WO2014083637A1

WO2014083637A1 - Method for separating target-receptor complex and free receptor

Info

Publication number: WO2014083637A1
Application number: PCT/JP2012/080748
Authority: WO
Inventors: 嘉哉佐藤
Original assignee: テルモ株式会社
Priority date: 2012-11-28
Filing date: 2012-11-28
Publication date: 2014-06-05
Also published as: WO2014083861A1

Abstract

[Problem] To provide a method for separating a target-receptor complex and a free receptor by electrophoresis. [Solution] A method for separating a complex of a target and a receptor that specifically binds to the target and a free receptor, wherein an isoelectric point of the receptor is higher than that of the complex, and electrophoresis is carried out under a complex non-denaturing condition in a buffer solution which has a pH that exceeds the isoelectric point of the complex and is +2 or less of the isoelectric point of the receptor.

Description

Method for separating target substance-receptor complex and free receptor

The present invention relates to a method for separating a target substance-receptor complex from a free receptor.

Some peptides present in the living body change in abundance depending on the disease, and these peptides can serve as markers for specific diseases. For example, blood C-peptide is a component of proinsulin, which is a pre-stage substance that synthesizes insulin, and is secreted into the blood at a rate similar to that of insulin, and circulates in the blood with almost no degradation. And discharged with urine. Therefore, the amount of insulin secreted can be determined by measuring C-peptide in blood or urine.

Therefore, it is possible to diagnose a disease by specifically detecting a peptide related to a specific disease, and the method for detecting these peptides can be an important clinical test.

On the other hand, a method using a substance (receptor) that specifically binds to a target substance is widely used for separating and quantifying the target substance. When the target substance is an antigen, an antigen-antibody reaction using an antibody (fragment) can be used. Since an antibody has a very high binding specificity to an antigen, the antigen can be specifically detected by utilizing an antigen-antibody reaction.

When a target substance-receptor complex is obtained using a receptor having a binding property specific to the target substance, it is generally necessary to separate the complex from the free receptor. As a separation method, after fixing an antigen to a well plate or a transfer membrane, an antibody is bound, and then an unreacted free antibody is washed and separated and removed, such as ELISA or Western blotting. Often used in However, this method takes time and requires some skill. Furthermore, it is difficult to automate a series of operations necessary for detection by this method, and it is very difficult to reduce the size for instant diagnosis at Point Of Care.

As a method for separating a target substance-receptor complex and a free receptor with a small number of work steps, there is an electrophoretic mobility shift assay (EMSA) (Non-patent Document 1). In the electrophoretic mobility shift assay (EMSA), a target substance and a receptor are reacted, and an increase in molecular weight (complex formation) caused thereby is detected by a molecular sieving effect of gel electrophoresis.

However, in order to detect an increase in molecular weight, the target substance and the receptor molecule must have the same size. Therefore, when the target substance is a relatively small molecule such as a peptide, the increase in the overall molecular weight including the receptor is small if the target substance is simply bound to the receptor. Therefore, even if the target substance-receptor complex is separated by electrophoresis, the target substance-receptor complex and the free receptor are not clearly separated, and the target substance-receptor complex and the free receptor are separated by the difference in molecular weight. It was very difficult to separate. That is, it was difficult to detect the antigen by ordinary electrophoresis.

Therefore, an object of the present invention is to provide a method for separating a target substance-receptor complex and a free antibody by electrophoresis.

The present invention is characterized in that electrophoresis is performed in a buffer solution in which a charge difference between a target substance-receptor complex and a free receptor is generated under non-denaturing conditions of the target substance-receptor complex. A method for separating the receptor complex from the free receptor.

It is a figure which shows the result of an Example. It is a figure which shows the result of a comparative example. It is a figure which shows the result of a comparative example.

A first embodiment of the present invention is a method for separating a target substance and a complex of a receptor that specifically binds to the target substance and a free receptor, wherein the isoelectric point of the receptor is the isoelectric point of the complex And the electrophoresis is performed in a buffer solution having a pH that exceeds the isoelectric point of the complex and not more than +2 of the isoelectric point of the receptor under the non-denaturing condition of the complex. A method for separating the receptor complex from 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 an extremely simple operation compared to the ELISA method.

