JP5324918B2 - HbA1c測定方法 - Google Patents
HbA1c測定方法 Download PDFInfo
- Publication number
- JP5324918B2 JP5324918B2 JP2008522139A JP2008522139A JP5324918B2 JP 5324918 B2 JP5324918 B2 JP 5324918B2 JP 2008522139 A JP2008522139 A JP 2008522139A JP 2008522139 A JP2008522139 A JP 2008522139A JP 5324918 B2 JP5324918 B2 JP 5324918B2
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- JP
- Japan
- Prior art keywords
- hba1c
- whole blood
- hemoglobin
- storage
- measuring
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/795—Porphyrin- or corrin-ring-containing peptides
- G01N2333/805—Haemoglobins; Myoglobins
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/19—Halogen containing
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
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- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
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- General Health & Medical Sciences (AREA)
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- Biotechnology (AREA)
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- Hematology (AREA)
- Genetics & Genomics (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
(A1)Hb含有物を、二酸化炭素の発生を抑制させた状態で保存する工程
(A2)保存後のHb含有物から、Hbに結合している二酸化炭素を減少させる工程
(A)本発明の製造方法により、保存後のHb含有試料を準備する工程
(B)保存後の前記Hb含有試料をプロテアーゼ処理して、前記Hb含有試料中のヘモグロビンを切断する工程
(C)前記(B)工程により得られたヘモグロビン断片の糖化部分に、フルクトシルアミンオキシダーゼを作用させる工程
(D)前記糖化部分とフルクトシルアミンオキシダーゼとの酸化還元反応を測定することにより、HbA1cの量を決定する工程
本発明の製造方法は、前述のように、HbA1cを測定する方法に使用するHb含有試料の製造方法であって、下記(A1)または(A2)工程を含むことを特徴とする。
(A1)Hb含有物を、二酸化炭素の発生を抑制させた状態で保存する工程
(A2)保存後のHb含有物から、Hbに結合している二酸化炭素を減少させる工程
本発明のHbA1c測定方法は、HbA1cを測定する方法であって、下記(A)〜(D)工程を含むことを特徴とする。
(A)本発明の製造方法により、保存後のHb含有試料を準備する工程
(B)保存後の前記全血試料をプロテアーゼ処理して、前記Hb含有試料中のヘモグロビンを切断する工程
(C)前記(B)工程により得られたヘモグロビン断片の糖化部分に、フルクトシルアミンオキシダーゼを作用させる工程
(D)前記糖化部分とフルクトシルアミンオキシダーゼとの酸化還元反応を測定することにより、HbA1cの量を決定する工程
本発明の第1のHbA1c測定方法は、下記(A1’)〜(D)工程を含む。
(A1’)解糖系阻害剤の存在下でHb含有物を保存する工程(保存後のHb含有試料の製造)
(B)保存後のHb含有試料をプロテアーゼ処理して、前記Hb含有試料中のヘモグロビンを切断する工程
