Method for evaluating detection molecule
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- SHIMADZU CORP
- Filing Date
- 2026-01-21
- Publication Date
- 2026-07-30
Smart Images
Figure 00000020_0000
Abstract
Description
Method for evaluating detection molecule
[0001] The present invention relates to a method for evaluating a detection molecule.
[0002] As analytical methods for quantitatively analyzing surface antigens of extracellular vesicles, flow cytometers, immunochromatography, mass spectrometry (e.g., ICP-MS), enzyme-linked immunosorbent assay (ELISA method), etc. are known. In these analytical methods, as a detection molecule for detecting a surface antigen, a probe molecule (e.g., a biomolecule such as an antibody) that recognizes the surface antigen and to which metal particles are bound is used (Patent Document 1).
[0003] Japanese Patent Application Laid-Open No. 2021-042983
[0004] The quality of the above detection molecule is affected by the affinity of the probe molecule for the surface antigen and the amount or position of the metal particles bound to the probe molecule. Also, the quality may vary for each production lot, and a method for quantitatively evaluating the quality of the detection molecule is desired.
[0005] The present invention has been made in view of the above circumstances, and an object thereof is to provide a method for evaluating a detection molecule that can quantitatively evaluate the quality.
[0006] A first aspect of the present invention is a method for evaluating a detection molecule comprising a probe molecule and metal particles bound to the probe molecule, the method comprising: preparing a detection target molecule immobilized on a support; applying the detection molecule to the detection target molecule; solubilizing the metal particles in the detection molecule bound to the detection target molecule; quantifying the solubilized metal particles by inductively coupled plasma mass spectrometry; and performing data correction based on the amount of the quantified metal particles. <00000![''15'']>
[0007] A second aspect of the present invention is a kit for use in the method for evaluating a detection molecule according to the first aspect, the kit comprising: a multi-well plate on which the detection target molecule is immobilized; the probe molecule; and the metal particles.
[0008] According to the present invention, it becomes possible to provide a method for evaluating detected molecules that can quantitatively assess quality.
[0009] Figure 1 is a graph showing the quantitative determination of the quality of detected molecules in each immobilized protein in Experiment 1, based on the amount of metal ions.
[0010] Hereinafter, one embodiment of the present invention (hereinafter referred to as "this embodiment") will be described. However, this embodiment is not limited thereto. In this specification, the notation in the form of "A to Z" means the upper and lower limits of the range (i.e., A or more and Z or less), and if no unit is specified for A and only a unit is specified for Z, the unit for A and the unit for Z are the same.
[0011] [Method for Evaluating Detected Molecules] One aspect of the present invention is a method for evaluating a detected molecule comprising a probe molecule and metal particles bound to the probe molecule, comprising: a step of preparing a target molecule to be detected immobilized on a support (preparation step); a step of applying the detected molecule to the target molecule to be detected (detected molecule application step); a step of solubilizing the metal particles in the detected molecule bound to the target molecule to be detected (metal particle solubilization step); a step of quantifying the solubilized metal particles by inductively coupled plasma mass spectrometry (quantification step); and a step of performing data correction based on the quantified amount of metal particles (data correction step).
[0012] <Preparation Step> In this step, the target molecule to be detected, which is immobilized on a support, is prepared. In this embodiment, "support" means a substance for immobilizing the target molecule to be detected. The support is not particularly limited, but examples include multi-well plates, beads, etc. In one aspect of this embodiment, the support is preferably a multi-well plate. When a multi-well plate (e.g., a 96-well plate) is used as the support, the throughput of the analysis can be improved.
[0013] In this embodiment, "target molecule" refers to a molecule detected by the detection molecule described later. The target molecule can be obtained by synthesis or isolation using known methods. Alternatively, the target molecule may be purchased commercially. The target molecule is not particularly limited, but examples include proteins, nucleic acids, and glycans. In one aspect of this embodiment, it is preferable that the target molecule includes at least one selected from the group consisting of proteins, nucleic acids, and glycans. Examples of proteins include surface antigens of extracellular vesicles (exosome markers, microendoplasmic reticulum markers, apoptotic body markers, etc.), membrane proteins, cell adhesion molecules, receptor proteins, proteins involved in membrane fusion or cleavage, major histocompatibility complex (MHC) molecules, heat shock proteins, disease-specific marker proteins, etc. Examples of nucleic acids include DNA and RNA. RNA includes messenger RNA, microRNA, non-coding RNA, etc. Examples of glycans include N-linked glycans and O-linked glycans.
