JP4240993B2 - Protein immobilization method - Google Patents

Description

【００４５】
図１から明らかなように、試薬としてSDSのみを含有する固定化用試料１を膜に固定化させた場合は、シグナル強度が全く測定できなかった（固定化用試料１）。また、SDSとTCAを含有する固定化用試料２を用いた場合は、少しシグナル強度が強くなった（測定できた）が、対照（精製水を用いた場合、従来の固定化法）ほど高いシグナル強度は得られなかった。
これに対し、SDSとエタノールを含有する固定化用試料３を用いた場合は、対照と同等若しくはそれ以上のシグナル強度が得られた。
また、TCAとエタノールを含有する固定化用試料５を用いた場合は、チトクロームcを固定化した場合以外は、すべて対照と比較して遙かに高いシグナル強度が得られた。
更に、TCAとエタノールとSDSを含有する固定化用試料４を用いた場合には、チトクロームcを固定化した場合も含めて、全ての場合で対照と比較して遙かに高いシグナル強度が得られた。
以上のことより、低級アルコールとハロゲノカルボン酸、又は更に長鎖アルキル硫酸塩の共存下で行う本発明に係る固定化法によれば、従来の水や緩衝液だけを用いて行っていた固定化法と比較して、膜への蛋白質の固定化率を飛躍的に向上させることができることが判る。
また、固定化用試料３及び４で、対照と同程度又はそれより高いシグナル強度が測定できたことから、本発明の固定化法によれば、予めSDS等を含有する試料を用いても、蛋白質を固定化することができることが判る。
【００４６】
実施例２．
[試料及び試液の調製]
(1)蛋白質試料
リゾチーム、チトクロームc、IgG、フィブリノーゲン（ヒト血漿由来、和光純薬工業(株)製）、BSA（牛血清アルブミン、和光純薬工業(株)製）、OVA、トリプシンインヒビター（大豆由来、和光純薬工業(株)製）、ヘモグロビンを夫々秤量し、精製水に溶解して250μg／mL溶液としたものを蛋白質試料として用いた。
尚、これらの蛋白質は等電点pIが4.0-11.4の幅広い範囲にあり、分子量は12000-150,000と広範囲のものである（久保ら,蛋白質 生化学ハンドブック,丸善株式会社,54-73 (1984)参照）。
(2)固定化用試液
2.5 W/V% TCA、45 V/V% エタノール、及び所定濃度（0〜0.4W/V%）のSDSを含有するように、精製水に溶解して調製したものを固定化用試液として用いた。
(3)固定化用試料
所定の蛋白質試料20μL（蛋白質量5μg）と固定化用試液300μLとを混合したものを調製し、固定化用試料とした（TCA終濃度2.34W/V%、エタノール終濃度42.2V/V%）。
[蛋白質の固定化及び測定]
実施例１と同様の方法で、各固定化用試料中の蛋白質をPVDF膜に固定化し、洗浄、染色処理し、次いでデンシトメーターで600nmの吸光度（シグナル強度）を測定した。
【００４７】
（結果）
結果を図２に示す。
図２に於いて、−△−はリゾチーム、−-−はチトクロームｃ、−○−はIgG、−□−はフィブリノーゲン、−●−はBSA、−◆−はOVA、−◇−はトリプシンインヒビターを含有する蛋白質試料を用いた場合の結果を夫々示す。また、横軸は固定化用試液中のSDS濃度を示す。更に、図２中の、各ポイントのバーは、±SDを示す。
図２から明らかな如く、殆どの蛋白質でSDS共存下にPVDF膜に固定化させると、一旦シグナル強度が増加するが、SDS濃度が0.1W/V%以上になるとシグナル強度がある一定の値を示す傾向を示した。このことから、固定化用試液中のSDS濃度を0.1W/V%以上（固定化用試料中の終濃度0.09W/V%以上）とすることにより、試料中の蛋白質のPVDF膜への固定化率が一定となると考えられる。
また、データのバラツキを示すCV値（CV値＝標準偏差／平均値（%））についてみると、どの蛋白質も固定化用試液中のSDS濃度が0.1 W/V%より低い濃度では、CV値が大きく、シグナル強度が安定していないことが分かる。それに対し、固定化用試液中のSDS濃度が0.1W/V%以上の場合は、CV値が比較的小さく、上述したようにこの濃度範囲でシグナル強度の値が安定であることが示されている。この結果は、試料中の蛋白質の固相膜に固定化される量を示していると考えられる。また、データは示していないが、この結果は再現性があることを確認している。一般に、SDS等の長鎖アルキル硫酸塩は、0.0025 W/V%といったかなりの低濃度でも蛋白質の構造崩壊作用を示し、構造崩壊の程度により蛋白質結合（染色）色素に対する反応性に違いを生ずるといわれている（Orsonneau, J-L et al., Clin.Chem., 35, 2233-2236 (1989)）。従って、ここでも、それが要因となって、SDS濃度が0.1 W/V%より低い固定化溶液を用いた場合で、シグナル強度が上昇する等の急激な変化を示し、且つ、それが変化の途中過程にあるため、CV値（＜15%）が大きくなったと推察される。また、このことは、0.1 W/V%より低いSDS濃度条件下では、試料中の蛋白質のPVDF膜への固定化率が変動している（一定でない）可能性をも示唆している。
【００４８】
以上のことより、図２に於いて、SDSの濃度変化の影響を受けずに吸光度（シグナル強度）が安定した時、始めて蛋白質のPVDF膜への固定化率が一定になっているのではないかと推察された。
そこで、図２の結果を基に、蛋白質試料としてBSAを用い、0.1 W/V% SDSを含有する固定化用試液で固定化した後測定を行った場合のシグナル強度を100（基準値）とした。そして、基準値に対する、その他の各蛋白質を蛋白質試料として用い、0.1W/V%SDS、0.2 W/V%SDS、0.3 W/V%SDS又は0.4 W/V%のSDS固定化用試液を用いて同様に固定化及び測定を行って得られたシグナル強度の相対値を夫々算出した。
【００４９】
表１に、SDS濃度が0.2W/V%〜0.4W/V%まで変化するまで、表２にはSDS濃度が0.1W/V%〜0.4W/V%まで変化するまでの、各ポイントのCV値を平均化した値（平均CV値（%））、各ポイントの相対値の平均値、そのポイント間の絶対偏差を平均した値（平均絶対偏差）、平均絶対偏差をその平均値で割りパーセント表示した値（ポイント間変動率（%））を夫々示す。ポイント間変動率とは、SDS濃度変化に伴うシグナル強度の変化を変動率として算出した値のことで、この数字が小さいほど、SDS濃度に影響されずにシグナル強度（測定結果）が一定であることを示している。
【００５０】
【表１】

【００５１】
【表２】

【００５２】
その結果、表１より、SDS濃度が0.2 W/V%−0.4W/V%間で、例えばフィブリノーゲン、OVA、IgG、チトクロームcは、そのポイント間変動率が最も安定し、1.4−2.7%を示した。また、これら蛋白質の平均CV値（0.8−1.9%）と比較しても遜色ない結果であり、SDS濃度が変化してもシグナル強度の変動が非常に少ないことを示している。
また、表２より、フィブリノーゲンを除く3つの蛋白質は、SDS濃度が0.1−0.4 W/V%の間でもポイント間変動率が2.3−3.3%であり、SDS濃度の影響によるシグナル強度の変動が少ないことがわかる。
また、SDS濃度が0.1−0.4 W/V%の間でポイント間変動率が10%を超えるフィブリノーゲン、リゾチーム、BSAについても、SDS濃度を0.2−0.4W/V%に限定すると安定した結果が得られることが判る。
以上のことより、SDS濃度が0.1W/V%以上でポイント間変動率が安定してくることから、この濃度範囲のSDSを含有する固定化用試液を用いて、蛋白質を固定化すると、試料中の蛋白質量の正確な定量が行えることが判る。
【００５３】
実施例３．低級アルコールの検討
低級アルコールとしてエタノールの代わりにメタノールを用いた場合の蛋白質試料の固定化及び測定を行った。
[試料及び試液の調製]
【００５４】
(1)蛋白質試料
BSA、OVA、ヘモグロビン、IgG、チトクロームc、リゾチームを夫々秤量し、精製水に溶解して250μg／mL溶液としたものを蛋白質試料として用いた。
(2)固定化用試液
精製水を用いて下記固定化用試液を調製した。
固定化用試液1：0.2 W/V% SDS、2.5 W/V% TCA
固定化用試液2：0.2 W/V% SDS、2.5 W/V% TCA、45% エタノール
固定化用試液3：0.2 W/V% SDS、2.5 W/V% TCA、45% メタノール（和光純薬工業(株)製、特級）
(3)固定化用試料
蛋白質試料20μL（蛋白質量5μg）と、所定の固定化用試液300mLとを混合したものを調製し、固定化用試料１,２,３とした。固定化用試料中の各試薬の終濃度は、夫々SDS 0.19W/V%、TCA 2.34W/V%、エタノール 42.2V/V%、メタノール 42.2V/V%である。
[蛋白質の固定化及び測定]
実施例１と同様の方法で、各固定化用試料中の蛋白質をPVDF膜に固定化し、洗浄、染色処理し、次いでデンシトメーターで600nmの吸光度（シグナル強度）を測定した。
【００５５】
[結果]
結果を図３に示す。図３に於いて、各バーは夫々下記固定化用試料を用いた場合の結果を示す。

【００５６】
図３から明らかな如く、メタノールを含有する固定化用試液を用いて調製した固定化用試料を用いた場合も、エタノールを用いた場合と同程度のシグナル強度が得られ、蛋白質をPVDF膜に固定化することができたことが判る。
【００５７】
実施例４．ハロゲノカルボン酸の検討
ハロゲノカルボン酸として、TCAの代わりにトリフルオロ酢酸（以下、TFAと略記する。）を用いた場合の蛋白質試料の固定化及び測定を行った。
[試料及び試液の調製]
(1)蛋白質試料
BSA、IgG、リゾチームを夫々秤量し、精製水に溶解して250μg／mL溶液としたものを蛋白質試料として用いた。
(2)固定化用試液
精製水を用いて、下記固定化用試液を調製した。
【００５８】
固定化用試液1：0.2 W/V% SDS、45 V/V% エタノール
固定化用試液2：0.2 W/V% SDS、45 V/V% エタノール、2.5 W/V% TCA
固定化用試液3：0.2 W/V% SDS、45 V/V% エタノール、2.5 W/V% TFA（和光純薬工業(株)製）
(3)固定化用試料
蛋白質試料20μL（蛋白質量5μg）と、所定の固定化用試液300mLとを混合したものを調製し、固定化用試料１,２,３とした。固定化用試料中の各試薬の終濃度は、夫々SDS 0.19W/V%、TCA 2.34W/V%、TFA 2.34W/V%、エタノール 42.2V/V%である。
[蛋白質の固定化および測定]
実施例１と同様の方法で、各固定化用試料中の蛋白質をPVDF膜に固定化し、洗浄、染色処理し、次いでデンシトメーターで600nmの吸光度（シグナル強度）を測定した。
【００５９】
[結果]
結果を図４に示す。図４に於いて、各バーは夫々下記固定化用試料を用いた場合の結果を示す。

