JP6399987B2 - Method for measuring anti-β2-GPI antibody - Google Patents
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Description
本発明は、抗β２−グリコプロテインI(β２−ＧＰＩ)抗体の測定法およびそれに使用するキットに関する。 The present invention relates to a method for measuring anti-β2-glycoprotein I (β2-GPI) antibody and a kit used therefor.
β２−ＧＰIを用いて抗カルジオリピン抗体などの自己抗体を測定する方法は既に知られている（特許文献１，２参照） Methods for measuring autoantibodies such as anti-cardiolipin antibodies using β2-GPI are already known (see Patent Documents 1 and 2).
しかしながら、β２−ＧＰIを用いて検体中の自己抗体を測定する場合、吸光度のバックグランドが上昇し、正確に測定できないという問題が度々生じることが明らかとなった。 However, when measuring autoantibodies in a specimen using β2-GPI, it has been clarified that the background of absorbance rises and the problem that it cannot be measured accurately often occurs.
発明者らは、この問題を克服すべく、鋭意検討を重ねた結果、β２−ＧＰIに混在するＩｇＧがバックグランド上昇の原因であることを突き止め、これを除去することで、バックグランドが上昇せず、ロット間差がなく、安定した測定法を提供できることを見出した。本発明はかかる新規の知見に基づくものである。従って、本発明は以下の発明を提供するものである。
（１）
β２−ＧＰＩを用いて検体中の抗β２−ＧＰＩ抗体を測定する方法であって、測定に用いるβ２−ＧＰＩとしてＩｇＧフリーのβ２−ＧＰＩを用いる抗β２−ＧＰＩ抗体の測定法。
（２）
ＩｇＧフリーのβ２−ＧＰＩが、プロテインＧ、プロテインＡ、抗ＩｇＧ抗体などで処理したβ２−ＧＰＩである、（１）記載の測定法。
（３）
ＩｇＧフリーのβ２−ＧＰＩを固相化された形態で用いる、（１）記載の測定法。
（４）
β２−ＧＰＩを用いて検体中の抗β２−ＧＰＩ抗体を測定するためのキットであって、測定に用いるβ２−ＧＰＩとしてＩｇＧフリーのβ２−ＧＰＩを用いる抗β２−ＧＰＩ抗体の測定キット。
（５）
ＩｇＧフリーのβ２−ＧＰＩが、プロテインＧ、プロテインＡ、抗ＩｇＧ抗体などで処理したβ２−ＧＰＩである、（４）記載のキット。
（６）
ＩｇＧフリーのβ２−ＧＰＩが、固相化されたものである、（４）記載のキット。
As a result of intensive studies to overcome this problem, the inventors have found that IgG mixed in β2-GPI is the cause of the background increase, and by removing this, the background is increased. It was found that there was no difference between lots and a stable measurement method could be provided. The present invention is based on such novel findings. Accordingly, the present invention provides the following inventions.
(1)
A method for measuring an anti-β2-GPI antibody in a specimen using β2-GPI, wherein IgG-free β2-GPI is used as β2-GPI used for the measurement.
(2)
The measurement method according to (1), wherein the IgG-free β2-GPI is β2-GPI treated with protein G, protein A, anti-IgG antibody or the like.
(3)
The measurement method according to (1), wherein IgG-free β2-GPI is used in a solid-phased form.
(4)
A kit for measuring an anti-β2-GPI antibody in a specimen using β2-GPI, and an anti-β2-GPI antibody measurement kit using IgG-free β2-GPI as β2-GPI used for the measurement.
(5)
The kit according to (4), wherein the IgG-free β2-GPI is β2-GPI treated with protein G, protein A, anti-IgG antibody or the like.
(6)
The kit according to (4), wherein IgG-free β2-GPI is solid-phased.
本発明の抗β２−ＧＰＩ抗体の測定法及びキットにおいては、プロテインＧ、プロテインＡ、抗ＩｇＧ抗体などで処理したβ２−ＧＰＩを用いる、すなわちＩｇＧフリーのβ２−ＧＰＩを用いるため、測定時におけるバックグランド上昇を抑制できる。さらに、使用するβ２−ＧＰIのロット間差もほとんどないキットを提供できるため、臨床検査において重要な安定した測定に大いに貢献することができる。 In the measurement method and kit of the anti-β2-GPI antibody of the present invention, β2-GPI treated with protein G, protein A, anti-IgG antibody or the like is used, that is, IgG-free β2-GPI is used. Ground rise can be suppressed. Further, since a kit with little difference in β2-GPI to be used can be provided, it can greatly contribute to stable measurement important in clinical examination.
