A method for resolving differences in antigen concentration in antigen solutions within containers containing antigen solutions.

Abstract

To provide a method for eliminating differences in antigen concentration in antigen solutions within a container containing the antigen solution. [Solution] An antigen solution containing a surfactant, used as a positive control in an immunoassay method.

Description

Technical Field

[0001] The present invention relates to a method for collecting an antigen solution with a uniform concentration without causing a difference in concentration due to the collection position of the antigen solution in a container containing the antigen solution by including a surfactant in the antigen solution.

Background Art

[0002] In an immunoassay method in which an antigen is a measurement target, usually, a liquid containing either an antigen derived from a specimen or a control antigen is prepared as an antigen solution, and it is necessary to use it for immunoassay. Methods for accurately measuring an antigen, such as a method for avoiding the influence of turbidity of an antigen solution (Patent Document 1), have been developed.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] The antigen solution prepared for antigen measurement may have a difference in antigen concentration depending on the position and location of the antigen solution in the container as time passes after preparation. In that case, since the antigen concentration obtained depends on the collection position of the antigen solution, there has been a problem that stable measurement results cannot be obtained.

[0005] An object of the present invention is to provide a method for eliminating the difference in antigen concentration of the antigen solution in a container containing the antigen solution. [[ID=۴۱]]

Means for Solving the Problems

[0006] In order to solve the above problems, the present inventors have found that by adding a surfactant to the antigen solution, it is possible to prevent a difference in antigen concentration in the solution from occurring over time, and have completed the present invention.

[0007] In other words, the present invention is as follows. [1] An antigen solution containing a surfactant, used as a positive control in an immunoassay. [2] The antigen solution of [1], wherein the surfactant is a nonionic surfactant. [3] The antigen solution of [1] or [2], wherein the surfactant is polyethylene glycol mono-4-octylphenyl ether. [4] The antigen solution of [2] or [3], comprising 0.006(w / v)% to 0.012(w / v)% of the nonionic surfactant. [5] An antigen solution of any of [1] to [4], wherein the antigen is derived from Mycoplasma pneumoniae. [6] An immunoassay method that measures antigens using one of the antigen solutions [1] to [5] as a positive control. [7] An immunoassay method for [6] in which the surfactant is a nonionic surfactant. [8] An immunoassay method for preparing an antigen solution containing a target antigen and measuring the antigen concentration in the antigen solution, wherein the antigen concentration is measured using an antigen solution containing a surfactant, thereby preventing differences in antigen concentration depending on the location of antigen solution collection in the container containing the prepared antigen solution, and enabling accurate measurement of the antigen concentration. [9] An immunoassay method for [8] in which the surfactant is a nonionic surfactant.

[10] The immunoassay method of [8] or [9], wherein the surfactant is polyethylene glycol mono-4-octylphenyl ether.

[11] An immunoassay method of any of [8] to

[10] in which the antigen is derived from Mycoplasma pneumoniae.

[12] An immunoassay method using any of [8] to

[10] , which uses an antigen preparation solution that has been prepared at least two hours prior to preparation.

[13] A method for preventing differences in the measured concentration of an antigen solution in a container containing an antigen solution after preparation of an antigen solution for measurement by an immunoassay, comprising preparing the antigen to be detected using an antigen dissolving solution containing a surfactant.

[14] The method of

[13] , wherein the surfactant is a nonionic surfactant.

[15] The method of

[13] or

[14] , wherein the surfactant is polyethylene glycol mono-4-octylphenyl ether.

[16] The antigen is derived from Mycoplasma pneumoniae, by any of the methods described in

[13] to

[15] .

[17] An immunoassay method for preparing an antigen solution containing a target antigen and measuring the antigen concentration in the antigen solution, wherein the antigen solution contains a surfactant and is used in an immunoassay method for accurately measuring the antigen concentration by preventing differences in antigen concentration depending on the location of antigen solution collection in a container containing the prepared antigen solution.

[18] The antigen solution of

[17] , wherein the surfactant is a nonionic surfactant.

[19] The antigen solution of

[18] , wherein the surfactant is polyethylene glycol mono-4-octylphenyl ether.

