JP2011047846A - Diagnostic method for prostate cancer - Google Patents
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本発明は、正確かつ簡便に前立腺癌の罹患可能性及び悪性度が判定できる前立腺癌の診断法に関する。 The present invention relates to a method for diagnosing prostate cancer that can accurately and easily determine the morbidity and malignancy of prostate cancer.
前立腺癌は、６５歳以上で罹患率が増加する男性特有の癌である。前立腺癌の死亡率は高くないが、進行すると骨に転移しやすい。前立腺癌の治療法としては、手術療法、放射線療法、内分泌療法等が主に行なわれている。 Prostate cancer is a cancer specific to men whose morbidity increases after age 65 years. Prostate cancer mortality is not high, but as it progresses, it tends to metastasize to bone. As treatment methods for prostate cancer, surgical treatment, radiation therapy, endocrine therapy and the like are mainly performed.
従来、前立腺癌の診断は直腸診、経直腸的前立腺超音波検査、生検が主であったが、最近前立腺特異抗原（prostate specific antigen：ＰＳＡ）の検出が行なわれるようになった。この血中ＰＳＡ値を用いた診断法の開発により、前立腺癌の検出率は増加し、早期診断が可能になった。しかしＰＳＡ検査値には、いわゆるグレーゾーンが多く、あくまで前立腺癌を発見するきっかけとなる指標にすぎないといわれている。 Conventionally, prostate cancer has been diagnosed mainly by rectal examination, transrectal ultrasonography, and biopsy, but recently, prostate specific antigen (PSA) has been detected. With the development of a diagnostic method using this blood PSA level, the detection rate of prostate cancer has increased, and early diagnosis has become possible. However, there are many so-called gray zones in the PSA test values, and it is said that the PSA test values are only an index that triggers the discovery of prostate cancer.
かかる観点から、さらに感度の高い前立腺癌マーカーが求められており、ハプトグロビン糖鎖構造の多岐化を検出する手段としてＰＨＡ−Ｌ4レクチンを用いる方法が報告されている（非特許文献１及び２）。また、ハプトグロビンβ鎖をＲＭ２抗体を用いて検出する手段も報告されている（特許文献１）。 From such a viewpoint, a prostate cancer marker with higher sensitivity has been demanded, and a method using PHA-L 4 lectin as a means for detecting diversification of haptoglobin sugar chain structures has been reported (Non-patent Documents 1 and 2). . A means for detecting haptoglobin β chain using RM2 antibody has also been reported (Patent Document 1).
しかしながら、ウエスタンブロット等の解析による従来のハプトグロビン糖鎖をマーカーとする前立腺癌の診断法では、感度が低く、また良性と悪性患者及び健常人との判別が困難であり、さらに高感度に再現よく前立腺癌を判別できる診断法が望まれていた。
従って、本発明は、キャリアタンパク質を修飾せずに高感度で分析し、正確かつ再現性の良い前立腺癌の診断法を提供することにある。
However, conventional methods for diagnosing prostate cancer using haptoglobin sugar chains as markers by analysis such as Western blotting have low sensitivity, and it is difficult to discriminate between benign and malignant patients and healthy individuals. A diagnostic method that can discriminate prostate cancer has been desired.
Accordingly, it is an object of the present invention to provide a method for accurately and reproducibly diagnosing prostate cancer by analyzing with high sensitivity without modifying a carrier protein.
そこで本発明者は、血液中のハプトグロビンの糖鎖構造変化に関し、β鎖やＰＨＡレクチン以外の糖鎖について種々検討した結果、ハプトグロビン糖鎖Ｎｅｕ α２−６量を特異的に検出すれば、前立腺癌の良性と悪性とが明確に判別できることを見出し、本発明を完成した。さらに、血液中のハプトグロビン糖鎖構造変化を解析するために、ハプトグロビンの抗体でそのキャリアタンパク質を捕獲し(第一段)、その糖鎖とある種のレクチン反応(第二段)を検出する表面プラズモン共鳴の多段階分析法を確立した。その結果、血液中のハプトグロビンとその修飾糖鎖に存在するＮｅｕ α２−６の量をほぼ同時に測定し、その比をもって前立腺癌の良性と悪性が明確に判別できることを見出した。 Therefore, the present inventor has made various studies on sugar chains other than β chain and PHA lectin with respect to changes in the sugar chain structure of haptoglobin in blood. As a result, if the amount of haptoglobin sugar chain Neu α2-6 is specifically detected, prostate cancer The present invention was completed by discovering that benign and malignant can be clearly distinguished. Furthermore, in order to analyze haptoglobin sugar chain structural changes in blood, the surface captures the carrier protein with a haptoglobin antibody (first stage) and detects some lectin reaction with the sugar chain (second stage). A multi-step analysis method of plasmon resonance was established. As a result, the amount of Neu α2-6 present in blood haptoglobin and its modified sugar chain was measured almost simultaneously, and it was found that the benign and malignant of prostate cancer could be clearly discriminated with the ratio.
すなわち、本発明は、被検者から単離した血液試料におけるハプトグロビン糖鎖Ｎｅｕ α２−６量を指標とする、前立腺癌の判定方法を提供するものである。 That is, the present invention provides a method for determining prostate cancer using the amount of haptoglobin sugar chain Neu α2-6 in a blood sample isolated from a subject as an index.
本発明方法によれば、悪性の前立腺癌が、正常者及び良性患者との間で有意差をもって判別できる。特に、検出手段として表面プラズモン共鳴法を採用した場合に、検出感度が高く、再現性も良好である。特に、抗ハプトグロビン抗体に結合したハプトグロビン量と、ハプトグロビン糖鎖Ｎｅｕ α２−６に結合したレクチン量とを、表面プラズモン共鳴法による多段階法で測定すれば、ハプトグロビン量とレクチン量とが同時に測定でき、高感度かつ再現性をさらに向上する。本発明方法とＰＳＡとを併用すれば、より高感度かつ正確に前立腺癌の診断が可能となる。 According to the method of the present invention, malignant prostate cancer can be discriminated with a significant difference between normal and benign patients. In particular, when the surface plasmon resonance method is adopted as the detection means, the detection sensitivity is high and the reproducibility is also good. In particular, if the amount of haptoglobin bound to the anti-haptoglobin antibody and the amount of lectin bound to the haptoglobin sugar chain Neu α2-6 are measured by a multi-step method using the surface plasmon resonance method, the amount of haptoglobin and the amount of lectin can be measured simultaneously. High sensitivity and reproducibility are further improved. If the method of the present invention and PSA are used in combination, prostate cancer can be diagnosed with higher sensitivity and accuracy.