The present invention utilizes the fact that a difference in charge occurs between the charge of the target substance-receptor complex and the charge of the free receptor in the electrophoresis buffer. Therefore, a difference occurs in the mobility of electrophoresis between the target substance-receptor complex and the free receptor, and the target substance-antibody complex and the free antibody can be clearly separated.

The present inventors paid attention to the isoelectric point of these compounds when detecting compounds having both an anion functional group and a cation functional group, such as peptides and proteins. In consideration of the isoelectric point of these compounds, the pH of the electrophoresis buffer for electrophoresis and the isoelectric point of the receptor that specifically binds to the target substance can be controlled or selected for electrophoresis. It has been found that there is a charge difference between the charge of the target substance-receptor complex (hereinafter also simply referred to as complex) and the charge of the free receptor in the buffer solution, and that the charge difference can separate the two. The present invention has been completed. Thus, according to the method of the present invention, the target substance can be detected by a very simple method. When the target substance is a substance related to a disease, the diagnosis of the disease can be easily performed by detecting the target substance.

Hereinafter, specific embodiments of the present invention will be described in detail.

[Samples used for electrophoresis]
The sample used for electrophoresis is a sample after performing a reaction (for example, 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. . Therefore, the sample used for electrophoresis contains a target substance-receptor complex and a free receptor.

Such a binding reaction is performed by a conventionally known method. As an aspect of the antigen-antibody reaction conditions, the reaction temperature is usually 4 to 50 ° C., preferably 4 to 42 ° C., more preferably 20 to 40 ° C., and the reaction time is usually 5 minutes to 24 hours. The reaction solvent is, for example, phosphate buffer, carbonate buffer, Tris buffer, glycine buffer, tricine buffer or the like. can do. The amount of the receptor to be reacted with the target antigen may be appropriately set, but usually a large excess of the receptor is added. Specifically, it is usually 1 to 100 moles per mole of antigen. When subjected to the reaction, the antigen and receptor are used after being dissolved in an appropriate solvent (for example, phosphate buffer, carbonate buffer, Tris buffer, Tricine buffer, glycine buffer, Tris-glycine buffer, etc.). be able to. The sample subjected to the antigen-antibody reaction may be pretreated before electrophoresis. Examples of the pretreatment include an operation of removing large aggregates by centrifugal operation or filter filtration.

[Target substance]
The target substance in the present invention is a substance having an isoelectric point. Since the isoelectric point of the complex is lower than the isoelectric point of the receptor, the isoelectric point of the target substance is naturally lower than the isoelectric point of the receptor.

The target substance is not particularly limited as long as it has an isoelectric point and the charge changes with pH, and examples thereof include proteins, peptides, sugar chains, and complexes thereof. Among these, the target substance is preferably an amphoteric substance, or is preferably a peptide or protein from the viewpoint of ease of operation and versatility.

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.

The molecular weight of the target substance is not particularly limited. In the case of a peptide having a low molecular weight, for example, a peptide, it is necessary to study various conditions for electrophoresis when trying to detect a peptide alone, and it is easier to perform electrophoresis with a peptide bound to a receptor such as an antibody. Can detect the target. Furthermore, a target substance having a low molecular weight is not clearly separated due to the small difference in molecular size between the free receptor and the complex even by separation with a molecular sieve. However, according to the separation method of the present invention, a free antibody and a complex can be easily and clearly separated even with a target substance having a relatively small molecular weight. From such a viewpoint, in the present invention, a target substance having a relatively small molecular weight is preferable. Specifically, the molecular weight is preferably 500 to 10,000, more preferably 1,000 to 10,000, and still more preferably 1,000 to 5,000. When the target substance is an unknown substance, the molecular weight can be determined by a known analysis method (SDS-PAGE, amino acid analysis, etc.).

Since the charge difference from the receptor can be easily controlled and separation by electrophoresis can be performed quickly, the isoelectric point of the target substance is preferably low to some extent. Specifically, the isoelectric point of the target substance is preferably 6 or less, more preferably 5 or less, and even more preferably 4 or less. Since the lower isoelectric point of the target substance is preferable, the lower limit is not particularly set, but the biological substance has an isoelectric point of usually 3 or more. The isoelectric point can be examined by staining the gel after performing isoelectric focusing. A conventionally known method can be adopted as the staining method, and examples thereof include a CBB (Coomassie Brilliant Blue) staining method and a silver staining method.

