Method for producing a gold colloid-labeled antibody suspension, a method for producing a conjugate pad, and a method for producing a testing instrument.

Abstract

A method for producing a gold colloid-labeled antibody suspension, comprising: a labeling step of obtaining a mixture by mixing a gold colloid solution and an antibody solution to obtain a gold colloid-labeled antibody in the mixture; a centrifugation step of centrifugating the mixture to obtain a precipitate of the gold colloid-labeled antibody and removing the supernatant containing antibody that does not have gold colloid adsorbed; and a suspension step of suspending the precipitate of the gold colloid-labeled antibody in a suspension solution containing lactalbumin to obtain a gold colloid-labeled antibody suspension.

Description

Technical Field

[0001] The present invention relates to a colloidal gold-labeled antibody suspension using lactalbumin, a method for producing the same, and an inspection instrument. This application claims priority from Japanese Patent Application No. 2024-087370 filed in Japan on May 29, 2024, the content of which is incorporated herein by reference.

Background Art

[0002] Conventionally, it has been widely practiced to label an antibody with colloidal gold and use it for medical examinations and academic research. For example, as a method for qualitatively or quantitatively measuring the presence of a test substance present in a biological sample such as urine or blood, immunochromatography, which is a kind of immunological measurement method, is well known (for example, Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] Colloidal gold is generally gold nanoparticles with an average particle diameter of less than 1 μm. When white light is irradiated on a solution in a dispersed state, the solution appears colored. When colloidal gold and an antibody are mixed in a solution, the colloidal gold and the antibody react physicochemically, and the antibody adsorbs onto the colloidal gold. As a result, the antibody can be made into a labeled state colored with colloidal gold. Thereafter, in order to prevent the colloidal gold-labeled antibody from adsorbing non-specifically to other substances, a blocking treatment is performed by contacting it with bovine serum albumin (BSA) or the like.

[0005] In the production of gold colloid-labeled antibodies, a process is performed to separate the labeled antibody adsorbed to the gold colloid from the unlabeled antibody by centrifuging the solution containing both to precipitate the gold colloid-labeled antibody and then removing the supernatant containing the unlabeled antibody. Subsequently, the precipitated gold colloid-labeled antibody is resuspended in the desired solution to obtain the desired gold colloid-labeled antibody suspension. However, a significant problem arose where a considerable amount of gold colloid-labeled antibody remained aggregated and could not be dispersed after resuspending. Since these aggregates cannot be used in subsequent processes, they worsen the production yield of gold colloid-labeled antibodies.

[0006] This invention provides a gold colloid-labeled antibody suspension with excellent dispersibility, a method for producing the same, and a testing device using the gold colloid-labeled antibody. [Means for solving the problem]

[0007] [1] A method for producing a gold colloid-labeled antibody suspension, comprising: a labeling step of obtaining a mixture by mixing a gold colloid solution and an antibody solution to obtain a gold colloid-labeled antibody in the mixture; a centrifugation step of centrifugating the mixture to obtain a precipitate of the gold colloid-labeled antibody and removing the supernatant containing antibody that does not have gold colloid adsorbed; and a suspension step of suspending the precipitate of the gold colloid-labeled antibody in a suspension solution containing lactalbumin to obtain a gold colloid-labeled antibody suspension. [2] A method for producing a gold colloid-labeled antibody suspension according to [1], wherein after the labeling step and before the centrifugation step, a solution containing BSA is added to the mixture to block the gold colloid-labeled antibody. [3] The method for producing a gold colloid-labeled antibody suspension according to [1] or [2], wherein the suspension solution does not contain BSA, or if it does contain BSA, its concentration is less than 1 mg / mL. [4] A method for producing a gold colloid-labeled antibody suspension according to any one of [1] to [3], wherein the concentration of lactalbumin contained in the suspension solution is 0.01 mg / mL or higher. [5] A gold colloid-labeled antibody suspension comprising a gold colloid-labeled antibody and lactalbumin at a concentration of 0.01 mg / mL or higher. [6] A testing instrument equipped with a pad for absorbing a solution to be tested, wherein a dried gold colloid-labeled antibody suspension containing a gold colloid-labeled antibody and 0.1 μg or more of lactalbumin is held on the pad. [7] The testing apparatus according to [6], wherein the pad is supported by an elongated support and is used for performing immunochromatography of the solution to be tested. [Effects of the Invention]

