JP2021051018A - Method of producing sample - Google Patents

Abstract

To provide a method for producing a sample capable of detecting the substance to be detected with high reproducibility in comparison with a conventional method.SOLUTION: In a method of producing a sample, a complex 40 is created by binding a first antibody 41 and a biotinylated antibody 45 obtained by joining biotin 44 to a second antibody 43 with the substance to be detected 42 in a solution dispensed on the substrate 10. The method of producing the sample includes: a drying step of performing drying against the substrate 10 to which the complex 40 is immobilized via a first antibody 41 until at least a part of the complex 40 is exposed; and a particle bonding step of binding particles 46 modified with avidin to the biotinylated antibody 45 of the composite 40 fixed to the substrate 10 after the drying step.SELECTED DRAWING: Figure 9

Description

The present invention relates to a sample production method.

An immunoassay method is known in which a biological substance such as a protein contained in a biological sample such as blood, serum, or plasma is analyzed as a substance to be detected. The biological substance is, for example, vesicle granules such as exosomes secreted from living cells.

In Patent Document 1, in a sample analysis device, a liquid containing an exosome to be detected is injected into a well having a bottom surface forming an uneven structure, and the antibody fixed in the recessed portion on the bottom surface of the well exists in the exosome. A method for capturing an exosome, which comprises an exosome immobilization step of binding an antigen and immobilizing the exosome in a recess, is described. In the above method, a buffer solution containing beads in which an antibody is immobilized on the surface is injected into an injection section, and an antibody immobilized on the beads is bound to an antigen possessed by the exosome to modify the exosome with beads. It is stated that additional steps may be included.

Japanese Unexamined Patent Publication No. 2014-219384

However, in the capture method according to Patent Document 1, the beads may come off due to the water flow of the cleaning liquid at the time of cleaning. As a result, when analyzing the same sample, the analysis results may vary, and the substance to be detected may not be analyzed with good reproducibility.

The present invention has been made in view of the problems of the prior art. An object of the present invention is to provide a sample production method capable of detecting a substance to be detected with high reproducibility as compared with a conventional method.

In order to solve the above problems, the sample production method according to the aspect of the present invention is biotinogenesis in which biotin is bound to a first antibody and a second antibody in a substance to be detected in a solution dispensed on a substrate. It comprises a drying step in which the complex produced by binding the antibody is dried to a substrate immobilized via the first antibody until at least a part of the complex is exposed. The sample preparation method includes a particle binding step of binding the avidin-modified particles to the biotinylated antibody of the complex immobilized on the substrate after the drying step.

According to the present disclosure, it is possible to provide a sample production method capable of detecting a substance to be detected with high reproducibility as compared with a conventional method.

It is a top view which shows the state which attached the cartridge to the substrate. It is sectional drawing which cut at the line II-II shown in FIG. It is a perspective view cut along the line III-III shown in FIG. It is sectional drawing which shows an example of the sample which the composite and the particle were bonded to the surface of a substrate. It is sectional drawing which shows an example of the state which fixed the 1st antibody on the surface of a substrate. It is sectional drawing which shows an example of the state in which the substance to be detected is bound to the 1st antibody. It is sectional drawing which shows an example of the state in which a biotinylated antibody is bound to the substance to be detected. It is sectional drawing which shows an example of the state which the surface of the substrate was dried. It is sectional drawing which shows an example of the state in which the particle modified with avidin is bound to the biotinylated antibody. It is sectional drawing which shows an example of the state which removed the cartridge from a substrate.

Hereinafter, the sample production method according to the present embodiment will be described. The dimensional ratios in the drawings are exaggerated for convenience of explanation and may differ from the actual ratios.

The sample production method according to the present embodiment is a method for producing a sample for detecting a substance to be detected. The sample obtained by the method according to the present embodiment can measure a specific antigen or antibody associated with a disease by measuring with an analyzer.