[0014] Examples of exosome markers include Alix, Tsg101, tetraspanin, and fluorolin, with tetraspanin including CD9, CD63, and CD81. Examples of microendoplasmic reticulum markers include integrins, selectins, and CD40. Examples of apoptotic body markers include phosphatidylserine and annexin V. Examples of disease-specific markers include Carcinoma Embryonic Antigen (CEA), Prostate specific antibody (PSA), and Carbohydrate antibody 19-9 (CA19-9). The probe molecules described later may be antibodies that specifically recognize these proteins. When exosomes are captured more selectively, the probe molecules are preferably selected from the group consisting of anti-CD9 antibodies, anti-CD63 antibodies, and anti-CD81 antibodies.
[0015] In this embodiment, when the support is a multi-well plate (96 wells), the amount of target molecules immobilized on the support may be 0.01 to 2 μg, 0.01 to 1 μg, 0.01 to 0.5 μg, or 0.01 to 0.1 μg per well.
[0016] The method for immobilizing the target molecule onto the support is not particularly limited; it can be immobilized by chemical bonding, hydrogen bonding, ionic bonding, complex formation, hydrophobic interactions, van der Waals interactions, electrostatic interactions, stereoselective interactions, etc.
[0017] For example, when immobilizing the target molecule by adding an aqueous solution containing the target molecule to a multiwell plate, the concentration of the target molecule in the aqueous solution may be 1 to 10 μg / mL or 0.1 to 1 μg / mL.
[0018] The aqueous solution used to dissolve the target molecule is preferably a buffer solution. Examples of buffer solutions include PBS, HEPES buffer, acetate buffer, phosphate buffer, citrate buffer, citrate-phosphate buffer, borate buffer, tartaric acid buffer, and Tris buffer. The aqueous solution may contain a salt. The concentration of the salt in the aqueous solution is preferably 150 mM or less, more preferably 10 mM to 150 mM, and even more preferably 15 mM to 100 mM. Examples of salts include sodium chloride (NaCl), potassium chloride (KCl), and calcium chloride (CaCl). ２ Examples include the above. The pH of the aqueous solution is not particularly limited as long as the target molecule can be stably present, but it is preferably between 6 and 10.
[0019] In one aspect of this embodiment, it is preferable to bring the target molecule to be detected into contact with the multiwell plate (support) in an environment of 20°C to 30°C. In other words, it is preferable that the temperature of the aqueous solution be 20°C to 30°C.
[0020] In one aspect of this embodiment, the time for which the molecule to be detected and the support are in contact may be, for example, 0.5 hours or more and 3 hours or 1 hour or more and 2 hours or less.
[0021] In another aspect of this embodiment, the support on which the target molecules to be detected are immobilized may be washed with PBS solution or the like to remove excess target molecules. Alternatively, the support on which the target molecules to be detected are immobilized may be blocked with a blocking agent. Examples of blocking agents include BSA, Lipidure BL802 (manufactured by NOF Corporation), and casein. The concentration of the blocking agent is preferably 1% by weight or more and 10% by weight or less, and more preferably 2% by weight or more and 5% by weight or less.
[0022] Two or more target molecules may be immobilized on a single support (for example, one well of a multiwell plate), or one target molecule may be immobilized on a single support. In one embodiment, one target molecule may be immobilized on one support, and another target molecule may be immobilized on another support.
[0023] <Detection Molecule Application Process> In this process, the detection molecule is applied to the target molecule. Here, the detection molecule consists of a probe molecule and metal particles bound to the probe molecule. Furthermore, "applying the detection molecule to the target molecule" is a concept that includes bringing the detection molecule into contact with the target molecule.
