Hapten measurement method

Abstract

To provide an immunoassay method and immunoassay reagent, which can improve hapten measurement sensitivity in hapten measurement by using the NHS ester method when binding a hapten to a carrier protein.SOLUTION: A hapten measurement method is provided, comprising binding a hapten to a carrier protein using the NHS ester method to produce a hapten-carrier protein conjugate, and measuring the hapten using the hapten-carrier protein conjugate.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for measuring a hapten using a hapten-carrier protein conjugate. [Background technology]

[0002] In the medical field, immunoassays that utilize antigen-antibody reactions are widely used as a method for detecting or quantifying specific substances in biological components. Immunoassays based on antigen-antibody reactions are highly specific and sensitive, making them an important means of clinical testing. Known immunoassays include enzyme immunoassay (EIA), radioimmunoassay (RIA), and turbidimetric immunoassay (TIA).

[0003] In carrying out these measurement methods, antibodies or antigens against the substance to be measured are bound to a water-insoluble solid phase such as a microtiter plate, a membrane filter, latex particles, or gold colloid. In order to produce diagnostic agents that are highly sensitive and stable, it is extremely important to have a technique for efficiently and stably binding antibodies or antigens to the above-mentioned water-insoluble solid phases.

[0004] Known methods for binding an antigen or antibody to the above-mentioned water-insoluble solid phase (carrier) mainly include methods using physical adsorption and covalent bonding. Compared with methods using covalent bonding, physical adsorption has the advantage of being simple and not impairing the activity of the antigen or antibody to be adsorbed. However, when the antigen is a low molecular weight substance such as a hapten, it is difficult to physically adsorb the antigen because of its weak interaction with the water-insoluble solid phase.

[0005] Conventionally, in order to solve the above problems, a method has been known in which a hapten is covalently bonded to a carrier protein (polymer carrier) using a crosslinking agent or a coupling agent. In particular, it is known to bond a hapten to a carrier protein when preparing an antibody against the hapten (see Patent Document 1). Examples of carrier proteins that can be used in this method include bovine serum albumin, human serum albumin, egg albumin, keyhole limpet hemocyanin, ovalbumin, thyroglobulin, fibrinogen, polylysine, etc.

[0006] Haptens and proteins are bonded to each other via their respective functional groups. Examples of functional groups of proteins used for bonding to haptens include ε- and α-amino groups (pKa 10.8), phenol groups, sulfhydryl groups, imidazole groups, and carboxyl groups. Crosslinking reagents (or methods) used to crosslink haptens and protein molecules include the mixed acid anhydride method, carbodiimide method, and NHS (N-hydroxysuccinimide) ester method as methods for activating carboxyl groups, the glutaraldehyde method (one step or two steps) and the NHS method (maleimide: succinimide, succinimide: succinimide, succinimide: 2-pyridylthiopropionate, etc.) as methods for activating amino groups, and the maleimide method as a method for activating thiol groups. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 2001-516567 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide an immunoassay and a reagent for the immunoassay, which can improve the measurement sensitivity of a hapten by using an NHS ester method when binding a hapten to a carrier protein. [Means for solving the problem]

[0009] As a result of intensive research, the present inventors have found that by using the NHS ester method when binding a carrier protein to a hapten, it is possible to improve the measurement sensitivity when measuring a hapten using a conjugate of a hapten and a carrier protein, and have completed the present invention.

