JP7421744B2 - Diagnosis of osteoarthritis - Google Patents
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Description
特許法第３０条第２項適用 平成３１年４月２２日 ｈｔｔｐ：／／ｓｑｕａｒｅ．ｕｍｉｎ．ａｃ．ｊｐ／ｊｓｍｂｍ２０１９／ｐｒｏｇｒａｍ．ｈｔｍｌ「第５１回日本結合組織学会学術大会 プログラム・抄録集第１５９頁」にて公開Application of Article 30, Paragraph 2 of the Patent Act April 22, 2019 http://square. umin. ac. jp/jsmbm2019/program. Published in html "51st Japanese Connective Tissue Society Academic Conference Program/Abstract Collection, page 159"
本発明は、変形性関節症の診断に関する。 The present invention relates to the diagnosis of osteoarthritis.
変形性関節症（Osteoarthritis：OA）は、遺伝的要因に加え、老化、肥満、外傷などが原因で引き起こされる関節疾患である。OAは、荷重関節である膝関節や股関節によくみられ、軟骨の変性及び摩耗や軟骨下骨の変化により関節の変形を生じ、歩行時に痛みを生じ移動機能が障害される。膝OAの国内患者数は2500万人に達すると推定されている。 Osteoarthritis (OA) is a joint disease caused by aging, obesity, trauma, etc. in addition to genetic factors. OA often occurs in the knee and hip joints, which are load-bearing joints, and deforms the joints due to cartilage degeneration and wear and changes in the subchondral bone, causing pain when walking and impairing mobility. The number of patients with knee OA in Japan is estimated to reach 25 million.
現在OAは問診、Ｘ線検査、CT、MRIなどにより診断されている。また、これまでに、尿や血液中の因子とOAとの関連について多く報告されている（非特許文献１）。例えば、特許文献１では、補体C4-A及び補体C3由来のペプチドフラグメントをOAの診断マーカーとして見出し、これらを質量分析または特異抗体で検出するOAの検査方法が報告されている。また、特許文献２では、IL6、MMP1、IL8などのタンパク質由来のポリペプチドを、質量分析や免疫測定法、その他の方法で検出するOA診断方法が報告されている。特に、骨、軟骨を構成する主要なタンパク質成分であるコラーゲンはOA疾患によって分解され、その分解産物であるコラーゲン由来のペプチドやアミノ酸が血液や尿中で増加するため、I型コラーゲンやII型コラーゲン由来のコラーゲン末端のペプチドはOAのバイオマーカーとしても利用されている。
しかし、これら既存の方法では初期段階で疾患を正確に検出することは難しく、より早期に、より正確にOAを診断するための技術が強く望まれている。
Currently, OA is diagnosed using medical interviews, X-ray examinations, CT, MRI, etc. Furthermore, there have been many reports on the relationship between factors in urine and blood and OA (Non-Patent Document 1). For example, Patent Document 1 reports an OA testing method in which peptide fragments derived from complement C4-A and complement C3 are discovered as diagnostic markers for OA, and these are detected using mass spectrometry or a specific antibody. Further, Patent Document 2 reports an OA diagnostic method in which polypeptides derived from proteins such as IL6, MMP1, and IL8 are detected by mass spectrometry, immunoassay, or other methods. In particular, collagen, which is a major protein component that makes up bones and cartilage, is degraded by OA disease, and collagen-derived peptides and amino acids, which are the degradation products, increase in the blood and urine. The derived collagen terminal peptide is also used as a biomarker for OA.
However, it is difficult to accurately detect the disease at an early stage with these existing methods, and there is a strong need for technology that can diagnose OA more accurately at an earlier stage.
本発明の課題は、OAの診断法を提供することにある。 An object of the present invention is to provide a method for diagnosing OA.
