JP3767232B2 - Method for measuring LDL-cholesterol - Google Patents

Description

【００５９】
〔測定条件〕
測定パラメータを以下のように設定して測定を行った。
測定方法；１ポイント エンド[３４]−[０]
試料量 ；２．５μｌ
試液量 ；３００μｌ
測定波長；７００／６００ｎｍ
測定温度；３７℃
〔結果〕
試液１〜１３を使用して得られた測定結果を、図１〜１３に夫々示す。
尚、図１〜13に於いて、−□−はＬＤＬ画分試料について得られた結果を、−○−はＨＤＬ画分試料について得られた結果を、−△−はＶＬＤＬ画分試料について得られた結果を、また、−●−は生理食塩水を試料として得られた結果を夫々示す。
図１の結果から明らかな如く、ポリアニオン及び両性界面活性剤を添加していない試液（試液１）では、試液と混合後すぐにＬＤＬ、ＨＤＬ及びＶＬＤＬとの反応が開始され、２〜５分以内で反応はほぼプラトーに達することが判る。
これに対して、図２〜１３から明らかなように、ポリアニオン及び両性界面活性剤の共存下でリポタンパク中のコレステロールの測定を行うと（試液２〜13）、ＬＤＬとの反応が阻害されること、即ち、ＨＤＬ、ＶＬＤＬとの反応が殆ど終了しているにも係わらずＬＤＬとの反応はいまだ進行中であったり（試液２及び３）、或いはＨＤＬ、ＶＬＤＬとの反応が実質的に終了した後に、ＬＤＬとの反応が始まる（試液４〜13）ことが判る。
以上のことから、ポリアニオンおよび両性界面活性剤を共存させることによりＬＤＬ−コレステロールの反応を遅延或いは一時的に停止させてＬＤＬ以外のリポタンパク中コレステロールの反応を先行させた後に、ＬＤＬ中コレステロールの反応を進行させることができること、即ち、ＬＤＬ−コレステロールを特異的に測定し得ることが判る。
【００６０】
実施例２
日立７１７０形自動分析装置〔（株）日立製作所製〕を使用して、本発明の測定法により、血清中のＬＤＬ−コレステロール量を測定した。
〔試料〕
新鮮血清10検体
〔試薬〕

〔測定条件〕
測定パラメータを以下のように設定して測定を行った。
測定方法；２ポイント エンド[１６]−[３４]
試料量 ；３μｌ
Ｒ−１量；２７０μｌ
Ｒ−２量；９０μｌ
測定波長；７００／６００ｎｍ
測定温度；３７℃
〔結果〕
結果を表２に示す。
【００６１】
参考例１
実施例２で用いた血清検体について、従来法のＦｒｉｅｄｅｗａｌｄ式によりＬＤＬ−コレステロール値を算出した。
尚、測定操作は、CLINICAL CHEMISTRY, Vol.18, No.6, p499-502,(1972)に従 って行った。
〔結果〕
測定結果を表２に併せて示す。
【００６２】
【表２】
表２

【００６３】
表２の結果から明らかな如く、本発明のポリアニオン及び両性界面活性剤を含有する試薬（試液１４）を用いて得られたＬＤＬ−コレステロール値は、従来法であるFriedewald式で算出したＬＤＬ−コレステロール値と良好な相関関係を示すこと、即ち、特異的にＬＤＬ−コレステロールを測定し得ることが判る。
【００６４】
実施例３
日立７１７０形自動分析装置〔（株）日立製作所製〕を使用して、本発明の測定法により、ＢＱ法（Beta quantification method）による測定値既知のコントロール血清〔ＰＢＩ（Pacific Biometrics，Inc．）社製）中のＬＤＬ−コレステロール量を測定した。
〔試料〕
コントロール血清５検体
〔試薬〕

〔測定条件〕
実施例２同じ。
〔結果〕
結果を表３に示す。
【００６５】
【表３】
表３

【００６６】
表３の結果から明らかな如く、本発明のポリアニオン及び両性界面活性剤を含有する試薬（試液１５及び試液１６）を用いて得られたＬＤＬ−コレステロール値は、ＢＱ法（Beta quantification method）による測定値が付されたコントロール血清の表示値と殆ど同等であることが判る。
【００６７】
実施例４
日立７１７０形自動分析装置〔（株）日立製作所製〕を使用して、本発明の測定法により、ＢＱ法（Beta quantification method）による測定値既知のコントロール血清〔ＰＢＩ（Pacific Biometrics，Inc．）社製）中のＬＤＬ−コレステロール量を測定した。
〔試料〕
実施例３と同じ。
〔試薬〕

〔測定条件〕
実施例２と同じ。
〔結果〕
結果を表４に示す。
【００６８】
【表４】
表４

【００６９】
表４の結果から明らかな如く、本発明のポリアニオン及び両性界面活性剤を含有する試薬（試液１７〜１９）を用いて得られたＬＤＬ−コレステロール値は、ＢＱ法（Beta quantification method）による測定値が付されたコントロール血清の表示値と殆ど同等であることが判る。
【００７０】
参考例２
自体公知の方法により修飾抗体の作成を行った。
（１）デキストラン修飾抗体の作製
羊由来の抗ＨＤＬ抗体（10mg protein Ab／ml）15mlとジアルデヒドデキストラン300mgとを混合し、50mM リン酸緩衝液（pH6.0）中、30℃で反応させた後、更に200mgのボランピリジンを加えて５時間反応させた。この反応液をセロハンチューブに詰めて脱塩後、限外濾過濃縮処理し、デキストラン修飾抗体（10mg protein Ab／ml×12.6ml）を得た。
【００７１】
（２）ヘパリン修飾抗体の作製
過ヨウ素酸酸化処理したヘパリン 100mgに、6-アミノカプロン酸 500mgを加えて透析した。これに羊由来の抗ＨＤＬ抗体（10mg protein Ab／ml）5mlと1-エチル-3-（3-ジメチルアミノプロピル）-カルボジイミド 300mgを加え、５℃で一晩反応させた。この反応液をセロハンチューブに詰めて脱塩後、限外濾過濃縮処理し、ヘパリン修飾抗体（10mg protein Ab／ml×3.6ml）を得た。
【００７２】
（３）アルギン酸修飾抗体の作製
アルギン酸 50mgを20mlの20mM リン酸緩衝液（pH7.0）に溶解し、これに羊由来の抗ＨＤＬ抗体（10mg protein Ab／ml）5mlと1-エチル-3-（3-ジメチルアミノプロピル）-カルボジイミド 500mgを加え、５℃で一晩反応させた。この反応液をセロハンチューブに詰めて脱塩後、限外濾過濃縮処理し、アルギン酸修飾抗体（10mg protein Ab／ml×3.4ml）を得た。
【００７３】
（４）グルクロン酸修飾抗体の作製
羊由来の抗ＨＤＬ抗体（10mg protein Ab／ml）10mlと400mgのグルクロン酸を0.1M リン酸緩衝液（pH7.0）に溶解し、250mgのシアンボロハイドライドを加えて37℃で５時間反応させた。この反応液をセロハンチューブに詰めて脱塩後、限外濾過濃縮処理し、グルクロン酸修飾抗体（10mg protein Ab／ml×7.6ml）を得た。
【００７４】
（５）ポリグルタミン酸修飾抗体の作製
ポリグルタミン酸 20mgを20mM リン酸緩衝液（pH6.5）４mlに溶解し、該溶解液に羊由来の抗ＨＤＬ抗体（10mg protein Ab／ml）１mlと1-エチル-3-（3-ジメチルアミノプロピル）-カルボジイミド40mgとを加え、５℃で一晩反応させた。この反応液をセロハンチューブに詰めて脱塩後、限外濾過濃縮処理し、ポリグルタミン酸修飾抗体を（10mg protein Ab／ml×0.72ml）を得た。
【００７５】
（６）グリチルリチン酸修飾抗体の作製
グリチルリチン酸 100mgを50mM リン酸緩衝液（pH6.5）２mlに溶解し、該溶解液にメタ過ヨウ素酸ナトリウム 100mgを加えて５℃で24時間反応させた後、セファデックスG-25のカラムで脱塩した。これと羊由来の抗ＨＤＬ抗体（10mg protein Ab ／ml）10mlを混合して30℃で48時間反応させた。この反応液をセロハンチューブに詰めて脱塩後、限外濾過濃縮処理し、グリチルリシン酸修飾抗体を（10mg protein Ab／ml×7.3ml）を得た。
【００７６】
実施例５
日立７１７０形自動分析装置〔（株）日立製作所製〕を使用して、本発明の測定法によりＬＤＬ−コレステロール測定値を求め、基準法であるＢＱ法でのＬＤＬ−コレステロール測定値との比較を行った。
〔試料〕
ＰＢＩ社（Pacific Biometrics，Inc．）より入手した、ＢＱ法（Beta quantification method）による測定値既知の人血清（トリグリセライド400mg／dl以下）15検体及び人血清（トリグリセライド400mg／dl以上）８検体を試料とした。
尚、血清中のトリグリセライド量は、該人血清に付された表示値による。
〔試薬〕

