JP2017078571A - Gel electrophoresis device and gel electrophoresis method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a gel electrophoresis device and a gel electrophoresis method which can excellently separate a target material contained in the whole blood.SOLUTION: A gel electrophoresis device includes: a well 20; a first buffer tank 30; a support part; a second buffer tank 50; a third buffer tank 30; and a power supply part 70. In the well 20, whole blood 90 is arranged. The support part is provided such that a migration gel 22 is mounted thereon and one end of the migration gel 22 is communicated with the other end of the well 20. A third buffer tank 60 is provided such that a third electrode 62 is arranged therein and a third buffer liquid 64 to be stored is communicated with an intermediate region 23 between the one end and the other end in the migration gel 22. The power supply part 70 stops voltage application to a first electrode 32 and a second electrode 52 when an electric migration started by the execution of the voltage application to the first electrode 32 and the second electrode 52 is unable to enter the migration gel 22 and prevented by blood cells contained in the whole blood 90 accumulated at the other end of the well 20, and re-starts the electric migration by executing the voltage application to the second electrode 52 and the third electrode 62.SELECTED DRAWING: Figure 11

Description

  The present invention relates to a gel electrophoresis apparatus and a gel electrophoresis method.

  Some peptides present in the living body change in abundance depending on the disease, and have the possibility of being used as a marker for a specific disease. For example, blood C-peptide is a component of proinsulin, which is a substance at the stage before insulin is synthesized, and is secreted into the blood at a rate similar to that of insulin, and circulates in the blood with almost no decomposition. And discharged with urine. For this reason, it is possible to diagnose a disease by measuring C-peptide in blood and grasping the amount of insulin secreted.

  As a method for measuring an antigen such as C-peptide, there is an electrophoretic mobility shift assay (EMSA) using specific binding between a nucleic acid and a protein (see, for example, Non-Patent Document 1).

Garner, M.M. M.M. And Revzin, A .; "Gel electrophoresis for quantification of protein binding to specific DNA regions: Application to components of the E. coli lactose operon regulatory system", Nucleic Acids Research, 9: 3047-3060 (1981).

  However, when nucleic acid is subjected to gel electrophoresis, a molecular sieving effect works and a band appears at a position corresponding to the molecular weight of the nucleic acid. However, when whole blood is used as a sample, a problem arises. For example, when whole blood is electrophoresed, blood cells contained in the whole blood clog the well and obstruct the flow of electricity. As a result, there is a possibility that the electrophoresis is inhibited and the separation performance is lowered.

  The present invention has been made in order to solve the above-described problems associated with the prior art, and an object thereof is to provide a gel electrophoresis apparatus and a gel electrophoresis method that can favorably separate a target substance contained in whole blood. To do.

  In order to achieve the above object, a uniform phase of the present invention is a gel electrophoresis apparatus for separating a target substance contained in whole blood by electrophoresis. The gel electrophoresis apparatus includes a well in which the whole blood as a specimen is disposed, a first electrode, and a first buffer tank provided so that a stored first buffer solution communicates with one end of the well. And a supporting portion provided so that one end of the electrophoresis gel is communicated with the other end of the well, a second electrode is disposed, and a second buffer solution to be stored is disposed on the electrophoresis gel. A second buffer tank provided to communicate with the other end and a third electrode are disposed, and the third buffer solution to be stored is provided to communicate with an intermediate portion between one end and the other end of the electrophoresis gel. And a power supply unit that controls voltage application to the first to third electrodes. The power supply unit is configured to apply the voltage applied to the first electrode and the second electrode. Electrophoresis started by performing voltage application on the first electrode and the second electrode cannot penetrate the electrophoresis gel and is deposited on the other end of the well. When inhibited by blood cells contained in blood, the voltage application to the first electrode and the second electrode is stopped, and the voltage application to the second electrode and the third electrode is performed, thereby performing electrophoresis. Let it resume.

  Another aspect of the present invention for achieving the above object is a gel electrophoresis method for separating a target substance contained in whole blood by electrophoresis. In the gel electrophoresis method, one end that communicates with the first buffer stored in the first buffer tank in which the first electrode is disposed, and the other end that communicates with one end of the electrophoresis gel placed on the support unit The whole blood is disposed as a specimen in a well having a first buffer electrode and a second buffer tank provided so that the first electrode and the stored second buffer solution communicate with the other end of the electrophoresis gel. A voltage application is performed on the second electrode, and electrophoresis started by executing the voltage application does not enter the electrophoresis gel, and the whole blood deposited on the other end of the well When inhibited by the blood cells contained, voltage application to the first electrode and the second electrode is stopped, and the second electrode and the stored third buffer solution are connected between one end and the other end of the electrophoresis gel. To communicate with the middle part A third electrode disposed on the third buffer tank kicked against performs voltage application to resume electrophoresis.