本発明者は、尿や血液などの生体試料を対象とし、骨と軟骨の主成分であるコラーゲンの分解産物に焦点を当てて鋭意検討したところ、コラーゲンの翻訳後修飾アミノ酸であるヒドロキシプロリン（Hyp）とヒドロキシリシン（Hyl）の量は非OA患者とOA患者との間で有意な差は見られなかった一方、Hylの糖鎖付加体であるガラクトシルヒドロキシリシン（GHL）とグルコシルガラクトシルヒドロキシリシン（GGHL）の量がOA患者で顕著に増加しており、当該GHLとGGHLの合計値を指標にOAを精度良く診断できることを見出し、本発明を完成した。 The present inventor conducted extensive research using biological samples such as urine and blood, focusing on the degradation products of collagen, which is the main component of bones and cartilage, and found that hydroxyproline (Hyp ) and hydroxylysine (Hyl) were not significantly different between non-OA patients and OA patients, whereas the amounts of glycosylated Hyl, galactosylhydroxylysine (GHL) and glucosylgalactosylhydroxylysine ( The present invention was completed based on the discovery that the amount of GGHL) is significantly increased in OA patients, and that OA can be diagnosed with high accuracy using the total value of GHL and GGHL as an index.
すなわち、本発明は、次の１）～３）を提供するものである。
１）GHL及びGGHLの組み合わせからなる、OAのバイオマーカー。
２）被験対象由来の試料中のGHL量及びGGHL量を指標とする、OAの検査方法。
３）被験対象由来の試料中のGHL量及びGGHL量を測定する試薬を含む、OAの検査キット。
That is, the present invention provides the following 1) to 3).
1) OA biomarker consisting of a combination of GHL and GGHL.
2) A testing method for OA using the amount of GHL and GGHL in a sample derived from a test subject as indicators.
3) An OA test kit containing a reagent for measuring the amount of GHL and GGHL in a sample derived from a test subject.
本発明によれば、OAの正確な診断が可能となる。本発明により早期にOAを診断できれば、OA患者に対し適切な処置を早期に行うことができる。 According to the present invention, accurate diagnosis of OA is possible. If OA can be diagnosed early according to the present invention, appropriate treatment can be performed early on for OA patients.
本発明のOAのバイオマーカーは、GHL及びGGHLの組み合わせからなる。ここで、GHLは、Hylにガラクトースが結合した分子式C12H24N2O8で表される化合物であり、GGHLはこれに更にグルコースが結合した分子式C18H34N2O13で表される化合物である。
斯かるバイオマーカーは、OAの検査に用いることができる。
The OA biomarker of the present invention consists of a combination of GHL and GGHL. Here, GHL is a compound represented by the molecular formula C 12 H 24 N 2 O 8 in which galactose is bound to Hyl, and GGHL is represented by the molecular formula C 18 H 34 N 2 O 13 in which glucose is further bound to this. It is a compound that
Such biomarkers can be used to test for OA.
本発明のOAの検査方法は、被験対象由来の試料中のバイオマーカーの量、すなわち、GHL量及びGGHL量を指標として行われる。
本発明の検査方法の対象としては、ヒト又は非ヒト動物が挙げられる。非ヒト動物としては、例えば、類人猿、その他霊長類、マウス、ラット、ハムスター、ウマ、ウシ、ブタ、ヒツジ、ヤギ、イヌ、ネコなどの非ヒト哺乳動物が挙げられる。好ましくは、ヒトである。ヒトは、関節周囲の疼痛や違和感などの症状を有する者が好ましく挙げられる。
被験対象由来の試料は、例えば、血液（全血）、血液に由来する血漿や血清、尿、関節液などが挙げられる。好ましくは、血液、尿である。
The OA testing method of the present invention is carried out using the amount of biomarkers in a sample derived from a test subject, that is, the amount of GHL and the amount of GGHL, as indicators.
Targets of the testing method of the present invention include humans and non-human animals. Examples of non-human animals include non-human mammals such as apes, other primates, mice, rats, hamsters, horses, cows, pigs, sheep, goats, dogs, and cats. Preferably it is a human. Preferably, the human being has symptoms such as pain or discomfort around the joints.
Samples derived from the test subject include, for example, blood (whole blood), plasma or serum derived from blood, urine, synovial fluid, and the like. Blood and urine are preferred.