【００６０】
図４から明らかな如く、TFAを含有する固定化用試試液用いて調製した固定化用試料を用いた場合も、TCAを用いた場合と同程度又はそれ以上のシグナル強度が得られ、蛋白質を有効に膜に固定化することができたことが判る。
【００６１】
実施例５．検量線の作成
[試料及び試液の調製]
(1)蛋白質試料
OVAを0〜20μg／20μLとなるように精製水に溶解して蛋白質試料とした。
(2)固定化用試液
0.2 V/V% SDS、2.5 V/V% TCA、45 V/V% エタノールとなるように精製水に溶解して調製したものを固定化用試液として用いた。
(3)固定化用試料
蛋白質試料20μL（蛋白質量5μg）と、固定化用試液300μLとを混合したものを調製し、固定化用試料とした。固定化用試料中の各試薬の終濃度は、夫々SDS 0.19W/V%、TCA 2.34W/V%、エタノール 42.2V/V%である。
[蛋白質の固定化及び測定]
実施例１と同様の方法で、各固定化用試料中の蛋白質をPVDF膜に固定化し、洗浄、染色処理し、次いでデンシトメーターで600nmの吸光度（シグナル強度）を測定した。
【００６２】
[結果]
その結果を基に、蛋白質量（μg）とシグナル強度との関係を示す検量線を作成した。
結果を図５に示す。図５に於いて、各プロットのバーは、±2SDを示す。
検量線から得られた結果は、測定範囲0.2−20μg／蛋白質試料、一致係数0.99以上、平均CV1.9％であった。
また、蛋白質濃度0.2−5μg／蛋白質試料の範囲で、直線性が得られた。測定結果を統計処理して得られた、この範囲での回帰直線式及び相関係数は下記の通りである。
回帰直線式：ｙ=0.12ｘ+0.01
相関係数（Ｒ^２）：0.99
ｘ：蛋白質量
ｙ：シグナル強度
図５から明らかな如く、本発明の方法によりOVAをPVDF膜に固定化し、蛋白質量の測定を行ったところ、良好な直線性を示す検量線が得られるので、本発明の固定化方法によれば、高精度のOVA（蛋白質）濃度の定量測定が行えることが判った。
尚、データは示していないが、実施例２で測定した他の蛋白質についても同様に測定を行った結果、OVAと同様に直線性のある検量線が得られ、これらの蛋白質についても定量測定が行えることが判った。
【００６３】
実施例６．
[試料及び試液の調製]
(1)蛋白質試料
精製水を用いて、BSA、トリプシンインヒビター、フィブリノーゲン、OVA、ヘモグロビン、IgG、チトクロームc、リゾチーム夫々の5μg／20μL溶液を調製し、蛋白質試料とした。
[固相化法による蛋白質の測定]
精製水を用いて0.2W/V%SDS、2.5W/V%TCA、45V/V% エタノールを含有する固定化用試液を調製した。次いで、調製した蛋白質試料20μLと固定化用試液300μLを混合し、得られた固定化用試料320μLを用いて実施例１と同様の方法で固定化用試料中の蛋白質をPVDF膜に固定化、洗浄、染色処理し、次いで、デンシトメーターで600nmの吸光度（シグナル強度）を測定した。固定化用試料中の各試薬の終濃度は、夫々SDS 0.19W/V%、TCA 2.34W/V%、エタノール 42.2V/V%である。
[液相法による蛋白質の測定]
上記で調製した蛋白質試料20μLに、1mL Pyromolex液を添加して、室温で20分間インキュベーションし、600nmの吸光度を測定した。
【００６４】
[結果]
蛋白質試料としてBSAを用い、0.2 W/V%SDSを含有する固定化用試料を調製して、BSAを固定化、測定を行った場合の吸光度（シグナル強度）を１（基準値）とした時の、基準値に対する各蛋白質について同様に固定化、測定を行って得られた吸光度の相対値を求めた結果を図６に示す。また、同様に蛋白質試料としてBSAを用い、液相法による測定を行った場合の吸光度を１（基準値）とした時の、基準値に対する各蛋白質について同様に液相法による測定を行って得られた吸光度の相対値を求めた結果も、図６に併せて示す。
尚、図６に於いて、各バーは夫々下記の方法により各蛋白質を測定した結果に基づいて得られた、上記相対値を示す。

【００６５】
図６より明らかな如く、液相法による測定では、蛋白質濃度は同じでも、蛋白質の種類によって、BSAに対する吸光度の相対値が大きく異なり、例えばフィブリノーゲンでは、0.46程度であった。
これに対し、本発明の固相法によって測定を行った場合のBSAに対するフィブリノーゲンの吸光度（シグナル強度）の相対値は0.75になり、BSAの場合との吸光度の差が少なくなっていることが判る。これは、測定した殆どの蛋白質についても言える。
また、測定した全蛋白質の平均吸光度の、BSAの場合のそれを１とした場合に対する相対値は、液相法の場合は0.65であるのに対して本発明の固相法の場合は0.94となり、蛋白質種による定量誤差が改善されたことが判る。これはタンパク質が変性状態で膜にトラップされることにより、液相法では反応できなかった、例えばチトクロームc、リゾチーム等の塩基性アミノ酸が、染色液と結合できる状態となり、本発明に係る固定化液で蛋白質が効率良く固定化され、より正確な測定結果を得られるようになったと考えられる。
【００６６】
実施例７．
[試料の調製と固定化]
精製水を用いて、IgG試料（蛋白質量0〜4μg／20μL）、及び2%SDS含有IgG試料（蛋白質量0〜4μg／20μL）を調製した。別に精製水を用いて0.25W/V%SDS、2.5W/V%TCA及び45V/V%エタノールを含有する固定化用試液を調製した。次いで、蛋白質試料20μLと固定化用試液300μLを混合し、得られた固定化用試料320μLを用いて実施例１と同様の方法で固定化用試料中の蛋白質をPVDF膜に固定化、洗浄、染色処理し、次いでデンシトメーターで600nmの吸光度（シグナル強度）を測定した。
尚、SDSを含有しないIgG試料を用いて得られた固定化用試料中の各試薬の終濃度は、夫々SDS 0.23W/V%、TCA 2.34 W/V%、エタノール 42.2V/V%である。また、2%SDS含有IgG試料を用いて得られた固定化用試料中の各試薬の終濃度は、夫々SDS 0.36 W/V%、TCA 2.34 W/V%、エタノール 42.2 V/V%である。
【００６７】
[結果]
得られた結果を基に、SDSを含有しないIgG試料を用いた場合と、2%SDS含有IgG試料を用いた場合夫々について、蛋白質量（μg）とシグナル強度との関係を示す検量線を作成した。
結果を図７に示す。図７に於いて、―◆―はSDSを含有しないIgG試料を用いた場合、―△―はSDSを含有するIgG試料を用いた場合の結果を夫々示す。また、各プロットのバーは、±SDを示す。
図７より明らかな如く、両方の検量線は、ほぼ一致した。
この結果から、本発明の固定化方法により蛋白質を固定化させれば、SDSが蛋白質試料中に存在していても、それに影響されることなく、目的の蛋白質を固相に固定化させ、蛋白質の定量を行うことができることが判る。
【００６８】
実施例８．
SDSを含有する種々の蛋白質試料を用いて本発明に係る蛋白質の固定化及び測定を行い、当該測定に及ぼすSDSの影響を、液相法による測定の場合と比較した。
[試料及び試液の調製]
(1)蛋白質試料
精製水を用いて、0W/V%SDS（対照）、0.2W/V%SDS又は2W/V%SDSを含有する蛋白質試料（BSA、トリプシンインヒビター、フィブリノーゲン、ヘモグロビン、ＯＶＡ、チトクロームc、リゾチーム、IgG、トリプシン（和光純薬工業(株)製）夫々250μg／mLを調製した。
[固定化法による蛋白質の測定]
精製水を用いて0.1W/V%SDS、2.5W/V%TCA、45V/V%エタノールを含有する固定化用試液を調製した。次いで調製した蛋白質試料20μLと固定化用試液300μLを混合し、得られた固定化用試料320μLを実施例１と同様の方法で固定化、洗浄、染色処理し、デンシトメーターで、600nmの吸光度（シグナル強度）を測定した。[液相法による測定]
上記で調製した蛋白質試料20μLに、1mL Pyromolex液（Protein Assay Rapid Kit wako、和光純薬工業(株)製）を添加して、20分室温でインキュベーションし、600nmに於ける吸光度を測定した。
【００６９】
[結果]
得られた結果を、夫々の対照（SDS含有していない試料）を用いて得られた結果を100とした相対値で表し、表３にまとめた。
【００７０】
【表３】

