JP3712963B2 - Aqueous solution containing peroxidase-labeled antibody - Google Patents

Description

【００１６】
比較例１
透析ヤギ血清の代わりに、透析を行なっていない正常ヤギ血清を同濃度で用いた以外は実施例１と同様の操作を行なった。表１に示したように、実施例１とは異なり、保存直後に活性が失われていることがわかる。
【００１７】
比較例２
第１試薬の作製において、透析ヤギ血清の代わりにＰＢＳを用いたこと以外は、実施例１と同様の操作を行なった。その結果を表１に示した。この結果からもわかるように、比較例１と同様に保存直後に活性が失われている。
【００１８】
実施例２
実施例１で梅毒トレポネーマ抗体陽性血清の代わりに、イムノティクルスオートＴＰ２（エイアンドティー社製）で陰性と判定された健常者血清１００検体を用いたこと以外は、実施例１と同様の操作を行なった。表２に示したように、１００検体全てが陰性となった。
【００１９】
【表２】

【００２０】
比較例３
５０ｖ／ｖ％透析ヤギ血清の代わりに１ｗ／ｖ％ＢＳＡを含むＰＢＳを用いた以外は実施例２と同様の操作を行なった。表２に示したように、１００検体中３検体が陽性と判定され、非特異反応が抑制されていないことがわかる。
【００２１】
実施例３および比較例４
実施例１および比較例３で調製した各第１試薬で、ＩＤ−１０００を使用して梅毒トレポネーマ抗体陽性血清を２０回繰り返し測定を行なった。表３に示したように、透析血清を用いた試薬の方がＢＳＡを用いた試薬よりも同時再現性が優れていることがわかる。
【００２２】
【表３】