本発明の特徴は、上述したように、プロテインＧ、プロテインＡ、抗ＩｇＧ抗体などで処理したβ２−ＧＰＩ、すなわちＩｇＧフリーのβ２−ＧＰＩを用いることにある。
本明細書において、「ＩｇＧフリー」とは、抗β２−ＧＰＩ抗体の測定に悪影響を及ぼさないことを意味し、ＩｇＧが極微量含まれていても、抗β２−ＧＰＩ抗体の測定に悪影響を及ぼさなければ、実質的にＩｇＧフリーの範疇に入る。具体的には、以下の方法によって抗β２−ＧＰＩ抗体を測定する際のバックグラウンドの吸光度（４５０ｎｍ）が０．２０以下、好ましくは０．１５以下であればよい。
（方法）
陰性荷電もしくは孤立電子対を有する官能基および／または遊離基を表面に導入した担体（たとえば、Ｎｕｎｃ社Ｍａｘｉｓｏｒｐ）に、β２−ＧＰＩを１〜２０μｇ／ｍＬの範囲の濃度で、１００μＬ／ウェルずつ加え、４℃で一晩静置して結合させる。作製したβ２−ＧＰＩ結合担体を３００μＬのＰＢＳで３回洗浄した後、３００μＬの１％ＢＳＡ含有ＰＢＳを加え、室温で２時間静置する。ＢＳＡ溶液を除き、３００μＬのＰＢＳ−Ｔで３回洗浄した後、西洋ワサビペルオキシダーゼ標識抗ヒトＩｇＧ抗体を１００μＬずつウェルに加え、室温で１時間反応させる。ＰＢＳ−Ｔで３回洗浄した後、基質溶液（３、３’、５、５’―テトラメチルベンジジン（ＴＭＢＺ）および過酸化水素含有溶液）を１００μＬ加え、室温で３０分反応させ、反応停止液（１Ｎ硫酸）を１００μＬ加え、吸光度（４５０ｎｍ）を測定する。
本発明に使用するβ２−ＧＰＩとしては、ヒトβ２−ＧＰＩ、ウシβ２−ＧＰＩなどを使用することができ、既に市販されており、購入して使用すればよい。
また、市販のヒトを含む動物の血清中にβ２−ＧＰＩが存在することから、公知の方法で血清から単離することも可能である。
さらに、β２−ＧＰＩのＤＮＡ配列は既に公知であり、公知の組換え法で調製することも可能である。
As described above, the present invention is characterized in that β2-GPI treated with protein G, protein A, anti-IgG antibody, or the like, that is, IgG-free β2-GPI is used.
In the present specification, “IgG-free” means that there is no adverse effect on the measurement of anti-β2-GPI antibody, and even if a trace amount of IgG is contained, the measurement of anti-β2-GPI antibody is adversely affected. If not, it falls into the category of substantially IgG free. Specifically, the background absorbance (450 nm) when measuring an anti-β2-GPI antibody by the following method is 0.20 or less, preferably 0.15 or less.
(Method)
Β2-GPI is added at a concentration of 1 to 20 μg / mL at a concentration of 100 μL / well to a carrier (for example, Nunc Maxisorp) having a negatively charged or lone pair of functional groups and / or free radicals introduced on the surface. Allow to bind overnight at 4 ° C. The prepared β2-GPI-binding carrier is washed with 300 μL of PBS three times, 300 μL of 1% BSA-containing PBS is added, and the mixture is allowed to stand at room temperature for 2 hours. After removing the BSA solution and washing three times with 300 μL of PBS-T, 100 μL of horseradish peroxidase-labeled anti-human IgG antibody is added to each well and allowed to react at room temperature for 1 hour. After washing 3 times with PBS-T, 100 μL of substrate solution (3,3 ′, 5,5′-tetramethylbenzidine (TMBZ) and hydrogen peroxide-containing solution) was added and reacted at room temperature for 30 minutes. 100 μL of (1N sulfuric acid) is added, and the absorbance (450 nm) is measured.
