JP4703008B2 - Use of at least one saposin level for diagnosing or monitoring lysosomal storage disorders in a subject - Google Patents

Description

表１は、種々のリソソーム貯蔵障害と４つのサポシンマーカー、Ｌａｍｐ−１およびα−グルコシダーゼとの間の相関性を示す。記号＋＋は、強い正の相関を示す（すなわち、この障害を患う患者のうち少なくとも８０％が、コントロール集団の少なくとも９５百分位数（９５ｔｈ ｐｅｒｃｅｎｔｉｌｅ）のマーカーレベルを有する）。記号＋は、より低いがなお正の相関を示す（すなわち、この障害を患う患者のうち少なくとも４０％が、コントロール集団のすくなくとも９５百分位数のマーカーレベルを有する）。記号「−」は、マーカーレベルと疾患の存在との間の負の相関を示す。記号「Ｏ」は、他の測定されたレベルに対して使用される。記号「ＮＤ」は、決定されていないことを意味する。
【００４１】
この表は、ゴシェ病が、試験した６つ全てのマーカーに対して強い正の相関を示す唯一のリソソーム貯蔵障害であることを示す。多数の疾患は、少なくとも１つのサポシンおよびＬａｍｐ−１について強い正の相関を示した。これらの疾患は、ゴシェ病に加えて、ＧＭ−１ガングリオシドーシス、Ｉ細胞病、ＭＰＳ Ｉ、ＭＰＳ ＩＩ、多発性スルファターゼ欠損症、ザントホフ病およびテイ−サックス病（Ａ／Ｂ）であった。これらの疾患のうちのいくつかは、少なくとも１つのサポシンについて強い正の相関を示したが、ＬＡＭＰ−１については強い正の相関を示さなかった。これらの疾患は、ニーマン−ピック病（Ａ／Ｂ）および．ニーマン−ピック病Ｃ、ポーンプ病ならびにシアル酸貯蔵疾患であった。他の疾患は、Ｌａｍｐ−１について強い正の相関を示したが、サポシンのいずれにも強い正の相関を示さなかった。これらの疾患は、ガラクトシアリドーシス、α−マンノシドーシス、ＭＰＳ ＩＩＩＡ、ＭＰＳ ＩＩＩＢ、ＭＰＳ ＩＩ、ＭＰＳ ＩＩＩＣ、ＭＰＳ ＩＩＩＤ、ＭＰＳ ＩＶＡ、およびＭＰＳ ＶＩを含んだ。従って、患者において示されるいくつかまたは全てのマーカーのレベルを試験して、そして表１または同様の表に示される相関と比較することによって、特定の疾患または疾患のサブセットを有するとして患者を分類することが可能である。
【００４２】
上記の診断試験は、リソソーム貯蔵障害に罹患していない患者のコントロール集団において決定されたベースラインレベルと、患者の検体の測定されたレベルとを比較することによって行われる。患者において測定されたレベルと罹患していないヒトにおけるベースラインレベルとの間の有意な逸脱は、診断試験の正の結果の合図である。測定値が、個体と実験誤差との間の固有の変動に起因して、罹患していない個体において代表的に観察される範囲の外側に入る場合、逸脱が有意であるとみなされる。例えば、測定レベルが、コントロール集団におけるレベルの平均＋一定の標準偏差に入らない場合、逸脱が有意であるとみなされ得る。いくつかの方法において、測定レベルとコントロールレベルとの間の逸脱は、測定レベルがコントロール集団の少なくとも７５、８０または９５百分位数のレベルである場合に、有意であると判定される。言い換えると、患者における測定レベルは、正常な個体のうち５０％、２５％、２０％または５％のみで生じる。検体の測定レベルがコントロール集団のベースラインレベルと有意に異ならない場合、診断試験の結果は、陰性であるとみなされる。
【００４３】
サポシンおよびＬａｍｐ−１に関して、陽性の結果が、代表的には、過剰の正常レベルにおける測定レベルによって示される。α−マンノシドーシスに関して、陽性の結果が、ベースラインを超えるレベルまたはベースライン未満のレベルのいずれかの測定レベルによって示され得る。α−グルコシダーゼの測定レベルが、ベースラインレベルを超えるかまたはそれ未満であるかは、リソソーム貯蔵障害に罹患している患者のサブタイプを示す（表１を参照のこと）。測定値とコントロール集団のベースライン値との間の逸脱の程度はまた、診断の潜在的な正確性、および／または患者が罹患している疾患の重篤度の指標を提供する。
【００４４】
診断試験が陽性の結果を与える場合に、患者は、リソソーム貯蔵障害に感受性であるか、またはこの障害の危険性があると最低でも示される。次いで、患者は、代表的には、さらなる試験またはスクリーニングに供される。このような試験またはスクリーニングは、まだ試験していないリソソーム貯蔵障害と相関するさらなる検体の分析を含み得る。このようなスクリーニングはまた、特定のリソソーム貯蔵障害と関連する酵素の欠損について生化学試験を行う工程を包含する。このようなアッセイは、代表的には、患者由来の尿、血液および皮膚線維芽細胞に対して行われる（Ｓｃｈｒｉｖｅｒら、前出）。例えば、αグルコシダーゼは、４−メチル−ウンベリフェリル−α−Ｄ−グルコピラノシドを基質として使用してアッセイされ得る（Ｖａｎ ｄｅｒ Ｐｌｏｅｇら、Ｐｅｄ．Ｒｅｓ．２４：９０〜９４（１９８８）を参照のこと）。さらなる試験はまた、リソソーム貯蔵障害の臨床上の症状（これは、小人症、角膜の混濁、肝脾腫大症、弁病変、冠状動脈の病変、骨格の変形、関節の硬直および進行性の精神遅滞のうちの１以上を含む）についてモニターする工程を包含し得る。さらなるスクリーニングもまた、リソソーム貯蔵障害を患う関連する家族のメンバーについての家族の病歴の分析、および／またはリソソーム貯蔵障害に関連する多型について患者からの、このような障害と関連する酵素をコードするＤＮＡの遺伝子分析を含む（例えば、米国５，２６６，４５９）。１以上のこれらのスクリーニングアッセイの結果として、検体レベルに基づく最初の診断が、確認され得、（またはそうでなければ）、そして特定のリソソーム貯蔵障害に罹患している患者が、同定され得る。
【００４５】
次いで、特定のリソソーム貯蔵障害を有すると同定された患者は、代表的には、この障害の処置を投与される。処置は、代表的には、特定の障害において損なわれるかまたは欠損している酵素を使用する、酵素置換療法の形式である。例えば、ポーンプ病は、同時継続出願ＵＳＳＮ０８／７００，７６０（１９９６年７月２９日出願）に記載されるようにα−グルコシダーゼで処置され得る。さらなる例としては、ゴシェ病は、グルコセンドロシダーゼ（ｇｌｕｃｏｃｅｎｄｏｒｏｓｉｄａｓｅ）を用いて処置され得る（例えば、米国５，８７９，６８０を参照のこと）。
【００４６】
本方法の第２の主な適用は、リソソーム貯蔵障害の処置を受けている患者の状態をモニターすることによる。首尾のよい処置結果は、検体（例えば、サポシン、Ｌａｍｐ−１、およびα−グルコシダーゼ）の異常なレベルから正常なレベルへの回復によって示される。代表的には、このような方法は、患者が処置を受ける前に、検体レベルについての初期値を測定する。次いで、繰り返しの測定が、経時的になされる。コントロール集団における平均レベルに対して初期レベルが評価される場合、その後の測定におけるレベルの有意な減少は、余生の処置結果を示す。同様に、検体の初期レベルが、コントロール集団の平均に対して減少している場合、初期レベルに対する測定レベルにおける有意な増大は、陽性の処置結果の合図である。その後に測定されたレベルが、初期レベルの繰り返しの測定の平均から一定の標準偏差程度を超えて異なる場合に、その後に測定されたレベルは、初期レベルに対して有意な変化を有するとみなされる。モニターすることによって陽性の処置結果が示される場合には、同じ処置レジメンが、継続され得るか、または低用量の処置レジメンに交換され得る。モニターすることによって陰性の処置結果が示される場合には、先行する処置レジメンは、代表的には、異なる治療剤の使用または以前の薬剤の投薬量の増加のいずれかに改変される。
【００４７】
（ＸＩＩ．診断キット）
本発明はさらに、上記の方法に使用するための試薬を含む診断キットを提供する。代表的には、このようなキットは、サポシン、Ｌａｍｐまたはα−グルコシダーゼに特異的に結合する、少なくとも１つの試薬を含む。いくつかのキットは、異なる検体に特異的に結合する複数の試薬を含む。例えば、キットは、サポシンに特異的に結合する試薬、Ｌａｍｐ−１に特異的に結合する試薬、およびα−グルコシダーゼに特異的に結合する試薬を含み得る。いくつかのキットは、２以上の異なるサポシンに対する複数の異なる試薬を含む。例えば、いくつかのキットは、他のサポシンに結合することなくサポシンＡに結合する試薬、他のサポシンに結合することなくサポシンＣに結合する試薬、および他のサポシンに結合することなくサポシンＤに結合する試薬を含む。この試薬は、代表的には、モノクローナル抗体またはポリクローナル抗体であるが、上記のような他の結合剤であり得る。いくつかのキットにおいて、結合剤は、固相に固定化されて提供される。いくつかのキットは、マイクロタイターディッシュのウェルに固定化された結合試薬を提供する。いくつかのキットはさらに、シグナルのレベルと検体の濃度とを相関付けるキットの較正に使用するためのキットによって、検出される検体のサンプルを含む。いくつかのキットはまた、上記のような１つの標識を含む。例えば、標識は、標識された抗イディオタイプ抗体の形態であり得る。キットはまた、代表的には、標識、または上記の診断アッセイの実行方法および／もしくはそれによって得られた結果を解釈する方法を記載する説明書を含む。
【００４８】
（実施例）
（１．リソソーム貯蔵疾患に対するマーカーとしてのサポシン）
本実施例は、罹患していない個体およびＬＳＤに罹患している個体から採取された血漿サンプル中のこれらのタンパク質レベルを決定することによって、ＬＳＤのスクリーニングマーカーとしての、サポシンＡ、Ｂ、ＣおよびＤの適合性を示す。
【００４９】
（材料および方法）
（患者サンプル）
使用された血漿サンプルは、ＬＳＤスクリーニングおよび慣用的な生化学のための処理のために、Ｎａｔｉｏｎａｌ Ｒｅｆｅｒｒａｌ Ｌａｂｏｒａｔｏｒｙ（Ｗｏｍｅｎ’ｓ ａｎｄ Ｃｈｉｌｄｅｒｎ’ｓ Ｈｏｓｐｉｔａｌ，Ａｄｅｌａｉｄｅ，Ａｕｓｔｒａｌｉａ）に提出されたサンプルに由来した。分画研究のための全血サンプルを、実験室内の健康な志願者から得た。
【００５０】
（ポリクローナル抗体）
抗サポシンＡ、Ｂ、ＣおよびＤポリクローナル抗体を産生して、そして以前に記載されたように特徴付けした［１５］。各抗体を２−ｍＬ Ｈｉｔｒａｐ^TMＰｒｏｔｅｉｎ Ｇカラム（Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ，Ｕｐｐｓａｌａ，Ｓｗｅｄｅｎ）で精製して、そして２８０ｎｍでの吸光度（１．０ｇ／Ｌに対して吸光度＝１．４）によって定量した。
【００５１】
（ポリクローナル抗体のユーロピウム標識）
精製された抗サポシンポリクローナル抗体を、ＤＥＬＦＩＡ（登録商標）標識キット（Ｗａｌｌａｃ，Ｎｏｒｔｈ Ｒｙｄｅ，Ａｕｓｔｒａｌｉａ）を使用して、Ｅｕ³⁺ランタニドで標識した。標識された抗体を、１．５×３０ｃｍ Ｓｕｐｅｒｏｓｅ １２ ｆａｓｔ−ｐｈａｓｅ液体クロマトグラフィーカラム（Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ，Ｕｐｐｓａｌａ，Ｓｗｅｄｅｎ）で以前に記載されたように精製した［１３］。各抗体分子に結合体化したＥｕ³⁺の量を、１ｎＭＥｕ³⁺標準溶液に比較して、既知の抗体の濃度の蛍光によって決定した。
【００５２】
（キャリブレータ（ｃａｌｉｂｒａｔｏｒ）および品質コントロール標準物の調製）
サポシンの免疫定量のための液体キャリブレータを、精製されたサポシンＡ、Ｂ、ＣおよびＤタンパク質を使用して調製した［２０］。精製されたサポシンＡタンパク質を、ＤＥＬＦＩＡ（登録商標）アッセイ緩衝液中に希釈して、最終濃度２．４３、１．２１、０．６１、０．３および０．１５μｇ／Ｌを得た。サポシンＡ免疫定量アッセイに関しては、低品質コントロール標準物および高品質コントロール標準物を、それぞれ０．２４および２．４３μｇ／ＬにサポシンＡタンパク質を希釈することによって調製した。精製されたサポシンＢタンパク質を、同じ緩衝液中に希釈して、最終濃度６．７８、３．３４、１．６７、０．８３および０．４２μｇ／Ｌを得た。サポシンＢ免疫定量アッセイに関しては、低品質コントロール標準物および高品質コントロール標準物を、それぞれ０．６７および６．７８μｇ／ＬにサポシンＢタンパク質を希釈することによって調製した。精製されたサポシンＣタンパク質を希釈して、最終濃度１．８８、０．９４、０．４７および０．２４μｇ／Ｌを得た。サポシンＣ免疫定量アッセイに関しては、低品質コントロール標準物および高品質コントロール標準物を、それぞれ０．４７および１．８８μｇ／ＬにサポシンＣタンパク質を希釈することによって調製した。精製されたサポシンＤタンパク質を希釈して、最終濃度０．９２、０．４６、０．２３および０．１１μｇ／Ｌを得た。サポシンＤ免疫定量アッセイに関しては、低品質コントロール標準物および高品質コントロール標準物を、それぞれ０．２３および０．９２μｇ／ＬにサポシンＤタンパク質を希釈することによって調製した。
【００５３】
サポシンＣおよびＤについてのキャリブレータおよびコントロールに、アッセイされているサンプルに等容量のコントロール血漿サンプルを補充して、サポシンＣおよびＤアッセイに対する血漿の阻害効果を補正した。全ての液体サポシンキャリブレータおよびコントロールを、４℃で保存した。
【００５４】
（サポシンの免疫定量）
サポシンの決定を、時間遅延型蛍光免疫アッセイを使用して行った。このアッセイ形式の基礎は、以前に概略されている［１３］。マイクロタイタープレート（Ｉｍｍｕｌｏｎ ４；Ｄｙｎａｔｅｃｈ Ｌａｂｓ，Ｃｈａｎｔｉｌｌｙ，ＶＡ）を、抗サポシンポリクローナル抗体（０．１Ｍ ＮａＨＣＯ₃（ｐＨ８．８）中に２．５ｍｇ／Ｌに希釈、１００μＬ／ウェル）で、４℃で一晩コーティングして、そしてＤＥＬＦＩＡ（登録商標）洗浄緩衝液で予備洗浄（×１）した。サンプルをＤＥＬＦＩＡ（登録商標）洗浄緩衝液（１００μＬ／ウェル）中に希釈して、２０℃で１０分間振盪して、そしてインキュベート（４℃で４時間）した。次いで、プレートを洗浄（×６）して、Ｅｕ−標識抗サポシンポリクローナル抗体を添加して（ＤＥＬＦＩＡ（登録商標）アッセイ緩衝液中に０．２５ｍｇ／Ｌに希釈、１００μＬ／ウェル）、そしてインキュベートした（４℃で一晩）。プレートを洗浄して（×６）、次いでＤＥＬＦＩＡ（登録商標）増強溶液（２００μＬ）を各ウェルに添加して、そしてプレートを振盪した（２℃で１０分間）。蛍光をＷａｌｌａｃ １２３４ ＤＥＬＦＩＡ（登録商標）Ｒｅｓｅａｒｃｈ Ｆｌｕｏｒｏｍｅｔｅｒで読み取った。各サポシンアッセイに関して、対応するサポシンキャリブレータを、無作為に分散された品質コントロールを含むマイクロタイタープレートの第１列にわたって配置した。全てのサポシンキャリブレータ、品質コントロールおよびサンプルを、二連でアッセイした。サポシン濃度を、線形回帰を使用して算定した。
【００５５】
（全血の分画）
６つのコントロール個体由来の全血サンプルを、以前に記載されたように、血漿、白血球および赤血球に分画した［２１］。
【００５６】
（結果）
（サポシン免疫定量アッセイの最適化）
抗サポシンポリクローナル抗体のユーロピウム標識は、各抗体分子に結合した４〜７個のＥｕ³⁺原子を伴って７５〜１００％の範囲の回収を生じた。免疫定量アッセイの最終パラメータを、試験した異なる条件下で一連の較正曲線を生成することによって最適化した。最適化されたパラメータとしては、コーティング抗体およびＥｕ−標識された抗体の濃度、ならびに抗体およびサンプルのインキュベーション時間が挙げられた。費用、時間の最小化、および各アッセイの再現性を、最終アッセイ条件を選択したときに考慮した。
【００５７】
最適化されたサポシンＡ、ＢおよびＣの較正曲線は、標準範囲に対して線形の応答を生じたが（Ｒ²＞０．９９）、サポシンＤの較正曲線は、減少した線形範囲を有した（Ｒ²＝０．９５７５）（図１）。コントロール血漿をサポシンＣおよびＤ較正曲線に添加した場合、シグナル強度の減少を観察した。２μＬの血漿は、２０％の減少を生じた。サポシンＣおよびＤの免疫定量アッセイに対する血漿の阻害効果を補正するために、アッセイされている血漿に対して等容量のコントロール血漿を、キャリブレータに添加した。ＬＳＤ罹患血漿はコントロール血漿と同じ様式でこのアッセイを阻害し、従ってコントロール血漿のみがキャリブレータをスパイクするために使用されたという仮説をたてた。
【００５８】
（コントロールおよびＬＳＤに罹患した個体の血漿中のサポシンレベル）
ＬＳＤについての新生児スクリーニングのマーカーとして各サポシンの適合性を評価するために、サポシンＡ、Ｂ、ＣおよびＤのレベルを、１１１人のコントロール個体（中央年齢＝７、範囲０〜６６）由来、および２８の異なる障害を示す、３３４人のＬＳＤに罹患した個体由来の血漿サンプルにおいて決定した（表１）。コントロール集団のサポシン濃度の９５百分位点（９５^th ｃｅｎｔｉｌｅ）を、このレベルを超えて上昇した血漿サポシンを有するＬＳＤに罹患した個体の集団全体とともに表１に示す。サポシンＡ、ＣおよびＤのレベルは、コントロール集団の血漿サンプルでは厳密な分布を示したが、一方、広範な範囲のサポシンＢレベルを観察した（図２）。かなりの割合のＬＳＤに罹患した個体が、コントロール集団の９５百分位点を超えるサポシンレベルを有し、ここで数人の個体は、コントロール集団の中央濃度の１０倍までの上昇を有することが見出された。２８の障害のうちの１５は、コントロール集団の９５百分位点を超える、１以上のサポシンレベルを有する８０％を超える個体を有した。
【００５９】
【表２】