GHL量及びGGHL量の測定は、適宜公知の手法を用いることができる。測定方法は、必ずしも厳密な定量方法でなくともよい。例えば、液体クロマトグラフィー質量分析（LC-MS）や液体クロマトグラフィー（HPLC）などの機器分析法、ELISA法やRIA法などの免疫学的測定法が挙げられる。簡便性及び正確性の観点から、好ましくは、GHL及びGGHLに特異的に結合する抗体を用いたELISA法（例えば、直接法、間接法、サンドイッチ法、競合法など）である。後記する実施例１に示すように、尿、血清中のHyl量はOA患者と非OA患者で有意な差がないため、いずれの分析方法であっても、GHL、GGHLの前駆体であり、かつ構造の類似したHylと識別してGHL及びGGHLを検出する必要がある。 The amount of GHL and the amount of GGHL can be measured by appropriately known methods. The measurement method does not necessarily have to be a strict quantitative method. Examples include instrumental analysis methods such as liquid chromatography mass spectrometry (LC-MS) and liquid chromatography (HPLC), and immunoassay methods such as ELISA and RIA methods. From the viewpoint of simplicity and accuracy, ELISA methods (eg, direct method, indirect method, sandwich method, competitive method, etc.) using antibodies that specifically bind to GHL and GGHL are preferred. As shown in Example 1 below, there is no significant difference in the amount of Hyl in urine and serum between OA patients and non-OA patients, so no matter which analysis method is used, Hyl is a precursor of GHL and GGHL. It is also necessary to detect GHL and GGHL by distinguishing it from Hyl, which has a similar structure.
GHL及びGGHLに特異的に結合する抗体は、両者で共通する骨格構造を持つGHLを免疫原として用い、適宜公知の手法により得ることができる。抗体は、免疫グロブリン（IgA、IgD、IgE、IgG、IgM、IgYなど）、Fabフラグメント、F（ab'）2フラグメント、一本鎖抗体フラグメント（scFv）、シングルドメイン抗体、diabodyなどが挙げられ、これらはヒト抗体、ヒト化抗体、キメラ抗体、マウス抗体、ラマ抗体、ニワトリ抗体などのモノクローナル抗体又はポリクローナル抗体が挙げられる。好ましくは、マウスモノクローナル抗体である。また、GHL及びGGHLに同等の力価で反応する抗体が好ましい。
GHL及びGGHLに特異的に結合する抗体として、国際公開第2004/040971号に記載の方法に従って、GANP（登録商標）マウスで作製した抗体を例として挙げることができる。
Antibodies that specifically bind to GHL and GGHL can be obtained by appropriately known techniques using GHL, which has a common skeleton structure for both, as an immunogen. Antibodies include immunoglobulins (IgA, IgD, IgE, IgG, IgM, IgY, etc.), Fab fragments, F(ab') 2 fragments, single chain antibody fragments (scFv), single domain antibodies, diabodies, etc. These include monoclonal or polyclonal antibodies such as human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies, llama antibodies, and chicken antibodies. Preferably, it is a mouse monoclonal antibody. Furthermore, antibodies that react with GHL and GGHL with equivalent titers are preferred.
An example of an antibody that specifically binds to GHL and GGHL is an antibody produced in GANP (registered trademark) mice according to the method described in WO 2004/040971.
後述する実施例に示すように、GHLとGGHLはOA患者で顕著に増加している。そして、GHLとGGHLを足し合わせた合計値は、非OA患者に比べて、OA患者において有意に高い値を示す。従って、本発明の検査方法によるGHL量及びGGHL量の測定結果に基づけば、OAの診断が可能である。
OAの診断にあたっては、例えば、被験対象由来の試料中のGHL及びGGHLの合計量と予め設定された閾値を比較して、当該被験対象由来の試料中の合計量が閾値よりも多い場合、被験対象はOAであると判定することができる。閾値は、例えば、健常者又は非OA患者の平均値や中央値を基に、統計解析ソフトウェアを用いたROC（Receiver Operating Characteristic）曲線を用いて算出するなど、適宜設定することができる。
As shown in the Examples below, GHL and GGHL are significantly increased in OA patients. The total value of GHL and GGHL is significantly higher in OA patients than in non-OA patients. Therefore, OA can be diagnosed based on the measurement results of GHL and GGHL levels using the testing method of the present invention.
