JP3573330B2 - Stabilized aqueous solution containing antigen - Google Patents
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Description
【０００１】
【産業上の利用分野】
本発明は、肺サーファクタント蛋白質における抗原活性の安定化法、安定化された抗原含有水溶液を使用した肺サーファクタント蛋白質の測定法、および安定化された抗原含有水溶液を構成試薬として含有する肺サーファクタント蛋白質測定用キットに関するものである。
【０００２】
【従来の技術】
肺サーファクタント蛋白質は肺サーファクタントに特異的なアポタンパク質（ＳＰ）であり、現在までに親水性のＳＰ−ＡとＳＰ−Ｄ、および疎水性のＳＰ−ＢとＳＰ−Ｃの４種類が報告されている。その中でも、ＳＰ−Ｄは気道−肺胞系における生体防御機構において重要な役割を果たしていると考えられており、抗原抗体反応を利用した免疫学的手法によりサンプル（血清など）中のＳＰ−Ｄを測定し、特発性間質性肺炎などの肺疾患を診断できることが報告されている（医学と薬学 ３６（４）：８０３−８０８（１９９６）参照）。
【０００３】
【発明が解決しようとする課題】
上記報告書で報告されている方法は、組換えＤＮＡ手法により取得されるリコンビナントＳＰ−Ｄ（ｒＳＰ−Ｄ）を標準物質として使用しており、このｒＳＰ−Ｄが２５℃以上になると抗原性を徐々に失ってしまうため、標準抗原溶液を低温で保存するとともに測定時の反応温度も４℃などの低温で行わなければならず、更なる操作性、安定性の向上が望まれていた。
【０００４】
【課題を解決するための手段】
本発明者らは、上記問題点を解決すべく鋭意検討した結果、まったく意外なことにＳＰ−Ｄとカルシウムなどの２価金属イオンを共存させるだけで、３７℃の条件下でも抗原活性が失活せずに安定化されること見いだし、本発明を完成させた。
【０００５】
すなわち本発明は、抗原抗体反応を利用した免疫学的手法によりサンプル中の肺サーファクタント蛋白質を測定する際、２価金属イオンを共存させ、肺サーファクタント蛋白質の抗原活性を安定化する方法に関するものである。
また、本発明は、抗原抗体反応を利用した免疫学的手法によりサンプル中の肺サーファクタント蛋白質を測定する際、抗原含有水溶液として肺サーファクタント蛋白質と２価金属イオンとからなる安定化された抗原含有水溶液を使用する方法に関するものである。
さらに、本発明は、抗原抗体反応を利用した免疫学的手法によりサンプル中の肺サーファクタント蛋白質を測定するためのキットであって、標準抗原溶液として肺サーファクタント蛋白質と２価金属イオンとからなる抗原含有水溶液を構成試薬として含有することを特徴とする、肺サーファクタント蛋白質測定用キットに関するものである。
【０００６】
【発明の実施の形態】
本発明における肺サーファクタント蛋白質とは、ＳＰ−Ａ、ＳＰ−Ｂ、ＳＰ−ＣおよびＳＰ−Ｄのいずれであってもかまわないが、特に断らない限り診断項目として有用性が確認されているＳＰ−ＡまたはＳＰ−Ｄを意味する。
【０００７】
最初に、本発明は、抗原抗体反応を利用した免疫学的手法によりサンプル中の肺サーファクタント蛋白質を測定する際、２価金属イオンを共存させ、肺サーファクタント蛋白質の抗原活性を安定化する方法に関するものである。
共存させる２価金属イオンとしては、周期表２族ａ亜族元素の２価金属イオンであればよく、具体的にはカルシウム、バリウム、マグネシウムなど各イオン例示することができ、特にカルシウムイオンが好適である。
このような２価金属イオンは、肺サーファクタント蛋白質と共存していればよく、必ずしも水溶液の状態である必要はない。後述の実施例に示すように、溶液状はもとより、肺サーファクタント蛋白質をプレートに結合させ、大方の水分を取り除いた状態であっても２価金属イオンを共存させることにより肺サーファクタント蛋白質を安定化させることが可能である。