[0010] That is, the present invention is as follows. [1] A hapten and a carrier protein are bound by the NHS ester method to prepare a hapten-carrier protein conjugate. A method for measuring a hapten, comprising measuring the hapten using the hapten-carrier protein conjugate. [2] In a method of binding a hapten to a carrier protein by the NHS ester method, the hapten has an amino group or an amino group has been introduced, and examples of the hapten that can be used include DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), BS3 (bis(sulfosuccinimidyl)suberate), TSAT (tris-(succinimidyl)aminotriacetate), BS(PEG)5 (PEGylated bis(sulfosuccinimidyl)suberate), BS(PEG)9 (PEGylated bis(sulfosuccinimidyl)suberate), DSP (dithiobis(succinimidyl propionate)), Lomant's Reagent, DST (disuccinimidyl tartrate), EGS (ethylene glycol bis(succinimidyl succinate)), and Sulfo-EGS (ethylene glycol bis(sulfosuccinimidyl The method for measuring a hapten according to [1], wherein a crosslinking agent selected from the group consisting of a succinate and a crosslinker is used. [3] A method for measuring the hapten of [1], wherein the hapten is 3-O-(2-Aminoethyl)-25-hydroxyvitamin D3. [4] A method for measuring the hapten of [1], which is an immunological assay. [5] A method for measuring the hapten of [4], which is a competitive immunoassay. [6] A method for measuring a hapten as described in [5], in which a hapten-carrier protein conjugate is immobilized on a carrier, a test sample containing a labeled anti-hapten antibody and the hapten is added to the carrier, and the hapten is measured by allowing the hapten-carrier protein conjugate to compete with the hapten for binding to the labeled anti-hapten antibody. [7] A method for measuring the hapten of [4], which is an agglutination method. [8] A method for measuring a hapten as described in [7], in which particles having a hapten-carrier protein conjugate bound thereto are mixed with a test sample containing the hapten and an anti-hapten antibody, the particles having the hapten-carrier protein conjugate bound thereto are agglutinated via the anti-hapten antibody, and the hapten in the test sample is measured based on the degree of agglutination. [9] A reagent for measuring a hapten, comprising a hapten-carrier protein bound by the NHS ester method.

[10] In a method for binding a hapten to a carrier protein by the NHS ester method, the hapten has an amino group or an amino group has been introduced, and examples of the hapten that can be used include DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), BS3 (bis(sulfosuccinimidyl)suberate), TSAT (tris-(succinimidyl)aminotriacetate), BS(PEG)5 (PEGylated bis(sulfosuccinimidyl)suberate), BS(PEG)9 (PEGylated bis(sulfosuccinimidyl)suberate), DSP (dithiobis(succinimidyl propionate)), Lomant's Reagent, DST (disuccinimidyl tartrate), EGS (ethylene glycol bis(succinimidyl succinate)), and Sulfo-EGS (ethylene glycol bis(sulfosuccinimidyl The reagent of [9], wherein the crosslinking agent is selected from the group consisting of succinate.

[11] The reagent of [9], in which the hapten is 3-O-(2-Aminoethyl)-25-hydroxyvitamin D3.

[12] Additionally, the reagent of [9], which is a reagent for immunoassays, contains an anti-hapten antibody.

[13] Additionally, the reagent [9] is a reagent for agglutination assay that contains an anti-hapten antibody. Effect of the Invention

[0011] By using the measurement method and reagent of the present invention, haptens can be measured with high sensitivity. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows the reactivity of vitamin D with BSA (carrier protein) when a vitamin D-BSA (carrier protein) conjugate is produced using DSG, AMAS, or EMCS. [Diagram 2] FIG. 1 shows the reactivity of vitamin D with and without DSG and BSA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention will be described in detail below. The present invention relates to a method for measuring a hapten. In the method of the present invention, a hapten is bound to a carrier protein and then bound to a solid phase.