[0024] In this embodiment, "probe molecule" means a molecule that specifically binds to the above-mentioned target molecule. The probe molecule may be a protein (antibody, antibody fragment, lectin, etc.) or a nucleic acid molecule (aptamer, nucleic acid molecule having a complementary sequence, etc.). The probe molecule may be a molecule that specifically binds to biomolecules contained in vesicles, such as extracellular vesicle markers including exosome markers, microendoplasmic reticulum markers, and apoptotic body markers, other membrane proteins, or glycans. The probe molecule may also be a molecule that specifically binds to disease-specific markers. In one aspect of this embodiment, it is preferable that the probe molecule includes at least one selected from the group consisting of antibodies, antibody fragments, aptamers, and lectins.
[0025] In this embodiment, "metal particles" means metal particles that are bound to the probe molecule and function as a label. The metal elements contained in the metal particles include gold (Au), silver (Ag), platinum (Pt), palladium (Pd), iridium (Ir), rhodium (Rh), ruthenium (Ru), aluminum (Al), copper (Cu), tellurium (Te), bismuth (Bi), lead (Pb), iron (Fe), cerium (Ce), molybdenum (Mo), niobium (Nb), tungsten (W), antimony (Sb), tin (Sn), vanadium (V), manganese (Mn), nickel ( Examples include Ni, cobalt (Co), zinc (Zn), lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), scandium (Sc), yttrium (Y), and titanium (Ti). From the viewpoint of material stability and ease of synthesis, the metal particles preferably contain at least one metal element selected from the group consisting of gold, platinum, iridium, palladium, silver, and copper, and preferably are composed of any of these metal elements. In one aspect of this embodiment, the metal particles may be elemental metals or alloys consisting of two or more metal elements.
[0026] For example, when using a first detection molecule (first probe molecule - first metal particle) and a second detection molecule (second probe molecule - second metal particle), and the first metal particle is an alloy consisting of metal element A and metal element B, and the second metal particle is an alloy consisting of metal element A and metal element C, the amount of metal element A corresponds to the total amount of the first and second detection molecules, and the amounts of metal element B and metal element C, respectively, correspond to the amounts of the first and second detection molecules. As will be described later, in the evaluation method according to this embodiment, the first and second metal particles are quantified simultaneously by ICP mass spectrometry or the like, so it becomes possible to simultaneously estimate the total amount of the first and second detection molecules, the amount of the first detection molecule, and the amount of the second detection molecule, and furthermore, to correct the amounts of the first and second metal particles using the total amount of the first and second detection molecules. The same applies when using four types of detection molecules (first to fourth detection molecules).
[0027] The metal particles may be metal nanoparticles, metal nanorods, or metal nanoplates. The metal particles are preferably small in order not to inhibit the binding between the target molecule and the probe molecule. The particle diameter (length of the maximum part of the particle) is usually 500 nm or less, preferably 10 nm to 500 nm, more preferably 10 nm to 200 nm, and even more preferably 10 nm to 100 nm. When the metal particles are approximately spherical, the diameter may be 500 nm or less, 200 nm or less, or 100 nm or less. The lower limit of the diameter may be 10 nm or more. When the metal particles are metal nanorods, the length of the short axis may be 100 nm or less, 50 nm or less, or 10 nm or less, and the length of the long axis may be 500 nm or less, 200 nm or less, or 100 nm or less. The lower limit of the length of the short axis may be 2 nm or more. The lower limit of the length of the long axis may be 5 nm or more. When the metal particles are metal nanoplates, their thickness may be 100 nm or less, 50 nm or less, or 30 nm or less, and the maximum length of the plane may be 500 nm or less, 200 nm or less, or 100 nm or less. The lower limit of the thickness may be 4 nm or more. The lower limit of the maximum length of the plane may be 10 nm or more. The size of the metal particles may be measured, for example, by scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM), or transmission electron microscopy (TEM). An example of the apparatus is the ultra-high resolution analytical scanning electron microscope SU-70 (manufactured by Hitachi, Ltd.). The maximum major axis may be measured using a dynamic light scattering particle size distribution analyzer (DLS). An example of the apparatus is the zeta potential, particle size, and molecular weight measurement system ELSZ-2000ZS (manufactured by Otsuka Electronics Co., Ltd.). Other devices for measuring the longest diameter include the laser diffraction particle size distribution analyzer SALD-7500nano (manufactured by Shimadzu Corporation) and the nanoparticle analysis system NanoSight LM10 (Malvern Panometric).When measuring the maximum major axis of anisotropic gold nanoparticles, such as metal nanorods or metal nanoplates, from SEM, STEM, or TEM observation images, the maximum major axis can be calculated by taking 125 data points (125 points total) from which the maximum major axis of 125 arbitrary anisotropic gold nanoparticles have been measured, excluding the upper and lower 10%, and then calculating the average value of these 100 data points.
