Colloidal gold-labeled antibody suspension, method for producing same, and inspection instrument

Abstract

This method for producing a colloidal gold-labeled antibody suspension includes: a labeling step for mixing a colloidal gold solution and an antibody solution to obtain a mixed solution, and obtaining a colloidal gold-labeled antibody in the mixed solution; a centrifugation step for centrifuging the mixed solution to obtain a precipitate of the colloidal gold-labeled antibody and removing a supernatant containing antibodies onto which colloidal gold does not adsorb; and a suspension step of suspending the precipitate of the colloidal gold-labeled antibody in a suspension solution containing lactalbumin, to obtain a colloidal gold-labeled antibody suspension.

Description

Gold colloid-labeled antibody suspension, its manufacturing method, and test equipment

[0001] The present invention relates to a gold colloid-labeled antibody suspension using lactalbumin, a method for producing the same, and a test instrument. This application claims priority to Japanese Patent Application No. 2024-087370, filed on May 29, 2024, the contents of which are incorporated herein by reference.

[0002] Antibodies labeled with gold colloids have been widely used in medical tests and academic research. For example, immunochromatography, a type of immunoassay method, is well known as a method for qualitatively or quantitatively measuring the presence of a test substance in a biological sample such as urine or blood (see, for example, Patent Document 1).

[0003] Japanese Patent Application Laid-Open No. 2002-148266

[0004] Gold colloids are generally gold nanoparticles with an average particle diameter of less than 1 μm. When dispersed in a solution, the solution appears colored when exposed to white light. When gold colloids and antibodies are mixed in the solution, they undergo a physicochemical reaction, causing the antibody to adsorb to the gold colloid. As a result, the antibody can be labeled with gold colloid and colored. A blocking process is then performed in which the antibody is exposed to bovine serum albumin (BSA) or similar to prevent nonspecific adsorption of the gold colloid-labeled antibody to other substances.

[0005] In the production of colloidal gold-labeled antibodies, to separate the labeled antibody adsorbed to the colloidal gold from the unlabeled antibody, a solution containing both is centrifuged to precipitate the colloidal gold-labeled antibody, and the supernatant containing the unlabeled antibody is removed. The precipitated colloidal gold-labeled antibody is then resuspended in the desired solution to obtain the desired colloidal gold-labeled antibody suspension. However, there is a problem in that a considerable amount of the colloidal gold-labeled antibody remains undispersed even after resuspension, forming aggregates. The aggregates cannot be used in subsequent processes, which reduces the production yield of colloidal gold-labeled antibodies.

[0006] The present invention provides a colloidal gold-labeled antibody suspension with excellent dispersibility, a method for producing the same, and a test instrument using the colloidal gold-labeled antibody.

[0007] [1] A method for producing a colloidal gold-labeled antibody suspension, comprising: a labeling step of mixing a colloidal gold solution with an antibody solution to obtain a mixture and obtaining a colloidal gold-labeled antibody in the mixture; a centrifugation step of centrifuging the mixture to obtain a precipitate of the colloidal gold-labeled antibody and removing a supernatant containing the antibody not adsorbed to the gold colloid; and a suspension step of suspending the colloidal gold-labeled antibody precipitate in a suspension solution containing lactalbumin to obtain a colloidal gold-labeled antibody suspension. [2] The method for producing a colloidal gold-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 perform a blocking treatment on the colloidal gold-labeled antibody. [3] The method for producing a colloidal gold-labeled antibody suspension according to [1] or [2], wherein the suspension solution does not contain BSA, or, if it contains BSA, its concentration is less than 1 mg / mL. [4] The method for producing a colloidal gold-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 more. [5] A colloidal gold-labeled antibody suspension containing a colloidal gold-labeled antibody and 0.01 mg / mL or more of lactalbumin. [6] A test device equipped with a pad for absorbing a test solution, wherein a dried form of a colloidal gold-labeled antibody suspension containing a colloidal gold-labeled antibody and 0.1 μg or more of lactalbumin is held in the pad. [7] The test device according to [6], wherein the pad is supported on an elongated support and is used for performing immunochromatography on the test solution.