First, the substrate 10 and the cartridge 20 attached to the substrate 10 will be described with reference to FIGS. 1 to 3. FIG. 1 is a plan view showing a state in which the cartridge 20 is attached to the substrate 10. FIG. 2 is a cross-sectional view taken along the line II-II shown in FIG. FIG. 3 is a perspective view cut along the line III-III shown in FIG.

As shown in FIGS. 1 and 2, the substrate 10 has a disk shape equivalent to that of an optical disc such as a Blu-ray disc (BD), a DVD, or a compact disc (CD). The substrate 10 is made of, for example, a resin material such as a polycarbonate resin or a cycloolefin polymer used for a general optical disc. The substrate 10 is not limited to the above-mentioned optical discs, and optical discs having other shapes or conforming to a predetermined standard can also be used.

The substrate 10 has a central hole 11 and a notch portion 12. The center hole 11 is a through hole provided in the central portion of the substrate 10 and penetrating in the thickness direction of the substrate 10. The notch portion 12 is provided on the outer peripheral portion of the substrate 10 and is cut out in a substantially rectangular shape.

The cartridge 20 has a plurality of through holes 21, a first convex portion 22, and a second convex portion 23. The plurality of through holes 21 are arranged at equal intervals on the same circumference with respect to the center of the substantially circular cartridge 20 in the plane direction of the cartridge 20. The through hole 21 is a hole that penetrates in the thickness direction perpendicular to the plane direction of the cartridge 20. The through hole 21 has an inner peripheral surface 24, and a region surrounded by the inner peripheral surface 24 forms a space. Note that FIG. 1 shows an example of a cartridge 20 provided with eight through holes 21 as an example, but the number of through holes 21 is not limited to this.

The first convex portion 22 is a protrusion provided at the center of the cartridge 20. The first convex portion 22 is formed so that it can be inserted into the central hole 11 of the substrate 10. The second convex portion 23 is a protrusion provided on the outer peripheral portion of the cartridge 20. The second convex portion 23 is formed so that it can be inserted into the notch portion 12 of the substrate 10. When the cartridge 20 is attached to the substrate 10, the first convex portion 22 is inserted into the center hole 11, and the second convex portion 23 is inserted into the notch portion 12. As a result, the movement of the substrate 10 and the cartridge 20 in the plane direction is prevented, and the substrate 10 and the cartridge 20 can be positioned.

When the cartridge 20 is attached to the substrate 10, the well 30 is formed by the surface 13 of the substrate 10 and the inner peripheral surface 24 of the through hole 21 of the cartridge 20. That is, the surface 13 of the substrate 10 forms the bottom surface of the well 30, and the inner peripheral surface 24 of the through hole 21 of the cartridge 20 forms the side surface of the well 30. The well 30 is a container for storing a sample containing a substance to be detected, a buffer solution, and a liquid such as a cleaning solution.

As shown in FIG. 3, a track region in which concave portions 14 and convex portions 15 are alternately arranged in the radial direction is formed on the surface 13 of the substrate 10. The concave portion 14 and the convex portion 15 are formed in a spiral shape from the inner peripheral portion to the outer peripheral portion of the substrate 10. The recess 14 corresponds to the groove of an optical disc. The convex portion 15 corresponds to a land of an optical disc. The track pitch of the substrate 10 corresponding to the track pitch of the optical disk is, for example, 320 nm.

At least a portion of the cartridge 20 in contact with the surface 13 of the substrate 10 may be formed of an elastic member. By forming such a portion with an elastic member, the elastic member is elastically deformed so as to fill the recess 14 in the track region when the cartridge 20 is attached to the substrate 10. Therefore, the substrate 10 and the cartridge 20 can be brought into close contact with each other, and the possibility of liquid leaking from between the surface 13 of the substrate 10 and the inner peripheral surface 24 of the cartridge 20 can be reduced. The elastic member may form at least a part of the cartridge 20 or may form the entire cartridge 20. The elastic member may be, for example, a resin such as silicone rubber, or a ring-shaped packing or the like.

