JP6879496B2 - 小分子のためのサンドイッチアッセイ - Google Patents
小分子のためのサンドイッチアッセイ Download PDFInfo
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- JP6879496B2 JP6879496B2 JP2018521922A JP2018521922A JP6879496B2 JP 6879496 B2 JP6879496 B2 JP 6879496B2 JP 2018521922 A JP2018521922 A JP 2018521922A JP 2018521922 A JP2018521922 A JP 2018521922A JP 6879496 B2 JP6879496 B2 JP 6879496B2
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- JP
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- Prior art keywords
- sirolimus
- antibody
- binding
- tacrolimus
- binding partner
- Prior art date
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- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 1
- PXQLVRUNWNTZOS-UHFFFAOYSA-N sulfanyl Chemical class [SH] PXQLVRUNWNTZOS-UHFFFAOYSA-N 0.000 description 1
- 150000003871 sulfonates Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 210000004243 sweat Anatomy 0.000 description 1
- 210000001179 synovial fluid Anatomy 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- 229940037128 systemic glucocorticoids Drugs 0.000 description 1
- 210000001138 tear Anatomy 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 150000007970 thio esters Chemical class 0.000 description 1
- 150000003556 thioamides Chemical class 0.000 description 1
- 150000003585 thioureas Chemical class 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 229940074409 trehalose dihydrate Drugs 0.000 description 1
- 150000005671 trienes Chemical group 0.000 description 1
- 150000003672 ureas Chemical class 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 125000005500 uronium group Chemical group 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2470/00—Immunochemical assays or immunoassays characterised by the reaction format or reaction type
- G01N2470/04—Sandwich assay format
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Description
本発明者らは、小分子の離間した部分に同時に特異結合する、例えば抗体等の、結合パートナーを設計することができることを発見した。これは驚くべき発見である。何故なら、小分子はハプテンであり、ハプテンは、比較的小さな分子(分子量(ダルトン)約2,500未満、又は約2,000未満、又は約1,500未満、又は約1,000未満)であり、抗体が結合できる部位を2つ以上有するとは考えられていないからである。本明細書に記載される原理に従えば、少なくとも2つの異なる結合パートナーを調製でき、これらは小分子の離間した部分に同時に結合する。第1の結合パートナーは、小分子の第1部分に特異的であってこれに結合する。第2の結合パートナーは、小分子の第1部分以外の第2部分で小分子に結合し、第2の結合パートナーは、小分子又は小分子の誘導体ではないハプテンを含有する免疫原から調製され、ここで、この免疫原のハプテンは、小分子の第2部分のそれと構造的に類似した部位を含んでいる。
[特定の例としてのシロリムス]
[シロリムスのサンドイッチアッセイのための抗体の調製]
[小分子のためのアッセイの概要]
[アッセイを実施するためのキット]
mg=ミリグラム
