JP7081861B1 - LIPに基づいてUMODを修飾した尿中のNeu5Gcを識別する尿路上皮癌の検査用キット、およびその製造方法 - Google Patents
LIPに基づいてUMODを修飾した尿中のNeu5Gcを識別する尿路上皮癌の検査用キット、およびその製造方法 Download PDFInfo
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Abstract
Description
活性化カルボキシル化ユーロピウム蛍光ナノ微小球を調製するステップと、
活性化カルボキシル化ユーロピウム蛍光ナノ微小球と濃度が1.41mg/mLのストレプトアビジンSAと濃度が5mg/mLのウサギIgGとを2:3:0.5の体積比で混合した後、結合緩衝液を加えて活性化カルボキシル化ユーロピウム蛍光ナノ微小球を2mg/mLの濃度に希釈して、37℃で2時間反応させるステップと、
反応液を4℃、15000×gの条件下で15分間遠心分離して沈殿物Aを取るステップと、
沈殿物Aに洗浄液を加えて超音波処理を行って再懸濁し、4℃、15000×gの条件下で15分間遠心分離して沈殿物Bを取るステップと、
沈殿物Bにブロッキング液を加えて超音波処理を行って再懸濁し、微小球懸濁液を得るステップと、
4℃、15000×gの条件下で微小球懸濁液を15分間遠心分離して沈殿物Cを取るステップと、
沈殿物Cに保存液を加えて超音波処理を行って再懸濁し、4℃、15000×gの条件下で15分間遠心分離して沈殿物Dを取るステップと、
沈殿物Dを保存液で2mg/mLの濃度に希釈するステップであって、前記保存液は、pHが7.8であり、再蒸留水を溶媒とし、25mmol/LのTris baseと、0.15mol/Lの塩化ナトリウムと、1wt%のウシ胎児アルブミンと、0.05wt%のTween-20と、5wt%のトレハロースとを含むステップと、を含む。
1mLのカルボキシル化ユーロピウム蛍光ナノ微小球の活性化緩衝液を試験管に加え、ボルテックスミキサーで十分に混合してカルボキシル化ユーロピウム蛍光ナノ微小球を懸濁し、4℃、15000×gの条件下で15分間遠心分離して上清液をマイクロピペットで吸い取り、このステップをもう1回繰り返す。カルボキシル化ユーロピウム蛍光ナノ微小球はThermo生物会社から購入した。
本発明により提供する尿路上皮癌の検査用キットを用いて、正常なヒト639例、良性疾患25例(膀胱炎症8例、膀胱嚢胞7例、膀胱ポリープ6例、腎炎4例)、尿路上皮癌患者99例(膀胱癌52例、腎臓癌20例、前立腺癌27例)の尿検体を検査測定した。結果を図1に示す。図1に示すように、正常なヒトの尿検体においてLIP特異的に識別される、UMOD糖タンパク質を修飾したNeu5Gcの含有量は67.63±9.31ng/mgであり、良性疾患25例の尿検体においてLIP特異的に識別される、UMOD糖タンパク質を修飾したNeu5Gcの含有量は70.71±8.20ng/mgであって、正常な人に比べて有意差はない。この結果は、泌尿器系癌のスクリーニングにおいて、尿路の炎症などの良性の問題がある場合、LIP特異的に識別される、UMOD糖タンパク質を修飾したNeu5Gcの含有量が正常な人に比べて差はなく、偽陽性の結果が発生しないことを示す。膀胱癌患者52例の尿検体においてLIP特異的に識別される、UMOD糖タンパク質を修飾したNeu5Gcの含有量が260.25±29.38ng/mgであり、腎癌患者20例の含有量は280.54±11.81ng/mg、前立腺癌患者27例の含有量は264.34±30.63ng/mgである。従って、本発明の検査キットは、尿路上皮癌の診断に優れた特異性を備える。
(付記1)
捕捉試薬と検出用緩衝液と試験紙とを備え、前記捕捉試薬は、ビオチン化結合のヤツメウナギ免疫タンパク質LIPであり、前記検出用緩衝液は、50mMのTris base、0.15MのNaCl、0.01%のTween-20及び7.5%のBSAを含んでなり、前記試験紙は、底板を有し、前記底板上には左から右の順で試料投入パッド、蛍光微小球結合釈放パッド、ニトロセルロース膜及び吸水パッドを段階状に順次固定され、前記蛍光微小球結合釈放パッドは、ユーロピウム蛍光ナノ粒子とストレプトアビジンSA及びウサギIgGの結合液をポリエステル繊維上に吹き付けることにより調製され、前記ニトロセルロース膜には左から右の順でUMODモノクローナル抗体から構成された検出バンドとヤギ抗ウサギIgGから構成された参照バンドが順次設けられる、ことを特徴とするLIPに基づいてUMODを修飾した尿中のNeu5Gcを識別する尿路上皮癌の検査用キット。
濃度が0.5mg/mLであるヤツメウナギ免疫タンパク質LIPの水溶液とビオチンを1mL:2mgの投与量の比率で室温で十分に混合して混合液を得、混合液を脱塩カラムで純化してビオチン化結合のヤツメウナギ免疫タンパク質LIPを得る、ことを特徴とする付記1記載のLIPに基づいてUMODを修飾した尿中のNeu5Gcを識別する尿路上皮癌の検査用キット。