本発明に用いる試料は、被検者から単離した血液試料である。被検者には、前立腺癌が疑われる者及び前立腺癌患者が含まれる。前立腺癌が疑われる者には、健常者も含まれる。また前立腺癌患者には、治療中の患者及び以前に治療した患者が含まれる。 The sample used in the present invention is a blood sample isolated from a subject. Subjects include those suspected of having prostate cancer and prostate cancer patients. Those suspected of having prostate cancer include healthy individuals. Prostate cancer patients also include those who are being treated and those who have been previously treated.
血液試料としては、ハプトグロビンを含む試料であればよく、全血、血漿、血清が含まれるが、血漿又は血清が好ましい。 The blood sample may be any sample containing haptoglobin and includes whole blood, plasma, and serum, with plasma or serum being preferred.
本発明の前立腺癌の判定方法は、血液中のハプトグロビン糖鎖Ｎｅｕ α２−６量を測定し、これを指標とするものである。ハプトグロビン糖鎖Ｎｅｕ α２−６量の測定としては、当該糖鎖を認識する抗体を用いる方法、当該糖鎖と特異的に結合するレクチンを用いる方法が挙げられるが、当該糖鎖と特異的に結合するレクチンを用いるのが好ましい。後述の実施例に示すように、従来用いられているハプトグロビン糖鎖構造の多岐化の検出のうち、ＰＨＡ−Ｌ4レクチンを用いる方法に比べて、Ｎｅｕ α２−６を検出する本発明方法は顕著に高感度である。 The method for determining prostate cancer of the present invention measures the amount of haptoglobin sugar chain Neu α2-6 in blood and uses this as an index. Examples of the measurement of the amount of haptoglobin sugar chain Neu α2-6 include a method using an antibody that recognizes the sugar chain and a method using a lectin that specifically binds to the sugar chain. Preferably, a lectin is used. As shown in the examples described later, among the conventionally used detection of diversification of haptoglobin sugar chain structures, the method of the present invention for detecting Neu α2-6 is remarkable compared to the method using PHA-L 4 lectin. High sensitivity.
Ｎｅｕ α２−６と特異的に結合するレクチンとしては、セイヨウニワトコ（Sambucus nigra(elderberry)）由来レクチン（ＳＮＡ）、ニワトコ（Sambucus sieboldiana(Japanese elderberry)）由来レクチン（ＳＳＡ）、及びキカラスウリ（Tricosanthes japonica）由来レクチン（ＴＪＡ−１）が挙げられるが、レクチンＳＮＡ及びレクチンＳＳＡが特に好ましい。 The lectins that specifically bind to Neu α2-6 include lectin (SNA) derived from elderberry (Sambucus nigra (elderberry)), lectin (SSA) derived from elderberry (Sambucus sieboldiana (Japanese elderberry)), and Tricosanthes japonica. ) Derived lectin (TJA-1), lectin SNA and lectin SSA are particularly preferred.
本発明方法においては、血液試料に抗ハプトグロビン抗体を反応させ、得られたハプトグロビン−抗ハプトグロビン複合体にレクチンを反応させてハプトグロビン糖鎖Ｎｅｕ α２−６に結合したレクチン量を測定するのが、簡便かつ高感度であり、より好ましい。 In the method of the present invention, it is easy to measure the amount of lectin bound to the haptoglobin sugar chain Neu α2-6 by reacting a blood sample with an anti-haptoglobin antibody and reacting the obtained haptoglobin-anti-haptoglobin complex with lectin. And it is highly sensitive and more preferable.
前記反応に用いられる抗ハプトグロビン抗体としては、市販の抗ハプトグロビン抗体であればよく、ポリクローナル抗体でも、モノクローナル抗体でもよい。この抗原抗体反応は、抗ハプトグロビン抗体を固定化した担体に血液試料を添加して行なうのが好ましい。抗原抗体反応により、血液試料中のハプトグロビン担体上に捕獲される。
次いで、前記糖鎖に特異的なレクチンを、前記担体上のハプトグロビンと反応させれば、糖鎖に特異的なレクチンが担体上のハプトグロビンに結合する。従って、結合したレクチン量を測定すれば、血液試料中のハプトグロビン糖鎖Ｎｅｕ α２−６量が定量できる。
The anti-haptoglobin antibody used in the reaction may be a commercially available anti-haptoglobin antibody, and may be a polyclonal antibody or a monoclonal antibody. This antigen-antibody reaction is preferably performed by adding a blood sample to a carrier on which an anti-haptoglobin antibody is immobilized. By the antigen-antibody reaction, it is captured on a haptoglobin carrier in the blood sample.
Next, when the lectin specific for the sugar chain is reacted with haptoglobin on the carrier, the lectin specific for the sugar chain binds to haptoglobin on the carrier. Therefore, by measuring the amount of bound lectin, the amount of haptoglobin sugar chain Neu α2-6 in the blood sample can be quantified.
ここで上記の反応は、マイクロセンサーチップ上で行なうのが好ましい。すなわち、抗ハプトグロビン抗体を固定化したセンサーチップに血液試料をミクロフローで流して抗原抗体反応させ、次いで前記レクチンをミクロフローで流してハプトグロビン糖鎖Ｎｅｕ α２−６にレクチンを結合させる。マイクロセンサーチップとしては、金膜にカルボキシル基を固定化したＣ１（ビアコア社）、或いは、金膜にポリエチレングリコールの先端にカルボキシル基を有するＳＡＭ（Self-assembled Monolayer）を固定化し使用する。 Here, the above reaction is preferably performed on a microsensor chip. That is, a blood sample is flowed through a microchip on a sensor chip on which an anti-haptoglobin antibody is immobilized to cause an antigen-antibody reaction, and then the lectin is flowed through the microflow to bind the lectin to the haptoglobin sugar chain Neu α2-6. As the microsensor chip, C1 (Biacore) having a carboxyl group immobilized on a gold film, or SAM (Self-assembled Monolayer) having a carboxyl group at the tip of polyethylene glycol is immobilized on the gold film.
ハプトグロビン糖鎖Ｎｅｕ α２−６に結合したレクチンの測定は、表面プラズモン共鳴法（ＳＰＲ法）、レクチンアレー等により行なうことができるが、簡便性、再現性、感度の点からＳＰＲ法が特に好ましい。ＳＰＲは金属と誘電体媒質との界面で生じる光学現象であり、金属表面上の薄い被検体層の密度や屈折率の変化に対して敏感である。従って、ＳＰＲバイオセンサは、センサ表面におけるリガンドと被検体との相互作用に起因する屈折率の変化又はＳＰＲシグナルのシフトを測定することができる。ＳＰＲ法は、市販のＳＰＲ装置を用いて行なえばよい。 The lectin bound to the haptoglobin sugar chain Neu α2-6 can be measured by a surface plasmon resonance method (SPR method), a lectin array or the like, but the SPR method is particularly preferred from the viewpoint of simplicity, reproducibility and sensitivity. SPR is an optical phenomenon that occurs at the interface between a metal and a dielectric medium, and is sensitive to changes in the density and refractive index of a thin specimen layer on the metal surface. Therefore, the SPR biosensor can measure a change in refractive index or SPR signal shift due to the interaction between the ligand and the analyte on the sensor surface. The SPR method may be performed using a commercially available SPR device.