[0008] According to the manufacturing method of the present invention, colloidal gold-labeled antibodies can be manufactured with high yield. In the colloidal gold-labeled antibody suspension of the present invention, the dispersibility of the colloidal gold-labeled antibodies is good, and specificity for the antigen is sufficiently maintained. In the test device of the present invention, the specificity of the colloidal gold-labeled antibodies is sufficiently maintained, and detection sensitivity is comparable to that of conventional devices.

[0009] <<Method for Producing a Colloidal Gold Labeled Antibody Suspension>> A first aspect of the present invention is a method for producing a colloidal gold labeled antibody suspension, which comprises a labeling step, a centrifugation step, and a suspension step, which are described below.

[0010] [Labeling Step] The labeling step is a step of mixing a colloidal gold solution with an antibody solution to obtain a mixed solution and obtaining a colloidal gold-labeled antibody in the mixed solution. This step can be performed in the same manner as conventional methods for obtaining colloidal gold-labeled antibodies.

[0011] The colloidal gold solution used in this step is not particularly limited, and any desired solution can be selected and used. Colloidal gold 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 colloidal gold in the mixed solution can be defined by its absorbance at its maximum absorption wavelength, and the absorbance ranges from 0.1 to 5, preferably 0.5 to 1.5.

[0012] The antibody solution used in this step is not particularly limited, and any desired antibody solution can be selected and used. The antibody may be either a monoclonal antibody or a 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 is derived is also not particularly limited. The antibody may be a full-length antibody or a fragment containing the antigen-binding site. The concentration of the antibody in the mixture is, for example, in the range of 0.01 to 100 μg / mL, preferably 0.1 to 10 μg / mL, and more preferably 0.5 to 5 μg / mL.

[0013] The dispersion medium for the gold colloid solution, antibody solution, and the mixed solution may be any dispersion medium that allows the antibody to be stably dispersed, and examples of such dispersion medium include those containing various commonly used pH buffers and inorganic salts. The pH of the mixed solution is preferably within a range in which the three-dimensional structure of the antibody is stably maintained, for example, 6.5 to 7.5.

[0014] The antibody and colloidal gold in the mixture come into physical contact and interact with each other, causing the colloidal gold to naturally adsorb to the antibody, resulting in a colloidal gold-labeled antibody. The reaction (adsorption) between the antibody and colloidal gold in the mixture is completed within, for example, several minutes to several tens of minutes at room temperature.

[0015] [Blocking Treatment] After the labeling step, in order to prevent nonspecific adsorption of the colloidal gold-labeled antibody formed in the mixed solution, it is preferable to carry out a blocking treatment in which a blocking agent is brought into contact with the colloidal gold-labeled antibody and the blocking agent is adsorbed to sites where nonspecific adsorption may occur.

[0016] Examples of blocking agents include proteins such as bovine serum albumin (BSA), gelatin, casein, ovalbumin, lactalbumin, sericin, and fetuin, as well as peptides, polyethylene glycol, polyanions, N,N-dialkylamides, lower alkyl sulfoxides, alkylcelluloses, and carboxyalkylcelluloses. Among these, BSA is preferred because it does not inhibit the specificity of the antibody or interfere with the performance of the gold colloid-labeled antibody.

[0017] The blocking treatment is preferably carried out by adding a solution containing a blocking agent, such as a solution containing BSA (BSA solution), to the mixture. The dispersion medium constituting the BSA solution may be any dispersion medium that stably disperses BSA, and examples thereof include those containing various commonly used pH buffers and inorganic salts. The pH of the BSA solution is preferably within a range that stably maintains the three-dimensional structure of BSA and antibodies, for example, 6.5 to 7.5. The BSA concentration in the mixture used in the blocking treatment is, for example, 1 to 100 mg / mL, preferably 5 to 20 mg / mL. Blocking agents other than BSA can also be used under similar conditions.