【００７１】
表３から明らかな如く、本発明に係る固相法による蛋白質の測定を行った場合には、2W/V%SDS含有試料でも殆どの蛋白質で、対照に対し±20%以内の測定結果が得られ、蛋白質試料中のSDSが蛋白質測定に及ぼす影響を回避できたことが判る。
これに対して、2W/V%SDS含有試料を用いて液相法によって測定を行った場合には、全く測定ができなかった。
以上の結果から、本発明の蛋白質の固定化法及び定量方法は、あらゆる蛋白質に適用することができることが判る。また、これまで知られていたタンパク定量阻害剤、特に蛋白質可溶化剤として汎用されるSDS含有試料中の蛋白質定量が可能になった点で、非常に有用でもあることが明らかとなった。
【００７２】
実施例９．
試料中に含まれる界面活性剤が、本発明の固定化用試液を用いた固定化方法及び蛋白質の測定に及ぼす影響を調べた。
[固相法による固定化及び蛋白質の測定]
（１）BSA又はIgG含有蛋白質試料中の蛋白質の測定
精製水で、表４記載の濃度となるように、各界面活性剤を含有するBSA試料又はIgG試料（蛋白質量 4μg／20μL）を調製し、蛋白質試料とした。
別に、精製水で0.1W/V%SDS、2.5 W/V% TCA、45 V/V% エタノールを含有する固定化用試液を調製した。
次いで蛋白質試料20μLと固定化用試液300μLを混合して固定化用試料を調製し、その320μLを用いて、実施例１と同様の方法で固定化用試料中の蛋白質をPVDF膜に固定化、洗浄、染色処理し、600nmの吸光度（シグナル強度）を測定した。
固定化用試料中の各試薬の終濃度は、SDSを含有しない蛋白質試料を用いた場合は、SDS 0.094W/V%、TCA 23.4W/V%、エタノール 42.2V/V%である。また、SDS含有蛋白質試料を用いた場合の各試薬の終濃度は、TCA及びエタノールの終濃度はSDSを含有しない蛋白質試料を用いた場合と同じであるが、SDSの終濃度は、１％SDS含有蛋白質試料を用いた場合は0.16W/V%、２%SDS含有試料を用いた場合は0.21W/V%、４%SDS含有試料を用いた場合は、0.34W/V%となる。
【００７３】
（２）OVA含有蛋白質試料中の蛋白質の測定
表４記載の濃度となるように、各界面活性剤を含有するOVA試料（蛋白質量 ５μg／20μL）を調製し、蛋白質試料とした。
別に、精製水で0.2W/V%SDS、2.5 W/V% TCA、45 V/V% エタノールを含有する固定化用試液を調製し、上記（1）と同様の方法で固定化用試料を調製し、実施例１と同様の方法でPVDF膜に固定化、洗浄、染色処理し、600nmの吸光度（シグナル強度）を測定した。
尚、固定化用試料中の各試薬の終濃度は、SDSを含有しない蛋白質試料を用いた場合は、SDS 0.19W/V%、TCA 23.4W/V%、エタノール 42.2V/V%である。また、2％ SDS含有蛋白質試料を用いた場合の各試薬の終濃度は、SDS 0.31W/V%、TCA 23.4%、エタノール 42.2W/V%となる。
また、界面活性剤（阻害物質）を含有しない以外は上記と同様に調製したBSA試料、IgG試料、OVA試料を用いて同様に固定化、測定を行い、対照とした。
[液相法による蛋白質の測定]
表４記載の濃度となるように各界面活性剤を含有するBSA試料（10μg／20μL）を調製したものを用い、1mL Pyromolex溶液を使って、実施例６と同様の方法で測定した。
また、界面活性剤（阻害物質）を含有しない以外は上記と同様に調製したBSA試料を用いて同様に液相法による測定を行い、対照とした。
【００７４】
[結果]
夫々の測定は３回ずつ行い、得られた吸光度の平均値を求めた。。対照を用いて得られた平均値を100として、それに対する界面活性剤を含有する試料を用いて得られた吸光度の平均値の相対値（%）を求め、その相関変位（CV）と併せて表４に示す。
表４に於いて、界面活性剤（阻害物質）の濃度は、蛋白質試料中の濃度を示す。また、液相法のデータ中、左側の値は界面活性剤の濃度を、右側の値は対照に対する、界面活性剤を含有する試料を用いた場合の平均測定値の相対値（%）±ＣＶを示す。
尚、表４に示したデータは、界面活性剤の最大許容濃度時のデータ、すなわち、添加剤として界面活性剤を用いた場合に、対照（添加剤なし）と比較して平均値が±20%となる結果が得られた時のデータを示している。
【００７５】
【表４】

【００７６】
SLS： N-ラウロイルサルコシン酸ナトリウム
Triton X-100（ローム アンド ハース社商品名）：ポリオキシエチレン(10)オクチルフェニルエーテル
NP-40（日本エマルジョン(株)商品名）：ポリオキシエチレン(9)オクチルフェニルエーテル
Tween 20（花王(株)商品名）：ポリオキシエチレンソルビタンモノラウレート
Tween 80（花王(株)商品名）：ポリオキシエチレンソルビタンモノオレエート
Brij 35（ICI社商品名）：ポリオキシエチレンラウリルエーテル
CHAPS：3-[(3-コラミドプロピル)ジメチルアンモニオプロパンスルホン酸]
CTAB：セチルトリメチルアンモニウムブロマイド
【００７７】
表４から明らかな如く、本発明の方法により蛋白質を固定化し測定した場合、蛋白質試料の調製時に一般に用いられる界面活性剤が高濃度共存していても、それに影響を受けずに蛋白質の測定が可能であることが判る。特に液相法と比較すると、液相法の10倍又はそれ以上の濃度の界面活性剤が蛋白質試料中に添加剤として共存していても、測定可能であることが判る。
以上のことより、本発明の蛋白質の固定化方法は、従来より添加剤として汎用されている界面活性剤に起因する問題、即ち蛋白質の定量を阻害するという問題を解決し得るものであることが判る。
【００７８】
実施例１０．イムノブロッティング
[試料及び試液の調製]
(1)蛋白質試料
マウスIgG（和光純薬工業(株)製）を秤量し、精製水に溶解して0~200μg／mL溶液としたものを蛋白質試料として用いた。
(2)固定化用試液
精製水で、0.2 W/V% SDS、2.5 W/V% TCA、45 V/V% エタノールを含有する固定化用試液を調製した。
(3)固定化用試料
上記で調製した各濃度の蛋白質試料夫々20μLと、固定化用試液300μLを混合したもの（SDS終濃度0.19W/V%、TCA終濃度2.34W/V%、エタノール終濃度42.2V/V%）を調製し、固定化用試料とした。
(4)ブロッキング溶液
ブロックエース（雪印乳業(株)製）を終濃度25%となるようにPBS（pH7.4）で希釈したものを用いた。
(5)抗体溶液
発光検出用抗体溶液：西洋ワサビペルオキシダーゼ標識抗マウスIgG抗体（アマシャムバイオサイエンス製）をブロッキング溶液で 1/10000希釈したものを用いた。
発色検出用抗体溶液：アルカリフォスファターゼ標識抗マウスIgG抗体（和光純薬工業(株)製）をブロッキング溶液で1/1000希釈したものを用いた。
(6)洗浄液
Tween 20を、終濃度0.05%となるようにPBS（pH7.4）で希釈したものを用いた。
(7)検出試薬
発光検出用：ECL Plus Western Blotting Starter Kit（アマシャムバイオサイエンス(株)製）
発色検出用：0.033% ニトロブルーテトラゾリウム（NBT,和光純薬工業(株)製）、0.0165% 5-ブロモ-4-クロロ-3-インドリルリン酸（BCIP、和光純薬工業(株)製）／100mM Tris-HCl pH9.5（100mM NaCl、5mM MgCl₂含有）
【００７９】
[蛋白質の固定化および測定]
実施例１と同様の方法で、上記した如く調製した固定化用試料をPVDF膜にアプライし、吸引濾過後、PBS (pH7.4) 300μLをアプライし、同様に吸引濾過を行った。PVDF膜を取り出し、ブロッキング溶液に浸し、ローテーションさせながら室温で1時間インキュベーションした（ブロッキング操作）。その後、発光検出用抗体溶液又は発色検出用抗体溶液に浸し、ローテーションさせながら室温1時間インキュベーションした（抗体反応）。抗体反応後の膜を洗浄液で5回洗浄した後、発光検出試薬又は発色検出試薬に浸し、検出反応を行った。発光検出は、PVDF膜を発光検出処理後、Ｘ線フィルム（アマシャムバイオサイエンス製）に感光させて検出を行った。
【００８０】
[結果]
結果を図８に示す。図８に於いて、ＡはPVDF膜に固定化した蛋白質試料の免疫検出を発光反応により行い、X線フィルムに感光させ検出したものである。Ｂは、PVDF膜に固定化した蛋白質試料の免疫検出を発色反応により行い、検出したものである。また、各ドットは、蛋白質試料を各蛋白質量として固定化後に検出した場合の結果を夫々示す。
図８より明らかな如く、イムノブロッティングにより発光検出、発色検出した場合の何れも、膜に固定化したマウスIgGを検出することができた。発光検出での検出限界は0.0625μg、発色検出での検出限界は0.5μgであった。従って、本発明の蛋白質の固定化方法により固定化すれば、高感度の免疫検出（イムノブロッティングによる検出）が行い得ることが判った。
【００８１】
【発明の効果】
本発明に係る蛋白質固定化方法によれば、従来の固定化方法よりも充分に蛋白質を固相に固定化できる。また、従来の固定化方法では正確に行えなかった蛋白質の定量も行うことができる。更に、本発明に係る蛋白質固定化方法を用いれば、イムノブロッティングを行った際の感度も高くなるという効果を奏する。
【図面の簡単な説明】
【図１】実施例１において得られた、各固定化用試料中の蛋白質を固定化したポリビニリデンジフロライド膜（PVDF膜）をPyromolex試液で染色した後、600nmにおける吸光度（シグナル強度）を測定した結果を示す。
【図２】実施例２に於いて得られた、各固定化用試料中の蛋白質を固定化したPVDF膜をPyromolex試液で染色した後、600nmにおける吸光度（シグナル強度）を測定した結果を示す。
【図３】実施例３に於いて得られた、各固定化用試料中の蛋白質を固定化したPVDF膜をPyromolex試液で染色した後、600nmに於ける吸光度（シグナル強度）を測定した結果を示す。
【図４】実施例４に於いて得られた、各固定化用試料中の蛋白質を固定化したPVDF膜をPyromolex試液で染色した後、600nmに於ける吸光度（シグナル強度）を測定した結果を示す。
【図５】実施例５に於いて得られた、蛋白質試料としてOVAを用いて、PVDF膜に固定化、定量を行って得られた検量線を示す。
【図６】実施例６に於いて得られた、固相法により蛋白質を測定した結果又は液相法により蛋白質を測定した結果に基づいて得られた相対値を示す。
【図７】実施例７に於いて得られた、SDSを含有しないIgG試料又はSDS含有するIgG試料を蛋白質試料として用い、本発明に係る固定化、蛋白質の定量を行って得られた検量線を示す。
【図８】実施例１０に於いて得られた、イムノブロッティングの結果を示し、ＡはPVDF膜に固定化した蛋白質試料の免疫検出を発光反応により行い、X線フィルムに感光させ検出したものである。Ｂは、PVDF膜に固定化した蛋白質試料の免疫検出を発色反応により行い、検出したものである。
【符号の説明】
図１に於いて、各バーは夫々下記固定化用試料を用いた場合の結果を示す。

図２に於いて、−△―はリソゾーム、−-−はチトクロームｃ、−○−はIgG、−◎−はフィブリノーゲン、−●−はBSA、−◆−はOVA、−◇−はトリプシンインヒビターを含有する蛋白質試料を用いた場合の結果を夫々示す。
図３に於いて、各バーは夫々下記固定化用試料を用いた場合の結果を示す。