Specific examples of target substances include the following peptides: C-peptide, corticotropin-like intermediate peptide, proenkephalin (PENK), gastrin, fibrinopeptide (Fibrinopeptide) Guanyli, Uroguanylin, Mammaglobin-A, Cholecystokinin, Cepeptin.

Note that, unless otherwise specified in the present specification, “protein” includes a peptide, but a polypeptide of less than 10 kDa may be particularly referred to as “peptide”. The protein may be a natural product or a synthetic product.

The target substance is a sample (blood, plasma, serum, tissue, joint fluid, urine, human or non-human mammal (eg, rat, mouse, rabbit, sheep, pig, cow, cat, dog, monkey, etc.) Lymph, etc.), cells (cultured cells, cell lines, etc.), culture supernatants thereof, extracts thereof, partially purified fractions thereof and the like. Moreover, you may extract from soil, water, etc. of various environments. At this time, the target substance can be produced by purification using a known protein purification method. Specifically, for example, mammalian tissues or cells are homogenized in the presence of a surfactant, and the resulting crude extract fraction of tissues is subjected to chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography and the like. It can be purified by attaching to a graph or the like. The target substance may be a commercial product.

Further, the test sample may be used as it is without purifying (extracting) the target substance. For example, the target substance-receptor binding reaction may be performed using a test sample such as a biological component such as blood, plasma, serum, tissue, joint fluid, urine, and lymph of the subject. At this time, a treatment such as dilution may be performed on the test sample.

[Receptor]
In the present invention, a receptor refers to a substance that can specifically bind to a target substance.

The isoelectric point of the receptor is higher than the isoelectric point of the complex. By setting the pH of the buffer as described above and setting the isoelectric point of the receptor in this way, the receptor has a negative charge smaller than the negative charge of the complex, or a positive charge, or It has no charge, and the mobility of the free receptor with respect to the positive electrode in electrophoresis is smaller than that of the complex. Therefore, separation of the free receptor and the complex by electrophoresis is easily and clearly performed.

The farther the isoelectric point of the receptor is from the isoelectric point of the target substance-receptor complex, the greater the difference in charge between the two due to pH adjustment of the buffer solution, and the easier it is to improve the resolution. Specifically, the isoelectric point of the receptor is preferably 0.1 or more higher than the isoelectric point of the target substance-receptor complex, more preferably 0.2 or more, and even more preferably 1 or more. It is particularly preferably 2 or more, and most preferably 3 or more. The isoelectric point of the receptor is preferably as far as possible from the isoelectric point of the target substance-receptor complex, but is usually 6 or less than the isoelectric point of the target substance-receptor complex.

The isoelectric point of the receptor is preferably 6 or more, more preferably 7 or more, and still more preferably 8 or more. When the isoelectric point is in such a range, the electrophoresis buffer can be set to the alkaline side, and the charge of the complex is more negatively charged. The separation of the free receptor and the complex can be performed more rapidly. Since the higher isoelectric point of the antibody is preferable, the upper limit is not particularly set, but it is usually 9 or less.

As a receptor, it is necessary to maintain the target substance binding activity in an electrophoresis buffer.

The isoelectric point of the receptor can be controlled by modification such as addition of a functional group. For example, when the isoelectric point of the receptor to be used is not a desired isoelectric point, an appropriate substance may be added so as to increase the isoelectric point. For example, the isoelectric point can be controlled by binding a carboxyl group, an amino group, or a sugar chain containing an amino sugar or uronic acid to a receptor molecule. Such a modification method of the modifying substance can be performed by a conventionally known method.

For example, the introduction of the modifying group can be performed using a cross-linking agent that can react with the functional group of the receptor before modification. Examples of the cross-linking agent include N-hydroxysuccinimide (NHS), N-succinimidyl (4-iodoacetyl) aminobenzoate (N-succinimidyl (4-iodoacetyl) aminobenzoate) (SIAB), dimaleimide, dithio-bis- Nitrobenzoic acid (dithio-bis-nitrobenzoic acid) (DTNB), N-succinimidyl-S-acetyl-thioacetate (N-succinimidyl-S-acetyl-thioacetate) (SATA), N-succinimidyl-3- (2-pyridyl) Dithio) propionate (N-succinimidyl-3- (2-pyridyldithio) propionate) (SP P), succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (succinimidyl) cyclohexane-1-carboxylate (SMCC), 6-hydrazinonicotideNIC ), M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBC), and the like.