[20] Antigen solution of

[18] or

[19] , wherein the antigen is derived from Mycoplasma pneumoniae. [Effects of the Invention]

[0008] This invention makes it possible to prevent differences in antigen concentration in the solution over time. [Modes for carrying out the invention]

[0009] The present invention will be described in detail below.

[0010] When preparing an antigen solution using an antigen diluent for antigen measurement, if the antigen does not dissolve uniformly and is instead distributed throughout the solution, the distribution of the antigen in the solution becomes uneven. In particular, as time passes after the preparation of the antigen solution, the antigen concentration in the solution becomes uneven, resulting in differences in antigen concentration depending on the location of the antigen solution in the container. For example, if an antigen solution is placed in a container and left standing, the antigen may move to the bottom of the container due to gravity, and the antigen concentration in the lower part of the antigen solution may be higher than that in the upper part of the container. In this case, differences in concentration occur depending on the location of the antigen solution sampled in the container. As a result, it becomes impossible to accurately measure the antigen concentration.

[0011] In this invention, the antigen solution can also be called the antigen preparation solution.

[0012] This invention provides a method for preventing differences in antigen concentration in the antigen solution within a container over time by adding a surfactant to the antigen solution.

[0013] The antigen of the present invention is not limited and is detectable by an antibody; it can be any substance contained in a sample such as a biological sample. For example, it can be any protein, sugar, or other compound, and may be a substance of biological origin. It may also be a complex of these, such as pathogenic microorganisms like bacteria or viruses, or a biological substance in a living organism or a substance in the environment. Specifically, examples include, but are not limited to, viral antigens such as influenza virus antigen, adenovirus antigen, RSV antigen, HA antigen, HBc antigen, HCV antigen, HIV antigen, EBV antigen, and NLV antigen; bacterial antigens such as Chlamydia trachomatis antigen, Streptococcus antigen, Bordetella pertussis antigen, Helicobacter pylori antigen, Leptospira antigen, Treponema pallidum antigen, Toxoplasma gondii antigen, Borrelia antigen, Bacillus anthrax antigen, and MRSA antigen; mycoplasma antigens such as Mycoplasma pneumoniae; peptide hormones such as human ciliary gonadotropins; steroids such as steroid hormones; physiologically active amines such as epinephrine and morphine; vitamins such as B vitamins; prostaglandins; antibiotics such as tetracycline; toxins produced by bacteria, etc.; and various tumor markers. Preferably, the method of the present invention is useful for measuring antigens that are prone to contamination of antigen preparation solutions with suspensions, such as viral antigens like influenza virus antigen, adenovirus antigen, RSV antigen, HA antigen, HBc antigen, HCV antigen, HIV antigen, EBV antigen, and NLV antigen, and bacterial antigens like Chlamydia trachomatis antigen, Streptococcus antigen, Bordetella pertussis antigen, Helicobacter pylori antigen, Leptospira antigen, Treponema pallidum antigen, Toxoplasma gondii antigen, Borrelia antigen, Bacillus anthrax antigen, and MRSA antigen.

[0014] The test sample includes body fluids such as blood, serum, plasma, urine, feces, saliva, tissue fluid, cerebrospinal fluid, and sweat, etc., or dilutions thereof, and blood, serum, plasma, urine, feces, cerebrospinal fluid, or dilutions thereof are preferred.

[0015] Also, the antigen solution of the present invention can be used as a positive control in immunoassay. In that case, it can also contain a plurality of the above antigens.

[0016] Any lysate or suspension can be used as the antigen solution as long as it can dissolve or suspend the antigen. Such suspensions include physiological saline, buffer solutions, etc. As the buffer solution, a buffer solution usually used in immunoassay can be used, and a phosphate buffer solution, a Tris buffer solution, etc. with a pH of about 6 to 9 can be used. The solution for dissolving or suspending the antigen can be called an antigen lysate or suspension. Also, the antigen lysate or suspension can be called an antigen lysate.

[0017] In the method of the present invention, a surfactant is present in the antigen solution. That is, a surfactant can be included in the above antigen lysate or suspension. Examples of the surfactant include anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants, and it is preferable to use a nonionic surfactant.