  According to the present invention, when the target substance contained in whole blood is separated by electrophoresis, if electrophoresis is inhibited by deposition of blood cells that cannot enter the electrophoresis gel, the electrode pair to which a voltage is applied is the first and The electrophoresis is restarted by switching from the second electrode to the second and third electrodes. The second buffer stored in the second buffer tank in which the second electrode is disposed is provided so as to communicate with the other end of the electrophoresis gel located on the opposite side of the well, and the third electrode is disposed. In the middle part of the electrophoresis gel communicating with the third buffer stored in the third buffer tank, blood cells that inhibit electrophoresis are not deposited. Therefore, the voltage application function of the second and third electrodes is not interfered by the accumulated blood cells. Therefore, when voltage is applied to the second and third electrodes and electrophoresis is resumed, electrophoresis on the target substance that has already moved (invaded) into the electrophoresis gel is not inhibited, and the target substance is well separated. be able to. That is, it is possible to provide a gel electrophoresis apparatus and a gel electrophoresis method that can favorably separate a target substance contained in whole blood.

  The electrophoresis gel has an introduction gel located on one end side of the electrophoresis gel, and a separation gel located on the other end side of the electrophoresis gel, and the introduction gel communicates with the other end of the well. When the separation gel is configured to communicate with the second buffer solution and the gel concentration of the introduced gel is smaller than the gel concentration of the separation gel, the blood cell accumulates and inhibits electrophoresis. Until the voltage application to the first electrode and the second electrode is stopped), the target substance contained in the whole blood can be efficiently moved to the introduction gel side.

  When a posterior gel is interposed between one end of the well and the first buffer solution, it is possible to suppress the whole blood arranged in the well from entering the first buffer solution side.

  The target substance is separated based on the difference in electrophoretic mobility caused by the difference in molecular weight between the complex of the target substance and receptor and the free receptor. In the case of using at least two kinds or more recognized in the above, the target substance can be separated and quantified with high accuracy.

  The molecular weight of the target substance is preferably 1000 to 30000. In this case, the effect of using two or more receptors is more exhibited.

  The molecular weight of the target substance is preferably 1/150 to 1/5 of the molecular weight of the receptor. In this case, the mobility difference in electrophoresis effectively occurs, and the target substance-receptor complex and the free receptor Can be easily and accurately separated.

  The number of electrodes applied to the electrophoresis is not limited to three. For example, the fourth electrode can be arranged in the third buffer tank. In this case, the power supply unit is configured to further control voltage application to the fourth electrode, and electrophoresis started by executing voltage application to the first electrode and the fourth electrode is applied to the electrophoresis gel. If it is blocked by blood cells contained in the whole blood deposited on the other end of the well without being able to enter, the voltage application to the first electrode and the fourth electrode is stopped, and the voltage application to the second electrode and the third electrode is stopped. By executing, it is possible to separate the target substance well by restarting electrophoresis.

It is a top view for demonstrating the gel electrophoresis apparatus which concerns on embodiment of this invention. It is a perspective view for demonstrating the apparatus main body part shown by FIG. It is sectional drawing for demonstrating the apparatus main body part shown by FIG. It is a perspective view for demonstrating the lower part of the apparatus main body part shown by FIG. It is a top view for demonstrating the lower part of the apparatus main-body part shown by FIG. It is the schematic for demonstrating inhibition of the electrophoresis by the blood cell contained in the whole blood. It is a flowchart for demonstrating the gel electrophoresis method which concerns on embodiment of this invention. It is a conceptual diagram for demonstrating the electrode connection process shown by FIG. It is a conceptual diagram for demonstrating the 1st electrophoresis process shown by FIG. It is a conceptual diagram for demonstrating the electrode change process shown by FIG. It is a conceptual diagram for demonstrating the 2nd electrophoresis process shown by FIG. It is an electrophoretic photograph concerning samples 1-4. 3 is an enlarged view of a main part of an electrophoretic photograph relating to Sample 1. FIG. 5 is an enlarged view of a main part of an electrophoretic photograph relating to sample 2. FIG. It is the schematic for demonstrating the modification 1 which concerns on embodiment of this invention. 10 is a schematic diagram for explaining a second electrophoresis step according to Modification Example 1. FIG. It is the schematic for demonstrating the modification 2 which concerns on embodiment of this invention. 10 is a schematic diagram for explaining a second electrophoresis step according to Modification 3. FIG. It is the schematic for demonstrating the modification 3 which concerns on embodiment of this invention. 10 is a schematic diagram for explaining a second electrophoresis step according to Modification 3. FIG.

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

  FIG. 1 is a plan view for explaining a gel electrophoresis apparatus according to an embodiment of the present invention. FIGS. 2 and 3 are a perspective view and a sectional view for explaining an apparatus main body shown in FIG. 4 and 5 are a perspective view and a plan view for explaining the lower part of the apparatus main body shown in FIG. 1, and FIG. 6 is a schematic diagram for explaining the inhibition of electrophoresis by blood cells contained in whole blood. It is.

  A gel electrophoresis apparatus 10 according to an embodiment of the present invention is used to separate a target substance contained in whole blood. As shown in FIGS. 1 to 5, an apparatus main body 12 and a power supply unit 70 are provided. The apparatus main body 12 includes a well 20, a first buffer tank 30, a support 40, a second buffer tank 50, and a third buffer tank 60.

  The well 20 is a cell in which whole blood as a specimen is placed (see FIG. 5).