[0008] The manufacturing method of the present invention allows for the production of gold colloid-labeled antibodies with a high yield. In the gold colloid-labeled antibody suspension of the present invention, the dispersibility of the gold colloid-labeled antibody is good, and its specificity for the antigen is sufficiently maintained. In the testing instrument of the present invention, the specificity of the gold colloid-labeled antibody is sufficiently maintained, and it has a detection sensitivity comparable to conventional instruments. [Modes for carrying out the invention]

[0009] Method for producing gold colloid-labeled antibody suspension A first aspect of the present invention is a method for producing a gold colloid-labeled antibody suspension, comprising a labeling step, a centrifugation step, and a suspension step, as described below.

[0010] [Labeling process] The labeling step involves obtaining a mixture by mixing a gold colloid solution and an antibody solution, thereby obtaining a gold colloid-labeled antibody in the mixture. This step can be carried out in the same manner as conventional methods for obtaining gold colloid-labeled antibodies.

[0011] The gold colloid solution used in this process is not particularly limited, and any desired solution can be selected and used. Gold colloid solutions for antibody labeling are commercially available, and examples include those with an average particle size of 5 to 500 nm as measured from TEM images using an electron microscope. The concentration of the gold colloid in the aforementioned mixture can be defined by its absorbance at its maximum absorption wavelength, and the absorbance can be in the range of, for example, 0.1 to 5, preferably 0.5 to 1.5.

[0012] The antibody solution used in this process is not particularly limited, and any desired solution can be selected and used. The antibody may be either a monoclonal or polyclonal antibody. The antibody subtype is also not particularly limited, and examples include IgG, IgA, IgM, IgD, and IgE. The animal species from which the antibody originates is also not particularly limited. The antibody may be full-length or a fragment containing an antigen-binding site. Examples of antibody concentrations in the mixture include 0.01 to 100 μg / mL, preferably 0.1 to 10 μg / mL, and more preferably 0.5 to 5 μg / mL.

[0013] The gold colloid solution, antibody solution, and dispersion medium of the mixture can be any medium that stably disperses the antibody, and examples include various commonly used pH buffers and inorganic salts. The pH of the mixture is preferably within a range that stably maintains the three-dimensional structure of the antibody, for example, 6.5 to 7.5.

[0014] In the aforementioned mixture, the antibody and the gold colloid physically come into contact and interact, causing the gold colloid to spontaneously adsorb to the antibody, resulting in a gold colloid-labeled antibody. The reaction (adsorption) between the antibody and the gold colloid in the aforementioned mixture is completed at room temperature in, for example, several minutes to several tens of minutes.

[0015] [Blocking process] After the labeling step, it is preferable to perform a blocking treatment in which a blocking agent is brought into contact with the gold colloid-labeled antibody formed in the mixture, and the blocking agent is adsorbed onto sites where nonspecific adsorption may occur, in order to prevent nonspecific adsorption of the gold colloid-labeled antibody.

[0016] Examples of blocking agents include proteins such as bovine serum albumin (BSA), gelatin, casein, ovalbumin, lactalbumin, sericin, fetuin, peptides, polyethylene glycol, polyanions, N,N-dialkylamide, lower alkyl sulfoxide, alkyl cellulose, carboxyalkyl cellulose, etc. Among these, BSA is preferred from the viewpoint of not inhibiting the specificity of the antibody and not interfering with the performance of the gold colloid-labeled antibody.

[0017] For the blocking treatment, a method of adding a solution containing a blocking agent, for example, a solution containing BSA (BSA solution), to the mixed solution is preferred. The dispersion medium constituting the BSA solution may be any one in which BSA is stably dispersed, and examples include those containing various commonly used pH buffers and inorganic salts. The pH of the BSA solution is preferably in the range where the three-dimensional structures of BSA and the antibody are stably maintained, for example, 6.5 to 7.5. Examples of the BSA concentration in the mixed solution in the blocking treatment include a range of 1 to 100 mg / mL, preferably 5 to 20 mg / mL. Blocking agents other than BSA can also be used under the same conditions.

