JPH08105890A - Preparation of reagent for measuring anti-phospholipid antibody - Google Patents
Preparation of reagent for measuring anti-phospholipid antibodyInfo
- Publication number
- JPH08105890A JPH08105890A JP24152494A JP24152494A JPH08105890A JP H08105890 A JPH08105890 A JP H08105890A JP 24152494 A JP24152494 A JP 24152494A JP 24152494 A JP24152494 A JP 24152494A JP H08105890 A JPH08105890 A JP H08105890A
- Authority
- JP
- Japan
- Prior art keywords
- latex
- reagent
- antiphospholipid antibody
- phospholipid
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、簡便かつ高感度な抗り
ん脂質抗体測定用試薬を製造する方法に関する。TECHNICAL FIELD The present invention relates to a method for producing a simple and highly sensitive reagent for measuring antiphospholipid antibody.
【０００２】[0002]
【従来の技術】医療の分野においては、従来から、血
清、血しょう、脳脊髄液等の検体中に含まれているカル
ジオライピン、フォスファチジルコリン、コレステロー
ル等のりん脂質に対応する抗りん脂質抗体を測定するこ
とにより、特定の疾病の診断をする方法が知られてい
る。このような方法のなかでも、高感度測定法として
は、酵素免疫測定法（ＥＬＩＳＡ）が開発され、梅毒、
エイズ等の診断方法として既に確立されている。しか
し、この方法は、迅速性、操作性に劣るので、抗りん脂
質抗体測定用として多くの検体を迅速に測定する場合に
は不適当なものであった。2. Description of the Related Art In the medical field, antiphospholipid antibodies corresponding to phospholipids such as cardiolipin, phosphatidylcholine and cholesterol contained in samples such as serum, plasma, cerebrospinal fluid, etc. A method for diagnosing a specific disease by measuring is known. Among these methods, enzyme-linked immunosorbent assay (ELISA) has been developed as a highly sensitive assay, and syphilis,
It has already been established as a diagnostic method for AIDS and the like. However, since this method is inferior in speed and operability, it was unsuitable for rapid measurement of many specimens for antiphospholipid antibody measurement.
【０００３】抗りん脂質抗体を測定する方法としては、
炭素粒子りん脂質抗原懸濁液を使用したＲＰＲ（Ｒａｐ
ｉｄ Ｐｌａｓｍａ Ｒｅａｇｉｎ）カード試験、ＶＤ
ＲＬ（Ｖｅｎｅｒｅａｌ Ｄｉｓｅａｓｅ Ｒｅｓｅａ
ｒｃｈ Ｌａｂ）、ＲＳＴ（Ｒｅａｇｉｎ Ｓｃｒｅｅ
ｎｉｎｇ Ｔｅｓｔ）等の迅速性、操作性に優れた測定
法が知られているが、これらは目視判定法であるので、
施術者、測定日により結果が異なる等の精度上の問題が
あった。As a method for measuring antiphospholipid antibody,
RPR (Rap using a carbon particle phospholipid antigen suspension)
id Plasma Reagin) card test, VD
RL (Venereal Disease Rease)
rch Lab), RST (Reaign Screen)
Ning Test) and other measurement methods that are excellent in quickness and operability are known, but since these are visual determination methods,
There was a problem in accuracy, such as the result depending on the practitioner and the measurement date.
【０００４】これに対し、検体中の抗りん脂質抗体を迅
速に測定する方法として、りん脂質をラテックス粒子等
の不溶性担体に固定化し、このものを緩衝液等に懸濁分
散させて試薬とする方法が提案されている。これらのう
ち、りん脂質のラテックス粒子等の不溶性担体への固定
化方法としては、例えば、次のような方法が知られてい
た。On the other hand, as a method for rapidly measuring an antiphospholipid antibody in a sample, phospholipids are immobilized on an insoluble carrier such as latex particles, and this is suspended and dispersed in a buffer or the like to prepare a reagent. A method has been proposed. Among these, the following methods have been known as methods for immobilizing phospholipids on insoluble carriers such as latex particles.
