JP2012073187A - Adapter for multi-well plate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To suppress consumption of multi-well plates as compared with the prior arts.SOLUTION: An adapter 1 for a multi-well plate is attachable to/detachable from an upper surface of the multi-well plate having a plurality of wells for accommodating a specimen. In the adapter 1 for the multi-well plate, a pore used for optically measuring the specimen is provided at a position corresponding to at least a part of the plurality of wells in the multi-well plate, and at least the upper surface thereof is white or black.

Description

  The present invention relates to a multiwell plate for chemiluminescence or fluorescence measurement.

  In the field of clinical examination and biotechnology, there is known a method for simultaneously measuring chemiluminescence or fluorescence emission in a plurality of samples using a multiwell plate capable of accommodating a large number of samples.

  In chemiluminescence measurement and fluorescence measurement, a multiwell plate colored in black or white is usually used. As described in Patent Document 1, in chemiluminescence measurement, since white has higher emission intensity and higher measurement sensitivity than black, a white plate is usually used. On the other hand, in fluorescence emission measurement, if a white plate is used for measurement, background fluorescence due to autofluorescence is generated and the measurement accuracy is lowered. Therefore, a black plate is usually used. As described above, the plates are properly used according to the conventional measurement.

Japanese Patent Laid-Open No. 3-40818

  However, using different plates for each measurement increases the consumption of the plates and is not preferable from the viewpoint of cost. In addition, since the multiwell plate is disposable, it is necessary to discard a large number of multiwell plates. Furthermore, depending on the measurement, both chemiluminescence measurement and fluorescence measurement may be required. At this time, in order to perform an appropriate measurement, a black plate is used for chemiluminescence measurement, and a white plate is used for fluorescence measurement. Thus, when performing both chemiluminescence measurement and fluorescence emission measurement, since both the white plate and the black plate are used, the operation is complicated and resources are wasted.

  This invention is made | formed in view of such a situation, The main objective is to suppress the consumption of a multiwell plate compared with the former.

  In order to solve the above-described problems, the multiwell plate adapter according to one aspect of the present invention is a multiwell plate adapter that can be attached to and detached from the upper surface of a multiwell plate having a plurality of storage portions for storing samples. And the hole used for the optical measurement of a sample is provided in the position corresponding to at least one part of the said accommodating part of the said multiwell plate, and at least an upper surface is provided with a plate-shaped part which is white or black.

  In this aspect, the plate-like portion may be white or black on the entire surface.

  In the above aspect, the multiwell plate is white or black. When the multiwell plate is black, the plate-like portion is white. When the multiwell plate is white, the plate-like portion is black. It may be.

  Moreover, the said aspect WHEREIN: The said said hole part may be provided in the said plate-shaped part at the same space | interval as the space | interval of the adjacent accommodating part in the said multiwell plate.

  Further, in the above aspect, the plate-like portion may be provided with the same number of holes as the accommodating portions provided in the multi-well plate.

  Further, in the above aspect, the apparatus further includes a cylindrical portion that protrudes from the periphery of the hole portion of the plate-shaped portion, has at least an inner peripheral surface of white or black, and is inserted into the accommodating portion of the multiwell plate. It may be.

  Further, in the above aspect, the multiwell plate is white or black, and when the multiwell plate is black, at least the inner peripheral surface of the cylindrical portion is white, and when the multiwell plate is white, At least the inner peripheral surface of the cylindrical portion may be black.

  Further, in the above aspect, an inner peripheral surface of the hole portion of the plate-like portion is formed in a female screw shape, and one end of the cylindrical portion is formed in a male screw shape, and the cylindrical portion And the cylindrical portion may be attached to the plate-like portion by screwing together the inner peripheral surface of the hole portion.

  According to the adapter for a multiwell plate according to the present invention, it is possible to suppress the consumption of the multiwell plate as compared with the conventional case.