[0028] In this embodiment, the surface of the metal particles may be coated with a surfactant. This provides stability to the metal particles in the aqueous solution. In one aspect of this embodiment, from the viewpoint of improving the stability of the metal particles in the aqueous solution, the aqueous solution may contain a dispersant. An example of a dispersant is sodium citrate.
[0029] In this embodiment, the method for binding the metal particles to the probe molecule is not particularly limited and any known method may be used. Examples include chemical modification methods using functional groups such as maleimide, and physical adsorption methods. Furthermore, the probe molecule (detection molecule) to which the metal particles are bound may be purchased commercially.
[0030] In one aspect of this embodiment, the detection molecules may be two or more types, three or more types, or four or more types. There is no particular upper limit, but for example, it may be 10 types or less. When using two or more types of detection molecules, the two or more detection molecules may be applied to the target molecule simultaneously, or the second detection molecule may be applied after the first detection molecule has been applied.
[0031] In this embodiment, there are preferably four or more types of detection molecules, and each of the four or more types of detection molecules preferably consists of a different probe molecule and different metal particles.
[0032] In other aspects of this embodiment, the detection molecule preferably comprises a first detection molecule, a second detection molecule, a third detection molecule, and a fourth detection molecule, wherein the first detection molecule consists of a first probe molecule and a first metal particle bound to the first probe molecule, the second detection molecule consists of a second probe molecule and a second metal particle bound to the second probe molecule, the third detection molecule consists of a third probe molecule and a third metal particle bound to the third probe molecule, and the fourth detection molecule preferably consists of a fourth probe molecule and a fourth metal particle bound to the fourth probe molecule. Preferably, the first probe molecule, the second probe molecule, the third probe molecule, and the fourth probe molecule are all different probe molecules. Preferably, the first metal particle, the second metal particle, the third metal particle, and the fourth metal particle are all different metal particles.
[0033] In this embodiment, the detection molecule is preferably applied to the target molecule in an aqueous solution containing a salt. The concentration of the salt in the aqueous solution is preferably 150 mM or less, more preferably 15 mM to 150 mM, even more preferably 20 mM to 150 mM, and even more preferably 20 mM to 100 mM. The salts mentioned above include sodium chloride (NaCl), potassium chloride (KCl), and calcium chloride (CaCl). ２ Examples include the following. In this case, the concentration of the detected molecule is preferably such that, when measuring 100 μL of the solution using a 96-well plate with a plate reader (Infinite M-200 pro, TECAN), the absorbance at the maximum absorption wavelength is 0.01 or more and 2 or less, and more preferably 0.5 or more and 1.5 or less. If there is no maximum absorption wavelength, the absorbance at the absorption wavelength of 550 nm is used.
[0034] In one aspect of this embodiment, the aqueous solution containing the salt is preferably a buffer solution. Examples of buffer solutions include PBS, HEPES buffer, acetate buffer, acetate phosphate buffer, citrate buffer, citrate phosphate buffer, borate buffer, tartaric acid buffer, Tris buffer, and the like. The pH of the aqueous solution containing the salt is not particularly limited as long as the detected molecule can be stably present, but it is more preferably between 6 and 10.
[0035] Furthermore, from the viewpoint of stabilizing the detected molecules, the aqueous solution containing the above salt may further contain a blocking agent. Examples of blocking agents include BSA, Lipidure BL802 (manufactured by NOF Corporation), and casein. The concentration of the above blocking agent is preferably 1% by weight or more and 10% by weight or less, and more preferably 2% by weight or more and 5% by weight or less.
[0036] In another aspect of this embodiment, it is preferable that the detection molecule be applied to the target molecule in an environment of 20°C to 30°C. In other words, it is preferable that the temperature of the aqueous solution containing the salt be 20°C to 30°C.
[0037] In this embodiment, the time for applying the detection molecule to the target molecule is not particularly limited as long as the interaction between the detection molecule and the target molecule reaches an equilibrium state, but for example, it may be 0.5 hours or more and 3 hours or 1 hour or more and 2 hours or less.