〔測定条件〕
実施例２と同じ。
〔結果〕
トリグリセライド400mg／dl以下の血清についての結果を表５に、また、トリグリセライド400mg／dl以上の血清についての結果を表６に夫々示す。
【００７７】
【表５】
表５

【００７８】
【表６】
表６

【００７９】
表５の結果から、トリグリセライドの値が400mg／dl以下の血清を検体とした場合は、試液２０及び試液２１の何れもそのＬＤＬ−コレステロール値は、ＢＱ法（Beta quantification method）により測定された表示値と殆ど同等であることが判る。また、表６の結果から、トリグリセライドの値が400mg／dl以上の血清を検体とした場合は、試液２１、即ち、ポリアニオン及び両性界面活性剤に、更にＬＤＬ以外のリポタンパクに結合する抗体を共存させて測定を行う方が、より表示値に近いこと、即ち、よりＬＤＬ−コレステロールを特異的に測定し得ることが判る。
【００８０】
【発明の効果】
以上述べた如く、本発明は生体由来試料中のＬＤＬ−コレステロ−ルを特異的に且つ精度良く測定し得る方法並びにそれに用いられる試薬を提供するものであり、本発明を利用することにより、従来の方法では不可能であったＬＤＬ−コレステロ−ルを直接、しかも汎用の自動分析装置を用いて測定し得る。
【図面の簡単な説明】
【図１】実施例１に於いて、試液１を用いて得られた、各種リポタンパク画分試料についての反応曲線を示す。
【図２】実施例１に於いて、試液２を用いて得られた、各種リポタンパク画分試料についての反応曲線を示す。
【図３】実施例１に於いて、試液３を用いて得られた、各種リポタンパク画分試料についての反応曲線を示す。
【図４】実施例１に於いて、試液４を用いて得られた、各種リポタンパク画分試料についての反応曲線を示す。
【図５】実施例１に於いて、試液５を用いて得られた、各種リポタンパク画分試料についての反応曲線を示す。
【図６】実施例１に於いて、試液６を用いて得られた、各種リポタンパク画分試料についての反応曲線を示す。
【図７】実施例１に於いて、試液７を用いて得られた、各種リポタンパク画分試料についての反応曲線を示す。
【図８】実施例１に於いて、試液８を用いて得られた、各種リポタンパク画分試料についての反応曲線を示す。
【図９】実施例１に於いて、試液９を用いて得られた、各種リポタンパク画分試料についての反応曲線を示す。
【図１０】実施例１に於いて、試液１０を用いて得られた、各種リポタンパク画分試料についての反応曲線を示す。
【図１１】実施例１に於いて、試液１１を用いて得られた、各種リポタンパク画分試料についての反応曲線を示す。
【図１２】実施例１に於いて、試液１２を用いて得られた、各種リポタンパク画分試料についての反応曲線を示す。
【図１３】実施例１に於いて、試液１３を用いて得られた、各種リポタンパク画分試料についての反応曲線を示す。
【符号の説明】
図１〜１３に於いて、−□−は低比重リポタンパク（ＬＤＬ）画分試料について得られた結果を、−○−は高比重リポタンパク（ＨＤＬ）画分試料について得られた結果を、−△−は超低比重リポタンパク（ＶＬＤＬ）画分試料について得られた結果を、また、−●−は生理食塩水を試料として得られた結果を夫々示す。[0001]
[Industrial application fields]
The present invention relates to a method for measuring cholesterol in a low density lipoprotein (hereinafter abbreviated as LDL) present in a biological sample such as serum or plasma, and a reagent and kit therefor.
[0002]
BACKGROUND OF THE INVENTION
The main components of serum lipids are cholesterol, triglycerides, phospholipids, etc., and these serum lipids bind to apoproteins to form lipoproteins and circulate in the blood. The lipoproteins are classified into high density lipoprotein (HDL), LDL, very low density lipoprotein (VLDL), chylomicron (CM), etc., depending on the specific gravity. Among these lipoproteins, HDL has the effect of transporting excess cholesterol deposited in tissues to the liver and has an anti-arteriosclerotic effect, while LDL is the main transporter of cholesterol from the liver to each tissue, The increase in LDL is considered to be closely related to the occurrence of arteriosclerosis.
Therefore, cholesterol in LDL (hereinafter abbreviated as LDL-cholesterol) is considered to be a risk factor for arteriosclerosis and ischemic heart disease (coronary artery disease), and the amount of LDL-cholesterol is the diagnosis / treatment of these diseases. And an important indicator of prevention.
[0003]
Conventionally, as a method for measuring LDL-cholesterol, a precipitation method, an ultracentrifugation method, an electrophoresis method, a calculation method using a calculation formula, and the like are known. Of these conventional methods, precipitation, ultracentrifugation and electrophoresis are pretreatment steps for separating LDL and unnecessary lipoproteins other than LDL by precipitation / centrifugation, ultracentrifugation or electrophoresis. Therefore, the operation is complicated, and there is a problem in that direct measurement cannot be performed only with an automatic analyzer widely spread in the field of clinical examination.
In addition, the calculation method calculated from the total cholesterol value, HDL-cholesterol value and triglyceride value known by the Friedewald formula is also accurate when a sample containing 400 mg / dl or more of triglyceride is used. There is a problem that the amount of LDL-cholesterol cannot be measured.
[0004]
In recent years, various methods have been developed in order to solve the above-described problems in the conventional method, for example, the method disclosed in Japanese Patent Application Laid-Open No. 7-280812.
That is, after aggregating LDL using an aggregating agent or / and an antibody, cholesterol contained in lipoproteins other than LDL is guided to another reaction system that does not participate in quantitative reaction, and then erased (consumed). This is a method of using an activator or / and an inorganic salt to dissolve the aggregated LDL to such an extent that a quantitative reaction is possible, and subjecting the LDL-cholesterol to the quantitative reaction and measuring the absorbance of the solution.
[0005]
However, since this method requires three or four types of test solutions, it can be applied to only a few automatic analyzers that can measure using three or four types of test solutions. However, there is a problem that measurement cannot be performed using an automatic analyzer that can use only up to two types of test solutions used in the above. In addition, this method has a problem that the reproducibility of the measured value is lowered because the measurement is performed using three or four kinds of test solutions.
[0006]
Furthermore, as a method for measuring LDL-cholesterol without a complicated pretreatment operation, there is a method disclosed in JP-A-58-165800. However, in this method, for example, the concentration of the surfactant or cholesterol esterase (hereinafter abbreviated as CHE) in the reagent is limited, so that the preparation of the reagent is complicated, and the pH at the time of measurement and the measurement time are further reduced. Since the measurement conditions such as the interval must be set strictly, and cholesterol in HDL reacts to some extent, LDL-cholesterol can be measured only by kinetic measurement, that is, by a late assay. It was difficult to say that it was a practical measurement method.
[0007]
[Problems to be solved by the invention]
In view of the situation as described above, the problem to be solved by the present invention is to separate LDL-cholesterol in a biological sample from LDL and unnecessary lipoproteins other than LDL, which were necessary in the conventional method. Therefore, it is possible to provide a method and a reagent used for the method that enable direct measurement using an automatic analyzer or the like without complicated pretreatment operations.
[0008]
[Means for Solving the Invention]
The present inventionIn the presence of polyanions and amphoteric surfactants,Biological samplesIn (1)CHE and cholesterol oxidase (hereinafter abbreviated as CO), or(2)CHE, cholesterol dehydrogenase (hereinafter abbreviated as CHD) and NAD (P)ActLetThe,Inhibits, delays, or temporarily stops the cholesterol reaction in the low density lipoprotein in the sample, erases (consumes) the cholesterol reaction in the lipoprotein other than the low density lipoprotein, and then in the low density lipoprotein. By the reaction of cholesterolMeasure cholesterol in biological samples based on hydrogen peroxide or NAD (P) H produced, Characterized byIt is an invention of a method for measuring cholesterol in low density lipoprotein.
[0009]
The present invention also provides:Inhibits, delays, or temporarily stops the reaction of cholesterol in low density lipoprotein in biological samples, erases (consumes) cholesterol reaction in lipoproteins other than low density lipoprotein, and then low density lipoprotein For measuring hydrogen peroxide or NAD (P) H produced by the reaction of cholesterol in (a) ~ (c) Kit for measuring cholesterol in any low-density lipoproteinIt is invention of this. (a) (1) Polyanions and amphoteric surfactants, (2) CHE, (3) CO, peroxidase (hereinafter abbreviated as POD), coupler or / and developer, and (Four) A kit for measuring cholesterol in a low-density lipoprotein, comprising a first reagent comprising an aqueous medium and a second reagent comprising an aqueous medium and, if necessary, a developer or a coupler.
(b) (1) Polyanions and amphoteric surfactants, (2) CHE, (3) CO, (Four) Catalase (hereinafter abbreviated as CAT), and (Five) A first reagent solution containing an aqueous medium and a second reagent solution containing a CAT inhibitor and an aqueous medium, wherein the POD, coupler, and developer are at least one of the first reagent solution and the second reagent solution, respectively. A kit for measuring cholesterol in low-density lipoprotein, which is contained in
(c) (1) Polyanions, (2) Amphoteric surfactants, (3) CHE, (Four) CHD, (Five) NAD (P), and (6) A first reagent comprising an aqueous medium; (1) A kit for measuring cholesterol in low-density lipoprotein, comprising a second test solution containing an aqueous medium.
[0013]
That is, the present inventors conducted extensive research to find a method that can measure LDL-cholesterol directly with an automatic analyzer without pretreatment for separating and separating unnecessary lipoproteins other than LDL, If the measurement of cholesterol in a biological sample is performed in the presence of a polyanion and an amphoteric surfactant, only the reaction of LDL-cholesterol is inhibited, in other words, it is delayed or temporarily stopped, and lipoproteins other than LDL It has been found that cholesterol can be preceded and eliminated, thus making it possible to specifically measure cholesterol in LDL without separating and separating unnecessary lipoproteins other than LDL. The invention has been completed.
[0014]
As a method for measuring cholesterol in a biological sample, in principle, the cholesterol in the biological sample is reacted with CHE to decompose it into free cholesterol and fatty acid, and then (1) is produced by reacting with CO. (2) A method of measuring NAD (P) H produced by reacting CHD and NAD (P) with (2) CHD and NAD (P) is mentioned. More specifically, an enzymatic reaction is used. ) For example, a cholesterol ester in a biological sample is decomposed into free cholesterol and a fatty acid by CHE, and oxidized with CO together with free cholesterol present from the beginning to cholest-4-en-3-one and hydrogen peroxide, In the presence of POD, the generated hydrogen peroxide is used to oxidize a color reagent (for example, a combination of a coupler and a developer; (2) For example, cholesterol in a biological sample is decomposed into free cholesterol and fatty acid by CHE, for example. In the presence of CHD together with the free cholesterol present from the above, it may be reacted with NAD (P) to measure the produced NAD (P) H at 340 nm.
[0015]
The present invention can be applied to any of these methods. That is, in the present invention, when performing these methods, the polyanion and the amphoteric surfactant may be present in the reaction system. More specifically, first, a biological sample is contacted with a polyanion and an amphoteric surfactant to inhibit the reaction of LDL-cholesterol in the sample, in other words, delayed or temporarily stopped, and as a result These are eliminated (consumed) by preceding the reaction of cholesterol in lipoproteins other than LDL, and then only the product produced by the reaction of LDL-cholesterol is measured.
[0016]
Specific measuring means of the method of the present invention include the following methods.
[0017]
[Method 1]
In the presence of a polyanion and an amphoteric surfactant, CHE and CO are allowed to act on a biological sample to generate hydrogen peroxide, and the resulting hydrogen peroxide is combined with a POD and an oxidizable color reagent (for example, a combination of a coupler and a developer). , A coloring agent that develops its own color by oxidation, etc.) to produce an oxidative dye, measure the absorbance at two different time points, and calculate the amount of LDL-cholesterol in the biological sample based on these .
[0018]
[Method 2]
In the presence of polyanion and amphoteric surfactant, CHE, CHD and NAD (P) are allowed to act on a biological sample to produce NAD (P) H, and the absorbance at two different time points is measured. Thus, the amount of LDL-cholesterol in the biological sample is calculated.
[0019]
[Method 3]
In the presence of a polyanion and an amphoteric surfactant, CHE and CO are allowed to act on a biological sample to produce hydrogen peroxide, and POD and a coupler (or developer) or CAT are allowed to act on the resulting hydrogen peroxide, and then the developer (Or a coupler), or a CAT inhibitor and an oxidizable color reagent are allowed to act to produce an oxidative dye, the absorbance is measured, and based on this, the amount of LDL-cholesterol in the biological sample is calculated.