[0018] In the mixed solution, the colloidal gold-labeled antibody and the blocking agent such as BSA come into contact and interact with each other, causing the blocking agent to spontaneously adsorb to the colloidal gold-labeled antibody, thereby completing the blocking process. The blocking process can be completed at room temperature in, for example, several minutes to several tens of minutes.

[0019] [Centrifugation step] The centrifugation step is a step in which the mixture is centrifuged to obtain a precipitate of the colloidal gold-conjugated antibody, and the supernatant containing the antibody not adsorbed to the colloidal gold is removed. This step can be performed in the same manner as in conventional methods for obtaining colloidal gold-conjugated antibodies.

[0020] The conditions for centrifugation are not particularly limited, as long as they are conditions that apply a centrifugal force sufficient to precipitate the colloidal gold-labeled antibody without precipitating the unlabeled antibody. Examples of centrifugal force include a range of 1,300 g to 15,000 g, and preferably a range of 5,000 g to 15,000 g. When using a centrifuge equipped with a rotor with a radius of approximately 5 cm to 30 cm, the rotation speed is, for example, a range of 5,000 to 20,000 rpm, and preferably a range of 8,000 to 12,000 rpm. At this rotation speed, the colloidal gold-labeled antibody can be precipitated by centrifugation for approximately 0.5 to 2 hours. During centrifugation, the mixture is generally cooled to approximately 4 to 10°C.

[0021] After centrifugation, the supernatant can be removed by a conventional method, such as decantation or aspirator suction.

[0022] [Suspension step] The suspension step is a step of suspending the precipitate of the colloidal gold-labeled antibody in a suspension solution containing lactalbumin to obtain a colloidal gold-labeled antibody suspension. The 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 the lactalbumin is derived is not particularly limited, and bovine-derived lactalbumin is preferred because it is commercially available at low cost.

[0023] The lactalbumin concentration in the suspension solution is preferably, for example, 0.01mg / mL or more, more preferably 0.10mg / mL or more, and even more preferably 0.50mg / mL or more.In a more preferred range, it may be 1.0mg / mL or more, 2.0mg / mL or more, or 4.0mg / mL or more.The upper limit of lactalbumin concentration is not particularly limited, and for example, 100mg / mL or less, 50mg / mL or less, 25mg / mL or less, 20mg / mL or less can be mentioned as a guideline.Since the suspension power of lactalbumin for gold colloid-labeled antibody is extremely high, it is sufficient if it is above the lower limit, and it is not necessary to use at a high concentration.

[0024] The dispersion medium constituting the suspension solution may be any one that allows lactalbumin to be stably dispersed, and examples thereof include various commonly used pH buffers. The dispersion medium may contain an inorganic salt. NaCl is preferred as the inorganic salt. Examples of the concentration of the inorganic salt include 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 allows the three-dimensional structure of lactalbumin to be stably maintained, 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 colloidal gold-conjugated antibody precipitate obtained in the centrifugation step can be converted into the amount of colloidal gold, since it is believed that almost all of the colloidal gold used in the labeling step is contained in the precipitate. When the volume of the mixture used in the labeling step at which the absorbance at the maximum absorption wavelength of the colloidal gold in the mixture is 1 is V1, and the volume of the suspending solution is V2, the volume ratio represented by V1 / V2 can be, for example, 1 to 100, preferably 10 to 80, and more preferably 15 to 80. In other words, the volume V2 of the suspending solution can be adjusted so that the absorbance at the maximum absorption wavelength of the colloidal gold in the colloidal gold-conjugated antibody suspension solution 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 precipitate is mixed with a suspending solution and suspended, the colloidal gold-labeled antibody and lactalbumin come into contact with each other and interact, resulting in a well-dispersed colloidal gold-labeled antibody suspension in which aggregation of the colloidal gold-labeled antibody is suppressed. The suspension of the precipitate in the suspending solution is completed at room temperature, for example, within a few minutes.