全てのサポシンアッセイは、１７〜２４の範囲の複製（ｒｅｐｌｉｃａｔｅ）数で行われた各アッセイに対するアッセイ間の変動係数を決定するために、低品質コントロール標準物および高品質コントロール標準物を含んだ。低品質コントロール標準物および高品質コントロール標準物の両方のサポシンＡアッセイの変動係数は、１０％未満であった。サポシンＢ、ＣおよびＤアッセイは、高品質コントロール標準物については１０％未満の変動係数、および低品質コントロール係数については１５〜２０％の変動係数を生じた。別々の実験を行い、ここで高標準物および低標準物の２４連のサンプルを、単一のプレート上でアッセイして、アッセイ間の変動係数を算定した。低品質コントロール標準物については６〜１６％の範囲の値を得て、そして高品質コントロール標準物については１０％未満の値を得た。
【００６０】
同じ血漿サンプル中のＬＡＭＰ−１レベルは、以前にアッセイされているので［１３］、サポシンレベルとＬＡＭＰ−１レベルとの間の直接的な比較を行い得た。ピヤソン相関を使用して、サポシンＢがサポシンＣおよびＤレベルの両方と低い相関性を示した（表３）ということを除いて、全てのサポシンの間で強い相関（ｐ＜０．０１）を観察した。サポシンとＬＡＭＰ−１との間に弱い相関（ｐ＜０．０５）が観察され、そしてコントロール集団においてサポシン濃度と年齢との間には相関性がみられなかった。
【００６１】
【表３】

（白血球におけるサポシンレベル）
６人の罹患していない個体由来の全血サンプルを分画して、そして血漿、白血球および赤血球におけるサポシンの分布を決定した（表３）。白血球における優勢なサポシンはサポシンＤ（濃度７５μｇ／Ｌ）であることが見出され、一方、より高い割合（７７％）のサポシンＢが、他のサポシンと比較して血漿画分に見出された。血漿、白血球および赤血球におけるサポシンＡ、ＣおよびＤの分布は、比較的等しい。
【００６２】
【表４】