図４に於いて、各バーは夫々下記固定化用試料を用いた場合の結果を示す。

図６に於いて、各バーは夫々下記の方法により各蛋白質を測定した結果に基づいて得られた、上記相対値を示す。

図７に於いて、−◆−はSDSを含有しない蛋白質試料を用いた場合、−△−はSDSを含有する蛋白質試料を用いた場合の結果を夫々示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel protein immobilization method on a solid phase, a protein quantification method using the same, an immunoblotting method, and a protein immobilization test solution.
[0002]
[Prior art]
Conventionally, protein quantification is performed by reacting a highly chemically reactive portion of a protein with a protein reaction reagent in a solution [Lowry, OH et al., J. Biol. Chem., 193: 265-275 (1951) Smith, PK et al., Anal. Biochem., 150: 76-85 (1985)] and reacting with a dye reagent that specifically adsorbs to proteins [Bradford, M., Anal. Biochem., 72: 248-254 (1976), Watanabe, N. et al., Clin. Chem., 32: 1551-1554 (1986)], a method of changing the absorbance of a solution and measuring based on the change in absorbance, so-called liquid phase The law was mainstream. However, biochemical samples usually contain many substances such as proteins, non-protein biological components, buffers, salts, antioxidants, chelating agents, saccharides, organic solvents, artificial polymers, and surfactants. In many samples, these coexisting substances inhibit the interaction (reaction / binding) between the protein and the protein measurement reagent, and accurate protein measurement is often impossible. Therefore, a method in which protein is adsorbed on a solid surface such as a membrane, an inhibitor for protein measurement such as the coexisting substance is washed away, and the protein staying on the solid surface is reacted and quantified with a protein measurement reagent, so-called solid phase immobilization. The method was developed [Kuno, H. et al., Nature, 215: 974-975 (1967), Gates, R., Anal. Biochem., 196: 290-295 (1991), Said-Fernandez, S. et al., Anal. Biochem. 191: 119-126 (1990), Ghosh, S. et al., Anal. Biochem. 169: 227-233 (1988), Lim, MK et al., BioTechniques 21: 888- 895 (1996)].
[0003]
However, these protein membrane immobilization methods can remove the inhibitor, but cannot fix the protein to the membrane at a certain rate, and the protein cannot be accurately quantified. It has not yet been resolved.
[0004]
On the other hand, as a protein immobilization method, a method using trichloroacetic acid, acetic acid solution or acetic acid / methanol solution having protein denaturation / charge neutralizing action as an immobilization solution has been used for a long time. However, protein cannot be measured quantitatively because sufficient immobilization may not be possible depending on the protein. Therefore, the development of a new protein immobilization / quantification method has been desired.
[0005]
[Non-Patent Document 1]
Lowry, OH et al., J. Biol. Chem., 193, 265-275 (1951), pp. 266-270)
[Non-Patent Document 1]
Smith, PK et al., Anal. Biochem., 150, 76-85 (1985), (p. 77-p. 80)
[Non-Patent Document 1]
Bradford, M., Anal. Biochem., 72, 248-254 (1976), (pp. 249-251)
[Non-Patent Document 1]
Watanabe, N. et al., Clin. Chem., 32, 1551-1554 (1986), (pages 1551 to 1552)
[Non-Patent Document 1]
Kuno, H. et al., Nature, 215, 974-975 (1967), (page 975)
[Non-Patent Document 1]
Gates, R., Anal. Biochem., 196, 290-295 (1991), (pp.291-294)
[Non-Patent Document 1]
Said-Fernandez, S. et al., Anal. Biochem., 191, 119-126 (1990), (p. 120-122, p. 125)
[Non-Patent Document 1]
Ghosh, S. et al. Anal. Biochem., 169, 227-233 (1988), (p. 229, p. 232)
[Non-Patent Document 1]
Lim, MKet al., BioTechniques, 21, 888-895 (1996), (p. 889-p. 890)
[0006]
[Problems to be solved by the invention]
The present invention has been made in view of the situation as described above. A protein in a sample that could not be easily immobilized by a conventional solid phase immobilization method can be immobilized on the solid phase, and an inhibitory substance coexisting in the sample can be obtained. An object of the present invention is to provide an immobilization method capable of quantitatively measuring / detecting a protein with reduced influence, a protein quantification method using the same, an immunoblotting method, and a protein immobilization reagent.
[0007]
[Means for Solving the Problems]
The present invention has been made for the purpose of solving the above-described problems, and has the following configuration.
(1) A method for immobilizing a protein on the solid phase, comprising contacting the protein with a solid phase having a hydrophobic surface in the presence of a lower alcohol and a halogenocarboxylic acid.
(2) A method for quantifying a protein, characterized in that a protein staining solution is brought into contact with a solid phase on which a protein is immobilized by the method (1) above, and the coloration produced thereby is performed.
(3) An immunoblotting method using a solid phase on which a protein is immobilized by the method of (1) above.
(4) containing a lower alcohol and a halogenocarboxylic acid, To solid phase with hydrophobic surface Protein immobilization reagent.
[0008]
In other words, the present inventors examined the factors that prevent immobilization of proteins by the conventional solid phase immobilization method, and the biggest factor is the stage of immobilizing the protein on the solid phase. It was found that the protein could not be quantitatively measured because the protein could not be sufficiently immobilized on the solid phase (at a certain rate).
[0009]
Therefore, as a result of further study on a method for efficiently performing immobilization, when a protein was immobilized in the presence of a lower alcohol such as ethanol and a halogenocarboxylic acid such as trichloroacetic acid, the protein was sufficiently immobilized on the solid phase. It was found that it can be immobilized.
[0010]
In addition, among the surfactants, we found that long-chain alkyl sulfates have unique properties for protein immobilization. That is, when a protein is immobilized on a long-chain alkyl sulfate in the presence of a lower alcohol or further halogenocarboxylic acid, it has some influence on the protein in the process of sucking and filtering the protein. It has been clarified that it promotes and stabilizes protein membrane adsorption while acting to exclude coexisting substances such as inhibitors from the membrane solid phase surface (suppressing the membrane retention of inhibitors). Such action of the long-chain alkyl sulfate can occur even when the protein-containing immobilization sample solution is allowed to stand on a solid surface such as a microplate well.
[0011]
In general, surfactants such as nonionic surfactants have the property of inhibiting the adsorption of hydrophobic substances, while the binding between proteins and membranes is thought to be due to hydrophobic bonds. For this reason, surfactants have not been favorably used for protein immobilization so far, and therefore the action of surfactants on proteins and the interaction of the affected proteins on the solid phase were discussed in detail. There are few reports. Therefore, it has not been known so far that a long-chain alkyl sulfate as a surfactant is effective for immobilizing proteins.
[0012]
Therefore, the present inventor is able to sufficiently immobilize a protein on a solid phase by coexisting a long-chain alkyl sulfate with lower alcohols and halogenocarboxylic acids conventionally used for protein immobilization. This is what they found for the first time. Furthermore, since the protein in the sample coexisting with the surfactant could not be immobilized efficiently in the conventional solid phase method, the protein could not be quantified, but according to the immobilization method of the present invention, The protein in such a sample can also be immobilized efficiently, and a highly effective and highly useful protein immobilization method has been completed.
[0013]
In the present invention, the term "protein immobilization method" refers to a method of immobilizing a protein on a solid phase, and the term "protein measurement or quantification by a solid phase method" refers to the method of immobilizing a protein by the immobilization method of the present invention. Is immobilized on a solid phase, and then protein is measured or quantified.
[0014]
Examples of the lower alcohol according to the present invention include methanol, ethanol, propanol, etc. Among them, ethanol or methanol is preferable.
[0015]
Examples of the halogen atom of the halogenocarboxylic acid according to the present invention include bromine, fluorine, chlorine, etc. Among them, chlorine is preferable. Examples of the carboxylic acid include acetic acid and propionic acid. Among them, acetic acid is preferable. Examples of such halogenocarboxylic acids include trichloroacetic acid (TCA) and trifluoroacetic acid (TFA).
[0016]
As the long-chain alkyl group of the long-chain alkyl sulfate according to the present invention, those having 7 to 25 carbon atoms are preferable, and among them, 8 to 15 is preferable. More preferably, it is a dodecyl group. Moreover, as a sulfate, a sodium salt, potassium salt, etc. are preferable, and a sodium salt is preferable especially. Specific examples of such long-chain alkyl sulfates include sodium dodecyl sulfate (SDS), among which SDS is preferred.
[0017]
In the immobilization method according to the present invention, the protein comprises a lower alcohol and a halogenocarboxylic acid. Or even As a method of coexisting with a long-chain alkyl sulfate, when the protein is brought into contact with a solid phase having a hydrophobic surface, the protein is converted into a lower alcohol and a halogenocarboxylic acid. Co-existing with the protein, or the protein is a lower alcohol, a halogenocarboxylic acid and a long-chain alkyl sulfate Any method can be used as long as it can coexist.
[0018]
For example, (1) a sample containing a protein, a solution containing a lower alcohol, and a solution containing a halogenocarboxylic acid Or even A method of mixing a solution containing a long-chain alkyl sulfate, (2) a sample containing a protein, a lower alcohol, and a halogenocarboxylic acid Or even Although the method etc. which mix a long-chain alkyl sulfate directly are mentioned, it is not specifically limited.
[0019]
Examples of the solution used when preparing a solution containing a lower alcohol, a solution containing a long-chain alkyl sulfate, and a solution containing a halogenocarboxylic acid include purified water and a buffer solution. Examples of the buffering agent include MOPS, HEPES and other good buffering agents, Tris buffering agent, phosphate buffering agent, veronal buffering agent, boric acid buffering agent and the like, which are usually used in this field. However, it is preferable to use purified water in order to avoid the influence on protein immobilization and measurement as much as possible.
[0020]
The preferred concentration of each reagent in the immobilization sample for contacting the protein with a solid phase having a hydrophobic surface is a lower alcohol concentration of 30-50 V / V% and a halogenocarboxylic acid concentration of 0.08-10 W / V%. The long-chain alkyl sulfate concentration is 0.1-1 W / V%.
[0021]
Examples of the solid phase having a hydrophobic surface according to the present invention include a film having a hydrophobic surface and a plate having a hydrophobic surface. Specific examples of membranes having a hydrophobic surface include, for example, polyvinylidene difluoride membranes (PVDF membranes), nitrocellulose membranes, filter paper, etc., which are hydrophobic membranes. Specific examples of plates having a hydrophobic surface include For example, a plastic plate or the like often used in ELISA or the like can be mentioned.
[0022]
As a method of bringing a protein into contact with a solid phase having a hydrophobic surface, the protein prepared by the above method, a lower alcohol, Halogenocarboxylic acid, or further long-chain alkyl sulfate What is necessary is just to contact the sample for fixation containing this with the solid phase which has the said hydrophobic surface. For example, there are methods such as dropping or applying the immobilization sample onto the solid phase.
[0023]
When a hydrophobic membrane is used as the solid phase, the immobilization sample is dropped on the hydrophobic membrane, and then allowed to stand to permeate the immobilization sample into the hydrophobic membrane, or A normal filtration method or a centrifugal filtration method in which the immobilization sample is suction filtered through the hydrophobic membrane may be used.
[0024]
The protein immobilization method by the filtration method will be specifically described below by taking a method using a commercially available dot blotter or slot blotter as an example.
[0025]
First, a hydrophobic membrane such as a PVDF membrane soaked in methanol and then distilled water and, if necessary, a filter paper soaked in distilled water are set on a dot blotter. Next, a certain amount of protein and lower alcohol And halogenocarboxylic acid, or further acid long chain alkyl sulfate When the sample for immobilization (up to 400 μL) containing is applied to the well of the dot blotter and slowly sucked (filtered) with a vacuum pump at a pressure of about 15 Kpa, the protein in the sample for immobilization is Adsorbed on PVDF membrane. After the immobilized sample is completely aspirated, the washing solution is applied to each well and aspirated. Next, the PVDF membrane is taken out from the dot blotter, placed on a paper towel, filter paper, etc., and vacuum-dried over about 30 minutes.
[0026]
Whether or not a protein is easily adsorbed depends on the relationship between the hydrophobicity of the protein and the hydrophobicity of the solid phase membrane surface. For example, a protein that is easily adsorbed under the conditions is sufficiently adsorbed even if it is rapidly aspirated, but the degree of adsorption of a protein that exhibits only moderate or weak absorptivity is greatly influenced by the aspiration rate. Therefore, in order to sufficiently adsorb the target protein, it is generally preferable to suck it slowly. For example, it is preferable to suck over 10 minutes or more.
[0027]
When a plate having a hydrophobic surface is used as the solid phase, for example, the immobilization sample may be dropped or coated on the plate and then allowed to stand and air dry.
[0028]
In order to quantify the protein, the solid phase on which the protein is immobilized may be subjected to an ordinary protein quantification method, and the amount of protein in the sample may be measured.
[0029]
The protein quantification method according to the present invention includes, for example, a method using the amino black, pyrogallol red-molybdate complex (Pyromolex) solution as a protein staining solution after immobilizing the protein on a solid phase by the above method, Coomassie A protein measuring method known per se, which is performed by staining with the Bradford method using brilliant blue (CBB) -G250, the method using bicine chrononic acid (BCA), etc., and measuring the degree of color development. Measurement may be performed according to
[0030]
For actual quantification, for each protein to be measured, a calibration curve is created by immobilizing, staining, and measuring in the same manner using a protein sample with a known protein concentration. Based on the calibration curve, the protein concentration in the sample is determined.
[0031]
For example, the quantification method using the pyromolex color development method will be described as an example. First, the protein in the protein sample is immobilized on the PVDF membrane by the method of the present invention, and then the PVDF membrane is washed with purified water or a buffer solution such as PBS. . If necessary, after vacuum drying at room temperature for about 30 minutes, immerse in Pyromolex-containing staining reagent for about 20 to 35 minutes to develop color. Thereafter, the absorbance at 600 nm is measured with a densitometer, a CCD camera or the like. The concentration of the protein may be determined from a calibration curve obtained by immobilizing and measuring the protein in the same manner using a protein sample with a known concentration in advance.
[0032]
As an immunoblotting method according to the present invention, the protein is measured / detected by an antigen-antibody reaction using an antibody against the protein or a labeled antibody, except that the protein is immobilized on a solid phase by the method of the present invention. A normal immunoblotting method can be applied. Since the protein can be efficiently immobilized on the solid phase by performing the immobilization method according to the present invention, the immunoblotting method according to the present invention can detect and analyze proteins with higher sensitivity than before. it can.
[0033]
Examples of the protein that can be immobilized by the immobilization method of the present invention include all proteins that have been immobilized by the conventional solid phase immobilization method. For example, various body fluids such as blood, serum, plasma, and cerebrospinal fluid, urine, and lymphocytes And proteins contained in biological samples such as blood cells and cells.
[0034]
Specifically, for example, enzymes such as lysozyme, cytochrome c and DNase, antibodies such as IgG, IgM and IgE, glycoproteins such as fibrinogen, serum proteins such as bovine serum albumin (BSA) and human serum albumin (HAS), egg white Examples include, but are not limited to, proteins such as albumin (OVA) and prion, inhibitors such as trypsin inhibitor, hormones such as insulin, and the like.
[0035]
The present invention is particularly effective in that, for example, a basic protein such as cytochrome c that cannot be sufficiently immobilized on a solid phase by a conventional solid phase immobilization method can be immobilized, and the protein can be quantified. is there.
[0036]
In the protein immobilization method according to the present invention, the upper limit of the concentration of the protein that can be immobilized on the solid phase is, for example, about 500 μg / cm when the solid phase is a membrane. ² About 10 μg / cm when the solid phase is a microplate ² Therefore, it is desirable that the amount of protein in the sample for immobilization is adjusted so as not to exceed the maximum retention capacity depending on the type of solid phase to be immobilized.
[0037]
Protein immobilization reagent according to the present invention Is , Containing a lower alcohol according to the present invention and a halogenocarboxylic acid, or further a long-chain alkyl sulfate A test solution for protein immobilization on a solid phase having a hydrophobic surface, That Configuration requirements Specific examples are as described above.
[0038]
The concentration of the lower alcohol in the protein immobilization test solution is 30-50 V / V% when the protein is immobilized on the solid phase, and the halogenocarboxylic acid concentration is 0.1-10 W / V%. The concentration of the long chain alkyl sulfate may be a concentration that is 0.1 to 1 W / V%. More preferably, the lower alcohol is 35 to 50 V / V%, the halogenocarboxylic acid is 0.5 to 5 W / V%, and the long-chain alkyl sulfate is 0.1 to 0.4 W / V%.
[0039]
Furthermore, the protein immobilization reagent according to the present invention contains salts, chelates, etc., as long as it does not affect the immobilization of the protein on the solid phase and the subsequent determination of the protein. Also good.
[0040]
The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples.
[0041]
【Example】
Example 1.
[Preparation of sample and test solution]
(1) Protein sample
Ovalbumin (hereinafter abbreviated as OVA, derived from chicken egg white, manufactured by Wako Pure Chemical Industries, Ltd.), hemoglobin (derived from bovine blood, manufactured by Wako Pure Chemical Industries, Ltd.), IgG (derived from bovine, Wako Pure Chemical Industries, Ltd. ( Co., Ltd.), cytochrome c (derived from horse heart muscle, manufactured by Wako Pure Chemical Industries, Ltd.), lysozyme (derived from chicken egg white, manufactured by Wako Pure Chemical Industries, Ltd.), respectively, weighed and dissolved in purified water, 250 μg / ML solution was used as a protein sample.
[0042]
(2) Immobilization reagent
Each reagent was dissolved in purified water to prepare the following immobilization reagent. Among these, the immobilization test solutions 3 to 5 are the immobilization test solution according to the present invention.
Each reagent is ethanol (manufactured by Wako Pure Chemical Industries, Ltd., special grade), trichloroacetic acid (hereinafter abbreviated as TCA, manufactured by Wako Pure Chemical Industries, Ltd., for chemical use), sodium dodecyl sulfate (hereinafter SDS). Abbreviated, Wako Pure Chemical Industries, Ltd., for chemical use) was used.
Control: Purified water
Immobilization reagent 1: 0.2 W / V% SDS,
Immobilization reagent 2: 0.2 W / V% SDS, 2.5 W / V% TCA
Immobilization reagent 3: 0.2 W / V% SDS, 45 V / V% ethanol
Immobilization reagent 4: 0.2 W / V% SDS, 2.5 W / V% TCA, 45 V / V% ethanol
Immobilization reagent 5: 2.5W / V% TCA, 45V / V% ethanol
(3) Immobilization sample
A mixture of 20 μL of protein sample (5 μg of protein) and 300 μL of a predetermined immobilization test solution was prepared and used as an immobilization sample. The final concentration of each reagent in the immobilization sample (concentration when contacting with the PVDF membrane; the same applies hereinafter) is as follows.
Immobilization sample 1: 0.19 W / V% SDS,
Immobilization sample 2: 0.19 W / V% SDS, 2.34 W / V% TCA
Sample for immobilization 3: 0.19 W / V% SDS, 42.2V / V% ethanol
Immobilization sample 4: 0.19 W / V% SDS, 2.34 W / V% TCA, 42.2 V / V% ethanol
Sample 5 for immobilization: 2.34W / V% TCA, 42.2V / V% ethanol
[0043]
[Immobilization and measurement of protein]
Polyvinylidene difluoride membrane (PVDF membrane, manufactured by Millipore, Immobilon P) with a hydrophilic treatment on the dot blotter ADVANTEC DP-96 (manufactured by Advantech) ^SQ 0.1 μm) was set. Next, 320 μL of immobilization sample was applied to the PVDF membrane, and suction filtration was performed at 15 KPa (10 cmHg) for 10 minutes with a vacuum pump (manufactured by Biocraft). Next, 300 μL of pH 7.4 phosphate buffered saline (PBS) was applied, and suction filtration was performed in the same manner to wash the PVDF membrane. After removing the PVDF membrane and vacuum-drying it, color it with Pyromolex reagent (Protein Assay Rapid Kit wako, manufactured by Wako Pure Chemical Industries, Ltd.), then 600 nm with densitometer SHIMADZU CS-9000 (manufactured by Shimadzu Corporation) The absorbance (signal intensity) of was measured.
[0044]
[result]
The results are shown in FIG. In FIG. 1, each bar shows the result when the following immobilization sample is used.