共存させる２価金属イオンの濃度は特に制限されるものではないが、通常０．００１〜１０００ｍＭ、好ましくは０．０１〜１００ｍＭの範囲から適宜選定すればよい。
【０００８】
次に、本発明は、抗原抗体反応を利用した免疫学的手法によりサンプル中の肺サーファクタント蛋白質を測定する際、抗原含有水溶液として肺サーファクタント蛋白質と２価金属イオンとからなる安定化された抗原含有水溶液を使用する方法に関するものである。
肺サーファクタント蛋白質と２価金属イオンは上述したものを使用することができる。ＳＰ−Ｄとカルシウムイオンを例に挙げ、具体的に説明すれば、塩化カルシウムなどの金属無機塩を水または適当な緩衝液に溶解させ、これに組換えＤＮＡ手法により調製したリコンビナントＳＰ−Ｄ（ｒＳＰ−Ｄ）を添加することで調製した安定化された抗原含有水溶液を使用することができる。
２価金属イオンの濃度は特に制限されるものではないが、通常０．００１〜１０００ｍＭ、好ましくは０．０１〜１００ｍＭの範囲から適宜選定すればよい。
【０００９】
さらに、本発明は、抗原抗体反応を利用した免疫学的手法によりサンプル中の肺サーファクタント蛋白質を測定するためのキットであって、標準抗原溶液として肺サーファクタント蛋白質と２価金属イオンとからなる抗原含有水溶液を構成試薬として含有することを特徴とする、肺サーファクタント蛋白質測定用キットに関するものである。
このようなキットを、ＥＬＩＳＡ法によるＳＰ−Ｄ測定キットを例に挙げ、具体的に説明すれば、たとえば以下のキットを例示することができる。
（キットＡ）
▲１▼固相化抗ＳＰ−Ｄ抗体試薬
▲２▼酵素標識化抗ＳＰ−Ｄ抗体試薬
▲３▼ＳＰ−Ｄ標準抗原溶液（カルシウムイオン含有）
さらに、上記キットに診断用キットに通常添付されている酵素反応基質液、酵素反応停止液、洗浄液などを添付してもよい。特に、２価金属イオンを含有する洗浄液、具体的には０．００１〜１０００ｍＭ、好ましくは０．０１〜１００ｍＭのカルシウムイオンを含有する洗浄液を添付することにより、測定中のＳＰ−Ｄの抗原活性をより強固に安定化することで測定精度を高めるとともに、操作性も向上させることができる。
このようなキットの使用法は、例えば、医学と薬学 ３６（４），１９９６ ８０４−８０８（１９９６）などに記載の公知の測定方法に準じて行えばよい。
【００１０】
【発明の効果】
肺サーファクタント蛋白質（たとえば、ＳＰ−Ｄ）と２価金属イオン（たとえば、カルシウムイオン）を共存させることで、肺サーファクタント蛋白質の抗原活性を安定化することは本発明者らによって初めて見いだされたことである。このことにより、従来低温保存を要求されていた肺サーファクタント蛋白質の標準抗原液は常温でも保存流通が可能になるとともに、測定時の反応温度も常温で行うことができ、肺サーファクタント蛋白質の免疫学的測定の操作性、汎用性を飛躍的に向上せしめることが可能となった。また、液状の安定性のみならず、プレート上に結合した肺サーファクタント蛋白質の安定性も２価金属イオンを共存させることで向上させることができ、精度および操作性が向上した肺サーファクタント蛋白質の測定が初めて可能となった。
【００１１】
【実施例】
以下、実施例により、本発明をさらに説明するが、本発明はこれに限定されるものではない。
【００１２】
実施例１ 溶液状での安定化の検討（１）
緩衝液（１０ｍＭ ＨＥＰＥＳ、１５０ｍＭ塩化ナトリウム、１．０％ウシ血清アルブミン、０．５％トライトンＸ−１００、ｐＨ７．２）に、最終濃度で１０ｍＭなるように塩化カルシウム、塩化マグネシウムまたは塩化カリウムを添加、あるいは５ｍＭ になるようにＥＤＴＡを添加した。この緩衝液を希釈用緩衝液とする。また、当該希釈用緩衝液にｒＳＰ−Ｄを一定量添加し、４℃または３７℃で３時間インキュベートし、これをサンプル液とした。このような希釈用緩衝液、サンプル液を用い、ヤマサ醤油株式会社製のＳＰ−Ｄ測定キットを使用して、医学と薬学 ３６（４），８０４−８０８（１９９６）記載の測定法（以下、標準操作法という）に従ってサンプル液中のＳＰ−Ｄを測定した。
