B is excited, a background light 1 from buffer B located higher than the light shielding part 1 is shielded by the light shielding part 1. Accordingly, in the fluorescence measurement from the bottom of each of the wells 550, high-accuracy fluorescence measurement becomes possible without being disturbed by the background light 1 from the buffer.
Even in this second embodiment, since fluorescence from excessive fluorescent dye in the buffer B is shielded by the light shielding part 1, replacement step of the buffer is unnecessary, thereby improving throughput of fluorescence measurement. Even a case where a desired reagent or the like is dispensed respectively to each well 550 while the masking member 200 is being provided, dispensed reagent reaches the object S to be measured via the light shielding part 1 having liquid permeability. Then, the reagent and object S to be measured cause a predetermined reaction and the result can be fluorescence observed from the bottom of each well 550. This allows various fluorescence measurements of all wells simultaneously.
Also in this embodiment, same as the first embodiment, when fluorescence measurement is performed using cells which express fluorescent protein, for example, green fluorescent protein (GFP) as the measurement object, a step of labeling cells with fluorescent dye may be eliminated. Further, same as the first embodiment, when light emission measurement is performed using cells which express gene of luminescent enzymes such as luciferases as the measurement object, similar effects will be developed.
(First Embodiment of Light Measuring Method)
Next, the first embodiment of the light measuring method according to the present invention will be explained in detail taking a case where the masking member 200 according to the above-mentioned second embodiment is used as an example.
Thus, since fluorescence from excessive fluorescent dyes in the buffer B is shielded by the masking member 200, there is no need for time-consuming washing step of removing excessive fluorescent dyes in the buffer. Besides, such a problem that objects to be measured such as cells or the like are detached from the bottom of the well 550 due to washing does not occur. Therefore, high-throughput fluorescence measurement becomes possible.
Following this, as shown in the area (d) of
In addition, although in this embodiment, fluorescent dye is taken into object S to be measured for fluorescence labeling, labeling of cells by the fluorescent dye is unnecessary for the case where the object S to be measured expresses fluorescent protein, for example, green fluorescent protein (GFP). It is possible also in this case to shield a background light originated from a test compound having self-fluorescence, and therefore, high-accuracy fluorescence measurement with less pseudopositive becomes possible. Further, washing step becomes unnecessary, thereby improving fluorescence measurement throughput.
Further, although in this embodiment, fluorescence labeled object S to be measured is used, in the case where cells which express gene of luminescent enzymes such as luciferases are used as the measurement object, labeling of cells by the fluorescent dye is unnecessary. It is possible also in this case to shield a background light originated from a test compound having self-fluorescence, and therefore, high-accuracy and high-throughput light emission measurement becomes possible.
(Second Embodiment of Light Measuring Method)
As mentioned above, even with a method for inserting a masking member before supplying fluorescent dye to the measurement object (light measuring method according to the second embodiment), high-accuracy and high-throughput fluorescence measurement becomes possible by shielding a background light in the buffer, without performing a washing step of removing excessive fluorescent dye in the buffer.
Note that, also in this embodiment, same as the first embodiment, when fluorescence measurement is performed using cells which express fluorescent protein, for example, green fluorescent protein (GFP) as the measurement object, a step of labeling cells with fluorescent dye may be eliminated. Further, same as the first embodiment, when light emission measurement is performed using cells which express gene of luminescent enzymes such as luciferases as the measurement object, similar effects will be developed.
(First to Third Embodiment of Light Measuring Kit)
As shown in the area (a) of
As shown in the area (b) of
As shown in the area (c) of
Note that, although in the above-mentioned first to third embodiments, embodiments in which ligands are fluorescence labeled are exemplified, the present invention is not limited thereto, and a fluorescence labeled second antibody that binds, for example, specifically to a ligand may be used. In this case, after a sample containing substances produced by antibodies and cells is added to in the well 550 where antibody S is fixed, the fluorescence labeled second antibody is added without involving a washing process. As it will be understood from the above, with light measuring kit (fluorescence measuring kit) according to the present invention, it is possible to remove, in various fluorescence immunoassay, background fluorescence from other than the sample adsorption plane thereby enabling improvement of detection sensitivity.
Further, although in the above-mentioned first to third embodiments, embodiments in which ligands are fluorescence labeled are exemplified, labeling may be made by luminescent enzymes such as peroxidase, alkali phosphatase or the like in lieu of fluorescence labeling. In this case, a substrate of luminescent enzymes (light emission reagents) such as luminol, hydrogen peroxide, luciferases or the like are added without involving a washing process. In this way, with light measuring kit (particularly fluorescence measuring kit) according to the present invention, it is possible to remove background light emission from other than the sample adsorption plane in various chemical and biological light emission immunoassay, thereby further enabling improvement of detection sensitivity.
