JP4487081B2 - Activity determination method and apparatus for renal disease - Google Patents

Activity determination method and apparatus for renal disease Download PDF

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JP4487081B2
JP4487081B2 JP2007512412A JP2007512412A JP4487081B2 JP 4487081 B2 JP4487081 B2 JP 4487081B2 JP 2007512412 A JP2007512412 A JP 2007512412A JP 2007512412 A JP2007512412 A JP 2007512412A JP 4487081 B2 JP4487081 B2 JP 4487081B2
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JPWO2006106598A1 (en
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実 坂爪
文武 下条
武英 松田
亮 久保田
麻 小川
大介 嵯峨
一衛 成田
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国立大学法人 新潟大学
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Description

【技術分野】
【0001】
本発明は、腎疾患の活動性判定法及びその装置に関する。
【背景技術】
【0002】
腎炎など腎臓の炎症性疾患の活動性判定は、従来、腎生検を行って病理学的に判断するか、あるいは、尿蛋白量の増加の有無をもってなされていた。しかし、腎生検は、侵襲的な検査法であり、疾患の経過中に複数回施行することは困難であった。また、尿蛋白は、腎臓の炎症の程度だけでなく、高血圧など様々な原因によって影響を受け、活動性の指標としての特異性は低かった。
【0003】
そこで、本発明者らは、多数の臨床例を検討した結果、腎臓に活動性炎症病変がある場合に、尿中に炎症性の活性型細胞が出現することから、免疫細胞特異的モノクローナル抗体とフローサイトメトリーを用いて炎症性免疫細胞の数の測定を行うことによって、腎臓の炎症の活動性を判定する方法を見出した(非特許文献1)。
【0004】
この方法は、腎臓の活動性炎症の有無を判定する手段として、蛍光標識抗CD3モノクローナル抗体と蛍光標識抗CD62Lモノクローナル抗体を用いて尿沈渣を処理し、フローサイトメトリー法でCD3陽性CD62L陰性のTリンパ球を検出し、その数が多数の場合と、蛍光標識抗CD3モノクローナル抗体と蛍光標識抗CD45ROモノクローナル抗体と蛍光標識抗CD45RAモノクローナル抗体を用いて尿沈渣を処理し、フローサイトメトリー法でCD3陽性CD45RO陰性CD45RA陽性のTリンパ球を検出し、その数が多数の場合に、腎臓の活動性炎症があると判定するものである。
【0005】
この方法によれば、従来の腎生検や尿蛋白による判定法に代わり、非侵襲的に腎疾患の活動性の判定を行うことができる。
【0006】
しかし、上記腎疾患の活動性の判定法は、Tリンパ球を検出するものであったが、判定結果の確実性が低いという欠点があった。
【0007】
すなわち、本発明者らが検討した結果、腎炎を伴わない原因不明の血尿を呈する特発性腎出血症例においては、末梢血型のCD3陽性CD62L陽性のTリンパ球が、CD3陽性CD62L陰性のTリンパ球に比べ優位に出現する。しかし、尿中CD3陽性CD62L陰性のTリンパ球も、尿中総Tリンパ球の30〜50%を占めることが分かった。また、活動性腎炎の尿にもCD3陽性CD62L陽性のTリンパ球が15〜40%認められた。したがって、CD3陽性CD62L陰性のTリンパ球の数は、腎炎活動性の判定には適さないことが分かった。
[0008]
なお、本発明者らは、CD62L陰性のTリンパ球と単球を含む免疫細胞が、糸球体障害を示すマーカーと考えられることを開示している(非特許文献2)が、これはCD3陽性CD62L陰性のTリンパ球を含んだ炎症細胞が活動性腎炎の症例で尿中に認められることを報告したものであり、これらの尿中炎症細胞の数から腎炎の活動性を判定する基準を示したものではなかった。
【非特許文献1】
J Am Soc Nephrol 12: 2636−2644,2001.
【非特許文献2】
日本腎臓学会誌 45(3):302,2003.
【発明の開示】
【発明が解決しようとする課題】
[0009]
以上のように、非侵襲的に腎疾患の活動性の判定を行うことができる腎疾患の活動性を知る方法としては、本発明者らが見出したTリンパ球を検出する方法が知られていた。しかし、この従来の判定法は、判定結果の確実性が低いという欠点があった。
[0010]
そこで、本発明は上記問題点に鑑み、より確実性の高い腎疾患の活動性判定法及びその装置を提供することを目的とする。
【課題を解決するための手段】
[0011]
本発明者らは、上記の課題を解決するために鋭意検討を行ったところ、活動性腎炎を伴わない特発性腎出血では、尿にほとんどCD14陽性の単球は検出できず、一方、活動性腎炎を伴った場合には、CD14陽性CD62L陰性の単球が尿中のCD14陽性総単球の90〜99%に認められることを見出し、本発明に想到した。
[0012]
すなわち、本発明の腎疾患の活動性判定法は、蛍光標識抗CD14モノクローナル抗体と蛍光標識抗CD62Lモノクローナル抗体を用いて腎炎が疑われるヒトから採取した尿沈渣を処理し、フローサイトメトリー法でCD14陽性CD62L陰性の単球を検出し、尿中のCD14陽性CD62L陰性の単球の数が30/ml以上の場合に腎臓の活動性炎症があると判定することを特徴とする。
[0013]
また、上記に加え、蛍光標識抗CD3モノクローナル抗体、蛍光標識抗CD62Lモノクローナル抗体、蛍光標識抗CD45RAモノクローナル抗体、蛍光標識抗CD45ROモノクローナル抗体を用いて腎炎が疑われるヒトから採取した尿沈渣を処理し、フローサイトメトリー法でCD3陽性CD62L陰性のTリンパ球とCD3陽性CD45RA陰性CD45RO陽性のTリンパ球を検出し、末梢血と比較してTリンパ球全体に占めるCD3陽性CD62L陰性のTリンパ球とCD3陽性CD45RA陰性CD45RO陽性のTリンパ球の割合が高いときに腎臓の活動性炎症があると判定することを特徴とする。
[0014]