[0045]
As is clear from FIG. 1, when immobilization sample 1 containing only SDS as a reagent was immobilized on a membrane, the signal intensity could not be measured at all (immobilization sample 1). In addition, when the sample 2 for immobilization containing SDS and TCA was used, the signal intensity was slightly increased (measured), but higher than the control (conventional immobilization method when using purified water). No signal intensity was obtained.
On the other hand, when the immobilization sample 3 containing SDS and ethanol was used, a signal intensity equal to or higher than that of the control was obtained.
In addition, when the sample 5 for immobilization containing TCA and ethanol was used, a signal intensity much higher than that of the control was obtained except when cytochrome c was immobilized.
Furthermore, when using the immobilization sample 4 containing TCA, ethanol and SDS, a signal intensity much higher than that of the control was obtained in all cases, including the case where cytochrome c was immobilized. It was.
From the above, lower alcohols and halogenocarboxylic acids Or even According to the immobilization method according to the present invention performed in the presence of a long-chain alkyl sulfate, the rate of protein immobilization on the membrane compared to the conventional immobilization method using only water and buffer solution. It can be seen that it can be improved dramatically.
In addition, in immobilization samples 3 and 4, signal intensity comparable to or higher than that of the control could be measured. Therefore, according to the immobilization method of the present invention, even if a sample containing SDS or the like was used in advance, It can be seen that the protein can be immobilized.
[0046]
Example 2
[Preparation of sample and test solution]
(1) Protein sample
Lysozyme, cytochrome c, IgG, fibrinogen (derived from human plasma, manufactured by Wako Pure Chemical Industries, Ltd.), BSA (bovine serum albumin, manufactured by Wako Pure Chemical Industries, Ltd.), OVA, trypsin inhibitor (derived from soybean, Wako Pure Chemical Industries, Ltd.) Kogyo Co., Ltd.) and hemoglobin were weighed and dissolved in purified water to give a 250 μg / mL solution, which was used as a protein sample.
These proteins have a wide range of isoelectric points pI of 4.0-11.4 and molecular weights of 12000-150,000 (Kubo et al., Protein Biochemistry Handbook, Maruzen Co., 54-73 (1984). reference).
(2) Immobilization reagent
A solution prepared by dissolving in purified water so as to contain 2.5 W / V% TCA, 45 V / V% ethanol, and SDS at a specified concentration (0 to 0.4 W / V%) is used as an immobilization reagent. It was.
(3) Immobilization sample
Prepare a mixture of 20 μL of the prescribed protein sample (5 μg protein mass) and 300 μL of the immobilization test solution as the immobilization sample (TCA final concentration 2.34 W / V%, ethanol final concentration 42.2 V / V%) .
[Immobilization and measurement of protein]
In the same manner as in Example 1, the protein in each immobilization sample was immobilized on a PVDF membrane, washed and stained, and then the absorbance (signal intensity) at 600 nm was measured with a densitometer.
[0047]
(result)
The results are shown in FIG.
In FIG. 2,-△-is lysozyme, --- is cytochrome c,-○-is IgG,-□-is fibrinogen,-●-is BSA,-◆-is OVA,-◇-is a trypsin inhibitor. The results when using the contained protein samples are shown. The horizontal axis represents the SDS concentration in the immobilization reagent. Furthermore, each point bar in FIG. 2 represents ± SD.
As is clear from FIG. 2, when most proteins are immobilized on a PVDF membrane in the presence of SDS, the signal intensity once increases. However, when the SDS concentration exceeds 0.1 W / V%, the signal intensity has a certain value. Showed a tendency to show. Therefore, by setting the SDS concentration in the immobilization test solution to 0.1 W / V% or more (final concentration in the immobilization sample of 0.09 W / V% or more), the protein in the sample is immobilized on the PVDF membrane. The conversion rate is considered to be constant.
In addition, looking at the CV value (CV value = standard deviation / average value (%)) indicating the variation in the data, the CV value of any protein is lower than 0.1 W / V% in the SDS concentration in the immobilization reagent. It can be seen that the signal intensity is not stable. In contrast, when the SDS concentration in the immobilization reagent is 0.1 W / V% or higher, the CV value is relatively small, and as described above, the signal intensity value is stable within this concentration range. Yes. This result is considered to indicate the amount of the protein in the sample immobilized on the solid phase membrane. Moreover, although data are not shown, it has confirmed that this result has reproducibility. In general, long-chain alkyl sulfates such as SDS show protein structural disruption even at very low concentrations of 0.0025 W / V%, and the difference in reactivity to protein binding (staining) dyes depends on the degree of structural disruption. (Orsonneau, JL et al., Clin. Chem., 35, 2233-2236 (1989)). Therefore, here too, due to that, when using an immobilization solution whose SDS concentration is lower than 0.1 W / V%, it shows a sudden change such as an increase in signal intensity, and It is presumed that the CV value (<15%) has increased because it is in the process. This also suggests that the immobilization rate of the protein in the sample to the PVDF membrane may fluctuate (is not constant) under SDS concentration conditions lower than 0.1 W / V%.
[0048]
From the above, in FIG. 2, when the absorbance (signal intensity) is stabilized without being affected by the change in SDS concentration, the rate of protein immobilization on the PVDF membrane is not constant for the first time. It was guessed.
Therefore, based on the results shown in FIG. 2, the signal intensity is 100 (reference value) when BSA is used as a protein sample and measurement is performed after immobilization with an immobilization reagent containing 0.1 W / V% SDS. did. Then, each other protein relative to the reference value is used as a protein sample, and 0.1 W / V% SDS, 0.2 W / V% SDS, 0.3 W / V% SDS, or 0.4 W / V% SDS immobilization reagent is used. Similarly, relative values of signal intensities obtained by immobilization and measurement were calculated.
[0049]
Table 1 shows the points until the SDS concentration changes from 0.2 W / V% to 0.4 W / V%, and Table 2 shows the points until the SDS concentration changes from 0.1 W / V% to 0.4 W / V%. The average value of CV values (average CV value (%)), the average value of the relative values of each point, the average value of the absolute deviation between the points (average absolute deviation), and the average absolute deviation divided by the average value The percentage values (variation rate between points (%)) are shown. The inter-point variation rate is a value calculated as the variation rate of the signal intensity accompanying the change in the SDS concentration. The smaller this number, the more constant the signal intensity (measurement result) without being affected by the SDS concentration. It is shown that.
[0050]
[Table 1]