As a modification method, for example, when the carboxyl group of the receptor is used, the carboxyl group is activated with carbodiimides such as 1-ethyl-3- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC). Then, it can be reacted with a compound having an amino group.

Specific examples of receptors include antibodies, antibody fragments, receptors, binding proteins, peptides and the like. Among them, it is preferable to use an antibody or an antibody fragment as a receptor because of its high specific binding property to an antigen.

Examples of antibodies include monoclonal antibodies, polyclonal antibodies, single chain antibodies, modified antibodies (eg, “humanized antibodies” in which only the antigen recognition site is humanized), chimeric antibodies, and bifunctionality capable of simultaneously recognizing two epitopes. An antibody etc. are mentioned. The antibody may be of any class such as IgA, IgD, IgE, IgG, IgM. From the viewpoint of specific binding to an antigen, it is more preferable to use a monoclonal antibody.

The receptor can be produced by a conventionally known method.

Commercially available antibodies may be used.

Such an antibody may be modified within a range where binding to an antigen is not inhibited. Examples of such modifications include fluorescent labeling in which a fluorescent labeling substance is bound by a chemical bond to improve detection efficiency, radioactive labeling in which the atoms constituting the antibody are labeled with a radioisotope, and a pairing substance Specific binding pair labeling in which a substance exhibiting specific binding property is chemically bound (for example, a label in which a lectin having binding property to a polysaccharide is chemically bound) and the like are exemplified. Examples of labeling substances used for labeling include fluorescent substances such as FITC (fluorescein isocyanate) or tetramethylrhodamine isocyanate, radioisotopes such as ¹²⁵ I, ³² P, ¹⁴ C, ³⁵ S or ³ H, alkaline phosphatase, peroxidase, β- Enzymes such as galactosidase or phycoerythrin are included. Alternatively, the antibody may be fused with a fluorescent protein such as green fluorescent protein (GFP).

The receptor may be an antibody fragment as long as it has reactivity with the antigen. Examples of the antibody fragment include Fab fragment, F (ab) ′ ₂ fragment, single chain antibody (scFv), scFv− Examples thereof include conjugate molecules prepared by genetic engineering such as Fc, minibody, and diabody, or derivatives thereof modified with a molecule having a protein stabilizing action such as polyethylene glycol (PEG).

[Electrophoresis]
In the present invention, since it is necessary for the target substance and the receptor to specifically bind even under electrophoresis, it is necessary to perform electrophoresis in an environment in which the receptor maintains the target substance binding activity. Therefore, electrophoresis is performed under non-denaturing conditions of the target substance-receptor complex. The “under non-denaturing target substance-receptor complex” means a condition under which the receptor maintains the target substance binding ability. When the receptor is a protein, specifically, “target substance-receptor complex non-denaturing condition” refers to the absence of a protein denaturing agent. Therefore, the sample is not heated prior to electrophoresis as in SDS-PAGE. Examples of the protein denaturant include an anionic surfactant (for example, SDS or LDS) or a chaotropic agent (urea, formamide, guanidine, potassium iodide, etc.). A native page, which is a kind of polyacrylamide gel electrophoresis (PAGE), is known as an electrophoresis method for electrophoresis of proteins without denaturation. The native page can also be used in the present invention. In general native pages, a buffer solution having a high pH condition using a tris-glycine system is used. In the present invention, a buffer solution adjusted to a specific pH is used.

The electrophoresis method may be a carrier-free electrophoresis method such as capillary (zone) electrophoresis or microchip electrophoresis, or an electrophoresis method using a carrier.

Examples of the carrier used for electrophoresis include agarose gel, acrylamide, dextran gel, filter paper, cellulose acetate membrane, and capillary polymers described below.

It is preferable to use acrylamide as a carrier because it is highly transparent and electrically neutral. In this case, the acrylamide gel may be a concentration gradient gel or a uniform concentration gel. Specifically, a concentration gradient gel of 4-16% (w / v), a concentration gradient gel of 5-20% (w / v), or a constant value of 8% (w / v) to 12% (w / v) A concentration gel can be used. In general, a band is sharper in a concentration gradient gel than in a constant concentration gel. The acrylamide gel may be appropriately adjusted, or a commercially available product may be used. Examples of the acrylamide gel include a tris-glycine gel (for example, Novex (registered trademark) Tris-glycine gel (Invitrogen), etc.), a tris-acetic acid gel (for example, NuPAGE Novex (registered trademark) (Invitrogen)), or a bistris gel (for example, And NativePAGE Novex (registered trademark) Bistris gel (Invitrogen), etc.). The pH of the buffer used when producing the acrylamide gel is preferably about 6-8.