本発明の腎疾患の活動性判定装置は、CD14陽性CD62L陰性の単球と、CD3陽性CD62L陰性のTリンパ球と、CD3陽性CD45RA陰性CD45RO陽性のTリンパ球とを検出する蛍光活性化セルソーターと、この蛍光活性化セルソーターで検出されたCD14陽性CD62L陰性の単球と、CD3陽性CD62L陰性のTリンパ球と、CD3陽性CD45RA陰性CD45RO陽性のTリンパ球の数をそれぞれ記録する記録手段と、この記録手段に記録されたCD14陽性CD62L陰性の単球と、CD3陽性CD62L陰性のTリンパ球と、CD3陽性CD45RA陰性CD45RO陽性のTリンパ球の数に基づき腎臓の活動性炎症の有無を判定する判定手段とを備え、前記判定手段は、尿中のCD14陽性CD62L陰性の単球の数が30/ml以上であって、末梢血と比較してTリンパ球全体に占めるCD3陽性CD62L陰性のTリンパ球とCD3陽性CD45RA陰性CD45RO陽性のTリンパ球の割合が高いときに腎臓の活動性炎症があると判定するコンピュータプログラムによって構成されたことを特徴とする。
[0015]
本発明の腎疾患の活動性判定キットは、上記の腎疾患の活動性判定法に用いられ、蛍光標識CD14モノクローナル抗体と、蛍光標識CD62Lモノクローナル抗体と、蛍光標識CD3モノクローナル抗体と、蛍光標識CD45ROモノクローナル抗体と、蛍光標識CD45RAモノクローナル抗体とを備えたことを特徴とする。
【発明の効果】
[0016]
本発明の腎疾患の活動性判定法によれば、尿沈渣中のCD14陽性CD62L陰性の単球を検出することにより、非侵襲的かつ特異的に腎疾患の活動性の判定を行うことができるとともに、より確実性の高い腎疾患の活動性判定を行うことができる。
[0017]
また、CD3陽性CD62L陰性のTリンパ球とCD3陽性CD45RA陰性CD45RO陽性のTリンパ球を検出することにより、より一層確実性の高い腎疾患の活動性判定を行うことができる。
[0018]
本発明の腎疾患の活動性判定装置によれば、CD14陽性CD62L陰性の単球と、CD3陽性CD62L陰性のTリンパ球と、CD3陽性CD45RA陰性CD45RO陽性のTリンパ球の数に基づき腎臓の活動性炎症の有無を判定することにより、侵襲的かつ特異的に腎疾患の活動性の判定を行うことができるとともに、より確実性の高い腎疾患の活動性判定を行うことができる。
【0019】
本発明の腎疾患の活動性判定キットによれば、CD14陽性CD62L陰性の単球と、CD3陽性CD62L陰性のTリンパ球と、CD3陽性CD45RA陰性CD45RO陽性のTリンパ球を検出するための、蛍光標識CD14モノクローナル抗体と、蛍光標識CD62Lモノクローナル抗体と、蛍光標識CD3モノクローナル抗体と、蛍光標識CD45ROモノクローナル抗体と、蛍光標識CD45RAモノクローナル抗体とを備えたことにより、簡便に、侵襲的かつ特異的に腎疾患の活動性の判定を行うことができるとともに、確実性の高い腎疾患の活動性判定を行うことができる。
【図面の簡単な説明】
【0020】
【図1】本発明の腎疾患の活動性判定法による検出結果を示すグラフである。
【図2】CD14陽性CD62L陰性の単球数と活動性炎症所見の相関を示すグラフである。
【発明を実施するための最良の形態】
【0021】
本願発明は、尿中活性型免疫細胞の検出とその数の測定により、非侵襲的に腎臓における炎症の活動性判定を行うものである。
【0022】
健常人の尿には免疫細胞は全く認められないが、腎臓に活動性の炎症病変が存在して炎症性免疫細胞が腎組織を破壊している場合には、この炎症性免疫細胞の一部が尿中に現れる。腎組織を破壊し尿に出現する炎症性免疫細胞の同定の手段として、尿を遠心分離して沈渣を得、免疫細胞の表面抗原に対する蛍光標識モノクローナル抗体を作用させ、フローサイトメトリー法で検出する。
【0023】
免疫細胞を検出するために用いるモノクローナル抗体は、CD3,CD14,CD45RO,CD45RA,CD62Lを対応抗原とする蛍光標識抗体である。フローサイトメトリーは多重染色が検出可能な機器を用いる。CD3はTリンパ球、CD14は単球・マクロファージ、CD45ROはメモリーTリンパ球、CD45RAは非活性型Tリンパ球のマーカーである。CD62Lは別名接着因子L−セレクチンである。CD62Lを発現している免疫細胞は非活性型であり、発現していないものは活性型あるいはエフェクター型である。
【0024】
これらの免疫細胞の表面マーカーを組み合わせることにより炎症性あるいは組織傷害性の免疫細胞を検出する。この方法によって同定する炎症性・組織傷害性の免疫細胞は、CD3陽性CD62L陰性のTリンパ球と、CD3陽性CD62L陰性CD45RO陽性CD45RA陰性、あるいはCD3陽性CD45RA陰性CD45RO陽性のTリンパ球と、CD14陽性CD62L陰性の単球・マクロファージである。炎症性あるいは組織傷害性の免疫細胞の数は、フローサイトメトリーの取り込みゲートの細胞総数に、CD3陽性CD62L陰性のTリンパ球、CD3陽性CD62L陰性CD45RO陽性CD45RA陰性、あるいはCD3陽性CD45RA陰性CD45RO陽性のTリンパ球と、CD14陽性CD62L陰性の単球・マクロファージの割合から求める。尿にこれらの細胞数が多数認められる場合に、腎臓における活動性炎症病変ありと判断する。
【0025】
本発明においては、尿蛋白や血尿が認められ腎炎が疑われる場合に、随時尿の解析により、より確実性の高い判定を行うために、特に、蛍光標識抗CD14モノクローナル抗体と蛍光標識抗CD62Lモノクローナル抗体を用いて尿沈渣を処理し、フローサイトメトリー法でCD14陽性CD62L陰性の単球を検出し、その数が30/ml以上の場合に、半月体形成や尿細管間質の炎症細胞浸潤など、腎臓の活動性炎症があると判定する。なお、ここでの単球の数は、尿の浸透圧で補正した数であり、補正値Y=実測値X×検体の浸透圧(mOsm/l)/290(mOsm/l)(290は血漿の浸透圧;この補正方法は非特許文献1に記載)の式から求められる。
【0026】
特発性腎出血では、尿にほとんどCD14陽性の単球は検出できず、一方、活動性腎炎を伴った場合には、CD14陽性CD62L陰性の単球が尿中のCD14陽性総単球の90〜99%に認められるため、確実に腎臓の活動性炎症の判定を行うことができる。
【0027】
さらに、補助的判定基準として、CD3陽性CD62L陰性のTリンパ球とCD3陽性CD45RA陰性CD45RO陽性のTリンパ球が尿に認められ、かつ、末梢血と比較して、Tリンパ球全体に占めるCD3陽性CD62L陰性のTリンパ球とCD3陽性CD45RA陰性CD45RO陽性のTリンパ球の割合が高いときに、腎臓の活動性炎症があると判定することによって、より一層確実性の高い腎疾患の活動性判定を行うことができる。
【0028】