（考察）
５９％の患者において、サポシンＡが、コントロール集団の９５百分位点を超えて上昇することが見出され、そしてサポシンＢ、ＣおよびＤは、それぞれ、２５％、６１％および７５％の患者で上昇した（表１）。本発明者らの研究において２８の示される障害のうち、６群におけるサポシンＡレベルが、コントロール群の９５百分位点を超える８０％よりも多くの個体を有し、サポシンＢは、２つの群において増大し、そしてサポシンＣおよびＤは、１０の群において各々上昇した（表２）。２８のうちの１５のＬＳＤ群において８０％よりも多くの個体で同定されたサポシンとともに、これらのうち６つ（すなわち、シスチン症、ファブリー病、ニーマン−ピック病（Ａ／Ｂ型およびＣ型）、ポーンプ病、およびウォルマン病）が、ＬＡＭＰ−１で上昇したことが以前に観察されなかった［１３］。Ｌａｍｐ−１に関して上昇しなかった残りのＬＳＤ（クラッベ病、異染性白質萎縮症、およびテイ−サックス病）は、サポシンレベルの増大を示すかなりの数の患者（４４〜７１％）を有した（表２）。約８５％のＬＳＤに罹患した個体が、ＬＡＭＰ−１およびサポシンの両方をスクリーニングマーカーとして使用して検出され得る。
【００６３】
スフィンゴ脂質またはスフィンゴ脂質誘導体を貯蔵するＬＳＤ群と、サポシンレベルの増大を示したＬＳＤ群との間には、相関性が存在した。例えば、スフィンゴ脂質が貯蔵されるニーマン−ピック病（Ａ／Ｂ型およびＣ型）は、全てのサポシンの上昇を示した。しかし、研究したＭＰＳ群のうちＭＰＳ ＩＩのみが、サポシンＣの上昇を示した。サポシンはまた、脱髄障害（例えば、ＭＬＤおよびクラッベ病）において上昇しなかった。従って、種々のＬＳＤ群で観察されたサポシンの上昇は、貯蔵に関与する組織および貯蔵される基質の型に関係し得る。さらに、サポシン増加の程度はまた、個体の重篤度を反映し得る。
【００６４】
ＬＳＤ群由来の血漿中の個々のサポシンレベルの比率もまた、試験して、そしてコントロール集団に比較して、この比率の値が良好なＬＳＤ検出パラメータであるか否かを決定した。しかし、サポシン間の強い相関（表３）は、個々のサポシンよりも低い推定値を生じる任意の２つのサポシンの比率を生じた。
【００６５】
サポシンＢは、コントロール個体の血漿中でかなり最も優勢なサポシンであった（表２）。サポシンＡ，ＣおよびＤ（それぞれ、３８、２３および３０％）と比較してより高い割合のサポシンＢ（７７％）が、血漿画分中に見出された。サポシンＤは、全血中で優勢なサポシン（７５μｇ／Ｌ）である。サポシンは同じ前駆体を起源とするので、それらの合成速度は等しいはずである。従って、観察された異なるレベルは、おそらく、異なる半減期から生じ、ここでサポシンＤは、細胞中でより長い半減期を有し、そしてサポシンＢは血漿中でより長い半減期を有した。
【００６６】
（実施例２）
血漿サンプルを、サポシン研究について記載されたのと同じ技術を使用する免疫定量アッセイによってα−グルコシダーゼタンパク質についてアッセイした。結果を、図３において囲いのプロットとして表す。結果をまた、表５に示す。この表５は、各障害の群における中央レベル、およびコントロール集団の９５百分位点を超えて上昇したα−グルコシダーゼレベルを有する患者の％を示す。患者の５５％全体が、α−グルコシダーゼの上昇を示した。全てのポーンプ患者は、コントロール集団の５百分位点未満のα−グルコシダーゼレベルを示した。
【００６７】
従って、α−グルコシダーゼは、血漿血清または全血中のこのタンパク質のレベルにおける上昇または減少のいずれかを検出することにより、一定範囲のＬＳＤの検出のために有用なマーカーである。
【００６８】
【表５】

（実施例３）
図２に示される実験を、サポシンＣに対するモノクローナル抗体およびポリクローナル抗体の組合せを使用して、いくつかのサンプルについて繰り返した。マイクロリッタープレートを、モノクローナル抗体７Ｂ２（４ｍｇ／Ｌ、１６時間、４℃）でコーティングして、洗浄して、次いでアッセイ緩衝液中で血漿サンプル（２μＬ）とともにインキュベートした（６時間、４℃）。このプレートを再度洗浄して、次いで示されるような液相ユーロピウム標識抗体を５００μｇ／ｍＬでインキュベートした（６時間、４℃）。このプレートを洗浄して、そして増強緩衝液（２００μＬ／ウェル）で発色させ、そして蛍光を読み取った。サポシンＣの濃度を、組換えサポシンＣを使用した較正曲線に基づいて算定した。
【００６９】
【表６】

７Ｂ２抗体は、捕捉工程のポリクローナル血清と同様に挙動した。しかし、３Ａ１抗体は、コントロールサンプルとの反応性の減少、ゴシェ病サンプルとの反応性の減少、ならびに異染性白質萎縮症およびニューロン性セロイド脂褐素沈着症のサンプルとの反応性の増加を示した。これらの結果は、免疫学的に識別可能なサポシンＣの複数のサブタイプが存在することを示す。
【００７０】
サポシンＡ、Ｂ、ＣおよびＤの４つのクラス全体における免疫学的に識別可能なサブタイプの存在は、サブタイプに特異的なモノクローナル抗体を使用する、より感度のよい診断アッセイを可能にする。例えば、モノクローナル抗体３Ａ１の使用は、コントロールに対する、異染性白質萎縮症およびニューロン性セロイド脂褐素沈着症サンプルのサポシンＣレベルの上昇を検出するための感度の改善を提供する。任意のサポシンに対する他のモノクローナルは、上記の手順を使用して、コントロールに対する感度の増大について試験され得る。
【００７１】
いくつかの方法において、１次スクリーニングを、１以上のサポシンに対するポリクローナル血清、およびサンプルを使用して行い、それによって同定された保障となるさらなる調査を、モノクローナル抗体を用いる二次スクリーニングに供する。いくつかの方法において、二次スクリーニングに使用されるモノクローナルを選択する。なぜなら、それらは、１次スクリーニングにより示唆される特定の疾患状態におけるサポシンの検出において感度の増強を示すからである。いくつかの方法において、サンプルを、同じサポシン型（例えば、サポシンＣ）を結合するが、この分類の異なるサブタイプに特異的に結合する、複数のモノクローナルを用いて試験する。特定のサブタイプに特異的なモノクローナル抗体（例えば、３Ａ１）を使用することによって、減少したシグナルとコントロールの患者との間のより高いレベルでの識別を達成することが可能になり、このことは、このアッセイの正確性を増大させる。
【００７２】
（参考文献）
【００７３】
【表７】