The present invention can also be applied to isoelectric focusing in which a pH gradient is formed in the electrophoresis gel.

In addition, a polymer that has a molecular sieving effect may be filled in a capillary used for capillary electrophoresis together with a buffer for electrophoresis (capillary polymer solution electrophoresis). In this case, the polymer (capillary polymer) filled in the capillary is not particularly limited as long as it is usually used in this field. Specifically, polyethylene oxide (polyethylene glycol), polypropylene oxide Polyalkyleneimines such as polyethyleneimine; Poly (meth) acrylic acid polymers such as poly (meth) acrylic acid and poly (meth) acrylic acid esters such as methyl poly (meth) acrylate; Polyacrylamide Polyamide polymers such as polymethacrylamide; Polyvinyl polymers such as polyvinyl acetate, polyvinyl pyrrolidone and polyvinyl oxazolidone; Water-soluble hydroxyl polymers such as pullulan, erucinane, xanthan, dextran and guar gum; Cellulose, hydroxyethyl cellulose, water-soluble cellulose such as hydroxypropyl cellulose; and derivatives thereof, and copolymers such as a monomer unit constituting these polymers.

The applied voltage in electrophoresis may be appropriately selected from the range usually used in this field, and is usually applied in the range of 5 to 2000 V / cm.

Electrophoresis time can usually be 30 to 180 minutes. From the viewpoint of rapid separation, the electrophoresis time is preferably about several minutes to 10 minutes. The electrophoresis temperature is not particularly limited, but is usually performed at room temperature (20 to 25 ° C.).

As a method for detecting the target substance-receptor complex and free antibody after electrophoresis, gel staining can be mentioned. As a staining method when the receptor is a protein, CBB (Coomassie Brilliant Blue) staining method, silver staining method, SYPRO (registered trademark) Ruby Protein Stein (manufactured by Takara Bio Inc.), Deep Purple Total Protein Stain (GE Healthcare) A dyeing method using a dye such as Japan). Alternatively, the amount of the protein can be quantified by calculating the signal intensity of the dye for the band of the antigen-receptor complex separated from the free antibody using image analysis software or the like. In the case of capillary electrophoresis, it can be directly detected using a UV / VIS absorptiometer (photodiode array) or the like.

(Buffer solution)
The buffer used for electrophoresis has a pH that exceeds the isoelectric point of the target substance-receptor complex and is not more than +2 of the isoelectric point of the receptor. By setting the pH of the buffer so as to exceed the isoelectric point of the target substance-receptor complex, a charge difference is generated between the complex and the receptor, and the complex and the free receptor can be separated in electrophoresis. . Further, when the pH of the buffer exceeds the isoelectric point +2 of the receptor, the negative charges of both the complex and the receptor in the buffer are increased, so that the charge difference with respect to the charge is reduced and the separability is deteriorated. More preferably, the pH of the buffer is +1.5 or less of the isoelectric point of the receptor and +1 or more of the isoelectric point of the complex. By setting the pH of the buffer to +1 or more of the isoelectric point of the complex, the complex has an appropriate charge and the separability is further improved. Here, “the pH of the buffer is +1.5 or less of the isoelectric point of the receptor and +1 or more of the isoelectric point of the complex” means that the isoelectric point of the receptor is 7, and the isoelectric point of the complex Is 5 means that the pH of the buffer is 6 to 8.5. The buffer for adjusting the pH is a buffer solution that comes into contact with the sample when electrophoresis is performed, that is, a buffer solution for running (running buffer).

From the viewpoint of maintaining the structure of the target substance, receptor and complex, it is not desirable that the pH of the buffer solution is a strong acidic region or a strong alkaline region. In the present invention, since the charge difference between the complex and the free receptor is used, the buffer solution does not need to be in the alkaline region. From such a viewpoint, the pH of the electrophoresis buffer is preferably 6-8.

The pH of the buffer solution may be adjusted as appropriate using an acidic substance such as hydrochloric acid or a basic substance such as sodium hydroxide.