腎臓の炎症性病変の存在診断あるいはその活動性判定は、腎疾患診療において治療法の選択、追加あるいは変更にあたって重要な意味がある。これまでは腎生検がそのための検査方法として採用されてきていた。しかし腎生検は侵襲的であり、実施後に死亡例も報告されている。そのため、病状によっては実施が困難な状況を経験することがあり、患者から実施の同意を得られないこともあった。また診療経過中に複数回実施することは難しかった。その他には、一般検尿検査による尿蛋白・血尿の程度を手がかりにしていたが、特異性が低く、活動性の判定が困難であった。そこで腎臓の炎症性病変の存在診断あるいは活動性判定のために、非侵襲的な新たな検査手段の導入が待たれていた。本願発明による検査は、このような腎疾患診療の問題点を解決することが可能となる。
【0029】
本願発明により、腎臓の炎症性疾患の病態の把握が簡便に行えるようになり、腎疾患の早期診断、早期治療に役立つ。
【0030】
また、治療経過中の治療効果判定や活動性変化の測定を迅速に行うことができるようになり、過度の治療を防ぐことができる。また逆に、治療の不足も防ぐことができ、最適な治療を行うことが可能となる。
【0031】
また、腎生検を行わずに、あるいはその結果を待たずに、腎臓疾患が活動性の高い炎症疾患であるか否かの判定に役立てることもできる。これは急性進行性糸球体腎炎症候群を呈する患者の早期診断に特に有用で、迅速な病態把握と早期治療に役立てることができる。
【0032】
本願発明により、腎疾患の病態把握が簡便かつ迅速に行うことができ、腎疾患の治療効率が飛躍的に向上し、結果として、毎年増加を続ける人口透析患者の増加に歯止めを掛けることも期待できる。
【0033】
また、本発明は、上記の腎疾患の活動性判定法に用いられる装置を提供する。この装置は、CD14陽性CD62L陰性の単球を検出する蛍光活性化セルソーターと、この蛍光活性化セルソーターで検出されたCD14陽性CD62L陰性の単球の数を記録する記録手段と、この記録手段に記録されたCD14陽性CD62L陰性の単球の数に基づき腎臓の活動性炎症の有無を判定する判定手段とを備えている。これら記録手段と判定手段は、コンピュータプログラムで構成されている。そして、判定手段は、記録手段に記録されたCD14陽性CD62L陰性の単球の数の補正値が予め決められた数となった場合に、腎臓の活動性炎症があると判定するように構成されている。さらに、判定手段によって判定された結果を表示する表示手段を設けてもよい。表示手段としては、コンピュータ等のディスプレイ装置で構成することができる。なお、蛍光活性化セルソーターは、一般に用いられるものと構成が同じであるので、その詳細な説明は省略する。
【0034】
この装置を用いて腎疾患の活動性判定を行う場合には、蛍光標識抗CD14モノクローナル抗体と蛍光標識抗CD62Lモノクローナル抗体を用いて尿沈渣を処理した後、これを蛍光活性化セルソーターへ導入する。蛍光活性化セルソーターでは、CD14陽性CD62L陰性の単球がソートされ、その数が記録手段に記録される。そして、判定手段は、記録手段に記録されたCD14陽性CD62L陰性の単球の数の補正値が予め決められた数を超えた場合に、腎臓の活動性炎症があると判定する。
【0035】
本発明の装置を用いることによって、簡便かつ確実に腎臓の活動性炎症の判定を行うことができる。
【0036】
また、本発明の装置は、CD14陽性CD62L陰性の単球を対象とした判定法に限らず、CD3陽性CD62L陰性のTリンパ球などの数に基づき腎臓の活動性炎症の判定を行う場合にも同様に用いることができる。具体的には、例えば、CD14陽性CD62L陰性の単球と、CD3陽性CD62L陰性のT細胞と、CD3陽性CD45RA陰性CD45RO陽性のT細胞とを検出する蛍光活性化セルソーターと、この蛍光活性化セルソーターで検出されたCD14陽性CD62L陰性の単球と、CD3陽性CD62L陰性のT細胞と、CD3陽性CD45RA陰性CD45RO陽性のT細胞の数をそれぞれ記録する記録手段と、この記録手段に記録されたCD14陽性CD62L陰性の単球と、CD3陽性CD62L陰性のT細胞と、CD3陽性CD45RA陰性CD45RO陽性のT細胞の数に基づき腎臓の活動性炎症の有無を判定する判定手段とから装置を構成する。そして、補助的判定基準として、CD3陽性CD62L陰性のTリンパ球とCD3陽性CD45RA陰性CD45RO陽性のTリンパ球が尿に認められ、かつ、末梢血と比較してTリンパ球全体に占めるCD3陽性CD62L陰性のTリンパ球とCD3陽性CD45RA陰性CD45RO陽性のTリンパ球の割合が高いときに、腎臓の活動性炎症があると判定するように構成することによって、より一層確実性の高い腎疾患の活動性判定を行うことができる。
【0037】
また、本発明は、上記の腎疾患の活動性判定法に用いられるキットを提供する。このキットは、蛍光標識CD14モノクローナル抗体と、蛍光標識CD62Lモノクローナル抗体と、蛍光標識CD3モノクローナル抗体と、蛍光標識CD45ROモノクローナル抗体と、蛍光標識CD45RAモノクローナル抗体とを備えている。このキットを本発明の腎疾患の活動性判定法に用いることで、簡便に、侵襲的かつ特異的に腎疾患の活動性の判定を行うことができるとともに、確実性の高い腎疾患の活動性判定を行うことができる。
【0038】
以下、具体的な実施例を例にとって説明するが、本発明は下記の実施例に限定されるものではなく、種々の変形実施が可能である。
【実施例1】
【0039】
患者の新鮮尿50mlを採取後、4℃で尿沈渣を遠心分離(1500×g)した。そして、この尿沈渣をリン酸緩衝液(PBS)で洗浄し、100μlの染色緩衝液(3%のウシ血清と0.05%のアジ化ナトリウムを含むリン酸緩衝液)に懸濁させ、5本の試験管(ベクトンディッキンソン社製ファルコン2052)に20μlずつ分けた。すぐに、10μlのFITCが結合したモノクローナル抗体(抗CD14モノクローナル抗体(M5E2、マウス、IgG1、κ)、抗CD62Lモノクローナル抗体(Dreg56、マウス、IgG1、κ))で懸濁液を4℃で30分間インキュベートした。アイソタイプの同等のFITCが結合した非特異的な抗体(マウス、IgG1、κ)を陰性コントロールに用いた。
【0040】
その後、1mlの溶血緩衝液(0.83%の塩化アンモニウムを含む20mlのトリス−HCl緩衝液)を加え、37℃で3分間インキュベートした後、3mlの染色緩衝液で2回洗浄した。これを遠心分離した後、ペレットを0.5mlの染色緩衝液に懸濁させ、フローサイトメトリー分析を行うまで冷暗所に保管した。
【0041】
そして、ベクトンディッキンソン社製の蛍光活性化セルソーター(FACScan)とソフトウェア(CellysisあるいはCell Quant)を用いて2色フローサイトメトリー分析を行い、CD14陽性CD62L陰性の単球を検出した。
【0042】
また、上記の比較として、FITCが結合したモノクローナル抗体として、抗CD3モノクローナル抗体(UCHT1、マウス、IgG1、κ)と、抗CD62Lモノクローナル抗体(Dreg56、マウス、IgG1、κ))を用いたほかは上記と同様の操作によって、尿中のCD3陽性CD62L陰性のTリンパ球を検出した。