The top view which shows the structure of the adapter for multiwell plates which concerns on embodiment. The front view which shows the structure of the adapter for multiwell plates which concerns on embodiment. The left view which shows the structure of the adapter for multiwell plates which concerns on embodiment. The right view which shows the structure of the adapter for multiwell plates which concerns on embodiment. The rear view which shows the structure of the adapter for multiwell plates which concerns on embodiment. The bottom view which shows the structure of the adapter for multiwell plates which concerns on embodiment. The partial expanded sectional view by the AA line in FIG. The partial expanded sectional view of the hole of a plate-shaped part. The side view of a cylindrical part. The perspective view which shows the structure of a multiwell filter plate. The perspective view which shows the adapter for multiwell plates of the state with which the multiwell filter plate was mounted | worn. Fluorescence emission when black adapter is not attached to white multiwell filter plate, adapter according to the embodiment is attached to white multiwell filter plate, and plate adapter is attached to white multiwell filter plate Graph comparing the measured background values. Fluorescence emission when black adapter is not attached to white multiwell filter plate, adapter according to the embodiment is attached to white multiwell filter plate, and plate adapter is attached to white multiwell filter plate The graph which compared S / N ratio of measurement. The graph which shows the experimental result when not attaching a black adapter to a white multiwell filter plate. The graph which shows the experimental result at the time of attaching the adapter which concerns on embodiment to a white multiwell filter plate. The graph which shows the experimental result by a fluorescence imager.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.

[Configuration of adapter for multiwell plate]
The adapter for a multiwell plate according to the present embodiment is mounted on the upper surface of a 96-well multiwell plate and is used for optical measurement of a sample accommodated in the multiwell plate in a measuring device such as a plate reader or an imager. .

  1 is a plan view showing the configuration of the multiwell plate adapter according to the present embodiment, FIG. 2 is a front view thereof, FIG. 3 is a left side view thereof, FIG. 4 is a right side view thereof, and FIG. FIG. 6 and FIG. 6 are bottom views thereof.

  As shown in the drawing, the multiwell plate adapter 1 according to the present embodiment includes a plate-like portion 2 having a rectangular plate shape with one corner missing in plan view. One corner of the plate-like portion 2, that is, the lower left corner in FIG. 1, has a missing shape, and a chamfered portion is formed by a straight line inclined at 45 degrees with respect to the X direction.

  The plate-like portion 2 is provided with a plurality of holes 21, 21, 21,. The holes 21, 21, 21,... Are arranged in a matrix at equal intervals, and are adjacent in the short side direction (X direction in FIG. 1) and the long side direction (Y direction) of the plate part 2. The interval between any two matching holes 21 and 21 (the distance between the centers of the holes 21) is 9 mm. .. 9 holes in the X direction and 12 holes in the Y direction are arranged in parallel, and a total of 98 hole parts 21, 21, 21,. ing.

  The plate-like portion 2 is made of a black polyacetal resin. That is, the entire surface of the plate-like portion 2 is colored black.

  From the bottom surface of the plate-like part 2, cylindrical parts 3, 3, 3,... Are provided so as to protrude from the peripheries of the holes 21, 21, 21,. The cylindrical portions 3, 3, 3,... Are provided corresponding to all the holes 21, 21, 21,... Of the plate-like portion 2, that is, 96 cylindrical portions 3, 3, 3,. It has been.

  FIG. 7 is a partially enlarged sectional view taken along line AA in FIG. As shown in FIG. 7, the cylindrical portion 3 has a cylindrical shape. Two rectangular missing portions 31 are provided at the front end of the cylindrical portion 3 so as to face each other.

  The cylindrical portion 3 is made of a black polyacetal resin. That is, the entire surface of the cylindrical portion 3 is colored black.

  FIG. 8 is a partially enlarged side sectional view of the hole 21 of the plate-like portion 2, and FIG. 9 is a side view of the tubular portion 3. As shown in FIG. 8, the inner peripheral surface 22 of the hole portion 21 of the plate-like portion 2 is formed in a female screw shape. On the other hand, the base end portion of the cylindrical portion 3 protrudes outward in a hook shape, and the outer peripheral surface of the hook portion 32 is formed in a male screw shape. The tubular portion 3 is attached to the plate-like portion by screwing the flange-like portion 32 of the tubular portion 3 into the hole portion 21 of the plate-like portion.