その結果を表１に記す。表１から明らかなように、２価金属イオン、特にカルシウムイオンがＳＰ−Ｄの抗原活性の安定に有効であることが明らかとなった。
【表１】
実施例２ 溶液状での安定化の検討（２）
カルシウム添加希釈用緩衝液（１０ｍＭ塩化カルシウム、１０ｍＭ ＨＥＰＥＳ、１５０ｍＭ塩化ナトリウム、０．５％トライトンＸ−１００、１．０％ウシ血清アルブミン）を用いてｒＳＰ−Ｄを希釈して標準抗原液を調製した。この標準抗原液ならびにカルシウム無添加標準抗原液を４℃または３７℃で３時間インキュベート後、標準操作法に従って、標準抗原液中のＳＰ−Ｄを測定した。その結果を図１に示す。
その結果、図１に示すように、カルシウム添加標準液は３７℃、３時間処理後も４℃の場合と同等の抗原性を保持することが認められた。一方、カルシウム無添加の場合は３７℃、３時間処理で５０％以上抗原性の失活が確認された。
【００１４】
実施例３ カルシウム添加標準液での温度安定性
実施例２で調製した、１０ｍＭ塩化カルシウムを添加した標準抗原液およびカルシウム無添加の標準抗原液を用いて２５℃、３０℃または３７℃で１、３、６時間または一晩インキュベート後、標準操作法に従って標準抗原液中のＳＰ−Ｄを測定した。カルシウムの添加により大幅に熱安定性が向上し、３０℃、３７℃でも６時間、２５℃ならば一晩処理しても９０％以上抗原活性が保持できることが明らかとなった。
【００１５】
実施例４ プレート上での安定性
抗ＳＰ−Ｄモノクローナル抗体を９６穴マイクロプレートに結合させ、この固相化抗体と実施例２で調製したカルシウム含有標準抗原液を４℃で一晩反応させた。次に、洗浄液（１０ｍＭ ＨＥＰＥＳ、１５０ｍＭ塩化ナトリウム、ｐＨ７．２）と、この洗浄液に１０ｍＭ塩化カルシウムを添加したカルシウム添加洗浄液にて各ウエルを３回洗浄後、紙タオル等の上で水分を完全に取り除き乾燥状態にした。その状態のまま５分間または１０分間放置した後、以後標準操作法に従ってＳＰ−Ｄを測定した。その結果を図２および図３に示す。
カルシウム無添加洗浄液を用いた場合は５分放置で１０％、１０分では１５％吸光度が低下した。しかしカルシウム添加洗浄液を用いた場合では１０分放置しても５％の低下にとどまり、プレート上でもカルシウムイオンの共存でＳＰ−Ｄが安定化することが明らかとなった。
【図面の簡単な説明】
【図１】図１は、カルシウムイオンの有無による溶液状でのＳＰ−Ｄ抗原活性の安定性を示すものである。縦軸は４℃での抗原活性を１００％としたときの相対活性（％）を、横軸は抗原液の濃度を示す。
【図２】図２は、カルシウムイオン無添加洗浄液を用いた場合のプレート上でのＳＰ−Ｄ抗原活性の安定性を示すものである。縦軸は洗浄直後の放置時間が０分の時の抗原活性を１００％としたときの相対活性（％）を、横軸は抗原液の濃度を示す。
【図３】図３は、カルシウムイオン添加洗浄液を用いた場合のプレート上でのＳＰ−Ｄ抗原活性の安定性を示すものである。縦軸は洗浄直後の放置時間が０分の時の抗原活性を１００％としたときの相対活性（％）を、横軸は抗原液の濃度を示す。[0001]
[Industrial applications]
The present invention relates to a method for stabilizing antigen activity in a lung surfactant protein, a method for measuring a lung surfactant protein using a stabilized aqueous solution containing an antigen, and a method for measuring a lung surfactant protein containing a stabilized aqueous solution containing an antigen as a constituent reagent It relates to a kit for use.