[0020]
[Method 4]
In the presence of a polyanion and an amphoteric surfactant, CHE, CHD and NAD (P) (or CHE, CO, POD and a coupler or / and developer) are allowed to act on a biological sample to produce NAD (P) H (or hydrogen peroxide). ), Then, CO, POD, an oxidizable color reagent and a CHD inhibitor (or CHD, NAD (P) and CO inhibitor) are allowed to act to produce an oxidation dye (or NAD ( P) H) is generated, the absorbance is measured, and based on this, the amount of LDL-cholesterol in the biological sample is calculated.
[0021]
In the above methods 1 and 2, the two different time points are specifically, the reaction of cholesterol in lipoproteins other than LDL is substantially finished and before the reaction of LDL-cholesterol is started. And two points of time when the reaction of LDL-cholesterol is substantially completed.
In the above methods 3 and 4, the amount of LDL-cholesterol in a biological sample can also be measured by measuring the absorbance only at one time point, that is, when the reaction of LDL-cholesterol is substantially completed. However, in order to further improve the measurement accuracy, it is preferable to measure the absorbance at two different time points similar to the methods 1 and 2 described above.
These two different points of time differ depending on the measurement method, the polyanion used, the type and concentration of the amphoteric surfactant, the type and concentration of other reagents such as enzymes, etc. The reactivity to the fraction (reaction curve etc.) may be examined and set as appropriate.
[0022]
The method of the present invention may be either a one-component method, a two-component method, or a method using a further test solution. However, the above methods 3 and 4 are practically a two-liquid method or a method using a reagent solution higher than that.
In the method using two or more test solutions, the polyanion and the amphoteric surfactant may be contained in a test solution that is directly mixed with a biological sample. In the above methods 1, 2, and 4, POD and a coupler or / and a developer or NAD (P) are contained in a test solution added before the reaction of LDL-cholesterol starts. In addition, in the above method 3, POD and coupler, POD and developer, or CAT may be contained in a test solution to be added before the reaction of LDL-cholesterol starts.
[0023]
Specific examples in the case of carrying out the method of the present invention by a two-component method are as follows, for example.
For example, when the above method 1 or 2 is carried out, a biological sample, (1) polyanion and amphoteric surfactant, (2) CHE, (3) CO, POD and oxidizable color reagent, (or CHD) And NAD (P)) and (4) the absorbance (OD) after mixing with the first reagent solution containing an aqueous medium.₁), And then mixing the mixed solution with a second reagent solution containing an aqueous medium, and then the absorbance (OD)₂) And the amount of LDL-cholesterol in the biological sample may be calculated based on these absorbances.
[0024]
For example, when the above method 3 is performed, a biological sample, (1) polyanion and amphoteric surfactant, (2) CHE, (3) CO, (4) POD and coupler (or developer) (or (4) CAT) and (5) Absorbance (OD) after mixing with the first test solution containing an aqueous medium.₁), And then the mixture (1) developer (or coupler) (or (1) CAT inhibitor) and (2) a second reagent solution containing an aqueous medium, and then the absorbance (OD)₂) And the amount of LDL-cholesterol in the biological sample may be calculated based on these absorbances. When CAT is contained in the first reagent, the POD, coupler, and developer are contained in at least one of the first reagent and the second reagent, respectively.
[0025]
Further, for example, when the above method 4 is performed, a biological sample, (1) polyanion and amphoteric surfactant, (2) CHE, (3) CHD and NAD (P) (or (3) CO, POD , And a coupler or / and developer), and (4) the absorbance (OD) after mixing with the first reagent solution containing an aqueous medium.₁), And then includes (1) an aqueous medium, (2) CO, POD, oxidizable color reagent, and CHD inhibitor (or (2) CHD, NAD (P), and CO inhibitor) Absorbance (OD) after mixing with the second reagent solution₂) And the amount of LDL-cholesterol in the biological sample may be calculated based on these absorbances.
[0026]
In the above method, OD₁And OD₂The measurement time differs depending on the measurement method, the polyanion used, the type and concentration of the amphoteric surfactant, the type and concentration of other reagents such as enzymes, etc. The property (reaction curve etc.) may be examined and set appropriately.
[0027]
When the method of the present invention is carried out by the one-liquid method (method 1 or 2 described above), more specifically, it may be carried out as follows.
That is, for example, a biological sample, (1) polyanion and amphoteric surfactant, (2) CHE, (3) CO, POD and oxidizable color reagent (or CHD and NAD (P)), and (4) A sample solution containing an aqueous medium is mixed, and the absorbance (OD) is reached at the time when the reaction of cholesterol in lipoproteins other than LDL is substantially finished and before the reaction of LDL-cholesterol is started.₁) And then the absorbance (OD) when the LDL-cholesterol reaction is substantially complete.₂) Is measured again, and the amount of LDL-cholesterol in the biological sample may be calculated based on these absorbances.
[0028]
In the above method, OD₁And OD₂To calculate the amount of LDL-cholesterol in a biological sample based on₂To OD₁[Or OD₁Value derived from (eg OD₁The value obtained by multiplying the liquid volume correction coefficient by the absorbance) (OD)_Three) And obtained OD_ThreeThe LDL-cholesterol concentration and OD were determined in the same manner as in the above method using a standard product such as a standard solution with a known LDL-cholesterol concentration as a sample._ThreeThe value of LDL-cholesterol in a sample derived from a living body may be calculated by applying a calibration curve indicating the relationship between
[0029]
In the present invention, in order to promote the cholesterol reaction, a nonionic surfactant, an anionic surfactant or the like may coexist, and the reaction of cholesterol in lipoproteins other than LDL is promoted. For this purpose, an antibody that binds to a lipoprotein other than LDL may coexist.
[0030]
Below, the preferable aspect of the structural requirements in this invention, its use density | concentration, etc. are demonstrated.
[0031]
The polyanion used in the present invention may be any polyanion that can inhibit the reaction of LDL-cholesterol by allowing it to coexist with an amphoteric surfactant and lead the reaction of cholesterol in lipoproteins other than LDL.
The term “inhibition” means that the reaction of LDL-cholesterol is delayed as compared with the reaction of cholesterol in lipoproteins other than LDL, or the reaction of LDL-cholesterol is temporarily stopped.
Specific examples of these polyanions include, for example, heparin, phosphotungstic acid, dextran sulfate, sulfated cyclodextrin, heparan sulfate, chondroitin sulfate, hyaluronic acid, sulfated oligosaccharides, sulfated polyacrylamide, carboxymethylated polyacrylamide, or these And the like. Examples of the salt include alkali metal salts such as Na salt and K salt, ammonium salt and the like.
Of the polyanions as described above, heparin, phosphotungstic acid, dextran sulfate, or salts thereof are preferred. The concentration of these polyanions may be any concentration that can inhibit the reaction of LDL-cholesterol by coexisting with an amphoteric surfactant and cause the reaction of cholesterol in lipoproteins other than LDL to precede. The concentration in the test solution directly mixed with the derived sample is usually 0.0001% to 10% (W / V), preferably 0.001% to 1% (W / V). These polyanions may be used alone or in appropriate combination of two or more.
[0032]
The amphoteric surfactant used in the present invention is not particularly limited as long as it can inhibit the reaction of LDL-cholesterol by coexisting with a polyanion to precede the reaction of cholesterol in lipoproteins other than LDL, For example, alkylbetaine derivatives (eg, lauryl betaine, stearyl betaine, lauryldimethylammonium betaine, coconut betaine, coconut oil fatty acid amidopropyl betaine, lauric acid amidopropyl betaine), imidazolinium betaine derivatives (eg, 2-lauryl-N-carboxyl) 2-alkyl-N-carboxymethyl-N-hydroxy such as methyl-N-hydroxyethylimidazolinium betaine, 2-undecyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine Tyrimidazolinium betaines such as 2-alkyl-N-carboxyethyl-N-hydroxyethylimidazolinium betaine, sulfobetaine derivatives such as N-octyl-N, N-dimethyl-3-ammonio-1-propanesulfone Acid, N-decyl-N, N-dimethyl-3-ammonio-1-propanesulfonic acid, N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonic acid, N-tetradecyl-N, N- Betaine derivatives such as dimethyl-3-ammonio-1-propanesulfonic acid, N-hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonic acid, and the like, such as alkylglycine, alkylbis (aminoethyl) glycine, Dioctyl polyaminoethyl glycine, N-alkyl polyaminoethyl Aminocarboxylic acid derivatives such as lysine and β-alanine derivatives, for example, bis (2-undecyl-N-hydroxyethylimidazoline) chloroacetic acid complexes, imidazoline derivatives such as alkylimidazoline derivatives, and amine oxide derivatives such as lauryldimethylamine oxide, etc. Can be mentioned.
Among the amphoteric surfactants as described above, lauryl betaine, coconut oil fatty acid amidopropyl betaine, lauric acid amidopropyl betaine, 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine and 2-alkyl-N- Carboxyethyl-N-hydroxyethylimidazolinium betaine, alkylglycine, alkylbis (aminoethyl) glycine, dioctylpolyaminoethylglycine, N-alkylpolyaminoethylglycine, and β-alanine derivatives are preferred.
The amphoteric surfactant may be used at a concentration that can inhibit the reaction of LDL-cholesterol by coexisting with a polyanion and precede the reaction of cholesterol in lipoproteins other than LDL. The concentration of the sample solution directly mixed with the derived sample is usually 0.0001% to 10% (W / V), preferably 0.001% to 1% (W / V). These amphoteric surfactants may be used alone or in combination of two or more.
[0033]
Examples of CO used in the present invention include those usually used in this field, such as those derived from microorganisms such as Nocardia and Pseudomonas, and those derived from animal organs such as bovine pancreas. . The amount of CO used is, for example, usually 0.03 to 330 u / ml, preferably 0.07 to 130 u / ml, more preferably 0.13 to 65 u / ml as the concentration in the first reagent in the two-liquid method, or The concentration in the final reaction solution is usually 0.02 to 250 u / ml, preferably 0.05 to 100 u / ml, more preferably 0.1 to 50 u / ml.
In the case of the one-liquid method, it is appropriately selected from the concentration range in the final reaction liquid. (The same applies hereinafter.)
Examples of CHE used in the present invention include those usually used in this field, such as those derived from microorganisms such as Candida and Pseudomonas, and those derived from animal organs such as bovine pancreas. . The amount of CHE used is, for example, usually 0.03 to 330 u / ml, preferably 0.07 to 130 u / ml, more preferably 0.13 to 65 u / ml as the concentration in the first reagent in the two-liquid method, or The concentration in the final reaction solution is usually 0.02 to 250 u / ml, preferably 0.05 to 100 u / ml, more preferably 0.1 to 50 u / ml.
[0034]
As the POD used in the present invention, those usually used in this field, for example, those derived from plants such as horseradish and radish, those derived from microorganisms such as mold and yeast, leukocytes of animals, Examples include those derived from the thyroid gland and the like. The amount of POD used is, for example, usually 0.1 to 1000 u / ml, preferably 0.25 to 400 u / ml, more preferably 0.5 to 200 u / ml as the concentration in the first reagent in the two-liquid method, or The concentration in the final reaction solution is usually 0.1 to 250 u / ml, preferably 0.25 to 100 u / ml, more preferably 0.5 to 50 u / ml.