g=グラム
ng=ナノグラム
mL=ミリリットル
μL=マイクロリットル
mmol=ミリモル
μmol=マイクロモル
℃=摂氏度
min=分
sec=秒
hr=時間
w/v=重量/体積
v/v=体積/体積
TLC=薄層クロマトグラフィ
HPLC=高速液体クロマトグラフィ
UV=紫外線
EtOAc=酢酸エチル
MeOH=メタノール
DMF=ジメチルホルムアミド
DI=脱イオン化された
THF=テトラヒドロフラン
NHS=N−ヒドロキシスクシンイミド
DCC=Ν,Ν−ジシクロヘキシルカルボジイミド
BSA=ウシ血清アルブミン
BGG=ウシガンマグロブリン
MS=質量分析法
SIRO=シロリムス
rotovap=ロータリーエバポレータ
(シロリムスに対するモノクローナル抗体の調製)
(C−32−シロリムスオキシム及びC−26−シロリムスオキシム(IVa及びIVb)の調製(図6))
シロリムス(I)(653.6mg、0.715mmol)及びカルボキシメトキシアミンヘミ塩酸塩(234.4mg、2.14mmol)のMeOH(20mL)中の溶液に酢酸ナトリウム(181.8mg、3.1mmol)を加える。反応混合物を窒素雰囲気下で室温(23℃)で一晩(18hr)撹拌する。TLC分析により反応の完了が示された(TLC、シリカゲルプレート、CH2Cl2/MeOH=9/1)。CH2C12(80mL)及びDI水(20mL)を混合物に加え、混合物を10分間攪拌する。CH2C12層を分離する。水層をCH2C12(3×30mL)で抽出する。CH2C12溶液を合一してDI水で洗浄し(2×40mL)Na2SO4で乾燥し、濾過しrotovapで濃縮してC−32−シロリムスオキシムとC−26−シロリムスオキシムとの混合物(IVa及びIVb、622mg)を得る。
C−32−シロリムスオキシムとC−26−シロリムスオキシムとを分離するための最適なTLC条件(シリカゲル、EtOAc/ヘキサン/MeOH=5/2/1、RfC−32−オキシム=0.59、RfC−26−オキシム=0.51)を開発し、BIOTAGE(登録商標)ISOLERA(商標)ワンフラッシュクロマトグラフィシステム(John Morris Scientific、ニューサウスウェールズ州チャッツウッド)に成功裡に適用する。C−32−シロリムスオキシムとC−26−シロリムスオキシムとの混合物(IVa及びIVb、622mg)をCH2C12(5mL)中に溶解する。このCH2C12溶液をBIOTAGE(登録商標)ISOLERA(商標)ワンフラッシュクロマトグラフィシステムに付随するカートリッジ(シリカ、50gSNAP Ultra)に溶出させる。このシステムを、混合溶媒を用い25mL/minの流速で、作動させる。カートリッジから回収した全ての画分をTLC(EtOAc/ヘキサン/MeOH=5/2/1)によりチェックする。TLC分析に基づき、より極性の高い純粋な画分(RfC−26−オキシム=0.51)を合一して濃縮し、C−26−シロリムスオキシム(IVa)(197mg)を得る。この化合物のHPLC−UV分析により95%の純度が示される。
IVa(167.97mg、0.17mmol)のTHF/DMF(8mLのTHF、0.4mLのDMF)中溶液に、NHS(41.8mg、0.35mmol)及びDCC(70.9mg、0.34mmol)を加える。反応混合物を窒素雰囲気下、室温で撹拌する。生成物のNHSエステルは、TLC分析において、化合物IVaよりもやや極性が低い。反応中に形成される白色固体を濾過し、次いでEtOAcで洗浄する。溶媒を除去したのち、反応混合物をEtOAcに再溶解して濾過する。溶媒を留去して淡黄色の固体を得、これを高真空下で1時間保持する。
シロリムス分子のドメインD1に結合するモノクローナル抗体を以下のように調製する。免疫原は上述のように調製したBSA接合体Vaである。この免疫原を用いてBalb/cマウスを免疫する。1回目の免疫は、腹腔内においてモノホスホリルリピドA及び合成トレハロースジコリノミコラートアジュバント(RIBI MPL+TDMエマルション、RIBI ImmunoChem Research Inc.、モンタナ州ハミルトン)を用い体積200μL中25μgで行なう。5週間後、200μLのモノホスホリルリピドA及び合成トレハロースジコリノミコラートアジュバント中の25μgの免疫原により腹腔内において追加免疫を与える。その後、更に8週間後、静脈内及び腹腔内で200μLのハンクス平衡塩類溶液中25μgの免疫原による融合前追加免疫(prefusion boost)を与える。
この前処理溶液は、5μg/mLのFK506、6.8mg/mLのPIPES(商標)1.5ナトリウム塩、0.3mg/mLのEDTAジナトリウム、1.0mg/mLのサポニン、0.2%のPROCLIN(登録商標)300、0.024mg/mLのネオマイシン硫酸塩、及び0.99mg/mLのNaN3を含有する(pH6.5)。最終反応混合物中のFK506の濃度は、1.1μg/mLである。
(タクロリムス−キーホールリンペットヘモシアニン接合体の調製)
タクロリムスモノオキシム(32.3mg、36.8μmol)の1.05mLの無水ジメチルホルムアミド中の溶液に1−エチル−3−(3−ジメチルアミノプロピル)カルボジイミド塩酸塩(EDAC)(11mg、57.4μΜ、1.5当量)及びN−ヒドロキシスクシンイミド(7.3mg、63.4μΜ、1.7当量)を加える。タクロリムス分子のC22位で連結が生ずる。この反応液を、アルゴン下、室温で1時間撹拌する。次いで、この混合物をシリンジを介してキーホールリンペットヘモシアニン(74mg、純度54%)の、5.0mLのホスフェート緩衝塩水(0.1M、pH8.0)及び0.25mLのジメチルホルムアミド中溶液に滴下添加する。室温で2時間撹拌した後、得られた懸濁液をPBS(ホスフェート緩衝塩水)(10mM、pH7.0)に対して透析する(1×4L、4℃、2時間)。