ここでＳＰＲを用いれば、抗ハプトグロビン抗体に結合したハプトグロビン量と、ハプトグロビン糖鎖Ｎｅｕ α２−６に結合したレクチン量とが、多段階分析により同時に測定可能である。すなわちＳＰＲを用いた多段階分析法によれば、ハプトグロビン量とレクチン量とが正確かつ同時に測定できるため特に好ましい。 If SPR is used here, the amount of haptoglobin bound to the anti-haptoglobin antibody and the amount of lectin bound to the haptoglobin sugar chain Neu α2-6 can be measured simultaneously by multi-step analysis. That is, the multistage analysis method using SPR is particularly preferable because the amount of haptoglobin and the amount of lectin can be measured accurately and simultaneously.
前記抗原抗体反応は、通常２０〜４０℃で、１〜１０分時間行なえばよい。また、糖鎖−レクチン結合反応は、通常２０〜４０℃で、１〜１０分時間行なえばよい。 The antigen-antibody reaction may be usually performed at 20 to 40 ° C. for 1 to 10 minutes. Moreover, what is necessary is just to perform sugar chain- lectin binding reaction normally at 20-40 degreeC for 1 to 10 minutes.
得られたＮｅｕ α２−６量を、健常者、良性患者、悪性患者、治療患者等で対比することにより前立腺癌の罹患可能性及び／又は悪性度を判定してもよいが、Ｎｅｕ α２−６量とハプトグロビン量との比を指標として判定するのがより好ましい。判定は、予め作成しておいた健常者、良性患者及び悪性患者のＮｅｕ α２−６量又はＮｅｕ α２−６とハプトグロビン量との比と、被検者のそれらとを対比することにより行なうのが好ましい。ここで良性患者には、前立腺肥大症患者が含まれる。 By comparing the obtained amount of Neu α2-6 with healthy subjects, benign patients, malignant patients, treated patients, etc., the morbidity and / or malignancy of prostate cancer may be determined. It is more preferable to determine the ratio between the amount and the haptoglobin amount as an index. The determination is performed by comparing the ratio of Neu α2-6 or Neu α2-6 and haptoglobin in healthy subjects, benign patients, and malignant patients prepared in advance with those of the subject. preferable. Here, benign patients include patients with benign prostatic hyperplasia.
また、本発明の判定方法に加えて、ＰＳＡとを併用すれば、より確実な前立腺の判定が可能となる。 In addition to the determination method of the present invention, if PSA is used in combination, the prostate can be determined more reliably.
Ａ．方法
（１）血清試料採取
順天堂医院泌尿器科外来を受診し、インフォームドコンセント後、同意書を提出した患者より血液を５ｍＬ採取した。採血後、数時間以内に研究基盤センター・生体分子研究部門において日立遠心分離器ＣＲ２２Ｅを用いて血清分離した（３０００ｒｐｍ，１５ｍｉｎ）。血清は１０本のエッペンドルフチューブに約０．３ｍＬずつ分注し、−８０℃で保存した。コントロールである健常人血清は学内掲示により募集したボランティアに対してインフォームドコンセント後、同意書の提出を求め、患者と同様に試料調製した。
A. Method (1) Collection of serum sample Visited Juntendo Clinic, Department of Urology, and after informed consent, 5 mL of blood was collected from the patient who submitted the consent form. Within several hours after blood collection, serum was separated using a Hitachi centrifuge CR22E (3000 rpm, 15 min) at the Research Infrastructure Center / Biomolecular Research Department. Serum was dispensed at approximately 0.3 mL into 10 Eppendorf tubes and stored at −80 ° C. The healthy human serum, which was a control, was asked to submit a consent form after informed consent to volunteers recruited by posting on campus, and samples were prepared in the same manner as patients.
（２）ＰＳＡ検査及び病理組織検査
患者血清に含まれるＴｏｔａｌ ＰＳＡ値（ｐｒｏｓｔａｔｅ ｓｐｅｃｉｆｉｃ ａｎｔｉｇｅｎ）は順天堂医院臨床検査部でＡｂｂｏｔ ＬａｂｏｒａｔｏｒｉｅｓのＡｘＳＹＭキットにより測定した。ボランティア血清も同部内で同様の検査を実施した。
また、前立腺の病態組織は病理診断部により検査・診断した。前立腺癌（Ｐｒｏｓｔａｔｅ Ｃａｎｃｅｒ）群においては悪性度をＧｌｅａｓｏｎ Ｓｃｏｒｅで表示した。また、組織過形成（Ｈｙｐｅｒｐｌａｓｉａ）が観察された良性組織（Ｂｅｎｉｇｎ Ｐｒｏｓｔａｔｉｃ Ｔｉｓｓｕｅ）は非癌群として分類した。後者には前立腺肥大（ＢＰＨ）が含まれた。健常人のボランティア群（Ｃｏｎｔｒｏｌ）を癌群（Ｃａｎｃｅｒ）、非癌群（Ｂｅｎｉｇｎ）との対照に用いた。
(2) PSA test and histopathological examination Total PSA value (prostate specific antigen) contained in patient serum was measured by AbbSY Laboratories' AxSYM kit at Juntendo Clinic's clinical laboratory. Volunteer serum was similarly tested in the same department.
The pathologic tissue of the prostate was examined and diagnosed by the pathological diagnosis department. In the prostate cancer (Prostate Cancer) group, the grade of malignancy was displayed as Gleason Score. Further, benign tissue (Benign Prosthetic Tissue) in which hyperplasia was observed was classified as a non-cancer group. The latter included benign prostatic hyperplasia (BPH). A group of healthy volunteers (Control) was used as a control for a cancer group (Cancer) and a non-cancer group (Benign).