【０００５】クリニック・エクスペリメンタル・イミュ
ノロジー（Ｃｌｉｎ．Ｅｘｐ．Ｉｍｍｕｎｏｌ．）６８
号、２１５〜２２２頁（１９８７年）、及び、公表平４
−５０３８６５号公報には、マイクロタイタープレート
のウェル又は磁性体粒子表面にりん脂質の有機溶媒液を
加え、溶媒を蒸発させてプレートの壁に物理吸着させる
方法（方法（１））が開示されている。公表昭６３−５
０１９２８号公報及び公表平４−５０３８６５号公報に
は、スペーサーを介した化学結合方法（方法（２））が
開示されている。特開昭５８−６１４６６号公報には、
メチル化タンパクを介した物理吸着方法（方法（３））
が開示されている。Clinic Experimental Immunology (Clin. Exp. Immunol.) 68
No., pp. 215-222 (1987), and Publication No. 4
Japanese Patent Publication No. -503865 discloses a method (method (1)) in which an organic solvent solution of phospholipid is added to the well of a microtiter plate or the surface of magnetic particles and the solvent is evaporated to physically adsorb on the wall of the plate. There is. Announcement Sho 63-5
No. 01928 and Japanese Patent Laid-Open No. 4-503865 disclose a chemical bonding method (method (2)) via a spacer. Japanese Patent Laid-Open No. 58-61466 discloses that
Physical adsorption method via methylated protein (method (3))
Is disclosed.
【０００６】しかしながら、方法（１）は、洗浄時にプ
レート壁に吸着したりん脂質がはがれ落ちやすいので，
測定値がばらつき、検出感度が低くなる等の問題があ
り、また、ラテックス粒子等の水溶性溶媒に懸濁されて
いる不溶性担体では、物理吸着後有機溶媒を蒸発させる
と、粒子自体が変性し、自己凝集を引き起こす等の問題
があり、診断用試薬として使用することができなかっ
た。また、方法（２）、方法（３）は、ラテックス粒子
を変性させることなくりん脂質を固定化することができ
るが、化学修飾されたりん脂質を用いるので、抗原とし
ての反応性が低下したり、更に化学結合の反応条件の設
定が複雑であること等により製造工程が複雑になる等の
問題があった。However, in the method (1), since the phospholipids adsorbed on the plate wall during washing are easily peeled off,
There are problems such as variations in measured values and low detection sensitivity.In addition, in an insoluble carrier suspended in a water-soluble solvent such as latex particles, when the organic solvent is evaporated after physical adsorption, the particles themselves are denatured. However, it cannot be used as a diagnostic reagent because of problems such as causing self-aggregation. In addition, the methods (2) and (3) can immobilize phospholipids without denaturing latex particles, but since chemically modified phospholipids are used, reactivity as an antigen decreases. Further, there is a problem that the manufacturing process becomes complicated due to complicated setting of reaction conditions for chemical bonding.
【０００７】更に、上述の方法はいずれもりん脂質の混
合液を抗原として用いるので、方法（１）のような物理
吸着法では、固相に対するりん脂質間の吸着しやすさの
差異から、温度、攪拌条件等により、個々のりん脂質の
吸着量、配向性が変化しやすく、試薬製造時のロット間
差を生じやすい欠点がある。また、方法（２）、方法
（３）についても同様に、架橋反応等を行う際のｐＨ、
試薬量比等の化学的条件も個々のりん脂質によって異な
るので、製造条件の最適化が困難である等の問題があっ
た。Further, in each of the above-mentioned methods, since a mixed solution of phospholipids is used as an antigen, in the physical adsorption method such as the method (1), the difference in easiness of adsorption between phospholipids on the solid phase causes However, there are drawbacks that the adsorption amount and orientation of individual phospholipids are likely to change depending on stirring conditions and the like, and a lot-to-lot difference is likely to occur during reagent production. Further, similarly for the method (2) and the method (3), the pH during the cross-linking reaction,
Since the chemical conditions such as the reagent amount ratio also differ depending on the individual phospholipid, there is a problem that it is difficult to optimize the production conditions.
【０００８】[0008]
【発明が解決しようとする課題】本発明は、上記に鑑
み、簡易に製造でき、かつ、高感度に抗りん脂質抗体を
検出できる抗りん脂質抗体検出用ラテックス試薬を提供
することを目的とする。SUMMARY OF THE INVENTION In view of the above, an object of the present invention is to provide a latex reagent for detecting antiphospholipid antibody which can be easily produced and can detect antiphospholipid antibody with high sensitivity. .
【０００９】[0009]
【課題を解決するための手段】本発明の要旨は、抗りん
脂質抗体検出用ラテックス試薬を、カルジオライピン、
フォスファチジルコリン及びコレステロールをそれぞれ
個別にラテックス粒子に固定化させてそれぞれのりん脂
質固定化ラテックス水性懸濁液を調製し、その後、これ
らの少なくとも１種を混合して得るところに存する。The gist of the present invention is to provide a latex reagent for detecting antiphospholipid antibody with cardiolipin,
Phosphatidylcholine and cholesterol are individually immobilized on latex particles to prepare respective phospholipid-immobilized latex aqueous suspensions, which are then obtained by mixing at least one of them.