  The first buffer tank 30 is provided with a first electrode 32 serving as a cathode, and stores a first buffer solution 34. The first buffer solution is provided so as to communicate with one end of the well 20.

  The support part 40 is a lane on which the well 20, the electrophoresis gel 22 and the back gel 26 are placed. In the present embodiment, two support parts 40 are installed (see FIGS. 4 and 5).

  The back gel 26 is interposed between one end of the well 20 and the first buffer solution 34, and suppresses the whole blood disposed in the well 20 from entering the first buffer solution side (first buffer tank side). Is provided to do. The gel concentration of the back gel 26 is, for example, 4% gel.

  The electrophoresis gel 22 is a member that separates a target substance contained in whole blood by electrophoresis, and includes an introduction gel 23 and a separation gel 24. The introduction gel 23 is located on one end side of the electrophoresis gel 22, communicates with the other end of the well 20, and constitutes an intermediate part between the one end and the other end of the electrophoresis gel 22, and is introduced at the initial stage of electrophoresis. This is the site where the target substance contained in blood moves (invades). The separation gel 24 is located on the other end side of the electrophoresis gel 22, communicates with the second buffer solution, and is a part that separates the target substance that has moved (invaded) into the introduction gel 23. The gel concentration of the introduction gel 23 is set to be smaller than that of the separation gel 24. For example, the electrophoresis gel 22 is a 7% gel, and the introduction gel 23 is a 4% gel.

  The second buffer tank 50 is provided with a second electrode 52 that is an anode, and stores a second buffer solution 54. The second buffer solution 54 is provided so as to communicate with the other end of the electrophoresis gel 22.

  The third buffer tank 60 is provided with a third electrode 62 serving as a second cathode, and stores a third buffer solution 64. The third buffer tank 60 is located above the support portion 40 and has a through hole 66 (see FIGS. 1 and 3). The through hole 66 is aligned with the introduction gel 23. Therefore, the third buffer solution 64 communicates with the introduction gel 23 via the through hole 66.

  The power supply unit 70 is provided to control voltage application to the first to third electrodes 32, 52, and 62, and includes a power supply 72, an electric circuit 74, and a switching mechanism 76 (see FIG. 1). The electric circuit 74 is configured such that the first to third electrodes 32, 52, 62 can be connected to the power source 72. The switching mechanism 76 is provided to switch between voltage application to the first and second electrodes 32 and 52 and voltage application to the second and third electrodes 52 and 62.

  For example, the applied voltage is 5 to 2000 V / cm, and the electrophoresis temperature is room temperature (20 to 25 ° C.). The electrophoresis time is 30 to 180 minutes, but is preferably about several to 10 minutes from the viewpoint of rapid separation.

  The blood cells 92 contained in the whole blood 90 migrate toward the introduction gel 23 when a voltage is applied to the first and second electrodes 32 and 52. Therefore, for example, after 3 to 4 minutes have elapsed from the start of electrophoresis, blood cells 92 that cannot enter the introduction gel 23 are deposited on the other end of the well 20 (the boundary between the well 20 and the introduction gel 23) (see FIG. 6). There is a possibility that the flow of electricity is obstructed, and as a result, electrophoresis is inhibited and the separation performance is lowered.

  Therefore, when the electrophoresis started by executing voltage application to the first and second electrodes 32 and 52 is inhibited by the accumulated blood cells 92, the power supply unit 70 uses the switching mechanism 76 to By stopping the voltage application to the first and second electrodes 32 and 52 and executing the voltage application to the second and third electrodes 52 and 62, the electrophoresis is set to resume.

  The second buffer solution 54 stored in the second buffer tank 50 in which the second electrode 52 is disposed is provided so as to communicate with the other end of the electrophoresis gel 22 located on the opposite side to the well 20. . A blood cell 92 that inhibits electrophoresis accumulates on the introduction gel 24 (intermediate portion of the electrophoresis gel 22) 24 that communicates with the third buffer solution 64 stored in the third buffer tank 60 in which the third electrode 62 is disposed. Not done. Therefore, the voltage application function of the second and third electrodes 52 and 62 is not interfered by the accumulated blood cells 92, and voltage application is performed on the second and third electrodes 52 and 62 to resume electrophoresis. In this case, electrophoresis with respect to the target substance moved to the electrophoresis gel 22 is not inhibited, and the target substance can be separated well. That is, it is possible to satisfactorily separate the target substance contained in whole blood.

  In addition, since the gel concentration of the introduction gel 23 is set to be smaller than that of the separation gel 24, the voltage is applied to the first and second electrodes 32 and 52 until the blood cell 92 is deposited and the electrophoresis is inhibited. The target substance contained in the whole blood 90 can be efficiently moved to the introduction gel side.

  For the electrophoresis method, for example, gel electrophoresis such as agarose gel electrophoresis, pulse-field gel electrophoresis (PFGE), polyacrylamide gel electrophoresis (Poly-Acrylamide Gel Electrophoresis; PAGE) can be applied. Is possible. Moreover, since it is highly transparent and electrically neutral, it is preferable to use polyacrylamide gel electrophoresis.