前述の発明は、理解を明解にする目的のために詳細に記載されているが、特定の改変が添付の特許請求の範囲内で行われ得ることが明らかである。本明細書中に引用される全ての刊行物および特許文献は、各々がそのように個別に示されるのと同じ程度に、全ての目的のためにそれらの全体において、本明細書によって参考として援用される。
【図面の簡単な説明】
【図１】 図１は、サポシンＡ、Ｂ、ＣおよびＤ免疫定量化アッセイについての較正曲線である。それぞれのタンパク質の免疫定量化における使用のための、サポシンＡ（パネルａ）、サポシンＢ（パネルｂ）、サポシンＣ（パネルｃ）およびサポシンＤ（パネルｄ）検量線を作製するために、最適な免疫定量化条件が使用された。マイクロタイタープレートを、一次抗サポシンポリクロナール抗体でコートし（２．５ｍｇ／Ｌ、４℃、終夜）、標準をインキュベートし（４℃、４時間）、そしてＥｕ標識した抗サポシンポリクロナール抗体を用いて検出した（０．２５ｍｇ／Ｌ、４℃、終夜）。
【図２】 図２は、コントロールおよびＬＳＤを発症した個体由来の血漿におけるサポシン濃度のボックスプロットである。サポシン濃度は、０．２５〜２μＬの血漿サンプルを用いることにより決定した。Ｎ＝各グループにおいて使用したサポシンの数。ボックス内の線は、平均レベルであり、平均の下および上の影を付けた領域は、２５百分位数（ｃｅｎｔｉｌｅ）および７５百分位数をそれぞれ表し、バーは範囲を表す。○は、アウトライヤーを示し、そして^*は、極度なアウトライヤーを示す。Ｇａｌａｃｔ＝ガラクトシアリドーシス；ＧＭ Ｉ＝ＧＭ Ｉガングリオシドーシス；Ｍａｎｎ＝α−マンノシドーシス；ＭＬＤ＝異染性白質萎縮症；ＭＳＤ＝多発性スルファターゼ欠損；ＮＣＬ＝ニューロンセロイド脂褐素症；Ｎ−Ｐ＝ニーマン−ピック病；ＳＡＳ＝シアリン酸貯蔵疾患；ＴＳＤ＝テイ−サックス病。
【図３】 図３は、コントロールおよびＬＳＤを発症した個体由来の血漿におけるα−グルコシダーゼレベルのボックスプロットである。α−グルコシダーゼ濃度は、５．０μＬの血漿サンプルを用いることにより決定した。Ｎ＝各グループにおいて使用したサポシンの数。ボックス内の線は、平均レベルであり、平均の下および上の影を付けた領域は、２５百分位数（ｃｅｎｔｉｌｅ）および７５百分位数をそれぞれ表し、バーは範囲を表す。○は、アウトライヤーを示し、そして^*は、極度なアウトライヤーを示す。Ｇａｌａｃｔ＝ガラクトシアリドーシス；ＧＭ Ｉ＝ＧＭ Ｉガングリオシドーシス；ＭＬＤ＝異染性白質萎縮症；ＭＳＤ＝多発性スルファターゼ欠損；Ｎ−Ｐ＝ニーマン−ピック病；ＳＡＳ＝シアリン酸貯蔵疾患；ＴＳＤ＝テイ−サックス病。Ｙ軸は、コントロールとＬＳＤ患者との間の差異を強調表示するために、拡大されている。[0001]
(Cross-reference of related applications)
This application has obtained priority from US Patent Application No. 60 / 124,864 (filed on March 17, 1999) and GB99 08190.3 (filed on April 9, 1999). It is also incorporated by reference in its entirety for all purposes.
[0002]
(background)
Lysosomal storage disorders (LSD) are a large family of genetic disorders that can cause symptoms of severe clinical symptoms. The frequency of each individual disease is relatively rare, but the overall incidence of all lysosomal storage disorders is about 1 in 7000 newborns. This is greater than the incidence of other diseases for which newborn screening methods are available (eg, phenylketonuria). Each lysosomal storage disorder results from a deficiency in a lysosomal enzyme, transporter or protein involved in lysosomal biosynthesis or function [1]. This deficiency leads to substrate accumulation (usually degraded in lysosomes) and an increase in the size and number of lysosomes in the cell.
[0003]
LSD, which primarily affects young children, has a wide range of clinical symptoms (mental retardation, skeletal abnormalities, organomegaly, corneal opacities, and coarse facial features, depending on the specific genotype involved. And can be very serious [2] [1]. In recent years, treatments for several LSDs have become possible, such as drug therapy [3-5], bone marrow transplantation [2, 6], and enzyme replacement therapy. [6, 7]. New treatment protocols are being developed due to the increased knowledge of the causes underlying certain disorders and the availability of animal models along with the development of new technologies. Animal models are particularly important for testing new treatments (eg, enzyme exchange therapy) [2, 6, 8, 9] and studies in animal models have also given treatment at an early stage of pathology. Cases have shown that in most cases maximum effectiveness is achieved [10, 11].
[0004]
In most disorders, clinical pathology is not obvious at birth but appears in the first few years of life. For current and proposed therapies to achieve maximum efficacy, especially if there is involvement of the central nervous system and / or bone pathology, the disorder is early, before the onset of irreversible pathology, It is important to be detected. Accordingly, there is a need for a method for screening newborns, preferably a screening method that can detect all or most lysosomal disorders using a minimal number of assays. The present invention fulfills this and other needs.
[0005]
(Definition)
The patient is a subject (typically a human), either alive or subjected to a postmortem analysis, and the patient has a disorder, susceptibility to the disorder or normal disorder You are undergoing a diagnostic test to determine if you have a risk that exceeds your risk. If the patient is currently showing the symptoms, the patient has the disease. If a patient is currently asymptomatic but has a genetic or other predisposition to obtain the disease at a later time, the patient is susceptible to the disease. If the patient carries a genetic or biochemical marker for the presence of the disease in an individual population, the patient is beyond the normal risk of having the disease. Some patients undergoing diagnostic tests are suspected of having the disease from symptoms, family history, or other previously performed tests. Other patients undergoing diagnostic tests do not have a known symptom or other risk factors for the disease, but will receive the test as a prophylactic screen, along with other tests as necessary.
[0006]
A diagnostic test is an extraordinary risk that a patient has, is susceptible to, or has a disability for such a disorder for the general population. Useful to show that you have
[0007]
Unless the context requires otherwise, the terms "comprise" or "comprises" or "comprising" variations are described as elements or integers or elements Or means the inclusion of a group of integers, but not any other element or integer or element or group of integers.
[0008]
Specific binding of the reagent to the analyte to form a complex means that the complex has a dissociation constant less than micromolar.
[0009]
(Summary of the Invention)
The present invention provides a method of diagnosing or monitoring lysosomal storage diseases in a patient. This method involves measuring the level of at least one saposin in a patient tissue sample, which level provides an indication of the presence or absence or extent of this disorder in the patient. In some methods, the sample is a plasma sample. In some methods, the presence of a disorder is indicated by a measured level of saposin that exceeds the average level of saposin in a control population of individuals without lysosomal storage disease. In some methods, the presence of a disorder is indicated when the measured level of saposin exceeds the 95 percentile level in the control population. In some methods, the patient is not known to have a lysosomal storage disorder prior to the measuring step. In some methods, the patient is less than 1 year old. In some methods, the patient is a fetus. The saposin detected by such a method can be either saposin A, B, C or D, prosaposin, any of these saposins or mRNA encoding these saposin subtypes. In some methods, saposin is detected using an antibody as a diagnostic reagent. In some methods, the lysosomal storage disease is cystinosis, Fabry disease, Niemann-Pick disease, Pomp disease or Wolman disease. Some methods also include informing the patient, parent or guardian of the outcome of the presence of the lysosomal storage disease indicated by the measuring step.
[0010]
The present invention further provides a method of diagnosing or monitoring a lysosomal storage disorder in a patient whose alpha-glucosidase level is measured in a tissue sample from the patient. The level of α-glucosidase provides an indication of the presence or extent of the disorder in the patient. In some methods, the sample is a plasma sample or a whole blood sample. In some methods, the step of measuring is from an increased α-glucosidase concentration relative to an average level in a control population of individuals without lysosomal storage disease, from acid lipase disease, mannosidosis, mucopolysaccharidosis. The presence of II (MPS II), MPS IIIA, MSD, mucolipidosis, NP (A / B), NP (C), Zandhof, SAS or TSD B1 is indicated. In some methods, the measuring step indicates the presence of MPS IVA or Pomp disease from a reduced level of α-glucosidase relative to an average level in a control population of individuals without lysosomal storage disease.
[0011]
The present invention further provides lysosomal storage diseases by measuring saposin levels, measuring LAMP-1 or LAMP-2 levels, and measuring α-glucosidase levels in the same or different tissue samples from patients. Provide a method of diagnosing. Presence of increased levels of saposin, LAMP-1 or LAMP-2, and either increased or decreased levels of α-glucosidase relative to corresponding levels in a control population of individuals without lysosomal storage disorders Is an indicator of lysosomal storage disorders.
[0012]
The present invention further provides a diagnostic kit. Some such kits include a first reagent that binds LAMP and a second reagent that binds saposin. Some kits further include a third reagent that binds to α-glucosidase.
[0013]
The present invention also provides a method for monitoring treatment of lysosomal storage diseases in a patient. Such a method involves determining a baseline level of saposin in a tissue sample from a patient having a lysosomal storage disorder prior to treatment with the drug. The baseline level is then compared to the level in the sample obtained after the patient has been treated with the drug. A decrease relative to baseline in the level of saposin after treatment indicates a positive treatment result.
[0014]
The present invention also monitors the treatment of acid lipase disease, mannosidosis, MPS II, MPS IIIA, MSD, mucolipidosis, NP (A / B), NP (C), Zanthof, SAS or TSD B1 Provide a way to do it. Such methods involve determining a baseline level of α-glucosidase in a tissue sample from a patient with this disorder prior to treatment with the drug. The baseline level is then compared to the level of α-glucosidase in a tissue sample from a patient with the disorder after treatment with the agent. A decrease relative to baseline indicates a positive treatment outcome.
[0015]
The present invention also provides a method of monitoring a patient having galactosialidosis, MPS IVA, or Pomp disease. Such methods involve determining a baseline level of α-glucosidase in a tissue sample from a patient with this disorder prior to treatment with the drug. The baseline level is then compared to the level of α-glucosidase in a tissue sample from the patient after treatment with the drug. An increase over baseline indicates a positive treatment result.
[0016]
(Detailed explanation)
(I. Introduction)
The present invention provides a method of diagnosing a patient for the presence of a lysosomal storage disorder. This method is performed by detecting a biological marker that correlates with at least one or a subset of such disorders. Some methods detect one or more naturally occurring polypeptides called saposins. Exceeding normal levels of such polypeptides in tissue samples from patients is associated with several types of lysosomal diseases (cystinosis, Fabry disease, Niemann-Pick disease (types A / B and C), pomp Disease, Wolman disease, Krabbe disease, metachromatic leukotrophy and Tay-Sachs disease). Another method involves determining the level of α-glycosidase in a tissue sample from a patient. The level of α-glycosidase is suppressed relative to the normal level in Pamp disease. The levels of α-glycosidase are acid lipase disease, mannosidosis, MPS II, MPS IIIA, multiple sulfatase deficiency, mucolipidosis, Niemann-Pick disease (A / B), Niemann-Pick disease (C), sialic acid In storage disease or Zandhoff disease, Tay-Sachs disease A / B, the level is elevated relative to the normal level.
[0017]
The method is suitable for large-scale screening of recently born infants or fetuses for the presence of a lysosomal storage disorder, optionally with additional biochemical and genetic markers of other disorders that may be present in the newborn. This method is also suitable for monitoring patients previously diagnosed with lysosomal storage diseases, particularly their response to treatment. The method for analyzing saposin and α-glucosidase is performed in combination, if necessary, in further combination with the step of detecting other biochemical markers that correlate with lysosomal storage disorders as described by WO 97/44668. . If desired, analysis of biochemical markers can also be combined with polymorphism analysis of genes encoding lysosomal enzymes for polymorphisms that correlate with disease.
[0018]
(II. Lysosome storage disorder)
There are over 30 lysosomal diseases, each usually resulting from the failure of a particular lysosomal protein as a result of a genetic mutation. See, for example, Cotran et al., Robbins Pathology Basis of Disease (4th edition, 1989), which is incorporated by reference in its entirety for all purposes. Failure in lysosomal proteins usually results in a detrimental accumulation of metabolites. For example, there is accumulation of mucopolysaccharides in Fuller syndrome, Hunter syndrome, Morquio syndrome and San Philip syndrome; accumulation of sphingolipids in Tay-Sachs syndrome, Gossy syndrome, Krabbe syndrome, Niemann-Pick syndrome, and Fabry syndrome And in fucose accumulation disease and mannosidosis there is accumulation of fucose-containing sphingolipid and glycoprotein fragments, and mannose-containing oligosaccharides, respectively.
[0019]
Type II glycogen storage disease (GSD II; Pomp disease; acid maltose deficiency) is caused by a failure of the lysosomal enzyme acid α-glucosidase (acid maltase). Three clinical forms are distinguished (infanthood, juvenile and adulthood). Infant GSDII has its onset shortly after birth and exhibits progressive muscle weakness and heart failure. This clinical variant is lethal within two years of birth. Symptoms in adult and juvenile patients occur later in life and involve only skeletal muscle. The patient eventually dies due to respiratory failure. Patients can exceptionally survive more than 60 years. The severity of the disease and the remaining acid alpha-glycosidase activity (this activity is 10-20% of the normal in the late-onset form of the disease and less than 2% in the early-onset form) There is a good correlation between them (see Hirschhom, The Metabolic and Molecular Bases of Inherited Disease, edited by Schriver et al. (7th edition) McGraw-Hill (1995), pages 2443-2464).
[0020]
Over 30 known genes encoding lysosomal enzymes (α-glucosidase, αL-iduronidase, iduronate-sulfate sulfatase, hexosaminidase A and B, ganglioside activator protein, arylsulfatase A and B, iduronate Sulfatase, heparin N-sulfatase, galacto-ceramidase, α-galactosylceramidase A, sphingomyelinase, α-fucosidase, α-mannosidase, aspartylglycosamininamide hydrolase, acid lipase, N-acetyl-α-D-glucosamine- 6-sulfate sulfatase, α-galactosidase and β-galactosidase, β-glucuronidase, β-mannosidase, ceramidase, galactose Transfected cerebroside mannosidase, alpha-N-acetylgalactosaminidase, and the like protective proteins include) exists. DNA clones containing the genomic or cDNA sequences of a number of known genes that encode lysosomal proteins are available. (Scott et al., Am. J. Hum. Genet. 47, 802-807 (1990); Wilson et al., PNAS 87, 8531-8535 (1990); Stein et al., J. Biol. Chem. 264, 1252-1259 (1989). Ginns et al., Biochem.Biophys.Res.Comm.123, 574-580 (1984); Hoefslot et al., EMBO J.7, 1697-1704 (1988); Hoefslot et al., Biochem J.272, 473-479 (1990). ); Meyerowitz & Proia, PNAS 81, 5384-5398 (1984); Scriver et al., Supra, part 12, 2427-2882, and references cited therein). Other examples of genomic and cDNA sequences are available from GenBank.
[0021]
(III. Saposin)