【００１０】上記カルジオライピン、フォスファチジル
コリン及びコレステロールとしては特に限定されず、市
販のものが用いられる。フォスファチジルコリンとして
は、例えば、レシチンが好ましい。これらは、適当な有
機溶媒に溶解して使用することができる。上記有機溶媒
としては、上記りん脂質を可溶化し、かつ、担体を溶
解、変性させないものであれば特に限定されず、メタノ
ール、エタノール、エーテル等が挙げられる。The above-mentioned cardiolipin, phosphatidylcholine and cholesterol are not particularly limited, and commercially available products are used. As the phosphatidylcholine, lecithin is preferable, for example. These can be used by dissolving them in a suitable organic solvent. The organic solvent is not particularly limited as long as it solubilizes the phospholipid and does not dissolve or denature the carrier, and examples thereof include methanol, ethanol, ether and the like.
【００１１】上記ラテックス粒子としては特に限定され
ず、合成ラテックス粒子、ゼラチン粒子、動物の赤血
球、カオリン、炭素末等が挙げられる。特にポリスチレ
ンラテックスが好ましい。また、りん脂質を担持させる
不溶性担体として、ラテックス粒子以外のものを用いる
ことができ、例えば、プラスチック性のマイクロタイタ
ープレート、試験管等が挙げられる。上記ラテックス粒
子の粒径は、０．０５〜１０μｍが好ましく、特に０．
１〜１μｍが好ましい。The latex particles are not particularly limited, and examples thereof include synthetic latex particles, gelatin particles, animal red blood cells, kaolin, carbon powder and the like. Polystyrene latex is particularly preferable. Further, as the insoluble carrier for supporting the phospholipid, other than the latex particles can be used, and examples thereof include a plastic microtiter plate and a test tube. The particle size of the latex particles is preferably 0.05 to 10 μm, and particularly preferably 0.
1 to 1 μm is preferable.
【００１２】本発明では、まず、上記カルジオライピ
ン、フォスファチジルコリン及びコレステロールをそれ
ぞれ上記有機溶媒に溶解させたものを、それぞれ個別
に、水性溶媒中に懸濁させたラテックス粒子とともに攪
拌して、ラテックス粒子に固定化させて、それぞれのり
ん脂質固定化ラテックスの水性懸濁液を調製する。本発
明では、その後、これらの少なくとも１種を混合して抗
りん脂質抗体検出用ラテックス試薬を得る。In the present invention, first, a mixture of the above-mentioned cardiolipin, phosphatidylcholine and cholesterol dissolved in the above organic solvent is stirred individually with latex particles suspended in an aqueous solvent to obtain a latex. Immobilizing on particles, an aqueous suspension of each phospholipid-immobilized latex is prepared. In the present invention, at least one of these is then mixed to obtain a latex reagent for detecting antiphospholipid antibody.
【００１３】本発明の抗りん脂質抗体検出用ラテックス
試薬は、通常の緩衝液に保存することができる。上記緩
衝液としては、りん酸等を成分とする既知のものを使用
することができる。また、上記緩衝液のｐＨ及びイオン
強度は、通常の生理学的な条件のものを使用することが
できる。本発明の抗りん脂質抗体検出用ラテックス試薬
を用いる場合には、非特異反応を抑制するとされている
既知物質、抗原抗体反応を促進するとされている既知物
質等を併用することができる。The latex reagent for detecting antiphospholipid antibody of the present invention can be stored in an ordinary buffer solution. As the buffer solution, a known buffer solution containing phosphoric acid or the like can be used. The pH and ionic strength of the buffer solution may be those under normal physiological conditions. When the latex reagent for detecting antiphospholipid antibody of the present invention is used, a known substance that suppresses nonspecific reaction, a known substance that promotes antigen-antibody reaction, and the like can be used in combination.
【００１４】本発明の抗りん脂質抗体検出用ラテックス
試薬は、懸濁液状態で長期間安定に保存することがで
き、また、凍結乾燥状態で保存することもできる。その
際、凍結乾燥時の安定剤として使用されている一般的な
薬剤を併用することができる。本発明の抗りん脂質抗体
検出用ラテックス試薬の凝集度を検出する手段として、
目視で判定する方法、反応液の吸光度の変化を機械によ
り測定する方法等を利用することができる。The latex reagent for detecting antiphospholipid antibody of the present invention can be stably stored in a suspension state for a long period of time, or can be stored in a freeze-dried state. At that time, a general drug used as a stabilizer during freeze-drying can be used in combination. As means for detecting the degree of aggregation of the latex reagent for detecting antiphospholipid antibody of the present invention,
A method of visually determining, a method of mechanically measuring a change in the absorbance of the reaction solution, or the like can be used.