  The buffer solutions used for the first to third buffer solutions 34, 54, 64 and the electrophoresis gel 22 are not particularly limited and are appropriately selected as necessary. For example, citric acid, succinic acid, tartaric acid, malic acid and the like Solution containing organic acids, salts thereof, amino acids such as glycine, taurine, arginine, inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, boric acid, acetic acid, salts thereof, etc. . Specific buffers are, for example, Tris-Glycine buffer, Tris buffer, Tris-Tricine buffer, Tris-HCl buffer, MES buffer, and MOSP buffer.

  Next, the target substance separated from whole blood by electrophoresis will be described.

  The target substance is, for example, a protein, peptide, sugar chain, nucleic acid, low molecular compound (preferably a low molecular compound having a molecular weight of 1 kDa or less), and a complex thereof. In the present specification, protein or peptide includes a free form, a phosphate group, a methyl group, an acetyl group, a sugar chain, a lipid, a modified body such as a lipid, a nitrile group, an acid (eg, inorganic acid, organic acid). Or a salt with a base (eg, alkali metal salt), a protein or peptide bound to another substance such as a nucleoprotein bound to a nucleic acid.

  Specific examples of the target substance include insulin, C-peptide, GLP-1 (glucagon-like peptide-1), GIP (glucose-dependent insulin secretion stimulating polypeptide), adiponectin, H-FABP (myocardial fatty acid binding protein), myoglobin, Proteins such as troponin I, troponin T, BNP (brain natriuretic peptide), NT-ProBNP (N-terminal pro brain natriuretic peptide).

  When the target substance is a marker related to some disease, it becomes a very useful means in clinical examination. For example, when the target substance is C-peptide, the diseases are insulinoma, obesity, liver disease, Cushing syndrome, acromegaly, abnormal insulinemia, insulin autoimmune syndrome, diabetes, hypoglycemia, undernutrition, brown cells And pituitary adrenal hypofunction.

  C-peptide is a peptide consisting of 31 amino acids and is a constituent of proinsulin, which is an insulin precursor. C-peptide is simultaneously released as a degradation product when proinsulin is cleaved by endopeptidase and insulin is released into the blood. Therefore, C-peptide plays a role as an indicator of insulin secretion kinetics, and the kinetics of blood C-peptide can be an important indicator for investigating endogenous insulin secretion ability in diabetic patients and the like. Therefore, if the subject's C-peptide level is high compared to the value of healthy subjects, the patient suffers from insulinoma, obesity, liver disease, Cushing's syndrome, acromegaly, abnormal insulinemia, insulin autoimmune syndrome, etc. If there is a possibility that the subject's C-peptide level is low, it should be judged that the subject may have diabetes, hypoglycemia, malnutrition, pheochromocytoma, hypopituitar adrenal function, etc. Can do.

  Next, separation and quantification of the target substance will be described.

  In order to separate and quantify the target substance with high accuracy, it is possible to use a substance (receptor) that specifically binds to the target substance. In this case, the sample used for electrophoresis is a substance after performing a reaction (for example, an antigen-antibody reaction) that binds a target substance (for example, an antigen) and a receptor (for example, an antibody) that specifically binds to the target substance. In whole blood, the separation of the target substance is based on the difference in electrophoretic mobility caused by the molecular weight difference between the target substance and receptor complex and the free receptor. The detection of the target substance-receptor complex by electrophoresis is a very useful means because it can be detected with high accuracy by a very simple operation as compared with the enzyme immunoassay (ELISA) of a heterogeneous measurement system.

  The target substance-receptor complex includes a complex in which all receptors bind to the target substance, and a complex in which only some of the receptors bind to the target substance. Among these, in the complex in which only some of the receptors are bound to the target substance, the complex and the free receptor may not be separated due to the difference in molecular weight. Therefore, it is preferable that all receptors as much as possible bind to the target substance to form a complex.

  The receptor is, for example, an antibody, an antibody fragment having an activity of binding to an antigen (antibody fragment), a nucleic acid such as a DNA aptamer, a protein, a receptor composed of a protein, a binding protein, a peptide, a biological receptor, or a sugar chain. . An antibody or an antibody fragment is preferable because it has a high specific binding property to a target substance (antigen).

  The antibody can be, for example, a monoclonal antibody, a polyclonal antibody, a single-chain antibody, a modified antibody (eg, a “humanized antibody” in which only the antigen recognition site is humanized), a chimeric antibody, and a dual function capable of simultaneously recognizing two epitopes. It is a sex antibody. From the viewpoint of specific binding to an antigen, a monoclonal antibody is preferable.

  The target substance and the receptor that specifically binds to the target substance are mixed and reacted. In terms of the antigen-antibody reaction conditions, the reaction temperature is 4 to 50 ° C., and the reaction time is 5 minutes to 24 hours. The reaction solvent is, for example, phosphate buffer (PBS), carbonate buffer, Tris buffer, glycine buffer, or tricine buffer. For example, the receptor may be added to and mixed with whole blood before being placed in the well 20, or the receptor may be preliminarily placed in the well 20 and mixed when the whole blood is placed in the well 20. The target substance and the receptor can be reacted with each other.