[0018] As the nonionic surfactant used in the method of the present invention, specifically, ester-type nonionic surfactants such as glycerin fatty acid esters, sorbitan fatty acid esters, and sucrose fatty acid esters; and surfactants such as fatty alcohol ethoxylates (alkyl polyethylene glycols) and polyoxyethylene alkyl phenyl ethers, etc. Among nonionic surfactants, polyoxyethylene myristyl ether, polyoxyethylene distyrylated phenyl ether, polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene octyldodecyl ether, polyoxyethylene alkylene alkyl ether, polyoxyethylene tribenzyl phenyl ether, polyoxyethylene polyoxypropylene glycol, polyoxyethylene glyceryl ether, and polyoxyethylene diglyceryl ether, etc. are preferred. Among these, polyoxyethylene alkyl phenyl ether is preferred, and among them, polyethylene glycol mono-4-octylphenyl ether (Triton X-100, Triton® X-100) is preferred.

[0019] In the present invention, when preparing an antigen solution containing the above antigen, a surfactant is added to the antigen solution. The antigen solution prepared by adding the surfactant in this way can be used as a positive control for the assay. The present invention includes a method of preventing a difference in the antigen concentration in the antigen solution used as a positive control in a container over time by adding a surfactant to the antigen solution used as a positive control. Further, the present invention includes an antigen solution containing a surfactant and used as a positive control in an immunoassay method, and a positive control used in immunoassay containing the antigen solution containing a surfactant.

[0020] In the present invention, immunoassay methods such as immunodiffusion, immunoturbidimetry, immunohistochemistry, hemagglutination, latex assay, enzyme immunoassay (EIA), and radioimmunoassay (RIA) can be applied. Among these, the sandwich method is preferred as the immunoassay method. In the sandwich method, a complex is formed by sandwiching an antigen between two antibodies, and the complex is detected. The sandwich method itself is well known in the field of immunoassay and can be performed by methods such as immunochromatography or ELISA. All of these sandwich methods are well known, and the method of the present invention can be performed by known sandwich methods, except that it uses a monoclonal antibody that recognizes the N protein as an antigen.

[0021] The sandwich method uses one or more antibodies that recognize the antigen (an antibody immobilized on the solid phase and a labeled antibody). Alternatively, the antigen may be sandwiched between two identical antibodies to form a complex.

[0022] In immunoassays using the sandwich method as the detection principle, any solid phase on which antibodies can be immobilized by known techniques can be used. For example, porous thin films (membranes) with capillary action, particulate matter, test tubes, resin plates, and other known materials can be arbitrarily selected. Furthermore, substances such as enzymes, radioisotopes, fluorescent substances, luminescent substances, colored particles, and colloidal particles can be used to label the antibodies. Among the various immunoassay methods using the aforementioned materials, immunochromatography, a lateral flow immunoassay method using a membrane, is particularly preferred from the viewpoint of simplicity and speed in clinical testing.

[0023] Immunochromatography can be performed using an immunoassay instrument consisting of a support having a detection region on which an antibody (antibody 1) that captures the target substance (antigen) is immobilized, a labeled region having a movable labeled antibody (antibody 2) labeled with a suitable labeling substance such as colored polystyrene particles or gold colloid, a sample pad on which the sample is dropped, an absorbent zone that absorbs the spread sample solution, and a backing sheet for bonding these components together. In this method, the prepared antigen solution is dropped onto the sample pad, and a complex of antibody 2, which can bind to the target substance (labeled reagent) labeled with a suitable labeling substance such as colored polystyrene particles or gold colloid, and the target substance is spread and moved on the solid support on which antibody 1 is immobilized using capillary action. As a result, a complex of immobilized substance-target antigen-labeled reagent is formed on the solid support, and the target antigen can be detected by detecting the signal of the labeled reagent emitted from the complex (in the case of gold colloid, the portion of the solid support on which the substance that can bind to the target antigen is immobilized turns red). This immunoassay method can be performed at 5 to 35°C, preferably at room temperature, and pretreatment with a sample processing solution should also be carried out within this temperature range.

[0024] Furthermore, the number of detection regions and the type of labeled antibody included in the labeled region are not limited to one. By using antibodies corresponding to multiple target substances, two or more antigens can be detected using the same immunoassay instrument.

[0025] In the method of the present invention, the sample is preferably diluted 5 to 20 times, more preferably 10 to 20 times, using an antigen lysis solution or suspension containing the above-mentioned surfactant, and then measured.