(C)前記(B)工程により得られたヘモグロビン断片の糖化部分に、フルクトシルアミンオキシダーゼを作用させる工程
(D)前記糖化部分とフルクトシルアミンオキシダーゼとの酸化還元反応を測定することにより、HbA1cの量を決定する工程
被検体から全血を採取し、HbA1cの測定時まで、前記解糖系阻害剤の存在下で保存する。なお、HbA1c測定において、全血の保存は必須ではないが、本発明の目的は、保存によるHbA1c値の変動を抑制することであるため、全血の保存が必要となる場合に、本発明を適用することが好ましい。
保存後の全血試料を溶血処理する。全血試料の溶血方法は、特に制限されず、例えば、浸透圧差を利用する方法、超音波による方法等が使用できる。浸透圧差を利用する場合、全血(または血球)に対して、例えば、2〜100倍体積量の精製水を添加して溶血させればよい。また、界面活性剤を試料に添加して溶血することもできる。
溶血した保存後全血試料をプロテアーゼ処理する。この処理で、前記試料中のHbのβ鎖N末端バリンや、前記N末端バリンを含むペプチド(β鎖N末端ペプチド)を切り出すことによって、後述するFAODを、Hbの糖化部分(β鎖N末端バリン)に効率良く作用させることができる。
R−X ・・・(I)
つぎに、前記プロテアーゼ処理後の反応液にFAODを添加する。これによって、Hb断片におけるN末端バリンの糖化部分にFAODを作用させ、酸化還元反応を行う。このFAOD処理によって、例えば、以下に示すように、N末端バリンに結合した糖が遊離し、過酸化水素が生成される。
R1−CO−CH2−NH−R2 + H2O + O2
→ R1−CO−CHO + NH2−R2 + H2O2 …(1)
−[CH(OH)]n−CH2OH
で示すことができ、nは、0〜6の整数である。
−CHR3−CO−R4 …(2)
−(NH−CHR3−CO)n−OH …(3)
つぎに、前記糖化部分とFAODとの酸化還元反応の測定を行う。この測定は、例えば、前記反応により生じる過酸化水素量の測定や、前記反応で消費される酸素量の測定があげられる。前記過酸化水素量は、例えば、ペルオキシダーゼ(POD)と酸化により発色する基質とを用いて、これらと過酸化水素との反応により前記基質を発色させ、この発色程度を測定することにより行うことができる。また、過酸化水素量は、POD等を用いた酵素的手法の他に、電気的手法等によって測定することもできる。
HbA1c%=(β鎖N末端バリンの糖化量/Hb量)×100
1 : 溶血処理+プロテアーゼ処理
2 : 溶血処理+プロテアーゼ処理+FAOD処理
3 : 溶血処理+プロテアーゼ処理+FAOD処理+POD処理
4 : プロテアーゼ処理+FAOD処理
5 : プロテアーゼ処理+FAOD処理+POD処理
6 : FAOD処理+POD処理
本発明の第2のHbA1c測定方法は、下記(A2’)〜(D)工程を含む。
(A2’)保存後の全血に、正イオンがK+、Na+およびMg2+からなる群から選択された少なくとも一つのイオンであり且つ負イオンがCl−、SO4 2−およびNO−からなる群から選択された少なくとも一つのイオンである強電解質物質を添加する工程(保存後の全血試料の製造)
(B)保存後の前記ヘモグロビン含有試料をプロテアーゼ処理して、前記ヘモグロビン含有試料中のヘモグロビンを切断する工程
(C)前記(B)工程により得られたヘモグロビン断片の糖化部分に、フルクトシルアミンオキシダーゼを作用させる工程
(D)前記糖化部分とフルクトシルアミンオキシダーゼとの酸化還元反応を測定することにより、HbA1cの量を決定する工程
まず、被検体から全血を採取して、HbA1cの測定時まで、これを保存する。なお、HbA1cの測定において、全血の保存は必須ではないが、本発明の目的は、保存によるHbA1c値の変動を抑制することであるため、全血の保存が必要となる場合に、本発明を適用することが好ましい。
保存後の全血を溶血処理する。溶血処理の条件は、例えば、前記第1のHbA1c測定方法と同様である。なお、前記強電解質物質は、溶血処理の前後いずれに添加してもよい。
前記溶血した全血に、前記強電解質物質を添加する。これによってHbA1c測定に供する保存後の全血試料を調製できる。
前記溶血した全血試料について、前記強電解質物質の存在下、プロテアーゼ処理を行う。
H管 :ヘパリンナトリウム採血管(テルモ社製)
DK管:EDTA−K採血管(テルモ社製)
FH管:フッ化ナトリウム+ヘパリンナトリウム採血管(テルモ社製)