FIG. 4 is a cross-sectional view showing an example of a sample in which the composite 40 and the particles 46 are bonded to the surface 13 of the substrate 10. As shown in FIG. 4, for example, when the composite 40 and the particles 46 are captured in the recess 14 of the surface 13 of the substrate 10 and the spot light collected by the objective lens 47 of the analyzer is scanned, the particles are captured in the recess 14. Where the 46 is captured, the phase of the reflected light reflected by the particles 46 changes. Therefore, the amount of light of the detection signal obtained by an optical detection unit such as an optical pickup changes. By counting the particles 46 captured on the surface 13 of the substrate 10 by utilizing this difference in the amount of light, the substance 42 to be detected contained in the sample can be quantified.

The complex 40 is composed of the first antibody 41, the substance to be detected 42, and the biotinylated antibody 45. The first antibody 41 is an antibody that specifically recognizes the substance to be detected 42 contained in the sample. The substance to be detected 42 is an antigen such as a specific protein associated with a disease. By using such an antigen as the substance to be detected 42, it is used for diagnosis of diseases, follow-up after treatment, diagnosis of prognosis, selection of therapeutic agents, companion diagnosis for obtaining treatment guidelines, monitoring of diseases or physical condition, etc. Can be useful.

For example, the substance to be detected 42, such as an exosome, can play a role as a biomarker because its concentration in the body fluid changes according to the disease state to be monitored. The substance to be detected 42 is, for example, as at least one protein selected from the group consisting of transmembrane membrane proteins known as antigens for identifying exosomes such as CD9, CD63, CD81, CEA and HER2. May be good. When the substance 42 to be detected is an exosome, the outer diameter of the exosome is often about 30 nm to 100 nm. Further, when the substance to be detected 42 is a protein, the outer diameter of the protein is often several nm to several hundred nm.

The biotinylated antibody 45 is an antibody in which biotin 44 is bound to the second antibody 43. Biotin 44 is a water-soluble vitamin belonging to the B vitamin group. Biotin 44 is not particularly limited as long as it can bind to the second antibody 43, and may be a biotin derivative in which the valerian acid side chain of biotin 44 is modified with a modifying group. The modifying group may be, for example, N-hydroxysuccinimidyl ester. In this case, the second antibody 43 and biotin 44 can be bound by the amide bond between the ester moiety of the N-hydroxysuccinimidyl ester and the amino group of the second antibody 43.

The second antibody 43 is not particularly limited as long as it can recognize the substance to be detected 42. For example, when the exosome is used as the detection target substance 42 as described above, the second antibody 43 may be an antibody that recognizes at least one protein selected from the group consisting of CD9, CD63, CD81, CEA, HER2, and the like. Good. The first antibody 41 and the second antibody 43 may recognize the same antigen or may be different. However, when the detection target substance 42 has only one target antigen, the second antibody 43 cannot bind to the detection target substance 42 if the recognition antigens are the same. Therefore, in such a case, it is necessary to enable the first antibody 41 and the second antibody 43 to recognize different antigens.

Avidin is a biotin-binding protein. The avidin may be a native avidin produced from birds, reptiles, amphibians, etc., a streptavidin isolated from Streptomyces avidin, or a neutravidin glycosylated native avidin. Native avidin is also simply referred to as avidin. An example of the mass of native avidin is about 66 kDa to 69 kDa, an example of the mass of streptavidin is about 53 kDa or about 75 kDa, and an example of the mass of neutravidin is about 60 kDa. These avidins may be used alone or in combination of two or more.

The particles 46 modified with avidin may be formed of at least one material selected from the group consisting of, for example, polymer particles, metal particles, silica particles and the like. The particles 46 may be magnetic beads or the like containing a magnetic material such as ferrite inside. When magnetic beads are used, the particles 46 can be induced on the surface 13 of the substrate 10 by magnetism, so that the time for binding biotin 44 and avidin can be shortened.