[0038] In this embodiment, after applying the detection molecule to the target molecule, it may be washed with a washing solution. This removes excess detection molecules that are not bound to the target molecule, thereby reducing background noise.
[0039] <Metal Particle Solubilization Process> In this process, the metal particles in the detection molecule that are bound to the detection target molecule are solubilized.
[0040] As a method for solubilizing the metal particles, there is no particular limitation as long as the metal particles can be completely solubilized. For example, a method of dissolving and extracting the metal particles bound to the probe molecule with an acid such as aqua regia (a mixed solution of concentrated nitric acid and concentrated hydrochloric acid), nitric acid, hydrochloric acid, sulfuric acid, etc. can be mentioned. Further, after separating the metal particles from the support with a stripping solution (for example, an aqueous solution of a surfactant such as SDS), the metal particles contained in the stripping solution may be dissolved with an acid. The concentration of aqua regia (concentrated hydrochloric acid: concentrated nitric acid = 3:1) may be 50% or more. The temperature for dissolving the metal particles is, for example, 25°C or higher, and the reaction time may be 10 minutes or more and 24 hours or less. From the viewpoint of detecting the metal particles with high sensitivity, it is preferable to dissolve the metal particles with aqua regia.
[0041] When a plurality of types of detection molecules (for example, a first detection molecule and a second detection molecule) are bound to one support, a solution containing a plurality of metal elements can be obtained by acid treatment. When the first detection molecule is bound to one support and the second detection molecule is bound to another support, a solution containing a plurality of metal elements can be obtained by mixing the solutions containing the metal elements obtained by acid treatment.
[0042] <Quantification step> In this step, the solubilized metal particles are quantified by inductively coupled plasma mass spectrometry (ICP-MS).
[0043] A solution containing a metal element (that is, solubilized metal particles) is introduced into an ICP-MS apparatus. In the case of ICP-MS, the metal elements in the solution are ionized by an inductively coupled plasma, separated by an electric field or a magnetic field, etc., and the concentration of each element is quantified. The inductively coupled plasma may be an argon gas plasma.
[0044] The quantified amount of metal ions correlates with the quality of the detection molecule with respect to the detection target molecule. Here, the "quality of the detection molecule" is a parameter that depends on the affinity of the detection molecule for the detection target molecule, the steric hindrance effect by the metal particles bound to the probe molecule, the amount of metal particles bound to the probe molecule, etc. It can be evaluated that the higher the quantified amount of metal ions, the higher the quality of the detection molecule. Further, according to ICP-MS, since the amounts of a plurality of types of metal ions can be quantified simultaneously, a plurality of detection molecules can be evaluated simultaneously.
[0045] Examples of the ICP-MS device include ICPMS-2030 (manufactured by Shimadzu Corporation).
[0046] <Data correction step> In this step, data correction is performed based on the amount of the above-mentioned metal particles quantified. By performing this data correction, it becomes possible to compare data obtained using different detection molecules for different production lots. Examples of the data correction method include the following. First, reference data (Std) is obtained by applying a detection molecule to a detection target molecule whose amount immobilized on a support is known. Next, analysis data (S) is obtained by applying the detection molecule to an analyte (including the above-mentioned detection target molecule) immobilized on another support. By dividing the above-mentioned analysis data (S) by the above-mentioned reference data (Std), analysis data (S') after data correction is obtained. Since the analysis data (S') thus obtained is a relative value with respect to the reference data (Std), it becomes possible to compare data even when obtained using different detection molecules for different production lots.
[0047] In the present embodiment, examples of the data to be corrected include the amount of the detection target molecule immobilized on a support, the amount of metal particles bound to the probe molecule, and the like.
[0048] [Kit for use in the method for evaluating a detection molecule] Another aspect of the present invention is a kit for use in the method for evaluating the above-mentioned detection molecule, which includes a multi-well plate on which the above-mentioned detection target molecule is immobilized, the above-mentioned probe molecule, and the above-mentioned metal particles.
[0049] The kit for use in the method for evaluating the above-mentioned detection molecule may further include one or more selected from the group consisting of a buffer for dissolving a reagent, a sample tube, and an instruction manual for the kit user.