[0018] In the mixed solution, the blocking agent such as BSA contacts and interacts with the gold colloid-labeled antibody, so that the blocking agent is naturally adsorbed on the gold colloid-labeled antibody and the blocking is completed. The blocking treatment is completed at room temperature in, for example, several minutes to several tens of minutes.

[0019] [Centrifugation step] The centrifugation step is a step of centrifuging the mixed solution to obtain a precipitate of the gold colloid-labeled antibody and removing the supernatant containing the antibody not adsorbed with gold colloid. This step can be carried out in the same manner as the conventional method for obtaining a gold colloid-labeled antibody.

[0020] The conditions for centrifugation are not particularly limited as long as they can precipitate the gold colloid-labeled antibody and apply a centrifugal force such that the unlabeled antibody does not precipitate. Examples of the centrifugal force include a range of 1300 g to 15000 g, preferably 5000 g to 15000 g. When using a centrifuge equipped with a rotor with a radius of about 5 cm to 30 cm, examples of the rotation speed include a range of 5000 to 20000 rpm, preferably 8000 to 12000 rpm. At this rotation speed, the gold colloid-labeled antibody can be precipitated by centrifugation for about 0.5 to 2 hours. It is common to cool to about 4 to 10 °C during centrifugation.

[0021] The supernatant after centrifugation can be removed by conventional methods, such as decantation or aspiration with an aspirator.

[0022] [Suspension step] The suspension step is a step of suspending the precipitate of the gold colloid-labeled antibody in a suspension solution containing lactalbumin to obtain a gold colloid-labeled antibody suspension. Lactalbumin may be α-lactalbumin or β-lactalbumin, but α-lactalbumin is preferred from the viewpoint of improving the yield by suspension. The animal species from which lactalbumin is derived is not particularly limited, and bovine-derived lactalbumin is preferred because commercially available products can be obtained at a low cost.

[0023] The concentration of lactalbumin in the suspension solution is preferably, for example, 0.01 mg / mL or more, more preferably 0.10 mg / mL or more, and even more preferably 0.50 mg / mL or more. In a more preferred range, it may be 1.0 mg / mL or more, 2.0 mg / mL or more, 4.0 mg / mL or more. The upper limit value of the lactalbumin concentration is not particularly limited, and examples of the guidelines include 100 mg / mL or less, 50 mg / mL or less, 25 mg / mL or less, 20 mg / mL or less, etc. Since the suspension power of lactalbumin for the gold colloid-labeled antibody is extremely high, it is sufficient if it is above the above lower limit value, and there is no need to use it at a high concentration.

[0024] The dispersion medium constituting the suspension solution can be any medium that stably disperses lactalbumin, and various commonly used pH buffers are examples. The dispersion medium may contain an inorganic salt. NaCl is preferred as the inorganic salt. Examples of inorganic salt concentrations include those in the range of 1 to 400 mM, 10 to 300 mM, and 100 to 200 mM. The pH of the suspension solution is preferably within a range that maintains the stable three-dimensional structure of lactalbumin, for example, 5.5 to 10.0, preferably 6.5 to 9.5, more preferably 7.0 to 9.0, and even more preferably 7.2 to 9.0.

[0025] The amount of gold colloid-labeled antibody precipitate obtained in the centrifugation step can be converted to the amount of gold colloid, since it is considered that almost all of the gold colloid used in the labeling step is contained in the precipitate. When the volume of the mixture used in the labeling step is V1 at which the absorbance at the maximum absorption wavelength of the gold colloid in the mixture is 1, and the volume of the suspension solution is V2, the volume ratio expressed as V1 / V2 can be, for example, 1 to 100, preferably 10 to 80, and more preferably 15 to 80. In other words, for a gold colloid-labeled antibody suspension solution, the volume V2 of the suspension solution should be adjusted so that the absorbance at the maximum absorption wavelength of the gold colloid is, for example, 1 to 100, preferably 10 to 50, more preferably 15 to 40, and even more preferably 20 to 30.

