JP4231721B2

JP4231721B2 - Complement value measuring reagent and method for stabilizing complement value measurement using the same

Info

Publication number: JP4231721B2
Application number: JP2003093994A
Authority: JP
Inventors: 雅行飯塚; 潤治松田; 広宣古島
Original assignee: Denka Seiken Co Ltd
Current assignee: Denka Seiken Co Ltd
Priority date: 2003-03-31
Filing date: 2003-03-31
Publication date: 2009-03-04
Anticipated expiration: 2023-03-31
Also published as: JP2004301612A

Description

【０００１】
【発明の属する技術分野】
本発明は、補体価測定用試薬及びそれを用いた補体価測定値の安定化方法に関する。
【０００２】
【従来の技術】
ウイルス、細菌等の病原体に対する補体結合性抗体は、病原体が感染するとその補体量は上昇または下降する。補体量の測定は、各種疾患における診断や治療効果の判断及び免疫能力の指標として重要なものとなっている。
【０００３】
従来の補体価測定方法では、ヒツジ赤血球に抗ヒツジ赤血球抗体を結合させた感作ヒツジ赤血球、および至適倍数に段階希釈した補体を含む溶液（血清など）とを混合して37℃60分間反応させ、補体の作用によって赤血球が破裂した結果溶液中に漏出する赤血球中のヘモグロビンを吸光度法により測定することによって、赤血球の溶血率をグラフより計算し補体価を求めるメイヤー法が一般的であった。近年では、ヘモグロビンを測定する替わりに、残存赤血球の濁度を光学的に測定することにより赤血球の溶血率を計算し補体価を求める自動化学分析装置で測定するオート法が一般的になってきている。オート法では自動化学分析装置に試薬をセットして測定するが、試薬をセットしてから測定までの時間が長い場合には、正確な測定を行うためには、測定直前に撹拌する必要があり、煩雑であった。
【０００４】
【発明が解決しようとする課題】
本発明の目的は、試薬の調製から測定までの時間が長い場合であっても、測定直前に撹拌しなくても正確な測定が可能である補体価測定用試薬を提供することである。
【０００５】
【課題を解決するための手段】
本願発明者らは、鋭意研究の結果、従来の補体価測定用試薬にシュクロースとエピクロロヒドリンの共重合体を配合することにより、試薬の調製から測定までの時間が長い場合あっても、測定直前に撹拌しなくても正確な測定が可能であることを見出し本発明を完成した。
【０００６】
すなわち、本発明は、緩衝液中に懸濁された感作赤血球または感作リポソームを含む補体価測定用試薬において、シュクロースとエピクロロヒドリンの共重合体を含むことを特徴とする補体価測定用試薬を提供する。また、本発明は、測定値安定化剤としてシュクロースとエピクロロヒドリンの共重合体を緩衝液に添加することから成る、緩衝液中に懸濁された感作赤血球を含む補体価測定用試薬を用いた補体価の測定値の安定化方法を提供する。
【０００７】
【発明の実施の形態】
上記の通り、本発明の補体価測定用試薬は、試薬の調製から測定までの時間が長い場合あっても、測定直前に撹拌しなくても正確な測定を可能とするために、緩衝液中にシュクロースとエピクロロヒドリンの共重合体を含むことを特徴とする。試薬の調製から測定までの時間が長い場合あっても、測定直前に撹拌しなくても正確な測定が可能となる理由は、シュクロースとエピクロロヒドリンの共重合体を配合することにより、感作赤血球の沈降が遅延されることに起因すると考えられる。シュクロースとエピクロロヒドリンの共重合体は、フィコール(Ficoll)の商品名でAmersham Biosciences社から市販されており、低粘度で高密度の水溶液を与えるため、培地や種々の生体反応の媒体中に配合されて広く用いられており、それ自体は周知の物質である。本発明に用いられるシュクロースとエピクロロヒドリンの共重合体の分子量は、特に限定されないが、１万〜１００万程度が好ましく、特に５万〜５０万程度が好ましい。
【０００８】
配合するシュクロースとエピクロロヒドリンの共重合体の量は、シュクロースとエピクロロヒドリンの共重合体を含む緩衝液の比重（赤血球を除く）が1.0〜1.3となる量が好ましく、特に、1.055〜1.060となる量が好ましい。
【０００９】
上記したシュクロースとエピクロロヒドリンの共重合体を含むこと以外は、試薬の組成及びそれを用いた測定方法は従来から使用されているものと同じでよい。すなわち、感作赤血球を緩衝液中に懸濁させた試薬に、補体を含む検体を添加し反応させる。検体中の補体量に依存した数の赤血球が溶血するので、緩衝液の吸光度を測定するか、又は、溶血した赤血球は波長６６０ｎｍでは検出されなくなるので波長６６０ｎｍにおける濁度を測定することにより、検体中の補体量を測定することができる。自動化装置では、後者の濁度を測定する方法が広く採用されている。なお、感作赤血球懸濁緩衝液としては、特に限定されないが、アルブミン及びグルコースを含むリン酸緩衝液が広く広く用いられており、本発明でもこの緩衝液を好ましく用いることができる。なお、感作赤血球に代えて感作リポソームを用いてもよい。感作リポソームを用いる方法もこの分野において周知である。
【００１０】
上記した本発明の補体価測定用試薬を用いて検体中の補体価を測定すると、試薬（赤血球懸濁液）の調製から測定までの間、長時間に亘って試薬が放置された場合でも、調製直後に測定を行った場合と測定値がほとんど変化しない。すなわち、上記シュクロースとエピクロロヒドリンの共重合体を配合することにより、測定値が安定化される。従って、本発明は、また、測定値安定化剤としてシュクロースとエピクロロヒドリンの共重合体を緩衝液に添加することから成る、緩衝液中に懸濁された感作赤血球を含む補体価測定用試薬を用いた補体価の測定値の安定化方法をも提供するものである。
【００１１】
実施例１
アルブミン（濃度0.5%)、グルコース（濃度1%）を含むリン酸緩衝液にフィコール(商品名）（分子量40万)を15重量%添加し完全に溶解させた。その時の比重は1.055〜1.060であった。赤血球に抗体を結合させた感作ヒツジ赤血球を、上記15%フィコール(商品名）添加緩衝液に懸濁させた。感作ヒツジ赤血球の含量は、1.4%であった。得られた、感作赤血球液を用い自動化学分析装置にて新鮮血清を試料とし、補体価を測定した。すなわち、試料３μｌにカルシウムイオン及びマグネシウムイオンを含むベロナール緩衝液（濃度：カルシウム0.0022%、マグネシウム0.011%）３５０μｌ加え試料を希釈し、更に上記赤血球懸濁液６０μｌを加え、３７℃で６分間反応させた後波長６６０ｎｍにおける濁度を測定（０時間）した。次に上記赤血球懸濁液を表１に示す所定の時間静置後、同様（０時間）な測定を行い波長６６０ｎｍにおける濁度を測定した。試料中の補体価（CH₅₀)につては既知濃度の補体を含む標準補体試料を用いて上記方法により測定して得られた検量線に基づき、検体中の補体価(CH₅₀)を決定した。結果を下記表１に示す。なお、補体価(CH₅₀)の１単位は、至適濃度の溶血素（ヒツジの赤血球をウサギに免疫して得られる抗血清）で感作されたヒツジ赤血球5 x 10⁸個の５０％を、7.5 mlの反応容量の中で３７℃で溶血させるのに必要な量である。
【００１２】
【表１】