（３）ＳＰＲ分析
（ｉ）センサーチップへのアミンカップリング法によるリガンド固定化
抗ヒトハプトグロビン抗体（ウサギ、ポリクローナル）をリガンドとしてセンサーチップＣ１にアミンカップリング法を用いて固定化した。ランニングバッファーにはＨＢＳ−Ｐ ｂｕｆｆｅｒ（１０ｍＭ ＨＥＰＥＳ ｐＨ７．４，０．１５Ｍ ＮａＣｌ，０．００５％ ｓｕｒｆａｃｔａｎｔ Ｐ−２０）を用い、流速は５μｌ／ｍｉｎとした。チップＣ１表面のカルボキシル基の活性化にＮＨＳ（Ｎ−ｈｙｄｒｏｘｙｓｕｃｃｉｎｉｍｉｄｅ）−ＥＤＣ（１−ｅｔｈｙｌ−３−（３−ｄｉｍｅｔｈｙｌａｍｉｎｏｐｒｏｐｙｌ）ｃａｒｂｏｄｉｉｍｉｄｅ ｈｙｄｒｏｃｈｌｏｒｉｄｅ）混合液を７分間（３５μｌ）インジェクションし、次いで抗ハプトグロビン抗体（２００μｇ／ｍＬ，１０ｍＭ 酢酸バッファーｐＨ５．５溶液）を７分間（３５μｌ）インジェクションしてリガンド抗体を結合させた。チップ上の未反応活性基のキャッピングをするために１Ｍ ｅｔｈａｎｏｌａｍｉｎｅ−ＨＣｌ ｐＨ８．５を１０分間（５０μｌ）インジェクションした。４つのフローセルのうち一番目に相当するＦｃ１はリガンドを固定化せずにブランクセルとした。
(3) SPR analysis (i) Ligand immobilized by amine coupling method to sensor chip Anti-human haptoglobin antibody (rabbit, polyclonal) was immobilized on sensor chip C1 using amine coupling method as a ligand. As the running buffer, HBS-P buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 0.005% surfactant P-20) was used, and the flow rate was 5 μl / min. NHS (N-hydroxysuccinimide) -EDC (1-ethyl-3- (3-dimethylaminopropyl) carbohydride hydrochloride) mixture was injected for 7 minutes (35 μl) to the activation of the carboxyl group on the surface of chip C1, followed by anti-haptoglobin (200 μg). / ML, 10 mM acetate buffer pH 5.5 solution) was injected for 7 minutes (35 μl) to bind the ligand antibody. In order to capping the unreacted active groups on the chip, 1M ethanolamine-HCl pH 8.5 was injected for 10 minutes (50 μl). Of the four flow cells, Fc1 corresponding to the first was used as a blank cell without immobilizing the ligand.
（ii）血清試料の調製
血清蛋白質の定量にはＢＣＡキット（Ｐｉｅｒｃｅ）を用い、ＢＳＡを標品として測定した。
血清、レクチン、抗体等の試料及び試薬の希釈にはランニングバッファー（０．９ｍＭ ＣａＣｌ2，０．５ｍＭ ＭｇＳＯ4・７Ｈ2Ｏ，０．１ｍＭ ＭｎＣｌ2・４Ｈ2Ｏ，０．２ｍｇ／ｍＬ ＢＳＡを含むＨＢＳ−Ｐ ｂｕｆｆｅｒ）を用いた。血清２μｌを１ｍｇ／ｍＬに希釈後、ウルトラフリーＭＣ ０．４５μｍでろ過し、得られたろ液を５０ｎｇ／μｌに希釈しアナライトとした。
(Ii) Preparation of serum sample For quantification of serum protein, BCA kit (Pierce) was used, and BSA was measured as a standard.
For dilution of samples and reagents such as serum, lectin and antibody, running buffer (0.9 mM CaCl 2 , 0.5 mM MgSO 4 .7H 2 O, 0.1 mM MnCl 2 .4H 2 O, 0.2 mg / mL BSA Containing HBS-P buffer). 2 μl of serum was diluted to 1 mg / mL, and then filtered through 0.45 μm of Ultra Free MC, and the obtained filtrate was diluted to 50 ng / μl to obtain an analyte.
（iii）レクチン及び糖鎖抗体の調製
表１に示したレクチン及び糖鎖抗体を上記のランニングバッファーで１００ｎｇ／μｌに調製し分析に用いた。
(Iii) Preparation of lectin and sugar chain antibody The lectin and sugar chain antibody shown in Table 1 were prepared to 100 ng / μl with the above running buffer and used for analysis.
（iv）ハプトグロビンの定量
シグマ社製ハプトグロビン標品（ｐｏｏｌｅｄ ｈｕｍａｎ ｐｌａｓｍａ）の濃度０．２５，０．５，１，５，１０μｇ／ｍＬを用いて検量線を作成し、血清中のハプトグロビン定量を行った。
(Iv) Quantification of haptoglobin A calibration curve was prepared using concentrations of 0.25, 0.5, 1, 5, 10 μg / mL of Sigma's haptoglobin preparation, and serum haptoglobin was quantified. It was.
（ｖ）ＢＩＡｃｏｒｅ分析法
ＳＰＲ分析にはＢＩＡｃｏｒｅ２０００（ＧＥヘルスケアバイオサイエンス）を用い、流速は１０μｌ／ｍｉｎとした。抗ハプトグロビン抗体を固定化したセンサーチップに各希釈血清を４分間（４０μｌ）、次いでレクチン又は糖鎖抗体を４分間（４０μｌ）インジェクションし、ハプトグロビン及びレクチンの結合量（ＲＵ）を順次測定した。センサーチップの再生には１０ｍＭ Ｇｌｙｃｉｎｅ−ＨＣｌ ｐＨ１．７／１Ｍ ＮａＣｌ／１％ Ｔｗｅｅｎ２０の１分間（１０μｌ）インジェクションを１０回連続的にサイクルさせるプログラムを用いた。
データはＢＩＡ ｅｖａｌｕａｔｉｏｎ ｓｏｆｔｗａｒｅ ４．１等により解析した。レクチンの結合量はハプトグロビン結合量に対する比｛（ｌｅｃｔｉｎ／Ｈａｐｔｏｇｌｏｂｉｎ）×１００｝に換算した。
(V) BIAcore analysis method BIAcore2000 (GE Healthcare Bioscience) was used for SPR analysis, and the flow rate was 10 μl / min. Each diluted serum was injected into a sensor chip on which an anti-haptoglobin antibody was immobilized for 4 minutes (40 μl), and then a lectin or sugar chain antibody was injected for 4 minutes (40 μl), and the binding amount (RU) of haptoglobin and lectin was sequentially measured. For regeneration of the sensor chip, a program that continuously cycles 10-minute injection of 1 mM (10 μl) of 10 mM Glycine-HCl pH 1.7 / 1 M NaCl / 1% Tween 20 was used.
Data was analyzed by BIA evaluation software 4.1 and the like. The amount of lectin binding was converted to the ratio {(lectin / Haptoglobin) × 100} to the amount of haptoglobin binding.