[0035]
The oxidizable color reagent used in the present invention is not particularly limited as long as it reacts with hydrogen peroxide in the presence of POD to produce a color. For example, 4-aminoantipyrine (hereinafter abbreviated as 4-AA). A combination of a coupler such as 4-AA and a phenol compound, a naphthol compound, or an aniline compound; for example, 3-methyl-2- Combinations of benzothiazolinone hydrazone and aniline compounds, such as 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid), triphenylmethane leuco dyes, diphenylamine derivatives, benzidine derivatives, triallylimidazole For oxidation of derivatives, leucomethylene blue derivatives, o-phenylenediamine derivatives, etc. Coloring agents and the like which is itself to color me.
Specific examples of the phenol compound as a developer include phenol, p-chlorophenol, 2,4-dichlorophenol, and the like. Specific examples of the naphthol compound include, for example, 1-naphthol and 1-naphthol-2. -Sulfonic acid, 1-naphthol-2-carboxylic acid and the like, and specific examples of aniline compounds include, for example, N, N-diethylaniline, N-ethyl-N- (β-hydroxyethyl) -m. -Toluidine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5 -Dimethoxy-4-fluoroaniline (FDAOS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimeth Cyaniline (HDAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS), N-ethyl-N- (3-methylphenyl) -N′-succinyl-ethylenediamine (EMSE) ) And the like.
When a combination of a coupler and a developer is used, the amount of coupler used varies depending on the type of coupler used and the type of developer to be combined. For example, the concentration in the first reagent in the two-component method is usually 0.01 to 400 mM. The concentration in the final reaction solution is usually in the range of 0.01 to 100 mM, preferably 0.1 to 10 mM, preferably 0.1 to 40 mM, more preferably 0.2 to 10 mM. The amount used when 4-AA is used as the coupler is, for example, usually 0.01 to 200 mM, preferably 0.1 to 40 mM, more preferably 0.2 to 10 mM as the concentration in the first reagent in the two-liquid method. The concentration in the final reaction solution is usually 0.01 to 50 mM, preferably 0.1 to 5 mM.
The amount of developer used varies depending on the type of developer to be used and the type of coupler to be combined, but cannot be generally stated. For example, the concentration in the first test solution in the two-component method is usually 0.01 to 200 mM, preferably Is 0.1 to 40 mM, more preferably 0.2 to 10 mM, and the concentration in the final reaction solution is usually 0.01 to 50 mM, preferably 0.1 to 5 mM.
Specific examples of the triphenylmethane leuco dye include leucomalachite green, bis (p-diethylaminophenyl) -2-sulfophenylmethane, bis (p-diethylaminophenyl) -3,4-disulfopropoxyphenylmethane di Specific examples of diphenylamine derivatives include bis [4-di (2-butoxyethyl) amino-2-methylphenyl] amine, N, N-bis (4-diethylamino-2-methylphenyl), and the like. ) -N′-p-toluenesulfonylurea and the like, and specific examples of the leucomethylene blue derivative include, for example, 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine / sodium salt, 10- [3- (methoxycarbonylaminomethyl) Phenylmethylaminocarbonyl] -3,7-bis (dimethylamino) phenothiazine and the like. Furthermore, specific examples of the benzidine derivative include benzidine, o-tolidine, o-dianisidine, 3,3′-diaminobenzidine, 3,3 ′, 5,5′-tetraaminobenzidine and the like, and triallylimidazole. Specific examples of the derivative include, for example, 2- (4-carboxyphenyl) -3-N-methylcarbamoyl-4,5-bis (4-diethylaminophenyl) imidazole, 2- (3-methoxy-4-diethylaminophenyl)- Examples include 3-N-methylcarbamoyl-4,5-bis (2-methyl-4-diethylaminophenyl) imidazole.
The amount of these color formers used is a concentration range usually used in this field.
[0036]
The CAT used in the present invention is usually used in this field, for example, those derived from animal organs such as cattle liver, those derived from plants such as chloroplast, Micrococcus, Rhodopseudomonas. Examples include those derived from microorganisms such as genera. The amount of CAT used is, for example, usually 10 to 50000 u / ml, preferably 100 to 5000 u / ml, more preferably 0.5 to 50 u / ml, as the concentration in the first test solution in the two-liquid method.
[0037]
The CHD used in the present invention includes those usually used in this field, such as those derived from the genus Nocardia. The amount of CHD used is, for example, usually 0.1 to 150 u / ml, preferably 0.3 to 100 u / ml, more preferably 0.5 to 60 u / ml as the concentration in the first test solution in the two-liquid method. The concentration in the reaction solution is usually 0.1 to 100 u / ml, preferably 0.5 to 50 u / ml.
[0038]
The NAD (P) used in the present invention includes those usually used in this field, for example, those derived from yeast. The amount of NAD (P) used is, for example, usually 0.2 to 70 mM, preferably 0.5 to 50 mM, more preferably 1 to 20 mM as the concentration in the first reagent in the two-liquid method. The concentration of is usually 0.2 to 50 mM, preferably 1 to 10 mM.
[0039]
Examples of the CAT inhibitor used in the present invention are those usually used in this field, such as NaN._Three, 3-amino-1,2,4-triazole and the like. The amount of these CAT inhibitors to be used varies depending on the type of CAT inhibitor to be used and cannot be generally stated. For example, the concentration in the second reagent in the two-component method is usually 0.01 to 10% (W / V ), Preferably 0.02 to 5% (W / V), more preferably 0.03 to 3% (W / V).
[0040]
Examples of the CO inhibitor used in the present invention include those usually used in this field, such as Ag + ion, Zn2 + ion, glutathione and the like. The amount of CO inhibitor used varies depending on the type of CO inhibitor used, and cannot be generally stated. For example, the concentration in the second reagent in the two-liquid method is usually 0.00002 to 40% (W / V ), Preferably 0.0002 to 4% (W / V), more preferably 0.002 to 0.4% (W / V).
[0041]
Examples of the CHD inhibitor used in the present invention include those usually used in this field, such as Ag + ion and Zn2 + ion. The amount of these CHD inhibitors used varies depending on the type of CHD inhibitor used and cannot be generally stated. For example, the concentration in the second reagent in the two-liquid method is usually 0.00001 to 70% (W / V ), Preferably 0.0001 to 7% (W / V), more preferably 0.001 to 0.7% (W / V).
[0042]
As the nonionic surfactant and the anionic surfactant used in the present invention, any nonionic surfactant may be used as long as it has a property capable of accelerating cholesterol reaction. Ethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene higher alcohol ether, polyoxyethylene fatty acid ester, polyoxyethylene sorbitan fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitol fatty acid ester, polyoxyethylene alkylamine, glycerin fatty acid Examples of the anionic surfactant include cholic acid and its derivatives. Furthermore, specific examples of polyoxyethylene alkyl ether include polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, etc. Specific examples of polyoxyethylene alkyl phenyl ether As polyoxyethylene octyl phenyl ether, polyoxyethylene nonyl phenyl ether and the like, specific examples of polyoxyethylene fatty acid ester include polyethylene glycol monolaurate, polyethylene glycol monostearate, polyethylene glycol distearate, Polyethylene glycol monooleate and the like. Specific examples of polyoxyethylene sorbitan fatty acid esters include polyoxyethylene. Examples include sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan tristearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan trioleate, and sorbitan fatty acids Specific examples of esters include sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan distearate, sorbitan tristearate, sorbitan monooleate, sorbitan trioleate, sorbitan sesquioleate, etc. Specific examples of polyoxyethylene sorbitol fatty acid esters include polyoxyethylene sorbitol tetraoleate and the like. Examples of Chi alkylene alkyl amine, polyoxyethylene lauryl amine, include polyoxyethylene stearyl amine and the like, Specific examples of the glycerin fatty acid ester, monoglyceride stearate, and monoglyceride oleate.
Specific examples of the anionic surfactant include cholic acid, deoxycholic acid, polyoxyethylene alkylphenol ether sulfate, dodecylbenzene sulfonate, lauroyl sarcosine and the like.
[0043]
The use concentration of the nonionic surfactant as described above is usually 0.0001% to 10%, preferably 0.001% to 1% as the concentration in the test solution when used in the one-component method, and is used in the two-component method. In this case, the concentration in the first reagent is usually 0.0001 to 5% (W / V), preferably 0.001 to 0.5% (W / V), although it varies depending on the ratio of the first reagent and the second reagent. The concentration in the second test solution is usually 0.01 to 20% (W / V), preferably 0.05 to 5% (W / V).
[0044]
The concentration of the anionic surfactant as described above is usually 0.0001% to 10%, preferably 0.001% to 1% as the concentration in the test solution when used in the one-component method, and is used in the two-component method. In this case, the concentration in the first reagent is usually 0.0001 to 5% (W / V), preferably 0.001 to 0.5% (W / V), although it varies depending on the ratio of the first reagent and the second reagent. The concentration in the second test solution is usually 0.01 to 20% (W / V), preferably 0.05 to 5% (W / V).
[0045]
In addition, as a use concentration when a nonionic surfactant and an anionic surfactant as described above are used in combination, when used in a one-component method, the total concentration of the surfactant in the test solution is usually 0.0001%. ~ 20%, preferably 0.001% ~ 2%. When used in the two-component method, the total concentration of the surfactant in the first reagent is different depending on the volume ratio of the first and second reagents. Usually, 0.0001 to 10% (W / V), preferably 0.001 to 1% (W / V), and the total concentration of the surfactant in the second reagent is usually 0.01 to 40% (W / V) Preferably, it is 0.05 to 10% (W / V).
The surfactants as described above may be used alone or in combination of two or more.
[0046]
The antibody that binds to lipoproteins other than LDL used in the present invention may be any antibody that has a property capable of promoting the reaction of cholesterol in lipoproteins other than LDL, such as anti-HDL antibody and anti-apolipoprotein A. Examples include antibodies, anti-apolipoprotein C antibodies, anti-apolipoprotein E antibodies, anti-α lipoprotein antibodies, anti-VLDL antibodies, anti-pre-β lipoprotein antibodies, and the like. These antibodies may be polyclonal antibodies or monoclonal antibodies, and may be used alone or in combination of two or more.
Further, these are enzymatically or chemically decomposed, for example, Fab, Fab ', F (ab')₂In addition, enzyme-labeled antibodies labeled with enzymes are also included in antibodies that bind to lipoproteins other than LDL.
In the present invention, it is preferable to use a modified antibody in which an appropriate compound is bound to the antibody as described above to add or enhance the property of promoting cholesterol reaction in lipoproteins other than LDL. .
Such compounds may be natural compounds or chemically synthesized compounds such as saccharides, fatty acids, various glycosides (alkaloid glycosides, steroid glycosides, terpenoid glycosides), water-soluble synthetic polymers, and the like. Can be mentioned. These compounds are covalently bonded to the amino group, carboxyl group, sulfhydryl group of the antibody and the amino group, imino group, carboxyl group, aldehyde group or 2,3-epoxypropyl group of the antibody by a method known per se. In the case where there is no active group for modification, the compound can be easily bound to an antibody by a method of activating and modifying these compounds or a method of modifying via a crosslinking agent.