[0027] The above steps result in the desired colloidal gold-labeled antibody suspension containing colloidal gold-labeled antibody and lactalbumin. The lactalbumin concentration in the colloidal gold-labeled antibody suspension depends on the volume V2 of the suspending solution used and the volume of the precipitate, but can be approximately the same as or slightly diluted with the lactalbumin concentration in the suspending solution, for example, 0.1 mg / mL or higher. The concentration of colloidal gold-labeled antibody in the colloidal gold-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 below, the suspending power of lactalbumin is much stronger than that of BSA, so the suspending solution does not need to contain BSA. The suspending solution may contain BSA, for example, at a concentration of less than 1 mg / mL, 0.1 mg / mL or less, or 0.01 mg / mL or less. If a blocking treatment with BSA is performed before the centrifugation step, a small amount of BSA will be contained in the precipitate of the colloidal gold-labeled antibody obtained in the centrifugation step, so a small amount of BSA may be contained in the colloidal gold-labeled antibody suspension.

[0029] <Colloidal Gold-Labeled Antibody Suspension> A second aspect of the present invention is a colloidal gold-labeled antibody suspension containing a colloidal gold-labeled antibody and 0.1 mg / mL or more of lactalbumin. The composition of the colloidal gold-labeled antibody suspension of this aspect may be the same as the composition of the colloidal gold-labeled antibody suspension obtained by the production method of the first aspect, and redundant explanations will be omitted.

[0030] <Test Instrument> A third aspect of the present invention is a test instrument equipped with a pad for absorbing a test solution containing an analyte. In this test instrument, a dried form of a colloidal gold-labeled antibody suspension containing a colloidal gold-labeled antibody that binds to the analyte and lactalbumin is retained in the pad. A pad that retains the colloidal gold-labeled antibody suspension and then dried to obtain a dried form is called a conjugate pad. In one example of a test instrument, the amount of lactalbumin contained in the conjugate pad is preferably 0.1 μg or more, more preferably 0.5 μg or more. More preferred ranges include 1.0 μg or more, 2.0 μg or more, and 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 glass fiber pad, filter paper, cotton, or nonwoven fabric. The composition of the colloidal gold-labeled antibody suspension may be the same as that of the colloidal gold-labeled antibody suspension obtained by the manufacturing method of the first aspect, and redundant description will be omitted.

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

[0032] The conjugate pad already contains a gold colloid-conjugated antibody. If the test solution contains an antigen of the gold colloid-conjugated antibody, the gold colloid-conjugated antibody and the antigen will bind to each other through an antigen-antibody reaction to form a complex when the test solution soaks into the conjugate pad. When the complex binds to the antibody on the antibody-immobilized membrane, a colored test line appears, allowing detection of a signal corresponding to the amount of antigen present in the test solution. Other agents may be used to obtain or enhance the detection signal. In the test instrument, the test solution penetrates the conjugate pad, the antibody-immobilized membrane, and the absorbent pad in this order. The absorbent pad is provided to finally receive and retain the test solution.

[0033] An example of an embodiment of this aspect is a test strip (immunochromatography test strip) based on immunochromatography. This strip includes the conjugate pad and an elongated support supporting the conjugate pad. The entire or a portion of the strip is capable of absorbing the test solution. By immersing one end of the strip in the test solution or by dripping the test solution onto the strip, the test solution permeates the strip from one end to the other. The conjugate pad is provided along the way, allowing a signal to be detected if the test solution reacts with the colloidal gold-conjugated antibody, as described above. Typically, this test is performed using a configuration in which an antibody-immobilized membrane is provided downstream adjacent to the conjugate pad, and an absorption pad is provided further downstream. This series of test methods is generally known as immunochromatography. In this aspect, the conjugate pad contains colloidal gold-conjugated antibody and lactalbumin, allowing the colloidal gold-conjugated antibody to be supported in a well-dispersed state on the conjugate pad. As a result, good detection sensitivity reflecting the well-dispersed state can be obtained.