Saposin is a small heat-stable glycoprotein, and there are four glycoproteins called saposins A, B, C and D. All four saposins are derived from a single 73 kD precursor protein called prosaposin. Mature saposins have a specific role in activating and enhancing their respective lysosomal hydrolysis activities [15, 16]. Saposins are important for the regulation of glycosphingolipid flow through the lysosomal hydrolysis pathway [15] and gene defects in sphingolipid hydrolases, and / or saposins are associated with the storage of sphingolipids [17]. All saposins contain about 80 amino acids and show a high degree of sequence identity with each other. Each saposin contains 6 cysteine residues in almost identical positions, all of which form 3 disulfide bridges to provide a hairpin structure. Each saposin also contains an N-glycoside type carbohydrate chain. They are bound to the same asparagine residue at position 21 from the N-terminus of each saposin. Of the four saposins, only saposin A has an additional carbohydrate chain linked to 42 asparagine residues. Structurally important proline residues are also conserved in almost identical positions. In all, 13 amino acids are conserved at the same position in each saposin domain. In addition, there are many amino acid residues of similar nature, particularly hydrophobic amino acid residues, at the same position in each domain. Despite such similarities, they are immunologically distinguishable and functionally different. Saposins are well conserved among different animal species, including humans, cows, rats, pigs, and even chickens.
[0022]
Saposins are required for the enzymatic hydrolysis of specific sphingolipids by specific lysosomal hydrolases. Although saposins are highly homologous to each other, they exhibit unique and different specificities. Saposin B, the first discovered saposin, stimulates hydrolysis reactions by arylsulfatase A, GM1 ganglioside β-galactosidase, and α-galactosidase A. Saposin C also catalyzes this reaction, but it has lower activity. Saposin D stimulates the activity of sphingomyelinase and ceramidase. In human tissue, there are probably three different pools of prosaposin (one for lysosomal targeting, one for secretion, and one for targeting to the plasma membrane).
[0023]
As described in Example 1, elevated levels of saposin positively correlate with the presence of several lysosomal storage disorders. Any saposin and mRNA encoding this saposin can be used as screening markers, but saposins A, C and / or D are preferred. This is because these saposins show the strongest correlation. Any or all of at least three mechanisms may account for the observed accumulation of saposin in tissue samples from LSD patients in Example 1. First, saposin synthesis can be stimulated by accumulation of either defective enzymes or lipids as a compensatory mechanism. Secondly, saposin is deposited with the substrate accumulating through the lack of processing by the defective enzyme. Thirdly, saposins cannot be degraded due to their inability to interact with non-functional lysosomal enzymes. However, the present invention does not depend on an understanding of the mechanism.
[0024]
(IV. Α-Glycosidase)
Acid α-glucosidase is an enzyme associated with a lysosomal storage disorder called Pomp disease. α-Glucosidase is first synthesized in a precursor form of about 100-110 kD (the apparent molecular weight or relative mobility of this precursor may vary to some extent depending on the analytical method used, but typically Is a range between 95 kD and 120 kD). The proteolytic processing of acid α-glucosidase is complex and involves a series of steps in addition to signal peptide cleavage that occurs at various subpleural locations. Polypeptides are cleaved at both the N-terminus and C-terminus, thereby increasing specific catalytic activity. The main species recognized are the 110/100 kD precursor, 95 kD intermediate and the mature forms of 76 kD and 70 kD (Hasicic et al., J. Biol. Chem. 255, 4937-4945 (1980); Ode Elferink et al., Eur J. Biochem.139, 489-495 (1984); Reuser et al., J. Biol.Chem. 260, 8336-8341 (1985); Hoefslot et al., EMBO J. 7, 1697-1704 (1988)).
[0025]
The present invention provides evidence that the level of acid α-glucosidase is suppressed in Pomp disease and in certain other lysosomal storage disorders. Conversely, the level of acid α-glucosidase is determined by certain other lysosomal storage disorders (mannosidosis, MPS II, MPS IIIA, MSD, mucolipidosis, Niemann-Pick disease (A / B), Niemann-Pick disease (C ), Zanthof disease, SAS or Tay-Sachs disease (A / B)). Thus, acid α-glucosidase levels are a useful biochemical marker for screening for lysosomal storage disorders. The methods of the invention detect each of the various molecular weight forms of acid α-glucosidase, combinations thereof, and mRNA encoding acid α-glucosidase.
[0026]
(V. Other biochemical markers)
WO 97/44668 describes a number of protein markers (including LAMP-1 and LAMP-2) that correlate with lysosomal storage disorders. LAMP-1 and LAMP-2 are lysosomal membrane glycoproteins (Dahlgren et al., Biochem. J. 311, 667-674 (1995). LAMP-1 is a preferred marker. Disease, galactosialidosis, GM-1-gangliosidosis, I cell disease, acid mannosidosis, MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIID, MPS IVA, MPS VI, multiple sulfatase deficiency, Correlates with Zandhof disease, sialic acid storage disease, and Tay-Sachs (AB) disease (13) Levels of LAMP-2 are also increased in many LSDs as LAMP-1 [14]. Some LSDs that do not correlate with saposin levels (See Table 1.) Thus, a combined screen for LAMP-1 and saposin identifies patients suffering from lysosomal storage disorders than either screen alone.
[0027]
(VI. Tissue sample)
Analytical samples can be obtained from any organ, tissue, fluid or other biological sample containing lysosomes or their component proteins. Preferred tissue samples are whole blood and products derived from whole blood (eg, plasma and serum). The blood sample can be obtained, for example, from a blood spot collected from a Guthrie card. Other sources of tissue samples are skin, hair, urine, saliva, semen, stool, sweat, milk, amniotic fluid, liver, heart, muscle, kidney and other body organs. Other tissue sources are cell lines grown from primary cells derived from patients. The tissue sample is typically lysed to release the protein content and / or nucleic acid content of the cells in the sample. The protein and / or nucleic acid fraction from such crude lysate is then subjected to partial or complete purification prior to analysis.
[0028]
Samples can be obtained from embryos, fetuses, newborns, young babies, or adults. Typically, samples can be obtained from newborns that are one day old, one week old, one month old, or less than six months old. Fetal samples can be obtained, for example, in the form of amniotic fluid from the mother's body or fetal blood. The amniotic fluid is preferably withdrawn from the womb of the pregnant woman using, for example, a 20 or 22 gauge needle under continuous ultrasound guidance. Methods for obtaining fetal blood are described in Daffos, The Union Patent-Prenatal Diagnosis and Treatment (Harrison et al. (Ed.), WB Sanders, Philadelphia, PA 1991), Chapter 11.
[0029]
In some methods, multiple diagnostic tests for multiple markers are performed on the same patient. Typically, multiple tests are performed on different aliquots of the same tissue sample. However, multiple assays can also be performed on separate samples from the same tissue source or on multiple samples from different tissue sources. For example, a test for one marker can be performed on a plasma sample and a test for a second marker is performed on a whole blood sample. In some methods, multiple samples are obtained from the same patient at different time points. In such a method, the multiple samples are typically from the same tissue (eg, all plasma).
[0030]
(VI. Protein assay format)
Polypeptide analytes (eg, saposin, α-glucosidase and LAMP-1) can be assayed by binding assays using antibodies or other reagents that have specific binding affinity for the analyte. Briefly, a sample containing the analyte is incubated with an antibody or other binding reagent, and the complex formed is detected and quantified. The conditions for incubating the antibody or other binding reagent with the test sample will vary depending on the format used in the assay, the detection method used in the assay and the type and nature of the binding reagent used. Any one of the commercially available immunological assay formats (eg, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), diffusion-based oakter ronnie, rocket-gel immunoelectrophoresis or in situ high Hybridization) can be used.
[0031]
Examples of such assay formats are US Pat. Nos. 3,791,932; 3,817,837; 3,839,153; 3,850,752; 850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; Uouchi 4,034,074; and 4,098,876; 3,791,932; No. 8,39,153; No. 3,850,752; No. 3,879,262; and No. 4,034,074, No. 4,016,043, No. 4,424,279. And in US Pat. No. 4,018,653. The result can be quantitative by simple observation of a visible signal or can be quantified by comparison with a control sample containing a known amount of analyte. Variations on this assay include simultaneous assays (where both sample and labeled antibody are added simultaneously to the bound antibody) or reverse assays (where the labeled antibody and the sample being tested are first combined). And then added to the bound antibody at the same time). The antibodies used above can be monoclonal or polyclonal.
[0032]
Substrates for use in solid phase assays are typically glass or polymers, and the most commonly used polymers are cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid support can be in the form of a tube, bead, disk, or microplate, or any other surface suitable for performing an immunoassay. The binding reagent can be bound to the substrate by covalent cross-linking. The binding process generally involves covalently cross-linking or physically adsorbing the molecule to an insoluble carrier.
[0033]
Labels for use in such assays include enzymes, fluorophores or radionuclide containing molecules (ie, radioisotopes). In the case of enzyme immunoassays, the enzyme is typically conjugated to the antibody by glutaraldehyde or periodate. Commonly used enzymes include horseradish peroxidase, glucose oxidase, β-galactosidase, and alkaline phosphatase, among others. The substrate used with a particular enzyme is generally selected for the occurrence of a detectable color change upon hydrolysis by the corresponding enzyme. It is also possible to use a fluorescent substrate that produces a fluorescent product. Alternatively, fluorescent compounds (eg, fluorescein, Eu ³⁺ Or other lanthanide metals, and rhodamine) can be chemically conjugated to the antibody without altering its binding ability. When activated by irradiation with light of a specific wavelength, an antibody labeled with a fluorescent dye absorbs light energy and has a unique color that can be visually detected by an excited state of the molecule, followed by an optical microscope. Induces light emission. Like ELISA, fluorescently labeled antibodies are allowed to bind to the primary antibody-hapten complex. After washing away unbound reagent, the remaining complex is then exposed to light of the appropriate wavelength and the fluorescence is observed. This indicates the presence of the target hapten. Other reporter molecules (eg, chemiluminescent or bioluminescent molecules) can also be used.
[0034]
VII. Binding reagents for use in protein assay formats
The above binding assays are typically performed using an antibody as a binding reagent. The antibody can be polyclonal or monoclonal. Production of non-human (eg, mouse, rat, etc.) monoclonal antibodies is well known and includes, for example, analyte polypeptides (eg, saposin, LAMP-1 or α-glucosidase) or immunogenic fragments thereof. This can be accomplished by immunizing the animal with (optionally including an adjuvant). Antibody producing cells obtained from the immunized animal can be immortalized and screened for the production of antibodies that bind to the specimen. See Harlow & Lane, Antibodies, A Laboratory Manual (CSH P. NY, 1988), which is incorporated by reference in its entirety for all purposes. Human antibodies and humanized antibodies can also be made, but are not superior to rodent antibodies for in vitro diagnostic assays. Intact antibodies and their binding fragments (eg Fv, Fab and (Fab ') ₂ Can be used in the method of the invention.
[0035]
Other suitable binding reagents can be screened from random libraries of peptides or other compounds that can also be screened for compatibility. Combinatorial libraries can be produced for many types of compounds that can be synthesized in a step-wise fashion. Such compounds include polypeptides, β-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, N-substituted glycine oligomers and oligocarbamates. It is done. A large combinatorial library of compounds is available from Affymax, WO95 / 12608, Affymax, WO93 / 06121, Columbia University, WO94 / 08051, Pharmacopeia, WO95 / 35503 and Scripps, WO95 / 30642 The code synthesis library (ESL) method described in US Pat. Peptide libraries can also be generated by phage display methods. See, for example, Devlin, WO 91/18980.
[0036]
(IX. MRNA assay)
Alternatively or in addition to protein analysis, the present invention provides diagnostic methods based on the detection and quantification of mRNA encoding saposin, LAMP-1 or α-glucosidase, or amplification products thereof. The RNA transcript for analysis is isolated from a biological sample obtained from a biological tissue or biological fluid in which the gene of interest is expressed. Samples include sputum, blood, blood cells (eg, white blood cells), biopsy samples from tissues or fine needles, urine, peritoneal fluid, and pleural fluid, or cells derived therefrom. Methods for isolating total mRNA are described in Chapter 3 of Laboratory Technologies in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, P.M. Tijsssen (edition). Elsevier, N.M. Y. (1993) and Chapter 3 of Laboratory Technologies in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, P.M. Tijsssen (edition). Elsevier, N.M. Y. (1993).
[0037]
Often it may be desirable to amplify RNA prior to hybridization. The amplification product can be single-stranded or double-stranded RNA or DNA. In one procedure, mRNA can be reverse transcribed using reverse transcriptase and primers consisting of oligo dT and a sequence encoding the phage T7 promoter to provide a single stranded DNA template. The second DNA strand can be polymerized using a DNA polymerase. After synthesis of double stranded cDNA, T7 RNA polymerase is added and RNA is transcribed from the cDNA template. Successful number of transcriptions from each single cDNA template results in amplified RNA. Alternatively, the cDNA can be amplified to produce a double stranded amplicon, and one strand of the amplicon uses a biotinylated primer that allows capture of the unwanted strand onto the streptavidin beads, ie Can be isolated. Alternatively, asymmetric PCR can be used to generate a single stranded target. If desired, the amplification product is labeled either during or after amplification. A variety of different fluorescent labels (including fluorescein and phycoerythrin) are available.
[0038]
RNA or its amplification product is typically detected by hybridization to a complementary probe. Either the analyte or the probe can be labeled. In some assay formats, the analyte is immobilized and in other formats, the probe is immobilized. If necessary, probes for multiple analytes are immobilized on the same support, and multiple analytes are detected and quantified simultaneously. Methods for transcript analysis and quantification using probe arrays are described in detail in WO 96/14839 and WO 97/01603.
[0039]
(X. Diagnosis method and monitoring method)
The above analysis of saposin, LAMP-1 and α-glucosidase levels has at least two main applications. First, such an analysis is useful in diagnosing patients who have not been clearly characterized as having lysosomal storage diseases. Analysis of saposin, LAMP-1 and α-glucosidase levels is an indication that the patient is suffering from, or susceptible to, or at risk for such a disease I will provide a. The relative levels of the different markers also sometimes provide an indication that a particular lysosomal storage disorder or subset of disorders is present (see Table 1).
[0040]
[Table 1]