[0047]
Specific examples of these compounds include dextran, pullulan, ficoll, dextrin, cyclodextrin, polyglutamic acid, polyaspartic acid, pectinic acid, prosberic acid, alginic acid, carboxymethyldextran, carboxymethylcellulose, polylysine, albumin, laminan, lichenan, lectin. Dextran sulfate, chondroitin sulfate, heparin, glucose, galactose, xylose, fructose, lactose, maltose, palatinose, trehalose, mannose, fucose, glucuronic acid, glucosamine, galactosamine, inositol, sialic acid, muramic acid, glycyrrhizic acid, arbutin, soybean in , Polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, etc. .
Of the above compounds, for example, those having an active group for the amino group, carboxyl group, sulfhydryl group, etc. of the antibody such as amino group, carboxyl group, imino group, aldehyde group, sulfhydryl group, etc., are directly subjected to the binding reaction with the antibody. Those having no active group may be activated by the following method for introducing an active group. The active group may be introduced through a spacer having an appropriate molecular length, such as an alkylene group having 1 to 6 carbon atoms.
As a method for introducing an active group into the above compound, for example, a method for introducing a cyanuryl group using cyanuric chloride (for example, J. Solid-phase Biochem., 4, 2128, 1976, etc.), metaperiodic acid, etc. Method for introducing aldehyde group using acid (for example, Proc. Nati. Acad. Sci. USA., 73, 2128, 1976, etc.), Method for introducing imino group using bromocyan (for example, Nature, 214, 1302) Page, 1967), 2,3-epoxypropyl group introduction method using epichlorohydrin (for example, J. Chromatog., 51, 479, 1970), carboxyl group introduction method using carboxylic acid anhydride (For example, Japanese Patent Application Laid-Open No. 5-268950) Use monobromocarboxylic acid That way the introduction of a carboxyl group (e.g., Arc.Biochem.Biophys., 147, pp. 788 pp., Etc. 1971) include known methods of introducing like.
Examples of a method for covalently binding a compound having an active group and an antibody include all known binding methods. For example, when the reactive group involved in the reaction is an amino group and a carboxyl group, for example, caribodiimide Method (for example, J. Biol. Chem., 245, 3059, 1970, etc.), activated ester method (for example, Cancer Biochem., 7, 175, 1984, etc.), acid anhydride method (for example, J. Biol. Chem., 237, 1825, 1962, etc.), azide method (for example, Eur. J. Biochem., 25, 129, 1972, etc.), carboxyl chloride method (for example, Angew). Chem, 67, 661, 1955, etc.), isocyanate method (eg, Nature, 210, 367, 196). 6 years, etc.), Woodward reagent method (eg, Biochim. Biophys. Acta, 178, 626, 1969, etc.), Ugi reaction (eg, Angew. Chem., 74, 9, 1962) Etc.). When the reactive group is an amino group, for example, a glutaraldehyde method (for example, Expertia, 28, 958, 1973), an alkylation method (for example, Biochim. Biophys. Acta, 198, 276, 1970, etc.), and when the reactive group is a hydroxyl group and an amino group, for example, an alkylation method (for example, Biochim. Biophys. Acta, 198, 276, 1970) Is mentioned. When the reactive group is an amino group and an aldehyde group, for example, a periodate oxidation method (for example, Proc. Natl. Acad. Sci. USA, 73, 2128, 1978, etc.) When the reactive group is an amino group or an imino group, for example, the bromocyan method (for example, Nature, 214, 1302, 1967) and the like can be mentioned.
[0048]
The concentration of the antibody as described above may be a concentration that can promote the reaction of cholesterol in lipoproteins other than LDL, and the concentration in a reagent that is directly mixed with a biological sample is usually 0.001 to 10 mg Ab / ml. Preferably, it is 0.01-1 mgAb / ml.
[0049]
Examples of the aqueous medium used in the present invention include water and a buffer solution. The buffer that constitutes the buffer may be any buffer that has a buffering action in the range of pH 5 to 11 and does not inhibit the cholesterol measurement reaction. For example, Tris (hydroxymethylaminomethane, Good's buffer, phosphate , Borate and the like, which are usually used in this field, and the concentration used is usually 1 mM to 2 M, preferably 10 mM to 1 M, and the pH is usually 5 to 11, preferably It is 6-8, More preferably, it is 7 vicinity.
[0050]
The LDL-cholesterol measurement reagent and kit of the present invention are used for measuring LDL-cholesterol in biological samples such as serum and plasma, and use a polyanion and an amphoteric surfactant. Except for reagents used in the method for measuring cholesterol in biological samples as described above, for example, CO, CHE, CHD, POD, NAD (P), oxidizable color reagent, CAT, CAT inhibitor, Prepared to contain CO inhibitor, CHD inhibitor, aqueous medium, if necessary nonionic surfactant, anionic surfactant, antibody, etc. in the concentration range normally used in this field The preferable aspect of the constituent requirements, the use concentration, and the like are as described above. Moreover, the LDL-cholesterol standard etc. may be combined with the said kit as needed. As the standard product, for example, standard serum prepared using serum from humans or animals, or one containing an LDL fraction derived therefrom can be used as a standard product.
[0051]
The LDL-cholesterol measurement method, measurement reagent and measurement kit of the present invention inhibit the reaction of LDL-cholesterol by coexisting a polyanion and an amphoteric surfactant, and thereby react with cholesterol in lipoproteins other than LDL. Since the LDL-cholesterol reaction is advanced after the preceding, only hydrogen peroxide or NAD (P) H produced by the reaction of LDL-cholesterol is specifically measured. This makes it possible to measure LDL-cholesterol in an endpoint assay using an analytical device.
[0052]
Preferred embodiments of the present invention include the following, for example.
That is, for example, biological samples such as serum and plasma, and (1) polyanion and amphoteric surfactant, (2) CHE, (3) CO, developer, coupler and POD (or CHD and NAD (P)) and (4) Absorbance (after mixing with a first reagent containing a buffering agent, if necessary, a nonionic surfactant or / and an anionic surfactant, antibody, etc., and reacting at 2-40 ° C. for 1-30 minutes. OD₁). Next, the reaction solution is mixed with a second reagent containing a nonionic surfactant or / and an anionic surfactant if necessary, and reacted at 2 to 40 ° C. for 1 to 60 minutes. Absorbance (OD₂). OD above₂To OD₁Values derived from (eg OD₁Absorbance (OD) obtained by subtracting the liquid volume correction coefficient from_Three) And obtained OD_ThreeThe LDL-cholesterol concentration and OD were determined in the same manner as described above using, for example, a standard product such as a standard solution with a known LDL-cholesterol concentration as a sample._ThreeThe value of LDL-cholesterol in a biological sample is obtained by applying a calibration curve indicating the relationship between
[0053]
In addition, for example, biological samples such as serum and plasma, and (1) polyanion and amphoteric surfactant, (2) CHE, (3) CO, developer (or coupler) and POD, and (4) buffer, if necessary Absorbance (OD) after mixing with a first reagent solution containing nonionic surfactant or / and anionic surfactant, antibody, etc. and reacting at 2-40 ° C. for 1-30 minutes₁). Next, the reaction solution is mixed with a second reagent containing a developer (or coupler) and a buffer, and if necessary, a nonionic surfactant or / and an anionic surfactant, and the mixture is mixed at 2 to 40 ° C. Absorbance (OD) after reaction for ~ 60 minutes₂). OD above₂To OD₁Values derived from (eg OD₁Absorbance (OD) obtained by subtracting the liquid volume correction coefficient from_Three) And obtained OD_ThreeThe LDL-cholesterol concentration and OD were determined in the same manner as described above using, for example, a standard product such as a standard solution with a known LDL-cholesterol concentration as a sample._ThreeThe value of LDL-cholesterol in a biological sample is obtained by applying a calibration curve indicating the relationship between
[0054]
Furthermore, for example, biological samples such as serum and plasma, and (1) polyanion and amphoteric surfactant, (2) CHE, (3) CO, developer (or coupler), (4) CAT and (5) buffer, If it mixes with the 1st test liquid containing a nonionic surfactant or / and an anionic surfactant, an antibody, etc., after making it react at 2-40 degreeC for 1 to 30 minutes, light absorbency (OD₁). Next, the reaction solution is mixed with a second reagent solution containing a coupler (or developer), POD, CAT inhibitor and buffer, and if necessary, a nonionic surfactant or / and an anionic surfactant, Absorbance (OD) after reaction at 2-40 ° C for 1-60 minutes₂). OD above₂To OD₁Values derived from (eg OD₁Absorbance (OD) obtained by subtracting the value obtained by multiplying by the correction factor_Three) And obtained OD_ThreeThe LDL-cholesterol concentration and OD were determined in the same manner as described above using, for example, a standard product such as a standard solution with a known LDL-cholesterol concentration as a sample._ThreeThe value of LDL-cholesterol in a biological sample is obtained by applying a calibration curve indicating the relationship between
In the case of the one-liquid method, for example, biological samples such as serum and plasma, and (1) polyanion and amphoteric surfactant, (2) CHE, (3) CO, POD, and oxidizable color reagent (or CHD) And NAD (P)), and (4) a buffer, if necessary, a nonionic surfactant or / and a test solution containing an anionic surfactant, an antibody, etc. Absorbance (OD) after reaction for ~ 30 minutes_{1 '}). Furthermore, after reacting at 2-40 ° C. for 1-60 minutes, the absorbance (OD_{2 '}). OD above_{2 '}To OD_{1 '}Absorbance (OD)_{Three '}) And obtained OD_{Three '}The LDL-cholesterol concentration and OD were determined in the same manner as described above using, for example, a standard product such as a standard solution with a known LDL-cholesterol concentration as a sample._{Three '}The value of LDL-cholesterol in a biological sample is obtained by applying a calibration curve indicating the relationship between
[0055]
In the above embodiment, OD₁(Or OD_{1 '}) And OD₂(Or OD_{2 '}) Is measured, for example, by examining the reactivity (reaction curve, etc.) to each lipoprotein fraction, and an optimal time point may be appropriately set from the above range.
[0056]
Hereinafter, the present invention will be described in more detail with reference to Examples and Reference Examples, but the present invention is not limited thereto.
[0057]
【Example】
Example 1
Using a Hitachi 7170 automatic analyzer (manufactured by Hitachi, Ltd.), reaction curves between the measurement reagent of the present invention and various lipoproteins fractionated by ultracentrifugation were measured.
〔sample〕
HDL fraction (84 mg / dl), LDL fraction (95 mg / dl) and VLDL fraction (33 mg / dl) obtained by fractionation from serum by a known ultracentrifugation method were used as samples.
〔reagent〕
(Reagents 1-13)
CO (code number: C00-321, manufactured by Toyobo Co., Ltd.) 1 U / ml, CHE (product number: T-18, manufactured by Asahi Kasei Kogyo Co., Ltd.) 1 U / ml, N- (2-hydroxy-3-sulfo) Propyl) -3,5-dimethoxyaniline (HDAOS) 0.5 mM, 4-AA 1 mM, POD (code number: PEO-302, manufactured by Toyobo Co., Ltd.) 1.5 U / ml, predetermined polyanion 0.04% (W / V) And 25 mM 2-hydroxy-N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPSO) -NaOH buffer (pH 7.0) containing 0.008% (W / V) of a given amphoteric surfactant A test solution was used.
Table 1 shows specific names of the polyanions and amphoteric surfactants used in the respective test solutions.
[0058]
[Table 1]
Table 1