[0026] When the aforementioned precipitate is mixed with the suspension solution and suspended, the gold colloid-labeled antibody and lactalbumin come into contact and interact, resulting in a gold colloid-labeled antibody suspension in a sufficiently dispersed state with suppressed aggregation of the gold colloid-labeled antibody. The suspension of the precipitate with the suspension solution can be completed at room temperature in, for example, a few minutes.

[0027] Through the above steps, a suspension of the desired gold colloid-labeled antibody, containing the gold colloid-labeled antibody and lactalbumin, is obtained. The lactalbumin concentration in the gold colloid-labeled antibody suspension depends on the volume V2 of the suspension solution used and the volume of the precipitate, but is generally the same as or slightly diluted to the lactalbumin concentration in the suspension solution, and can be, for example, 0.1 mg / mL or higher. The concentration of the gold colloid-labeled antibody in the gold colloid-labeled antibody suspension can be defined by the absorbance at the maximum absorption wavelength of the gold colloid, and can be, for example, 1 to 100, preferably 10 to 80, and more preferably 15 to 80.

[0028] As shown in the examples described later, lactalbumin has a much higher suspending power than BSA, so the suspension solution does not need to contain BSA. However, the suspension solution may contain BSA, for example, less than 1 mg / mL, 0.1 mg / mL or less, or 0.01 mg / mL or less. If blocking with BSA is performed before the centrifugation step, a small amount of BSA may be present in the gold colloid-labeled antibody suspension, as the precipitate of gold colloid-labeled antibody obtained in the centrifugation step will contain some BSA.

[0029] Gold colloid-labeled antibody suspension A second aspect of the present invention is a gold colloid-labeled antibody suspension comprising a gold colloid-labeled antibody and 0.1 mg / mL or more of lactalbumin. The composition of the gold colloid-labeled antibody suspension in this embodiment may be the same as the composition of the gold colloid-labeled antibody suspension obtained by the production method in the first embodiment, and redundant explanations will be omitted.

[0030] <<Inspection Equipment>> A third aspect of the present invention is a testing device equipped with a pad that absorbs a test solution containing the substance to be detected. In the testing device of this embodiment, a dried gold colloid-labeled antibody suspension containing a gold colloid-labeled antibody bound to the substance to be detected and lactalbumin is held in the pad. The pad that holds the gold colloid-labeled antibody suspension and is then dried is called a conjugate pad. In one example of the testing device, the amount of lactalbumin contained in the conjugate pad is preferably 0.1 μg or more, and more preferably 0.5 μg or more. In a more preferred range, it may be 1.0 μg or more, 2.0 μg or more, or 4.0 μg or more. The material of the conjugate pad is not particularly limited as long as it is capable of absorbing and retaining water, such as a fiberglass pad, filter paper, cotton, or nonwoven fabric. The composition of the gold colloid-labeled antibody suspension may be the same as that of the gold colloid-labeled antibody suspension obtained by the production method of the first embodiment, and redundant explanations will be omitted.

[0031] The testing device of this embodiment preferably has a structure in which a conjugate pad, an antibody-immobilized membrane, and an absorbent pad are arranged in order on a support such as a plastic adhesive sheet, and may also include components other than these elements. The antibody-immobilized membrane includes a test line on which an antibody that binds to the substance to be detected is immobilized, and optionally a control line for confirming that the test has proceeded appropriately.

[0032] Since the conjugate pad already contains a gold colloid-labeled antibody, if the antigen of the gold colloid-labeled antibody is present in the test solution, when the test solution soaks into the conjugate pad, the gold colloid-labeled antibody and the antigen bind to each other via an antigen-antibody reaction to form a complex. When the complex binds to the antibody on the antibody-immobilized membrane, the test line changes color, allowing for the detection of a signal corresponding to the amount of antigen present in the test solution. Other drugs may be used to obtain or enhance the detection signal. In the aforementioned testing device, the solution to be tested permeates the conjugate pad, the antibody-immobilized membrane, and the absorbent pad in that order. The absorbent pad is provided to ultimately receive and hold the solution to be tested.