Table 1 shows the correlation between various lysosomal storage disorders and the four saposin markers, Lamp-1 and α-glucosidase. The symbol ++ indicates a strong positive correlation (ie, at least 80% of patients suffering from this disorder have a marker level of at least 95th percentile of the control population). The sign + indicates a lower but still positive correlation (ie, at least 40% of patients with this disorder have at least 95 percentile marker levels in the control population). The symbol “-” indicates a negative correlation between the marker level and the presence of the disease. The symbol “O” is used for other measured levels. The symbol “ND” means not determined.
[0041]
This table shows that Gaucher disease is the only lysosomal storage disorder that shows a strong positive correlation for all 6 markers tested. A number of diseases showed a strong positive correlation for at least one saposin and Lamp-1. These diseases were GM-1 gangliosidosis, I cell disease, MPS I, MPS II, multiple sulfatase deficiency, Zandhof disease and Tay-Sachs disease (A / B) in addition to Gosche disease. Some of these diseases showed a strong positive correlation for at least one saposin, but no strong positive correlation for LAMP-1. These diseases include Neiman-Pick disease (A / B) and. Neiman-Pick disease C, Pomp disease and sialic acid storage disease. Other diseases showed a strong positive correlation for Lamp-1, but no strong positive correlation for any of the saposins. These diseases included galactosialidosis, α-mannosidosis, MPS IIIA, MPS IIIB, MPS II, MPS IIIC, MPS IIID, MPS IVA, and MPS VI. Therefore, classify a patient as having a particular disease or subset of diseases by testing the level of some or all markers shown in the patient and comparing to the correlation shown in Table 1 or a similar table It is possible.
[0042]
The diagnostic test described above is performed by comparing baseline levels determined in a control population of patients not suffering from lysosomal storage disorders with measured levels of patient specimens. A significant deviation between the level measured in the patient and the baseline level in an unaffected human is a sign of a positive result of the diagnostic test. Deviations are considered significant if the measured values fall outside the range typically observed in unaffected individuals due to inherent variations between individuals and experimental errors. For example, if the measured level does not fall within the mean of the level in the control population + a certain standard deviation, the deviation can be considered significant. In some methods, the deviation between the measurement level and the control level is determined to be significant if the measurement level is at least the 75, 80 or 95 percentile level of the control population. In other words, measurement levels in patients occur in only 50%, 25%, 20% or 5% of normal individuals. A diagnostic test result is considered negative if the measured level of the specimen is not significantly different from the baseline level of the control population.
[0043]
For saposin and Lamp-1, a positive result is typically indicated by a measured level at an excessive normal level. For α-mannosidosis, a positive result can be indicated by a measured level of either a level above baseline or a level below baseline. Whether the measured level of α-glucosidase is above or below the baseline level indicates the subtype of the patient suffering from a lysosomal storage disorder (see Table 1). The degree of deviation between the measured value and the baseline value of the control population also provides an indication of the potential accuracy of the diagnosis and / or the severity of the disease the patient is suffering from.
[0044]
If the diagnostic test gives a positive result, the patient is at least shown to be susceptible to, or at risk for, a lysosomal storage disorder. The patient is then typically subjected to further testing or screening. Such testing or screening may include analysis of additional analytes that correlate with lysosomal storage disorders that have not yet been tested. Such screening also includes performing biochemical tests for enzyme deficiencies associated with specific lysosomal storage disorders. Such assays are typically performed on urine, blood and dermal fibroblasts from patients (Schriver et al., Supra). For example, α-glucosidase can be assayed using 4-methyl-umbelliferyl-α-D-glucopyranoside as a substrate (see Van der Ploeg et al., Ped. Res. 24: 90-94 (1988)). ). Further trials also included clinical symptoms of lysosomal storage disorders (including dwarfism, corneal opacity, hepatosplenomegaly, valve lesions, coronary artery lesions, skeletal deformities, joint stiffness and progressive mentality Monitoring for including one or more of the delays). Further screening also encodes a family history analysis for members of related families suffering from lysosomal storage disorders and / or polymorphisms associated with lysosomal storage disorders from the patient for enzymes associated with such disorders Includes genetic analysis of DNA (eg, US 5,266,459). As a result of one or more of these screening assays, an initial diagnosis based on analyte levels can be confirmed (or otherwise) and patients suffering from a particular lysosomal storage disorder can be identified.
[0045]
Patients identified as having a particular lysosomal storage disorder are then typically administered treatment for this disorder. Treatment is typically a form of enzyme replacement therapy that uses an enzyme that is impaired or missing in a particular disorder. For example, Pomp disease can be treated with α-glucosidase as described in co-pending application USSN 08 / 700,760 (filed July 29, 1996). As a further example, Gaucher's disease can be treated with glucocendorosidase (see, eg, US 5,879,680).
[0046]
The second major application of this method is by monitoring the condition of patients undergoing treatment for lysosomal storage disorders. Successful treatment outcome is indicated by recovery from abnormal levels to normal levels of specimens (eg, saposin, Lamp-1, and α-glucosidase). Typically, such methods measure an initial value for the analyte level before the patient is treated. Repeated measurements are then made over time. If the initial level is assessed relative to the mean level in the control population, a significant decrease in the level in subsequent measurements indicates a residual treatment outcome. Similarly, if the initial level of the sample is decreasing relative to the mean of the control population, a significant increase in the measured level relative to the initial level is a signal for a positive treatment outcome. If the subsequently measured level differs by more than a certain standard deviation from the average of repeated measurements of the initial level, the subsequently measured level is considered to have a significant change relative to the initial level. . If monitoring shows a positive treatment result, the same treatment regimen can be continued or replaced with a lower dose treatment regimen. If monitoring indicates a negative treatment outcome, the preceding treatment regimen is typically modified to either use a different therapeutic agent or increase the dosage of the previous agent.
[0047]
(XII. Diagnostic kit)
The present invention further provides a diagnostic kit comprising reagents for use in the above method. Typically, such kits include at least one reagent that specifically binds to saposin, Lamp, or α-glucosidase. Some kits contain multiple reagents that specifically bind to different analytes. For example, the kit can include a reagent that specifically binds to saposin, a reagent that specifically binds to Lamp-1, and a reagent that specifically binds to α-glucosidase. Some kits contain multiple different reagents for two or more different saposins. For example, some kits bind reagents to saposin A without binding to other saposins, reagents to bind saposin C without binding to other saposins, and saposin D without binding to other saposins. Contains a binding reagent. This reagent is typically a monoclonal or polyclonal antibody, but can be other binding agents as described above. In some kits, the binding agent is provided immobilized on a solid phase. Some kits provide binding reagents immobilized on the wells of a microtiter dish. Some kits further comprise a sample of the analyte detected by the kit for use in calibration of the kit that correlates the signal level with the analyte concentration. Some kits also include one label as described above. For example, the label can be in the form of a labeled anti-idiotype antibody. The kit also typically includes a label or instructions describing how to perform the diagnostic assay described above and / or how to interpret the results obtained thereby.
[0048]
(Example)
(1. Saposin as a marker for lysosomal storage diseases)
This example shows saposins A, B, C and as screening markers for LSD by determining these protein levels in plasma samples taken from unaffected individuals and individuals suffering from LSD. The suitability of D is shown.
[0049]
(Materials and methods)
(Patient sample)
The plasma samples used were derived from samples submitted to the National Referral Laboratory (Women's and Children's Hospital, Adelaide, Australia) for processing for LSD screening and conventional biochemistry. Whole blood samples for fractionation studies were obtained from healthy volunteers in the laboratory.
[0050]
(Polyclonal antibody)
Anti-saposin A, B, C and D polyclonal antibodies were produced and characterized as previously described [15]. Each antibody was treated with 2-mL Hitrap ^TM Purified on a Protein G column (Pharmacia Biotech, Uppsala, Sweden) and quantified by absorbance at 280 nm (absorbance = 1.4 vs 1.0 g / L).
[0051]
(Europium labeling of polyclonal antibody)
Purified anti-saposin polyclonal antibody was purified using the DELFIA® labeling kit (Wallac, North Ryde, Australia) using Eu. ³⁺ Labeled with lanthanide. The labeled antibody was purified as previously described on a 1.5 x 30 cm Superose 12 fast-phase liquid chromatography column (Pharmacia Biotech, Uppsala, Sweden) [13]. Eu conjugated to each antibody molecule ³⁺ The amount of 1 nMEu ³⁺ Determined by fluorescence at known antibody concentrations compared to standard solutions.
[0052]
(Preparation of calibrator and quality control standard)
A liquid calibrator for immunoassay of saposin was prepared using purified saposin A, B, C and D proteins [20]. Purified saposin A protein was diluted in DELFIA® assay buffer to give final concentrations of 2.43, 1.21, 0.61, 0.3 and 0.15 μg / L. For the saposin A immunoassay assay, a low quality control standard and a high quality control standard were prepared by diluting saposin A protein to 0.24 and 2.43 μg / L, respectively. Purified saposin B protein was diluted in the same buffer to give final concentrations of 6.78, 3.34, 1.67, 0.83 and 0.42 μg / L. For the saposin B immunoassay assay, a low quality control standard and a high quality control standard were prepared by diluting saposin B protein to 0.67 and 6.78 μg / L, respectively. Purified saposin C protein was diluted to give final concentrations of 1.88, 0.94, 0.47 and 0.24 μg / L. For the saposin C immunoassay assay, a low quality control standard and a high quality control standard were prepared by diluting saposin C protein to 0.47 and 1.88 μg / L, respectively. Purified saposin D protein was diluted to give final concentrations of 0.92, 0.46, 0.23 and 0.11 μg / L. For the saposin D immunoassay assay, a low quality control standard and a high quality control standard were prepared by diluting saposin D protein to 0.23 and 0.92 μg / L, respectively.
[0053]
Calibrators and controls for saposins C and D were supplemented with an equal volume of control plasma sample to the sample being assayed to correct the inhibitory effect of plasma on saposin C and D assays. All liquid saposin calibrators and controls were stored at 4 ° C.
[0054]
(Immunoquantification of saposin)
Saposin determination was performed using a time-delayed fluorescent immunoassay. The basis for this assay format has been outlined previously [13]. Microtiter plates (Immulon 4; Dynatech Labs, Chantilly, VA) were added to anti-saposin polyclonal antibody (0.1 M NaHCO 3). _Three (2.5 μl / L diluted in pH 8.8, 100 μL / well), coated overnight at 4 ° C. and prewashed (× 1) with DELFIA® wash buffer. Samples were diluted in DELFIA® wash buffer (100 μL / well), shaken at 20 ° C. for 10 minutes, and incubated (4 hours at 4 ° C.). The plates are then washed (x6), Eu-labeled anti-saposin polyclonal antibody is added (diluted to 0.25 mg / L in DELFIA® assay buffer, 100 μL / well) and incubated (Overnight at 4 ° C.). Plates were washed (x6), then DELFIA® enhancement solution (200 μL) was added to each well, and plates were shaken (2 ° C. for 10 minutes). Fluorescence was read on a Wallac 1234 DELFIA® Research Fluorometer. For each saposin assay, a corresponding saposin calibrator was placed across the first row of microtiter plates containing randomly distributed quality controls. All saposin calibrators, quality controls and samples were assayed in duplicate. Saposin concentration was calculated using linear regression.
[0055]
(Fractionation of whole blood)
Whole blood samples from six control individuals were fractionated into plasma, white blood cells and red blood cells as previously described [21].
[0056]
(result)
(Optimization of saposin immunoassay assay)
The europium labeling of the anti-saposin polyclonal antibody is based on 4-7 Eu bound to each antibody molecule. ³⁺ Recoveries in the range of 75-100% with atoms were produced. The final parameters of the immunoquantitative assay were optimized by generating a series of calibration curves under the different conditions tested. Optimized parameters included coating antibody and Eu-labeled antibody concentrations, and antibody and sample incubation times. Cost, time minimization, and reproducibility of each assay were considered when selecting the final assay conditions.
[0057]
The optimized saposins A, B and C calibration curves produced a linear response to the standard range (R ² > 0.99), the calibration curve for saposin D had a reduced linear range (R ² = 0.9575) (Figure 1). When control plasma was added to the saposin C and D calibration curves, a decrease in signal intensity was observed. 2 μL of plasma produced a 20% decrease. To correct for the inhibitory effect of plasma on the saposins C and D immunoquantitative assay, an equal volume of control plasma relative to the plasma being assayed was added to the calibrator. It was hypothesized that LSD-affected plasma inhibited this assay in the same manner as control plasma, so only control plasma was used to spike the calibrator.
[0058]
(Saposin levels in plasma of individuals suffering from control and LSD)
To assess the suitability of each saposin as a marker for newborn screening for LSD, the levels of saposins A, B, C and D were derived from 111 control individuals (median age = 7, range 0-66), and Determined in plasma samples from 334 individuals with LSD showing 28 different disorders (Table 1). 95th percentile of control population saposin concentration (95 ^th (centile) is shown in Table 1 along with the entire population of individuals suffering from LSD with plasma saposin elevated above this level. Saposin A, C and D levels showed a tight distribution in plasma samples of the control population, while a broad range of saposin B levels were observed (Figure 2). Individuals affected by a significant proportion of LSD have saposin levels above the 95th percentile of the control population, where several individuals have an increase of up to 10 times the median concentration of the control population Was found. Fifteen of the 28 disorders had over 80% individuals with one or more saposin levels above the 95th percentile of the control population.
[0059]
[Table 2]