[0050] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
[0051] (Experiment 1) Aqueous solutions (100 μL) of each antigen protein were separately added to each well of a 96-well microtiter plate (MTP), and various antigens (target molecules) were immobilized on each well (support) (preparation step). The concentrations of the aqueous solutions of each antigen protein were as follows: Recombinant CD9 protein (rCD9): 3 μg / mL, Recombinant CD63 protein (rCD63): 0.1 μg / mL, BSA-conjugate CD81 peptide (BSA-CD81 peptide): 1 μg / mL.
[0052] Next, detection molecules were prepared by metal-labeling anti-CD9 antibody with gold (Au) particles (particle size 100 nm) (Au-CD9 Ab), anti-CD63 antibody with platinum (Pt) particles (particle size 30 nm) (Pt-CD63 Ab), and anti-CD81 antibody with palladium (Pd) particles (particle size 100 nm) (Pd-CD81 Ab). After the reaction of each metal-labeled antibody (detection molecule) with the various antigens immobilized in each well, excess antibody was removed by washing (detection molecule application step). Aqua regia was added to each well to elute the metal particles that had bound to each antigen via the antibody (metal particle solubilization step). The solution containing the metal particles was subjected to ICP-MS to quantify Au ions, Pt ions, and Pd ions (quantification step). This quantified the amount of metal labeled on the antibody bound to the antigen. On the other hand, wells without immobilized antigens were prepared separately, reacted with metal-labeled antibodies, and data was obtained from these wells to create blank data.
[0053] The results are shown in Figure 1. In Figure 1, the horizontal axis represents the target molecule immobilized on the support, and the vertical axis represents the amount of metal detected. Upon examining the binding amount to each antigen, it was confirmed that the amount of gold (Au) was significantly higher for rCD9, the amount of platinum (Pt) was significantly higher for rCD63, and the amount of palladium (Pd) was significantly higher for rCD81. These findings suggest that each antibody specifically recognizes the antigen. Furthermore, it was suggested that by correcting the data based on the amount of the quantified metal particles, it would be possible to compare data obtained using detection molecules from different manufacturing lots. From the above, it was found that the quality of detection molecules can be quantitatively evaluated according to the detection molecule evaluation method of the present invention.
[0054] [Aspects] The above-described exemplary embodiments and examples will be understood by those skilled in the art to be specific examples of the following aspects.
[0055] (Section 1) A method for evaluating a detection molecule according to one embodiment is a method for evaluating a detection molecule comprising a probe molecule and metal particles bound to the probe molecule, comprising the steps of: preparing a target molecule to be detected immobilized on a support; applying the detection molecule to the target molecule to be detected; solubilizing the metal particles in the detection molecule bound to the target molecule to be detected; quantifying the solubilized metal particles by inductively coupled plasma mass spectrometry; and performing data correction based on the amount of metal particles quantified. The method for evaluating a detection molecule described in Section 1 provides a method for evaluating a detection molecule that can quantitatively evaluate its quality.
[0056] (Section 2) In the method for evaluating detection molecules described in Section 1, there are four or more types of detection molecules, and each of the four or more types of detection molecules consists of a probe molecule that is different from each other and a metal particle that is different from each other. According to the method for evaluating detection molecules described in Section 2, the quality of multiple detection molecules can be evaluated simultaneously.
[0057] (Section 3) In the method for evaluating a detection molecule described in Section 2, the detection molecule includes a first detection molecule, a second detection molecule, a third detection molecule, and a fourth detection molecule, wherein the first detection molecule consists of a first probe molecule and a first metal particle bound to the first probe molecule, the second detection molecule consists of a second probe molecule and a second metal particle bound to the second probe molecule, the third detection molecule consists of a third probe molecule and a third metal particle bound to the third probe molecule, and the fourth detection molecule consists of a fourth probe molecule and a fourth metal particle bound to the fourth probe molecule. According to the method for evaluating a detection molecule described in Section 3, the quality of multiple detection molecules can be evaluated simultaneously.
[0058] (Clause 4) In the method for evaluating a detection molecule described in any of Clauses 1 to 3, the probe molecule includes at least one selected from the group consisting of antibodies, antibody fragments, aptamers, and lectins. The method for evaluating a detection molecule described in Clause 4 provides a more suitable method for evaluating a detection molecule that can quantitatively evaluate its quality.