  FIG. 10 is a perspective view showing the configuration of the multiwell filter plate, and FIG. 11 is a perspective view showing the multiwell plate adapter mounted on the multiwell filter plate. The multiwell filter plate 4 has a rectangular trapezoidal shape with one corner missing in plan view, and a plurality of wells 41, 41, 41,. That is, the shape of the upper surface of the multiwell filter plate 4 corresponds to the shape of the adapter 1 according to the present embodiment, and when the adapter 1 is mounted on the upper surface of the multiwell filter plate 4 as shown in FIG. The plate-like portion 2 of the adapter 1 is slightly smaller than the upper surface of the multiwell filter plate 4 so that the upper surface of the multiwell filter plate 4 protrudes around the plate-like portion 2 with a uniform width in plan view. Yes. In other words, when the adapter 1 is placed on the surface of the multiwell filter plate 4, the plate-like portion 2 of the adapter 1 does not protrude from the upper surface of the multiwell filter plate 4.

  The multiwell filter plate 4 is provided with eight wells 41, 41, 41,... Arranged at equal intervals in the short side direction and twelve wells 41, 41, 41,. That is, a total of 96 wells 41, 41, 41,... Are arranged in a matrix on the multiwell filter plate 4. The intervals at which the wells 41, 41, 41,... Are arranged are the same as the intervals at which the holes 21, 21, 21,. That is, when the adapter 1 is attached to the multi-well filter plate 4, the positions of the holes 21, 21, 21,... Coincide with the positions of the wells 41, 41, 41,.

  Each of the cylindrical portions 3, 3, 3,... Of the adapter 1 is inserted into the wells 41, 41, 41,. The amount of protrusion of the cylindrical portions 3, 3, 3,... From the bottom surface of the plate-like portion 2 is slightly smaller than the depth of the wells 41, 41, 41,. ..., the tips of the cylindrical parts 3, 3, 3, ... are in contact with the bottoms (filters) of the wells 41, 41, 41, ... so that the plate-like part 2 is separated from the upper surface of the multiwell filter plate 4. Absent.

  The multiwell filter plate 4 shown in FIG. 10 is white, and the white multiwell filter plate 4 is suitable for chemiluminescence measurement, but is not suitable for fluorescence emission measurement. This is due to the following reasons. In the case of chemiluminescence, light from chemiluminescence is reflected on the side surface of the well of the white multi-well filter plate 4, so that weak chemiluminescence can be easily measured. In this case, weak fluorescence is emitted from the multiwell filter plate 4 and has a slight influence on the chemiluminescence measurement result as a noise component. However, the above-described effect of reflection is more general than the influence of this noise component. Therefore, a white multiwell filter plate is used for chemiluminescence measurement.

  On the other hand, in the case of fluorescence emission, fluorescence is emitted from the white multi-well plate 4 by light irradiating the well, and this fluorescence is measured together with the fluorescence emitted from the sample as a noise component. The ratio is low. If it is a black multiwell filter plate, fluorescence will not generate | occur | produce substantially from a multiwell filter plate, but the fluorescence emitted from a sample can be measured with sufficient sensitivity. For this reason, a black multiwell filter plate is used for measuring fluorescence.

  The adapter 1 according to the present embodiment is used by being mounted on a white multiwell plate. When the adapter 1 is attached to the multiwell filter plate 4, the upper surface of the multiwell filter plate 4 is covered with the black adapter 1. Therefore, when the fluorescence emission is measured, the fluorescence from the multiwell filter plate 4 is not mixed as a noise component, and the S / N ratio of the measurement result is improved.

  Further, by providing the adapter 1 with the cylindrical portions 3, 3, 3, ..., the side surfaces of the wells 41, 41, 41, ... of the multiwell filter plate 4 are hidden by the cylindrical portions 3, 3, 3, ..., It is possible to prevent fluorescence generated from the side surfaces of the wells 41, 41, 41,... From being measured as noise components. Therefore, the S / N ratio can be further improved.

