JP4574066B2 - Blood sample for Preβ1-HDL measurement - Google Patents
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Description
【０００１】
【発明の属する技術分野】
本発明は、保存安定性の高いＰｒｅβ１−ＨＤＬ測定用血液検体及び当該検体を使用したＰｒｅβ１−ＨＤＬの免疫学的測定法に関する。
【０００２】
【従来の技術】
アポＡ−ＩはＨＤＬを構成する主なアポ蛋白質であり、ＨＤＬの末梢細胞から肝臓へのコレステロールを逆転送する機能において中心的な役割を果たしているものである。（Philips M. C. et al. Biochem. Biophys. Acta, 906：p. 223(1987)）。このことから、動脈硬化症の診断にアポＡ−Ｉを測定することが行われている。
【０００３】
近年、アポリポ蛋白質Ａ−II（以下、「アポＡ−II」という）を持たないアポＡ−Ｉ含有ＨＤＬ（石塚ら：医学と薬学、39巻５号、1041頁、1988）が、アポＡ−Ｉ及びアポＡ−II含有ＨＤＬより細胞からのコレステロール引き抜き作用が強いことや、脂質とは結合せずに存在するアポＡ−Ｉや小粒子で脂質含量の少ないＰｒｅβ１−ＨＤＬ（T. Miida. et al. Biochemistry, 29：p. 10469(1990)）に存在するアポＡ−Ｉが細胞からのコレステロールの逆転送系において重要な役割を演じていることが判明したことから、これら特定のアポＡ−Ｉを測定することが重要となってきた。アポＡ−IIを持たないアポＡ−Ｉ含有ＨＤＬのうち、Ｐｒｅβ１−ＨＤＬは、細胞表面との特異的な相互作用を介して末梢細胞からコレステロールを引き抜き（Fielding, C. et al, Lipid Res., 36：p211-228(1995)）、その作用はＨＤＬよりも効率的であることから、特に注目されている。そしてＰｒｅβ１−ＨＤＬに対する抗体を用いるＰｒｅβ１−ＨＤＬの免疫学的測定法も開発されている（特開2000-239300）。
【０００４】
【発明が解決しようとする課題】
しかしながら、採血後の血液中におけるＰｒｅβ１−ＨＤＬは非常に不安定で、血液検体を通常の冷所保存条件である４℃や、室温で保存すると、Ｐｒｅβ１−ＨＤＬ濃度は、採血直後の測定値に比べて１日後で３０％程度、４日後で５０％程度高値となってしまうという問題があることが判明した。また、このような保存安定性を防止する手段として検体の凍結保存がある。しかし、凍結保存後に融解して測定すると、この測定値もまた採血時の値に比べて高値となることが判明した。
従って、本発明の目的はＰｒｅβ１−ＨＤＬ測定用の血液検体の安定化手段を提供することにある。
【０００５】
【課題を解決するための手段】
そこで、本発明者は、採血後の血液、血漿又は血清検体中のＰｒｅβ１−ＨＤＬの安定化を図るべく、種々検討した結果、血液検体に２５〜８０重量％となるように単糖類、２糖類又はグリセロールを添加すれば、４℃で５日間経過後も検体中のＰｒｅβ１−ＨＤＬ濃度はほとんど変化せず、また凍結融解後の検体中のＰｒｅβ１−ＨＤＬ濃度もほとんど変化せず、安定性の高いＰｒｅβ１−ＨＤＬ測定用血液検体が得られることを見出し、本発明を完成するに至った。
【０００６】
すなわち、本発明は、単糖類、２糖類又はグリセロールを２５〜８０重量％含有するＰｒｅβ１−ＨＤＬ測定用血液検体を提供するものである。
また本発明は、当該Ｐｒｅβ１−ＨＤＬ測定用血液検体に抗Ｐｒｅβ１−ＨＤＬ抗体を反応させることを特徴とするＰｒｅβ１−ＨＤＬの免疫学的測定法を提供するものである。
【０００７】
【発明の実施の形態】
