JP6787053B2 - Dispersibility-stabilized complex in which proteins are adsorbed on colloidal gold - Google Patents
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Description
本発明は、分散性安定化された、金コロイドにタンパク質が吸着した複合体に関するものである。 The present invention relates to a dispersion-stabilized complex in which proteins are adsorbed on colloidal gold.
イムノクロマト等の体外診断における染色材料として、金コロイドが多く用いられる。金コロイドは表面プラズモン共鳴に起因した赤色の強い発色を有しており、視覚による簡易的な診断を可能にする。金コロイドへのタンパク質固定化は、一般的に金コロイド表面が負に帯電したことを利用した静電的な物理吸着により行われる。しかし、吸着させるタンパク質の性質によっては、金コロイドの凝集が進行してしまうことがあった。 Colloidal gold is often used as a dyeing material in in vitro diagnosis such as immunochromatography. Colloidal gold has a strong red color due to surface plasmon resonance, which enables simple visual diagnosis. Protein immobilization on colloidal gold is generally performed by electrostatic physical adsorption utilizing the negative charge on the surface of colloidal gold. However, depending on the nature of the protein to be adsorbed, the aggregation of gold colloid may proceed.
金コロイド粒子の凝集を防ぐタンパク質固定化方法として、例えば特許文献１には、チオール基を有する非架橋アミノデキストランにより金コロイドを覆う方法が示されている。この方法では、金コロイドをコアに有し、周辺をアミノデキストランで覆うコアーシェル構造を形成することで、金コロイドの分散安定性を向上している。
As a protein immobilization method for preventing the aggregation of gold colloid particles, for example,
また、特許文献２にはジスルフィド基又はチオール基を有する化合物を用いて金コロイドを修飾する方法が示されている。 Further, Patent Document 2 discloses a method of modifying colloidal gold using a compound having a disulfide group or a thiol group.
これらの方法はどちらも金コロイドをコアに有するコアーシェル型の粒子を作製した後に、シェルの表面に存在する官能基を利用してタンパク質を導入する必要があり、物理吸着によるタンパク質固定化に比べ、操作が煩雑であった。 Both of these methods require the introduction of proteins using the functional groups present on the surface of the shell after producing core-shell type particles having gold colloid in the core, compared to protein immobilization by physical adsorption. The operation was complicated.
本発明の目的は、分散性安定化された、金コロイドにタンパク質が吸着した複合体を提供することにある。 An object of the present invention is to provide a dispersion-stabilized complex in which a protein is adsorbed on colloidal gold.
上記課題に鑑みてなされた本発明は、以下の態様を包含する。
（１）金コロイドにタンパク質が吸着した複合体であって、金コロイドに吸着している部分以外のタンパク質表面の一部又は全部がポリエチレングリコールで修飾されていることを特徴とする前記複合体。
（２）前記タンパク質が抗体である、請求項１に記載の複合体。
（３）前記ポリエチレングリコールの鎖長が３０Å以上である、請求項１または２に記載の複合体。
（４）金コロイドの表面にタンパク質を吸着させた後、前記タンパク質の表面に対しポリエチレングリコールで修飾を行う請求項１〜３に記載の複合体の製造方法。
The present invention made in view of the above problems includes the following aspects.
(1) The complex in which a protein is adsorbed on colloidal gold, wherein a part or all of the protein surface other than the portion adsorbed on colloidal gold is modified with polyethylene glycol.
(2) The complex according to
(3) The composite according to claim 1 or 2, wherein the polyethylene glycol has a chain length of 30 Å or more.