（４）ＳＤＳ−ＰＡＧＥ及びＬｅｃｔｉｎ Ｂｌｏｔ、Ｗｅｓｔｅｒｎ Ｂｌｏｔ分析
（ｉ）ＳＤＳ−ＰＡＧＥ
患者血清のＳＤＳ−ＰＡＧＥは下記の条件で実施した。濃縮ゲル４％、分離ゲル１２％（１４０ｍｍ×８０ｍｍ×１ｍｍ）を用いた。ただし、ＳＮＡ−１レクチン解析には前出のゲルに加え、精度を上げるためにミニゲル（８０ｍｍ×８０ｍｍ×１ｍｍ）を使用して、濃縮ゲル４％、分離ゲル１０％を使用した。ランニングバッファーには２４．８ｍＭ Ｔｒｉｓ／１９２ｍＭ Ｇｌｙｃｉｎｅ／３．５ｍＭ ＳＤＳを用いた。泳動条件は濃縮ゲル１２ｍＡ／ｇｅｌ、分離ゲル２４ｍＡ／ｇｅｌで実施した。分子量マーカーとしてＰｒｅｃｉｓｉｏｎ ｐｌｕｓ ｐｒｏｔｅｉｎ ｓｔａｎｄａｒｄ（Ｂｉｏ−Ｒａｄ）を１０μｌをゲルに添加した。
Ｌｅｃｔｉｎ Ｂｌｏｔ及び Ｗｅｓｔｅｒｎ Ｂｌｏｔ法に用いる試料量及びハプトグロビン標品量は検出に用いるレクチンあるいは糖鎖抗体の最適検出量とした（表２）。
(4) SDS-PAGE and Lectin Blot, Western Blot analysis (i) SDS-PAGE
SDS-PAGE of patient serum was performed under the following conditions. Concentrated gel 4% and separation gel 12% (140 mm × 80 mm × 1 mm) were used. However, for the SNA-1 lectin analysis, in addition to the gel described above, a mini gel (80 mm × 80 mm × 1 mm) was used to increase accuracy, and a concentrated gel 4% and a separated gel 10% were used. As a running buffer, 24.8 mM Tris / 192 mM Glycine / 3.5 mM SDS was used. The electrophoresis conditions were a concentrated gel 12 mA / gel and a separation gel 24 mA / gel. 10 μl of Precision plus protein standard (Bio-Rad) was added to the gel as a molecular weight marker.
The sample amount and the haptoglobin sample amount used in the Lectin Blot and Western Blot methods were the optimum detection amounts of lectin or sugar chain antibody used for detection (Table 2).
（ii）Ｌｅｃｔｉｎ Ｂｌｏｔ及びＷｅｓｔｅｒｎ Ｂｌｏｔ法
電気泳動後、室温においてゲルはＰＶＤＦ膜（Ｉｍｍｏｂｉｌｏｎ Ｐ，０．４５μｍ，Ｍｉｌｌｉｐｏｒｅ）に、５０Ｖ、４５分間転写した。転写バッファーは１０ｍＭ ＣＡＰＳ ｐＨ１１／１０％ ＭｅＯＨを用いた。Ｂｌｏｔ法において非特異的タンパク質のＰＶＤＦ膜への吸着を阻止するブロッキング用バッファーにはＴ−ＴＢＳ（−）（２０ｍＭ Ｔｒｉｓ−ＨＣｌ／１５０ｍＭ ＮａＣｌ／０．０５％ Ｔｗｅｅｎ２０）を用い、レクチンと糖鎖結合反応には必須な２価イオンを含むＴ−ＴＢＳ（＋）（０．９ｍＭ ＣａＣｌ2／０．５ｍＭ ＭｇＳＯ4／０．１ｍＭ ＭｎＣｌ2・４Ｈ2Ｏ ｉｎ Ｔ−ＴＢＳ）を用いた。
転写終了後１時間ブロッキングを実施したが、Ｗｅｓｔｅｒｎ Ｂｌｏｔ法においては抗ハプトグロビン抗体及び糖鎖抗体のいずれにおいても５％スキムミルク／Ｔ−ＴＢＳ（−）を用い、Ｌｅｃｔｉｎ Ｂｌｏｔ法においては３％ ＢＳＡ／Ｔ−ＴＢＳ（−）を用いた。ブロッキング液を洗浄後、レクチン（２μｇ／ｍＬ ＰＨＡ−Ｌ4−ＨＲＰ、ＳＮＡ−１−ＨＲＰ、ＭＡＡ−ＨＲＰ，ＡＡＡ−ＨＲＰ，ＡＡＬ−ｂｉｏｔｉｎ ｉｎ Ｔ−ＴＢＳ（＋））又は一次抗体（２μｇ／ｍＬ ａｎｔｉ−Ｈａｐｔｏｇｌｏｂｉｎ（ｐｏｌｙｃｌｏｎａｌ，ｒａｂｂｉｔ）、ａｎｔｉ−ｓｉａｌｙｌ−Ｌｅａ（ｍｏｎｏｃｌｏｎａｌ，ｍｏｕｓｅ））を４℃において、一昼夜反応させた。反応液を除去、洗浄後、Ｗｅｓｔｅｒｎ Ｂｌｏｔは二次抗体（ａｎｔｉ−ｒａｂｂｉｔ ＩｇＧ−ＨＲＰ（１０，０００倍希釈），ａｎｔｉ−ｍｏｕｓｅ ＩｇＧ−ＨＲＰ（５，０００倍希釈））を、ＡＡＬ−ｂｉｏｔｉｎにはｓｔｒｅｐｔａｖｉｄｉｎ−ＨＲＰ（５，０００倍希釈）を室温、１時間反応させた。転写膜を洗浄後ＥＣＬ−ｐｌｕｓ（ＧＥヘルスケア）を用いて発色し、画像を撮影、解析した。ハプトグロビン標品の染色濃度から検量線を作成し各試料中のハプトグロビン及び糖鎖濃度を解析した。
(Ii) After electrophoresis on Lectin Blot and Western Blot , the gel was transferred to a PVDF membrane (Immobilon P, 0.45 μm, Millipore) at 50 V for 45 minutes at room temperature. As a transfer buffer, 10 mM CAPS pH 11/10% MeOH was used. T-TBS (-) (20 mM Tris-HCl / 150 mM NaCl / 0.05% Tween 20) is used as a blocking buffer to block adsorption of non-specific proteins to the PVDF membrane in the Blot method, and lectin and sugar chain binding T-TBS (+) (0.9 mM CaCl 2 /0.5 mM MgSO 4 /0.1 mM MnCl 2 .4H 2 O in T-TBS) containing essential divalent ions was used for the reaction.
Although blocking was performed for 1 hour after the completion of transcription, 5% skim milk / T-TBS (−) was used for both the anti-haptoglobin antibody and the sugar chain antibody in the Western Blot method, and 3% BSA / T in the Lectin Blot method. -TBS (-) was used. After washing the blocking solution, lectin (2 μg / mL PHA-L 4 -HRP, SNA-1-HRP, MAA-HRP, AAA-HRP, AAL-biotin in T-TBS (+)) or primary antibody (2 μg / mL) anti-Haptoglobin (polyclonal, rabbit) and anti-sialyl-Lea (monoclonal, mouse)) were reacted at 4 ° C. overnight. After removing the reaction solution and washing, Western Blot is secondary antibody (anti-rabbit IgG-HRP (10,000-fold dilution), anti-mouse IgG-HRP (5,000-fold dilution)), AAL-biotin is streptavidin-HRP (5,000-fold dilution) was allowed to react at room temperature for 1 hour. After the transfer film was washed, color was developed using ECL-plus (GE Healthcare), and an image was taken and analyzed. A calibration curve was created from the staining concentration of the haptoglobin preparation, and the haptoglobin and sugar chain concentrations in each sample were analyzed.