タクロリムス及びシロリムスの両方に結合するモノクローナル抗体を以下のようにして調製する。免疫原は、上述のように調製したタクロリムス−キーホールリンペットヘモシアニン接合体である。この免疫原を使用してBalb/cマウスを免疫する。1回目の免疫は、腹腔内において、モノホスホリルリピドA及び合成トレハロースジコリノミコラートアジュバント(RIBI MPL+TDMエマルション、RIBI ImmunoChem Research Inc.、モンタナ州ハミルトン)を用い体積200μL中、25μgで行なう。5週間後、200μLのモノホスホリルリピドA及び合成トレハロースジコリノミコラートアジュバント中、25μgの免疫原により腹腔内において追加免疫を与える。その後、更に8週間後、静脈内及び腹腔内で200μLのハンクス平衡塩類溶液中、25μgの免疫原による融合前追加免疫(prefusion boost)を与える。
(自動化クローム粒子サンドイッチアッセイを用いたシロリムスの判定)
シロリムスのドメインD1に結合するモノクローナル抗体を用いた抗シロリムスF(ab’)2−β−ガラクトシダーゼ接合体の調製
シロリムスのドメインD1に結合するモノクローナル抗シロリムス抗体(実施例1において上述したように調製した。)を、リジル−エンドペプチダーゼ(Wako、ヴァージニア州リッチモンド)消化を用いてF(ab’)2に断片化した後、既知の技術に従い標準的ヘテロ二機能性SMCC(スクシンイミジルトランス−4−(N−マレイミジルメチル)シクロヘキサン−1−カルボキシレート)リンカーを用いて、β−ガラクトシダーゼに接合する。抗体接合体溶液は、およそ2.0μg/mLの抗シロリムス抗体−β−ガラクトシダーゼ接合体、30mg/mLのプロテアーゼ非含有ウシ血清アルブミン、0.126mg/mLのMgCl2、0.03mL/mLのエチレングリコール、24.5mg/mLのHEPES、38.5mg/mLのNaHEPES、50mg/mLのNaCl及びβ−galムテイン(不活性化ベータガラクトシダーゼ)を含有する(pH7.8)。
シロリムスのドメインD1に結合するモノクローナル抗体(免疫原としてC−26−シロリムスオキシム−BSA接合体(Va)(図6中R5=BSA)を使用して実施例1において上述したようにして調製した。)を、グルタルアルデヒドを被着した二酸化クロム粒子に接合することによって、クローム粒子(イムノアッセイ固相)を調製する。このクローム試薬は、クローム粒子、60.4mg/mLのトレハロース二水和物及び7.2mg/mLのポリエチレングリコール(PEG)8000を含有する。本検討においては、3つのクローム粒子濃度、即ち、5、2.5及び1.67mg/mLを使用する。
シロリムスのためのサンドイッチアッセイの原理及び操作は以下のとおりである:DIMENSION(登録商標)RxL分析器上の反応容器中で、シロリムス含有全血試料(50μL)を、上述したようにして調製した溶血性の前処理試薬と組み合わせる。この全血は、超音波サンプルプローブを用いて最初に血液を混合することによってスタンダードカップから採取する。全血試料を前処理溶液と混合することにより、全血の溶血及びタンパク質結合シロリムス分子のそれらの結合ドメインからの除去置換が確実に行なわれる。
(自動化ELISAサンドイッチアッセイを用いたシロリムスの判定)
(シロリムスのためのサンドイッチ酵素結合免疫吸着アッセイ(ELISA))
以下の工程を採用する。工程1:シロリムスのドメインD1に結合する精製モノクローナル抗体(免疫原としてC−26−シロリムスオキシム−BSA接合体(Va)(図6中、R5=BSA)を使用し実施例1において上述したようにして調製した。)(PBS中10μg/mL)50μLを4℃で一晩ELISAプレート上にコーティングする。これらのプレートを、0.05%のTWEEN(登録商標)20を含有するMILLI−Q(登録商標)水(Millipore Corporation、マサチューセッツ州ビレリカ)を用いて洗浄する。工程2:200μLのPCTブロッカー溶液(0.05%のTWEEN(登録商標)20を含有するホスフェートバッファ中の0.5%カゼイン(乳タンパク質))を各ウェルに加えて、媒体を室温で30分間インキュベートする。プレートを0.05%のTWEEN(登録商標)20を含有するMILLI−Q(登録商標)水を用いて洗浄する。工程3:PBS中に希釈した所望の濃度のシロリムス50μLを各ウェルに加えて媒体を室温で30分間インキュベートする。プレートを0.05%のTWEEN(登録商標)20を含有するMILLI−Q(登録商標)水を用いて洗浄する。試験されるシロリムス薬物濃度は、それぞれ0、0.01、0.02、0.04、0.08、0.16、0.31、0.63、1.25、2.50、5.0、及び10.0ng/mLとする。工程4:タクロリムス及びシロリムスの両方に結合するモノクローナル抗体を用いて調製した抗シロリムスF(ab’)2−β−ガラクトシダーゼ接合体(上述したのと同様にして調製した。)(PCTブロッカー溶液中1:300希釈)を加えて、媒体を室温で30分間インキュベートする。プレートを0.05%のTWEEN(登録商標)20を含有するMILLI−Q(登録商標)水を用いて洗浄する。工程5:β−ガラクトシダーゼ基剤溶液CPRGを各ウェルに加える(100μL/ウェル)。工程6:これらのウェルを、プレートリーダーで577nmで1分毎に20分間測定する。結果はシロリムスの良好な検出を示す。