[0033] An example of an embodiment of this design is a test strip (immunochromatography test strip) based on the immunochromatography method. This strip comprises the conjugate pad and an elongated support that supports the conjugate pad. The entire or a portion of the strip is capable of absorbing the solution to be tested. By immersing one end of the strip in the solution to be tested, or by dropping the solution onto the strip, the solution to be tested permeates and progresses from one end of the strip to the other. Since the conjugate pad is located along this path, if the solution to be tested reacts with the gold colloid-labeled antibody, a signal can be detected as described above. Typically, the test is carried out with an antibody-immobilized membrane located downstream of the conjugate pad, and an absorption pad located further downstream. This series of test methods is generally known as immunochromatography and is well-known. In this embodiment, the conjugate pad contains a gold colloid-labeled antibody and lactalbumin, so that the gold colloid-labeled antibody is supported on the conjugate pad in a well-dispersed state. As a result, good detection sensitivity can be obtained, reflecting the good dispersion state. [Examples]

[0034] In the following, unless otherwise specified, units such as "%" and "parts" refer to "mass percent" and "parts by mass" based on mass.

[0035] (1) Preparation of anti-SARS-CoV-2 antibody The anti-SARS-CoV-2 antibodies used in the following tests were obtained by immunizing mice with recombinant SARS-CoV-2 nucleoprotein as the antigen, using methods commonly used by those skilled in the art to produce monoclonal antibodies.

[0036] (2) Preparation of gold colloid solution To 500 mL of purified water heated to 93°C, 1 mL of 7% (w / v) triammonium citrate aqueous solution was added and stirred. Subsequently, 1 mL of 5% (w / v) tetrachlorogold(III) aqueous solution was added and the mixture was reacted for 10 minutes while stirring, after which the reaction solution was brought to a boil. After this, it was cooled in ice water to prepare a gold colloid solution with an average particle size of 60 nm.

[0037] (3) Preparation of gold colloid-labeled anti-SARS-CoV-2 antibody solution 1 To 20 mL of a 60 nm gold colloid solution prepared using purified water and 1N potassium carbonate aqueous solution to achieve an absorbance of 1 at the maximum absorption wavelength and a pH of 7.0, 1 mL of 2 mM phosphate buffer (pH 7.0) containing 25 μg / mL of anti-SARS-CoV-2 antibody was added and the mixture was stirred at room temperature for 10 minutes. Subsequently, 2 mL of 10% BSA (bovine serum albumin) solution was added to the gold colloid solution and the mixture was stirred at room temperature for 5 minutes. The resulting solution was centrifuged at 10°C and 10,000 rpm for 45 minutes, and the supernatant was removed. The gold colloid-labeled antibody remaining as precipitate was suspended in 0.8 mL of a gold colloid-labeled antibody suspension solution (20 mM Tris, 150 mM NaCl, 20 mg / mL lactalbumin (Sigma-Aldrch), pH 7.4) to obtain the gold colloid-labeled anti-SARS-CoV-2 antibody solution of Example 1.

[0038] The 20 mg / mL lactalbumin (Example 1) in the gold colloid-labeled antibody suspension solution was replaced with 10 mg / mL lactalbumin (Example 2), 5 mg / mL lactalbumin (Example 3), 1 mg / mL lactalbumin (Example 4), 20 mg / mL BSA (Comparative Example 1), 10 mg / mL BSA (Comparative Example 2), 5 mg / mL BSA (Comparative Example 3), and 1 mg / mL BSA (Comparative Example 4) to obtain the gold colloid-labeled anti-SARS-CoV-2 antibody solutions of Examples 2-4 and Comparative Examples 1-4.

[0039] (4) Confirmation of the amount of gold colloid-labeled anti-SARS-CoV-2 antibody recovered. Each solution of gold colloid-labeled anti-SARS-CoV-2 antibody was diluted 41-fold. The absorbance of each solution at 400 nm to 900 nm was measured using a spectrophotometer U-3900H (HITACHI), and the maximum absorption wavelength and the absorbance at the maximum absorption wavelength were calculated. The results are shown in Table 1.