[0051]
[Table 2]

[0052]
As a result, from Table 1, when the SDS concentration is between 0.2 W / V%-0.4 W / V%, for example, fibrinogen, OVA, IgG, and cytochrome c have the most stable variability between points, 1.4-2.7%. Indicated. Moreover, even if it compares with the average CV value (0.8-1.9%) of these proteins, it is inferior result and it has shown that the fluctuation | variation of signal intensity is very small even if SDS concentration changes.
In addition, from Table 2, the three proteins excluding fibrinogen have a point-to-point variation rate of 2.3-3.3% even when the SDS concentration is between 0.1 and 0.4 W / V%, and there is little variation in signal intensity due to the effect of the SDS concentration. I understand that.
For fibrinogen, lysozyme, and BSA, where the SDS concentration is between 0.1-0.4 W / V% and the inter-point variability exceeds 10%, stable results are obtained when the SDS concentration is limited to 0.2-0.4 W / V%. You can see that
From the above, the SDS-to-point variability is stabilized when the SDS concentration is 0.1 W / V% or more, so when the protein is immobilized using the immobilization reagent containing SDS in this concentration range, the sample It can be seen that the amount of protein in the sample can be accurately quantified.
[0053]
Example 3 FIG. Examination of lower alcohol
The protein sample was immobilized and measured when methanol was used as the lower alcohol instead of ethanol.
[Preparation of sample and test solution]
[0054]
(1) Protein sample
BSA, OVA, hemoglobin, IgG, cytochrome c, and lysozyme were weighed and dissolved in purified water to give a 250 μg / mL solution, which was used as a protein sample.
(2) Immobilization reagent
The following immobilization test solution was prepared using purified water.
Immobilization reagent 1: 0.2 W / V% SDS, 2.5 W / V% TCA
Immobilization reagent 2: 0.2 W / V% SDS, 2.5 W / V% TCA, 45% ethanol
Immobilization reagent 3: 0.2 W / V% SDS, 2.5 W / V% TCA, 45% methanol (made by Wako Pure Chemical Industries, Ltd., special grade)
(3) Immobilization sample
A sample prepared by mixing 20 μL of a protein sample (5 μg protein mass) and 300 mL of a predetermined immobilization test solution was prepared as immobilization samples 1, 2, and 3. The final concentration of each reagent in the immobilization sample is SDS 0.19 W / V%, TCA 2.34 W / V%, ethanol 42.2 V / V%, and methanol 42.2 V / V%, respectively.
[Immobilization and measurement of protein]
In the same manner as in Example 1, the protein in each immobilization sample was immobilized on a PVDF membrane, washed and stained, and then the absorbance (signal intensity) at 600 nm was measured with a densitometer.
[0055]
[result]
The results are shown in FIG. In FIG. 3, each bar shows the result when the following immobilization sample is used.

[0056]
As is clear from FIG. 3, when using the immobilization sample prepared using the immobilization reagent containing methanol, the same signal intensity as that obtained using ethanol was obtained, and the protein was applied to the PVDF membrane. It can be seen that the immobilization was possible.
[0057]
Example 4 Investigation of halogenocarboxylic acids
The protein sample was immobilized and measured when trifluoroacetic acid (hereinafter abbreviated as TFA) was used as the halogenocarboxylic acid instead of TCA.
[Preparation of sample and test solution]
(1) Protein sample
BSA, IgG and lysozyme were weighed and dissolved in purified water to give a 250 μg / mL solution, which was used as a protein sample.
(2) Immobilization reagent
The following immobilization test solution was prepared using purified water.
[0058]
Immobilization reagent 1: 0.2 W / V% SDS, 45 V / V% ethanol
Immobilization reagent 2: 0.2 W / V% SDS, 45 V / V% ethanol, 2.5 W / V% TCA
Immobilization reagent 3: 0.2 W / V% SDS, 45 V / V% ethanol, 2.5 W / V% TFA (manufactured by Wako Pure Chemical Industries, Ltd.)
(3) Immobilization sample
A sample prepared by mixing 20 μL of a protein sample (5 μg protein mass) and 300 mL of a predetermined immobilization test solution was prepared as immobilization samples 1, 2, and 3. The final concentration of each reagent in the immobilization sample is SDS 0.19 W / V%, TCA 2.34 W / V%, TFA 2.34 W / V%, and ethanol 42.2 V / V%, respectively.
[Immobilization and measurement of protein]
In the same manner as in Example 1, the protein in each immobilization sample was immobilized on a PVDF membrane, washed and stained, and then the absorbance (signal intensity) at 600 nm was measured with a densitometer.
[0059]
[result]
The results are shown in FIG. In FIG. 4, each bar shows the result when the following immobilization sample is used.