【００１３】
表１に示されるように、本発明の補体価測定用試薬を用いれば、試薬調製後、１２時間もの長時間試薬を静置した後に測定を行っても、試薬調製直後に測定した場合とほとんど同じ測定値が得られることがわかる。
【００１４】
比較例１
フィコール（商品名）を添加しないことを除き、実施例１と同じ操作を行った。ただし、静置時間は２時間３０分で既に大きな誤差を生じていたので、２時間３０分で打ち切った。結果を下記表２に示す。
【００１５】
【表２】

【００１６】
表２から明らかなように、フィコール（商品名）を添加しない従来の方法では、試薬の調製から測定までの静置時間が僅か２時間３０分であっても、調製直後に測定した場合の測定値とは７〜１１％もの誤差が生じることがわかる。
【００１７】
【発明の効果】
上記実施例及び比較例により実験的に示された通り、本発明の補体価測定用試薬を用いると、試薬の調製から測定までの時間が長い場合であっても、測定直前に撹拌しなくても正確な測定が可能となる。従って、本発明の試薬を用いると、測定直前に人手で撹拌する必要がなくなり、補体価の自動測定に有利である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a reagent for measuring complement value and a method for stabilizing a measured value of complement value using the same.
[0002]
[Prior art]
Complement-binding antibodies against pathogens such as viruses and bacteria increase or decrease the amount of complement when the pathogen is infected. The measurement of the amount of complement is important as an indicator of diagnosis and therapeutic effect in various diseases and immune ability.
[0003]
In the conventional complement titration method, a sensitized sheep erythrocyte in which an anti-sheep erythrocyte antibody is bound to a sheep erythrocyte and a solution (such as serum) containing complement diluted serially to an optimal multiple are mixed at 37 ° C. 60 The Meyer method is generally used to calculate the hemolysis rate of erythrocytes from the graph and determine the complement value by measuring the hemoglobin in the erythrocytes leaked into the solution as a result of the rupture of the erythrocytes by the action of complement and the leakage of red blood cells by the action of complement. It was the target. In recent years, instead of measuring hemoglobin, an automatic method has been used in which an automatic chemical analyzer that calculates the hemolysis rate of red blood cells by optically measuring the turbidity of the remaining red blood cells to determine the complement value is used. ing. In the auto method, the reagent is set in the automatic chemical analyzer and the measurement is performed. However, if the time from setting the reagent to the measurement is long, it is necessary to stir immediately before the measurement to perform accurate measurement It was cumbersome.
[0004]
[Problems to be solved by the invention]
An object of the present invention is to provide a reagent for measuring complement number, which enables accurate measurement even when the time from preparation of the reagent to measurement is long, without stirring immediately before the measurement.
[0005]
[Means for Solving the Problems]
As a result of diligent research, the inventors of the present application have found that it takes a long time from preparation of a reagent to measurement by adding a copolymer of sucrose and epichlorohydrin to a conventional reagent for measuring complement value. However, the present invention was completed by finding that accurate measurement is possible without stirring immediately before measurement.
[0006]
That is, the present invention provides a complement titration-measuring reagent comprising sensitized red blood cells or sensitized liposomes suspended in a buffer solution, comprising a sucrose and epichlorohydrin copolymer. A reagent for measuring valence is provided. The present invention also relates to a complement number measurement comprising sensitized red blood cells suspended in a buffer solution, comprising adding a copolymer of sucrose and epichlorohydrin as a measurement value stabilizer to the buffer solution. A method for stabilizing a measured value of complement value using a reagent for use is provided.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
As described above, the reagent for measuring complement value of the present invention uses a buffer solution in order to enable accurate measurement even if the time from the preparation of the reagent to the measurement is long or without stirring just before the measurement. It contains a copolymer of sucrose and epichlorohydrin . Even if the time from the preparation of the reagent to the measurement is long, the reason why accurate measurement is possible without stirring immediately before the measurement is by blending a copolymer of sucrose and epichlorohydrin , This is thought to be due to the delayed sedimentation of sensitized red blood cells. A copolymer of sucrose and epichlorohydrin is commercially available from Amersham Biosciences under the trade name Ficoll and provides a low-viscosity, high-density aqueous solution that can be used in media and various biological reaction media. It is a well-known substance per se. The molecular weight of the sucrose and epichlorohydrin copolymer used in the present invention is not particularly limited, but is preferably about 10,000 to 1,000,000, and particularly preferably about 50,000 to 500,000.