[0034] Hereinafter, unless otherwise specified, the units "%" and "parts" refer to "% by mass" and "parts by mass" based on mass.

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

[0036] (2) Preparation of gold colloid solution: 1 mL of a 7% (w / v) aqueous solution of triammonium citrate was added to 500 mL of purified water heated to 93°C and mixed with stirring. Next, 1 mL of a 5% (w / v) aqueous solution of gold tetrachloro(III) was added, and the mixture was allowed to react for 10 minutes with stirring. The reaction solution was then boiled. The mixture was then cooled in ice water to prepare a gold colloid solution with an average particle size of 60 nm.

[0037] (3) Preparation of colloidal gold-labeled anti-SARS-CoV-2 antibody solution 1. 20 mL of 60 nm colloidal gold solution was 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 stirred at room temperature for 10 minutes. Subsequently, 2 mL of 10% BSA (bovine serum albumin) solution was added to the colloidal gold solution and 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 colloidal gold-labeled antibody remaining as a precipitate was suspended in 0.8 mL of a colloidal gold-labeled antibody suspension solution (20 mM Tris, 150 mM NaCl, 20 mg / mL lactalbumin (Sigma-Aldrich), pH 7.4) to obtain the colloidal gold-labeled anti-SARS-CoV-2 antibody solution of Example 1.

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

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

[0040] (Results) The theoretical value of absorbance at the maximum absorption wavelength derived from the gold colloid used to prepare the gold colloid-labeled anti-SARS-CoV-2 antibody solution was 25, and the absorbance at the maximum wavelength of the gold colloid-labeled anti-SARS-CoV-2 antibody solution was 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, which were suspended in a gold colloid-labeled antibody suspension solution containing lactalbumin, had absorbances at the maximum absorption wavelength close to the theoretical value, which was good. On the other hand, the gold colloid-labeled anti-SARS-CoV-2 antibody solutions of Comparative Examples 1 to 4, which were suspended in a gold colloid-labeled antibody suspension solution containing BSA, had absorbances at the maximum absorption wavelength that were less than half the theoretical value, which was poor.

[0041]

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

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

[0044] (7) Preparation of test strips: A conjugate pad, antibody-immobilized membrane, and absorbent pad were attached to a plastic adhesive sheet, which was then cut to a width of 5 mm to obtain an immunochromatography test strip for measuring SARS-CoV-2. 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) Sample Preparation SARS-CoV-2 Nucleocapsid Recombinant Protein (Icosagen) was added to the specimen diluent included with Rapid Tester FLU-NEXT (Sekisui Medical Co., Ltd.) at a concentration of 450 TCID50 / mL to prepare a sample.

[0046] (9) Immunochromatography Test Strip for Measuring SARS-CoV-2 120 μL of sample was dropped onto an immunochromatography test strip for measuring SARS-CoV-2 and allowed to stand for 20 minutes. The absorbance at the test line was measured using a densitometry analyzer (Rapid Tester (registered trademark) Reader, Sekisui Medical Co., Ltd.). The results are shown in Table 2. SARS-CoV-2 antigen was detected in all test strips of Examples 1 to 4 and Comparative Example 1, regardless of the protein type and protein concentration in the colloidal gold-labeled antibody suspension solution. On the other hand, the concentration of colloidal gold-labeled anti-SARS-CoV-2 antibody in the colloidal gold-labeled anti-SARS-CoV-2 antibody solution in Comparative Examples 2 to 4 was low, and a conjugate pad could not be prepared. These results show that the amount of colloidal gold-labeled anti-SARS-CoV-2 antibody in the colloidal gold-labeled anti-SARS-CoV-2 antibody solutions in Examples 1 to 4 was sufficient for preparing a conjugate pad, whereas the amount of colloidal gold-labeled anti-SARS-CoV-2 antibody in Comparative Examples 2 to 4 was insufficient, and the amount of colloidal gold-labeled anti-SARS-CoV-2 antibody in Comparative Example 1 was near the lower limit of the allowable amount. In other words, this shows that the lactalbumin used in Examples 1 to 4 has a significantly superior ability to suspend colloidal gold-labeled antibodies compared to BSA.