[Verification of the effect of installing the adapter]
(1) Preparation of measurement plate <Membrane pretreatment and washing>
A 70% ethanol solution was dispensed into each well of a 96-well multiwell filter plate (manufactured by Millipore) equipped with a hydrophobic PVDF membrane and molded with white acrylic. After dispensing, the solution was removed by suction from the bottom of the filter. Next, purified water and TBS buffer (Tris 25 mM, physiological saline 150 mM, pH 7.4) were dispensed into each well. After dispensing, the solution was removed by suction from the bottom of the filter.

<Preparation of measurement sample CDK2>
As a measurement sample, a recombinant CDK2 solution (manufactured by Santa Cruz) was diluted with a TBS buffer and prepared so that the concentration of CDK2 was 7.5 U / mL (2.71 μg / mL = 1 U). A sample with a CDK2 concentration of 0 U / mL (TBS buffer only) was also prepared.

<Immobilization of CDK2>
100 μL of each prepared measurement sample was dispensed into each well. After dispensing, suction was performed from the lower part of the filter, and CDK2 contained in the measurement sample was adsorbed and immobilized on the membrane. After CDK2 was immobilized, 300 μL of TBS buffer was dispensed into each well, and washing was performed by aspirating and removing the buffer from the bottom of the filter after dispensing.

<Blocking process>
A blocking solution in which BSA (bovine serum albumin) was added to the TBS buffer so as to have a concentration of 4% was prepared. 100 μL of the prepared blocking solution was dispensed into each well. After dispensing, the blocking solution was removed by suction from the lower part of the filter, and blocking treatment was performed.

<Reaction between CDK2 and anti-CDK2 antibody>
100 μL of 4 μg / mL anti-CDK2 rabbit polyclonal antibody solution (Santa Cruz) was dispensed into each well, and CDK2 and anti-CDK2 antibody were reacted. After dispensing, the antibody solution was removed by suction from the bottom of the filter. After the antibody solution was removed by suction, 300 μL of TBS buffer was dispensed into each well, and the filter was washed by removing the buffer from the lower part of the filter after dispensing.

<Reaction between anti-CDK2 antibody and secondary antibody>
100 μL of a secondary antibody 4 μg / mL biotinylated anti-rabbit IgG polyclonal antibody solution (Santa Cruz) was dispensed into each well, and the anti-CDK2 antibody and biotinylated anti-rabbit IgG polyclonal antibody were reacted. After dispensing, the antibody solution was removed by suction from the bottom of the filter. After the antibody solution was removed by suction, 300 μL of TBS buffer was dispensed into each well, and the filter was washed by removing the buffer from the lower part of the filter after dispensing.

<Binding of labeling substance>
As a fluorescent labeling reagent, 100 μL of 10 μg / mL FITC-streptavidin solution (manufactured by Vector) was dispensed into each well, and a fluorescent labeling substance was bound to the biotinylated anti-rabbit IgG polyclonal antibody. After dispensing, the solution was removed by suction from the bottom of the filter. After removing the solution by suction, 300 μL of TBS buffer solution was dispensed into each well, and the filter was washed by sucking and removing the buffer solution from the bottom of the filter after dispensing. After washing, the plate was dried (20 minutes at room temperature).

(2) Fluorescence measurement “A black adapter that covers the upper part of the plate and the inner wall of the well (adapter according to the above embodiment)” and “a black plate that covers only the upper part of the plate on the measurement plate” Fluorescence emission was measured under the conditions of “with adapter attached” and “without adapter”.

  A plate reader InfiniteF200 (manufactured by TECAN) was used for fluorescence emission measurement. The measurement was performed according to the instruction manual of InfiniteF200 under the conditions of Ex (excitation light): 535 nm and Em (emission): 485 nm.

  As a reference example, fluorescence emission measurement was performed using pharosFX (manufactured by Bio-Rad), which is a fluorescence imager, instead of a plate reader. The measurement was performed under the conditions of Ex (excitation light): 530 nm and Em (emission): 488 nm according to the instruction manual of pharosFX. In the measurement with a fluorescence imager, a measurement plate without an adapter was used.