[0014] A hapten is a low molecular weight substance having a molecular weight of several hundred or less that does not have the antigenicity to produce an antibody, but shows antigenicity by binding to a carrier protein. The hapten in the present invention is not particularly limited, and includes hormones, biochemical messengers, steroids, drugs, drug metabolites, polypeptides, vitamins, alkaloids, etc., and particularly includes thyroid hormones such as triiodothyronine, thyroxine, and 3,5-diiodo-L-thyronine, and steroid hormones such as estrone, estradiol (hereinafter referred to as E2), estriol, progesterone, and cortisol. In particular, vitamin D3-O-(2-aminoethyl)-25-hydroxyvitamin D3 is preferred in the present invention. In the present invention, the hapten and the carrier protein are bound to each other using an inter-amino group crosslinking agent as described below, so that the hapten may originally have an amino group or an amino group may be introduced therein. The introduction of an amino group may be performed by a known method such as reductive amination. For example, when vitamin D is used as a hapten, it may be bound to a carrier protein using 3-O-(2-aminoethyl)-25-hydroxyvitamin D3. When 3-O-(2-aminoethyl)-25-hydroxyvitamin D3 is used as a hapten and bound to a carrier protein to prepare a hapten-carrier protein conjugate, the hapten to be detected is vitamin D.

[0015] Examples of carrier proteins to be bound to haptens include serum albumin (hereinafter referred to as BSA), ovalbumin, keyhole limpet hemocyanin, thyroglobulin, immunoglobulin, and the like.

[0016] The hapten of the present invention is bound to a carrier protein by the NHS ester (N-hydroxyester) method. The NHS ester method is a method in which a hapten is bound to a carrier protein by an NHS ester-activated crosslinker. The hapten is bound via the α-amino group at the N-terminus or the ε-amino group of the lysine side chain on the carrier protein. Examples of the crosslinking agent used in this case include DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), BS3 (bis(sulfosuccinimidyl)suberate), TSAT (tris-(succinimidyl)aminotriacetate), BS(PEG)5 (PEGylated bis(sulfosuccinimidyl)suberate), BS(PEG)9 (PEGylated bis(sulfosuccinimidyl)suberate), DSP (dithiobis(succinimidyl propionate)), Lomant's Reagent, DST (disuccinimidyl tartrate), EGS (ethylene glycol bis(succinimidyl succinate)), Sulfo-EGS (ethylene glycol bis(sulfosuccinimidyl succinate)), etc., which are known as crosslinking agents between amino groups. Among these, DSG (disuccinimidyl glutarate) is preferred.

[0017] The crosslinking reaction of NHS ester is carried out by mixing the hapten, the carrier protein and the above-mentioned crosslinking agent in a phosphate, carbonate, bicarbonate, HEPES or borate buffer at room temperature or on ice at pH 7.2 to 8.5, and reacting for 0.5 to 4 hours. In this case, the mixing ratio of the hapten to the carrier protein is 1 to 100, preferably 2 to 50, more preferably 5 to 20, more preferably 5 to 15, and even more preferably 10, in terms of molar ratio. After the reaction, Tris buffer or glycine buffer may be added to stop the reaction. Then, the buffer may be replaced with the buffer used during the reaction.

[0018] In this way, the hapten and the carrier protein can be bound together. The bound hapten and the carrier protein are called a hapten-carrier protein conjugate.

[0019] A carrier protein has multiple ε-amino groups in its side chains, and multiple hapten molecules can be bound to one molecule of the carrier protein.

[0020] In the present invention, a hapten is measured using a hapten-carrier protein conjugate.

[0021] The hapten-carrier protein conjugate of the present invention can be applied to any immunological measurement method for measuring a hapten using an antigen-antibody reaction. That is, it can be applied to, for example, an immunodiffusion method, an immunoturbidimetric method, an immunonephelometry method, a hemagglutination method, a latex method, an enzyme immunoassay (EIA method), a radioimmunoassay (RIA method), an immunochromatography method, etc. It can also be applied to a competitive immunoassay method in which a conjugate of a substance to be measured and a substance to be measured compete with each other in a reaction between the substance to be measured and an antibody. In these measurement methods, when an antigen is measured by a normal antigen-antibody reaction, a hapten-carrier protein conjugate may be used instead of the antigen.

[0022] A specific method for measuring a hapten using the hapten-carrier protein conjugate of the present invention will be described below.