Ｂ．結果
（１）前立腺疾患患者のＰＳＡ値及び病理検査（Ｇｌｅａｓｏｎ ｓｃｏｒｅ）
表３に分析した１５検体の前立腺癌群（Ｃａｎｃｅｒ）、２０検体良性前立腺組織群（Ｂｅｎｉｇｎ）及び９検体の健常人ボランテイア群（Ｃｏｎｔｒｏｌ）のＰＳＡ値、病理組織結果、血清タンパク質量、ハプトグロビン量及び血清タンパク質量に対するハプトグロビン％値等を示した。
研究に用いた患者血清はＣａｎｃｅｒが１５例（４３％）で、そのうちＧｌｅａｓｏｎ ｓｃｏｒｅは７以上が１１例、Ｇｌｅａｓｏｎ Ｓｃｏｒｅ５及び６が４例であった。また、Ｂｅｎｉｇｎは２０例（５７％）（６例はＢＰＨ）であった。
Ｔｏｔａｌ ＰＳＡ値は癌化で著しく高値を示す症例が存在したが、一方ではＧｌｅａｓｏｎ Ｓｃｏｒｅが高い、すなわち悪性度の高い症例においてＰＳＡ値１０以下の検体が４症例存在した（２７％）。ＢｅｎｉｇｎにおいてＰＳＡ値２０を超える症例も存在した。コントロールはＰＳＡ値４以下であり、明らかに正常であった。
ＰＳＡ値が前立腺癌の有用な指標ではあるが、病理組織検査の有用性は高く最終診断の判定に用いられていた。早期癌診断に関連して、Ｂｅｎｉｇｎで観察されるグレイゾーンと言われるＰＳＡ値４−１０は注目される数値範囲であった。
B. Results (1) PSA level and pathological examination (Gleason score) of patients with prostate disease
PSA values, histopathological results, serum protein amount, haptoglobin amount and 15 samples of prostate cancer group (Cancer), 20 samples of benign prostate tissue group (Benign) and 9 samples of healthy volunteer group (Control) analyzed in Table 3 The haptoglobin% value etc. with respect to serum protein amount were shown.
The patient serum used in the study was Cancer (15 cases (43%)), of which Gleason score was 7 or more, 11 cases, and Gleason Score 5 and 6 were 4 cases. Benign was 20 cases (57%) (6 cases were BPH).
There were cases in which the total PSA value showed a markedly high value due to canceration, but on the other hand, there were 4 samples (27%) having a high Gleason Score, that is, a high malignancy and a PSA value of 10 or less. There were also cases where the PSA value exceeded 20 in Benign. The control had a PSA value of 4 or less and was clearly normal.
Although the PSA value is a useful index for prostate cancer, the usefulness of histopathological examination is high and it has been used for the determination of the final diagnosis. In connection with early cancer diagnosis, the PSA value 4-10, referred to as the gray zone observed at Benign, was a noted numerical range.
（２）ＳＰＲによる血清中ハプトグロビンの定量
図１に抗ハプトグロビン抗体の固定化センサーグラムを示した。
(2) Quantification of serum haptoglobin by SPR FIG. 1 shows an immobilized sensorgram of an anti-haptoglobin antibody.
図２の（１）に多段階相互作用の模式図を、図２の（２）に試料分析のセンサーグラムを示した。ＳＰＲ法の第一段目では血清中のハプトグロビンを選択的に捕捉するためアフィニティークロマトグラフィーがセンサーチップ上で実施され、第二段目でハプトグロビンの糖鎖に対するレクチンの結合が示された。 FIG. 2 (1) shows a schematic diagram of multi-stage interaction, and FIG. 2 (2) shows a sample analysis sensorgram. In the first stage of the SPR method, affinity chromatography was performed on the sensor chip to selectively capture haptoglobin in the serum, and in the second stage, the binding of lectin to the sugar chain of haptoglobin was shown.
図３の（ａ）に標品を用いたハプトグロビン検量線を示した。検量線は再現性があり、ＳＰＲ分析法によるキャリアタンパク質検出法であるハプトグロビン検出の精度が確認された。また、本法は血清２μｌを希釈後フィルターでろ過し、更なる希釈で最終濃度５０ｎｇ／μｌとした。分析に用いた総血清タンパク質量は２μｇであった。 A haptoglobin calibration curve using the standard is shown in FIG. The calibration curve was reproducible, and the accuracy of haptoglobin detection, which is a carrier protein detection method by SPR analysis, was confirmed. In this method, 2 μl of serum was diluted and filtered through a filter, and further diluted to a final concentration of 50 ng / μl. The total serum protein amount used for analysis was 2 μg.
図３の（ｂ）にＣａｎｃｅｒ，Ｂｅｎｉｇｎ，Ｃｏｎｔｒｏｌのハプトグロビン量及び（ｃ）に血清タンパク質総量に対するハプトグロビン含有量（％）の症例別分布を示した。
表３に示す血清タンパク質量は、ＣｏｎｔｒｏｌとＣａｎｃｅｒでは平均値で約６％の相違がありＣａｎｃｅｒにおいて有意に増加していた。ハプトグロビン量はほぼ正常値範囲であり、血清蛋白質量の増加により、血清タンパク質総量に対するハプトグロビン含有量の平均値（％）はＣａｎｃｅｒ １．４８％、Ｂｅｎｉｇｎ ２．２７％、Ｃｏｎｔｒｏｌ ３．６７％を示した。健常人血清タンパク質量はやや増加し、その全タンパク質に対する含量もわずかに増加した。
FIG. 3 (b) shows the haptoglobin content of Cancer, Benign, and Control, and (c) shows the distribution of haptoglobin content (%) with respect to the total serum protein by case.
The serum protein amount shown in Table 3 was significantly increased in Cancer with a difference of about 6% in the average value between Control and Cancer. The amount of haptoglobin is almost in the normal value range, and the average value (%) of the haptoglobin content relative to the total amount of serum protein is Cancer 1.48%, Benign 2.27%, and Control 3.67% due to the increase in serum protein mass. It was. The amount of healthy human serum protein increased slightly, and its content relative to total protein also increased slightly.