In addition, it is necessary to adjust the pH of the buffer solution in a pH range where the buffer action of the buffer solution is not lost, and the buffer solution may be appropriately selected according to the pH of the desired buffer solution. For example, hydrochloric acid-potassium chloride buffer solution, glycine-hydrochloric acid buffer solution, citrate-phosphate buffer solution, citrate buffer solution, etc. in the acidic region of pH 7 or lower, and tris-hydrochloric acid buffer solution in the neutral region of about pH 6-8. Solution, phosphate buffer, tris-glycine buffer, tris-tricine buffer solution, bis-tris buffer solution, MES buffer solution, MOPS buffer solution, TES buffer solution, PIPES buffer solution, etc. Glycine-sodium hydroxide buffer or the like may be used.

The buffer solution is not particularly limited as long as it is a solution containing a conventionally known buffer solution composition having a buffer capacity, and examples thereof include organic acids such as citric acid, succinic acid, tartaric acid, malic acid, and salts thereof. Solutions containing amino acids; amino acids such as glycine, taurine and arginine; and solutions containing inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, boric acid and acetic acid, and salts thereof. As a buffer for electrophoresis when the receptor is a protein, for example, Tris-Glycine buffer, Tris buffer, Tris-Tricine buffer, Tris-HCl buffer, etc., and generally used as a buffer for protein electrophoresis The buffer solution etc. which are provided in the commercially available kit for protein electrophoresis can also be used. The electrophoresis buffer can be used at a concentration generally used as an electrophoresis buffer.

The target substance-receptor complex and the free receptor can be clearly separated by the separation method of the present invention. Therefore, if the separation method of the present invention is used, a target substance that is a target substance can be detected.

Specifically, in the modification of the first embodiment of the present invention, a target substance and a receptor that specifically binds to the target substance are bound to form a target substance-receptor complex, and electrophoresis is performed. And detecting a target substance, wherein the isoelectric point of the receptor is higher than the isoelectric point of the complex, and the electrophoresis is performed under the non-denaturing condition of the complex. This is a method for detecting a target substance, which is carried out in a buffer solution having a pH exceeding the electric point and +2 or less of the isoelectric point of the receptor. Here, “detection” means “detecting the presence, absence or concentration of the target substance”. By such a method, the target substance can be detected simply and specifically.

The step of forming the target substance-receptor complex and the step of performing electrophoresis are as described in the first embodiment.

As a method for detecting the target substance, any method can be used as long as it is a method for visualizing the target substance-receptor complex. Enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), chemiluminescence immunoassay (CLIA) can be used. At this time, a receptor (for example, primary antibody) that reacts directly with the target substance may be labeled (direct method), or the first antigen-receptor reaction is performed using an unlabeled primary antibody or the like, and the primary antibody itself. Alternatively, another antibody (secondary antibody) (indirect method) may be labeled.

As the labeling substance used for labeling, FITC (fluorescein isothiocyanate), FITC (Fluorescein Isothiocyanate), PE (phycoerythrin), APC (Allophycocyanin), Cy-3, Cy-5, the fluorescent substances such as tetramethylrhodamine ^{isocyanate, 125} Examples include radioisotopes such as I, ³² P, ¹⁴ C, ³⁵ S or ³ H, alkaline phosphatase, peroxidase (eg, peroxidase), enzymes such as β-galactosidase or phycoerythrin, and chemical substances such as acridinium esters. When an enzyme is used as a labeling substance, a second signal that depends on the enzyme (for example, formation of a colored product from a colorless substrate) may be generated. In this case, a substrate other than the labeling enzyme is required. . For example, 3,3 ′, 5,5′-tetramethylbenzidine or the like is used in the case of peroxidase, and sodium paranitrophenyl phosphate or the like is used in the case of alkaline phosphatase.

As a method of labeling with a labeling substance, a conventionally known method can be used. For example, an active group capable of forming a bond with a specific functional group and a labeling substance active group in which a labeling substance such as a fluorescent substance is bound, and a receptor. There are a method of labeling by mixing, a method of labeling by cross-linking a labeling substance and a receptor through a crosslinker using various crosslinkers having active groups at both ends.

Examples of the indirect method include LAB (Linked Avidin-Biotin) method, ABC (Avidin-Biotin Complex) method, LSAB (Linked Streptavidin-Biotin) method using biotinylated antibody.