[0040] (result) The theoretical absorbance at the maximum absorption wavelength derived from the gold colloid used to prepare the gold colloid-labeled anti-SARS-CoV-2 antibody solution is 25. The absorbance at the maximum absorption wavelength of the gold colloid-labeled anti-SARS-CoV-2 antibody solution is the sum of the absorbance derived from the gold colloid and the absorbance derived from the protein. The gold colloid-labeled anti-SARS-CoV-2 antibody solutions of Examples 1 to 4, suspended in a gold colloid-labeled antibody suspension solution containing lactalbumin, showed good results, with absorbance at the maximum absorption wavelength being close to the theoretical value. On the other hand, the gold colloid-labeled anti-SARS-CoV-2 antibody solutions of Comparative Examples 1 to 4, suspended in a gold colloid-labeled antibody suspension solution containing BSA, showed poor results, with absorbance at the maximum absorption wavelength being less than half of the theoretical value.

[0041] [Table 1]

[0042] (5) Fabrication of conjugate pads The gold colloid-labeled anti-SARS-CoV-2 antibody solutions of Examples 1-4 and Comparative Example 1, prepared in (3) above, were diluted with their respective gold colloid-labeled antibody suspension solutions to obtain an absorbance of 9 at the maximum absorption wavelength. The diluted gold colloid-labeled anti-SARS-CoV-2 antibody solutions were then diluted with 1.33% casein and 4% sucrose solution (pH 7.5) to obtain an absorbance of 3.4 at the maximum absorption wavelength to prepare conjugate pad coating solutions. Using an immunochromatographic dispenser (dispensing platform XYZ3060; BIO DOT), each labeling reagent solution was applied to a glass fiber pad at a rate of 10 μL / cm along a line, and the labeling reagent solution was dried to obtain a conjugate pad.

[0043] (6) Preparation of antibody-immobilized membranes A PBS (phosphate-buffered saline) solution containing 0.75 mg / mL of anti-SARS-CoV-2 antibody and 2.5% sucrose was prepared to obtain a detection layer coating solution. A PBS solution containing 1.0 mg / mL of goat anti-mouse IgG monoclonal antibody and 2.5% sucrose was prepared to obtain a control layer coating solution. Using an immunochromatography dispenser (dispensing platform XYZ3050, BIO DOT), the detection layer coating solution and the control layer coating solution were applied at 1.0 μL / cm to two locations on a nitrocellulose membrane, and the membrane was dried to obtain an antibody-immobilized membrane.

[0044] (7) Preparation of test strips An immunochromatographic test strip for SARS-CoV-2 measurement was obtained by attaching a conjugate pad, antibody-immobilized membrane, and absorbent pad to a plastic adhesive sheet and cutting it into 5 mm wide strips. The test strip was 5 mm wide and 70 mm long, with the conjugate pad, antibody-immobilized membrane (test line, control line), and absorbent pad arranged in that order from one end to the other.

[0045] (8) Preparation of the sample The sample used was prepared by adding SARS-CoV-2 Nucleocapsid Recombinant Protein (Icosagen) at a concentration of 450 TCID50 / mL to the sample diluent provided with the Rapid Tester FLU·NEXT (Sekisui Medical Co., Ltd.).

[0046] (9) Immunochromatography test strips for SARS-CoV-2 measurement 120 μL of the sample was added to an immunochromatographic test strip for SARS-CoV-2 measurement and allowed to stand for 20 minutes. The absorbance at the test line was measured using a densitometry analyzer (RapidTester® Reader, Sekisui Medical Co., Ltd.). The results are shown in Table 2. Regardless of the protein species and protein concentration in the gold colloid-labeled antibody suspension solution, SARS-CoV-2 antigen was detected in all test strips of Examples 1-4 and Comparative Example 1. On the other hand, the concentration of gold colloid-labeled anti-SARS-CoV-2 antibody in the gold colloid-labeled anti-SARS-CoV-2 antibody solutions of Comparative Examples 2-4 was low, and conjugate pads could not be prepared. These results indicate that the amount of gold colloid-labeled anti-SARS-CoV-2 antibody in the gold colloid-labeled anti-SARS-CoV-2 antibody solutions of Examples 1-4 was sufficient to create a conjugate pad, while the amount of gold colloid-labeled anti-SARS-CoV-2 antibody in Comparative Examples 2-4 was insufficient, with the amount of gold colloid-labeled anti-SARS-CoV-2 antibody in Comparative Example 1 being near the lower limit of the acceptable level. In other words, the lactalbumin used in Examples 1-4 has significantly superior ability to suspend gold colloid-labeled antibodies compared to BSA.