As β2-GPI used in the present invention, human β2-GPI, bovine β2-GPI and the like can be used, which are already on the market and may be purchased and used.
In addition, since β2-GPI is present in the serum of animals including commercially available humans, it can be isolated from the serum by a known method.
Furthermore, the DNA sequence of β2-GPI is already known and can be prepared by a known recombinant method.
β２−ＧＰＩ精製に使用するプロテインＧ、プロテインＡ、抗ＩｇＧ抗体は市販されており、市販品を使用すれば良く、特にセファロースなどの担体に結合され、カラム等に充填されたものを使用することが、β２−ＧＰＩの精製に便宜である。 Protein G, protein A, and anti-IgG antibody used for β2-GPI purification are commercially available, and commercially available products may be used, particularly those that are bound to a carrier such as Sepharose and packed in a column or the like. Is convenient for purification of β2-GPI.
プロテインＧ、プロテインＡ、抗ＩｇＧ抗体などを用いたβ２−ＧＰＩの精製処理は、カラムなどを利用した連続法でも、接触後分離するバッチ法であってもかまわない。
精製処理自体は、リン酸緩衝生理食塩水（ＰＢＳ）などのｐＨ ４．０〜９．０の緩衝液を用い、プロテインＧ、プロテインＡ、抗ＩｇＧ抗体などとβ２−ＧＰＩを接触させることにより実施することができる。
The purification process of β2-GPI using protein G, protein A, anti-IgG antibody or the like may be a continuous method using a column or the like, or a batch method in which separation is performed after contact.
The purification treatment itself is performed by contacting β2-GPI with protein G, protein A, anti-IgG antibody, etc. using a buffer solution of pH 4.0-9.0 such as phosphate buffered saline (PBS). can do.
このようなプロテインＧ、プロテインＡ、抗ＩｇＧ抗体などで処理したβ２−ＧＰＩは実質的にＩｇＧフリーであり、抗β２−ＧＰＩ抗体の測定に悪影響を及ぼさないため、抗β２−ＧＰＩ抗体の測定に最適である。 Β2-GPI treated with such protein G, protein A, anti-IgG antibody and the like is substantially IgG-free and does not adversely affect the measurement of anti-β2-GPI antibody. Is optimal.
ＩｇＧフリーのβ２−ＧＰＩを結合させる担体としては、陰性荷電もしくは孤立電子対を有する官能基および／または遊離基を表面に導入したものであれば特に限定されない。また、担体の形状は、平板状（マイクロタイタープレートなど）、粒子状（磁性ビーズ、ラテックスなど）などが例示され、測定法に応じて適宜選択すればよい。
抗β２−ＧＰＩ抗体の測定自体は、既に多くの方法が報告されており、適宜公知の方法（例えば、特許文献１及び２記載の方法など）を適用すればよい。
The carrier for binding IgG-free β2-GPI is not particularly limited as long as it has a functional group having a negative charge or a lone electron pair and / or a free group introduced on the surface. Examples of the shape of the carrier include a flat plate shape (such as a microtiter plate) and a particle shape (such as magnetic beads and latex), and may be appropriately selected depending on the measurement method.
Many methods have already been reported for measurement of anti-β2-GPI antibodies, and known methods (for example, methods described in Patent Documents 1 and 2) may be applied as appropriate.
以下、実施例を示し、本発明を具体的に説明するが、本発明がこれに限定されないのは明らかである。 Hereinafter, the present invention will be described in detail with reference to examples, but it is clear that the present invention is not limited thereto.