The average particle size of the particles 46 is not particularly limited, but may be 100 nm to 1 μm. By setting the average particle size of the particles 46 to 100 nm or more, the detection target substance 42 can be easily detected. Further, by setting the average particle size of the particles 46 to 1 μm or less, the accuracy of counting the substance 42 to be detected can be improved. The average particle size of the particles 46 may be 100 nm or more, or 200 nm or less. The average particle size of the particles 46 can be obtained by observing the particles 46 with a scanning electron microscope or the like and calculating the average value of the particle sizes.

Next, a method for producing a sample according to the present embodiment will be described. The sample production method according to the present embodiment includes a drying step and a particle bonding step. As described below, the sample production method may further include a fixation step, a detection target substance binding step, and a biotinylated antibody binding step. A method for producing a sample according to the present embodiment will be described with reference to FIGS. 5 to 10 using the substrate 10 and the cartridge 20.

(Fixing process)
As shown in FIG. 5, in the fixing step, the first antibody 41 is fixed on the surface 13 of the substrate 10. Specifically, a solution containing the first antibody 41 is placed (dispensed) in the well 30. The first antibody 41 is fixed to the surface 13 of the substrate 10 by a hydrophobic bond. For example, a solution containing the first antibody 41 is placed in the well 30 and reacted at a predetermined temperature for a predetermined time to fix the first antibody 41 on the surface 13 of the substrate 10. The predetermined reaction temperature and the predetermined reaction time are not particularly limited because they vary depending on the state of the surface 13 of the substrate 10, the type of the first antibody 41, and the like. The first antibody 41 may be fixed to the surface 13 of the substrate 10 by covalent bonding by chemically treating the surface 13 of the substrate 10.

The solution may contain the first antibody 41. The solution is not particularly limited because it varies depending on the type of the first antibody 41 and the like, but may contain, for example, the first antibody 41 and a buffer solution. As the buffer solution, known ones can be used, and for example, Phosphate Buffered Saline (PBS) may be used.

After fixing the first antibody 41 on the surface 13 of the substrate 10, the solution in the well 30 may be removed and washed with a washing liquid. As the cleaning liquid, known ones can be used, and for example, Phosphate Buffered Saline with Tween® 20 (PBS-T) may be used. After cleaning the inside of the well 30 with the cleaning liquid, the cleaning liquid is removed from the inside of the well 30. However, it is preferable that the surface 13 of the substrate 10 is not completely dried so that the first antibody 41 is not denatured.

(Detection target substance binding process)
As shown in FIG. 6, in the detection target substance binding step, the detection target substance 42 is bound to the first antibody 41 immobilized on the surface 13 of the substrate 10. Specifically, a sample containing the substance to be detected 42 is placed in the well 30 having the substrate 10 on which the first antibody 41 is immobilized. The first antibody 41 specifically recognizes the substance to be detected 42. Therefore, in the liquid 48, the substance to be detected 42 binds to the first antibody 41 by an antigen-antibody reaction and is captured on the surface 13 of the substrate 10.

The sample is a solution containing the substance to be detected 42. The sample is not particularly limited because it varies depending on the type of the detection target substance 42 and the like, but may contain, for example, the detection target substance 42 and the buffer solution. The buffer solution may be the same as that used in the fixing step, or may be different.

After binding the substance to be detected 42 to the first antibody 41, the solution in the well 30 may be removed and washed with a washing solution. After cleaning the inside of the well 30 with the cleaning liquid, the cleaning liquid is removed from the inside of the well 30. However, it is preferable that the surface 13 of the substrate 10 is not completely dried so that the first antibody 41 and the substance to be detected 42 are not denatured. As the cleaning liquid, the same one used in the fixing step may be used, or a different one may be used.