[0002]
[Prior art]
Lung surfactant protein is an apoprotein (SP) specific to lung surfactant, and four types of hydrophilic SP-A and SP-D and hydrophobic SP-B and SP-C have been reported so far. I have. Among them, SP-D is considered to play an important role in the host defense mechanism in the airway-alveolar system, and SP-D in a sample (such as serum) is determined by an immunological technique utilizing an antigen-antibody reaction. Has been reported to be able to diagnose pulmonary diseases such as idiopathic interstitial pneumonia (see Medicine and Pharmaceutical Sciences 36 (4): 803-808 (1996)).
[0003]
[Problems to be solved by the invention]
The method reported in the above report uses a recombinant SP-D (rSP-D) obtained by a recombinant DNA technique as a standard substance. Since the standard antigen solution is gradually lost, the standard antigen solution must be stored at a low temperature and the reaction temperature at the time of measurement must be performed at a low temperature such as 4 ° C., and further improvement in operability and stability has been desired.
[0004]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to solve the above-mentioned problems. As a result, surprisingly, the antigen activity was lost even at 37 ° C only by allowing SP-D and a divalent metal ion such as calcium to coexist. The present invention has been found to be stabilized without being utilized, and the present invention has been completed.
[0005]
That is, the present invention relates to a method for stabilizing the antigen activity of a lung surfactant protein in the presence of a divalent metal ion when measuring a lung surfactant protein in a sample by an immunological technique utilizing an antigen-antibody reaction. .
Further, the present invention provides a method for measuring a lung surfactant protein in a sample by an immunological technique utilizing an antigen-antibody reaction, wherein the stabilized antigen-containing aqueous solution comprising a lung surfactant protein and a divalent metal ion is used as the antigen-containing aqueous solution. About how to use .
Furthermore, the present invention provides a kit for measuring a lung surfactant protein in a sample by an immunological technique utilizing an antigen-antibody reaction, wherein the kit contains an antigen comprising a lung surfactant protein and a divalent metal ion as a standard antigen solution. The present invention relates to a kit for measuring a lung surfactant protein, which comprises an aqueous solution as a constituent reagent.
[0006]
BEST MODE FOR CARRYING OUT THE INVENTION
The pulmonary surfactant protein in the present invention may be any of SP-A, SP-B, SP-C, and SP-D. Unless otherwise specified, SP- has been confirmed to be useful as a diagnostic item. A or SP-D.
[0007]
First, the present invention relates to a method for stabilizing the antigen activity of a lung surfactant protein in the presence of a divalent metal ion when measuring a lung surfactant protein in a sample by an immunological technique utilizing an antigen-antibody reaction. It is.
The coexisting divalent metal ion may be a divalent metal ion of a group 2a subgroup element of the periodic table, and specific examples thereof include calcium, barium, and magnesium. Particularly, calcium ion is preferable. It is.
Such a divalent metal ion only needs to coexist with the lung surfactant protein, and does not necessarily need to be in the form of an aqueous solution. As shown in the examples described below, the lung surfactant protein is bound to the plate as well as in the form of a solution, and the lung surfactant protein is stabilized by coexisting with divalent metal ions even when most of the water is removed. It is possible.
The concentration of the coexisting divalent metal ion is not particularly limited, but may be appropriately selected usually from the range of 0.001 to 1000 mM, preferably from 0.01 to 100 mM.
[0008]
Next, the present invention relates to a method for measuring a lung surfactant protein in a sample by an immunological technique utilizing an antigen-antibody reaction, wherein the antigen-containing aqueous solution contains a stabilized antigen containing a lung surfactant protein and a divalent metal ion. The present invention relates to a method using an aqueous solution .
