JP5752688B2 - 抗原賦活プロセスのための方法及び装置 - Google Patents
抗原賦活プロセスのための方法及び装置 Download PDFInfo
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- JP5752688B2 JP5752688B2 JP2012526689A JP2012526689A JP5752688B2 JP 5752688 B2 JP5752688 B2 JP 5752688B2 JP 2012526689 A JP2012526689 A JP 2012526689A JP 2012526689 A JP2012526689 A JP 2012526689A JP 5752688 B2 JP5752688 B2 JP 5752688B2
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N1/31—Apparatus therefor
- G01N1/312—Apparatus therefor for samples mounted on planar substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Pathology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Sampling And Sample Adjustment (AREA)
- Peptides Or Proteins (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Description
「抗体」は、別の分子に特異的に結合する免疫グロブリンをいい、その分子の特定の空間及び極性構造と相補的なものと定義される。抗体は、モノクローナル抗体であっても又はポリクローナル抗体であってもよく、当技術分野で周知の技法、例えば、宿主の免疫化と血清(ポリクローナル)の回収、或いは連続ハイブリッド細胞系の調製と分泌タンパク質(モノクローナル)の回収)、或いは天然抗体の特異的結合に必要なアミノ酸配列を少なくともコードするヌクレオチド配列又はそれらの突然変異誘発型のクローニングと発現などによって調製できる。抗体は、完全な免疫グロブリン又はその断片を含むことができ、それらの免疫グロブリンとして、種々のクラス及びアイソタイプ、例えば、IgA、IgD、IgE、IgG1、IgG2a、IgG2b、及びIgG3、IgMが挙げられる。機能性の抗体断片は、完全長の抗体に類似する親和性で結合性を保持することが可能である抗体の部分(例えば、Fab、Fv及びF(ab’)2又はFab’)を含むことができる。さらに、免疫グロブリン又はそれらの断片の凝集体、高分子及びコンジュゲートも、特定の分子に対する結合親和性が実質的に維持される限り、適切な場合には使用することができる。
方法
システムの感受性を反映する信号対ノイズの比に影響を及ぼす変数を、実験計画法(DOE)のアプローチを使用して分析した。変数には、温度、曝露時間、pH、及び洗浄の順序を含めた。画像を、Zeiss Axilmager Z1顕微鏡を使用して倍率20×又は63×で獲得した。画像を、GNU Image Manipulationプログラム(GIMP)ソフトウエアを使用して定量化して、未染色の領域からのバックグラウンドのピクセル強度を減じた後の、面積当たりの平均ピクセル強度を計算した。平均ピクセル強度を、同じ組織の10個の画像の平均として計算した。
ヒト保存前立腺組織切片中のARを検出するためにアンドロゲン受容体の抗体を使用することを、最初に選択して分析した。この組合せは、エピトープのアンマスキングなしで染色すると、検出可能な信号が発生しない(図示せず)ことに起因して選んだ。抗原のアンマスキングのための2種類の一般的な緩衝液、すなわち、クエン酸又はTrisを使用し、試料を圧力釜中で20分間処理すると、蛍光色素を使用して中等度の強度の染色が得られた。用いたクエン酸アンマスキング条件は、様々な抗体によるARの検出に常用されているプロトコールである。クエン酸単独(セット1)、Tris単独(セット2)、順次Trisとクエン酸(セット3)、及び順次クエン酸とTris(セット4)を含めて、種々の条件を試験した。ベースラインの測定を確立するために10枚のスライドを染色した対照試料を除き、全ての条件を、スライド上で三つ組で試験した。
また、自動化プロセスを、Discovery XT自動染色装置を使用しても評価した。自動染色装置と手動の二段階プロセスとの間には、機器の操作に関連して処理条件に差があった。例えば、自動染色装置は、試料を脱ろうするために熱及び洗剤を使用し、一方、手動プロセスは、無毒性のHistochoice(商標)ろう洗浄試薬(Amresco)を使用した。