Claims (5)
- 500〜2,500の分子量を有する15〜50員環の免疫抑制薬を含有することが疑われる試料中における当該免疫抑制薬の存在及び/又は量を判定する方法であって、当該免疫抑制薬がシロリムスであり、
(a)媒体中で、
(i)前記試料、
(ii)前記免疫抑制薬の第1部分に特異的であって該第1部分に結合する第1の結合パートナーであって、タクロリムスに対して実質的に結合親和性を示さない第1のモノクローナル抗体である第1の結合パートナー、及び
(iii)前記免疫抑制薬の前記第1部分以外の第2部分で前記免疫抑制薬に結合する第2の結合パートナーであって、前記免疫抑制薬又はその誘導体ではないハプテンであって前記免疫抑制薬の前記第2部分のものと構造的に類似した部位を含有するハプテンを含有する免疫原から調製される、抗タクロリムス抗体であってタクロリムス及びシロリムスの双方に結合するように調製され選択される第2のモノクローナル抗体である第2の結合パートナー
を組み合わせて提供する工程、
(b)前記第1の結合パートナー及び前記第2の結合パートナーを前記免疫抑制薬に結合させるための条件下で、前記媒体をインキュベートする工程、及び
(c)前記媒体について、前記免疫抑制薬と前記第1の結合パートナーと前記第2の結合パートナーとを含有する免疫複合体の存在を試験する工程であって、前記免疫複合体の存在及び/又は量が前記試料中の前記免疫抑制薬の存在及び/又は量を示すものである工程
を含む、方法。 - 前記免疫原のハプテンがタクロリムス又はその誘導体である請求項1に記載の方法。
- 前記第1の結合パートナー及び前記第2の結合パートナーのうちの一方がシグナル生成系の構成要素を含有する請求項1又は2に記載の方法。
- 前記第1の結合パートナー及び前記第2の結合パートナーのうちの一方が支持体に結合される請求項1〜3のいずれか1項に記載の方法。
- 前記第1部分がトリエン部分から成る請求項1〜4のいずれか1項に記載の方法。
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EP3234607B1 (en) * | 2014-12-17 | 2021-04-28 | Siemens Healthcare Diagnostics Inc. | Sandwich assay design for small molecules |
US10408764B2 (en) | 2017-09-13 | 2019-09-10 | Applied Materials Israel Ltd. | System, method and computer program product for object examination |
CN114008457A (zh) * | 2019-06-28 | 2022-02-01 | 美国西门子医学诊断股份有限公司 | 使用颗粒增强凝集检测的夹心免疫测定试剂及其生产和使用方法 |
CN110907649A (zh) * | 2019-12-23 | 2020-03-24 | 山东莱博生物科技有限公司 | 一种检测短链脂肪酸的试剂盒及其应用 |
CN113176403A (zh) * | 2021-04-21 | 2021-07-27 | 佛山职业技术学院 | 一种检测啶虫脒的双抗夹心elisa试剂盒及其应用 |
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EP3234607B1 (en) * | 2014-12-17 | 2021-04-28 | Siemens Healthcare Diagnostics Inc. | Sandwich assay design for small molecules |
CN112912094A (zh) * | 2018-11-02 | 2021-06-04 | 美国西门子医学诊断股份有限公司 | 用于结合巨菲蛋白的药物测定中的结合竞争剂及其使用方法 |
CN114008457A (zh) * | 2019-06-28 | 2022-02-01 | 美国西门子医学诊断股份有限公司 | 使用颗粒增强凝集检测的夹心免疫测定试剂及其生产和使用方法 |
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EP3368900B1 (en) | 2020-12-23 |
EP3368900A1 (en) | 2018-09-05 |
US11913965B2 (en) | 2024-02-27 |
US20210199677A1 (en) | 2021-07-01 |
US20240159781A1 (en) | 2024-05-16 |
US20180306828A1 (en) | 2018-10-25 |
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EP3368900A4 (en) | 2018-09-05 |
WO2017074703A1 (en) | 2017-05-04 |
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