本発明において単糖類、２糖類及びグリセロールは、血液検体中のＰｒｅβ１−ＨＤＬの安定化剤として作用するものであり、単糖類としては、グルコース、フルクトース、ガラクトース等が挙げられる。２糖類としては、サッカロース、マルトース、ラクトース等が挙げられる。これらのうちサッカロース及びグリセロールがより好ましく、サッカロースが特に好ましい。
【０００８】
これら安定化剤の検体中の濃度は、安定化作用の点から２５〜８０重量％である必要があるが、３０〜８０重量％、特に３０〜７０重量％が好ましい。２５重量％未満では安定化作用がない。
【０００９】
検体としては、全血、血漿、血清のいずれも挙げられるが、血漿が好ましい。
また、検体には、血液凝固防止の目的で、クエン酸（好ましくは０．１〜１．０％）、ＥＤＴＡ（好ましくは０．０５〜１０mM）等が含まれていてもよい。
【００１０】
本発明の血液検体は、例えば血漿の場合には、患者又は被検者からＥＤＴＡ採血管等を用いて採血し、冷却後遠心分離して血漿を得た後、これに、濃度が２５〜８０重量％になるように単糖類、２糖類若しくはグリセロール又はこれらの水溶液を添加することにより調製できる。
【００１１】
かくして得られた本発明の血液検体はＰｒｅβ１−ＨＤＬが安定化されているので、５日間程度まで冷所保存（通常２〜１０℃）した後に抗Ｐｒｅβ１−ＨＤＬ抗体を用いて免疫測定しても安定したＰｒｅβ１−ＨＤＬ濃度の測定が可能である。
【００１２】
本発明血液検体に抗Ｐｒｅβ１−ＨＤＬ抗体を反応させてＰｒｅβ１−ＨＤＬを測定するには、通常の免疫学的測定法、通常の競合法、サンドイッチ法によるＲＩＡ又はＥＩＡ等が挙げられる。これらの方法の実施にあたっては、抗Ｐｒｅβ１−ＨＤＬ抗体の標識体を用いることもできる。ここで標識物質としては、パーオキシダーゼ、アルカリホスファターゼ、グルコアミラーゼ、β−ガラクトオキシダーゼ等の酵素；125Ｉ、131Ｉ、トリチウム等の放射性物質が挙げられる。
また、抗体を固相化するための単体としては、各種プラスチックウェル、各種プラスチックビーズ等が挙げられる。
【００１３】
抗Ｐｒｅβ１−ＨＤＬ抗体としてはポリクローナル抗体でもよいが、モノクローナル抗体を用いるのが好ましく、当該モノクローナル抗体としては特開２０００−２３９３００記載のモノクローナル抗体５５２０１が特に好ましい。
【００１４】
例えば、ＥＬＩＳＡ法で測定する場合には、精製したアポＡ−Ｉを標準品として次のような方法で定量することができる。すなわち、抗Ｐｒｅβ１−ＨＤＬモノクローナル抗体を固相化したＥＬＩＳＡプレートに、希釈した試料を添加し反応させた後、酵素標識した抗アポＡ−Ｉポリクローナル抗体を反応させ、発色後吸光度の変化から試料中に存在するＰｒｅβ１−ＨＤＬを定量する方法が挙げられる。
【００１５】
なお、これらの測定は、通常の免疫学的測定法と同様に０〜４０℃のいずれの温度で行うこともできる。
【００１６】
【実施例】
次に実施例を挙げて本発明をさらに詳細に説明するが、本発明は何らこれらに限定されるものではない。
【００１７】
実施例１
（１）ＥＤＴＡ（４mM）採血管を用いて採血した血液を氷冷した後、３０００rpm、１５分遠心分離して血漿を得た。氷冷下血漿０．１mLに表１の濃度になるようにサッカロース水溶液２mLを加え、Ｐｒｅβ１−ＨＤＬ測定用血漿検体を得た。
【００１８】
（２）得られた検体を用いて、採血当日、４℃に１日保存後、及び４℃に５日間保存後のＰｒｅβ１−ＨＤＬ濃度を測定した。また、サッカロースを添加しない血漿についても同様にしてＰｒｅβ１−ＨＤＬ濃度を測定した。