【００２３】
【発明の効果】
本発明の水溶液を用いることにより、ペルオキシダーゼ標識抗体が長期間安定で、かつ非特異反応が抑制され測定再現性が良好な組成物が提供される。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an aqueous solution containing a peroxidase-labeled antibody used for immunoassay and the like.
[0002]
[Prior art]
In recent years, measurement methods using immune reactions are frequently used in research fields such as clinical examinations, immunology, biochemistry, and molecular biology because of their simple operation. Among them, enzyme immunoassay (hereinafter referred to as EIA), chemiluminescence immunoassay (hereinafter referred to as CLIA) and the like are frequently used from the viewpoint of increasing sensitivity. In the measurement method, an enzyme-labeled antibody is usually used. Peroxidase is often used as one of the enzymes to be labeled, but peroxidase-labeled antibodies are often unstable, especially at low concentrations, and are known to deteriorate during storage. Several techniques are known to solve this problem. For example, labeling by maleimide hinge method (enzyme immunoassay 3rd edition, page 99), use of citrate buffer (Clin. Chem. Vol. 28, No. 12, p. 2423, (1982)), labeled antibody Lyophilized (US Pat. No. 4,265,552), addition of aminopyrine (Japanese Patent Publication No. 1-295566), addition of anilinonaphthalenesulfonic acid (Japanese Patent Publication No. 58-5668), addition of calcium chloride (US Patent) No. 4782023) has been reported. In addition, a method of allowing free peroxidase to coexist in a solution containing a peroxidase-labeled antibody has been proposed (Japanese Patent Laid-Open No. 7-222600). As described above, various attempts have been made to stabilize peroxidase-labeled antibodies, but few have sufficient storage stability to withstand use as commercially available reagents that require stability of at least six months to one year or more. . Further, although stability is maintained if lyophilized, another problem arises that the operation becomes complicated during use, and further improvement has been desired.
[0003]
Further, in the above-described measurement method, a solid phase that carries an antigen or an antibody against the substance to be measured is used, but the labeling substance may adsorb nonspecifically to the solid phase. Therefore, it is important to add a nonspecific adsorption inhibitor before adding the labeling substance and cover the adsorption point on the solid phase in order to suppress nonspecific adsorption. Nonspecific adsorption inhibitors used when performing immunoassays such as EIA and CLIA adsorb antibodies that are not specific to antigens (or antibodies) to the solid phase, or labeling substances that are not specific to the solid phase. It has an effect of preventing adsorption. Conventionally, as this non-specific adsorption inhibitor, those obtained by dissolving bovine serum albumin (BSA), milk protein or the like in an appropriate buffer solution have been often used. However, the anti-biotin-labeled antibody is supported on a solid phase, and in the sandwich EIA method via the antibody, the effect is not sufficient, or conversely, these nonspecific adsorption inhibitors induce nonspecific adsorption. There was a problem.
[0004]
[Problems to be solved by the invention]
An object of the present invention is to provide an aqueous solution containing a peroxidase-labeled antibody that is stable for a long period of time, suppresses non-specific reactions, and has good measurement reproducibility. Other objects and advantages of the present invention will become apparent from the following description.
[0005]
[Means for Solving the Problems]
As a result of diligent research to solve the above problems, the present inventors have made the stability of peroxidase-labeled antibody in an aqueous solution coexisting with animal serum that has been dialyzed in an aqueous solution containing a peroxidase-labeled antibody, The inventors have found that suppression of non-specific reaction and measurement reproducibility are improved, and the present invention has been completed.
[0006]
That is, the present invention is an aqueous solution characterized by containing a peroxidase-labeled antibody and dialyzed serum.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The peroxidase-labeled antibody used in the present invention refers to an antibody in which peroxidase is chemically bound. The peroxidase-labeled antibody may be commercially available or may be prepared from peroxidase and an antibody, and is not particularly limited. When peroxidase is chemically bound to an antibody, for example, a known method such as periodate method, glutaraldehyde method, maleimide method, or the like can be used. The peroxidase used in the present invention may be derived from either animals or plants, but is preferably derived from plants. More preferred is horseradish peroxidase (hereinafter sometimes abbreviated as HRP). The antibody labeled with peroxidase in the present invention may be a polyclonal antibody or a monoclonal antibody. The subtype is not particularly limited, and may be any of IgG, IgM, IgE, IgA, and IgD. Further, it is not limited by the type of antigen recognized by the antibody. The animal species used for obtaining the antibody is not particularly limited, but those usually used in immunological measurement such as goat, rabbit and mouse are preferred.
[0008]
In the present invention, dialyzed serum is used. Although a known method can be used for dialysis without any problem, it is preferable to use a dialysis membrane having a molecular weight cutoff of 5,000 to 50,000, such as a cellophane membrane. Even if a dialysis membrane having a higher molecular weight cutoff or a dialysis membrane having a lower molecular weight cutoff is used, the effect of using dialyzed serum is weakened. Serum is not particularly limited, but is usually selected from the group consisting of goats, donkeys, cattle, fetal bovines, mice, sheep, rabbits, horses, chickens, monkeys, rats, guinea pigs, dogs and humans, which are usually used for immunoassays It is preferred to use at least one serum derived from animals. Serum derived from such animals can be used without any particular limitation. It is preferable to use serum from the same animal species as the animal from which the antibody used for the immune reaction was obtained. For example, when immobilizing an immune complex consisting of a biotin-labeled antibody, an antigen recognized by the antibody, and an enzyme-labeled antibody on a solid phase carrying an anti-biotin antibody, the same animal as the animal species from which the anti-biotin antibody was obtained It is preferred that the dialyzed serum of the species coexist in a solution containing the enzyme labeled antibody. The concentration of dialyzed serum is not particularly limited, but is preferably 0.1 to 50 v / v%. If the concentration is lower than that, the preservation of the peroxidase-labeled antibody and the effect of suppressing the nonspecific reaction are small. If the concentration is higher than that, measurement errors are likely to occur, such as an increase in the viscosity of the solution, which is not preferable.
[0009]
The dialysis used in the present invention and the buffer for diluting the serum are generally used, for example, phosphate buffer, Tris-HCl buffer, barbital buffer, citrate buffer, acetate buffer, Good buffer, etc. Can be used. The concentration and pH of the buffer in the buffer solution are not particularly limited, but if the concentration is too high, the reaction system may be affected, and if it is too low, the reaction tends to become unstable. Since biological proteins are present in the reaction system, they are preferably neutral to weakly alkaline, and most preferably in the range of pH 7.0 to 8.5. Other additives may be present in the solution of the present invention. Examples of such other additives include inorganic salts such as sodium chloride and potassium chloride, and preservatives such as sodium azide and microcide.
[0010]
【Example】
In order to describe the present invention in more detail, examples and comparative examples will be described below, but the present invention is not limited to these examples.
[0011]
Example 1
1. Preparation of dialyzed goat serum 10 mL of normal goat serum was filled into a dialysis tube (manufactured by Wako Pure Chemical Industries, Ltd.) having a molecular weight cut off of 12,000 to 14,000, and 20 mmol / L containing 0.15 mol / L sodium chloride. The sample was immersed in 1,000 mL of a phosphate buffer (pH 7.4) (hereinafter abbreviated as PBS) at 4 ° C. for 5 hours. This operation was performed three times to prepare dialyzed serum. Subsequently, the dialyzed serum was diluted with PBS to prepare 50 v / v% dialyzed goat serum.
[0012]
2. Preparation of Biotin-labeled Syphilis Treponema Antigen Solution As syphilis treponema antigens, 15 kDa (abbreviated as antigen A), 17 kDa (abbreviated as antigen B) (hereinafter, Bios Pacific), 47 kDa (abbreviated as antigen C) (manufactured by Capricorn) A seed was used. 3.4 mg of biotinamido caproic acid N-hydroxysuccinimide ester (manufactured by Nacalai Tesque) was dissolved in 100 μL of DMSO (the solution prepared here is hereinafter referred to as a biotin solution). 1 mg of each antigen was dissolved in 500 μL of 10 mmol / L HEPES buffer. 7 μL, 6 μL, or 2 μL of the prepared biotin solution was added thereto and mixed well, followed by reaction at 25 ° C. for 4 hours. In order to remove the unreacted biotin labeling agent, NAP-5 (manufactured by Pharmacia) was used, and gel filtration was performed with PBS according to the instruction manual. The biotin-labeled antigen solution recovered by gel filtration was diluted with PBS so that antigen A was 1 μg / mL, antigen B was 1.1 μg / mL, and antigen C was 3.1 μg / mL. Sodium azide was added to this so that it might become 0.095 w / v%. This was used as a biotin-labeled syphilis treponema antigen solution (hereinafter abbreviated as the first reagent).
[0013]
3. Preparation of HRP-labeled antibody solution Using 50 v / v% dialyzed goat serum, HRP-labeled anti-human IgG mouse monoclonal antibody (manufactured by Southern Biotechnology) was diluted to 60 ng / mL and adjusted to 0.095 w / v%. Sodium chloride was added. This was used as an HRP-labeled antibody solution (hereinafter abbreviated as second reagent).
[0014]
4). Measurement First, diluted serum was prepared by mixing 50 μL of syphilis treponema antibody-positive serum and 10 μL of the first reagent, and incubated at 40 ° C. for 5 minutes. On the other hand, the anti-biotin antibody carrying | support reaction agent (made by Toyobo Co., Ltd.) was incubated at 40 degreeC. This incubation was continued until photometry was completed. 50 μL of ID-1000 block solution (Toyobo Co., Ltd.) was added dropwise to the reactant. Next, 50 μL of diluted serum after incubation was dropped, and 10 μL of the second reagent was further dropped. Two minutes later, 200 μL of ID-1000 cleaning liquid (manufactured by Toyobo Co., Ltd.) was added dropwise in an amount of 100 μL, and after another minute, ID-1000 color developing solution (manufactured by Toyobo Co., Ltd.) was added dropwise. The reflection intensity was measured when a laser beam having a wavelength of 670 nm was irradiated on the solid phase surface for 2 seconds after 5.5 to 7.5 seconds and 2 seconds after 40.5 to 42.5 seconds from the bottom of the colored droplet. . For actual measurement, ID-1000 (manufactured by Toyobo Co., Ltd.) was used. The 1st reagent was preserve | saved at 12 degreeC and the said measurement was performed 0, 1, 6, and 12 months after preservation | save. Table 1 shows the results. Here, the relative value when the measured value at the start of storage stability is 100 is shown. As shown in Table 1, it can be seen that the activity is substantially maintained during the storage period.
[0015]
[Table 1]

