JP5616851B2 - イヌのproBNPの決定 - Google Patents
イヌのproBNPの決定 Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Brain natriuretic peptide [BNP, proBNP]; Cardionatrin; Cardiodilatin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
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- Molecular Biology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Description
下記のステップ;
−ネコまたはイヌのproBNPサンプルを提供するステップ、
−該サンプルと、ネコのproBNPまたはそのフラグメントを決定する場合に、ネコのproBNPのアミノ酸20〜42を含む領域および/またはアミノ酸57〜80を含む領域において少なくとも一つのエピトープに結合する少なくとも一つの抗体を接触させるステップ、またイヌのproBNPまたはそのフラグメントを決定する場合に、イヌのproBNPのアミノ酸20〜86を含む領域において少なくとも一つのエピトープに結合する少なくとも一つの抗体を接触させるステップ、
そして
−サンプル中に存在するネコまたはイヌのproBNPまたはそのフラグメントの存在および/または濃度を決定するステップ、
を含む、ネコまたはイヌのproBNPまたはそのフラグメントを決定する方法を提供するものである。
ネコまたはイヌのproBNP各々の開示された領域におけるエピトープに結合し得る抗体が、proBNPを特異的に決定するために十分に適当であることが判った。
本発明の少なくとも一つの抗体または本発明の少なくとも一つの抗体混合物、
少なくとも一つの抗体のまたは少なくとも一つの抗体混合物が、ネコまたはイヌのproBNPまたはそのフラグメントに結合することを定性的および/または定量的に検出するための手段、および
所望により、ポジティブコントロールまたは濃度決定のための標準として、本発明のペプチドおよび/またはネコまたはイヌのproBNPまたはそのフラグメント、
を含む、
ネコまたはイヌのproBNPまたはそのフラグメントを決定するためのキットに関する。
エピトープは、例えば、Fraga S.("Theoretical prediction of protein antigenic determinants from amino acid sequences.", 1982; Can. J. Chem. 60:2606-2610)のアルゴリズムのProtScale(http://www.expasy.org/tools/protscale.html)を用いる計算によって決定できる。
ヒツジの血液から回収されるproBNPのペプチド/エピトープに対する抗体または血清の各反応性をELISA試験によってアッセイした。最初に、マイクロタイタープレートを、4℃でストレプトアビジン(0.5μg/ml, 200 μl/ウェル)にて終夜被覆し、洗浄し、0.25% Tween 20 を含有する0.1 M PBS(pH 7.5)の1% BSAでブロッキングし、1回以上洗浄し、3時間4℃でビオチン(0.25 μg/ml, 200 μl/ウェル)と結合した合成proBNPペプチド配列と共にインキュベートした。さらなる洗浄ステップの後、血清サンプルを、3%BSAを含有する0.1Mリン酸緩衝液にて1:1000/1:10000/1:100000希釈し、マイクロタイタープレートに加えた。プレート上のペプチド/エピトープに対する抗体の結合は、セイヨウワサビペルオキシダーゼと結合した抗ヒツジIgG抗体およびTMB(テトラメチル-ベンジジン)を含有する基質溶液の添加によって決定した。セイヨウワサビペルオキシダーゼとTMBとの反応を、0.9% 硫酸の添加により停止させた。顕色展開を、マイクロタイタープレートを分析し得る分光計を用いて追跡した。
本発明の抗体の一つを用いて被覆したマイクロタイタープレートのウェル中に、ネコまたはイヌ血清(20 μl)をピペットで移し、0.1Mリン酸緩衝液(pH 7)中のさらなるペルオキシダーゼ標識した本発明の第二の抗体(200 μl)と共に4〜16時間、室温でインキュベートした。次に、マイクロタイタープレートを、0.1% トライトンx100を有する0.1Mリン酸緩衝液(300 μl, pH 7)を用いて5回洗浄し、テトラメチルベンジジン(200 μl)を基質として添加した。20〜30分間の顕色展開の後、該反応を0.9%硫酸(50 μl)を添加することによって停止し、Nt-proBNPの量に直接的に比例する色調強度を、マイクロタイタープレート分光計によって測定した。正確な濃度は、組換え体ネコまたはイヌのNt-proBNP由来の校正曲線との比較によって決定した。