[0023] Haptens are usually measured by an immunoassay method called a competitive method. Therefore, in the present invention, it is preferable to measure haptens by a competitive method using a hapten-carrier protein conjugate. The competitive method is a method in which a hapten contained in a sample and a hapten in a measurement reagent are competitively reacted with a certain amount of anti-hapten antibody. When the amount of hapten contained in a sample increases, the amount of hapten in the measurement reagent that binds to the anti-hapten antibody decreases. From this, the amount of hapten contained in a sample can be estimated based on the ratio of hapten in the measurement reagent that binds to the anti-hapten antibody. The method of measuring haptens using the hapten-carrier protein conjugate of the present invention is preferably a method that uses an anti-hapten antibody against the hapten, which is the substance to be detected, and utilizes the fact that the binding of the hapten-carrier protein conjugate and the hapten to the anti-hapten antibody competes. In other words, a competitive immunoassay is preferable.

[0024] Examples of methods for measuring a hapten using the hapten-carrier protein conjugate of the present invention include a method in which the hapten-carrier protein conjugate is immobilized on a carrier, a method in which an anti-hapten antibody is immobilized on a carrier, and a method that utilizes particle agglutination.

[0025] In these methods, the anti-hapten antibody or an antibody against the anti-hapten antibody (secondary antibody) may be labeled with a labeling substance. Here, the antibody can be labeled using an enzyme such as alkaline phosphatase or horseradish peroxidase, a fluorescent dye such as fluorescein isothiocyanate (FITC), a radioisotope, or the like. When an enzyme is used, the enzyme is reacted with a corresponding substrate and the dye generated by the enzyme reaction is detected. When a fluorescent dye is used, the fluorescence emitted from the fluorescent dye is detected. When a radioisotope is used, radiation from the radioisotope is detected, and the hapten can be measured based on the measured value of the signal from the labeling substance.

[0026] (1) Method of immobilizing a hapten-carrier protein conjugate on a support When a hapten-carrier protein conjugate is immobilized on a carrier and a competitive immunoassay is performed, it is preferable to use a labeled anti-hapten antibody against the hapten or a secondary antibody against the anti-hapten antibody. When the amount of hapten (substance to be detected) in the test sample is large, the amount of labeled anti-hapten antibody or labeled secondary antibody that binds to the immobilized hapten-carrier protein conjugate is reduced, resulting in a weak signal from the labeled substance.

[0027] The carrier may be any of those used in conventional immunoassays, such as the wells of a plastic microtiter plate, a nitrocellulose membrane, etc. The immobilization of the hapten-carrier protein conjugate on the carrier may be carried out by a method conventionally known in this field, and for example, in the case of binding to the wells of a microtiter plate, a solution of the hapten-carrier protein conjugate at about 3 to 10 μg / mL (preferably about 5 μg / mL) may be added to the solid phase, and the solution may be left at 4° C. overnight (preferably 12 hours or more).

[0028] Specifically, a hapten-carrier protein conjugate is immobilized on a carrier, a test sample containing a labeled anti-hapten antibody and a hapten (substance to be detected) is added to the carrier, and the hapten-carrier protein conjugate and the hapten (substance to be detected) are allowed to compete with each other for binding to the labeled anti-hapten antibody. This reaction can be carried out at 4°C to 45°C, more preferably 20°C to 40°C, and even more preferably 25°C to 38°C, and the reaction time is about 10 minutes to 18 hours, more preferably 10 minutes to 1 hour, and even more preferably 30 minutes to 1 hour. When the amount of hapten (substance to be detected) in the test sample is large, the amount of labeled anti-hapten antibody that binds to the immobilized hapten-carrier protein conjugate is reduced, and the signal from the labeled substance is reduced.