【0043】
さらに、患者の末梢血についても、上記とほぼ同様の操作によって、CD14陽性CD62L陰性の単球と、CD3陽性CD62L陰性のTリンパ球を検出した。
【0044】
その結果を図1に示す。尿(図右)のCD3陽性リンパ球はCD62L陰性、CD14陽性単球・マクロファージはCD62L陰性であり、いずれも活性型であった。同一患者の末梢血(図左)の同じ細胞群では、CD3陽性リンパ球のほとんどがCD62L陽性で、CD14陽性単球・マクロファージはCD62L陽性であり、非活性型であった。これら尿の活性型免疫細胞数は尿1ml当たり200個と多数認められ、腎炎の活動性は高いと判定された。
【実施例2】
【0045】
尿中のCD14陽性CD62L陰性の細胞の数と、腎生検で確認した活動性炎症所見との相関を調べた。なお、細胞数は尿浸透圧で補正し、活動性炎症所見として細胞性半月体形成あるいは間質尿細管細胞湿潤の程度を見た。
【0046】
102例の症例の内訳は、IgA腎症54名、ANCA関連腎炎17名、メサンギウム増殖性腎症(非IgA腎症)14名、膜性腎症7名、アミロイド腎1名、微小変化型8名、高血圧腎症1名である。
【0047】
尿中に検出される尿浸透圧で補正したCD14陽性CD62L陰性の単球の数が30/ml以上の場合に、針生検による腎生検で半月体形成や尿細管間質の炎症細胞湿潤などの腎炎活動性所見が確認できる感度は、85%、特異度は89%であった。すなわち、カットオフ値を30/mlとしたとき、感度85%、特異度89%であった。
【0048】
以上の結果より、CD14陽性CD62L陰性の単球の数と、臨床診断の結果との間には非常に高い相関性が認められ、本発明の腎疾患の活動性判定法が確実性の高い方法であることが確認された。そして、CD14陽性CD62L陰性の単球の数が30/ml以上の場合に、腎臓の活動性炎症があると判定することができることが確認された。
【Technical field】
[0001]
The present invention relates to a method for determining the activity of a renal disease and an apparatus therefor.
[Background]
[0002]
The determination of the activity of inflammatory diseases of the kidney such as nephritis has been conventionally performed by performing a renal biopsy to determine pathologically or by the presence or absence of an increase in the amount of urine protein. However, renal biopsy is an invasive test and it has been difficult to perform multiple times during the course of the disease. Urine protein was affected not only by the degree of inflammation of the kidneys but also by various causes such as hypertension, and its specificity as an activity index was low.
[0003]
Therefore, as a result of examining a large number of clinical cases, the present inventors have found that inflammatory active cells appear in urine when there is an active inflammatory lesion in the kidney. The present inventors have found a method for determining the activity of renal inflammation by measuring the number of inflammatory immune cells using flow cytometry (Non-patent Document 1).
[0004]
In this method, as a means for determining the presence or absence of active inflammation of the kidney, urinary sediment is treated with a fluorescently labeled anti-CD3 monoclonal antibody and a fluorescently labeled anti-CD62L monoclonal antibody, and a CD3 positive CD62L negative T is detected by flow cytometry. When lymphocytes are detected and the number is large, urine sediment is treated with fluorescently labeled anti-CD3 monoclonal antibody, fluorescently labeled anti-CD45RO monoclonal antibody and fluorescently labeled anti-CD45RA monoclonal antibody, and CD3 positive by flow cytometry When CD45RO negative CD45RA positive T lymphocytes are detected and the number is large, it is determined that there is active inflammation of the kidney.
[0005]
According to this method, the activity of renal disease can be determined non-invasively instead of the conventional determination method using renal biopsy or urine protein.