As the lung surfactant protein and the divalent metal ion, those described above can be used. Taking SP-D and calcium ions as an example, specifically, a metal inorganic salt such as calcium chloride is dissolved in water or a suitable buffer, and the recombinant SP-D (recombinant DNA-prepared by recombinant DNA technique) is added thereto. A stabilized antigen-containing aqueous solution prepared by adding rSP-D) can be used.
The concentration of the divalent metal ion is not particularly limited, but may be appropriately selected usually from the range of 0.001 to 1000 mM, preferably from 0.01 to 100 mM.
[0009]
Furthermore, the present invention provides a kit for measuring a lung surfactant protein in a sample by an immunological technique utilizing an antigen-antibody reaction, wherein the kit contains an antigen comprising a lung surfactant protein and a divalent metal ion as a standard antigen solution. The present invention relates to a kit for measuring a lung surfactant protein, which comprises an aqueous solution as a constituent reagent.
Such a kit is exemplified by a kit for measuring SP-D by ELISA, and specifically described, for example, the following kits can be exemplified.
(Kit A)
(1) Immobilized anti-SP-D antibody reagent (2) Enzyme-labeled anti-SP-D antibody reagent (3) SP-D standard antigen solution (containing calcium ions)
Further, an enzyme reaction substrate solution, an enzyme reaction stop solution, a washing solution, and the like which are usually attached to the diagnostic kit may be attached to the kit. In particular, by attaching a washing solution containing divalent metal ions, specifically a washing solution containing calcium ions of 0.001 to 1000 mM, preferably 0.01 to 100 mM, the antigen activity of SP-D during measurement is attached. By more stabilizing, the measurement accuracy can be improved and the operability can be improved.
Such a kit can be used according to a known measurement method described in, for example, Medicine and Pharmaceutical Sciences 36 (4), 1996 804-808 (1996).
[0010]
【The invention's effect】
It has been found by the present inventors for the first time to stabilize the antigen activity of a lung surfactant protein by coexisting a lung surfactant protein (eg, SP-D) and a divalent metal ion (eg, calcium ion). is there. As a result, the standard antigen solution of lung surfactant protein, which had conventionally been required to be kept at low temperature, can be stored and distributed at room temperature, and the reaction temperature at the time of measurement can be performed at room temperature. It has made it possible to dramatically improve the operability and versatility of measurement. Further, not only the stability of the liquid but also the stability of the lung surfactant protein bound on the plate can be improved by coexisting divalent metal ions, and the measurement of the lung surfactant protein with improved accuracy and operability can be performed. It became possible for the first time.
[0011]
【Example】
Hereinafter, the present invention will be further described by way of examples, but the present invention is not limited thereto.
[0012]
Example 1 Study of stabilization in a solution state (1)
Add calcium chloride, magnesium chloride or potassium chloride to a buffer (10 mM HEPES, 150 mM sodium chloride, 1.0% bovine serum albumin, 0.5% Triton X-100, pH 7.2) to a final concentration of 10 mM. Alternatively, EDTA was added to a concentration of 5 mM. This buffer is used as a dilution buffer. In addition, a certain amount of rSP-D was added to the buffer for dilution, and the mixture was incubated at 4 ° C. or 37 ° C. for 3 hours to obtain a sample solution. Using such a buffer for dilution and sample solution, using a SP-D measurement kit manufactured by Yamasa Shoyu Co., Ltd., a measurement method described in Medicine and Pharmacy 36 (4), 804-808 (1996) (hereinafter, referred to as “ SP-D in the sample solution was measured according to a standard operation method).
Table 1 shows the results. As is clear from Table 1, it was revealed that divalent metal ions, particularly calcium ions, are effective for stabilizing the antigen activity of SP-D.
[Table 1]
Example 2 Examination of stabilization in a solution state (2)
A standard antigen solution is prepared by diluting rSP-D with a buffer solution for adding calcium (10 mM calcium chloride, 10 mM HEPES, 150 mM sodium chloride, 0.5% Triton X-100, 1.0% bovine serum albumin). did. After incubating the standard antigen solution and the calcium-free standard antigen solution at 4 ° C. or 37 ° C. for 3 hours, SP-D in the standard antigen solution was measured according to a standard operation method. The result is shown in FIG.