[0029] Alternatively, a hapten-carrier protein conjugate may be immobilized on a carrier, and a test sample containing an anti-hapten antibody, a labeled secondary antibody (antibody against the hapten antibody), and a hapten (substance to be detected) may be added to the carrier, allowing the hapten-carrier protein conjugate and the hapten (substance to be detected) to compete for binding to the anti-hapten antibody. The reaction conditions are the same as above. When the amount of hapten (substance to be detected) in the test sample is large, the amount of labeled secondary antibody that binds to the immobilized hapten-carrier protein conjugate decreases, resulting in a smaller signal from the labeled substance.

[0030] In these methods, it is preferable to include a washing step after adding a test sample containing a labeled anti-hapten antibody or an anti-hapten antibody and a labeled secondary antibody, and a hapten (substance to be detected) to the solid-phase hapten-carrier protein conjugate on the carrier, and the washing step removes the labeled antibody that does not bind to the hapten-carrier protein conjugate, or the complex between the hapten and the anti-hapten antibody that does not bind to the carrier protein in the test sample.

[0031] (2) Method of immobilizing anti-hapten antibodies An anti-hapten antibody is immobilized on a carrier, and the above-mentioned hapten-carrier protein conjugate and labeled anti-hapten antibody are added to the immobilized anti-hapten antibody. The immobilization of the anti-hapten antibody on the carrier can be carried out by a known method. A complex of the anti-hapten antibody: hapten-carrier protein conjugate: labeled anti-hapten antibody (":" indicates a bond) is formed on the carrier. This reaction can be carried out at 4°C to 45°C, more preferably 20°C to 40°C, and even more preferably 25°C to 38°C, and the reaction time is about 10 minutes to 18 hours, more preferably 10 minutes to 1 hour, and even more preferably about 30 minutes to 1 hour. In this case, if a test sample containing a hapten (substance to be detected) is added, the hapten binds to the immobilized anti-hapten antibody, but the hapten-carrier protein conjugate does not bind to the immobilized anti-hapten antibody, and a complex of anti-hapten antibody: hapten-carrier protein conjugate: labeled anti-hapten antibody is not formed. In other words, when the amount of hapten in the test sample is large, a complex of anti-hapten antibody: hapten-carrier protein conjugate: labeled anti-hapten antibody (":" indicates binding) is less likely to be formed on the carrier, and the signal from the labeled substance becomes smaller.

[0032] (3) Agglomeration method In the agglutination method, particles to which a hapten-carrier protein conjugate is bound are used, and the particles are agglutinated via an anti-hapten antibody, and the hapten in the test sample can be detected based on the degree of agglutination. Examples of particles used in this method include latex particles having a diameter of 0.05 to 10 μm, preferably 0.1 to 0.4 μm, gelatin particles having a diameter of 0.5 to 10 μm, and animal red blood cells. Binding a hapten-carrier protein conjugate to a particle is called sensitizing the particle with the hapten-carrier protein conjugate. The method of sensitizing the hapten-carrier protein conjugate to the carrier is not particularly limited and may be according to a known method. For example, the hapten-carrier protein conjugate may be physically adsorbed to the carrier or chemically bound to the carrier. More specifically, for example, the hapten-carrier protein conjugate and the carrier are mixed, and then heated and shaken at 30 to 37° C. for 1 to 2 hours, whereby the hapten-carrier protein conjugate can be sensitized to the carrier. The carrier sensitized with the hapten-carrier protein conjugate is preferably kept as a medium dispersion until it is reacted with a test sample. In this case, the medium may be, for example, a phosphate buffer, a glycine buffer, or the like.

[0033] When particles sensitized with a hapten-carrier protein conjugate are mixed with an anti-hapten antibody, the particles aggregate via the anti-hapten antibody to form an aggregate. If a test sample containing a hapten (substance to be detected) is added at this time, the anti-hapten antibody binds to the unconjugated hapten in the test sample, and no aggregates are formed. In other words, the more hapten there is in the test sample, the less aggregates are formed.