[0006]
However, although the method for determining the activity of the renal disease detects T lymphocytes, there is a drawback that the certainty of the determination result is low.
[0007]
That is, as a result of the examination by the present inventors, in a case of idiopathic renal bleeding exhibiting hematuria of unknown cause not accompanied by nephritis, peripheral blood type CD3-positive CD62L-positive T lymphocytes are CD3-positive CD62L-negative T lymphocytes. Appears superior to. However, urinary CD3-positive CD62L-negative T lymphocytes were also found to account for 30-50% of total urinary T lymphocytes. Moreover, 15 to 40% of CD3 positive CD62L positive T lymphocytes were also found in urine of active nephritis. Therefore, it was found that the number of CD3-positive CD62L-negative T lymphocytes is not suitable for determining nephritic activity.
[0008]
The present inventors have disclosed that immune cells including CD62L-negative T lymphocytes and monocytes are considered as markers showing glomerular damage (Non-Patent Document 2), which is CD3-positive. It is reported that inflammatory cells containing CD62L-negative T lymphocytes are found in urine in cases of active nephritis, and the criteria for judging the activity of nephritis from the number of urinary inflammatory cells are shown. It was not.
[Non-Patent Document 1]
J Am Soc Nephrol 12: 2636-2644, 2001.
[Non-Patent Document 2]
Japanese Journal of Nephrology 45 (3): 302,2003.
DISCLOSURE OF THE INVENTION
[Problems to be solved by the invention]
[0009]
As described above, a method for detecting T lymphocytes found by the present inventors is known as a method for knowing the activity of a renal disease that can determine the activity of the renal disease non-invasively. It was. However, this conventional determination method has a drawback that the certainty of the determination result is low.
[0010]
In view of the above problems, an object of the present invention is to provide a more reliable method for determining the activity of renal diseases and an apparatus therefor.
[Means for Solving the Problems]
[0011]
As a result of diligent studies to solve the above-mentioned problems, the present inventors hardly detected CD14-positive monocytes in urine in idiopathic renal hemorrhage without active nephritis. In the case of nephritis, the inventors found that CD14-positive CD62L-negative monocytes are found in 90 to 99% of urine CD14-positive total monocytes, and have arrived at the present invention.
[0012]
That is, the renal disease activity determination method of the present invention uses a fluorescently labeled anti-CD14 monoclonal antibody and a fluorescently labeled anti-CD62L monoclonal antibody to treat urine sediment collected from a human suspected of nephritis, and then uses CD14 by flow cytometry. Positive CD62L-negative monocytes are detected, and when the number of CD14-positive CD62L-negative monocytes in urine is 30 / ml or more, it is determined that there is active inflammation of the kidney .
[0013]
In addition to the above, a urinary sediment collected from a human suspected of having nephritis using a fluorescently labeled anti-CD3 monoclonal antibody, a fluorescently labeled anti-CD62L monoclonal antibody, a fluorescently labeled anti-CD45RA monoclonal antibody, or a fluorescently labeled anti-CD45RO monoclonal antibody, CD3-positive CD62L-negative T lymphocytes and CD3-positive CD45RA-negative CD45RO-positive T lymphocytes were detected by flow cytometry, and CD3-positive CD62L-negative T lymphocytes and CD3 occupying the entire T lymphocytes compared to peripheral blood. It is characterized by determining that there is active inflammation of the kidney when the proportion of positive CD45RA negative CD45RO positive T lymphocytes is high .
[0014]
The apparatus for determining renal disease activity according to the present invention comprises a CD14-positive CD62L-negative monocyte, a CD3-positive CD62L-negative T lymphocyte, and a fluorescence-activated cell sorter for detecting CD3-positive CD45RA-negative CD45RO-positive T lymphocytes; Recording means for recording the number of CD14 positive CD62L negative monocytes, CD3 positive CD62L negative T lymphocytes, and CD3 positive CD45RA negative CD45RO positive T lymphocytes detected by the fluorescence activated cell sorter, Determination of the presence or absence of active inflammation of the kidney based on the number of CD14 positive CD62L negative monocytes, CD3 positive CD62L negative T lymphocytes and CD3 positive CD45RA negative CD45RO positive T lymphocytes recorded in the recording means and means, the determining means, CD14 positive CD62L negative urine When the number of monocytes is 30 / ml or more and the proportion of CD3 positive CD62L negative T lymphocytes and CD3 positive CD45RA negative CD45RO positive T lymphocytes in the total T lymphocytes is higher than the peripheral blood It is constituted by a computer program for judging that there is active inflammation of the kidney .
[0015]
The renal disease activity determination kit of the present invention is used for the above-described renal disease activity determination method, and comprises a fluorescently labeled CD14 monoclonal antibody, a fluorescently labeled CD62L monoclonal antibody, a fluorescently labeled CD3 monoclonal antibody, and a fluorescently labeled CD45RO monoclonal. An antibody and a fluorescently labeled CD45RA monoclonal antibody are provided.
【The invention's effect】
[0016]
According to the renal disease activity determination method of the present invention, the activity of renal disease can be determined noninvasively and specifically by detecting CD14-positive CD62L-negative monocytes in the urine sediment. At the same time, it is possible to determine the activity of kidney disease with higher certainty.
[0017]
Further, by detecting CD3-positive CD62L-negative T lymphocytes and CD3-positive CD45RA-negative CD45RO-positive T lymphocytes, it is possible to determine the activity of kidney disease with higher certainty.
[0018]
According to the renal disease activity determination apparatus of the present invention, the activity of the kidney based on the number of CD14 positive CD62L negative monocytes, CD3 positive CD62L negative T lymphocytes, and CD3 positive CD45RA negative CD45RO positive T lymphocytes. By determining the presence or absence of inflammatory inflammation, it is possible to determine the activity of kidney disease invasively and specifically, and to determine the activity of kidney disease with higher certainty.
[0019]
According to the renal disease activity determination kit of the present invention, fluorescence for detecting CD14-positive CD62L-negative monocytes, CD3-positive CD62L-negative T lymphocytes, and CD3-positive CD45RA-negative CD45RO-positive T lymphocytes. By providing a labeled CD14 monoclonal antibody, a fluorescently labeled CD62L monoclonal antibody, a fluorescently labeled CD3 monoclonal antibody, a fluorescently labeled CD45RO monoclonal antibody, and a fluorescently labeled CD45RA monoclonal antibody, it is simple, invasive and specific for kidney disease. The activity determination of the renal disease with high certainty can be performed.