(Biotinylated antibody binding step)
As shown in FIG. 7, in the biotinylated antibody binding step, the biotinylated antibody 45 is bound to the substance to be detected 42. Specifically, the solution containing the biotinylated antibody 45 is placed in the well 30 having the substrate 10 in which the first antibody 41 and the substance to be detected 42 are captured. The second antibody 43 contained in the biotinylated antibody 45 specifically recognizes the substance to be detected 42. Therefore, in the liquid 48, the biotinylated antibody 45 binds to the substance to be detected 42 by an antigen-antibody reaction and is captured on the surface 13 of the substrate 10. In this way, the first antibody 41, the substance to be detected 42, and the biotinylated antibody 45 are bound to form the complex 40.

The solution may contain the biotinylated antibody 45. The solution is not particularly limited because it varies depending on the type of the biotinylated antibody 45 and the like, but may contain, for example, the biotinylated antibody 45 and the buffer solution. The buffer solution may be the same as that used in the fixing step, or may be different.

After binding the biotinylated antibody 45 to the substance to be detected 42, the solution in the well 30 may be removed and washed with a washing solution. After cleaning the inside of the well 30 with the cleaning liquid, the cleaning liquid is removed from the inside of the well 30. As the cleaning liquid, the same one used in the fixing step may be used, or a different one may be used.

(Drying process)
As shown in FIG. 8, in the drying step, the substrate 10 on which the complex 40 is fixed in the liquid 48 via the first antibody 41 is composed of the substance to be detected 42, the first antibody 41, and the biotinylated antibody 45. Dry until at least one substance selected from the group, i.e. at least a portion of the complex 40, is exposed. The complex 40 contains a detection target substance 42, a first antibody 41 bound to the detection target substance 42, and a biotinylated antibody 45 in which biotin 44 is bound to a second antibody 43 bound to the detection target substance 42. ..

The surface 13 of the substrate 10 may be dried until only one of the first antibody 41, the substance to be detected 42, or the biotinylated antibody 45 is exposed. Further, the surface 13 of the substrate 10 has at least one of the first antibody 41 and the detection target substance 42, at least one of the first antibody 41 and the biotinylated antibody 45, or the detection target substance 42 and the biotinylated antibody 45. It may be dried until at least one of the substances is exposed. Further, the surface 13 of the substrate 10 may be dried until all of the first antibody 41, the substance to be detected 42, and the biotinylated antibody 45 are exposed. Further, it may be dried from the surface 13 of the substrate 10 until the liquid 48 is completely eliminated.

By the drying step as described above, at least one of the binding between the first antibody 41 and the detection target substance 42 and the binding between the detection target substance 42 and the biotinylated antibody 45 becomes stronger. It is presumed that the reason why the bond is strong is physical adsorption due to electrostatic interaction. As the bond becomes stronger, the composite 40 is less likely to separate from the surface 13 of the substrate 10 with a water flow generated by cleaning or the like.

Since the protein used as the substance to be detected 42 and the antibodies such as the first antibody 41 and the second antibody 43 are usually easily denatured when dried, a reaction such as binding is carried out in the liquid 48. If at least a part of the complex 40 is denatured by the drying step, specific binding such as an antigen-antibody reaction may not be possible, and accurate measurement of the substance to be detected 42 may be difficult. However, biotin 44 is bound to the second antibody 43, and since biotin 44 is a small molecule as described above, it is not easily denatured and loses its activity even when the surface 13 of the substrate 10 is dried, and it binds to avidin. Is possible.

The method for drying the surface 13 of the substrate 10 is not particularly limited. For example, the substrate 10 may be placed in a commercially available oven and the substrate 10 may be dried at a predetermined temperature for a predetermined time. The predetermined temperature may be, for example, 25 ° C. or higher, 30 ° C. or higher, or 35 ° C. or higher. Further, the predetermined temperature may be 40 ° C. or lower. The predetermined time may be, for example, 5 minutes or more, 10 minutes or more, or 15 minutes or more. Further, the predetermined time may be 60 minutes or less, or 30 minutes or less.