(3) Results FIG. 12 is a graph showing the background value obtained from the results of fluorescence emission measurement under each condition. The result when the adapter is not attached to the white measurement plate (multiwell filter plate) is “PL”, and the result when the black adapter that covers the top of the plate and the inner wall of the well is attached to the white measurement plate is “PL + A”. The result when a black adapter that covers only the top of the plate is attached to the white measurement plate is indicated by “PL + B”.

  From FIG. 12, it can be seen that the background value is greatly decreased in PL + A compared with the background value of PL. This suggests that the use of a black adapter covering the upper part of the plate and the inner wall of the well suppresses the background value and increases the measurement sensitivity.

  FIG. 13 is a graph showing the S / N ratio obtained from the results of fluorescence emission measurement under each condition. The result when the adapter was not attached to the white measurement plate was measured with “PL”, and the result when the black adapter covering the top of the plate and the inner wall of the well was attached to the white measurement plate was measured with “PL + A”, a fluorescence imager. The result is indicated by “Im”.

  From FIG. 12 and FIG. 13, the S / N ratio was significantly improved with PL + A compared with the S / N ratio of PL, and a result higher than the S / N ratio of the fluorescence imager measurement was obtained. This suggests that the use of a black adapter that covers the upper part of the plate and the inner wall of the well improves the S / N ratio and increases the measurement sensitivity.

[Measurement of detection limit (minimum detection sensitivity)]
Measured under each condition (when measured with a plate reader using a black plate adapter covering the upper part of the plate and the inner wall of the well, when measured with a plate reader without using a plate adapter, and when measured with a fluorescence imager) The detection limit (minimum detection sensitivity) was investigated.

(1) Preparation of measurement plate In preparation of measurement sample CDK2 of Example 1 and immobilization of CDK2, as a measurement sample, a recombinant CDK2 solution (manufactured by Santa Cruz) was diluted with TBS buffer, and the concentration of CDK2 was 0.5, 1.0. , 2.5, 5.0, and 7.5 U / mL, and prepared in the same manner as in Example 1 except that 100 μL of each prepared measurement sample was dispensed into each well. Using.

(2) Fluorescence measurement Fluorescence measurement was performed in the same manner as in Example 1.

(3) Results The concentration of CDK2 at the detection limit was calculated by the 2SD method (detection limit measurement method) from the fluorescence measurement results obtained under each condition. Graphs showing the calculated results are shown in FIGS. FIG. 14 is a graph showing experimental results when the black adapter is not attached to the white multiwell filter plate. FIG. 15 is a graph when the adapter according to the above embodiment is attached to the white multiwell filter plate. It is a graph which shows an experimental result, and FIG. 16 is a graph which shows the result of the experiment by the fluorescence imager implemented as reference. The detection limit was the concentration of CDK2 when the range of 2SD values when the concentration of CDK2 was 0 U / mL did not overlap.

  14-16, by using an adapter, a detection limit improves compared with the case where an adapter is not used, and a detection limit comparable to the case where it measures with a fluorescence imager can be obtained. From this, it was suggested that detection sensitivity increases by using an adapter.

(Other embodiments)
In the above embodiment, the configuration in which the adapter 1 includes the cylindrical portions 3, 3, 3,... Has been described, but is not limited thereto. It is good also as a plate-shaped adapter provided with the some hole part, without providing cylindrical part 3,3,3 ....

  Moreover, in embodiment mentioned above, although the adapter 1 which made the whole surface black was described, it is not limited to this. The entire surface of the adapter may be white. In this case, when chemiluminescence is measured using a black multiwell plate, the chemiluminescence can be accurately measured by attaching a white adapter to the multiwell plate.

  Also, the top surface of the adapter can be black and the other parts can be in other colors. If at least the upper surface is black, it is possible to suppress the fluorescence emitted from the adapter in the fluorescence measurement from being included in the measurement result as a noise component.