[0059]
〔Measurement condition〕
Measurement was performed with the measurement parameters set as follows.
Measuring method: 1 point End [34]-[0]
Sample volume: 2.5 μl
Reagent volume: 300 μl
Measurement wavelength: 700 / 600nm
Measurement temperature: 37 ° C
〔result〕
The measurement results obtained using the test solutions 1 to 13 are shown in FIGS.
1 to 13,-□-indicates the result obtained for the LDL fraction sample,-○-indicates the result obtained for the HDL fraction sample, and -Δ- indicates the result obtained for the VLDL fraction sample. The results obtained and-●-show the results obtained using physiological saline as a sample, respectively.
As is apparent from the results of FIG. 1, in the test solution (sample solution 1) to which no polyanion or amphoteric surfactant was added, the reaction with LDL, HDL and VLDL was started immediately after mixing with the test solution, and within 2 to 5 minutes. It can be seen that the reaction almost reaches a plateau.
On the other hand, as is apparent from FIGS. 2 to 13, when cholesterol in lipoprotein is measured in the presence of a polyanion and an amphoteric surfactant (samples 2 to 13), the reaction with LDL is inhibited. That is, although the reaction with HDL and VLDL is almost completed, the reaction with LDL is still in progress (reagents 2 and 3), or the reaction with HDL and VLDL is substantially completed. After that, it can be seen that the reaction with LDL starts (Reagents 4 to 13).
From the above, the reaction of cholesterol in LDL precedes the reaction of cholesterol in lipoproteins other than LDL by delaying or temporarily stopping the reaction of LDL-cholesterol by coexisting a polyanion and an amphoteric surfactant. It can be seen that LDL-cholesterol can be specifically measured.
[0060]
Example 2
Using a Hitachi 7170 type automatic analyzer (manufactured by Hitachi, Ltd.), the amount of LDL-cholesterol in the serum was measured by the measurement method of the present invention.
〔sample〕
10 samples of fresh serum
〔reagent〕