プロテインＧ処理によるＩｇＧフリーβ２−ＧＰＩの調製
方法
ＰｒｏｔｅｉｎＧ Ｓｅｐｈａｒｏｓｅ ４ Ｆａｓｔ Ｆｌｏｗ（ＧＥ Ｈｅａｌｔｈｃａｒｅ社）を５ｍＬ用いて、プロテインＧカラムを作製、ＰＢＳを１５ｍＬ流してカラムを平衡化した。平衡化したプロテインＧカラムに精製β２−ＧＰＩ溶液（１１ｍｇ／ｍＬ）５ｍＬを流し、さらにＰＢＳを５〜１０ｍＬ流して素通り画分を回収した。０．１Ｍグリシン緩衝液（０．１Ｍ Ｇｌｙｃｉｎｅ−ＨＣｌ，ｐＨ ２．８）をプロテインＧカラムに１０ｍＬ流し、吸着したタンパク質を溶出して溶出画分を得た。素通り画分と溶出画分の吸光度２８０ｎｍを測定し、各画分におけるタンパク質濃度を確認して素通り画分をプロテインＧ処理画分とした。
９６ウェルイムノモジュールプレート（Ｎｕｎｃ社、Ｍａｘｉｓｏｒｐ）にプロテインＧ未処理のヒト精製β２−ＧＰＩおよびプロテインＧ処理画分（１μｇ／ｍＬ：ＰＢＳ）を１００μＬ／ウェルずつ加え、４℃で一晩静置した。３００μＬのＰＢＳで３回洗浄した後、３００μＬの１％ウシ精製アルブミン（ＢＳＡ）含有ＰＢＳを加え、室温で２時間静置した。ＢＳＡ溶液を除き、西洋ワサビペルオキシダーゼ標識抗ヒトＩｇＧ抗体を１００μＬずつウェルに加え、室温で１時間反応させた。３００μＬの０．０５％Ｔｗｅｅｎ２０含有ＰＢＳ（ＰＢＳ−Ｔ）で３回洗浄した後、基質溶液（３、３’、５、５’―テトラメチルベンジジン（ＴＭＢＺ）および過酸化水素含有溶液）を１００μＬ加え、室温で３０分反応させ、反応停止液（１Ｎ硫酸）を１００μＬ加え、吸光度（４５０ｎｍ）を測定した。
Preparation Method of IgG-Free β2-GPI by Protein G Treatment A protein G column was prepared using 5 mL of Protein G Sepharose 4 Fast Flow (GE Healthcare), and the column was equilibrated by flowing 15 mL of PBS. 5 mL of purified β2-GPI solution (11 mg / mL) was allowed to flow through the equilibrated protein G column, and further 5 to 10 mL of PBS was allowed to flow to collect the flow-through fraction. 10 mL of 0.1 M glycine buffer (0.1 M Glycine-HCl, pH 2.8) was passed through the protein G column, and the adsorbed protein was eluted to obtain an elution fraction. The absorbance at 280 nm of the flow-through fraction and the elution fraction was measured, the protein concentration in each fraction was confirmed, and the flow-through fraction was used as the protein G-treated fraction.
Protein G-untreated human purified β2-GPI and protein G-treated fraction (1 μg / mL: PBS) were added to a 96-well immunomodule plate (Nunc, Maxisorp) at 100 μL / well and allowed to stand overnight at 4 ° C. . After washing 3 times with 300 μL of PBS, 300 μL of 1% bovine albumin (BSA) -containing PBS was added and left at room temperature for 2 hours. The BSA solution was removed, and horseradish peroxidase-labeled anti-human IgG antibody was added to each well in an amount of 100 μL and allowed to react at room temperature for 1 hour. After washing 3 times with 300 μL of 0.05% Tween20-containing PBS (PBS-T), 100 μL of substrate solution (3,3 ′, 5,5′-tetramethylbenzidine (TMBZ) and hydrogen peroxide-containing solution) was added. The mixture was reacted at room temperature for 30 minutes, 100 μL of a reaction stop solution (1N sulfuric acid) was added, and the absorbance (450 nm) was measured.
結果
その結果を表１に示す。表中のＰｒｏｔｅｉｎＧ（−）はプロテインＧ未処理のβ２−ＧＰＩを用いたとき、ＰｒｏｔｅｉｎＧ（＋）はプロテインＧ処理したβ２−ＧＰＩ画分を用いたときを示している。表１からプロテインＧ処理により、β２−ＧＰＩに含まれていたＩｇＧが減少し、ＥＩＡ法での吸光度レベルが劇的に低減したことが明らかとなった。なお、溶出画分を用いた測定では、吸光度４５０ｎｍの検出限界以上（吸光度４．０）となった。したがって、上記方法によるβ２ＧＰＩのプロテインＧ処理画分をＩｇＧフリーβ２−ＧＰＩとした。
Results The results are shown in Table 1. Protein G (-) in the table indicates that when β2-GPI untreated with protein G is used, and Protein G (+) indicates when the β2-GPI fraction treated with protein G is used. From Table 1, it was revealed that IgG contained in β2-GPI was decreased by protein G treatment, and the absorbance level by the EIA method was dramatically reduced. In the measurement using the eluted fraction, the absorbance exceeded the detection limit of 450 nm (absorbance 4.0). Therefore, the protein G-treated fraction of β2GPI by the above method was IgG-free β2-GPI.