The surface 13 of the substrate 10 may be dried until at least one substance selected from the group consisting of the substance to be detected 42, the first antibody 41, and the biotinylated antibody 45 is exposed. In the drying step, the surface 13 of the substrate 10 may be dried until the height from the surface 13 of the substrate 10 to the liquid surface of the solution is 40 nm or less. In the drying step, the surface 13 of the substrate 10 may be dried until the height from the surface 13 of the substrate 10 to the liquid surface is 20 nm or less, or may be dried until the height is 10 nm or less, or 5 nm or less. May be dried to.

(Particle bonding process)
As shown in FIG. 9, in the particle binding step, after the drying step, the avidin-modified particles 46 are bound to the biotinylated antibody 45 bound to the substance to be detected 42. Since biotin 44 and avidin have a high affinity and a small dissociation constant, even if the particles 46 modified with avidin are large, they can be prevented from being dissociated in a step such as washing and the particles 46 being separated. ..

Even if the particles 46 modified with avidin are put into the liquid 48 without going through the above-mentioned drying step, the particles 46 are captured on the surface 13 of the substrate 10 by the binding of biotin 44 and avidin. To. However, the binding between the first antibody 41 and the detection target substance 42 and the binding between the detection target substance 42 and the second antibody 43 are weaker than the binding between biotin 44 and avidin. Further, the particles 46 have a predetermined mass and volume, and are easily flown together with the fluid flowing around the particles 46. Therefore, when the surface 13 of the substrate 10 is washed after the particles 46 are bound to the detection target substance 42 without going through the drying step, the binding between the first antibody 41 and the detection target substance 42 and the detection target substance are observed. At least one of the bonds between 42 and the second antibody 43 is easily broken. As a result, the particles 46 are likely to separate from the surface 13 of the substrate 10 due to cleaning or the like, which may reduce the measurement sensitivity or increase the variation in the measurement results.

However, in the present embodiment, at least one of the binding between the first antibody 41 and the detection target substance 42 and the binding between the detection target substance 42 and the second antibody 43 is strengthened by the drying step as described above. .. As a result, it is possible to prevent the particles 46 from being separated from the surface 13 of the substrate 10. Therefore, it is possible to reduce the possibility that the measurement sensitivity is lowered and the variation in the measurement results is large.

To bind the particles 46 to the biotinylated antibody 45, for example, a solution containing the particles 46 is placed in the well 30. Particle 46 binds to biotinylated antibody 45 by a specific reaction between biotin 44 and avidin. Therefore, the particles 46 can be bound to the biotinylated antibody 45 by putting a solution containing the particles 46 in the well 30 and reacting the particles 46 at a predetermined temperature for a predetermined time. As a result, the substance to be detected 42 can be labeled with the particles 46. The predetermined reaction temperature and the predetermined reaction time are not particularly limited because they vary depending on the types of the biotinylated antibody 45 and the particles 46.

The solution may contain particles 46. The solution is not particularly limited because it varies depending on the type of the particles 46 and the like, but may be the same as or different from the solution containing the first antibody 41 described above.

After binding the particles 46 to the substance to be detected 42, the solution in the well 30 may be removed and washed with a washing liquid. As the cleaning liquid, known ones can be used, and they may be the same as those used in the fixing step described above, or may be different. After cleaning the inside of the well 30 with the cleaning liquid, the cleaning liquid is removed from the inside of the well 30.

As shown in FIG. 10, the cartridge 20 is removed from the substrate 10 after the particle bonding step. The substance 42 to be detected can be indirectly counted by setting the substrate 10 from which the cartridge 20 has been removed in the analyzer and measuring the number of particles 46 captured on the surface 13 of the substrate 10.