As a result, as shown in FIG. 1, it was confirmed that the calcium-containing standard solution maintained the same antigenicity as that at 4 ° C. even after treatment at 37 ° C. for 3 hours. On the other hand, when calcium was not added, inactivation of antigenicity of 50% or more was confirmed after treatment at 37 ° C. for 3 hours.
[0014]
Example 3 Temperature stability in a standard solution containing calcium Prepared at 25 ° C., 30 ° C. or 37 ° C. using the standard antigen solution containing 10 mM calcium chloride and the standard antigen solution containing no calcium prepared in Example 2, After incubation for 3, 6 hours or overnight, SP-D in the standard antigen solution was measured according to the standard procedure. It has been clarified that the addition of calcium greatly improves the thermal stability, and that the antigen activity can be maintained at 90% or more even when treated at 30 ° C. and 37 ° C. for 6 hours and at 25 ° C. overnight.
[0015]
Example 4 Stable anti-SP-D monoclonal antibody on a plate was bound to a 96-well microplate, and the immobilized antibody was reacted with the calcium-containing standard antigen solution prepared in Example 2 at 4 ° C. overnight. . Next, each well was washed three times with a washing solution (10 mM HEPES, 150 mM sodium chloride, pH 7.2) and a calcium-added washing solution obtained by adding 10 mM calcium chloride to the washing solution, and then water was completely removed on a paper towel or the like. Removed and left dry. After leaving it in that state for 5 or 10 minutes, SP-D was measured according to the standard operation method. The results are shown in FIGS.
When the washing solution without calcium was used, the absorbance decreased by 10% when left for 5 minutes and by 15% after 10 minutes. However, in the case of using the calcium-added washing solution, the decrease was only 5% even after being left for 10 minutes, and it was clarified that SP-D was stabilized on the plate by the coexistence of calcium ions.
[Brief description of the drawings]
FIG. 1 shows the stability of SP-D antigen activity in a solution depending on the presence or absence of calcium ions. The vertical axis indicates the relative activity (%) when the antigen activity at 4 ° C. is defined as 100%, and the horizontal axis indicates the concentration of the antigen solution.
FIG. 2 shows the stability of SP-D antigen activity on a plate when a calcium ion-free washing solution was used. The vertical axis represents the relative activity (%) when the antigen activity when the standing time immediately after washing is 0 minutes is defined as 100%, and the horizontal axis represents the concentration of the antigen solution.
FIG. 3 shows the stability of SP-D antigen activity on a plate when a calcium ion-added washing solution was used. The vertical axis represents the relative activity (%) when the antigen activity when the standing time immediately after washing is 0 minutes is defined as 100%, and the horizontal axis represents the concentration of the antigen solution.
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| WO2006121064A1 (en) * | 2005-05-11 | 2006-11-16 | Yamasa Corporation | Method of stabilizing pulmonary surfactant protein |
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| CN112119088A (en) * | 2018-03-29 | 2020-12-22 | 气道治疗公司 | Methods and compositions comprising surfactant protein D (SP-D) |
| JP7425459B2 (en) * | 2019-10-11 | 2024-01-31 | 株式会社シノテスト | Stabilized HMGB1-containing solution |
| WO2023145915A1 (en) * | 2022-01-31 | 2023-08-03 | 積水メディカル株式会社 | Immunoassay method for pulmonary surfactant protein and immunoassay reagent |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2006121064A1 (en) * | 2005-05-11 | 2006-11-16 | Yamasa Corporation | Method of stabilizing pulmonary surfactant protein |
| US7776619B2 (en) | 2005-05-11 | 2010-08-17 | Yamasa Corporation | Method of stabilizing pulmonary surfactant protein |
| JP4734328B2 (en) * | 2005-05-11 | 2011-07-27 | ヤマサ醤油株式会社 | Stabilization of pulmonary surfactant protein |
| KR101243867B1 (en) * | 2005-05-11 | 2013-03-20 | 야마사 쇼유 가부시키가이샤 | Method of stabilizing pulmonary surfactant protein |
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