In diagnosing OA, for example, the total amount of GHL and GGHL in a sample derived from a test subject is compared with a preset threshold, and if the total amount in the sample derived from the subject is greater than the threshold, the test subject It can be determined that the target is OA. The threshold value can be set as appropriate, for example, by calculating it using an ROC (Receiver Operating Characteristic) curve using statistical analysis software, based on the average value or median value of healthy subjects or non-OA patients.
また、被験対象由来の試料中のGHL量及びGGHL量を指標に、OAの治療効果判定や、OA治療薬のスクリーニングを行うこともできる。 Furthermore, the therapeutic effect of OA can be determined and OA therapeutic drugs can be screened using the amount of GHL and GGHL in a sample derived from a test subject as indicators.
本発明のOAの検査キットは、被験対象由来の試料中のGHL量及びGGHL量を測定する試薬を含む。当該測定試薬としては、例えば、LC-MSに用いる試薬、HPLCに用いる試薬、免疫学的測定試薬などが挙げられる。本発明のOAの検査キットは、簡便性及び正確性の観点から、好ましくは免疫学的測定試薬を含み、より好ましくはELISA試薬を含む。
ELISA試薬は、GHL及びGGHLに特異的に結合する抗体を含む。抗体は、既に説明したとおりである。
検査キットには、上記測定試薬の他、必要に応じて測定用プロトコールなどを含めることができる。
The OA test kit of the present invention includes reagents for measuring the amount of GHL and GGHL in a sample derived from a test subject. Examples of the measurement reagent include reagents used for LC-MS, reagents used for HPLC, and immunological measurement reagents. From the viewpoint of convenience and accuracy, the OA test kit of the present invention preferably includes an immunoassay reagent, more preferably an ELISA reagent.
ELISA reagents include antibodies that specifically bind GHL and GGHL. Antibodies are as described above.
In addition to the above-mentioned measurement reagents, the test kit can also include a measurement protocol and the like, if necessary.
次に実施例を挙げて本発明を更に詳細に説明する。 Next, the present invention will be explained in more detail with reference to Examples.
実施例１
Ａ．方法
（１）測定試料の調製
対象は、人工膝関節置換術を受けた膝OA患者（尿：男4名・女4名、平均71.5歳。血液：男4名・女3名、平均70.1歳)と、コントロール群として膝前十字靭帯再建術を受けた膝OAのない患者(男3名・女2名、平均26.6歳)である。手術日当日の朝に採血と採尿を行い、全血から血清試料を調製した。
尿サンプルは、安定同位体標識コラーゲンをアルカリまたは酸で加水分解して作製した各翻訳後修飾アミノ酸の内部標準（特許第6301298号に記載）を添加し、アセトニトリル及びギ酸をそれぞれ50％、0.1％となるように添加して希釈し測定試料とした。
血清サンプルは、同様に内部標準を添加してからエタノール及びギ酸をそれぞれ75％、0.1％となるように添加して除タンパク処理を行い、遠心分離後の上清を測定試料とした。調製した尿、血清試料について、以下の条件でHyp、Hyl、GHL、GGHLのLC-MS分析を行った。測定結果は、有意水準を0.05に設定したスチューデントのt検定で統計的に評価した。
Example 1
A. Method (1) Preparation of measurement samples Subjects were knee OA patients who underwent total knee arthroplasty (urine: 4 males and 4 females, average age 71.5 years; blood: 4 males and 3 females, average age 70.1 years old) ) and a control group of patients without knee OA who underwent anterior cruciate ligament reconstruction (3 males and 2 females, mean age 26.6 years). Blood and urine were collected on the morning of the surgery day, and serum samples were prepared from the whole blood.
Urine samples were prepared by adding an internal standard of each post-translationally modified amino acid (described in Patent No. 6301298) prepared by hydrolyzing stable isotope-labeled collagen with alkali or acid, and adding acetonitrile and formic acid at 50% and 0.1%, respectively. It was added and diluted to give a measurement sample.