All saposin assays included a low quality control standard and a high quality control standard to determine the inter-assay coefficient of variation for each assay performed at a replicate number ranging from 17-24. . The coefficient of variation of both the low quality control standard and the high quality control standard saposin A assay was less than 10%. Saposin B, C and D assays yielded a coefficient of variation of less than 10% for high quality control standards and 15-20% for low quality control factors. Separate experiments were performed where 24 samples of high and low standards were assayed on a single plate to calculate the inter-assay coefficient of variation. Values in the range of 6-16% were obtained for the low quality control standard and values less than 10% for the high quality control standard.
[0060]
Since LAMP-1 levels in the same plasma sample have been previously assayed [13], a direct comparison between saposin and LAMP-1 levels could be made. Using Pyason correlation, a strong correlation (p <0.01) was observed between all saposins except that saposin B showed a low correlation with both saposin C and D levels (Table 3). Observed. A weak correlation (p <0.05) was observed between saposin and LAMP-1, and there was no correlation between saposin concentration and age in the control population.
[0061]
[Table 3]

(Saposin level in leukocytes)
Whole blood samples from 6 unaffected individuals were fractionated and the distribution of saposin in plasma, leukocytes and erythrocytes was determined (Table 3). The dominant saposin in leukocytes was found to be saposin D (concentration 75 μg / L), while a higher proportion (77%) of saposin B was found in the plasma fraction compared to other saposins. It was. The distribution of saposins A, C and D in plasma, leukocytes and erythrocytes is relatively equal.
[0062]
[Table 4]

(Discussion)
In 59% of patients, saposin A was found to rise above the 95th percentile of the control population, and saposins B, C and D were 25%, 61% and 75% of patients, respectively. (Table 1). Of the 28 indicated disorders in our study, saposin A levels in 6 groups have more than 80% individuals above the 95th percentile of the control group, and saposin B Increased in the group and saposins C and D increased in each of the 10 groups (Table 2). Six of these (ie, cystinosis, Fabry disease, Niemann-Pick disease (types A / B and C)), with saposins identified in more than 80% of the 15 LSD groups out of 28 Have not previously been observed to be elevated with LAMP-1 [13]. The remaining LSD (Krabbe disease, metachromatic leukotrophy, and Tay-Sachs disease) that did not rise for Lamp-1 had a significant number of patients (44-71%) showing increased saposin levels. (Table 2). Individuals suffering from about 85% LSD can be detected using both LAMP-1 and saposin as screening markers.
[0063]
There was a correlation between the LSD group that stores sphingolipids or sphingolipid derivatives and the LSD group that showed increased saposin levels. For example, Niemann-Pick disease (types A / B and C) in which sphingolipids are stored showed an increase in all saposins. However, only MPS II of the MPS groups studied showed an increase in saposin C. Saposin was also not elevated in demyelinating disorders such as MLD and Krabbe disease. Thus, the increase in saposin observed in the various LSD groups may be related to the tissue involved in storage and the type of substrate stored. Furthermore, the degree of saposin increase can also reflect the severity of the individual.
[0064]
The ratio of individual saposin levels in plasma from the LSD group was also tested and compared to the control population to determine if this ratio value was a good LSD detection parameter. However, a strong correlation between saposins (Table 3) resulted in a ratio of any two saposins that yielded lower estimates than the individual saposins.
[0065]
Saposin B was the most prevalent saposin in the plasma of control individuals (Table 2). A higher proportion of saposin B (77%) was found in the plasma fraction compared to saposins A, C and D (38, 23 and 30%, respectively). Saposin D is the predominant saposin (75 μg / L) in whole blood. Since saposins originate from the same precursor, their synthesis rates should be equal. Thus, the different levels observed probably resulted from different half-lives, where saposin D had a longer half-life in cells and saposin B had a longer half-life in plasma.
[0066]
(Example 2)
Plasma samples were assayed for α-glucosidase protein by an immunoquantitative assay using the same technique described for the saposin study. The results are represented as a box plot in FIG. The results are also shown in Table 5. This Table 5 shows the percent of patients with elevated levels of α-glucosidase above the median level in each disorder group and the 95th percentile of the control population. All 55% of patients showed an increase in α-glucosidase. All pamp patients showed α-glucosidase levels below the 5th percentile of the control population.
[0067]
Thus, α-glucosidase is a useful marker for the detection of a range of LSD by detecting either an increase or decrease in the level of this protein in plasma serum or whole blood.
[0068]
[Table 5]

(Example 3)
The experiment shown in FIG. 2 was repeated for several samples using a combination of monoclonal and polyclonal antibodies against saposin C. Microliter plates were coated with monoclonal antibody 7B2 (4 mg / L, 16 hours, 4 ° C.), washed and then incubated with plasma samples (2 μL) in assay buffer (6 hours, 4 ° C.). The plate was washed again and then incubated with 500 μg / mL of liquid phase europium labeled antibody as indicated (6 hours, 4 ° C.). The plate was washed and developed with enhancement buffer (200 μL / well) and the fluorescence read. The concentration of saposin C was calculated based on a calibration curve using recombinant saposin C.
[0069]
[Table 6]

The 7B2 antibody behaved similarly to the polyclonal serum in the capture step. However, the 3A1 antibody has decreased reactivity with control samples, decreased reactivity with Gossy disease samples, and increased reactivity with samples of metachromatic leukodystrophy and neuronal ceroid lipoxinosis. Indicated. These results indicate that there are multiple subtypes of saposin C that are immunologically distinguishable.
[0070]
The presence of immunologically distinguishable subtypes across all four classes of saposins A, B, C and D allows for a more sensitive diagnostic assay using monoclonal antibodies specific for the subtype. For example, the use of monoclonal antibody 3A1 provides an improvement in sensitivity to detect elevated saposin C levels in metachromatic leukotrophy and neuronal ceroid sebaceous brown disease samples relative to controls. Other monoclonals for any saposin can be tested for increased sensitivity to the control using the procedure described above.
[0071]
In some methods, a primary screen is performed using polyclonal sera against one or more saposins and samples, and the further investigation identified thereby is subjected to a secondary screen using monoclonal antibodies. In some methods, monoclonals used for secondary screening are selected. Because they show enhanced sensitivity in the detection of saposin in the particular disease state suggested by the primary screen. In some methods, samples are tested with multiple monoclonals that bind the same saposin type (eg, saposin C) but specifically bind to different subtypes of this class. By using a monoclonal antibody (eg 3A1) specific for a particular subtype, it becomes possible to achieve a higher level of discrimination between the reduced signal and the control patient, Increase the accuracy of this assay.
[0072]
(References)
[0073]
[Table 7]