[Brief description of the drawings]
[0020]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing the detection results of the renal disease activity determination method of the present invention.
FIG. 2 is a graph showing the correlation between the number of CD14-positive CD62L-negative monocytes and active inflammatory findings.
BEST MODE FOR CARRYING OUT THE INVENTION
[0021]
In the present invention, the activity of inflammation in the kidney is determined non-invasively by detecting urinary active immune cells and measuring the number thereof.
[0022]
In healthy human urine, there are no immune cells, but if there are active inflammatory lesions in the kidney and the inflammatory immune cells destroy the kidney tissue, some of these inflammatory immune cells Appears in the urine. As a means of identifying inflammatory immune cells appearing in urine by destroying renal tissue, urine is centrifuged to obtain a precipitate, and a fluorescent-labeled monoclonal antibody against the surface antigen of immune cells is allowed to act and detected by flow cytometry.
[0023]
Monoclonal antibodies used for detecting immune cells are fluorescently labeled antibodies having CD3, CD14, CD45RO, CD45RA, and CD62L as corresponding antigens. Flow cytometry uses an instrument capable of detecting multiple staining. CD3 is a T lymphocyte, CD14 is a monocyte / macrophage, CD45RO is a memory T lymphocyte, and CD45RA is an inactive T lymphocyte marker. CD62L is also known as adhesion factor L-selectin. Immune cells expressing CD62L are inactive, and those not expressing are active or effector.
[0024]
Inflammatory or tissue-damaging immune cells are detected by combining these immune cell surface markers. Inflammatory / tissue-damaged immune cells identified by this method are CD3 positive CD62L negative T lymphocytes, CD3 positive CD62L negative CD45RO positive CD45RA negative, CD3 positive CD45RA negative CD45RO positive T lymphocytes, and CD14 positive It is a CD62L-negative monocyte / macrophage. The number of inflammatory or tissue-damaging immune cells is the number of cells in the uptake gate of flow cytometry, CD3 positive CD62L negative T lymphocytes, CD3 positive CD62L negative CD45RO positive CD45RA negative, or CD3 positive CD45RA negative CD45RO positive. It is determined from the proportion of T lymphocytes and CD14 positive CD62L negative monocytes / macrophages. If these urine counts are large, it is determined that there is an active inflammatory lesion in the kidney.
[0025]
In the present invention, when urine protein or hematuria is observed and nephritis is suspected, in order to make a more reliable determination by analyzing urine at any time, in particular, a fluorescent labeled anti-CD14 monoclonal antibody and a fluorescent labeled anti-CD62L monoclonal antibody are used. Treat urinary sediment with antibody, detect CD14-positive CD62L-negative monocytes by flow cytometry, and when the number is 30 / ml or more, meniscus formation, inflammatory cell infiltration of tubulointerstitium, etc. Determine that there is active inflammation of the kidneys. Here, the number of monocytes is a number corrected by osmotic pressure of urine, and correction value Y = actual value X × specimen osmotic pressure (mOsm / l) / 290 (mOsm / l) (290 is plasma This correction method can be obtained from the equation described in Non-Patent Document 1.
[0026]
In idiopathic renal hemorrhage, almost no CD14-positive monocytes can be detected in urine, while in the case of active nephritis, CD14-positive CD62L-negative monocytes are 90 to 90% of CD14-positive total monocytes in urine. Since it is found in 99%, the active inflammation of the kidney can be reliably determined.
[0027]
Further, as auxiliary criteria, CD3-positive CD62L-negative T lymphocytes and CD3-positive CD45RA-negative CD45RO-positive T lymphocytes are found in the urine, and CD3 positive occupies the entire T lymphocytes compared to peripheral blood. By determining that there is active inflammation of the kidney when the proportion of CD62L-negative T lymphocytes and CD3-positive CD45RA-negative CD45RO-positive T lymphocytes is high, it is possible to determine the activity of kidney disease with more certainty. It can be carried out.
[0028]
Diagnosing the presence of inflammatory lesions in the kidney or determining their activity is important in selecting, adding, or changing treatment methods in renal disease medical care. Until now, renal biopsy has been adopted as an inspection method. However, renal biopsy is invasive, and deaths have been reported after it has been performed. For this reason, depending on the medical condition, the patient may experience a situation that is difficult to implement, and the patient may not be able to obtain consent for the implementation. Also, it was difficult to carry out several times during the course of medical treatment. In addition, although it was based on the level of urine protein and hematuria by general urinalysis, the specificity was low and it was difficult to judge the activity. Therefore, in order to diagnose the presence of inflammatory lesions in the kidney or to determine the activity, it has been awaited to introduce new noninvasive examination means. The examination according to the present invention can solve such problems in medical care for kidney diseases.
[0029]
According to the present invention, it becomes possible to easily grasp the pathology of inflammatory diseases of the kidney, which is useful for early diagnosis and early treatment of renal diseases.
[0030]
In addition, it becomes possible to quickly determine the treatment effect and measure the change in activity during the course of treatment, thereby preventing excessive treatment. Conversely, lack of treatment can be prevented and optimal treatment can be performed.
[0031]
It can also be used to determine whether a kidney disease is a highly active inflammatory disease without performing a renal biopsy or waiting for the result. This is particularly useful for the early diagnosis of patients with acute progressive glomerulonephritis syndrome, and can be used for prompt understanding of disease state and early treatment.
[0032]
According to the present invention, it is possible to easily and quickly grasp the disease state of kidney disease, and the treatment efficiency of kidney disease is dramatically improved. As a result, it is expected that the increase in the number of artificial dialysis patients that continue to increase every year will be stopped. it can.