  Moreover, in embodiment mentioned above, although the case where the adapter 1 was formed using the black material was described, it is not limited to this. For example, it is also possible to form the adapter 1 using a material of a color other than black, such as white, and apply a black paint or the like to the surface.

  In the above-described embodiment, the case where the adapter 1 covers almost the entire upper surface of the multiwell plate has been described. However, the present invention is not limited to this. The adapter 1 can be used for multi-well plates of various sizes, such as a 384-well multi-well plate. In this case, a part of the upper surface of the multiwell plate is covered with the adapter, and the other part is exposed. In this way, fluorescent light emission can be measured at the part where the adapter is used, and chemiluminescence can be measured at the part where the multi-well plate is exposed. it can.

  Further, the adapter 1 can have a size other than the size described in the above embodiment. For example, the adapter can be configured to cover a part of the upper surface of the 96-well multiwell plate, such as an adapter having a configuration in which the plate-like portion 2 is half the size and 48 holes are provided. Even in this case, since a part of the upper surface of the multiwell plate is covered with the adapter and the other part is exposed, various measurements are possible.

  Moreover, in embodiment mentioned above, although the plate-shaped part 2 and the cylindrical part 3 were comprised as a separate component, the structure which manufactures an adapter by attaching the cylindrical part 3 to the plate-shaped part 2 was described. However, the present invention is not limited to this. You may manufacture the adapter provided with the plate-shaped part 2 and the cylindrical part 3 by injection molding etc. by integral molding. Moreover, although the female screw was provided in the plate-shaped part 2, the male screw was provided in the cylindrical part, and the structure joined by screwing both together was described, it is not limited to this, Other joining methods, For example, it is good also as a structure which joins a cylindrical part to a plate-shaped part by fitting the base end of a cylindrical part to a plate-shaped part.

  The adapter for a multiwell plate according to the present invention is useful as an adapter used for a multiwell plate for chemiluminescence or fluorescence measurement.

DESCRIPTION OF SYMBOLS 1 Adapter for multiwell plates 2 Plate-shaped part 21 Hole part 22 Inner peripheral surface (female screw part)
3 cylindrical part 31 missing part 32 hooked part (male screw part)
4 Multiwell filter plate 41 well

Claims (8)

A multi-well plate adapter that can be attached to and detached from the upper surface of a multi-well plate having a plurality of storage portions for storing samples,
A hole used for optical measurement of a sample is provided at a position corresponding to at least a part of the accommodating portion of the multi-well plate, and at least an upper surface includes a white or black plate-shaped portion.
Adapter for multiwell plates. The plate-like portion is white or black on the entire surface,
The adapter for multiwell plates according to claim 1. The multiwell plate is white or black,
When the multiwell plate is black, the plate-like portion is white,
When the multiwell plate is white, the plate-like portion is black,
The adapter for multiwell plates according to claim 1 or 2. The plate-like portion is provided with the adjacent hole portions at the same interval as the interval between adjacent accommodating portions in the multi-well plate,
The multiwell plate adapter according to any one of claims 1 to 3. The plate-like portion is provided with the same number of holes as the accommodating portions provided in the multiwell plate.
The adapter for multiwell plates in any one of Claims 1 thru | or 4. A cylindrical portion protruding from the periphery of the hole of the plate-like portion, at least the inner peripheral surface being white or black, further comprising a cylindrical portion inserted into the accommodating portion of the multiwell plate;
The multiwell plate adapter according to any one of claims 1 to 5. The multiwell plate is white or black,
When the multiwell plate is black, at least the inner peripheral surface of the cylindrical portion is white,
When the multiwell plate is white, at least the inner peripheral surface of the cylindrical portion is black,
The adapter for multiwell plates according to claim 6. The inner peripheral surface of the hole of the plate-like part is formed in a female screw shape,
One end of the cylindrical part is formed in a male screw shape,
The cylindrical part is attached to the plate-like part by screwing the cylindrical part and the inner peripheral surface of the hole part,
The adapter for multiwell plates according to claim 6 or 7.

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