The serum sample was subjected to protein removal treatment by adding an internal standard and ethanol and formic acid to 75% and 0.1%, respectively, and the supernatant after centrifugation was used as the measurement sample. LC-MS analysis of Hyp, Hyl, GHL, and GGHL was performed on the prepared urine and serum samples under the following conditions. The measurement results were statistically evaluated using Student's t test with the significance level set at 0.05.
（２）LC-MS測定条件
高速液体クロマトグラフ：1200 Series(Agilent Technologies)、
質量分析装置：3200 QTRAP(AB Sciex)、
分析カラム：TSKgel Amide-80 5μm, 2.0mmi.d.×150mm(Tosoh)、
カラム温度：40℃
移動相：Ａ液；0.1％ギ酸、Ｂ液；100％アセトニトリル、
グラジエント条件：0～3分：Ａ液10％；Ｂ液90％、3～5分：Ａ液10～30％；Ｂ液90～70％、5～15分：Ａ液30～60％；Ｂ液70～40％、15～17分：Ａ液90％；Ｂ液10％、17～20分：Ａ液10％；Ｂ液90％、
流速：0.5mL/min、
イオン化：ESI、ポジティブ、
分析モード：Multiple Reaction Monitoring(MRM)モード、
イオンスプレー電圧：4kV、
イオンソース温度：500℃
(2) LC-MS measurement conditions High performance liquid chromatograph: 1200 Series (Agilent Technologies),
Mass spectrometer: 3200 QTRAP (AB Sciex),
Analytical column: TSKgel Amide-80 5μm, 2.0mmi.d.×150mm (Tosoh),
Column temperature: 40℃
Mobile phase: A solution: 0.1% formic acid, B solution: 100% acetonitrile,
Gradient conditions: 0 to 3 minutes: A liquid 10%; B liquid 90%, 3 to 5 minutes: A liquid 10 to 30%; B liquid 90 to 70%, 5 to 15 minutes: A liquid 30 to 60%; B Liquid 70-40%, 15-17 minutes: A liquid 90%; B liquid 10%, 17-20 minutes: A liquid 10%; B liquid 90%,
Flow rate: 0.5mL/min,
Ionization: ESI, positive,
Analysis mode: Multiple Reaction Monitoring (MRM) mode,
Ion spray voltage: 4kV,
Ion source temperature: 500℃
Ｂ．結果
尿サンプルの測定結果を図１に示す。Hyp及びHylの尿検体中濃度は、OA患者と膝前十字靭帯損傷患者（Control）の間で有意な差は見られなかった一方、GHLの尿検体中濃度はOA検体で有意に増加していた。さらに、GHLとGGHLを足し合わせた合計値（GHL＋GGHL）はOA検体において有意に高く、その値は2.7 μmol/mmol creatinineを境界としてControl検体とOA検体で完全に分離された。
同様に、図２に示すように、血清サンプルでもGHL及びGHL＋GGHL値がOA検体で有意に高く、GHL＋GGHL値は0.34 nmol/mLを境界としてControl検体とOA検体で完全に分離された。
B. Results The measurement results of the urine samples are shown in Figure 1. There was no significant difference in the concentrations of Hyp and Hyl in urine samples between OA patients and patients with anterior cruciate ligament injury (control), whereas the concentration of GHL in urine samples was significantly increased in OA samples. Ta. Furthermore, the total value of GHL and GGHL (GHL + GGHL) was significantly higher in the OA sample, and the value was completely separated between the control sample and the OA sample with a boundary of 2.7 μmol/mmol creatinine.
Similarly, as shown in FIG. 2, in serum samples, GHL and GHL+GGHL values were significantly higher in OA samples, and GHL+GGHL values were completely separated between control samples and OA samples with a boundary of 0.34 nmol/mL.