[0060]
As is clear from FIG. 4, when using the immobilization sample prepared using the immobilization test solution containing TFA, a signal intensity equal to or higher than that obtained using TCA was obtained, and the protein It can be seen that the membrane was effectively immobilized on the membrane.
[0061]
Embodiment 5 FIG. Creating a calibration curve
[Preparation of sample and test solution]
(1) Protein sample
A protein sample was prepared by dissolving OVA in purified water at 0 to 20 μg / 20 μL.
(2) Immobilization reagent
A solution prepared by dissolving in purified water so as to be 0.2 V / V% SDS, 2.5 V / V% TCA, and 45 V / V% ethanol was used as an immobilization reagent.
(3) Immobilization sample
A mixture of 20 μL protein sample (protein mass 5 μg) and 300 μL immobilization test solution was prepared and used as an immobilization sample. The final concentration of each reagent in the immobilization sample is SDS 0.19 W / V%, TCA 2.34 W / V%, and ethanol 42.2 V / V%, respectively.
[Immobilization and measurement of protein]
In the same manner as in Example 1, the protein in each immobilization sample was immobilized on a PVDF membrane, washed and stained, and then the absorbance (signal intensity) at 600 nm was measured with a densitometer.
[0062]
[result]
Based on the results, a calibration curve showing the relationship between the protein mass (μg) and the signal intensity was prepared.
The results are shown in FIG. In FIG. 5, the bar of each plot indicates ± 2SD.
The results obtained from the calibration curve were a measurement range of 0.2-20 μg / protein sample, a coincidence coefficient of 0.99 or more, and an average CV of 1.9%.
In addition, linearity was obtained in the range of protein concentration 0.2-5 μg / protein sample. The regression linear equation and the correlation coefficient in this range obtained by statistically processing the measurement results are as follows.
Regression linear equation: y = 0.12x + 0.01
Correlation coefficient (R ² ): 0.99
x: Protein mass
y: signal intensity
As is apparent from FIG. 5, when the OVA was immobilized on the PVDF membrane by the method of the present invention and the protein amount was measured, a calibration curve showing good linearity was obtained, so that according to the immobilization method of the present invention. As a result, it was found that the OVA (protein) concentration can be measured with high accuracy.
In addition, although data is not shown, as a result of measuring similarly about the other protein measured in Example 2, the linear calibration curve was obtained similarly to OVA, and quantitative measurement is also carried out about these proteins. I found that I can do it.
[0063]
Example 6
[Preparation of sample and test solution]
(1) Protein sample
Using purified water, 5 μg / 20 μL solutions of BSA, trypsin inhibitor, fibrinogen, OVA, hemoglobin, IgG, cytochrome c, and lysozyme were prepared as protein samples.
[Measurement of protein by solid phase method]
An immobilization reagent solution containing 0.2 W / V% SDS, 2.5 W / V% TCA, and 45 V / V% ethanol was prepared using purified water. Next, 20 μL of the prepared protein sample and 300 μL of the immobilization test solution were mixed, and the protein in the immobilization sample was immobilized on the PVDF membrane by the same method as in Example 1 using the obtained immobilization sample 320 μL. After washing and staining, the absorbance (signal intensity) at 600 nm was measured with a densitometer. The final concentration of each reagent in the immobilization sample is SDS 0.19 W / V%, TCA 2.34 W / V%, and ethanol 42.2 V / V%, respectively.
[Measurement of protein by liquid phase method]
To 20 μL of the protein sample prepared above, 1 mL Pyromolex solution was added, incubated at room temperature for 20 minutes, and the absorbance at 600 nm was measured.
[0064]
[result]
When BSA is used as a protein sample, a sample for immobilization containing 0.2 W / V% SDS is prepared, and the absorbance (signal intensity) when BSA is immobilized and measured is 1 (reference value) FIG. 6 shows the results of obtaining the relative values of the absorbance obtained by immobilizing and measuring the respective proteins with respect to the reference values in the same manner. Similarly, BSA is used as a protein sample, and when the absorbance by the liquid phase method is 1 (reference value), each protein relative to the reference value is similarly measured by the liquid phase method. The result of obtaining the relative value of the obtained absorbance is also shown in FIG.
In FIG. 6, each bar represents the relative value obtained based on the result of measuring each protein by the following method.

[0065]
As apparent from FIG. 6, in the measurement by the liquid phase method, the relative value of the absorbance with respect to BSA varies greatly depending on the type of protein even when the protein concentration is the same. For example, it is about 0.46 for fibrinogen.
On the other hand, the relative value of the absorbance (signal intensity) of fibrinogen with respect to BSA when measured by the solid phase method of the present invention is 0.75, which shows that the difference in absorbance with BSA is small. . This is also true for most proteins measured.
Further, the relative value of the average absorbance of all the measured proteins with respect to the case where BSA is set to 1 is 0.65 in the liquid phase method, whereas it is 0.94 in the solid phase method of the present invention. It can be seen that the quantitative error due to the protein species was improved. This is because the protein is trapped on the membrane in a denatured state, and a basic amino acid such as cytochrome c or lysozyme, which could not be reacted by the liquid phase method, can be bound to the staining solution. It is considered that the protein was efficiently immobilized with the solution, and a more accurate measurement result was obtained.
[0066]
Example 7
[Sample preparation and immobilization]
Using purified water, IgG samples (protein mass 0-4 μg / 20 μL) and 2% SDS-containing IgG samples (protein mass 0-4 μg / 20 μL) were prepared. Separately, an immobilizing reagent solution containing 0.25 W / V% SDS, 2.5 W / V% TCA and 45 V / V% ethanol was prepared using purified water. Next, 20 μL of the protein sample and 300 μL of the immobilization test solution were mixed, and the protein in the immobilization sample was immobilized on the PVDF membrane and washed in the same manner as in Example 1 using the obtained 320 μL of the immobilization sample. After staining, the absorbance (signal intensity) at 600 nm was measured with a densitometer.
The final concentration of each reagent in the immobilization sample obtained using the IgG sample not containing SDS is SDS 0.23 W / V%, TCA 2.34 W / V%, and ethanol 42.2 V / V%, respectively. . The final concentration of each reagent in the immobilization sample obtained using the 2% SDS-containing IgG sample is SDS 0.36 W / V%, TCA 2.34 W / V%, and ethanol 42.2 V / V%, respectively. .
[0067]
[result]
Based on the results obtained, a calibration curve showing the relationship between protein mass (μg) and signal intensity was prepared for each of the cases where an IgG sample containing no SDS and an IgG sample containing 2% SDS were used. did.
The results are shown in FIG. In FIG. 7,-♦-indicates the results when using an IgG sample not containing SDS, and -Δ- indicates the results when using an IgG sample containing SDS. The bar of each plot indicates ± SD.
As is clear from FIG. 7, both calibration curves almost coincided.
From this result, if the protein is immobilized by the immobilization method of the present invention, even if SDS is present in the protein sample, the target protein is immobilized on the solid phase without being affected by it. It can be seen that quantification can be performed.
[0068]
Example 8 FIG.
The protein according to the present invention was immobilized and measured using various protein samples containing SDS, and the influence of SDS on the measurement was compared with that measured by the liquid phase method.
[Preparation of sample and test solution]
(1) Protein sample
Using purified water, a protein sample containing 0 W / V% SDS (control), 0.2 W / V% SDS or 2 W / V% SDS (BSA, trypsin inhibitor, fibrinogen, hemoglobin, OVA, cytochrome c, lysozyme, IgG 250 μg / mL of trypsin (manufactured by Wako Pure Chemical Industries, Ltd.) was prepared.
[Measurement of protein by immobilization method]
An immobilization test solution containing 0.1 W / V% SDS, 2.5 W / V% TCA, and 45 V / V% ethanol was prepared using purified water. Next, 20 μL of the prepared protein sample and 300 μL of the immobilization test solution are mixed, and 320 μL of the obtained immobilization sample is immobilized, washed and stained in the same manner as in Example 1, and the absorbance at 600 nm is measured with a densitometer. (Signal intensity) was measured. [Measurement by liquid phase method]
To 20 μL of the protein sample prepared above, 1 mL Pyromolex solution (Protein Assay Rapid Kit wako, manufactured by Wako Pure Chemical Industries, Ltd.) was added, incubated at room temperature for 20 minutes, and the absorbance at 600 nm was measured.
[0069]
[result]
The results obtained are expressed as relative values with the results obtained using the respective controls (samples not containing SDS) as 100, and are summarized in Table 3.
[0070]
[Table 3]

[0071]
As is apparent from Table 3, when the protein was measured by the solid phase method according to the present invention, the measurement results within ± 20% of the control were obtained for the 2W / V% SDS-containing sample for most proteins. It can be seen that the influence of SDS in protein samples on protein measurement could be avoided.
On the other hand, when measurement was performed by a liquid phase method using a sample containing 2 W / V% SDS, measurement could not be performed at all.
From the above results, it can be seen that the protein immobilization method and quantification method of the present invention can be applied to all proteins. In addition, it became clear that the protein quantification inhibitors known so far, in particular, the protein quantification in SDS-containing samples that are widely used as protein solubilizers became possible, which proved very useful.
[0072]
Example 9
The influence of the surfactant contained in the sample on the immobilization method and the protein measurement using the immobilization reagent of the present invention was examined.
[Immobilization by solid-phase method and measurement of protein]
(1) Measurement of proteins in BSA or IgG-containing protein samples
BSA samples or IgG samples (protein mass: 4 μg / 20 μL) containing each surfactant were prepared with purified water so as to have the concentrations shown in Table 4, and used as protein samples.
Separately, an immobilization reagent solution containing 0.1 W / V% SDS, 2.5 W / V% TCA, and 45 V / V% ethanol was prepared with purified water.
Next, 20 μL of the protein sample and 300 μL of the immobilization test solution are mixed to prepare an immobilization sample. Using the 320 μL, the protein in the immobilization sample is immobilized on the PVDF membrane in the same manner as in Example 1. After washing and staining, the absorbance (signal intensity) at 600 nm was measured.
The final concentration of each reagent in the immobilization sample is SDS 0.094 W / V%, TCA 23.4 W / V%, ethanol 42.2 V / V% when a protein sample not containing SDS is used. The final concentration of each reagent when using an SDS-containing protein sample is the same as that when using a protein sample containing no SDS, but the final concentration of TCA and ethanol is 1% SDS. When using a protein sample containing 0.16 W / V%, when using a sample containing 2% SDS, 0.21 W / V%, and when using a sample containing 4% SDS, it is 0.34 W / V%.
[0073]
(2) Measurement of protein in OVA-containing protein samples
OVA samples (protein mass 5 μg / 20 μL) containing each surfactant were prepared so as to have the concentrations shown in Table 4, and used as protein samples.
Separately, prepare an immobilization test solution containing 0.2 W / V% SDS, 2.5 W / V% TCA, 45 V / V% ethanol in purified water, and prepare the immobilization sample in the same manner as in (1) above. The sample was prepared, immobilized on a PVDF membrane in the same manner as in Example 1, washed and stained, and the absorbance (signal intensity) at 600 nm was measured.
The final concentration of each reagent in the immobilization sample is SDS 0.19 W / V%, TCA 23.4 W / V%, ethanol 42.2 V / V% when a protein sample not containing SDS is used. The final concentration of each reagent when using a 2% SDS-containing protein sample is SDS 0.31 W / V%, TCA 23.4%, ethanol 42.2 W / V%.
Further, a BSA sample, an IgG sample, and an OVA sample prepared in the same manner as above except that the surfactant (inhibitory substance) was not contained were immobilized and measured in the same manner as a control.
[Measurement of protein by liquid phase method]
Measurement was performed in the same manner as in Example 6 using a 1 mL Pyromolex solution prepared by preparing BSA samples (10 μg / 20 μL) containing each surfactant so as to have the concentrations shown in Table 4.
In addition, a BSA sample prepared in the same manner as described above except that it did not contain a surfactant (inhibitory substance) was similarly measured by the liquid phase method and used as a control.
[0074]
[result]
Each measurement was performed three times, and the average value of the obtained absorbance was determined. . Taking the average value obtained using the control as 100, the relative value (%) of the average value obtained using the surfactant-containing sample was obtained, and the relative displacement (CV) was also calculated. Table 4 shows.
In Table 4, the concentration of the surfactant (inhibitory substance) indicates the concentration in the protein sample. In the liquid phase method data, the value on the left is the concentration of the surfactant, and the value on the right is the relative value (%) ± CV of the average measured value when the sample containing the surfactant is used relative to the control. Indicates.
The data shown in Table 4 is the data at the maximum allowable concentration of the surfactant, that is, when the surfactant is used as the additive, the average value is ± 20 compared to the control (no additive). The data when% results are obtained is shown.
[0075]
[Table 4]