Although the foregoing invention has been described in detail for purposes of clarity of understanding, it will be apparent that certain modifications may be made within the scope of the appended claims. All publications and patent documents cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each were so individually indicated. Is done.
[Brief description of the drawings]
FIG. 1 is a calibration curve for saposins A, B, C and D immunoquantification assays. Optimal to generate a calibration curve for saposin A (panel a), saposin B (panel b), saposin C (panel c) and saposin D (panel d) for use in immunoquantification of the respective proteins Immunoquantification conditions were used. Microtiter plates are coated with primary anti-saposin polyclonal antibody (2.5 mg / L, 4 ° C., overnight), standards are incubated (4 ° C., 4 hours), and Eu-labeled anti-saposin polyclonal antibody is used. (0.25 mg / L, 4 ° C., overnight).
FIG. 2 is a box plot of saposin concentration in plasma from controls and individuals who developed LSD. Saposin concentration was determined by using 0.25-2 μL of plasma sample. N = number of saposins used in each group. The line in the box is the average level, the shaded areas below and above the average represent the 25th percentile and 75th percentile, respectively, and the bar represents the range. ○ indicates an outlier, and ^* Indicates an extreme outlier. Galact = Galactosialidosis; GM I = GM I gangliosidosis; Mann = α-mannosidosis; MLD = metachromatic leukotrophy; MSD = multiple sulfatase deficiency; -P = Niemann-Pick disease; SAS = sialic acid storage disease; TSD = Tay-Sachs disease.
FIG. 3 is a box plot of α-glucosidase levels in plasma from individuals who developed control and LSD. The α-glucosidase concentration was determined by using 5.0 μL of plasma sample. N = number of saposins used in each group. The line in the box is the average level, the shaded areas below and above the average represent the 25th percentile and 75th percentile, respectively, and the bar represents the range. ○ indicates an outlier, and ^* Indicates an extreme outlier. Galact = Galactosia Ridosis; GM I = GM I gangliosidosis; MLD = metachromatic leukodystrophy; MSD = multiple sulfatase deficiency; NP = Niemann-Pick disease; SAS = sialic acid storage disease; TSD = Tay -Sachs disease. The Y axis is magnified to highlight differences between controls and LSD patients.

Claims (13)

A method of using the level of at least one saposin for diagnosis or monitoring of lysosomal storage disorder in a subject,
Measuring the level of at least said one saposin in a sample obtained from the subject;
Comparing the level to a baseline level, wherein the baseline level is at least the level of the saposin as determined in a control population of subjects not afflicted with the lysosomal storage disorder. Detecting whether the level does not exceed or exceeds the 95th percentile of the baseline level of at least the saposin in the control population;
Where (i) the relative amount of each saposin measured at a level not exceeding 95 percentile is an indication of the absence of the lysosomal storage disorder in the subject;
(Ii) the relative amount of each saposin measured at a level above the 95 percentile is an indication of the presence or extent of the lysosomal storage disorder in the subject;
(Iii) the sample is a sample of plasma, serum, or whole blood; and (iv) the saposin contains saposin A and the lysosomal storage disorder is cystinosis, Fabry disease, Gaucher disease, GM- 1-gangliosidosis, I-cell disease, Krabbe disease, MPS I, MPS II, MPS IVA, MPS VI, Niemann-Pick disease (type A / B), Niemann-Pick disease (type C), Zanthof disease, sialic acid Selected from the group consisting of storage disease, type 1 Tay-Sachs disease and Wolman disease; or the saposin comprises saposin B and the lysosomal storage disorder is Fabry disease, Goshe disease, Niemann-Pick disease (A / B), Pomp disease and Zandhof disease; or the saposin comprises saposin C, and Lysosomal storage disorders include cystinosis, Fabry disease, Goshe disease, GM-1-gangliosidosis, I cell disease, MPS I, MPS
Group consisting of II, MPS IIID, MPS VI, multiple sulfatase deficiency, Niemann-Pick disease (A / B type), Niemann-Pick disease (type C), Pomp disease, Zandhof disease, sialic acid storage disease and Wolman disease Or the saposin comprises saposin D and the lysosomal storage disorder is cystinosis, Fabry disease, Goshe disease, GM-1-gangliosidosis, I cell disease, α-mannosidosis, Metachromatic leukotrophy, MPS I, MPS VI, multiple sulfatase deficiency, Niemann-Pick disease (type A / B), Niemann-Pick disease (type C), Pomp disease, Tay-Sachs (A / B) A method selected from the group consisting of Wolman disease and Wolman disease. Measuring a second level of second saposin in a second sample from said subject, wherein said saposin and said second saposin are the same, and said sample and said second sample are Obtained at different times; and comparing the level in the sample with the second level to monitor the progress of the lysosomal storage disorder;
Detecting whether the second level does not exceed or exceeds the 95th percentile of the baseline level of at least the saposin in the control population, wherein (i) the A comparison of the level to the second level is an indicator of progression of the lysosomal storage disorder in the subject; and (ii) the second sample is a sample of plasma, serum or whole blood;
The method of claim 1.   2. The method of claim 1, further comprising selecting the subject undergoing treatment for the lysosomal storage disorder.   2. The method of claim 1, wherein the subject is not known to have a lysosomal storage disorder.   2. The method of claim 1, further comprising selecting the subject to be an infant under 1 year of age.   The method of claim 1, further comprising selecting the subject being a fetus, and wherein the sample is a fetal blood sample.   3. The method of claim 2, wherein a change in the level of the saposin indicates progression or recovery of the disorder in the subject known to have a lysosomal storage disorder.   3. The method of claim 2, wherein a change in the level of the saposin indicates a response to treatment of the lysosomal storage disorder in the subject being treated for a lysosomal storage disorder.   The method of claim 1, wherein the measuring comprises detecting binding between a saposin polypeptide and an antibody.   The method of claim 9, wherein the antibody is a monoclonal antibody.   The method of claim 9, wherein the antibody is immobilized on a solid phase. A method of using a pre-treatment baseline level of saposin and a post-treatment baseline level of saposin for monitoring treatment of a lysosomal storage disorder in a patient comprising the steps of :
Prior to treatment with the drug, the step of measuring a baseline level before said treatment of said saposin in a sample from said patient with a lysosomal storage disorder;
After treatment with the agent, a step of measuring a baseline level after said treatment of said saposin that put the sample from the patient with the lysosomal storage disorder; and and baseline levels prior the treatment of said saposin, Comparing the post-treatment baseline level of the saposin;
Where (i) the sample is a sample of plasma, serum, whole blood, or a mixture thereof;
(Ii) the saposin comprises saposin A and the lysosomal storage disorder is cystinosis, Fabry disease, Goshe disease, GM-1-gangliosidosis, I cell disease, Krabbe disease, MPS I, MPS II, MPS Is it selected from the group consisting of IVA, MPS VI, Niemann-Pick disease (type A / B), Niemann-Pick disease (type C), Zandhof disease, sialic acid storage disease, type 1 Tay-Sachs disease and Wolman disease Or the saposin comprises saposin B, and the lysosomal storage disorder is selected from the group consisting of Fabry disease, Gaucher disease, Niemann-Pick disease (type A / B), Pomp disease and Zandhof disease; The saposin comprises saposin C, and the lysosomal storage disorder is cystinosis, Fabry disease, Gaucher disease, GM-1-Ga Griot Sydow cis, I-cell disease, MPS I, MPS
Group consisting of II, MPS IIID, MPS VI, multiple sulfatase deficiency, Niemann-Pick disease (A / B type), Niemann-Pick disease (type C), Pomp disease, Zandhof disease, sialic acid storage disease and Wolman disease Or the saposin comprises saposin D and the lysosomal storage disorder is cystinosis, Fabry disease, Goshe disease, GM-1-gangliosidosis, I cell disease, α-mannosidosis, Metachromatic leukotrophy, MPS I, MPS VI, multiple sulfatase deficiency, Niemann-Pick disease (type A / B), Niemann-Pick disease (type C), Pomp disease, Tay-Sachs (A / B) (Iii) a baseline level after the treatment relative to a baseline level before the treatment; Decrease Le indicates a positive treatment outcome, methods. A method of using the level of the saposin for diagnosis or monitoring of lysosomal storage disorder in a subject,
Measuring the level of saposin in a sample obtained from the subject;
Comparing the level to a baseline level, wherein the baseline level is the level of the saposin as determined in a control population of subjects not afflicted with the lysosomal storage disorder; and the level to detect whether it exceeds or does not exceed the 95th percentile of the baseline level of the saposin in the control population, step,
Where (i) the relative amount of each saposin measured at a level not exceeding 95 percentile is an indication of the absence of the lysosomal storage disorder in the subject;
(Ii) the relative amount of each saposin measured at a level above the 95 percentile is an indication of the presence or extent of the lysosomal storage disorder in the subject;
(Iii) the saposin comprises saposin A and the lysosomal storage disorder is cystinosis, Fabry disease, Goshe disease, GM-1-gangliosidosis, I cell disease, Krabbe disease, MPS I, MPS II, MPS Is it selected from the group consisting of IVA, MPS VI, Niemann-Pick disease (type A / B), Niemann-Pick disease (type C), Zandhof disease, sialic acid storage disease, type 1 Tay-Sachs disease and Wolman disease Or the saposin comprises saposin B, and the lysosomal storage disorder is selected from the group consisting of Fabry disease, Gaucher disease, Niemann-Pick disease (type A / B), Pomp disease and Zandhof disease; The saposin comprises saposin C, and the lysosomal storage disorder is cystinosis, Fabry disease, Gaucher disease, GM-1- Packaging Rio Sydow cis, I-cell disease, MPS I, MPS
Group consisting of II, MPS IIID, MPS VI, multiple sulfatase deficiency, Niemann-Pick disease (A / B type), Niemann-Pick disease (type C), Pomp disease, Zandhof disease, sialic acid storage disease and Wolman disease Or the saposin comprises saposin D and the lysosomal storage disorder is cystinosis, Fabry disease, Goshe disease, GM-1-gangliosidosis, I cell disease, α-mannosidosis, Metachromatic leukotrophy, MPS I, MPS VI, multiple sulfatase deficiency, Niemann-Pick disease (type A / B), Niemann-Pick disease (type C), Pomp disease, Tay-Sachs (A / B) And (iv) the sample is plasma; and (v) the baseline level and The level is approximately equal to the percentage increase in the level of the lysosomal storage disorders,
Method.

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