[0016]
Comparative Example 1
The same operation as in Example 1 was performed except that normal goat serum not dialyzed was used at the same concentration instead of dialyzed goat serum. As shown in Table 1, it can be seen that, unlike Example 1, the activity was lost immediately after storage.
[0017]
Comparative Example 2
In preparing the first reagent, the same operation as in Example 1 was performed except that PBS was used instead of dialyzed goat serum. The results are shown in Table 1. As can be seen from these results, the activity is lost immediately after storage as in Comparative Example 1.
[0018]
Example 2
The same operation as in Example 1 was carried out except that 100 healthy subject sera determined to be negative by Immunotic Auto TP2 (manufactured by A & T) were used instead of syphilis treponema antibody-positive sera in Example 1. It was. As shown in Table 2, all 100 samples were negative.
[0019]
[Table 2]

[0020]
Comparative Example 3
The same operation as in Example 2 was performed except that PBS containing 1 w / v% BSA was used instead of 50 v / v% dialyzed goat serum. As shown in Table 2, it can be seen that 3 out of 100 samples were determined to be positive, and the nonspecific reaction was not suppressed.
[0021]
Example 3 and Comparative Example 4
With each first reagent prepared in Example 1 and Comparative Example 3, syphilis treponema antibody-positive serum was repeatedly measured 20 times using ID-1000. As shown in Table 3, it can be seen that the reagent using dialysis serum has better simultaneous reproducibility than the reagent using BSA.
[0022]
[Table 3]

[0023]
【The invention's effect】
By using the aqueous solution of the present invention, a composition is provided in which the peroxidase-labeled antibody is stable for a long period of time, non-specific reaction is suppressed, and measurement reproducibility is good.

Claims (3)

An aqueous solution comprising a peroxidase-labeled antibody and dialyzed serum. 2. The serum is at least one serum derived from an animal selected from the group consisting of goat, donkey, cow, fetal bovine, mouse, sheep, rabbit, horse, chicken, monkey, rat, guinea pig, dog and human. The aqueous solution described in 1. The aqueous solution according to claim 1, wherein the dialyzed serum is serum dialyzed with a cellophane membrane.