抗ＩｇＧ抗体結合担体処理によるＩｇＧフリーβ２−ＧＰＩの調製
（１）方法
抗ヒトＩｇＧ抗体（３．７ｍｇ）をＣＮＢｒ−ａｃｔｉｖａｔｅｄ Ｓｅｐｈａｒｏｓｅ ４Ｂ（ＧＥ Ｈｅａｌｔｈｃａｒｅ社）にカップリングさせ、抗ヒトＩｇＧ抗体アフィニティーカラムを２ｍＬ作製した。
作製した抗ヒトＩｇＧアフィニティーカラム２ｍＬに、ＰＢＳを４ｍＬ流してカラムを平衡化した。平衡化したカラムに精製β２−ＧＰＩ溶液（１０．２ｍｇ／ｍＬ）０．５ｍＬを流し、さらにＰＢＳを２〜４ｍＬ流して素通り画分を回収した。０．１Ｍグリシン緩衝液（０．１Ｍ Ｇｌｙｃｉｎｅ−ＨＣｌ，ｐＨ ２．８）をアフィニティーカラムに１０ｍＬ流し、吸着したタンパク質を溶出して溶出画分を得た。素通り画分と溶出画分の吸光度２８０ｎｍを測定し、各画分におけるタンパク質濃度を確認して素通り画分を抗ヒトＩｇＧ抗体アフィニティー処理画分とした。
上記１）と同様のＥＩＡ法で、抗ヒトＩｇＧ抗体アフィニティーカラム処理画分に含まれるＩｇＧを検出した。
Preparation of IgG-free β2-GPI by treatment with anti-IgG antibody binding carrier (1) Method An anti-human IgG antibody (3.7 mg) was coupled to CNBr-activated Sepharose 4B (GE Healthcare), and an anti-human IgG antibody affinity column was used. 2 mL was prepared.
The column was equilibrated by flowing 4 mL of PBS into 2 mL of the prepared anti-human IgG affinity column. 0.5 mL of purified β2-GPI solution (10.2 mg / mL) was allowed to flow through the equilibrated column, and further 2 to 4 mL of PBS was allowed to flow to collect the flow-through fraction. 10 mL of 0.1 M glycine buffer (0.1 M Glycine-HCl, pH 2.8) was passed through the affinity column, and the adsorbed protein was eluted to obtain an elution fraction. The absorbance at 280 nm of the flow-through fraction and the elution fraction was measured, the protein concentration in each fraction was confirmed, and the flow-through fraction was used as an anti-human IgG antibody affinity-treated fraction.
IgG contained in the fraction treated with the anti-human IgG antibody affinity column was detected by the same EIA method as in 1) above.
結果
その結果を表２に示す。表中の抗ＩｇＧアフィニティー（−）は抗ヒトＩｇＧ抗体アフィニティーカラム未処理のβ２−ＧＰＩを用いたとき、抗ＩｇＧアフィニティー（＋）は抗ヒトＩｇＧ抗体アフィニティーカラム処理したβ２−ＧＰＩ画分を用いたときを示している。表２から明らかなように、抗ヒトＩｇＧ抗体アフィニティーカラム処理により、β２−ＧＰＩに含まれていたＩｇＧが減少し、ＥＩＡ法での吸光度レベルが劇的に低減したことが明らかとなった。
Results The results are shown in Table 2. When anti-IgG affinity (-) in the table used β2-GPI that was not treated with an anti-human IgG antibody affinity column, anti-IgG affinity (+) used was a β2-GPI fraction treated with anti-human IgG antibody affinity column. Showing the time. As is apparent from Table 2, it was revealed that the IgG contained in β2-GPI was reduced by the anti-human IgG antibody affinity column treatment, and the absorbance level by the EIA method was dramatically reduced.