As a method for detecting the labeling substance, a conventionally known method can be used. The detection and / or quantification of the product produced by the enzyme can be performed by measuring the absorbance of the product. For example, when 3,3 ′, 5,5′-tetramethylbenzidine is used as the enzyme substrate, the absorbance at 655 nm may be measured. In addition, when the labeling substance is a radioisotope, it may be measured with a radiation counter. In the case of a fluorescent substance, it is visualized by fluorescence excitation, or when quantification is performed, a fluorescence measuring instrument or an image processing system is used. Just measure.

The target substance can be quantified by, for example, creating a calibration curve using the fluorescence intensity obtained using a standard sample containing a known concentration of the target substance, thereby simplifying the amount of target substance contained in the sample. It can be quantified.

The above detection method is a very useful means in clinical examination especially when the target substance is a marker related to some disease.

For example, human 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 blood C-peptide kinetics can be an important indicator for examining the secretory ability of endogenous insulin in diabetic patients and the like. In fact, the measurement of C-peptide is used for the diagnosis or treatment of diabetes, and is also useful for the diagnosis of insulinoma, insulin autoimmune syndrome and the like. According to the separation method of the first embodiment of the present invention or the detection method of this modified example, even a low molecular weight peptide such as C-peptide can be separated and detected. Can be quantified. Furthermore, the amount of insulin secreted can be examined by comparing with normal values. It can be seen that if the amount of C-peptide in the subject is lower than the normal value, the subject's ability to secrete insulin is reduced. Therefore, in a preferred embodiment of the present invention, the target substance is a C-peptide.

In addition, another embodiment of the present invention is a method for diagnosing a disease related to a target substance using the separation / detection method of the first embodiment. Specifically, collecting a sample from a subject, binding a target substance in the sample and a receptor that specifically binds to the target substance to form a target substance-receptor complex, Performing electrophoresis, detecting a target substance, and determining whether the subject suffers from a disease related to the target substance by comparing with a value of a healthy target substance. The isoelectric point of the receptor is higher than the isoelectric point of the complex, and the electrophoresis exceeds the isoelectric point of the complex under the non-denaturing condition of the complex and the isoelectric point of the receptor. This is a method for diagnosing a disease involving a target substance, which is performed in a buffer solution having a pH of +2 or less. Here, the subject or the healthy person is preferably a human or a non-human mammal. Examples of the disease include when the target substance is C-peptide, insulinoma, obesity, liver disease, Cushing syndrome, acromegaly, abnormal insulinemia, insulin autoimmune syndrome, diabetes, hypoglycemia, undernutrition, Examples include pheochromocytoma and hypopituitar adrenal function. If the subject's C-peptide level is higher than that of healthy subjects, they may have insulinoma, obesity, liver disease, Cushing's syndrome, acromegaly, abnormal insulinemia, insulin autoimmune syndrome, etc. If the subject's C-peptide level is lower than that of healthy subjects, the patient may be suffering from diabetes, hypoglycemia, malnutrition, pheochromocytoma, hypopituitar adrenal function, etc. .

Furthermore, another embodiment of the present invention provides a receptor that specifically binds to a target substance and has an isoelectric point higher than the isoelectric point of the target substance, and exceeds the isoelectric point of the target substance-receptor complex. And a set for electrophoresis containing a buffer solution having a pH that is +2 or less of the isoelectric point of the receptor, and a test kit for a disease involving a target substance.

The receptor included in the test kit preferably includes a container containing the receptor. The receptor may be in a state preliminarily dissolved in a buffer solution. The electrophoresis set includes an assay buffer as described above or a concentrated stock solution thereof. The electrophoresis set includes other electrophoresis apparatuses such as electrophoresis tanks, glass plates for electrophoresis, buffer tanks, spacers, combs, clips, power supplies, or peristaltic pumps; reagents for electrophoresis; gels, etc. A carrier; a detection reagent and the like.