ユーロピウム蛍光ナノ粒子とストレプトアビジンSA及びウサギIgGの結合液は以下の調製方法に従って調製され、前記調製方法は、
活性化カルボキシル化ユーロピウム蛍光ナノ微小球を調製するステップと、
前記活性化カルボキシル化ユーロピウム蛍光ナノ微小球と濃度が1.41mg/mLのストレプトアビジンSAと濃度が5mg/mLのウサギIgGとを2:3:0.5の体積比で混合した後、結合緩衝液を加えて前記活性化カルボキシル化ユーロピウム蛍光ナノ微小球を2mg/mLの濃度に希釈して、37℃で2時間反応させるステップと、
反応液を4℃、15000×gの条件下で15分間遠心分離して沈殿物Aを取るステップと、
前記沈殿物Aに洗浄液を加えて超音波処理を行って再懸濁し、4℃、15000×gの条件下で15分間遠心分離して沈殿物Bを取るステップと、
前記沈殿物Bにブロッキング液を加えて超音波処理を行って再懸濁し、微小球懸濁液を得るステップと、
4℃、15000×gの条件下で前記微小球懸濁液を15分間遠心分離して沈殿物Cを取るステップと、
前記沈殿物Cに保存液を加えて超音波処理を行って再懸濁し、4℃、15000×gの条件下で15分間遠心分離して沈殿物Dを取るステップと、
前記沈殿物Dを保存液で2mg/mLの濃度に希釈するステップであって、前記保存液は、pHが7.8であり、再蒸留水を溶媒とし、25mmol/Lの Tris baseと、0.15mol/Lの塩化ナトリウムと、1wt%のウシ胎児アルブミンと、0.05wt%のTween-20と、5wt%のトレハロースとを含むステップと、を含む、ことを特徴とする付記1又は付記2記載のLIPに基づいてUMODを修飾した尿中のNeu5Gcを識別する尿路上皮癌の検査用キット。
Claims (3)
- 捕捉試薬と検出用緩衝液と試験紙とを備え、前記捕捉試薬は、ビオチン化結合のヤツメウナギ免疫タンパク質LIPであり、前記検出用緩衝液は、50mMのTris base、0.15MのNaCl、0.01%のTween-20及び7.5%のBSAを含んでなり、前記試験紙は、底板を有し、前記底板上には左から右の順で試料投入パッド、蛍光微小球結合釈放パッド、ニトロセルロース膜及び吸水パッドを段階状に順次固定され、前記蛍光微小球結合釈放パッドは、ユーロピウム蛍光ナノ粒子とストレプトアビジンSA及びウサギIgGの結合液をポリエステル繊維上に吹き付けることにより調製され、前記ニトロセルロース膜には左から右の順でUMODモノクローナル抗体から構成された検出バンドとヤギ抗ウサギIgGから構成された参照バンドが順次設けられる、ことを特徴とするLIPに基づいてUMODを修飾した尿中のNeu5Gcを識別する尿路上皮癌の検査用キット。
- 濃度が0.5mg/mLであるヤツメウナギ免疫タンパク質LIPの水溶液とビオチンを1mL:2mgの投与量の比率で室温で十分に混合して混合液を得、前記混合液を脱塩カラムで純化して、前記ビオチン化結合のヤツメウナギ免疫タンパク質LIPを得る、ことを特徴とする請求項1に記載のLIPに基づいてUMODを修飾した尿中のNeu5Gcを識別する尿路上皮癌の検査用キットの製造方法。
- ユーロピウム蛍光ナノ粒子とストレプトアビジンSA及びウサギIgGの前記結合液は以下の調製方法に従って調製され、前記調製方法は、
活性化カルボキシル化ユーロピウム蛍光ナノ微小球を調製するステップと、
前記活性化カルボキシル化ユーロピウム蛍光ナノ微小球と濃度が1.41mg/mLのストレプトアビジンSAと濃度が5mg/mLのウサギIgGとを2:3:0.5の体積比で混合した後、結合緩衝液を加えて前記活性化カルボキシル化ユーロピウム蛍光ナノ微小球を2mg/mLの濃度に希釈して、37℃で2時間反応させるステップと、
反応液を4℃、15000×gの条件下で15分間遠心分離して沈殿物Aを取るステップと、
前記沈殿物Aに洗浄液を加えて超音波処理を行って再懸濁し、4℃、15000×gの条件下で15分間遠心分離して沈殿物Bを取るステップと、
前記沈殿物Bにブロッキング液を加えて超音波処理を行って再懸濁し、微小球懸濁液を得るステップと、
4℃、15000×gの条件下で前記微小球懸濁液を15分間遠心分離して沈殿物Cを取るステップと、
前記沈殿物Cに保存液を加えて超音波処理を行って再懸濁し、4℃、15000×gの条件下で15分間遠心分離して沈殿物Dを取るステップと、
前記沈殿物Dを保存液で2mg/mLの濃度に希釈するステップであって、前記保存液は、pHが7.8であり、再蒸留水を溶媒とし、25mmol/Lの Tris baseと、0.15mol/Lの塩化ナトリウムと、1wt%のウシ胎児アルブミンと、0.05wt%のTween-20と、5wt%のトレハロースとを含むステップと、を含む、ことを特徴とする請求項1に記載のLIPに基づいてUMODを修飾した尿中のNeu5Gcを識別する尿路上皮癌の検査用キットの製造方法。
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