ＩｇＧフリーβ２−ＧＰＩを用いた抗β２−ＧＰＩ抗体測定におけるバッググランド低減の検討
（１）目的
プロテインＧ処理によるバックグラウンド低減効果の確認。
（２）方法
９６ウェルイムノモジュールプレート（Ｎｕｎｃ社、Ｍａｘｉｓｏｒｐ）にプロテインＧ未処理およびプロテインＧ処理したヒト精製β２−ＧＰＩ（１０μｇ／ｍＬ：ＰＢＳ、純度９５％以上）を１００μＬ／ウェルずつ加え、４℃で一晩静置した。３００μＬのＰＢＳで３回洗浄した後、３００μＬの１％ＢＳＡ含有ＰＢＳを加え、室温で２時間静置した。ＢＳＡ溶液を除き、１％ＢＳＡ含有ＰＢＳで適宜希釈した被験血清を１００μＬずつウェルに加え、室温で１時間静置した。３００μＬのＰＢＳ−Ｔで３回洗浄した後、西洋ワサビペルオキシダーゼ標識抗ヒトＩｇＧ抗体を１００μＬずつウェルに加え、室温で１時間反応させた。ＰＢＳ−Ｔで３回洗浄した後、基質溶液（３、３’、５、５’―テトラメチルベンジジン（ＴＭＢＺ）および過酸化水素含有溶液）を１００μＬ加え、室温で３０分反応させ、反応停止液（１Ｎ硫酸）を１００μＬ加え、吸光度（４５０ｎｍ）を測定した。
Examination of background reduction in measurement of anti-β2-GPI antibody using IgG-free β2-GPI (1) Confirmation of background reduction effect by target protein G treatment.
(2) Method A 96-well immunomodule plate (Nunc, Maxisorp) was added with 100 μL / well of protein G-untreated and protein G-treated human purified β2-GPI (10 μg / mL: PBS, purity 95% or more). Allowed to stand overnight at ° C. After washing 3 times with 300 μL of PBS, 300 μL of 1% BSA-containing PBS was added and allowed to stand at room temperature for 2 hours. The BSA solution was removed, 100 μL of test serum appropriately diluted with 1% BSA-containing PBS was added to each well and allowed to stand at room temperature for 1 hour. After washing with 300 μL of PBS-T three times, 100 μL of horseradish peroxidase-labeled anti-human IgG antibody was added to each well and allowed to react at room temperature for 1 hour. After washing 3 times with PBS-T, 100 μL of substrate solution (3,3 ′, 5,5′-tetramethylbenzidine (TMBZ) and hydrogen peroxide-containing solution) was added and reacted at room temperature for 30 minutes. 100 μL of (1N sulfuric acid) was added, and the absorbance (450 nm) was measured.
（３）結果
その結果を表３に示す。表中のＰｒｏｔｅｉｎＧ（−）はプロテインＧ未処理のβ２−ＧＰＩを用いたとき、ＰｒｏｔｅｉｎＧ（＋）はプロテインＧ処理したβ２−ＧＰＩを用いたときを示している。表３からプロテインＧ処理により、被験血清非含有のバックグラウンドの吸光度が低下し、被験血清の各希釈倍率における吸光度の低下も確認された。
(3) Results Table 3 shows the results. Protein G (-) in the table indicates when Protein G-untreated β2-GPI is used, and Protein G (+) indicates when Protein G-treated β2-GPI is used. From Table 3, the absorbance of the background containing no test serum was reduced by the protein G treatment, and a decrease in absorbance at each dilution of the test serum was also confirmed.
（４）考察
抗β２−ＧＰＩ抗体測定系は上記精製β２−ＧＰＩを固相化し抗原として用いるため、精製β２−ＧＰＩに混在するＩｇＧは、２次反応において酵素標識抗ヒトＩｇＧ抗体によって自己抗体の介在しない複合体を形成し、バックグラウンドとして検出されてしまうと考えられた。そこで、精製ヒトβ２−ＧＰＩをプロテインＧカラムに接触させることによって、精製β２−ＧＰＩに微量に混在するＩｇＧをプロテインＧで吸着除去したところ、表３の結果から明らかなように、抗β２−ＧＰＩ抗体の測定におけるバックグラウンドを低減できることが明らかとなった。
(4) Discussion Since the anti-β2-GPI antibody measurement system uses the above-mentioned purified β2-GPI as a solid phase and uses it as an antigen, IgG mixed in the purified β2-GPI is converted into an autoantibody by an enzyme-labeled anti-human IgG antibody in the secondary reaction. It was thought that a non-intervening complex was formed and detected as background. Then, when purified human β2-GPI was brought into contact with a protein G column, IgG mixed in a trace amount in purified β2-GPI was adsorbed and removed with protein G. As is apparent from the results in Table 3, anti-β2-GPI It was found that the background in antibody measurement can be reduced.