【００１５】[0015]
【作用】りん脂質は、一分子中に親水基と疎水基を有す
る両親媒性分子であり、平均分子量が８００と小さく、
スペーサー等を介して固定化しなければ抗原としての反
応性が得られないとされていた。しかし、本発明は、そ
れぞれのりん脂質のラテックス表面への吸着力に差があ
ることから、それぞれのりん脂質をポリスチレンラテッ
クス上に個別に固定化させてラテックス水性懸濁液を調
製し、その後それぞれのラテックス水性懸濁液を混合さ
せることにより、ロット間差も少なく、安定した性能を
示す抗りん脂質抗体検出用ラテックス試薬を得ることが
できる。[Function] Phospholipid is an amphipathic molecule having a hydrophilic group and a hydrophobic group in one molecule, and has a small average molecular weight of 800,
It was considered that the antigenic reactivity could not be obtained unless it was immobilized via a spacer or the like. However, the present invention is different in the adsorptivity of each phospholipid to the latex surface, so that each phospholipid is individually immobilized on polystyrene latex to prepare a latex aqueous suspension, and then each is prepared. By mixing the latex aqueous suspension described in (1) above, a latex reagent for detecting antiphospholipid antibody showing stable performance with little difference between lots can be obtained.
【００１６】[0016]
【実施例】以下に実施例を掲げて本発明を更に詳しく説
明するが、本発明はこれら実施例のみに限定されるもの
ではない。The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.
【００１７】実施例１ （１）試薬及び血清の調製 （ａ）１０％（Ｗ／Ｖ）ポリスチレンラテックス液 ポリスチレンラテックス（積水化学工業社製、固形分１
０％（Ｗ／Ｖ）、平均粒径０．４００μｍ）をそのまま
用いた。 （ｂ）抗原液 カルジオライピンのエタノール溶液（シグマ社製、試薬
特級、５ｍｇ／ｍｌ）、フォスファチジルコリン（ナカ
ライテスク社製、試薬特級）をエタノール（ナカライテ
スク社製、試薬特級、９９％）に溶解し、１０ｍｇ／ｍ
ｌとしたもの、及び、コレステロール（ナカライテスク
社製、試薬特級）を同じく１０ｍｇ／ｍｌエタノール溶
液にしたものを用いた。Example 1 (1) Preparation of Reagent and Serum (a) 10% (W / V) Polystyrene Latex Solution Polystyrene latex (Sekisui Chemical Co., Ltd., solid content 1
0% (W / V), average particle size 0.400 μm) was used as it was. (B) Antigen solution An ethanol solution of cardiolipin (Sigma, special reagent, 5 mg / ml) and phosphatidylcholine (Nacalai Tesque, special reagent) were added to ethanol (Nacalai Tesque, special reagent, 99%). Dissolves, 10 mg / m
1 was used, and cholesterol (manufactured by Nacalai Tesque, Inc., reagent grade) was also used in a 10 mg / ml ethanol solution.
【００１８】（ｃ）ブロッキング用緩衝液 １００ｍＭりん酸水素ナトリウム（１２水和物）及び１
００ｍＭりん酸水素ナトリウム（２水和物）を混合して
ｐＨを７．４０に調整したりん酸緩衝液（以下１００ｍ
Ｍりん酸緩衝液という）に、牛血清アルブミン（Ｂｏｖ
ｉｎｅ ｓｅｒｕｍ ａｌｂｕｍｉｎ、Ｍｉｌｅｓ社
製、Ｆｒａｃｔｉｏｎ Ｖ、試薬特級、以下「ＢＳＡ」
という）を１％（Ｗ／Ｖ）、アジ化ナトリウム（ナカラ
イテスク社製、試薬特級）を０．１％（Ｗ／Ｖ）になる
ようにしたものをブロッキング用緩衝液とした。(C) Blocking buffer 100 mM sodium hydrogen phosphate (12 hydrate) and 1
Phosphate buffer solution adjusted to pH 7.40 by mixing 00 mM sodium hydrogen phosphate (dihydrate) (hereinafter 100 m
Bovine serum albumin (Bov)
ine serum albumin, manufactured by Miles, Fraction V, reagent special grade, hereinafter "BSA"
1% (W / V) and sodium azide (Nacalai Tesque, Inc., reagent grade) at 0.1% (W / V) were used as blocking buffers.