〔Measurement condition〕
Measurement was performed with the measurement parameters set as follows.
Measurement method: 2 points End [16]-[34]
Sample volume: 3 μl
R-1 amount: 270 μl
R-2 amount: 90 μl
Measurement wavelength: 700 / 600nm
Measurement temperature: 37 ° C
〔result〕
The results are shown in Table 2.
[0061]
Reference example 1
About the serum sample used in Example 2, the LDL-cholesterol value was calculated by the conventional Friedewald equation.
The measurement operation was performed according to CLINICAL CHEMISTRY, Vol. 18, No. 6, p499-502, (1972).
〔result〕
The measurement results are also shown in Table 2.
[0062]
[Table 2]
Table 2

[0063]
As is apparent from the results in Table 2, the LDL-cholesterol value obtained using the reagent containing the polyanion of the present invention and the amphoteric surfactant (sample solution 14) was calculated using the conventional Friedewald equation. It can be seen that the value shows a good correlation, that is, LDL-cholesterol can be specifically measured.
[0064]
Example 3
Using a Hitachi 7170 automatic analyzer (manufactured by Hitachi, Ltd.), a control serum (PBI (Pacific Biometrics, Inc.)) with a known measurement value by the BQ method (Beta quantification method) according to the measurement method of the present invention. The amount of LDL-cholesterol in the product was measured.
〔sample〕
5 samples of control serum
〔reagent〕

〔Measurement condition〕
Same as Example 2.
〔result〕
The results are shown in Table 3.
[0065]
[Table 3]
Table 3

[0066]
As is apparent from the results in Table 3, the LDL-cholesterol value obtained using the reagent (sample 15 and sample 16) containing the polyanion and the amphoteric surfactant of the present invention was measured by the BQ method (Beta quantification method). It turns out that it is almost equivalent to the display value of the control serum to which the value was given.
[0067]
Example 4
Using a Hitachi 7170 automatic analyzer (manufactured by Hitachi, Ltd.), a control serum (PBI (Pacific Biometrics, Inc.)) with a known measurement value by the BQ method (Beta quantification method) according to the measurement method of the present invention. The amount of LDL-cholesterol in the product was measured.
〔sample〕
Same as Example 3.
〔reagent〕

〔Measurement condition〕
Same as Example 2.
〔result〕
The results are shown in Table 4.
[0068]
[Table 4]
Table 4

[0069]
As is apparent from the results in Table 4, the LDL-cholesterol value obtained using the reagent containing the polyanion of the present invention and the amphoteric surfactant (samples 17 to 19) is a value measured by the BQ method (Beta quantification method). It can be seen that it is almost equivalent to the indicated value of the control serum marked with.
[0070]
Reference example 2
A modified antibody was prepared by a method known per se.
(1) Preparation of dextran modified antibody
After mixing 15 ml of sheep-derived anti-HDL antibody (10 mg protein Ab / ml) and 300 mg of dialdehyde dextran and reacting at 50 ° C. in 50 mM phosphate buffer (pH 6.0), 200 mg of borane pyridine was further added. In addition, the reaction was allowed to proceed for 5 hours. This reaction solution was packed in a cellophane tube, desalted, and then subjected to ultrafiltration concentration treatment to obtain a dextran-modified antibody (10 mg protein Ab / ml × 12.6 ml).
[0071]
(2) Preparation of heparin-modified antibody
500 mg of 6-aminocaproic acid was added to 100 mg of heparin treated with periodate and dialyzed. To this, 5 ml of sheep-derived anti-HDL antibody (10 mg protein Ab / ml) and 300 mg of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide were added and reacted at 5 ° C. overnight. This reaction solution was packed in a cellophane tube, desalted, and then subjected to ultrafiltration concentration treatment to obtain a heparin-modified antibody (10 mg protein Ab / ml × 3.6 ml).
[0072]
(3) Preparation of alginate-modified antibody
50 mg of alginic acid is dissolved in 20 ml of 20 mM phosphate buffer (pH 7.0), and 5 ml of anti-HDL antibody (10 mg protein Ab / ml) derived from sheep and 1-ethyl-3- (3-dimethylaminopropyl)- Carbodiimide 500 mg was added and reacted at 5 ° C. overnight. This reaction solution was packed in a cellophane tube, desalted, and then subjected to ultrafiltration concentration treatment to obtain an alginate-modified antibody (10 mg protein Ab / ml × 3.4 ml).
[0073]
(4) Production of glucuronic acid-modified antibody
Dissolve 10 ml of sheep-derived anti-HDL antibody (10 mg protein Ab / ml) and 400 mg of glucuronic acid in 0.1 M phosphate buffer (pH 7.0), add 250 mg of cyanborohydride, and react at 37 ° C for 5 hours. It was. This reaction solution was packed in a cellophane tube, desalted, and then subjected to ultrafiltration concentration treatment to obtain a glucuronic acid-modified antibody (10 mg protein Ab / ml × 7.6 ml).
[0074]
(5) Preparation of polyglutamic acid-modified antibody
  20 mg of polyglutamic acid is dissolved in 4 ml of 20 mM phosphate buffer (pH 6.5), 1 ml of anti-HDL antibody derived from sheep (10 mg protein Ab / ml) and 1-ethyl-3- (3-dimethylaminopropyl) are dissolved in the solution. ) -Carbodiimide 40 mg was added and reacted at 5 ° C. overnight. This reaction solution was packed in a cellophane tube and desalted, and then subjected to ultrafiltration concentration treatment to obtain a polyglutamic acid-modified antibody (10 mg protein Ab / ml × 0.72 ml).
[0075]
(6) Preparation of glycyrrhizic acid-modified antibody
  Dissolve 100 mg of glycyrrhizic acid in 2 ml of 50 mM phosphate buffer (pH 6.5), add 100 mg of sodium metaperiodate to the solution and react for 24 hours at 5 ° C. Then, use Sephadex G-25 column. Desalted. This was mixed with 10 ml of sheep-derived anti-HDL antibody (10 mg protein Ab / ml) and reacted at 30 ° C. for 48 hours. This reaction solution was packed in a cellophane tube, desalted, and then subjected to ultrafiltration concentration treatment to obtain a glycyrrhizic acid-modified antibody (10 mg protein Ab / ml × 7.3 ml).
[0076]
Example 5
Using a Hitachi 7170 type automatic analyzer (manufactured by Hitachi, Ltd.), the LDL-cholesterol measurement value is obtained by the measurement method of the present invention, and the comparison with the LDL-cholesterol measurement value by the BQ method which is the reference method is performed. went.
〔sample〕
Samples obtained from PBI (Pacific Biometrics, Inc.) 15 samples of human serum (triglyceride 400 mg / dl or less) with known measurement values by the BQ method (beta quantification method) and 8 samples of human serum (triglyceride 400 mg / dl or more) It was.
The amount of triglyceride in the serum depends on the displayed value given to the human serum.
〔reagent〕