[0034] The agglutination reaction can be carried out using a slide glass, a 96-well flat-bottom plate, a 96-well U-bottom plate, a 96-well V-bottom plate, or the like.

[0035] In the agglutination method, the degree of agglutination can be measured visually or by measuring absorbance. In the agglutination method of the present invention, the measurement is preferably performed using an automatic analyzer. The automatic analyzer is an automatic analyzer for biochemical testing applicable to immunoassays, and various types of such instruments are commercially available, such as Hitachi Autoanalyzer 7180, Hitachi Autoanalyzer 7250, Autoanalyzer 7450, and Autoanalyzer 7350, all of which are manufactured by Hitachi, Ltd.

[0036] For example, in a method using an automatic analyzer, the hapten is measured by optically capturing the agglutinates formed in the reaction vessel. That is, the agglutinates are irradiated with light ranging from visible light to near infrared light, and the degree of agglutination of the carrier particles is measured by detecting the change in absorbance or the change in the intensity of the scattered light. In this case, the measurement method includes the One Point method, the Two Point method, the Three Point method, the Rate method, etc., and the method of the present invention can be applied to any of the measurement methods. The test sample is dispensed in advance into the reaction vessel, and the first and second reagents are dispensed therein to cause a reaction and measure the degree of agglutination, so that the hapten to be measured in the test sample can be quantified using the degree of agglutination as an index. The order of addition of the first and second reagents is not limited, but preferably the first reagent is dispensed before the second reagent, or the first and second reagents are dispensed simultaneously. In this case, a standard curve relating the concentration of the hapten to be measured to the degree of agglutination is prepared in advance using a plurality of samples whose concentrations of the hapten to be measured are known, and the concentration of the hapten to be measured in the test sample can be calculated based on the standard curve.

[0037] The first reagent may include a buffer solution, and may contain other stabilizing reagents, protective reagents, etc. The second reagent may include the above-mentioned particles sensitized with an antibody or antigen that specifically binds to the antigen or antibody to be measured, and may contain other stabilizing reagents, protective reagents, etc.

[0038] In the detection by the agglutination method using sensitized particles, several μL to several tens of μL of physiological saline or a suitable buffer solution is added to several μL to several tens of μL of a test sample in a suitable reaction vessel, mixed, and incubated for several minutes, and then several tens of μL to several hundreds of μL of sensitized particles bound to an antibody or antigen are added and incubated for several minutes. Then, the turbidity caused by the agglutination of the sensitized particles caused by the antigen-antibody reaction can be measured by measuring the absorbance at a suitable measurement wavelength (for example, 570 nm). In addition, several tens of μL to several hundreds of μL of a solution of the sensitized particles suspended in a buffer solution may be added to several μL of the reaction sample. Examples of the buffer solution include a suitable buffer solution of pH 5.0 to 10, such as a phosphate buffer solution, a borate buffer solution, and a Tris buffer solution.

[0039] The test sample to be subjected to the immunoassay method of the present invention is not particularly limited, but is preferably a biological sample, and in particular, bodily fluids such as blood, serum, plasma, urine, stool, saliva, tissue fluid, cerebrospinal fluid, swab fluid, etc., or dilutions thereof are preferred, and blood, serum and plasma, or dilutions thereof are even more preferred. EXAMPLES

[0040] The present invention will now be described in detail with reference to examples, although the present invention is not limited to the following examples.

[0041] Example 1 Vitamin D was used as the hapten and bovine serum albumin was used as the carrier protein.