In the method according to the present embodiment, the surface 13 of the substrate 10 on which the complex 40 is fixed in the liquid 48 via the first antibody 41 is composed of the substance to be detected 42, the first antibody 41, and the biotinylated antibody 45. Dry until at least one substance selected from the group is exposed. Therefore, at least one of the binding between the first antibody 41 and the detection target substance 42 and the binding between the detection target substance 42 and the biotinylated antibody 45 can be strengthened.

Further, in the method according to the present embodiment, after the drying step, the particles 46 modified with avidin are bound to the biotinylated antibody 45 bound to the substance to be detected 42. Since biotin 44 of the biotinylated antibody 45 is a small molecule, it is difficult to be denatured by the drying step, and avidin can specifically recognize biotin 44 even after the drying step. Therefore, the detection target substance 42 sandwiched between the first antibody 41 and the biotinylated antibody 45 can be specifically labeled with the particles 46.

The particles 46 captured on the surface 13 of the substrate 10 by such a method are difficult to separate from the surface 13 of the substrate 10. Then, the substance 42 to be detected is indirectly measured by scanning the number of particles 46 captured on the surface 13 of the substrate 10 with an optical pickup and counting them.

Therefore, the detection limit of the detection target substance 42 is lowered, and the detection target substance 42 can be detected with high sensitivity. In addition, since the detachment of the particles 46 is reduced, the coefficient of variation can be reduced, and exosomes can be detected with high reproducibility.

As described above, the sample production method may further include a fixing step of fixing the first antibody 41 on the surface 13 of the substrate 10. The sample production method may further include a detection target substance binding step of binding the detection target substance 42 to the first antibody 41 immobilized on the substrate 10. The sample preparation method may further include a biotinylated antibody binding step of binding the biotinylated antibody 45 to the substance to be detected 42. By such a method, the complex 40 containing the substance to be detected 42 and the particles 46 can be more reliably captured on the surface 13 of the substrate 10.

However, the sample production method according to the present embodiment is not limited to the above, and may include a drying step and a particle bonding step. For example, after forming the complex 40 containing the first antibody 41, the substance to be detected 42, and the biotinylated antibody 45, the first antibody 41 may be fixed to the surface 13 of the substrate 10 by covalent bonding or the like. Further, after binding the detection target substance 42 and the biotinylated antibody 45, it may be bound to the first antibody 41 immobilized on the surface 13 of the substrate 10.

The sample production method is produced by binding the first antibody 41 and the biotinylated antibody 45 in which biotin 44 is bound to the second antibody 43 to the substance to be detected 42 in the solution dispensed on the substrate 10. It comprises a drying step of drying the substrate 10 on which the complex 40 is immobilized via the first antibody 41 until at least a part of the complex 40 is exposed. The sample preparation method includes a particle binding step of binding the avidin-modified particles 46 to the biotinylated antibody 45 of the complex 40 immobilized on the substrate 10 after the drying step. Therefore, it is possible to provide a sample production method capable of detecting the substance to be detected with high reproducibility as compared with the conventional method.

Hereinafter, the present disclosure will be described in more detail with reference to Examples and Comparative Examples, but the present disclosure is not limited to these Examples.

[Example]
An analysis disk (sample) was prepared as follows, and the number of exosomes in the sample was measured using ExoCounter (registered trademark) provided by JVC KENWOOD Corporation. The disc was produced as follows.

(Fixing process)
First, a dedicated well was attached to the disk (board). Next, a PBS buffer solution containing an anti-human CD9 (manufactured by BioLegend) antibody in the well was dropped into the well, and the CD9 antibody was fixed on the surface of the disk. Then, after sucking and removing the liquid in the well, the well was washed three times with PBS-T buffer, and the PBS-T buffer was sucked and removed.