15日放置した全血を精製水で26倍希釈し、この希釈液6.5μLに精製水6.5μLを添加したものをサンプルとした。このサンプル13μLに前記第1試薬78μLを混合して37℃で5分間インキュベートした。ここで、この反応液について、波長571nm/751nmにおける吸光度測定(B1)、および、波長694nm/751nmにおける吸光度測定(A1)を行った。次に、前記反応液に、さらに前記第2試薬19.5μLを添加して37℃で5分間インキュベートした後、前記反応液について、再度、波長694nm/751nmにおける吸光度測定(A2)を行った。そして、下記式に示すように、2回目の吸光度(A2)から、1回目の吸光度(A1)に容量変化を補正する値[(13+78)/(13+78+19.5)]を乗じた値を差し引き、この値を、全血試料のHbA1c濃度に相当する吸光度(HbA1c吸光度)とした。なお、1回目の波長571nm/751nmにおける吸光度(B1)は、試料中のHb濃度に相当する(Hb吸光度)。測定は、生化学自動分析装置(商品名JCA−BM8:日本電子社製)を用いて行った。また、コントロールとして、採血直後の全血、ならびに、全血に代えて精製水を使用し、同様にして吸光度測定を行った。また、この吸光度を、予め作成した検量線に代入して、HbA1c%を求めた。前記検量線は、HbA1c%が既知である標準試料を用いて同様に吸光度測定を行い、前記吸光度とHbA1c%の値とのプロットから作成した。これらの結果を下記表2に示す。
HbA1c吸光度=A2−[A1×(13+78)/(13+78+19.5)]
採血直後 5.45
1日後 5.35
5日後 5.61
7日後 5.50
9日後 5.56
12日後 5.51
Claims (8)
- HbA1cを測定する方法であって、
下記(A)〜(D)工程を含むことを特徴とするHbA1c測定方法。
(A)下記(A1)または(A2)工程により、保存後のヘモグロビン含有試料を準備する工程
(A1)ヘモグロビン含有物を、フッ化ナトリウムおよびフッ化カリウムの少なくとも一方である解糖系阻害剤の存在下、保存して、これを保存後のヘモグロビン含有試料とする工程
(A2)保存後のヘモグロビン含有物に、KCl、K2SO4、NaClおよびMgSO4からなる群から選択された少なくとも一つである強電解質物質を添加して、これを保存後のヘモグロビン含有試料とする工程
(B)保存後の前記ヘモグロビン含有試料をプロテアーゼ処理して、前記ヘモグロビン含有試料中のヘモグロビンを切断する工程
(C)前記(B)工程により得られたヘモグロビン断片の糖化部分に、フルクトシルアミンオキシダーゼを作用させる工程
(D)前記糖化部分とフルクトシルアミンオキシダーゼとの酸化還元反応を測定することにより、HbA1cの量を決定する工程 - 前記(A)工程が、前記(A1)工程により、保存後の前記ヘモグロビン含有試料を準備する工程であって、
前記(B)工程において、プロテアーゼ処理の反応液における前記解糖系阻害剤の濃度が、0.01〜10mol/Lである、請求項1記載のHbA1c測定方法。 - 前記(A)工程が、前記(A2)工程により、保存後の前記ヘモグロビン含有試料を準備する工程であって、
前記(B)工程において、前記強電解質物質の存在下、プロテアーゼ処理を行う、請求項1記載のHbA1c測定方法。 - 前記(B)工程において、プロテアーゼ処理の反応液中の前記強電解質物質の濃度が、10〜3000mmol/Lである、請求項3記載のHbA1c測定方法。
- 前記(D)工程において、前記糖化部分とフルクトシルアミンオキシダーゼとの酸化還元反応を測定することにより、前記ヘモグロビン含有試料中のヘモグロビンにおけるHbA1c量を算出し、さらに、前記ヘモグロビン含有試料中のヘモグロビン量と前記HbA1c量から、HbA1c比率を算出する、請求項1から4のいずれか一項に記載のHbA1c測定方法。
- 前記ヘモグロビン含有試料が、全血試料または血球試料である、請求項1から5のいずれか一項に記載のHbA1c測定方法。
- 請求項1記載のHbA1c測定方法に使用する試薬キットであって、
フルクトシルアミンオキシダーゼおよび強電解質物質を含む第1試薬と、プロテアーゼを含む第2試薬とを含み、
前記強電解質物質が、KCl、K2SO4、NaClおよびMgSO4からなる群から選択された少なくとも一つである測定キット。 - 前記第1試薬が、さらに酸化酵素を含み、前記第2試薬が、さらに酸化により発色する基質を含む、請求項7記載の測定キット。
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