すなわち、特開２０００−２３９３００の実施例１で得たモノクローナル抗体５５２０１を２０mMリン酸緩衝生理食塩水（PBS；pH7.2）で３μｇ／mLの濃度に調整後、９６穴ＥＬＩＳＡプレート（ヌンク社製）に５０μＬ／ウェル加え、４℃で一夜インキュベートした。プレートをＰＢＳで３回洗浄後、ブロッキング液（１％BSA-PBS）を１００μＬ／ウェル加え、１時間ブロッキングした。ブロッキング液を除去後、ブロッキング液にて希釈した前記検体又は精製アポＡ−Ｉを５０μＬ／ウェル加え、室温で１時間インキュベートした。ブロッキング液で３回洗浄した後、アポＡ−Ｉを山羊に免疫して得たヤギ抗アポＡ−Ｉ抗体を過ヨウ素酸法にてペルオキシダーゼ標識したペルオキシダーゼ標識ヤギ抗アポＡ−Ｉ抗体を５０μＬ／ウェル加え、室温で１時間インキュベートした。同様にブロッキング液で３回洗浄した後、ペルオキシダーゼ基質溶液を５０μＬ／ウェル加えた。１０分後、１．５Ｎ硫酸を５０μＬ／ウェル加え、４９２nmにおける吸光度を測定し、精製アポＡ−Ｉを標準品として、各検体中のＰｒｅβ１−ＨＤＬ量を算出した。
【００１９】
その結果、表１に示すように、サッカロースを添加しない血漿、４．８重量％及び９．５重量％のサッカロースを含む血漿はいずれも採血当日のＰｒｅβ１−ＨＤＬ濃度に比べて１日後及び５日後では高くなってしまった。これに対し２８．６〜６６．７重量％のサッカロースを含む血漿は、５日後までＰｒｅβ１−ＨＤＬ濃度の変化がなかった。
【００２０】
【表１】
実施例２
実施例１と同様にして、検体として、血漿、１％ＢＳＡ−ＰＢＳ含有血漿、４７．６重量％サッカロース含有血漿、及び４７．６重量％グリセロール含有血漿を用いてＰｒｅβ１−ＨＤＬ濃度を測定した。その結果を図１に示す。図１から明らかなように、血漿及びＢＳＡ−ＰＢＳ含有血漿は１日後（４℃）からＰｒｅβ１−ＨＤＬ濃度が高くなっていたが、４７．６重量％グリセロール又は４７．６重量％サッカロース含有血漿は４日後（４℃）までＰｒｅβ１−ＨＤＬ濃度が変化しなかった。
【００２２】
実施例３
高脂血症患者の血漿及びこれに４７．６重量％となるようにサッカロースを添加した血漿を用いて、実施例１と同様にしてＰｒｅβ１−ＨＤＬ濃度を測定した。その結果、図２に示すように、サッカロース４７．６重量％含有血漿は４℃で５日間保存してもＰｒｅβ１−ＨＤＬ濃度が変化しなかった。
【００２３】
実施例４
検体を４℃で保存して後に測定するかわりに、−８０℃に凍結して保存した後融解して測定した。その結果を図３に示す。図３から明らかなように、血漿をそのまま凍結融解した検体はＰｒｅβ１−ＨＤＬ濃度が高くなった。これに対し、４７．６重量％サッカロースを含有する血漿は凍結融解して測定してもＰｒｅβ１−ＨＤＬ濃度に変化がみられなかった。
【００２４】
【発明の効果】
本発明の血液検体を用いれば、冷所保存後又は凍結保存後に測定してもＰｒｅβ１−ＨＤＬ濃度が測定できる。従来法では、正確に採血後直ちにＰｒｅβ１−ＨＤＬを測定しなければならず、実際に病院で採血した検体を検査センター等で測定することはできなかった。本発明の血液検体を用いれば冷所で５日間保存してもＰｒｅβ１−ＨＤＬが安定に保持されるので、病院で採血した血液を用いた検査センターでの測定が可能となった。
【図面の簡単な説明】
【図１】血液検体の４℃での保存日数とＰｒｅβ１−ＨＤＬ濃度との関係を示す図である。
【図２】高脂血症患者の血液検体の４℃での保存日数とＰｒｅβ１−ＨＤＬ濃度との関係を示す図である。
【図３】凍結前、凍結血漿及び４７．６重量％サッカロース含有凍結血漿のＰｒｅβ１−ＨＤＬ濃度を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a blood sample for measurement of Preβ1-HDL having high storage stability and an immunological measurement method for Preβ1-HDL using the sample.