実施例２
Ａ．方法
（１）抗体作製
GHL及びGGHLに特異的に結合する抗体は、国際公開第2004/040971号に記載の方法に従って、GANP（登録商標）マウスを使って作製した（トランスジェニック社）。キャリアタンパク質Keyhole limpet hemocyanin（KLH）に結合させたGHL（KLH-GHL；糖鎖工学研究所社製）を免疫原として用い、GANPマウス3匹に対して背部皮下へカクテル免疫した。3回免疫後に抗血清力価の上昇を確認し、高力価マウス1匹から脾臓を摘出してミエローマ細胞（P3U1）と融合した。融合脾細胞の培養上清をサンプルとして、Hyl標準品（Sigma-Aldrich社製）、GHL標準品（糖鎖工学研究所社製）、GGHL標準品（糖鎖工学研究所社製）による競合ELISAスクリーニングを行った。その結果を元に、Hylに反応せず、GHL及びGGHLに同等の力価で反応する抗体を産生するクローンを選別した。得られたクローンの融合脾細胞を使ってマウス腹水を採取し（免疫生物研究所社）、HiTrap Protein A HPカラム（GE Healthcare社製）を用いて抗体（クローン1、クローン2）を精製した。
Example 2
A. Method (1) Antibody production
Antibodies that specifically bind to GHL and GGHL were produced using GANP (registered trademark) mice according to the method described in International Publication No. 2004/040971 (Transgenic). Three GANP mice were cocktail-immunized subcutaneously on the back using GHL (KLH-GHL; manufactured by Glycochemistry Institute, Inc.) bound to the carrier protein Keyhole limpet hemocyanin (KLH) as an immunogen. After three immunizations, an increase in antiserum titer was confirmed, and the spleen was removed from one high-titer mouse and fused with myeloma cells (P3U1). Competitive ELISA using Hyl standard product (manufactured by Sigma-Aldrich), GHL standard product (manufactured by Glyco Engineering Research Institute), and GGHL standard product (manufactured by Glyco Engineering Research Institute) using the culture supernatant of fused splenocytes as a sample. Screening was conducted. Based on the results, clones were selected that produced antibodies that did not react with Hyl but reacted with GHL and GGHL with equivalent titers. Mouse ascites was collected using the fused splenocytes of the obtained clones (Immune Biological Laboratories), and antibodies (clone 1, clone 2) were purified using a HiTrap Protein A HP column (GE Healthcare).
（２）標準品による競合ELISA（クローン1）
96穴プレートに基質として0.01 μg/mL濃度のKLH-GHLを室温で1時間コートし、1％ BSA/PBS-Tで4℃、一晩ブロッキングした。洗浄後、0.01％ BSA/PBS-Tで64000倍希釈した抗GHL・GGHL抗体（クローン1）を添加し、そこに競合物質として0～1600 nmol/mLのHyl、GHL又はGGHLを加えて室温で1時間反応させた。洗浄後、0.01％ BSA/PBS-Tで10000倍希釈したHRP標識ヤギ抗マウスIgG二次抗体（Jackson ImmunoResearch社製）を添加し、室温で1時間反応させて洗浄した後、TMB溶液（Kirkegaard & Perry Laboratories社製）で発色させ、プレートリーダーで450 nmの波長で測定を行った。
(2) Competitive ELISA using standard products (clone 1)
A 96-well plate was coated with KLH-GHL at a concentration of 0.01 μg/mL as a substrate for 1 hour at room temperature, and blocked with 1% BSA/PBS-T at 4°C overnight. After washing, add anti-GHL/GGHL antibody (clone 1) diluted 64,000 times with 0.01% BSA/PBS-T, add 0 to 1,600 nmol/mL of Hyl, GHL, or GGHL as a competitor and incubate at room temperature. The reaction was allowed to proceed for 1 hour. After washing, HRP-labeled goat anti-mouse IgG secondary antibody (manufactured by Jackson ImmunoResearch) diluted 10,000 times with 0.01% BSA/PBS-T was added and reacted for 1 hour at room temperature. After washing, TMB solution (Kirkegaard & Perry Laboratories, Inc.) and measured using a plate reader at a wavelength of 450 nm.