[0033]
Moreover, this invention provides the apparatus used for the activity determination method of said renal disease. This apparatus comprises a fluorescence activated cell sorter for detecting CD14 positive CD62L negative monocytes, a recording means for recording the number of CD14 positive CD62L negative monocytes detected by the fluorescence activated cell sorter, and recording on the recording means. Determination means for determining the presence or absence of active inflammation of the kidney based on the number of CD14-positive CD62L-negative monocytes. These recording means and determination means are constituted by computer programs. The determining means is configured to determine that there is active inflammation of the kidney when the correction value for the number of CD14-positive CD62L-negative monocytes recorded in the recording means reaches a predetermined number. ing. Further, display means for displaying the result determined by the determination means may be provided. The display means can be composed of a display device such as a computer. Since the fluorescence activated cell sorter has the same configuration as that generally used, detailed description thereof will be omitted.
[0034]
When this apparatus is used to determine the activity of kidney disease, the urinary sediment is treated with a fluorescently labeled anti-CD14 monoclonal antibody and a fluorescently labeled anti-CD62L monoclonal antibody and then introduced into a fluorescence activated cell sorter. In the fluorescence activated cell sorter, the CD14 positive CD62L negative monocytes are sorted and the number is recorded in the recording means. Then, the determination means determines that there is active inflammation of the kidney when the correction value of the number of CD14 positive CD62L negative monocytes recorded in the recording means exceeds a predetermined number.
[0035]
By using the apparatus of the present invention, it is possible to easily and reliably determine the active inflammation of the kidney.
[0036]
The apparatus of the present invention is not limited to the determination method for CD14 positive CD62L negative monocytes, but also when determining the active inflammation of the kidney based on the number of CD3 positive CD62L negative T lymphocytes and the like. It can be used similarly. Specifically, for example, a fluorescence activated cell sorter for detecting CD14 positive CD62L negative monocytes, CD3 positive CD62L negative T cells, and CD3 positive CD45RA negative CD45RO positive T cells, Recording means for recording the number of detected CD14 positive CD62L negative monocytes, CD3 positive CD62L negative T cells, CD3 positive CD45RA negative CD45RO positive T cells, and CD14 positive CD62L recorded in this recording means The apparatus comprises a negative monocyte, a CD3 positive CD62L negative T cell, and a determination means for determining the presence or absence of active inflammation of the kidney based on the number of CD3 positive CD45RA negative CD45RO positive T cells. As an auxiliary criterion, CD3-positive CD62L-negative T lymphocytes and CD3-positive CD45RA-negative CD45RO-positive T lymphocytes are found in urine, and occupy the entire T lymphocytes compared to peripheral blood. More reliable kidney disease activity by configuring to determine that there is active inflammation of the kidney when the proportion of T lymphocytes negative and CD3 positive CD45RA negative CD45RO positive T lymphocytes is high Sex determination can be performed.
[0037]
The present invention also provides a kit for use in the above-described method for determining the activity of renal diseases. This kit includes a fluorescently labeled CD14 monoclonal antibody, a fluorescently labeled CD62L monoclonal antibody, a fluorescently labeled CD3 monoclonal antibody, a fluorescently labeled CD45RO monoclonal antibody, and a fluorescently labeled CD45RA monoclonal antibody. By using this kit for the renal disease activity determination method of the present invention, the activity of the renal disease can be easily and invasively and specifically determined, and the activity of the renal disease is highly reliable. Judgment can be made.
[0038]
Hereinafter, although a specific example is described as an example, the present invention is not limited to the following example, and various modifications can be made.
[Example 1]
[0039]
After collecting 50 ml of fresh urine from the patient, the urine sediment was centrifuged (1500 × g) at 4 ° C. The urine sediment is washed with phosphate buffer (PBS), suspended in 100 μl of staining buffer (phosphate buffer containing 3% bovine serum and 0.05% sodium azide), and 5 20 μl each was divided into two test tubes (Falcon 2052 manufactured by Becton Dickinson). Immediately, 10 μl of FITC-conjugated monoclonal antibody (anti-CD14 monoclonal antibody (M5E2, mouse, IgG1, κ), anti-CD62L monoclonal antibody (Dreg56, mouse, IgG1, κ)) was suspended at 4 ° C. for 30 minutes. Incubated. Non-specific antibodies (mouse, IgG1, κ) conjugated with equivalent isotype FITC were used as negative controls.
[0040]
Thereafter, 1 ml of hemolysis buffer (20 ml of Tris-HCl buffer containing 0.83% ammonium chloride) was added, incubated at 37 ° C. for 3 minutes, and then washed twice with 3 ml of staining buffer. After centrifugation, the pellet was suspended in 0.5 ml staining buffer and stored in a cool dark place until flow cytometric analysis was performed.
[0041]
Then, two-color flow cytometry analysis was performed using a fluorescence activated cell sorter (FACScan) manufactured by Becton Dickinson and software (Cellysis or Cell Quant) to detect CD14 positive CD62L negative monocytes.
[0042]
In addition, as a comparison above, the above except that an anti-CD3 monoclonal antibody (UCHT1, mouse, IgG1, κ) and an anti-CD62L monoclonal antibody (Dreg56, mouse, IgG1, κ)) were used as monoclonal antibodies bound to FITC. In the same manner as above, CD3-positive CD62L-negative T lymphocytes in urine were detected.
[0043]
Further, in the peripheral blood of the patient, CD14-positive CD62L-negative monocytes and CD3-positive CD62L-negative T lymphocytes were detected by substantially the same operation as described above.
[0044]
The result is shown in FIG. Urinary (right) CD3-positive lymphocytes were CD62L-negative, and CD14-positive monocytes / macrophages were CD62L-negative, both of which were active. In the same cell group of peripheral blood (left in the figure) of the same patient, most of the CD3-positive lymphocytes were CD62L-positive, and CD14-positive monocytes / macrophages were CD62L-positive and inactive. The number of active immune cells in urine was as large as 200 per 1 ml of urine, and the activity of nephritis was determined to be high.
[Example 2]
[0045]
The correlation between the number of CD14 positive CD62L negative cells in urine and active inflammatory findings confirmed by renal biopsy was examined. The number of cells was corrected by urine osmotic pressure, and the degree of cellular meniscus formation or interstitial tubule cell wetting was observed as active inflammatory findings.
[0046]
The breakdown of the 102 cases is as follows: 54 IgA nephropathy, 17 ANCA-associated nephritis, 14 mesangial proliferative nephropathy (non-IgA nephropathy), 7 membranous nephropathy, 1 amyloid kidney, 8 minute changes One person is hypertensive nephropathy.