[0026] The concentration of the surfactant used in the present invention is preferably 0.006(w / v)% or higher, for example, 0.006(w / v)% to 0.012(w / v)%.

[0027] After preparing an antigen solution using an antigen dissolving solution or suspension, the antigen concentration in the antigen solution becomes non-uniform over time, resulting in differences in antigen concentration depending on the location of the antigen solution in the container. In particular, the antigen concentration is higher in the lower part of the container than in the upper part. The method of the present invention can eliminate the difference in antigen concentration depending on the location of the antigen solution in the container. The method of the present invention is effective even immediately after antigen preparation, but is particularly effective when measured 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours or more after antigen solution preparation. Preferably, the antigen is measured within 9 hours, 10 hours, 12 hours, 18 hours, or 24 hours after antigen solution preparation. Here, the upper antigen solution refers to the antigen solution located above a certain height from the bottom of the container, and the lower antigen solution refers to the antigen solution located below a certain height from the bottom of the container. Furthermore, the upper antigen solution refers to the antigen solution present in the portion of the container that is above 50%, 60%, 70%, 80%, 90%, or 95% of the total height from the bottom of the container, while the lower antigen solution refers to the antigen solution present in the portion of the container that is below 95%, 90%, 80%, 70%, 60%, or 50% of the total height from the bottom of the container. For example, if a container contains 1 ml of antigen solution, 75 μl of the supernatant can be called the upper antigen solution, and the remainder can be called the lower antigen solution.

[0028] Furthermore, the present invention also encompasses antigen solutions containing surfactants and immunoassay kits containing said antigen solutions. The immunoassay kit includes an antigen solution containing a surfactant. It may also include a positive control, a negative control, instructions, etc. [Examples]

[0029] [Example 1] Mycoplasma pneumoniae (Myco) inactivated antigen was diluted in 1 mL of antigen diluent in a multi-tube to prepare an antigen suspension, which was then used as the antigen solution.

[0030] Mycoplasma pneumoniae (Myco) inactivated antigen was prepared as follows: First, the bacterial strain was inoculated into PPLO-BROTH containing 17 vol% horse serum and 2.6 (w / v)% yeast extract, and continuously rotated cultured at 37°C for 7 days. The culture solution was then transferred to a centrifuge tube and centrifuged at 12,000 × g for 50 minutes. The supernatant was removed, a predetermined amount of barbiturate buffer was added, and the suspension was resuspended and centrifuged at 12,000 × g for 50 minutes. This process was repeated to wash the sediment. A predetermined amount of barbiturate buffer was added to the sediment after removing the supernatant, and the suspension was resuspended. This bacterial suspension was sonicated and centrifuged at 1,690 × g for 30 minutes. The supernatant was collected, and a 5(w / v)% BSA solution was added to achieve a final concentration of 1.25(w / v)%. Inactivation treatment was carried out at 60°C for 20 minutes to obtain an inactivated Mycoplasma pneumoniae cell suspension. This was further diluted with 2(w / v)% BSA-added PBS(-) and freeze-dried. The freeze-dried inactivated Mycoplasma pneumoniae cells were re-lysed to obtain the inactivated antigen.

[0031] In a 1 ml multi-tube, 75 μl of the supernatant was used as the upper antigen solution, and the remainder as the lower antigen solution. Antigen solutions were prepared in multi-tubes after 0 hours and 2 hours, respectively. The upper and lower antigen solutions were collected and diluted 16-fold (40 μL / 640 μL) with 0.3% (w / v) BSA and PBS.

[0032] The intensity of the color of the test line in each dilution was visually determined using an immunochromatographic test kit (QuickNavi-Mycoplasma (Denka Co., Ltd.)) corresponding to the Mycoplasma pneumoniae antigen.

[0033] The intensity of the test line color was judged on a four-point scale from "++++" to "+", as follows. Dark:++++>+++>++>+: Light

[0034] The measurement results are shown in Table 1.

[0035] [Table 1]

[0036] As shown in Table 1, for Mycoplasma pneumoniae antigen measured with QuickNavi-Mycoplasma, there was no difference in the results between the upper and lower antigen solutions after 0 hours.