〔Measurement condition〕
Same as Example 2.
〔result〕
The results for sera with a triglyceride of 400 mg / dl or less are shown in Table 5, and the results for sera with a triglyceride of 400 mg / dl or more are shown in Table 6, respectively.
[0077]
[Table 5]
Table 5

[0078]
[Table 6]
Table 6

[0079]
From the results shown in Table 5, when serum having a triglyceride value of 400 mg / dl or less was used as a sample, the LDL-cholesterol values of both the test solution 20 and the test solution 21 were measured by the BQ method (Beta quantification method). It turns out that it is almost equivalent to the value. From the results shown in Table 6, when serum having a triglyceride value of 400 mg / dl or more is used as a sample, the test solution 21, that is, a polyanion and an amphoteric surfactant, and an antibody that binds to a lipoprotein other than LDL coexist. It can be seen that the measurement is closer to the displayed value, that is, LDL-cholesterol can be measured more specifically.
[0080]
【The invention's effect】
As described above, the present invention provides a method capable of specifically and accurately measuring LDL-cholesterol in a sample derived from a living body, and a reagent used therefor. LDL-cholesterol, which was impossible with this method, can be measured directly and using a general-purpose automatic analyzer.
[Brief description of the drawings]
FIG. 1 shows reaction curves for various lipoprotein fraction samples obtained using sample solution 1 in Example 1. FIG.
FIG. 2 shows reaction curves for various lipoprotein fraction samples obtained using the test solution 2 in Example 1.
FIG. 3 shows reaction curves for various lipoprotein fraction samples obtained using the test solution 3 in Example 1.
FIG. 4 shows reaction curves for various lipoprotein fraction samples obtained using the test solution 4 in Example 1.
FIG. 5 shows reaction curves for various lipoprotein fraction samples obtained using the sample solution 5 in Example 1.
FIG. 6 shows reaction curves for various lipoprotein fraction samples obtained using the test solution 6 in Example 1.
FIG. 7 shows reaction curves for various lipoprotein fraction samples obtained using sample solution 7 in Example 1.
FIG. 8 shows reaction curves of various lipoprotein fraction samples obtained using the test solution 8 in Example 1.
FIG. 9 shows reaction curves for various lipoprotein fraction samples obtained using the test solution 9 in Example 1.
FIG. 10 shows reaction curves for various lipoprotein fraction samples obtained using the test solution 10 in Example 1.
FIG. 11 shows reaction curves for various lipoprotein fraction samples obtained using the test solution 11 in Example 1.
FIG. 12 shows reaction curves for various lipoprotein fraction samples obtained using the test solution 12 in Example 1.
FIG. 13 shows reaction curves for various lipoprotein fraction samples obtained using the test solution 13 in Example 1.
[Explanation of symbols]
1 to 13,-□-indicates the result obtained for the low density lipoprotein (LDL) fraction sample,-○-indicates the result obtained for the high density lipoprotein (HDL) fraction sample, -Δ- indicates the results obtained for the ultra-low density lipoprotein (VLDL) fraction sample, and-●-indicates the results obtained using physiological saline as a sample.

Claims (11)

The presence of a polyanion and an amphoteric surfactant, a biological sample (1) cholesterol esterase and cholesterol oxidase, or (2) cholesterol esterase, by the action of cholesterol dehydrogenase and NAD (P), low density lipoprotein in the sample Inhibits, delays or temporarily stops the reaction of cholesterol in the medium, eliminates (consumes) the reaction of cholesterol in lipoproteins other than low-density lipoprotein, and then generates excess cholesterol by reaction in cholesterol in low-density lipoprotein. A method for measuring cholesterol in a low density lipoprotein, comprising measuring cholesterol in a biological sample based on hydrogen oxide or NAD (P) H. In the presence of a polyanion and an amphoteric surfactant , (1) cholesterol esterase and cholesterol oxidase, or (2) cholesterol esterase, cholesterol dehydrogenase and NAD (P) are allowed to act on a biological sample at two different times . The method according to claim 1, wherein the absorbance is measured, and the amount of cholesterol in the low density lipoprotein in the biological sample is calculated based on the absorbance. Before contacting the biological sample with polyanion and amphoteric surfactant, the reaction of cholesterol in lipoproteins other than low density lipoprotein is substantially completed, and before the reaction of cholesterol in low density lipoprotein is initiated Measure the absorbance of the reaction system, and measure the absorbance of the reaction system again after the cholesterol reaction in the low density lipoprotein is substantially completed. Based on these results, measure the absorbance of the low density lipoprotein in the biological sample. The method according to claim 2, wherein the amount of cholesterol is calculated. The method according to any one of claims 1 to 3, wherein the measurement is carried out by a two-solution method. The method according to claim 4, wherein the first test solution contains catalase and the second test solution contains a catalase inhibitor. Furthermore, the measuring method in any one of Claims 1-5 which measures in coexistence of a nonionic surfactant or / and an anionic surfactant. Furthermore, the method in any one of Claims 1-6 which performs a measurement in the coexistence of the antibody couple | bonded with lipoproteins other than low density lipoprotein. Inhibits, delays, or temporarily stops the reaction of cholesterol in low density lipoprotein in biological samples, erases (consumes) cholesterol reaction in lipoproteins other than low density lipoprotein, and then low density lipoprotein A kit for measuring cholesterol in a low-density lipoprotein according to any one of (a) to (c) below for measuring hydrogen peroxide or NAD (P) H produced by the reaction of cholesterol therein .
(a) (1) a polyanion and an amphoteric surfactant, (2) cholesterol esterase, (3) cholesterol oxidase, peroxidase, and a coupler or / and developer, and (4) a first reagent solution comprising an aqueous medium and an aqueous solution A kit for measuring cholesterol in low-density lipoprotein, comprising a medium and, if necessary, a second test solution comprising a developer or a coupler.
(b) (1) polyanion and amphoteric surfactant, (2) cholesterol esterase, (3) cholesterol oxidase, (4) catalase, and (5) a first reagent solution containing an aqueous medium, a catalase inhibitor and an aqueous solution A kit for measuring cholesterol in low-density lipoprotein, comprising a second reagent containing a medium, wherein peroxidase, coupler, and developer are contained in at least one of the first reagent and the second reagent, respectively. .
(c) (1) a polyanion, (2) an amphoteric surfactant, (3) cholesterol esterase, (4) cholesterol dehydrogenase, (5) NAD (P), and (6) a first reagent solution comprising an aqueous medium; (1) A kit for measuring cholesterol in low-density lipoprotein, comprising a second reagent solution containing an aqueous medium. (1) Polyanions and amphoteric surfactants, (2) Cholesterol esterase, (3) Cholesterol oxidase, (Four) Catalase, and (Five) A first test solution containing an aqueous medium and a second test solution containing a catalase inhibitor and an aqueous medium, wherein the peroxidase, coupler and developer are at least one of the first test solution and the second test solution, respectively. In The kit for measuring cholesterol in low specific gravity lipoprotein according to claim 8, which is contained. Furthermore, the kit of Claim 8 or 9 in which the nonionic surfactant or / and the anionic surfactant are each contained in at least one of the 1st test solution and the 2nd test solution. The kit according to any one of claims 8 to 10, wherein the first test solution further comprises an antibody that binds to a lipoprotein other than the low density lipoprotein.

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