[0002]
[Prior art]
ApoA-I is the main apoprotein constituting HDL, and plays a central role in the function of reversely transferring cholesterol from peripheral cells of HDL to the liver. (Philips MC et al. Biochem. Biophys. Acta, 906 : p. 223 (1987)). For this reason, apoA-I is measured for the diagnosis of arteriosclerosis.
[0003]
In recent years, apoA-I-containing HDL without apolipoprotein A-II (hereinafter referred to as “apoA-II”) (Ishizuka et al .: Medicine and pharmacy, Vol. 39, No. 5, page 1041, 1988) has been developed. Pre-β1-HDL (T. Miida. Et al., Which has a stronger cholesterol-extracting action from cells than HDL containing I and Apo A-II, and apo A-I that is not bound to lipids or small particles and has low lipid content. al. Biochemistry, 29 : p. 10469 (1990)), it has been found that apo A-I plays an important role in the reverse transfer system of cholesterol from cells. It has become important to measure I. Among apoA-I-containing HDL without apoA-II, Preβ1-HDL extracts cholesterol from peripheral cells through specific interactions with the cell surface (Fielding, C. et al, Lipid Res. , 36 : p211-228 (1995)), which is particularly noted because its action is more efficient than HDL. An immunoassay for Preβ1-HDL using an antibody against Preβ1-HDL has also been developed (Japanese Patent Laid-Open No. 2000-239300).
[0004]
[Problems to be solved by the invention]
However, Preβ1-HDL in the blood after blood collection is very unstable, and if the blood sample is stored at 4 ° C. which is a normal cold storage condition or at room temperature, the Preβ1-HDL concentration becomes a measured value immediately after blood collection. In comparison, it has been found that there is a problem that the price becomes about 30% after 1 day and about 50% after 4 days. In addition, as a means for preventing such storage stability, there is cryopreservation of a specimen. However, it was found that when measured after thawing after cryopreservation, this measured value was also higher than that at the time of blood collection.
Accordingly, an object of the present invention is to provide a means for stabilizing a blood sample for measuring Preβ1-HDL.
[0005]
[Means for Solving the Problems]
Accordingly, the present inventor has made various studies to stabilize Preβ1-HDL in blood, plasma or serum samples after blood collection. As a result, the present inventors have obtained monosaccharides and disaccharides so that the blood samples may be 25 to 80% by weight. Alternatively, if glycerol is added, the Preβ1-HDL concentration in the sample hardly changes even after 5 days at 4 ° C., and the Preβ1-HDL concentration in the sample after freezing and thawing hardly changes, which is highly stable. The present inventors have found that a blood sample for measuring Preβ1-HDL can be obtained, and have completed the present invention.
[0006]
That is, the present invention provides a blood sample for measuring Preβ1-HDL containing 25 to 80% by weight of monosaccharide, disaccharide or glycerol.
The present invention also provides a method for immunoassay of Preβ1-HDL, characterized in that an anti-Preβ1-HDL antibody is reacted with the blood sample for measurement of Preβ1-HDL.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, monosaccharides, disaccharides, and glycerol act as stabilizers for Preβ1-HDL in a blood sample, and examples of monosaccharides include glucose, fructose, and galactose. Examples of the disaccharide include saccharose, maltose, and lactose. Of these, sucrose and glycerol are more preferable, and saccharose is particularly preferable.
[0008]
The concentration of these stabilizers in the specimen needs to be 25 to 80% by weight from the viewpoint of the stabilizing action, but is preferably 30 to 80% by weight, particularly preferably 30 to 70% by weight. If it is less than 25% by weight, there is no stabilizing action.
[0009]
Examples of the specimen include whole blood, plasma, and serum, with plasma being preferred.