[0047] [Table 2]

[0048] (9) Preparation of gold colloid-labeled anti-SARS-CoV-2 antibody solution 2 To 750 mL of a 60 nm gold colloid solution prepared to have an absorbance of 1 at the maximum absorption wavelength, 10 mL of 2 mM phosphate buffer (pH 7.0) containing 60 μg / mL of anti-SARS-CoV-2 antibody was added, and the mixture was stirred at room temperature for 10 minutes. Subsequently, 56 mL of 10% BSA (bovine serum albumin) solution was added to the gold colloid solution, and the mixture was stirred at room temperature for 5 minutes. The resulting solution was centrifuged at 10°C and 10,000 rpm for 45 minutes, and the supernatant was removed. The gold colloid-labeled antibody remaining as precipitate was divided into 30 equal parts and dispensed. The dispensed gold colloid-labeled antibody was suspended in the protein for gold colloid-labeled antibody suspension shown in Table 3, in a pH-adjusted buffer, and in a solution containing NaCl, respectively, and the maximum absorption wavelength and the absorbance at the maximum absorption wavelength were measured using the method described in (3). The theoretical absorbance at the maximum absorption wavelength derived from the gold colloid used to prepare the gold colloid-labeled anti-SARS-CoV-2 antibody solution is 25. When the gold colloid-labeled anti-SARS-CoV-2 antibody was suspended in a gold colloid-labeled antibody suspension solution containing lactalbumin, it showed a recovery rate of over 50% of the theoretical absorbance, indicating a good recovery rate of the gold colloid-labeled antibody. On the other hand, when using a gold colloid-labeled antibody suspension solution containing casein, a high-wavelength shift in the maximum absorption wavelength and poor recovery rate of the gold colloid-labeled antibody were observed.

[0049] [Table 3]

[0050] From the above, it is clear that in the method for producing a gold colloid-labeled antibody suspension according to the present invention, since a suspension solution containing lactalbumin is used when suspending the precipitate of the antibody adsorbed with gold colloid, antibody aggregation is easily prevented and the antibody recovery rate is significantly improved. Furthermore, it is clear that the antigen-binding function of the obtained gold colloid-labeled antibody is sufficiently maintained. The effects of the present invention are astonishing. This is because BSA, which has been conventionally used as a general blocking agent to prevent nonspecific adsorption of proteins, is a type of albumin, and it has been believed that the blocking function of lactalbumin is equivalent to that of BSA. Through diligent research by the inventors, it has become clear that lactalbumin shows a particularly remarkable effect among known blocking agents such as BSA and casein.

Claims

1. A method for producing a gold colloid-labeled antibody suspension, A labeling step is to obtain a mixture by mixing a gold colloid solution and an antibody solution, and to obtain a gold colloid-labeled antibody in the mixture. The mixture is centrifuged to obtain a precipitate of the gold colloid-labeled antibody, and the supernatant containing the antibody that does not adsorb gold colloid is removed in a centrifugation step. The process includes a suspension step of suspending the precipitate of the gold colloid-labeled antibody in a suspension solution containing lactalbumin to obtain a gold colloid-labeled antibody suspension, The aforementioned suspension solution does not contain BSA, or if it does contain BSA, its concentration is less than 0.01 mg / mL. A method for producing a gold colloid-labeled antibody suspension, wherein the concentration of lactalbumin contained in the suspension solution is 0.10 mg / mL or higher.

2. A method for producing a gold colloid-labeled antibody suspension according to claim 1, wherein, after the labeling step and before the centrifugation step, a solution containing BSA is added to the mixture to block the gold colloid-labeled antibody.

3. A step of obtaining the gold colloid-labeled antibody suspension by the manufacturing method described in Claim 1, A method for producing a conjugate pad, comprising the steps of: holding the gold colloid-labeled antibody suspension on a pad and drying the pad to obtain a conjugate pad.

4. A step of obtaining the conjugate pad by the method of Claim 3, A method for manufacturing an inspection instrument, comprising the step of obtaining an inspection instrument equipped with the conjugate pad on a support.

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