[0076]
SLS: Sodium N-lauroyl sarcosinate
Triton X-100 (Rohm and Haas brand name): Polyoxyethylene (10) octylphenyl ether
NP-40 (Nippon Emulsion Co., Ltd. trade name): Polyoxyethylene (9) Octylphenyl ether
Tween 20 (trade name of Kao Corporation): polyoxyethylene sorbitan monolaurate
Tween 80 (trade name of Kao Corporation): polyoxyethylene sorbitan monooleate
Brij 35 (trade name of ICI): Polyoxyethylene lauryl ether
CHAPS: 3-[(3-Colamidopropyl) dimethylammoniopropanesulfonic acid]
CTAB: Cetyltrimethylammonium bromide
[0077]
As is apparent from Table 4, when the protein is immobilized and measured by the method of the present invention, the measurement of the protein can be carried out without being affected by the presence of a surfactant generally used at the time of preparation of the protein sample. It turns out that it is possible. In particular, as compared with the liquid phase method, it can be seen that measurement is possible even when a surfactant having a concentration 10 times or more that of the liquid phase method coexists as an additive in the protein sample.
From the above, the protein immobilization method of the present invention can solve the problem caused by surfactants that have been widely used as additives, that is, the problem of inhibiting protein quantification. I understand.
[0078]
Example 10 Immunoblotting
[Preparation of sample and test solution]
(1) Protein sample
Mouse IgG (Wako Pure Chemical Industries, Ltd.) was weighed and dissolved in purified water to give a 0 to 200 μg / mL solution, which was used as a protein sample.
(2) Immobilization reagent
An immobilization reagent solution containing 0.2 W / V% SDS, 2.5 W / V% TCA, and 45 V / V% ethanol was prepared using purified water.
(3) Immobilization sample
20 μL of protein samples of each concentration prepared above and 300 μL of immobilization reagent (SDS final concentration 0.19 W / V%, TCA final concentration 2.34 W / V%, ethanol final concentration 42.2 V / V%) Was prepared as a sample for immobilization.
(4) Blocking solution
Block Ace (manufactured by Snow Brand Milk Products Co., Ltd.) diluted with PBS (pH 7.4) to a final concentration of 25% was used.
(5) Antibody solution
Luminescence detection antibody solution: A horseradish peroxidase-labeled anti-mouse IgG antibody (manufactured by Amersham Biosciences) diluted 1/10000 with a blocking solution was used.
Color detection antibody solution: Alkaline phosphatase-labeled anti-mouse IgG antibody (manufactured by Wako Pure Chemical Industries, Ltd.) diluted 1/1000 with a blocking solution was used.
(6) Cleaning solution
Tween 20 diluted with PBS (pH 7.4) to a final concentration of 0.05% was used.
(7) Detection reagent
For luminescence detection: ECL Plus Western Blotting Starter Kit (manufactured by Amersham Biosciences)
For color detection: 0.033% nitro blue tetrazolium (NBT, manufactured by Wako Pure Chemical Industries, Ltd.), 0.0165% 5-bromo-4-chloro-3-indolyl phosphate (BCIP, manufactured by Wako Pure Chemical Industries, Ltd.) / 100mM Tris-HCl pH9.5 (100mM NaCl, 5mM MgCl ₂ Contains)
[0079]
[Immobilization and measurement of protein]
In the same manner as in Example 1, the immobilization sample prepared as described above was applied to a PVDF membrane, filtered with suction, 300 μL of PBS (pH 7.4) was applied, and suction filtration was performed in the same manner. The PVDF membrane was taken out, immersed in a blocking solution, and incubated for 1 hour at room temperature while rotating (blocking operation). Thereafter, the sample was immersed in an antibody solution for luminescence detection or an antibody solution for color development, and incubated for 1 hour at room temperature while rotating (antibody reaction). The membrane after the antibody reaction was washed 5 times with a washing solution, and then immersed in a luminescence detection reagent or a color development detection reagent to carry out a detection reaction. Luminescence was detected by exposing the PVDF film to an X-ray film (manufactured by Amersham Bioscience) after luminescence detection processing.
[0080]
[result]
The results are shown in FIG. In FIG. 8, A is a detection of a protein sample immobilized on a PVDF membrane by immunodetection by a luminescence reaction and exposure to an X-ray film. B shows the detection by immunodetection of the protein sample immobilized on the PVDF membrane by a color reaction. Each dot indicates the result when the protein sample is detected after immobilization as the amount of each protein.
As is clear from FIG. 8, mouse IgG immobilized on the membrane could be detected in both cases where luminescence detection and color development were detected by immunoblotting. The detection limit for luminescence detection was 0.0625 μg, and the detection limit for color development detection was 0.5 μg. Therefore, it was found that high-sensitivity immunodetection (detection by immunoblotting) can be performed by immobilization by the protein immobilization method of the present invention.
[0081]
【The invention's effect】
According to the protein immobilization method of the present invention, the protein can be immobilized on the solid phase more sufficiently than the conventional immobilization method. In addition, it is possible to quantify proteins that could not be accurately performed by conventional immobilization methods. Furthermore, if the protein immobilization method according to the present invention is used, there is an effect that the sensitivity upon immunoblotting is also increased.
[Brief description of the drawings]
FIG. 1 shows the absorbance (signal intensity) at 600 nm after staining a polyvinylidene difluoride membrane (PVDF membrane) obtained by immobilizing proteins in each immobilization sample obtained in Example 1 with a Pyromolex test solution. The measurement results are shown.
FIG. 2 shows the results of measuring the absorbance (signal intensity) at 600 nm after staining the PVDF membrane on which the protein in each immobilization sample obtained in Example 2 was immobilized with Pyromolex test solution.
FIG. 3 shows the results of measuring the absorbance (signal intensity) at 600 nm after staining the PVDF membrane on which the protein in each immobilization sample obtained in Example 3 was immobilized with Pyromolex test solution. Show.
FIG. 4 shows the results of measuring the absorbance (signal intensity) at 600 nm after staining the PVDF membrane on which the protein in each immobilization sample obtained in Example 4 was immobilized with Pyromolex test solution. Show.
5 shows a calibration curve obtained by immobilizing and quantifying a PVDF membrane using OVA as a protein sample obtained in Example 5. FIG.
FIG. 6 shows the relative values obtained in Example 6 based on the results of measuring proteins by the solid phase method or the results of measuring proteins by the liquid phase method.
FIG. 7 is a calibration curve obtained by performing immobilization and protein quantification according to the present invention using the IgG sample not containing SDS or the IgG sample containing SDS as a protein sample obtained in Example 7. Indicates.
FIG. 8 shows the result of immunoblotting obtained in Example 10, where A is a protein sample immobilized on a PVDF membrane, immunodetected by a luminescent reaction, exposed to an X-ray film and detected. is there. B shows the detection by immunodetection of the protein sample immobilized on the PVDF membrane by a color reaction.
[Explanation of symbols]
In FIG. 1, each bar shows the result when the following immobilization sample is used.

In FIG. 2,-△-is lysosome, --- is cytochrome c,-○-is IgG,-◎-is fibrinogen,-●-is BSA,-◆-is OVA,-◇-is trypsin inhibitor. The results when using the contained protein samples are shown.
In FIG. 3, each bar shows the result when the following immobilization sample is used.

In FIG. 4, each bar shows the result when the following immobilization sample is used.

In FIG. 6, each bar shows the relative value obtained based on the result of measuring each protein by the following method.

In FIG. 7,-♦-indicates the result when a protein sample not containing SDS is used, and -Δ- indicates the result when a protein sample containing SDS is used.

Claims (20)

A method for immobilizing a protein on the solid phase, comprising contacting the protein with a solid phase having a hydrophobic surface in the presence of a lower alcohol and a halogenocarboxylic acid. The immobilization method according to claim 1, wherein the lower alcohol is ethanol or methanol. The immobilization method according to claim 1 or 2, wherein the halogenocarboxylic acid is trichloroacetic acid (hereinafter abbreviated as TCA) or trifluoroacetic acid (hereinafter abbreviated as TFA). The immobilization method according to any one of claims 1 to 3, wherein a lower alcohol concentration in contacting the protein with a solid phase having a hydrophobic surface is 30 to 50 V / V%. The immobilization method according to any one of claims 1 to 4, wherein the concentration of halogenocarboxylic acid when the protein is brought into contact with a solid phase having a hydrophobic surface is 0.08 to 10 W / V%. Furthermore, the immobilization method according to claim 1, wherein the protein is brought into contact with a solid phase having a hydrophobic surface in the presence of a long-chain alkyl sulfate. The immobilization method according to claim 6, wherein the long-chain alkyl sulfate is sodium dodecyl sulfate (hereinafter abbreviated as SDS). The immobilization method according to claim 6 or 7, wherein the concentration of the long-chain alkyl sulfate when the protein is brought into contact with the solid phase having a hydrophobic surface is 0.1 to 1 W / V%. The immobilization method according to claim 1, wherein the solid phase is a hydrophobic membrane. A method for quantifying a protein, characterized in that a protein staining solution is brought into contact with a solid phase on which a protein is immobilized by the method according to any one of claims 1 to 9, and the coloration produced thereby is performed. An immunoblotting method using a solid phase on which a protein is immobilized by the method according to claim 1. A test solution for immobilizing a protein on a solid phase having a hydrophobic surface, comprising a lower alcohol and a halogenocarboxylic acid. The reagent solution according to claim 12, wherein the lower alcohol is ethanol or methanol. The reagent solution according to claim 12 or 13, wherein the halogenocarboxylic acid is TCA or TFA. The test solution according to any one of claims 12 to 14, wherein the lower alcohol concentration is 30 to 50 V / V%. The reagent solution according to any one of claims 12 to 15, wherein the halogenocarboxylic acid concentration is 0.1 to 10 W / V%. Furthermore, the test liquid in any one of Claims 12-16 containing a long-chain alkyl sulfate. The test solution according to claim 17, wherein the long-chain alkyl sulfate is SDS. The test solution according to claim 17 or 18, wherein the long-chain alkyl sulfate concentration is 0.1 to 1 W / V%. The reagent solution according to any one of claims 11 to 19, wherein the solid phase is a hydrophobic membrane.

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