ＩｇＧフリーβ２−ＧＰＩのロット間差の検討
目的
プロテインＧ処理によるβ２−ＧＰＩロット間差の低減効果の確認。
方法
上記１）と同様の方法で、プロテインＧ処理によるＩｇＧフリーβ２−ＧＰＩを２ロット作製し、上記３）と同様の方法で抗β２ＧＰＩ抗体を測定した。
Examination of differences between lots of IgG-free β2-GPI Objective: Confirmation of the effect of reducing differences between β2-GPI lots by protein G treatment.
Method Two lots of IgG-free β2-GPI by protein G treatment were prepared in the same manner as in 1) above, and anti-β2GPI antibody was measured in the same manner as in 3) above.
結果
その結果を表４に示す。表中のＬｏｔ１およびＬｏｔ２はＩｇＧフリーβ２−ＧＰＩのロットを示す。表４より、０Ｕ／ｍＬのバックグラウンドはいずれのロットにおいても極めて低値を示し、各標準液の吸光度も同等の値を示した。また、複数の患者血清の吸光度も同等の値であった。したがって、ＩｇＧフリーβ２−ＧＰＩを固相抗原として使用することで、β２−ＧＰＩのロット間差の影響を抑制できることが確認された。
Results The results are shown in Table 4. Lot1 and Lot2 in the table indicate lots of IgG-free β2-GPI. From Table 4, the background of 0 U / mL showed an extremely low value in any lot, and the absorbance of each standard solution also showed an equivalent value. In addition, the absorbances of multiple patient sera were equivalent. Therefore, it was confirmed that the effect of the difference between β2-GPI lots can be suppressed by using IgG-free β2-GPI as a solid phase antigen.
考察
抗β２−ＧＰＩ抗体測定系の抗原としてβ２−ＧＰＩを使用するとき、β２−ＧＰＩのロットごとに標準曲線、特にバックグランドの吸光度レベルが異なることが問題となっていた。そこで、バックグラウンドの原因のＩｇＧをプロテインＧ処理によりβ２−ＧＰＩから除き、ＩｇＧフリーβ２−ＧＰＩを抗原として使用したところ、表４の結果から明らかなように、抗β２−ＧＰＩ抗体の測定において、ＩｇＧフリーβ２−ＧＰＩの２つのロット間差はほとんど観察されなかった。したがって、ＩｇＧフリーのβ２−ＧＰＩを抗β２−ＧＰＩ抗体測定の抗原に用いることで、ロット間差を極めて小さくできることが明らかとなった。
Discussion When β2-GPI is used as an antigen of an anti-β2-GPI antibody measurement system, the standard curve, particularly the background absorbance level, differs for each β2-GPI lot. Therefore, when IgG that caused background was removed from β2-GPI by protein G treatment and IgG-free β2-GPI was used as an antigen, as is apparent from the results in Table 4, in the measurement of anti-β2-GPI antibody, Little difference between the two lots of IgG-free β2-GPI was observed. Therefore, it became clear that the difference between lots can be made extremely small by using IgG-free β2-GPI as an antigen for anti-β2-GPI antibody measurement.
Claims (4)
（方法）陰性荷電もしくは孤立電子対を有する官能基および／または遊離基を表面に導入した担体（たとえば、Ｎｕｎｃ社Ｍａｘｉｓｏｒｐ）に、β２−ＧＰＩを１〜２０μｇ／ｍＬの範囲の濃度で、１００μＬ／ウェルずつ加え、４℃で一晩静置して結合させる。作製したβ２−ＧＰＩ結合担体を３００μＬのＰＢＳで３回洗浄した後、３００μＬの１％ＢＳＡ含有ＰＢＳを加え、室温で２時間静置する。ＢＳＡ溶液を除き、３００μＬのＰＢＳ−Ｔで３回洗浄した後、西洋ワサビペルオキシダーゼ標識抗ヒトＩｇＧ抗体を１００μＬずつウェルに加え、室温で１時間反応させる。ＰＢＳ−Ｔで３回洗浄した後、基質溶液（３、３’、５、５’―テトラメチルベンジジン（ＴＭＢＺ）および過酸化水素含有溶液）を１００μＬ加え、室温で３０分反応させ、反応停止液（１Ｎ硫酸）を１００μＬ加え、吸光度（４５０ｎｍ）を測定する。 A method for measuring an anti-β2-GPI antibody in a specimen using β2-glycoprotein I (β2-GPI), and measuring the anti-β2-GPI antibody by the following method as β2-GPI used for the measurement Method for measuring anti-β2-GPI antibody using IgG-free β2-GPI treated with protein G, protein A or anti-IgG antibody, wherein the background absorbance (450 nm) is 0.20 or less.