（３）ハプトグロビン糖鎖に対するレクチン結合量の比較
ＳＰＲ法はハプトグロビン測定濃度とその糖鎖測定用ハプトグロビン濃度が一致しており、血清の希釈が一律であり、ハプトグロビン量及び糖鎖量を順次測定することに適していた。ハプトグロビン量（ＲＵ）に対する糖鎖構造を反映するレクチン結合量（ＲＵ）を１００％表示し、Ｃａｎｃｅｒ、Ｂｅｎｉｇｎ、Ｃｏｎｔｒｏｌ群間の有意差検定を実施し、比較した結果を図４に示す。
(3) Comparison of the amount of lectin binding to haptoglobin sugar chains In the SPR method, the haptoglobin measurement concentration and the haptoglobin concentration for sugar chain measurement match, the serum dilution is uniform, and the haptoglobin amount and sugar chain amount are measured sequentially. It was suitable. The lectin binding amount (RU) reflecting the sugar chain structure relative to the haptoglobin amount (RU) is displayed as 100%, a significant difference test is performed between the Cancer, Benign, and Control groups, and the comparison results are shown in FIG.
糖鎖の多岐化を検出するＰＨＡ−Ｌ4，ＮｅｕＡｃ α２−６結合を検出するＳＮＡ−１，ＮｅｕＡｃ α２−３結合を検出するＭＡＡ，Ｆｕｃ α１−６結合を検出するＡＡＬ，Ｆｕｃα１−２及びＦｕｃ α１−３結合を検出するＡＡＡのレクチンを用いてハプトグロビンに存在する糖鎖解析を実施した。
ＣａｎｃｅｒとＣｏｎｔｒｏｌ間で有意差が認められたのはＳＮＡ−１及びＡＡＬであった。特にＳＮＡ−１はレスポンスが大きく、前立腺癌検出マーカーとしての有用性が期待される結果を得た。さらにこれらはＣａｎｃｅｒとＢｅｎｉｇｎ間においても有意差を示し、Ｃａｎｃｅｒのみならず、Ｂｅｎｉｇｎとの識別の可能性が示唆された。
PHA-L 4 for detecting sugar chain diversification, SNA-1 for detecting NeuAc α2-6 bond, MAA for detecting NeuAc α2-3 bond, AAL, Fucα1-2 and Fuc for detecting Fuc α1-6 bond Analysis of sugar chains present in haptoglobin was performed using AAA lectin that detects α1-3 binding.
It was SNA-1 and AAL that a significant difference was recognized between Cancer and Control. In particular, SNA-1 has a large response, and the results are expected to be useful as a prostate cancer detection marker. Furthermore, these also showed a significant difference between Cancer and Benign, suggesting the possibility of discriminating not only Cancer but also Benign.
すでに癌化におけるハプトグロビン糖鎖構造の多岐化（ＰＨＡ−Ｌ4レクチン）は、ＳＤＳ−ＰＡＧＥ−Ｍａｓｓ Ｓｐｅｃｔｒｏｍｅｔｒｙ法（非特許文献１）及び、ＰＨＡ−Ｌ4カラム精製−Ｍａｓｓ ｓｐｅｃｔｒｏｍｅｔｒｙで報告されている（非特許文献２）。
しかしながら今回のＳＰＲ法ではＰＨＡ−Ｌ4の結合量が殆ど検出されず、Ｃｏｎｔｒｏｌに対する有意な差が認められなかった。また、ＳＤＳ−ＰＡＧＥ法においては、ＳＤＳの可溶化によりハプトグロビンの高次構造が完全に破壊され、サブユニットに分離され、レクチン結合部位が暴露されていると考えられた。それに対して、ＳＰＲ法においてハプトグロビンは未変性タンパク質と抗体との反応であり、レクチンとのハプトグロビン糖鎖の結合部位と、ハプトグロビンの抗原抗体反応の部位の関係が不明であった。その結果、レクチンとハプトグロビン抗体間において糖鎖周辺の競合関係、あるいは構造阻害関係が存在している可能性も否定できない。従って、ＳＰＲ法におけるＰＨＡ−Ｌ4結合解析は試料量の検討のみならず、反応性の解明が求められた。
Diversification of haptoglobin sugar chain structures in canceration (PHA-L 4 lectin) has already been reported by SDS-PAGE-Mass Spectrometry method (Non-patent Document 1) and PHA-L 4 column purification-Mass spectrometry ( Non-patent document 2).
However, in this SPR method, the binding amount of PHA-L 4 was hardly detected, and a significant difference with respect to Control was not recognized. In the SDS-PAGE method, it was considered that the higher-order structure of haptoglobin was completely destroyed by solubilization of SDS, separated into subunits, and the lectin binding site was exposed. In contrast, in the SPR method, haptoglobin is a reaction between a native protein and an antibody, and the relationship between the binding site of a haptoglobin sugar chain to a lectin and the site of an antigen-antibody reaction of haptoglobin is unknown. As a result, it cannot be denied that there is a competitive relationship or a structure-inhibiting relationship around the sugar chain between the lectin and the haptoglobin antibody. Therefore, PHA-L 4 binding analysis in the SPR method required not only examination of the sample amount but also elucidation of reactivity.
ｓＬｅａ糖鎖を検出する抗体ａｎｔｉ−ｓＬｅａ及びＦｕｃ α１−２及びＦｕｃ α１−３に対するＡＡＡレクチンはＳＰＲ法においてハプトグロビンとの結合が検出されなかった。これらのレクチン及び糖鎖抗体に関してもＰＨＡ−Ｌ4レクチン同様の検討が求められた。 AAA lectin against the antibodies anti-sLea, Fuc α1-2, and Fuc α1-3, which detect sLea sugar chains, was not detected to bind to haptoglobin in the SPR method. Regarding these lectins and sugar chain antibodies, examinations similar to those of PHA-L 4 lectins were required.
図５にＳＮＡ−１及びＡＡＬのＣａｎｃｅｒ，Ｂｅｎｉｇｎ，ＣｏｎｔｒｏｌのＬｅｃｔｉｎ／Ｈａｐｔｏｇｌｏｂｉｎ比の分布図を示した。 FIG. 5 shows a distribution diagram of the Lectin / Haptoglobin ratio of Cancer, Benign, and Control of SNA-1 and AAL.
ＳＮＡ−１の分布はＡＡＬに比較してその有意差検定からも明らかなように、病態における結合量が増加し、Ｃａｎｃｅｒ、Ｂｅｎｉｇｎ及びＣｏｎｔｒｏｌの判別が可能となった。 As is apparent from the significance test for the distribution of SNA-1 as compared with AAL, the amount of binding in the disease state increased, and Cancer, Benign and Control could be discriminated.
（４）Ｗｅｓｔｅｒｎ Ｂｌｏｔ法による血清中ハプトグロビンの検出
図６の（ａ）にハプトグロビン抗体によるＷｅｓｔｅｒｎ Ｂｌｏｔ法のパターンを示した。各試料のハプトグロビンβ鎖バンドはハプトグロビン標品を用いた検量線より血清中の濃度を求めた。ＣａｎｃｅｒとＣｏｎｔｒｏｌ間でハプトグロビン量の大きな差異は認められなかった。
(4) Detection of haptoglobin in serum by Western Blot method FIG. 6A shows the pattern of Western Blot method using a haptoglobin antibody. The serum concentration of the haptoglobin β chain band of each sample was determined from a calibration curve using a haptoglobin sample. There was no significant difference in the amount of haptoglobin between Cancer and Control.