（３）尿検体の測定（クローン1）
人工膝関節置換術を受けた膝OA患者22名（男9名・女13名、平均69.3歳）と、コントロール群として年齢をマッチさせた膝OAのない31名(男11名・女20名、平均65.5歳)から尿検体を採取した。この尿検体を4 mmol/Lクレアチニン濃度に希釈し、抗GHL・GGHL抗体（クローン１）を用いて上記（２）と同様の方法で競合ELISAを行った。他方、同サンプルについて実施例１と同様に、LC-MSを用いてGHL及びGGHLの測定を行った。測定結果は、有意水準を0.05に設定したスチューデントのt検定で統計的に評価した。
(3) Measurement of urine sample (clone 1)
22 patients with knee OA who underwent total knee arthroplasty (9 males and 13 females, average age 69.3 years) and a control group of 31 age-matched patients without knee OA (11 males and 20 females) , average age 65.5 years). This urine sample was diluted to a concentration of 4 mmol/L creatinine, and competitive ELISA was performed using anti-GHL/GGHL antibody (clone 1) in the same manner as in (2) above. On the other hand, GHL and GGHL were measured using LC-MS on the same sample in the same manner as in Example 1. The measurement results were statistically evaluated using Student's t test with the significance level set at 0.05.
Ｂ．結果
抗GHL・GGHL抗体とこれに対する２次抗体を用いた競合ELISA測定系における、GHL、GGHL及びHylの標準品による反応阻害率を図３に示す。Hylでは阻害反応が起こらなかった一方、濃度依存性はやや低かったが、GHL、GGHLによって顕著かつ両者でほぼ同等の競合阻害が観察され、この結果から、ここで樹立した競合ELISA測定系は検体中のGHL、GGHLを特異的に検出可能であることが確認された。
この競合ELISA測定系におけるOA患者及び健常者の尿検体による阻害率を図４に示し、LC-MSで測定した同検体のGHL＋GGHL濃度を図５に示す。ELISA測定系において、健常者に比べてOA患者で有意に阻害率が上昇することが確認され、同様に、LC-MSで評価したGHL＋GGHL量もOA患者で有意に上昇することが確認された。
B. Results Figure 3 shows the reaction inhibition rate of GHL, GGHL and Hyl standard products in a competitive ELISA measurement system using anti-GHL/GGHL antibodies and secondary antibodies thereto. While no inhibitory reaction occurred with Hyl, and the concentration dependence was somewhat low, significant and almost equal competitive inhibition was observed with GHL and GGHL.From these results, the competitive ELISA measurement system established here can be used to It was confirmed that it was possible to specifically detect GHL and GGHL.
FIG. 4 shows the inhibition rates of urine samples from OA patients and healthy individuals in this competitive ELISA measurement system, and FIG. 5 shows the GHL+GGHL concentrations of the same samples measured by LC-MS. In the ELISA measurement system, it was confirmed that the inhibition rate was significantly increased in OA patients compared to healthy subjects, and similarly, it was confirmed that the amount of GHL + GGHL evaluated by LC-MS was also significantly increased in OA patients.
実施例３
Ａ．方法
（１）標準品による競合ELISA（HRP標識クローン２）
96穴プレートに基質として0.01 μg/mL濃度のKLH-GHLを室温で1時間コートし、1％ BSA/PBS-Tで4℃、一晩ブロッキングした。洗浄後、0.01％ BSA/PBS-Tで32000倍希釈した抗GHL・GGHL抗体（HRP標識クローン２：Peroxidase Labeling kit-NH2（同仁化学研究所社製）を用いHRP標識した）を添加し、そこに競合物質として0～4000 nmol/mLのHyl、GHL又はGGHLを加えて25℃で2時間反応させた。洗浄後、TMB溶液で発色させ、プレートリーダーで450 nmの波長で測定を行った。
Example 3
A. Method (1) Competitive ELISA using standard products (HRP-labeled clone 2)
A 96-well plate was coated with KLH-GHL at a concentration of 0.01 μg/mL as a substrate for 1 hour at room temperature, and blocked with 1% BSA/PBS-T at 4°C overnight. After washing, anti-GHL/GGHL antibody (HRP-labeled clone 2: HRP-labeled using Peroxidase Labeling kit-NH2 (manufactured by Dojindo Laboratories)) diluted 32,000 times with 0.01% BSA/PBS-T was added. 0 to 4000 nmol/mL of Hyl, GHL, or GGHL was added as a competitive substance and reacted at 25°C for 2 hours. After washing, color was developed with TMB solution, and measurement was performed using a plate reader at a wavelength of 450 nm.