(Method) β2-GPI was added at a concentration of 1 to 20 μg / mL at a concentration of 100 μL / mL to a carrier (for example, Nunc Maxisorp) having a functional group having a negative charge or a lone electron pair and / or a free radical introduced on the surface. Add wells and let stand at 4 ° C overnight to bind. The prepared β2-GPI-binding carrier is washed with 300 μL of PBS three times, 300 μL of 1% BSA-containing PBS is added, and the mixture is allowed to stand at room temperature for 2 hours. After removing the BSA solution and washing three times with 300 μL of PBS-T, 100 μL of horseradish peroxidase-labeled anti-human IgG antibody is added to each well and allowed to react at room temperature for 1 hour. After washing 3 times with PBS-T, 100 μL of substrate solution (3,3 ′, 5,5′-tetramethylbenzidine (TMBZ) and hydrogen peroxide-containing solution) was added and reacted at room temperature for 30 minutes. 100 μL of (1N sulfuric acid) is added, and the absorbance (450 nm) is measured.
（方法）陰性荷電もしくは孤立電子対を有する官能基および／または遊離基を表面に導入した担体（たとえば、Ｎｕｎｃ社Ｍａｘｉｓｏｒｐ）に、β２−ＧＰＩを１〜２０μｇ／ｍＬの範囲の濃度で、１００μＬ／ウェルずつ加え、４℃で一晩静置して結合させる。作製したβ２−ＧＰＩ結合担体を３００μＬのＰＢＳで３回洗浄した後、３００μＬの１％ＢＳＡ含有ＰＢＳを加え、室温で２時間静置する。ＢＳＡ溶液を除き、３００μＬのＰＢＳ−Ｔで３回洗浄した後、西洋ワサビペルオキシダーゼ標識抗ヒトＩｇＧ抗体を１００μＬずつウェルに加え、室温で１時間反応させる。ＰＢＳ−Ｔで３回洗浄した後、基質溶液（３、３’、５、５’―テトラメチルベンジジン（ＴＭＢＺ）および過酸化水素含有溶液）を１００μＬ加え、室温で３０分反応させ、反応停止液（１Ｎ硫酸）を１００μＬ加え、吸光度（４５０ｎｍ）を測定する。 A kit for measuring an anti-β2-GPI antibody in a specimen using β2-glycoprotein I (β2-GPI), and measuring the anti-β2-GPI antibody as β2-GPI used for the measurement by the following method An anti-β2-GPI antibody measurement kit using IgG-free β2-GPI treated with protein G, protein A or anti-IgG antibody, which has a background absorbance (450 nm) of 0.20 or less.
(Method) β2-GPI was added at a concentration of 1 to 20 μg / mL at a concentration of 100 μL / mL to a carrier (for example, Nunc Maxisorp) having a functional group having a negative charge or a lone electron pair and / or a free radical introduced on the surface. Add wells and let stand at 4 ° C overnight to bind. The prepared β2-GPI-binding carrier is washed with 300 μL of PBS three times, 300 μL of 1% BSA-containing PBS is added, and the mixture is allowed to stand at room temperature for 2 hours. After removing the BSA solution and washing three times with 300 μL of PBS-T, 100 μL of horseradish peroxidase-labeled anti-human IgG antibody is added to each well and allowed to react at room temperature for 1 hour. After washing 3 times with PBS-T, 100 μL of substrate solution (3,3 ′, 5,5′-tetramethylbenzidine (TMBZ) and hydrogen peroxide-containing solution) was added and reacted at room temperature for 30 minutes. 100 μL of (1N sulfuric acid) is added, and the absorbance (450 nm) is measured.
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