  In general, when two or more types of receptors having relatively large molecular weights (recognizing different sites) are bound to a low molecular weight target substance, the receptors that bind to the target substance are very close to each other. Therefore, it is presumed that the target substance-receptor complex does not have stability sufficient to withstand electrophoresis in a sterically unstable state. That is, at the time of electrophoresis, at least one kind of receptor may be detached from the target substance. In this case, the target to be detected by electrophoresis is a target substance to which one kind of receptor binds, and the signal of the receptor free substance and the signal of the target substance to which one kind of receptor bind cannot be separated, and the target substance Concentration cannot be measured accurately.

  On the other hand, when the target substance has a low molecular weight, the target substance-receptor complex is stably present during electrophoresis even when two or more receptors that specifically recognize different parts of the target substance are used. The target substance-receptor complex and the free receptor can be clearly separated by electrophoresis. Therefore, the molecular weight of the target substance is preferably 1000 to 30000, more preferably 1000 to 10000, and more preferably 1000 to 5000 from the viewpoint of more exerting the effect of using two or more receptors. Is more preferable.

  The molecular weight of the target substance is preferably 1/150 to 1/5 of the molecular weight of the receptor. In this case, based on the molecular weight difference between the target substance-receptor complex and the free receptor, a difference in mobility in electrophoresis is effectively generated, and the target substance-receptor complex and the free receptor can be easily and accurately measured. This is because they can be separated well.

  The pH of the buffer solution should be around neutral to weakly basic, that is, 6 to 9, considering the stability and reactivity of the reagent in an environment where the receptor maintains the target substance binding activity and is negatively charged. preferable.

  Next, a method for detecting a target substance-receptor complex and a free receptor will be described.

  In the electrophoresis gel (separation gel) after electrophoresis, a method for detecting the target substance-receptor complex and the free receptor is, for example, gel staining. As a staining method when the receptor is a protein, CBB (Coomassie Brilliant Blue) staining method, silver staining method, SYPRO (registered trademark) Ruby Protein Stain (manufactured by Takara Bio Inc.), Deep Purple Total Protein Stain (GE Healthcare) A dyeing method using a dye such as Japan).

  The quantification of the target substance is performed based on, for example, the signal intensity (staining density) of the dye in the band obtained by the staining method. For example, the band of the target substance-receptor complex is detected from the result of staining after electrophoresis (for example, silver staining). The signal intensity of the dye in the band is proportional to the amount of the target substance. Therefore, a calibration curve with the target substance amount can be prepared for the signal intensity of the target substance-receptor complex band measured in advance, and the detected target substance-receptor complex band is determined based on the calibration curve. By comparing the signal intensities, the amount of the target substance in the sample can be accurately measured. The signal intensity of the band dye can be calculated by image analysis software such as imageJ, NIHimage, or SCIONimage.

  The quantification of the target substance can be performed based on visualizing the target substance-receptor complex using the labeling substance. For example, enzyme immunoassay (EIA), radioimmunoassay using the labeling substance is possible. A measurement method (RIA), a fluorescence immunoassay method (FIA), a chemiluminescence immunoassay method (CLIA), or the like can be applied. At this time, the direct method and the indirect method can be applied. In the direct method, a primary receptor (for example, a primary antibody) that reacts directly with a target substance is used as a label. In the indirect method, a first target substance-receptor binding reaction (for example, antigen-antibody reaction) is performed using a primary receptor (for example, primary antibody), and the primary receptor (primary antibody) itself is used as a target substance (for example, antigen). Another receptor (secondary receptor) (for example, an antibody (secondary antibody)) is used as a label.

Labeling substances used for labeling include, for example, Alexa Floor (Life), Hilite Floor (Ana spec), IRDye (LI-COR), FITC (fluorescein isocyanate), FITC (Fluorescein Isothiocyanate), PE (phycoy), thyr Fluorescent substances such as APC (Allophycocyanin), Cy-3, Cy-5, tetramethylrhodamine isocyanate, radioisotopes such as ¹²⁵ I, ³² P, ¹⁴ C, ³⁵ S or ³ H, alkaline phosphatase, peroxidase (eg, peroxidase) , Β-galactosidase or an enzyme such as phycoerythrin, and a chemical substance such as an acridinium ester.

  Next, the gel electrophoresis method according to the embodiment of the present invention will be described.

  FIG. 7 is a flowchart for explaining a gel electrophoresis method according to an embodiment of the present invention. FIGS. 8, 9, 10 and 11 are an electrode connection process and a first electrophoresis process shown in FIG. FIG. 5 is a conceptual diagram for explaining an electrode changing step and a second electrophoresis step.

  The gel electrophoresis method according to the present embodiment is used to separate a target substance contained in whole blood by electrophoresis. As shown in FIG. 7, the sample placement step, the electrode connection step, the first electrophoresis A process, an electrode changing process, and a second electrophoresis process.

  In the specimen placement step, whole blood 90 is placed in the well 20. Thereby, the whole blood 90 is communicated with the first buffer solution 34 via the back gel 26, and is communicated with the second buffer solution 54 via the electrophoresis gel 22 (the introduction gel 23 and the separation gel 24).

  In the electrode connection step, as shown in FIG. 8, the switching mechanism 76 is controlled, the power source 72 and the first and second electrodes 32 and 52 are connected, and voltage application to the first and second electrodes 32 and 52 is performed. Is set to be possible.