（２）尿検体の測定（HRP標識クローン２）
人工膝関節置換術を受けた膝OA患者31名（男12名・女19名、平均68.9歳）と、コントロール群として年齢をマッチさせた膝OAのない43名(男17名・女26名、平均66.7歳)から尿検体を採取した。この尿検体を2 mmol/Lクレアチニン濃度に希釈し、抗GHL・GGHL抗体（HRP標識クローン２）を用いて上記（１）と同様の方法で競合ELISAを行った。他方、同サンプルについて実施例１と同様に、LC-MSを用いてGHL及びGGHLの測定を行った。測定結果は、有意水準を0.05に設定したスチューデントのt検定で統計的に評価した。
(2) Measurement of urine sample (HRP-labeled clone 2)
31 patients with knee OA (12 men, 19 women, average age 68.9 years) who underwent total knee arthroplasty and 43 age-matched patients without knee OA (17 men, 26 women) served as a control group. , average age 66.7 years). This urine sample was diluted to a concentration of 2 mmol/L creatinine, and competitive ELISA was performed using an anti-GHL/GGHL antibody (HRP-labeled clone 2) in the same manner as in (1) above. On the other hand, GHL and GGHL were measured using LC-MS on the same sample in the same manner as in Example 1. The measurement results were statistically evaluated using Student's t test with the significance level set at 0.05.
Ｂ．結果
HRP標識抗GHL・GGHL抗体を用いた競合ELISA測定系における、GHL、GGHL及びHylの標準品による反応阻害率を図６に示す。実施例２と同様に、GHL、GGHL特異的かつ両者でほぼ同等な反応阻害が確認され、このELISA測定系においてはGHL、GGHL濃度依存的な阻害率の上昇も示された。
この競合ELISA測定系におけるOA患者及び健常者の尿検体による阻害率を図７に示し、LC-MSで測定した同検体のGHL＋GGHL濃度を図８に示す。HRP標識抗GHL・GGHL抗体を用いることで、実施例２で使用した4 mmol/Lクレアチニン濃度よりも低い、2 mmol/Lクレアチニン濃度の尿検体でも測定が可能となり、このELISA測定系においても、健常者に比べてOA患者で有意に阻害率が上昇することが確認された。また、LC-MSで評価したGHL＋GGHL量もOA患者で有意に上昇することが確認された。
以上の結果から、GHLとGGHLの組み合わせはOAの検査用バイオマーカーとして有用であり、当該バイオマーカーを測定することでより精度の高いOAの診断が可能であることが確認された。
B. result
Figure 6 shows the reaction inhibition rate of GHL, GGHL, and Hyl standard products in a competitive ELISA measurement system using HRP-labeled anti-GHL/GGHL antibodies. As in Example 2, GHL- and GGHL-specific and substantially equivalent reaction inhibition was confirmed for both, and this ELISA measurement system also showed an increase in GHL and GGHL concentration-dependent inhibition rate.
The inhibition rates of urine samples from OA patients and healthy individuals in this competitive ELISA measurement system are shown in FIG. 7, and the GHL+GGHL concentrations of the same samples measured by LC-MS are shown in FIG. By using HRP-labeled anti-GHL/GGHL antibodies, it is possible to measure urine specimens with a 2 mmol/L creatinine concentration, which is lower than the 4 mmol/L creatinine concentration used in Example 2, and with this ELISA measurement system, It was confirmed that the inhibition rate was significantly increased in OA patients compared to healthy subjects. Furthermore, it was confirmed that the amount of GHL + GGHL evaluated by LC-MS was also significantly increased in OA patients.
From the above results, it was confirmed that the combination of GHL and GGHL is useful as a biomarker for testing OA, and that measuring this biomarker enables more accurate diagnosis of OA.
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| JP2019517007A (en) | 2016-04-15 | 2019-06-20 | イーエスエム テクノロジーズ エルエルシーＥＳＭ Ｔｅｃｈｎｏｌｏｇｉｅｓ， ＬＬＣ | Method for evaluating joint treatment |
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