[0047]

[0048] (9) Preparation of Colloidal Gold-Labeled Anti-SARS-CoV-2 Antibody Solution 2: 10 mL of 2 mM phosphate buffer (pH 7.0) containing 60 μg / mL of anti-SARS-CoV-2 antibody was added to 750 mL of 60 nm colloidal gold solution prepared to have an absorbance of 1 at the maximum absorption wavelength, 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 colloidal gold 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 colloidal gold-labeled antibody remaining as a precipitate was dispensed into 30 equal aliquots. The dispensed colloidal gold-labeled antibody was suspended in the protein for suspending the colloidal gold-labeled antibody shown in Table 3, a pH-adjusted buffer, and a solution containing NaCl, 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 colloidal gold used to prepare the colloidal gold-labeled anti-SARS-CoV-2 antibody solution is 25. When the colloidal gold-labeled anti-SARS-CoV-2 antibody was suspended in a colloidal gold-labeled antibody suspension solution containing lactalbumin, the absorbance was 50% or more of the theoretical absorbance, indicating a good recovery rate of the colloidal gold-labeled antibody. On the other hand, when a colloidal gold-labeled antibody suspension solution containing casein was used, a high-wavelength shift in the maximum absorption wavelength and poor recovery rate of the colloidal gold-labeled antibody were observed.

[0049]

[0050] From the above, it is clear that in the method for producing a colloidal gold-labeled antibody suspension according to the present invention, a suspension solution containing lactalbumin is used when suspending the precipitate of an antibody adsorbed with colloidal gold, which makes it easier to prevent antibody aggregation and significantly improves the recovery rate of the antibody. It is also clear that the antigen-binding function of the resulting colloidal gold-labeled antibody is fully maintained. The effects of the present invention are surprising. This is because BSA, which has traditionally been used as a common 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 extensive research by the present inventors, it has been revealed that lactalbumin exhibits particularly remarkable effects compared to known blocking agents such as BSA and casein.

Claims

1. A method for producing a colloidal gold-labeled antibody suspension, comprising: a labeling step of mixing a colloidal gold solution with an antibody solution to obtain a mixed solution and obtaining a colloidal gold-labeled antibody in the mixed solution; a centrifugation step of centrifuging the mixed solution to obtain a precipitate of the colloidal gold-labeled antibody and removing a supernatant containing antibody not adsorbed to the gold colloid; and a suspension step of suspending the colloidal gold-labeled antibody precipitate in a suspension solution containing lactalbumin to obtain a colloidal gold-labeled antibody suspension.

2. The 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 perform a blocking treatment on the gold colloid-labeled antibody.

3. The method for producing a gold colloid-labeled antibody suspension according to claim 2, wherein the suspension solution does not contain BSA, or if it does contain BSA, the concentration of BSA is less than 1 mg / mL.

4. A method for producing a gold colloid-labeled antibody suspension according to any one of claims 1 to 3, wherein the concentration of lactalbumin contained in the suspension solution is 0.01 mg / mL or more.

5. A colloidal gold-labeled antibody suspension comprising a colloidal gold-labeled antibody and 0.01 mg / mL or more of lactalbumin.

6. A test device having a pad for absorbing a test solution, wherein a dried suspension of a gold colloid-labeled antibody containing a gold colloid-labeled antibody and 0.1 μg or more of lactalbumin is held in the pad.

7. The test device according to claim 6, wherein the pad is supported on an elongated support, and the test device is used to perform immunochromatography on the test solution.

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