15 ホルムアルデヒド固定組織試料
20 試料取扱サブシステム
30 試薬分注サブシステム
40 信号検出サブシステム
Claims (15)
- ホルムアルデヒド固定組織試料の抗原を賦活する方法であって、当該方法が、
ホルムアルデヒド固定組織試料を第1の抗原賦活化溶液中で90℃超の温度でインキュベートする段階と、
ホルムアルデヒド固定組織試料を第2の抗原賦活化溶液に移す段階と、
ホルムアルデヒド固定組織試料を第2の抗原賦活化溶液中で90℃超の温度でインキュベートする段階と
を含んでおり、
第1の抗原賦活化溶液がpH範囲5〜7の緩衝液を含んでいて、第2の抗原賦活化溶液がpH範囲7.5〜11の緩衝液を含んでいるか、或いは第1の抗原賦活化溶液がpH範囲7.5〜11の緩衝液を含んでいて、第2の抗原賦活化溶液がpH範囲5〜7の緩衝液を含んでおり、
pH範囲5〜7の緩衝液が、クエン酸、リン酸二水素カリウム、ホウ酸、ジエチルバルビツール酸、ピペラジン−N,N’−ビス(2−エタンスルホン酸)、ジメチルアルシン酸、2−(N−モルホリノ)エタンスルホン酸又はそれらの組合せから選択され、
pH範囲7.5〜11の緩衝液が、トリス(ヒドロキシメチル)メチルアミン(TRIS)、2−(N−モルホリノ)エタンスルホン酸(TAPS)、N,N−ビス(2−ヒドロキシエチル)グリシン(Bicine)、N−トリス(ヒドロキシメチル)メチルグリシン(Tricine)、4−2−ヒドロキシエチル−1−ピペラジンエタンスルホン酸(HEPES)、2−{[トリス(ヒドロキシメチル)メチル]アミノ}エタンスルホン酸(TES)又はそれらの組合せから選択され、
第1の抗原賦活化溶液と第2の抗原賦活化溶液とを用いた処理が、第2の抗原賦活化溶液を用いずに第1の抗原賦活化溶液を用いた場合及び第1の抗原賦活化溶液を用いずに第2の抗原賦活化溶液を用いた場合に比べて、組織試料における抗原賦活化を増大させる、方法。 - pH範囲5〜7の緩衝液がクエン酸を含む、請求項1記載の方法。
- pH範囲7.5〜11の緩衝液がTRISを含む、請求項1又は請求項2記載の方法。
- ホルムアルデヒド固定組織試料をパラフィン中に包埋する、請求項1乃至請求項3のいずれか1項記載の方法。
- ホルムアルデヒド固定組織試料が、組織の切片部分である、請求項1乃至請求項4のいずれか1項記載の方法。
- 第1の抗原賦活化溶液と共にインキュベートする段階及び第2の抗原賦活化溶液と共にインキュベートする段階が、加熱デバイス中で10分超かけてインキュベートすることを含む、請求項1乃至請求項5のいずれか1項記載の方法。
- 加熱デバイスが、圧力釜、オートクレーブ、水浴、加熱板、マイクロ波、蒸気加熱又はそれらの組合せである、請求項6記載の方法。
- 抗原を免疫染色する段階をさらに含む、請求項1乃至請求項7のいずれか1項記載の方法。
- 免疫染色することが、免疫ペルオキシダーゼで標識すること及び標識を消去することを順次に行うことを含む、請求項8記載の方法。
- ホルムアルデヒド固定組織試料に対して、FISH又はCISH分析を行う、請求項1乃至請求項9のいずれか1項記載の方法。
- 追加の処理を行わずに前記組織試料を第2の抗原賦活化溶液に移す、請求項1乃至請求項10のいずれか1項記載の方法。
- ホルムアルデヒド固定組織試料中の抗原の賦活化用キットであって、当該キットが、
試料中の未賦活抗原の少なくとも一部を賦活する第1の抗原賦活化溶液、及び
試料中の未賦活抗原の別の部分の少なくとも一部を賦活する第2の抗原賦活化溶液
を含んでおり、
第1の抗原賦活化溶液がpH範囲5〜7の緩衝液であって、第2の抗原賦活化溶液がpH範囲7.5〜11の緩衝液であり、
第1の抗原賦活化溶液が、クエン酸、リン酸二水素カリウム、ホウ酸、ジエチルバルビツール酸、ピペラジン−N,N’−ビス(2−エタンスルホン酸)、ジメチルアルシン酸、2−(N−モルホリノ)エタンスルホン酸又はそれらの組合せを含んでおり、
第2の抗原賦活化溶液が、トリス(ヒドロキシメチル)メチルアミン(TRIS)、2−(N−モルホリノ)エタンスルホン酸(TAPS)、N,N−ビス(2−ヒドロキシエチル)グリシン(Bicine)、N−トリス(ヒドロキシメチル)メチルグリシン(Tricine)、4−2−ヒドロキシエチル−1−ピペラジンエタンスルホン酸(HEPES)、2−{[トリス(ヒドロキシメチル)メチル]アミノ}エタンスルホン酸(TES)又はそれらの組合せを含んでいる、キット。 - 第1の抗原賦活化溶液がクエン酸を含む、請求項12記載のキット。
- 第2の抗原賦活化溶液がTRISを含む、請求項12又は請求項13記載のキット。
- 1種以上の免疫染色試薬をさらに含む、請求項12乃至請求項14のいずれか1項記載のキット。
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