  In the first electrophoresis step, as shown in FIG. 8, voltage application to the first and second electrodes 32 and 52 is started, and the target substance and blood cell 92 contained in the whole blood 90 are directed toward the introduction gel 23. Run. At this time, the target substance moves to the electrophoresis gel 22 (introduction gel 23), while the blood cell 92 is deposited on the other end of the well 20 (the boundary between the well 20 and the introduction gel 23), thereby obstructing the flow of electricity. As a result, electrophoresis is inhibited (see FIG. 6).

  In the electrode changing step, the connection between the power source 72 and the first and second electrodes 32 and 52 is released by controlling the switching mechanism 76 as shown in FIG. The voltage application to the first and second electrodes 32 and 52 is stopped, and the voltage application to the second and third electrodes 52 and 62 is performed by connecting the power source 72 to the second and third electrodes 52 and 62. Is set to be possible.

  In the second electrophoresis step, voltage application to the second and third electrodes 52 and 62 is started. Since the third buffer solution 64 in which the third electrode 62 is disposed and stored in the third buffer tank 60 communicates with the introduction gel 23, as shown in FIG. 11, the migration gel 22 (the introduction gel 23 and the separation gel 23 is separated). Electrophoresis in gel 24) is resumed normally. That is, since blood cells 92 that inhibit electrophoresis are not deposited on the introduction gel 23, when applying voltage to the second and third electrodes 52, 62 to resume electrophoresis, the electrophoresis gel 22. Electrophoresis with respect to the target substance that has already been transferred to is not inhibited, and the target substance can be separated well.

  Next, the results of target substance separation by gel electrophoresis will be described.

  FIG. 12 is an electrophoretic photograph relating to samples 1 to 4, and FIGS. 13 and 14 are enlarged views of essential parts of the electrophoretic photographs relating to sample 1 and sample 2.

  The target substance is C-peptide contained in whole blood. Whole blood was fasting and 6 μL was used.

  Sample 1 and sample 2 relate to the present embodiment, and are common in that electrophoresis is resumed by switching the electrode pair from the first and second electrodes to the second and third electrodes during the electrophoresis. However, sample 1 is applied as it is with whole blood as a specimen, and sample 2 is added by adding C-peptide standard (Wako Pure Chemical Industries, Ltd.) to the concentration of C-peptide contained in whole blood. It is adjusted to increase by 4 ng / mL.

  Sample 3 and sample 4 relate to the comparative example and are common in that the electrode pair is not switched. However, sample 3 is applied as it is as a whole blood sample, and sample 4 is a C contained in whole blood. -Peptide concentration is adjusted to increase by +2.4 ng / mL by adding C-peptide standard (Wako Pure Chemical Industries, Ltd.).

  Visualization was performed by labeling Anti-h-Ceptide 9101 (MedixBiochemical) with the fluorescent substance IRDye800CW (MESTEKNO Co., Ltd.) and detected by the Odyssey (registered trademark) imaging system (MESTEKNO Corporation).

  As shown in FIG. 12, in the sample 3 and the sample 4 according to the comparative example, the electrophoretic image was disturbed, and the complex band and the unreacted labeled antibody band were not well separated. On the other hand, in the sample 1 according to the present embodiment, the unreacted labeled antibody bands are well separated as clearly shown in FIG. In Sample 2 according to the present embodiment, as clearly shown in FIG. 14, the complex band and the unreacted labeled antibody band are well separated. Thus, it was shown that switching electrode pairs during electrophoresis is effective for analyzing whole blood.

  Next, Modifications 1 to 3 according to the present embodiment will be sequentially described.

  FIG. 15 is a schematic diagram for explaining the first modification according to the embodiment of the present invention, FIG. 16 is a schematic diagram for explaining the second electrophoresis step according to the first modification, and FIG. FIG. 18 is a schematic diagram for explaining a second electrophoresis step according to Modification Example 3. FIG. 18 is a schematic diagram for explaining Modification Example 2 according to the embodiment of the invention.

  The third electrode 62 (third buffer tank 60) is not limited to a configuration in which the third electrode 62 (third buffer tank 60) is disposed between the first electrode 32 (first buffer tank 30) and the second electrode 52 (second buffer tank 50). Accordingly, the arrangement can be variously modified.

  For example, as shown in FIG. 15, the third electrode 62 (third buffer tank 60) is opposite to the second electrode 52 (second buffer tank 50) with respect to the first electrode 32 (first buffer tank 30). It is also possible to arrange them. Also in this case, in the second electrophoresis step, as shown in FIG. 16, when the voltage application is executed to the second and third electrodes 52 and 62 and the electrophoresis is restarted, Since the blood cells 92 that inhibit the migration are not deposited, the electrophoresis of the target substance that has moved to the introduction gel 23 is not inhibited, and the target substance can be separated well.

  Further, as shown in FIG. 17, the third electrode 62 (third buffer tank 60) can be juxtaposed with the first electrode 32 (first buffer tank 30). Also in this case, in the second electrophoresis step, as shown in FIG. 18, when the voltage application is executed to the second and third electrodes 52 and 62 and the electrophoresis is restarted, Since the blood cells 92 that inhibit the migration are not deposited, the electrophoresis of the target substance that has moved to the introduction gel 23 is not inhibited, and the target substance can be separated well.