The specimen may contain citric acid (preferably 0.1 to 1.0%), EDTA (preferably 0.05 to 10 mM), etc. for the purpose of preventing blood coagulation.
[0010]
For example, in the case of plasma, the blood sample of the present invention is collected from a patient or a subject using an EDTA blood collection tube or the like, cooled and centrifuged to obtain plasma, and then the concentration thereof is 25 to 80. It can be prepared by adding monosaccharides, disaccharides or glycerol, or an aqueous solution thereof to a weight percentage.
[0011]
Since the thus obtained blood sample of the present invention is stabilized in Preβ1-HDL, it can be stored in a cold place for about 5 days (usually 2 to 10 ° C.) and then immunoassayed using an anti-Preβ1-HDL antibody. Stable measurement of Preβ1-HDL concentration is possible.
[0012]
In order to measure Preβ1-HDL by reacting the blood sample of the present invention with an anti-Preβ1-HDL antibody, RIA or EIA by a normal immunological measurement method, a normal competition method, a sandwich method, and the like can be mentioned. In carrying out these methods, a label of an anti-Preβ1-HDL antibody can also be used. Here, examples of the labeling substance include enzymes such as peroxidase, alkaline phosphatase, glucoamylase, and β-galactooxidase; radioactive substances such as 125 I, 131 I, and tritium.
Examples of the simple substance for immobilizing the antibody include various plastic wells and various plastic beads.
[0013]
A polyclonal antibody may be used as the anti-Preβ1-HDL antibody, but a monoclonal antibody is preferably used, and the monoclonal antibody 55201 described in JP-A-2000-239300 is particularly preferable as the monoclonal antibody.
[0014]
For example, when measuring by ELISA, purified ApoA-I can be quantified by the following method using a standard product. That is, after the diluted sample was added to the ELISA plate on which the anti-Preβ1-HDL monoclonal antibody was immobilized and reacted, the enzyme-labeled anti-apo AI polyclonal antibody was reacted, and the change in absorbance after color development For example, a method of quantifying Preβ1-HDL present.
[0015]
In addition, these measurements can also be performed at any temperature of 0-40 degreeC similarly to the normal immunological measuring method.
[0016]
【Example】
EXAMPLES Next, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these at all.
[0017]
Example 1
(1) Blood collected using an EDTA (4 mM) blood collection tube was ice-cooled, and then centrifuged at 3000 rpm for 15 minutes to obtain plasma. Under ice-cooled plasma (0.1 mL), 2 mL of an aqueous saccharose solution was added so as to have the concentration shown in Table 1 to obtain a plasma sample for Preβ1-HDL measurement.
[0018]
(2) Using the obtained specimen, the Preβ1-HDL concentration was measured on the day of blood collection, after storage at 4 ° C. for 1 day, and after storage at 4 ° C. for 5 days. In addition, the Preβ1-HDL concentration was measured in the same manner for plasma not added with saccharose.
That is, after adjusting the monoclonal antibody 55201 obtained in Example 1 of JP-A-2000-239300 to a concentration of 3 μg / mL with 20 mM phosphate buffered saline (PBS; pH 7.2), a 96-well ELISA plate (manufactured by NUNK) 50 μL / well, and incubated at 4 ° C. overnight. After the plate was washed 3 times with PBS, blocking solution (1% BSA-PBS) was added at 100 μL / well and blocked for 1 hour. After removing the blocking solution, 50 μL / well of the specimen diluted with the blocking solution or purified apoA-I was added and incubated at room temperature for 1 hour. After washing 3 times with blocking solution, goat anti-apo A-I antibody obtained by immunizing goats with apo A-I was peroxidase-labeled peroxidase-labeled goat anti-apo A-I antibody at 50 μL / Wells were added and incubated for 1 hour at room temperature. Similarly, after washing 3 times with blocking solution, 50 μL / well of peroxidase substrate solution was added. Ten minutes later, 50 μL / well of 1.5N sulfuric acid was added, the absorbance at 492 nm was measured, and the amount of Preβ1-HDL in each sample was calculated using purified apo AI as a standard.