[0037] However, after 2 hours, a difference in the results emerged between the upper and lower antigen solutions. Compared to the results after 0 hours, the test line for the upper antigen solution became fainter, while the test line for the lower antigen solution became darker, resulting in significantly different measurement results.

[0038] [Example 2] A Mycoplasma pneumoniae (Myco) antigen solution was prepared in the same manner as in Example 1, and an antigen solution containing polyethylene glycol mono-4-octylphenyl ether (Triton X-100, Tx-100) in the range of 0.006 to 0.0012 (w / v)% was prepared.

[0039] Next, the Mycoplasma pneumoniae antigen solution was left to stand for 8 hours and measured in the same manner as in Example 1.

[0040] [Table 2]

[0041] As shown in Table 2, when the concentration of polyethylene glycol mono-4-octylphenyl ether in the Mycoplasma pneumoniae antigen solution was 0.0 to 0.004 (w / v)%, a difference in the determination of the upper and lower antigen solutions occurred.

[0042] However, when the concentration of polyethylene glycol mono-4-octylphenyl ether in the Mycoplasma pneumoniae antigen solution was between 0.006 (w / v)% and 0.012 (w / v)%, there was no difference in the determination of the upper and lower antigen solutions.

[0043] The antigen solution prepared by adding a surfactant in this manner can be used as a positive control. [Industrial applicability]

[0044] The method of the present invention allows for the accurate measurement of the antigen concentration in an antigen solution after a period of time has elapsed since preparation.

Claims

1. An antigen solution containing a surfactant, used as a positive control in immunoassay methods.

2. The antigen solution according to claim 1, wherein the surfactant is a nonionic surfactant.

3. The antigen solution according to claim 1 or 2, wherein the surfactant is polyethylene glycol mono-4-octylphenyl ether.

4. The antigen solution according to claim 2 or 3, wherein the nonionic surfactant is contained in an amount of 0.006 (w / v)% to 0.012 (w / v)%.

5. The antigen solution according to any one of claims 1 to 4, wherein the antigen is derived from Mycoplasma pneumoniae.

6. An immunoassay method for measuring an antigen, using the antigen solution described in any one of claims 1 to 5 as a positive control.

7. The immunoassay method according to claim 6, wherein the surfactant is a nonionic surfactant.

8. An immunoassay method for preparing an antigen solution containing a target antigen and measuring the antigen concentration in the antigen solution, wherein the method uses an antigen solution containing a surfactant to measure the antigen concentration, thereby preventing differences in antigen concentration depending on the sampling location of the antigen solution in the container containing the prepared antigen solution, and enabling accurate measurement of the antigen concentration.

9. The immunoassay method according to claim 8, wherein the surfactant is a nonionic surfactant.

10. The immunoassay method according to claim 8 or 9, wherein the surfactant is polyethylene glycol mono-4-octylphenyl ether.

11. An immunoassay method according to any one of claims 8 to 10, wherein the antigen is derived from Mycoplasma pneumoniae.

12. An immunoassay method according to any one of claims 8 to 10, wherein the antigen preparation solution is used after two hours or more have elapsed since the preparation of the antigen solution.

13. A method for preventing differences in the measured concentration of an antigen solution in a container containing an antigen solution after preparation, which is measured by an immunoassay method, including preparing the antigen to be detected using an antigen dissolving solution containing a surfactant.

14. The method according to claim 13, wherein the surfactant is a nonionic surfactant.

15. The method according to claim 13 or 14, wherein the surfactant is polyethylene glycol mono-4-octylphenyl ether.

16. The method according to any one of claims 13 to 15, wherein the antigen is derived from Mycoplasma pneumoniae.

17. An immunoassay method for preparing an antigen solution containing a target antigen and measuring the antigen concentration in the antigen solution, wherein the antigen solution contains a surfactant and is used in an immunoassay method for accurately measuring the antigen concentration by preventing differences in antigen concentration depending on the location of antigen solution collection in the container containing the prepared antigen solution.

18. The antigen solution according to claim 17, wherein the surfactant is a nonionic surfactant.

19. The antigen solution according to claim 18, wherein the surfactant is polyethylene glycol mono-4-octylphenyl ether.

20. The antigen solution according to claim 18 or 19, wherein the antigen is derived from Mycoplasma pneumoniae.

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