(4) The method for producing a complex according to
以下、本発明について詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明に用いられる金コロイドは、体外診断、バイオセンサー等の分析で用いられるものであればよく、特に限定されない。金コロイドは表面プラズモン効果による赤色が確認可能な大きさであればよく、サイズは特に限定されない。本発明における金コロイドの直径は好ましくは１０〜２００ｎｍ、更に好ましくは２０〜６０ｎｍである。 The gold colloid used in the present invention is not particularly limited as long as it is used for in vitro diagnosis, analysis of a biosensor or the like. The size of the gold colloid is not particularly limited as long as the red color due to the surface plasmon effect can be confirmed. The diameter of the gold colloid in the present invention is preferably 10 to 200 nm, more preferably 20 to 60 nm.
本発明に用いられるタンパク質は、金コロイド表面に吸着可能なタンパク質であり、例えば、ＢＳＡ（ウシ血清アルブミン）、ＫＬＨ（キーホールリンペットヘモシアニン）、レクチン、ストレプトアビジンなどが挙げられるが、抗体であることが特に好ましい。タンパク質表面にはポリエチレングリコール（ＰＥＧ）修飾可能な官能基を有している必要があり、ＰＥＧ修飾可能な具体的な官能基には、アミノ基、チオール基などが挙げられる。 The protein used in the present invention is a protein that can be adsorbed on the surface of colloidal gold, and examples thereof include BSA (bovine serum albumin), KLH (keyhole limpet hemocyanin), lectin, and streptavidin, which are antibodies. Is particularly preferred. The protein surface must have a polyethylene glycol (PEG) modifiable functional group, and specific PEG-modifiable functional groups include amino groups and thiol groups.
金コロイドにタンパク質が吸着した複合体とは、金コロイド表面にタンパク質が物理吸着して一体となった状態を指す。 The complex in which proteins are adsorbed on colloidal gold refers to a state in which proteins are physically adsorbed on the surface of colloidal gold and integrated.
本発明の複合体は、金コロイドに吸着している部分以外のタンパク質表面の一部又は全部がポリエチレングリコールで修飾されていることを特徴とする。金コロイドに吸着している部分以外のタンパク質表面は修飾可能な官能基の半数以上がポリエチレングリコールで修飾されていることが好ましく、また、その鎖長は、金コロイドの凝集を抑えることが可能な鎖長であればよく、３０Å以上の鎖長を有することが好ましい。 The complex of the present invention is characterized in that a part or all of the protein surface other than the portion adsorbed on the gold colloid is modified with polyethylene glycol. It is preferable that more than half of the modifiable functional groups of the protein surface other than the portion adsorbed on the gold colloid are modified with polyethylene glycol, and the chain length thereof can suppress the aggregation of the gold colloid. The chain length may be long, and it is preferable to have a chain length of 30 Å or more.
次に、本発明の複合体の製造方法について説明するが、本発明はこれに限定されるものではない。 Next, the method for producing the composite of the present invention will be described, but the present invention is not limited thereto.
本発明の複合体は、金コロイドの表面にタンパク質を吸着させた後、前記タンパク質の表面に対しポリエチレングリコールで修飾を行うことで製造可能である。製造方法について、更に工程ごとに詳細に説明する。 The complex of the present invention can be produced by adsorbing a protein on the surface of colloidal gold and then modifying the surface of the protein with polyethylene glycol. The manufacturing method will be described in more detail for each step.
まず、金コロイド溶液にｐＨ調整用の緩衝液を加えてｐＨを６〜９に調整する（最適なｐＨはタンパク質により異なるため、適宜最適なｐＨを検討することが好ましい）。 First, a buffer solution for pH adjustment is added to the colloidal gold solution to adjust the pH to 6 to 9 (the optimum pH differs depending on the protein, so it is preferable to appropriately examine the optimum pH).
次に、ｐＨを調整した金コロイド溶液にタンパク質溶液を添加し、室温で１０〜３０分間静置することで、金コロイド表面にタンパク質を物理吸着させる。 Next, the protein solution is added to the pH-adjusted gold colloidal solution and allowed to stand at room temperature for 10 to 30 minutes to physically adsorb the protein on the gold colloidal surface.