Furthermore, the kit may include a target substance having a known concentration as a standard sample and a container containing the target substance. The receptor is preferably labeled with a labeling substance such as a fluorescent substance because diagnosis is easy. Each reagent included in the kit may be provided by being dispensed into a container for each sample measurement, or a plurality of sample measurements may be provided for each reagent in a separate container. . In the latter case, each reagent is dispensed into a predetermined measurement container before use. When the reagent is provided for each sample measurement, the container containing each reagent may be integrally formed as a cartridge, and each reagent may be stored in a different section of the cartridge. When the receptor is included in the kit as a lyophilized product, a buffer as described above suitable for dissolving them may be further included in the kit. Other containers for assays that contain these receptors, or that may be included in kits, do not interact with receptors and target substances and do not interfere with the reactions used in the assay, such as enzyme reactions and chemiluminescent reactions Any material can be used. If necessary, the surface may be pre-treated to avoid such interaction. An instruction manual is usually attached to the test kit.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

(Example 1)
1. Anti h-C-peptide antibody [7E10] (Abcam, Mouse monoclonal) was diluted to 0.08 mg / mL with PBS buffer solution (10 mM pH 7.4) to prepare an antibody solution. The isoelectric point of the anti hC-peptide antibody was pI6.1 as measured by capillary isoelectric focusing gel electrophoresis.

2. C-Peptide (human) (manufactured by Bachem) was diluted to 4, 2, 1, 0.5, 0.25 μM with a PBS buffer solution (10 mM pH 7.4) to prepare an antigen solution.

3.1. 1. 20 μL of antibody solution prepared in 1. 4.7 μL of the antigen solution of each concentration prepared in the above and 20 μL of PBS buffer solution (10 mM pH 7.4) were mixed. Then, it left still at room temperature (25 degreeC) for 1 hour. Here, the isoelectric points of the antigen-antibody complex were pI5.9 (one antigen binding) and pI5.6 (two antigen bindings) as measured by capillary isoelectric focusing gel electrophoresis.

4. Next, 3. 14.9 μL of Loading Buffer (40% (w / v) glycerin, 0.4% (w / v) bromophenol blue (BPB), 0.2 M Tris-HCl pH 7.4) was added to the mixed solution prepared in step 1 above. .

5.4. 10 μL of the mixed solution prepared in (1) was applied to the gel well, and electrophoresis was performed at 200 V constant voltage and room temperature (20 to 25 ° C.) for 120 minutes. 2. The amount of 0.25, 0.5, 1, 2, 4 μM C-Peptide prepared in 1 in each lane was 0.6, 1.2, 2.4, 4.8, and 9.5 ng, respectively. . The equipment and solutions used are as follows.

・ Electrophoresis device XCell SureLock Mini-Cell (manufactured by Invitrogen)
Acrylamide gel NativePAGE 4-16% Vistris gel, 1.0 mm, 10 wells (Invitrogen)
・ Cathode Buffer (running buffer) 25 mM Tris, 192 mM Glycine, pH 7.4
-Anode Buffer 100 mM Tris, pH 7.8

6. The gel after electrophoresis was stained with a silver staining kit WAKO (manufactured by Wako Pure Chemical Industries, Ltd.). The operation was performed according to the operation procedure attached to the kit.

The results are shown in FIG. From the results in FIG. 1, it can be seen that the antibody band and the antigen-antibody complex band were clearly separated. The band of the antigen-antibody complex becomes deeper as the amount of C-peptide increases, while the band of the antibody becomes thinner as the amount of C-peptide increases.

(Comparative Example 1)
Electrophoresis was performed in the same manner as in Example 1 except that the Cathode Buffer used for electrophoresis was changed to 100 mM succinate buffer (pH 5.4). The result is shown in FIG.
(Comparative Example 2)
The Cathode Buffer used for electrophoresis was changed to 25 mM Tris, 192 mM Glycine buffer (pH 8.6). The amount of C-peptide in each lane was 2.4, 4.8, and 9.5 ng. For other operations, electrophoresis was performed in the same manner as in Example 1.

In the comparative example, as shown in FIG. 2, the antibody band and the antigen-antibody complex band were not clearly separated, and the antigen-antibody complex band could not be clearly detected. The result is shown in FIG.

Claims

A method of separating a target substance and a complex of a receptor that specifically binds to the target substance and a free receptor,
The isoelectric point of the receptor is higher than the isoelectric point of the complex;
The electrophoresis is performed in a buffer solution having a pH that exceeds the isoelectric point of the complex and not more than +2 of the isoelectric point of the receptor under non-denaturing conditions of the complex.
A method for separating a target substance-receptor complex from a free receptor.
The method according to claim 1, wherein the molecular weight of the target substance is 500 to 10,000.
The method according to claim 1 or 2, wherein the pH of the buffer solution is +1.5 or less of the isoelectric point of the receptor and +1.0 or more of the isoelectric point of the complex.