[0042] 1.Material Vitamin D: 3-O-(2-aminoethyl)-25-hydroxyvitamin D3 (MedChemExpress) Bovine serum albumin: BSA (Boval) ·Disuccinimidyl glutarate:DSG(Thermo Fisher Scientific)

[0043] [ka]

[0044] 2. Preparation of Vitamin D-Carrier Protein Conjugates 1) Vitamin D and BSA (1 mg / mL) were mixed in PBS (pH 7.2) at a ratio of 1 to 40:1. 2) 30 μL of DSG (3.26 mg / mL) was added. 3) Incubate at RT (room temperature) for 30 minutes or on ice for 2 hours. 4) 1M Tris (pH 7.5) was added to a final concentration of 20 to 50 mM, and the mixture was incubated for 15 minutes. 5) The buffer was replaced with PBS.

[0045] 3.Immunological measurements The obtained conjugate was used as a sample (0.33 mg / mL) and measured with Diazyme EZ Vitamin D Assay (Diazyme Laboratories Inc.), an anti-hapten antibody solid-phase latex, to confirm the reactivity between the conjugate and the antibody from the change in absorbance due to latex agglutination. AU5800 (Beckman Coulter) was used for the measurement.

[0046] Comparative Example 1 1.Material Vitamin D: 3-O-(2-aminoethyl)-25-hydroxyvitamin D3 (MedChemExpress) Bovine serum albumin: BSA (Boval) Iminothiolane hydrochloride: 2-IT (FUJIFILM Wako Pure Chemical Industries, Ltd.) ·AMAS:N-α-maleimidoacet-oxysuccinimide ester(Thermo Fisher Scientific) AMAS has an NHS ester group and a maleimide reactive group at both ends, respectively.

[0047] [ka]

[0048] ·EMCS:N-ε-maleimidocaproyl-oxysuccinimide ester(Thermo Fisher Scientific) EMCS has an NHS ester group and a maleimide reactive group at both ends, respectively.

[0049] [ka]

[0050] 2. Preparation of Vitamin D-Carrier Protein Conjugates 2-1.BSA thiolation 1) BSA was dissolved in PBS (pH 7.2) to a concentration of 1 mg / mL. 2) 110 μL of iminothiolane hydrochloride (2 mg / mL) was added. 3) Incubate at RT (room temperature) for 1 hour. 4) The buffer was replaced with PBS (pH 7.2).

[0051] 2-2.BSA NHS esterification 1) 75 μL of AMAS 2.52 mg / mL or EMCS 3.08 mg / mL was added to the preparation 2-1. 2) Incubate at RT (room temperature) for 30 minutes or at 4°C for 2 hours. 3) The buffer was replaced with PBS (pH 7.2).

[0052] 2-3.Join 1) The preparation 2-2 obtained by reacting the maleimide group of AMAS or EMCS with the thiol group of thiolated BSA was mixed with vitamin D (3-O-(2-Aminoethyl)-25-hydroxyvitamin D3) in a molar ratio to BSA of 1 to 40:1. 2) Incubate in the dark at room temperature for 30 minutes or at 4°C for 2 hours. 3) The buffer was replaced with PBS.

[0053] 3. Test Example (Immunological Assay) The procedure was as described in Example 1-3. The results are shown in FIG. As a result, the conjugate crosslinked with DSG (2-1) showed the highest reactivity with anti-hapten antibodies. In addition, the bond between AMAS and carrier proteins is called a maleimide reaction, which is said to be susceptible to hydrolysis due to pH. On the other hand, the bond with DSG is an ester bond, so it is expected that hydrolysis due to pH will be less likely to occur.

[0054] Comparative Example 2 To confirm whether the conjugate prepared in Example 1 reacted with the antibody via DSG, a comparison was made with the method of Example 1 in which DSG was not added.

[0055] 1.Material Vitamin D: 3-O-(2-aminoethyl)-25-hydroxyvitamin D3 (MedChemExpress) Bovine serum albumin: BSA (Boval)

[0056] 2. Preparation of Vitamin D-Carrier Protein Conjugates 1) Vitamin D and BSA (1 mg / mL) were mixed in PBS (pH 7.2) at a ratio of 1 to 40:1. 2) Incubate at RT (room temperature) for 30 minutes or on ice for 2 hours. 3) 1M Tris (pH 7.5) was added to a final concentration of 20 to 50 mM, and the mixture was incubated for 15 minutes. 4) The buffer was replaced with PBS.