[0008]
The amount of the sucrose and epichlorohydrin copolymer to be blended is preferably such that the specific gravity (excluding erythrocytes) of the buffer containing the sucrose and epichlorohydrin copolymer is 1.0 to 1.3. An amount of 1.055 to 1.060 is preferred.
[0009]
The composition of the reagent and the measurement method using the same may be the same as those conventionally used except that the copolymer of sucrose and epichlorohydrin is included. That is, a specimen containing complement is added to a reagent in which sensitized red blood cells are suspended in a buffer solution, and reacted. Since the number of red blood cells depending on the amount of complement in the sample is hemolyzed, the absorbance of the buffer solution is measured, or the hemolyzed red blood cells are not detected at a wavelength of 660 nm, so by measuring the turbidity at a wavelength of 660 nm, The amount of complement in the sample can be measured. In the automation apparatus, the latter method of measuring turbidity is widely adopted. The sensitized red blood cell suspension buffer is not particularly limited, but a phosphate buffer containing albumin and glucose is widely used, and this buffer can also be preferably used in the present invention. Sensitized liposomes may be used in place of the sensitized red blood cells. Methods using sensitized liposomes are also well known in the art.
[0010]
When the complement value in a sample is measured using the above-described reagent for measuring complement value of the present invention, the reagent is left for a long time from the preparation of the reagent (erythrocyte suspension) to the measurement. However, the measured values are almost the same as when measured immediately after preparation. That is, the measurement value is stabilized by blending the sucrose and epichlorohydrin copolymer . Accordingly, the present invention also provides a complement comprising sensitized red blood cells suspended in a buffer comprising adding a copolymer of sucrose and epichlorohydrin to the buffer as a measurement value stabilizer. The present invention also provides a method for stabilizing the measured value of complement value using a valence measuring reagent.
[0011]
Example 1
To a phosphate buffer containing albumin (concentration 0.5%) and glucose (concentration 1%), 15% by weight of Ficoll (trade name) (molecular weight 400,000) was added and completely dissolved. The specific gravity at that time was 1.055 to 1.060. Sensitized sheep erythrocytes in which antibodies were bound to erythrocytes were suspended in the above 15% Ficoll (trade name) addition buffer. The content of sensitized sheep erythrocytes was 1.4%. Complement titer was measured using the obtained sensitized erythrocyte solution, using fresh serum as a sample with an automatic chemical analyzer. That is, 350 μl of veronal buffer solution (concentration: calcium 0.0022%, magnesium 0.011%) containing calcium ions and magnesium ions was added to 3 μl of the sample, the sample was diluted, and 60 μl of the erythrocyte suspension was further added, and reacted at 37 ° C. for 6 minutes. After that, the turbidity at a wavelength of 660 nm was measured (0 hour). Next, the erythrocyte suspension was allowed to stand for a predetermined time as shown in Table 1, and then the same (0 hour) measurement was performed to measure turbidity at a wavelength of 660 nm. Complement activity in the sample (CH ₅₀₎ connexion based on the calibration curve obtained by measuring by the above method using a standard complement sample containing the complement of a known concentration, in the sample complement activity (CH ₅₀ )It was determined. The results are shown in Table 1 below. One unit of complement value (CH ₅₀ ) is 50% of 5 × 10 ⁸ sheep erythrocytes sensitized with an optimal concentration of hemolysin (antiserum obtained by immunizing rabbits with sheep erythrocytes). Is required to hemolyze at 37 ° C. in a 7.5 ml reaction volume.
[0012]
[Table 1]