  FIG. 19 is a schematic diagram for explaining the third modification according to the embodiment of the present invention, and FIG. 20 is a schematic diagram for explaining the second electrophoresis step according to the third modification.

  The number of electrodes applied to the electrophoresis is not limited to three. For example, the fourth electrode 68 as the second anode can be disposed in the third buffer tank 60. In this case, the power supply unit 70 is configured to control voltage application to the fourth electrode 68.

  Specifically, as illustrated in FIG. 19, the power supply unit 70 includes power supplies 72 and 73, electric circuits 74 and 75, and switching mechanisms 76 and 77. The power source 72, the electric circuit 74, and the switching mechanism 76 are used to control voltage application to the second and third electrodes 52 and 62. The power source 73, the electric circuit 75, and the switching mechanism 77 are used to control voltage application to the first and fourth electrodes 32 and 68.

  Next, a gel electrophoresis method according to Modification 3 will be described.

  In the specimen placement step, whole blood 90 is placed in the well 20. As a result, the whole blood 90 is communicated with the first buffer solution 34 via the rear gel 26 and is communicated with the third buffer solution 64 via the introduction gel 23.

  In the electrode connection step, the switching mechanism 77 is controlled, the power source 73 and the first and fourth electrodes 32 and 68 are connected, and voltage application to the first and fourth electrodes 32 and 68 is set to be possible. At this time, the power source 72 and the second and third electrodes 52 and 62 are not connected.

  In the first electrophoresis step, voltage application to the first and fourth electrodes 32 and 68 is started, and the target substance and blood cells 92 contained in the whole blood 90 migrate toward the introduction gel 23 (see FIG. 19). . At this time, the target substance moves to the electrophoresis gel 22 (introduction gel 23), while the blood cell 92 is deposited on the other end of the well 20 (the boundary between the well 20 and the introduction gel 23), thereby obstructing the flow of electricity. As a result, electrophoresis is inhibited.

  In the electrode changing step, in order to cope with the inhibition of electrophoresis, the switching mechanism 77 is controlled to disconnect the power source 73 from the first and fourth electrodes 32 and 68, and the first and fourth electrodes 32. , 68 is stopped and the switching mechanism 76 is controlled to connect the power source 72 to the second and third electrodes 52, 62, so that the voltages to the second and third electrodes 52, 62 are connected. Application is set to be possible.

  In the second electrophoresis step, voltage application to the second and third electrodes 52 and 62 is started. Since the third buffer solution 64 in which the third electrode 62 is disposed and stored in the third buffer tank 60 communicates with the introduction gel 23, as shown in FIG. 20, the migration gel 22 (the introduction gel 23 and the separation gel 23 is separated). Electrophoresis in gel 24) is resumed normally. That is, since blood cells 92 that inhibit electrophoresis are not deposited on the introduction gel 23, when applying voltage to the second and third electrodes 52, 62 to resume electrophoresis, the electrophoresis gel 22. Electrophoresis with respect to the target substance that has already been transferred to is not inhibited, and the target substance can be separated well.

  As described above, in this embodiment, when the target substance contained in whole blood is separated by electrophoresis, if electrophoresis is inhibited by the accumulation of blood cells that cannot enter the electrophoresis gel, an electrode to which a voltage is applied is applied. The pair is switched from the first and second electrodes to the second and third electrodes, and electrophoresis is resumed. The second buffer stored in the second buffer tank in which the second electrode is disposed is provided so as to communicate with the other end of the electrophoresis gel located on the opposite side of the well, and the third electrode is disposed. In the middle part of the electrophoresis gel communicating with the third buffer stored in the third buffer tank, blood cells that inhibit electrophoresis are not deposited. Therefore, the voltage application function of the second and third electrodes is not interfered by the accumulated blood cells. Therefore, when voltage is applied to the second and third electrodes and electrophoresis is resumed, electrophoresis on the target substance that has already moved (invaded) into the electrophoresis gel is not inhibited, and the target substance is well separated. be able to. That is, it is possible to provide a gel electrophoresis apparatus and a gel electrophoresis method that can favorably separate a target substance contained in whole blood.

  The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims. For example, a target substance contained in whole blood can be separated and quantified without using a substance (receptor) that specifically binds to the target substance. It is also possible to omit the back gel as needed, or to appropriately omit the introduction gel by giving the separation gel the function of the introduction gel.

10 gel electrophoresis apparatus,
12 device body,
20 wells,
22 electrophoresis gel,
23 Introducing gel,
24 separation gel,
26 Back gel,
30 First buffer tank,
32 first electrode;
34 first buffer,
40 support,
50 second buffer tank,
52 second electrode,
54 second buffer solution,
60 third buffer tank,
62 third electrode,
64 third buffer,
66 through holes,
68 4th electrode,
70 power supply,
72, 73 power supply,
74,75 electric circuit,
76, 77 switching mechanism,
90 whole blood,
92 Blood cells.