（５）Ｌｅｃｔｉｎ Ｂｌｏｔ及びＷｅｓｔｅｒｎ Ｂｌｏｔ法によるハトグロビン糖鎖に対する結合量の比較
図６の（ｂ）＆（ｃ）にＳＮＡ−１及びＡＡＬのＬｅｃｔｉｎ Ｂｌｏｔ法のパターンを示した。血清中にはレクチン結合糖タンパク質が多数存在し、染色バンドが多く、正確なる再現性、定量性を求めるには困難があった。
図７にハプトグロビン１分子あたりのレクチン及び糖鎖抗体の結合量を病態別に比較した。ｔ−Ｔｅｓｔの結果、Ｐ値は低いが、ＳＰＲ法と同様にＣａｎｃｅｒとＣｏｎｔｒｏｌ間に有意差が生じたのはＡＡＬ、ＳＮＡ−１であった。また、Ｗｅｓｔｅｒｎ Ｂｌｏｔ法ではａｎｔｉ−ｓＬｅａも有意差を示した。今回、ｓＬｅａはＳＮＡ−１の５倍量のタンパク質量を用いた。
(5) Comparison of binding amount to pigeon saccharide sugar chain by Lectin Blot and Western Blot method FIGS. 6B and 6C show patterns of the SNA-1 and AAL Lectin Blot methods. There are many lectin-binding glycoproteins in serum, and there are many stained bands, making it difficult to obtain accurate reproducibility and quantitativeness.
FIG. 7 compares the binding amount of lectin and sugar chain antibody per molecule of haptoglobin according to pathological condition. As a result of t-Test, although the P value was low, it was AAL and SNA-1 that caused a significant difference between Cancer and Control as in the SPR method. Moreover, anti-sLea showed a significant difference in Western Blot method. This time, sLea used 5 times as much protein as SNA-1.
（６）ＳＰＲ法とＬｅｃｔｉｎ Ｂｌｏｔ及びＷｅｓｔｅｒｎ Ｂｌｏｔ法の比較
Ｌｅｃｔｉｎ Ｂｌｏｔ及びＷｅｓｔｅｒｎ Ｂｌｏｔ法の電気泳動法（ＳＤＳ−ＰＡＧＥ）はタンパク質変性し、サブユニット分離しているために、ＰＶＤＦ膜上でタンパク質糖鎖とレクチン或いは糖鎖抗体との反応が容易になり、その反応性は上昇したことが予測された。ただし、その方法はｉ）ＳＤＳ−ＰＡＧＥ、ii）ＰＶＤＦ膜への転写、iii）非特異的な結合を防ぐためのブロッキング、iv）レクチン或いは糖鎖抗体との反応、さらに、ｖ）ＥＣＬその他を用いたタンパク質或いは糖鎖の発色、vi）Ｘ線フィルムへの焼き付け、vii）デンシトメーターへの取り込み、viii）定量とその手技の多さに加え、必要とする時間は最低３日間を必要とした。その上、手技の熟練度により品質の異なるデータが見られ、データの再現性を得るためにはかなりの時間を要した。但し、Ｌｅｃｔｉｎ Ｂｌｏｔ及びＷｅｓｔｅｒｎ Ｂｌｏｔ法は一回の操作で同時に多検体の結果が得られるという利点があり、試料間の相対的な比較検討には適していた。また、血清の全タンパク質が同時に観察される様相から各レクチンで染色されるタンパク質の分子量分布が明らかとなった。
(6) Comparison between SPR method and Lectin Blot and Western Blot method Since Lectin Blot and Western Blot electrophoresis (SDS-PAGE) is protein-denatured and subunit-separated, protein sugar chains on the PVDF membrane It was predicted that the reaction with lectin or sugar chain antibody became easier and the reactivity increased. However, the methods are i) SDS-PAGE, ii) transcription to PVDF membrane, iii) blocking to prevent non-specific binding, iv) reaction with lectin or sugar chain antibody, and v) ECL and others. Color development of the protein or sugar chain used, vi) Baking on X-ray film, vii) Incorporation into densitometer, viii) In addition to quantification and the procedure, the time required is at least 3 days did. In addition, data of different quality was seen depending on the skill level of the procedure, and it took a considerable amount of time to obtain data reproducibility. However, the Lectin Blot and Western Blot methods have the advantage that multiple sample results can be obtained simultaneously in a single operation, and are suitable for relative comparative studies between samples. In addition, the molecular weight distribution of proteins stained with each lectin was clarified from the aspect in which all serum proteins were observed simultaneously.
それに反して、ＳＰＲ分析は第一段目をタンパク質の抗体で目的とする糖タンパク質を捕獲するアフィニティークロマトグラフィーを利用するために、希釈試料を流すことで血清中から容易にハプトグロビンを分離精製した。次いで他の血清タンパク質成分を洗浄し、さらに第二段目のレクチン溶液とハプトグロビン糖鎖との反応を観察し、その結合量を測定することが可能であった。すべてのステップが自動化されており２４時間自動分析によって効率的な解析が可能であった。解析ソフトプログラムは機器に内蔵していた。 On the other hand, in the SPR analysis, haptoglobin was easily separated and purified from serum by flowing a diluted sample in order to use affinity chromatography in which the target glycoprotein was captured with the protein antibody in the first stage. Subsequently, it was possible to wash other serum protein components and observe the reaction between the second-stage lectin solution and the haptoglobin sugar chain to measure the amount of binding. All steps were automated, and efficient analysis was possible by 24-hour automatic analysis. The analysis software program was built into the equipment.
シアル酸（ＮｅｕＡｃ α２−６）を認識するレクチンＳＮＡ−１はＣａｎｃｅｒとＣｏｎｔｒｏｌ間のみならず、ＣａｎｃｅｒとＢｅｎｉｇｎ間でも有意差が認められ、現在、疾患マーカーとして広く用いられているＰＳＡと併用し、前立腺癌診断を補完しうる可能性が示唆された。ＳＰＲ 法はＬｅｃｔｉｎ Ｂｌｏｔ及びＷｅｓｔｅｒｎ Ｂｌｏｔ法に比して、特異性、再現性、定量性、簡便性において格段の優位性を示した。 The lectin SNA-1 which recognizes sialic acid (NeuAc α2-6) is recognized not only between Cancer and Control, but also between Cancer and Benign, and is used in combination with PSA currently widely used as a disease marker, This suggests the possibility of complementing prostate cancer diagnosis. The SPR method showed a significant advantage in specificity, reproducibility, quantitativeness, and simplicity compared with the Lectin Blot and Western Blot methods.
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