[0013]
As shown in Table 1, if the reagent for measuring complement value of the present invention is used, even if the measurement is carried out after leaving the reagent for 12 hours after the reagent preparation, It can be seen that almost the same measurement value is obtained.
[0014]
Comparative Example 1
The same operation as in Example 1 was performed except that Ficoll (trade name) was not added. However, the standing time was already 2 hours and 30 minutes, and a large error had already occurred. The results are shown in Table 2 below.
[0015]
[Table 2]

[0016]
As is clear from Table 2, in the conventional method in which Ficoll (trade name) is not added, even when the standing time from preparation to measurement of the reagent is only 2 hours and 30 minutes, measurement is performed immediately after preparation. It can be seen that an error of 7 to 11% occurs from the value.
[0017]
【The invention's effect】
As experimentally shown by the above Examples and Comparative Examples, when the complement value measuring reagent of the present invention is used, even if the time from the preparation of the reagent to the measurement is long, it is not stirred immediately before the measurement. However, accurate measurement is possible. Therefore, the use of the reagent of the present invention eliminates the need for manual stirring immediately before the measurement, and is advantageous for automatic measurement of complement value.

Claims

In complement activity measurement reagent containing sensitized red blood cells or sensitized liposomes were suspended in buffer, complement activity measurement reagents comprising a copolymer of sucrose and epichlorohydrin.

The reagent according to claim 1, wherein the specific gravity of the buffer solution is 1.0 to 1.3.

The reagent according to claim 2 , wherein the specific gravity is 1.055 to 1.060.

A reagent for measuring complement value comprising sensitized red blood cells or sensitized liposomes suspended in a buffer, comprising adding a copolymer of sucrose and epichlorohydrin to the buffer as a measurement value stabilizer A method for stabilizing the measured value of complement value using sucrose.

The method according to claim 4 , wherein the specific gravity of the buffer solution is 1.0 to 1.3.

The method of claim 5, wherein the specific gravity of from 1.055 to 1.060.