[0047]
When the number of CD14-positive CD62L-negative monocytes corrected by urine osmotic pressure detected in urine is 30 / ml or more, renal biopsy by needle biopsy, crescent formation, wet inflammatory cells of tubulointerstitium, etc. The sensitivity for confirming the nephritis activity findings was 85%, and the specificity was 89%. That is, when the cutoff value was 30 / ml, the sensitivity was 85% and the specificity was 89%.
[0048]
From the above results, a very high correlation is recognized between the number of CD14-positive CD62L-negative monocytes and the results of clinical diagnosis, and the method for determining the activity of renal disease according to the present invention is highly reliable. It was confirmed that. It was confirmed that it was possible to determine that there was active inflammation of the kidney when the number of CD14 positive CD62L negative monocytes was 30 / ml or more.

Claims (4)

蛍光標識抗CD14モノクローナル抗体と蛍光標識抗CD62Lモノクローナル抗体を用いて腎炎が疑われるヒトから採取した尿沈渣を処理し、フローサイトメトリー法でCD14陽性CD62L陰性の単球を検出し、尿中のCD14陽性CD62L陰性の単球の数が30/ml以上の場合に腎臓の活動性炎症があると判定することを特徴とする腎疾患の活動性判定法。Urine sediment collected from humans suspected of having nephritis is treated with a fluorescently labeled anti-CD14 monoclonal antibody and a fluorescently labeled anti-CD62L monoclonal antibody, and CD14-positive CD62L-negative monocytes are detected by flow cytometry, and CD14 in urine is detected. An activity determination method for renal disease, characterized by determining that there is active inflammation of the kidney when the number of positive CD62L-negative monocytes is 30 / ml or more . 蛍光標識抗CD3モノクローナル抗体、蛍光標識抗CD62Lモノクローナル抗体、蛍光標識抗CD45RAモノクローナル抗体、蛍光標識抗CD45ROモノクローナル抗体を用いて腎炎が疑われるヒトから採取した尿沈渣を処理し、フローサイトメトリー法でCD3陽性CD62L陰性のTリンパ球とCD3陽性CD45RA陰性CD45RO陽性のTリンパ球を検出し、末梢血と比較してTリンパ球全体に占めるCD3陽性CD62L陰性のTリンパ球とCD3陽性CD45RA陰性CD45RO陽性のTリンパ球の割合が高いときに腎臓の活動性炎症があると判定することを特徴とする請求の範囲1に記載の腎疾患の活動性判定法。Urine sediment collected from humans suspected of nephritis is treated with fluorescently labeled anti-CD3 monoclonal antibody, fluorescently labeled anti-CD62L monoclonal antibody, fluorescently labeled anti-CD45RA monoclonal antibody, and fluorescently labeled anti-CD45RO monoclonal antibody, and CD3 is obtained by flow cytometry. Positive CD62L negative T lymphocytes and CD3 positive CD45RA negative CD45RO positive T lymphocytes are detected , and compared to peripheral blood, CD3 positive CD62L negative T lymphocytes and CD3 positive CD45RA negative CD45RO positive The method for determining the activity of a renal disease according to claim 1, wherein it is determined that there is active inflammation of the kidney when the proportion of T lymphocytes is high . CD14陽性CD62L陰性の単球と、CD3陽性CD62L陰性のT細胞と、CD3陽性CD45RA陰性CD45RO陽性のT細胞とを検出する蛍光活性化セルソーターと、この蛍光活性化セルソーターで検出されたCD14陽性CD62L陰性の単球と、CD3陽性CD62L陰性のT細胞と、CD3陽性CD45RA陰性CD45RO陽性のT細胞の数をそれぞれ記録する記録手段と、この記録手段に記録されたCD14陽性CD62L陰性の単球と、CD3陽性CD62L陰性のT細胞と、CD3陽性CD45RA陰性CD45RO陽性のT細胞の数に基づき腎臓の活動性炎症の有無を判定する判定手段とを備え、前記判定手段は、尿中のCD14陽性CD62L陰性の単球の数が30/ml以上であって、末梢血と比較してTリンパ球全体に占めるCD3陽性CD62L陰性のTリンパ球とCD3陽性CD45RA陰性CD45RO陽性のTリンパ球の割合が高いときに腎臓の活動性炎症があると判定するコンピュータプログラムによって構成されたことを特徴とする腎疾患の活動性判定装置。Fluorescence activated cell sorter for detecting CD14 positive CD62L negative monocytes, CD3 positive CD62L negative T cells, CD3 positive CD45RA negative CD45RO positive T cells, and CD14 positive CD62L negative detected by this fluorescent activated cell sorter Recording means for recording the number of CD3 positive CD62L negative T cells, CD3 positive CD45RA negative CD45RO positive T cells, CD14 positive CD62L negative monocytes recorded in the recording means, CD3 Determination means for determining the presence or absence of active inflammation of the kidney based on the number of CD3 positive CD45RA negative CD45RO positive T cells, and the determination means comprises CD14 positive CD62L negative in urine The number of monocytes is 30 / ml or more, and T And characterized in that it is constituted by a determined computer program is active inflammation of the kidney when the proportion of T lymphocytes T lymphocytes and CD3 positive CD45RA - CD45RO positive CD3 positive CD62L negative in the whole path sphere is high To determine the activity of kidney disease. 請求の範囲2に記載の腎疾患の活動性判定法に用いられ、蛍光標識CD14モノクローナル抗体と、蛍光標識CD62Lモノクローナル抗体と、蛍光標識CD3モノクローナル抗体と、蛍光標識CD45ROモノクローナル抗体と、蛍光標識CD45RAモノクローナル抗体とを備えたことを特徴とする腎疾患の活動性判定キット。 A fluorescently labeled CD14 monoclonal antibody, a fluorescently labeled CD62L monoclonal antibody, a fluorescently labeled CD3 monoclonal antibody, a fluorescently labeled CD45RO monoclonal antibody, and a fluorescently labeled CD45RA monoclonal, which are used for the activity determination method for renal disease according to claim 2. An activity determination kit for renal disease, comprising an antibody.
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