次に、金コロイドに物理吸着させたタンパク質表面をＰＥＧ修飾するため、アミノ基又はチオール基に反応性を有するＰＥＧ化試薬（例えばＮＨＳ−ＰＥＧ２４−ＯＭｅ、Ｍａｌｅｉｍｉｄｅ−ＰＥＧ２４−ＯＭｅ、いずれもＴｈｅｒｏｍｏＦｉｓｈｅｒＳＣＩＥＮＴＩＦＩＣ社製）を添加し、室温で３０〜６０分間静置する。 Next, in order to PEG-modify the surface of the protein physically adsorbed on colloidal gold, PEGylation reagents having reactivity with an amino group or a thiol group (for example, NHS-PEG24-OMe and Maleimide-PEG24-OMe, all manufactured by Theromo Fisher SCIENTIFIC). ) Is added, and the mixture is allowed to stand at room temperature for 30 to 60 minutes.
なお、前記タンパク質によって金コロイド表面が完全には覆えていない場合があるので、タンパク質表面のＰＥＧ修飾が終了した後、金コロイド表面の覆えていない箇所をＢＳＡ、スキムミルク、ブロッキングワン（ナカライテスク社製）などのブロッキング試薬で覆い、非特異吸着を抑える操作を行ってもよい。 Since the gold colloid surface may not be completely covered by the protein, after the PEG modification of the protein surface is completed, the uncovered portion of the gold colloid surface is covered with BSA, skim milk, or blocking one (manufactured by Nacalai Tesque). ) And other blocking reagents may be used to suppress nonspecific adsorption.
次に、金コロイドを沈殿させるため、金コロイドの沈殿は形成されるが凝集は進行しない条件（６０ｎｍの金コロイドの場合は、８，０００ｇ、９分）で遠心操作を行い、透明になった上清を廃棄する。この操作は精製度を上げるために、ブロッキング操作と組み合わせて複数回行っても問題ない。 Next, in order to precipitate the gold colloid, centrifugation was performed under the condition that the precipitation of the gold colloid was formed but the aggregation did not proceed (8,000 g, 9 minutes in the case of the gold colloid of 60 nm), and the gold colloid became transparent. Discard the supernatant. This operation may be performed multiple times in combination with the blocking operation in order to increase the degree of purification.
以下、本発明を特許５８１０５１４号公報に記載のＢＮＰ（ｂｒａｉｎ ｎａｔｒｉｕｒｅｔｉｃ ｐｅｐｔｉｄｅ）認識抗体（ＢＣ２３−１１）を用いた実施例によって具体的に示すが、本発明はこれに限定されない。 Hereinafter, the present invention will be specifically shown by an example using a BNP (brain natriuretic peptide) recognition antibody (BC23-11) described in Japanese Patent No. 5810514, but the present invention is not limited thereto.
実施例１
（１）金コロイド表面への抗体吸着
金コロイドは、ワインレッドケミカル社製の直径６０ｎｍの金コロイド（ＷＲＧＨ１−６０ＮＭ）を用いた２５０μＬの金コロイド溶液にｐＨ９．２の１０ｍＭ Ｔｒｉｓ−ＨＣｌ溶液を２５０μＬ添加しｐＨを調整し、０．１ｍｇ／ｍＬのＢＣ２３−１１溶液（ｐＨ９．２、１０ｍＭ Ｔｒｉｓ−ＨＣｌ）を５００μＬ添加し、１５分間静置した。
（２）抗体表面のＰＥＧ修飾
次に、（１）で得られた溶液に、２５０ｍＭのＭｅｔｈｙｌ−ＰＥＧ−ＮＨＳ２４−Ｅｓｔｅｒ（ＴｈｅｒｏｍｏＦｉｓｈｅｒＳＣＩＥＮＴＩＦＩＣ社製）のジメチルスルホキシド（ＤＭＳＯ）溶液１０μＬを添加し、３０分静置した。
（３）ブロッキング
次に、（２）で得られた溶液に、牛血清アルブミンとポリエチレングリコール２０，０００（和光純薬社製）の混合液を１０００μＬ添加し、１５分静置した。
（４）精製
次に、（３）で得られた溶液に対して、８，０００ｇで９分間の遠心操作を行い、金コロイドを沈殿させ、透明になった上清を廃棄した後、残った沈殿に対して（３）で使用したのと同一の混合液を１０００μＬ添加し、同様の遠心操作を繰り返した。
Example 1
(1) Adsorption of antibody on the surface of gold colloid For gold colloid, 250 μL of 10 mM Tris-HCl solution of pH 9.2 was added to 250 μL of gold colloid solution using gold colloid (WRGH1-60NM) having a diameter of 60 nm manufactured by Wine Red Chemical Co., Ltd. The pH was adjusted by adding 500 μL of a 0.1 mg / mL BC23-11 solution (pH 9.2, 10 mM Tris-HCl), and the mixture was allowed to stand for 15 minutes.