[0059] (Clause 5) In the method for evaluating a detection molecule described in any of paragraphs 1 to 4, the metal particles include at least one metal element selected from the group consisting of gold, platinum, iridium, palladium, silver, and copper. Since the metal element is excellent in stability and ease of synthesis, the method for evaluating a detection molecule described in paragraph 5 can provide a more suitable method for evaluating a detection molecule in which quality can be quantitatively evaluated.
[0060] (Clause 6) In the method for evaluating a detection molecule described in any of Clauses 1 to 5, the detection target molecule includes at least one selected from the group consisting of proteins, nucleic acids, and sugar chains. The method for evaluating a detection molecule described in Clause 6 provides a more suitable method for evaluating a detection molecule that can quantitatively evaluate its quality.
[0061] (Section 7) In the method for evaluating a detected molecule described in any of Sections 1 to 6, the support is a multiwell plate. The method for evaluating a detected molecule described in Section 7 can improve the throughput of the evaluation.
[0062] (Clause 8) In the method for evaluating the detected molecule described in any of Clauses 1 to 7, the particle size of the metal particles is 500 nm or less. According to the method for evaluating the detected molecule described in Clause 8, it is less likely to inhibit the binding between the target molecule and the detected molecule.
[0063] (Clause 9) A kit according to one embodiment is a kit for use in the method for evaluating a detection molecule described in any of Clauses 1 to 8, comprising: a multiwell plate on which the target molecule to be detected is immobilized; the probe molecule; and the metal particles. According to the kit described in Clause 9, a method for evaluating a detection molecule that can quantitatively evaluate its quality can be performed.
[0064] As described above, embodiments and examples of the present invention have been explained, but it is also intended from the outset that the configurations of each of the above embodiments and examples may be combined as appropriate.
[0065] The embodiments and examples disclosed herein should be considered in all respects to be illustrative and not restrictive. The scope of the present invention is indicated by the claims rather than the embodiments and examples described above, and all modifications within the scope of the claims are intended to be included in the meaning of equivalents and within the scope.
Claims
1. A method for evaluating a detection molecule comprising a probe molecule and metal particles bound to the probe molecule, comprising: a step of preparing a target molecule to be detected immobilized on a support; a step of applying the detection molecule to the target molecule to be detected; a step of solubilizing the metal particles in the detection molecule bound to the target molecule to be detected; a step of quantifying the solubilized metal particles by inductively coupled plasma mass spectrometry; and a step of performing data correction based on the amount of the quantified metal particles.
2. The method for evaluating a detection molecule according to claim 1, wherein the detection molecule comprises four or more types, and each of the four or more detection molecules comprises a probe molecule and a metal particle that is different from each other.
3. The method for evaluating a detection molecule according to claim 2, wherein the detection molecule comprises a first detection molecule, a second detection molecule, a third detection molecule, and a fourth detection molecule, the first detection molecule consists of a first probe molecule and a first metal particle bound to the first probe molecule, the second detection molecule consists of a second probe molecule and a second metal particle bound to the second probe molecule, the third detection molecule consists of a third probe molecule and a third metal particle bound to the third probe molecule, and the fourth detection molecule consists of a fourth probe molecule and a fourth metal particle bound to the fourth probe molecule.
4. The method for evaluating a detection molecule according to any one of claims 1 to 3, wherein the probe molecule comprises at least one selected from the group consisting of antibodies, antibody fragments, aptamers, and lectins.
5. The method for evaluating a detected molecule according to any one of claims 1 to 3, wherein the metal particles include at least one metal element selected from the group consisting of gold, platinum, iridium, palladium, silver, and copper.
6. The method for evaluating a detection molecule according to any one of claims 1 to 3, wherein the detection target molecule includes at least one selected from the group consisting of proteins, nucleic acids, and sugar chains.
7. The method for evaluating a detected molecule according to any one of claims 1 to 3, wherein the support is a multiwell plate.
8. The method for evaluating a detected molecule according to any one of claims 1 to 3, wherein the particle size of the metal particles is 500 nm or less.
9. A kit for use in a method for evaluating a detection molecule according to any one of claims 1 to 3, comprising: a multiwell plate on which the target molecule to be detected is immobilized; the probe molecule; and the metal particles.