[0019]
As a result, as shown in Table 1, the plasma without sucrose added and the plasma containing 4.8% by weight and 9.5% by weight saccharose were both 1 day and 5 days later than the Preβ1-HDL concentration on the day of blood collection. Then it became high. In contrast, plasma containing 28.6-66.7% by weight of saccharose did not change the Preβ1-HDL concentration until 5 days later.
[0020]
[Table 1]
Example 2
In the same manner as in Example 1, Preβ1-HDL concentration was measured using plasma, plasma containing 1% BSA-PBS, plasma containing 47.6 wt% saccharose, and plasma containing 47.6 wt% glycerol. The result is shown in FIG. As is clear from FIG. 1, the plasma and BSA-PBS-containing plasma had a higher Preβ1-HDL concentration after 1 day (4 ° C.), but 47.6% by weight glycerol or 47.6% by weight saccharose-containing plasma The Preβ1-HDL concentration did not change until 4 days (4 ° C.).
[0022]
Example 3
Preβ1-HDL concentration was measured in the same manner as in Example 1 using the plasma of a hyperlipidemic patient and plasma with sucrose added to 47.6 wt%. As a result, as shown in FIG. 2, the plasma containing 47.6% by weight of saccharose did not change the Preβ1-HDL concentration even after being stored at 4 ° C. for 5 days.
[0023]
Example 4
Instead of storing the specimen at 4 ° C. for later measurement, the specimen was frozen at −80 ° C. for storage and then thawed for measurement. The result is shown in FIG. As is clear from FIG. 3, the Preβ1-HDL concentration of the specimen obtained by freezing and thawing plasma as it was increased. In contrast, plasma containing 47.6% by weight saccharose did not change in Preβ1-HDL concentration even when measured by freezing and thawing.
[0024]
【The invention's effect】
If the blood sample of the present invention is used, the Preβ1-HDL concentration can be measured even after measurement after cold storage or after cryopreservation. In the conventional method, Preβ1-HDL must be measured immediately after blood collection accurately, and a sample actually collected at a hospital cannot be measured at a laboratory or the like. When the blood sample of the present invention is used, Preβ1-HDL is stably maintained even after being stored in a cold place for 5 days, so that measurement at a laboratory center using blood collected at a hospital becomes possible.
[Brief description of the drawings]
FIG. 1 is a graph showing the relationship between the number of days a blood sample is stored at 4 ° C. and the Preβ1-HDL concentration.
FIG. 2 is a graph showing the relationship between the storage days of a blood sample of a hyperlipidemic patient at 4 ° C. and the Preβ1-HDL concentration.
FIG. 3 is a graph showing the Preβ1-HDL concentration of frozen plasma and frozen plasma containing 47.6 wt% saccharose before freezing.
Claims (4)
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| JP2000009730A (en) * | 1998-06-19 | 2000-01-14 | Cosmo Sogo Kenkyusho:Kk | Measuring method and measuring kit for lipoprotein a-i cholesterol |
| JP2000239300A (en) * | 1998-12-22 | 2000-09-05 | Dai Ichi Pure Chem Co Ltd | Monoclonal antibody against apolipoprotein a-i |
| JP2001066314A (en) * | 1999-06-23 | 2001-03-16 | E Miller Norman | Detecting method for lipid metabolic error and detecting method for disease using it |
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| DE3445010A1 (en) * | 1984-12-10 | 1986-06-19 | Boehringer Mannheim Gmbh | CONTROL OR OAK SERUM FOR LIPID DIAGNOSTICS |
| JPH09169799A (en) * | 1995-12-19 | 1997-06-30 | Cosmo Sogo Kenkyusho:Kk | Lipoprotein composition |
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| JP2000009730A (en) * | 1998-06-19 | 2000-01-14 | Cosmo Sogo Kenkyusho:Kk | Measuring method and measuring kit for lipoprotein a-i cholesterol |
| JP2000239300A (en) * | 1998-12-22 | 2000-09-05 | Dai Ichi Pure Chem Co Ltd | Monoclonal antibody against apolipoprotein a-i |
| JP2001066314A (en) * | 1999-06-23 | 2001-03-16 | E Miller Norman | Detecting method for lipid metabolic error and detecting method for disease using it |
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