Next, usage of the light measuring kit according to the present invention will be explained specifically taking a case of fluorescence measurement where fluorescence measuring kit is used as the light measuring kit according to above-mentioned third embodiment.
Note that the light measuring kit according to the present invention allows various fluorescence measurements, while, without attaching/detaching a masking member as illustrated in
Note that although the above example of use exemplifies the case of fluorescence labeling of a second antibody, the present invention is not limited thereto, and for example, a ligand may be fluorescence labeled. When this is the case, a process for adding a second antibody becomes unnecessary. In accordance with fluorescence measurement using a fluorescence measuring kit as the light measuring kit according to the present invention, in various fluorescence immunoassay, it is possible to remove background fluorescence from other than sample adsorption plane, thereby further enabling improvement of detection sensitivity.
In addition, although the above example of use exemplifies the case of fluorescence labeling of a second antibody, labeling may be made by luminescent enzymes such as peroxidase, alkali phosphatase or the like in lieu of fluorescence labeling. In this case, a substrate of luminescent enzymes (light emission reagents) such as luminol, hydrogen peroxide, luciferases or the like are added without involving a washing process. As such, with light emission measurement using a light emission measuring kit as the light measuring kit according to the present invention, in various chemical and biological light emission immunoassay, it is possible to remove background light emission from other than sample adsorption plane, thereby further enabling improvement of detection sensitivity.
(First Embodiment of Light Measuring Container)
The light measuring container according to the first embodiment is used while a masking member having light shielding effect and liquid permeability for shielding lights from other than objects to be measured existing in the container is being inserted in the container. As shown in the areas (a) and (b) of
As shown in the area (c) of
Note that, although fluorescence labeled object S to be measured is used in the first embodiment, light emission measurement may be carried out using cells expressing gene of luminescent enzymes such as luciferases as the measurement object. In light emission measurement, irradiation of an exciting light exciting fluorescent dye is unnecessary. In the case of light emission measurement, a masking member is capable of shielding a background light from a liquid containing light emission reagent or the like located higher than the measurement object to the bottom of a container, separation between light emission originated from the measurement object and background light becomes possible, thereby allowing high-accuracy and high-throughput light emission measurement without giving influences to the measurement object.
The light measuring container according to the first embodiment can be utilized, in addition to fluorescence assay using cells, as a tool for various light emission assay, fluorescence immunoassay, and chemical and biological light emission assay.
(Second Embodiment of Light Measuring Container)
As shown in the area (a) of
As shown in the area (d) of
Note that, in this embodiment, although fluorescence labeled object S to be measured is used, light emission measurement may be carried out using cells expressing gene of luminescent enzymes such as luciferases as the measurement object. In light emission measurement, irradiation of an exciting light exciting fluorescent dye is unnecessary. At light emission measurement, since a masking member is capable of shielding a background light from a liquid containing light emission reagent located higher than the measurement object to the bottom of a container, separation between light emission originated from the measurement object and background light becomes possible, thereby allowing high-accuracy and high-throughput light emission measurement without giving influences to the measurement object.
The light measuring container according to the second embodiment can be utilized, in addition to fluorescence assay using cells, as a tool for various light emission assay, fluorescence immunoassay, and chemical and biological light emission assay.
The inventors, in order to verify effects of the masking member according to the present invention, carried out the following fluorescence measurements using the masking member shown in
Two microplates with CHO cells adhered to the bottom of each of wells were prepared. Buffer containing fluorescent dye (Fluo 3) was supplied to CHO cells of the microplates to effect CHO cells to take-in fluorescent dye. On this occasion, removal of the buffer containing fluorescent dye was not performed and washing of the fluorescent dye was not performed. In addition, 1 μM of FITC was added as an alternative of the test compound in each of the wells of both microplates.
Next, a masking member as shown in
As a result, with microplates to which is inserted the masking member, detection of fluorescence originated from cells was possible. On the other hand, for microplates to which no masking member is inserted, 1 μM of FITC was too large fluorescence amount and hence camera range used for fluorescence measurement soon caused saturation, and detection of fluorescence originated from cells was not possible.
It is to be noted that it is evident that the masking member, the light measuring method, the light measuring kit and the light measuring container according to the present invention are not limited to above-mentioned examples, and the present invention may be modified in various ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended for inclusion within the scope of the following claims.
The masking member, the light measuring method, the light measuring kit and the light measuring container according to the present invention are applicable to fluorescence measurement performed at screening of chemical library in drug discovery.
Number | Date | Country | Kind |
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2004-100964 | Mar 2004 | JP | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/JP05/06110 | 3/30/2005 | WO | 00 | 1/2/2008 |