[0057] 3.Immunological measurements The procedure was as described in Example 1-3. The results are shown in FIG. As a result, it was confirmed that without DSG, vitamin D would not be cross-linked with the carrier protein and would not react, suggesting that the inclusion of DSG contributes to the creation of conjugates that react well with antibodies. [Industrial Applicability]

[0058] According to the method of the present invention, haptens such as vitamin D can be measured.

Claims

1. The hapten and carrier protein are bound by the NHS ester method to prepare a hapten-carrier protein conjugate. A method for measuring a hapten, comprising measuring the hapten using the hapten-carrier protein conjugate.

2. In the method of binding a hapten to a carrier protein by the NHS ester method, the hapten has an amino group or an amino group has been introduced therein, and examples of the hapten that can be used include DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), BS3 (bis(sulfosuccinimidyl)suberate), TSAT (tris-(succinimidyl)aminotriacetate), BS(PEG)5 (PEGylated bis(sulfosuccinimidyl)suberate), BS(PEG)9 (PEGylated bis(sulfosuccinimidyl)suberate), DSP (dithiobis(succinimidyl propionate)), Lomant's Reagent, DST (disuccinimidyl tartrate), EGS (ethylene glycol bis(succinimidyl succinate)), and Sulfo-EGS (ethylene glycol bis(sulfosuccinimidyl 2. The method for measuring a hapten according to claim 1, wherein a crosslinking agent selected from the group consisting of succinates is used.

3. The method for measuring a hapten according to claim 1, wherein the hapten is 3-O-(2-aminoethyl)-25-hydroxyvitamin D3.

4. The method for measuring a hapten according to claim 1, which is an immunological measurement method.

5. The method for measuring a hapten according to claim 4, which is a competitive immunoassay.

6. The method for measuring a hapten according to claim 5, wherein the hapten-carrier protein conjugate is immobilized on a carrier, a test sample containing a labeled anti-hapten antibody and the hapten is added to the carrier, and the hapten is measured by allowing the hapten-carrier protein conjugate and the hapten to compete with each other for binding to the labeled anti-hapten antibody.

7. 5. The method for measuring a hapten according to claim 4, which is an agglutination method.

8. The method for measuring a hapten according to claim 7, which comprises mixing particles having a hapten-carrier protein conjugate bound thereto with a test sample containing the hapten and an anti-hapten antibody, agglutinating the particles having the hapten-carrier protein conjugate bound thereto via the anti-hapten antibody, and measuring the hapten in the test sample based on the degree of agglutination.

9. A reagent for measuring a hapten, comprising a hapten-carrier protein bound by the NHS ester method.

10. The reagent according to claim 9, wherein the method for binding a hapten to a carrier protein by the NHS ester method uses a crosslinking agent selected from the group consisting of DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), BS3 (bis(sulfosuccinimidyl)suberate), TSAT (tris-(succinimidyl)aminotriacetate), BS(PEG)5 (PEGylated bis(sulfosuccinimidyl)suberate), BS(PEG)9 (PEGylated bis(sulfosuccinimidyl)suberate), DSP (dithiobis(succinimidyl propionate)), Lomant's Reagent, DST (disuccinimidyl tartrate), EGS (ethylene glycol bis(succinimidyl succinate)), and Sulfo-EGS (ethylene glycol bis(sulfosuccinimidyl succinate)).

11. The reagent according to claim 9, wherein the hapten is 3-O-(2-aminoethyl)-25-hydroxyvitamin D3.

12. The reagent according to claim 9, which is a reagent for immunological assay, further comprising an anti-hapten antibody.

13. The reagent according to claim 9, which is a reagent for an agglutination method, further comprising an anti-hapten antibody.

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