Claims (14)

A gel electrophoresis apparatus for separating a target substance contained in whole blood by electrophoresis,
A well in which the whole blood is placed as a specimen;
A first buffer tank in which a first electrode is arranged and a first buffer solution to be stored communicates with one end of the well;
A supporting part on which an electrophoresis gel is placed and provided so that one end of the electrophoresis gel communicates with the other end of the well;
A second buffer tank in which a second electrode is disposed and a second buffer solution to be stored communicates with the other end of the electrophoresis gel;
A third buffer tank in which a third electrode is disposed and provided so that a third buffer solution to be stored communicates with an intermediate portion between one end and the other end of the electrophoresis gel;
A power supply unit that controls voltage application to the first to third electrodes,
The power supply unit is
Electrophoresis initiated by applying a voltage to the first electrode and the second electrode is inhibited by blood cells contained in the whole blood deposited on the other end of the well without being able to enter the electrophoresis gel. Then, the voltage application to the first electrode and the second electrode is stopped, and the voltage application to the second electrode and the third electrode is executed to resume electrophoresis. Gel electrophoresis device. The electrophoresis gel has an introduction gel located on one end side of the electrophoresis gel, and a separation gel located on the other end side of the electrophoresis gel,
The introduction gel communicates with the other end of the well, and constitutes the intermediate portion of the electrophoresis gel;
The gel electrophoresis apparatus according to claim 1, wherein the separation gel communicates with the second buffer solution, and the gel concentration of the introduced gel is smaller than the gel concentration of the separation gel. A back gel interposed between one end of the well and the first buffer,
The gel electrophoresis apparatus according to claim 2, wherein the back gel suppresses the whole blood disposed in the well from entering the first buffer solution. The separation of the target substance is based on the difference in mobility of electrophoresis caused by the difference in molecular weight between the complex of the target substance and receptor and the free receptor,
The gel electrophoresis apparatus according to any one of claims 1 to 3, wherein at least two or more kinds that specifically recognize different portions of the target substance are used as the receptor.   The gel electrophoresis apparatus according to claim 4, wherein the target substance has a molecular weight of 1000 to 30000.   The gel electrophoresis apparatus according to claim 4 or 5, wherein the molecular weight of the target substance is 1/150 to 1/5 of the molecular weight of the receptor. A fourth electrode disposed in the third buffer tank;
The power supply unit is
Configured to control voltage application to the fourth electrode;
Electrophoresis started by executing voltage application to the first electrode and the fourth electrode is inhibited by blood cells contained in the whole blood deposited on the other end of the well without entering the electrophoresis gel. Then, the voltage application to the first electrode and the fourth electrode is stopped, and the voltage application to the second electrode and the third electrode is executed to resume electrophoresis. The gel electrophoresis apparatus according to any one of claims 1 to 6. A gel electrophoresis method for separating a target substance contained in whole blood by electrophoresis,
As a sample, a well having one end where the first buffer stored in the first buffer tank in which the first electrode is arranged communicates and the other end where one end of the electrophoresis gel placed on the support portion communicates Placing the whole blood,
A voltage is applied to the first electrode and the second electrode disposed in a second buffer tank provided so that the stored second buffer solution communicates with the other end of the electrophoresis gel,
When electrophoresis started by executing the voltage application is inhibited by blood cells contained in the whole blood deposited on the other end of the well without entering the electrophoresis gel, the first electrode and the Stop applying voltage to the second electrode,
For the second electrode and the third electrode disposed in a third buffer tank provided so that the third buffer solution to be stored communicates with an intermediate portion between one end and the other end of the electrophoresis gel. And applying a voltage to restart the electrophoresis. The electrophoresis gel has an introduction gel located on one end side of the electrophoresis gel, and a separation gel located on the other end side of the electrophoresis gel,
The introduction gel communicates with the other end of the well, and constitutes the intermediate portion of the electrophoresis gel;
The gel electrophoresis method according to claim 8, wherein the separation gel is in communication with the second buffer solution, and the gel concentration of the introduced gel is smaller than the gel concentration of the separation gel. A back gel interposed between one end of the well and the front first buffer,
The gel electrophoresis method according to claim 9, wherein the back gel suppresses the whole blood arranged in the well from entering the first buffer solution. The separation of the target substance is based on the difference in mobility of electrophoresis caused by the difference in molecular weight between the complex of the target substance and receptor and the free receptor,
The gel electrophoresis method according to any one of claims 8 to 10, wherein at least two or more kinds that specifically recognize different sites of the target substance are used as the receptor.   The gel electrophoresis method according to claim 11, wherein the molecular weight of the target substance is 1000 to 30000.   The gel electrophoresis method according to claim 11 or 12, wherein the molecular weight of the target substance is 1/150 to 1/5 of the molecular weight of the receptor. A fourth electrode is further disposed in the third buffer tank;
Electrophoresis started by executing voltage application to the first electrode and the fourth electrode is inhibited by blood cells contained in the whole blood deposited on the other end of the well without entering the electrophoresis gel. Then, the voltage application to the first electrode and the fourth electrode is stopped, and the voltage application to the second electrode and the third electrode is executed to resume electrophoresis. The gel electrophoresis method according to any one of claims 8 to 13.