(Detection target substance binding process)
Next, a sample solution containing 0.1 μg of HCT116-derived exosomes manufactured by HanzaBioMed was dropped into the well, and the exosomes were captured with the above CD9 antibody. Then, after sucking and removing the liquid in the well, the well was washed three times with PBS-T buffer, and the PBS-T buffer was sucked and removed.

(Biotinylated antibody binding step)
A PBS buffer containing a biotinylated anti-human CD9 (BioLegend) antibody was added dropwise into the wells to bind the CD9 antibody to exosomes. Then, after the liquid in the well was sucked and removed, the well was washed three times with PBS-T buffer, the well was washed three times with pure water, and then the pure water was sucked and removed.

(Drying process)
The disc was left in an oven at 37 ° C. for 20 minutes to dry. When the dried disc was taken out of the oven and the inside of the well was visually inspected, no liquid could be confirmed on the surface of the disc.

(Particle bonding process)
Avidin was bound to particles having an average particle diameter of about 180 nm (FG beads (registered trademark) COOH beads manufactured by Tamagawa Seiki Co., Ltd.) to prepare avidin beads. Then, a liquid containing avidin beads was dropped into the well and bound to the biotinylated CD9 antibody. Then, after sucking and removing the liquid in the well, the well was washed three times with PBS-T buffer, the buffer was sucked and removed, and the well was removed from the disc.

(measurement)
A total of 5 discs were prepared by the same method as described above, and the number of beads fixed on each disc was measured by ExoCounter®. In addition, the average value (Ave.), Standard deviation (σ), and coefficient of variation (CV) of these measurement results were calculated. These results are shown in Table 1.

[Comparison example]
In the drying step, the number of particles was measured in the same manner as in Example 1 except that the particles were not dried. These results are shown in Table 1.

As shown in Table 1, in the method of the example, the average value of the counts, which is an index of sensitivity, was increased by 20% or more as compared with the method of the comparative example. That is, in the method of the example, the LOD (detection limit) was low and the exosome could be detected with high sensitivity as compared with the method of the comparative example.

Further, in the method of the example, as compared with the method of the comparative example, the CV, which is an index of the variation in the measurement results, was reduced from 13.9% to 8.6%, and the exosome could be detected with high reproducibility. ..

The reason why such high sensitivity and high reproducibility could be realized is that in the method of the example, the binding of the exosome and the antibody was strong due to the provision of the drying step, and the avidin beads were detached during washing. It is presumed that the number was small. On the other hand, in the method of the comparative example, since the drying step was omitted, the binding between the exosome and the antibody was weak, and it is considered that the number of avidin beads detached during washing was large.

Although the contents of the present disclosure have been described above according to the embodiments, it is obvious to those skilled in the art that the present disclosure is not limited to these descriptions and various modifications and improvements can be made.

10 Substrate 13 Surface 40 Complex 41 First antibody 42 Detectable substance 43 Second antibody 44 Biotin 45 Biotinylated antibody 46 Particles

Claims (4)

In the solution dispensed on the substrate, a complex produced by binding the first antibody and the biotinylated antibody in which biotin is bound to the second antibody to the detection target substance is formed via the first antibody. A drying step of drying the fixed substrate until at least a part of the composite is exposed.
After the drying step, a particle binding step of binding particles modified with avidin to the biotinylated antibody of the complex immobilized on the substrate, and a particle binding step.
A method for producing a sample, including. A fixing step of fixing the first antibody on the surface of the substrate, and
A detection target substance binding step of binding the detection target substance to the first antibody immobilized on the surface of the substrate, and a detection target substance binding step.
A biotinylated antibody binding step of binding the biotinylated antibody to the detection target substance, and
The sample production method according to claim 1, further comprising. The sample production method according to claim 1 or 2, wherein in the drying step, the surface of the substrate is dried until the height from the surface of the substrate to the liquid surface of the solution is 40 nm or less. The sample production method according to any one of claims 1 to 3, wherein the average particle size of the particles is 100 nm to 1 μm.

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