(2) PEG modification of antibody surface Next, 10 μL of a 250 mM Methyl-PEG-NHS24-Ester (manufactured by Theromo FisherSCIETIFIC) dimethyl sulfoxide (DMSO) solution was added to the solution obtained in (1), and the mixture was allowed to stand for 30 minutes. Placed.
(3) Blocking Next, 1000 μL of a mixed solution of bovine serum albumin and polyethylene glycol 20,000 (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the solution obtained in (2), and the mixture was allowed to stand for 15 minutes.
(4) Purification Next, the solution obtained in (3) was centrifuged at 8,000 g for 9 minutes to precipitate gold colloid, and the clear supernatant was discarded and remained. To the precipitate, 1000 μL of the same mixed solution used in (3) was added, and the same centrifugation operation was repeated.
比較例１
（２）の工程（抗体表面のＰＥＧ修飾）を行わなかった以外は実施例１と同様の条件で抗体吸着した金コロイドを作製した。
Comparative Example 1
A gold colloid adsorbed with an antibody was prepared under the same conditions as in Example 1 except that the step (2) (PEG modification of the antibody surface) was not performed.
（ＵＶ−Ｖｉｓスペクトル測定）
実施例１と比較例１で作製した抗体吸着した金コロイドを保存用の緩衝液３００μＬに、ペレットを懸濁して、ＵＶ−Ｖｉｓスペクトル測定用サンプルとした。図１に結果を示す。分散している金コロイドは可視光領域（４００ｎｍ〜５００ｎｍ）に強い吸収帯を有するが、凝集が進行すると、ピーク強度の低下、吸収ピークのブロードニングを起こすことが知られている。実施例１の金コロイドでは可視光領域に強い吸収帯を有するが、比較例１の金コロイドでは可視光領域のピークが低下し、ピークのブロードニングが起こっている。この結果から比較例１の金コロイドは凝集が進行していることが確認された。
(UV-Vis spectrum measurement)
The antibody-adsorbed gold colloid prepared in Example 1 and Comparative Example 1 was suspended in 300 μL of a buffer solution for storage to prepare a sample for UV-Vis spectrum measurement. The results are shown in FIG. The dispersed gold colloid has a strong absorption band in the visible light region (400 nm to 500 nm), but it is known that when aggregation progresses, the peak intensity decreases and the absorption peak broadens. The gold colloid of Example 1 has a strong absorption band in the visible light region, but the gold colloid of Comparative Example 1 has a reduced peak in the visible light region, causing peak broadening. From this result, it was confirmed that the gold colloid of Comparative Example 1 was agglomerated.
以上の結果から、金コロイドに物理吸着したタンパク質表面をＰＥＧ修飾することで、金コロイドの分散安定性を向上できることが確認された。 From the above results, it was confirmed that the dispersion stability of the gold colloid can be improved by PEG-modifying the surface of the protein physically adsorbed on the gold colloid.
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