組換え体ネコ、イヌおよびヒトNt-proBNPを、マイクロタイタープレート(250 ng/ml, 200 μl/ウェル, 終夜室温)上に被覆した。次に、このプレートを洗浄し、抗ヒト、抗ネコおよび抗イヌの抗血清(10-100 μg/ml, 0.1 M リン酸緩衝液中で, pH 7)の希釈物と接触させた。洗浄工程の後に、結合抗体の量を適当な二次抗体(ペルオキシダーゼ標識化抗ヒツジ抗体)を用いて測定した。各抗体は、対応するNt-proBNP分子(即ち、ネコ Nt-proBNPを用いる抗ネコ抗血清)とは十分反応するが、驚くべきことに各々他種のNt-proBNP分子と反応しないかまたは非常にわずかな程度反応することが示された。
Claims (8)
- イヌのサンプル中において、イヌのproBNPまたはそのフラグメントを決定する方法であって、
(a)イヌの血液または尿サンプルを提供し、
(b)該サンプルと、配列番号2または配列番号5のアミノ酸配列を含むイヌのproBNPの領域にあるエピトープに結合する少なくとも一つの抗体を接触させ、
(c)該サンプル中に存在するイヌの該エピトープまたはそのフラグメントの存在および/または濃度を決定すること
を含む方法。 - 少なくとも一つの抗体がポリクローナルであることを特徴とする、請求項1記載の方法。
- 少なくとも一つの抗体がモノクローナルであることを特徴とする、請求項1記載の方法。
- 少なくとも一つのさらなる抗体が、該少なくとも一つの抗体または該エピトープに結合することを特徴とする、請求項1〜3のいずれかに記載の方法。
- 少なくとも一つの抗体および/または少なくとも一つのさらなる抗体が標識されることを特徴とする、請求項1〜4のいずれかに記載の方法。
- 少なくとも一つの抗体および/または少なくとも一つのさらなる抗体が、ペルオキシダーゼ、ビオチン、蛍光染料、金コロイドまたは放射性核種で標識されることを特徴とする、請求項5記載の方法。
- 少なくとも一つの抗体または少なくとも一つのさらなる抗体が、固体相に結合することを特徴とする、請求項1〜6のいずれかに記載の方法。
- イヌのproBNPまたはそのフラグメントの決定が、ラジオイムノアッセイ、免疫結合アッセイ、ウェスタンブロット、免疫組織化学、酵素イムノアッセイ、側方流動装置、およびその組合せ物からなる群から選択される方法によって行われることを特徴とする、請求項1〜7のいずれかに記載の方法。
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AT500800B1 (de) * | 2004-09-08 | 2008-07-15 | Biomedica Medizinprodukte Gmbh | Verfahren zur bestimmung von probnp |
WO2008058004A2 (en) * | 2006-11-02 | 2008-05-15 | Winnik Mitchell A | Particles containing detectable elemental code |
CA2669024C (en) * | 2006-11-10 | 2016-01-12 | Hytest Ltd. | Stable standards for bnp immunoassays |
AT504942B1 (de) * | 2007-03-07 | 2009-08-15 | Biomedica Medizinprodukte Gmbh | Verfahren zur bestimmung von probnp |
WO2012019012A1 (en) * | 2010-08-04 | 2012-02-09 | Idexx Laboratories, Inc. | Detection of degradation products of canine nt-probnp |
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JP2014210761A (ja) * | 2013-04-05 | 2014-11-13 | 株式会社シバヤギ | 抗イヌn末端プロ心房性ナトリウム利尿ペプチド抗体ならびにそれを用いた免疫学的測定方法、および免疫学的測定用キット |
CN103529224B (zh) * | 2013-10-17 | 2016-09-07 | 天津中新科炬生物制药有限公司 | 同时检测人脑钠肽和n末端脑钠肽前体的快速定量检测装置及检测方法 |
WO2018074533A1 (ja) * | 2016-10-18 | 2018-04-26 | 積水メディカル株式会社 | 抗ヒトbnp断片(4-32)抗体を用いた免疫測定方法 |
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CN111781385B (zh) * | 2020-08-19 | 2023-07-18 | 武汉生之源生物科技股份有限公司 | 一种NT-proBNP检测试剂盒及其制备方法 |
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