【００１９】（ｄ）ラテックス保存用緩衝液 １００ｍＭりん酸緩衝液にＢＳＡを１％（Ｗ／Ｖ）、ア
ジ化ナトリウム（ナカライテスク社製、試薬特級）０．
１％（Ｗ／Ｖ）、ＥＤＴＡ（ナカライテスク社製、試薬
特級）を１０ｍＭ、塩化コリン（ナカライテスク社製、
試薬、特級）を５００ｍＭになるようにしたものをラテ
ックス保存液用緩衝液とした。 （ｅ）検体希釈用緩衝液 １００ｍＭりん酸緩衝液にグリコシルエチルメタクリレ
ートのホモポリマー（日本精化社製、平均分子量２７
万）を１％（Ｗ／Ｖ）、ＢＳＡを０．２５％（Ｗ／
Ｖ）、アジ化ナトリウムを０．１％（Ｗ／Ｖ）になるよ
うにしたものを検体希釈用緩衝液とした。(D) Latex storage buffer 1% BSA (W / V) in 100 mM phosphate buffer, sodium azide (Nacalai Tesque, special grade reagent)
1% (W / V), EDTA (Nacalai Tesque, special grade reagent) 10 mM, choline chloride (Nacalai Tesque,
The buffer solution for the latex storage solution was prepared by adjusting the amount of the reagent (special grade) to 500 mM. (E) Buffer for sample dilution Homopolymer of glycosyl ethyl methacrylate in 100 mM phosphate buffer (manufactured by Nippon Seika Co., Ltd., average molecular weight 27
10,000) for 1% (W / V) and BSA for 0.25% (W / V)
V) and sodium azide adjusted to 0.1% (W / V) were used as the sample dilution buffer.
【００２０】（ｆ）梅毒陰性血清 正常家兎より採取した血清で、ＲＰＲカードテスト法
（化学及び血清療法研究所社製）及びセロディアＴＰＨ
Ａキット（富士レビオ社製）の両方により陰性と判定さ
れたものを用いた。 （ｇ）梅毒陽性血清 実験的に梅毒菌を接種し、梅毒にした家兎より採取した
血清で、ＲＰＲカードテスト法（化学及び血清療法研究
所社製）及びセロディアＴＰＨＡキット（富士レビオ社
製）の両方により陽性と判定されたものを用いた。(F) Syphilis-negative serum Serum collected from normal rabbits, using the RPR card test method (Chemical and Serum Therapy Research Institute) and Cerrodia TPH
A kit that was determined to be negative by both A kits (manufactured by Fujirebio) was used. (G) Syphilis-positive serum Serum collected from rabbits that had been experimentally inoculated with syphilis and had syphilis, RPR card test method (Chemical and Serum Therapy Research Laboratories) and Cellodia TPHA kit (Fujirebio) Those that were determined to be positive by both of the above were used.
【００２１】（２）試薬の調製 １０％（Ｗ／Ｖ）ポリスチレンラテックス液０．１ｍｌ
をポリカーボネートチューブ中で攪拌させながら、カル
ジオライピンエタノール溶液０．０３３ｍｌを添加し引
き続き室温で１時間攪拌した後、ブロッキング用緩衝液
２．０ｍｌを一気に添加し、１．５時間、室温で攪拌し
た。次に、高速冷却遠心機（日立ＨＲ２６型）で、１９
０００×ｇ、１０℃で３０分遠心洗浄を行った。得られ
たラテックスの沈渣にラテックス保存用緩衝液５．０ｍ
ｌを添加し、タッチミキサーにてよく攪拌した後、高速
冷却遠心機で、１９０００×ｇ、１０℃で３０分遠心洗
浄を行った。この洗浄操作を３回繰り返した。最終的に
得られた沈渣にラテックス保存用緩衝液２．０ｍｌを添
加し、タッチミキサーにてよく攪拌し、超音波破砕機
（アストラソン社製、マイクロチップ、出力目盛３、５
０％サイクル）にて氷浴中、１分間ソニケートし、分散
させた。この後、更にラテックス保存用緩衝液６．０ｍ
ｌを添加し、タッチミキサーで充分混合させた後、固形
分０．１２５％（Ｗ／Ｖ）のラテックス懸濁液として４
℃にて保存した。(2) Preparation of reagent 0.1% of 10% (W / V) polystyrene latex solution
While stirring in a polycarbonate tube, add 0.033 ml of a cardiolipin ethanol solution and subsequently stir at room temperature for 1 hour, then add 2.0 ml of blocking buffer at once and stir at room temperature for 1.5 hours. did. Next, with a high-speed cooling centrifuge (Hitachi HR26 type),
Centrifugal washing was performed at 000 xg and 10 ° C for 30 minutes. 5.0m of buffer solution for latex storage in the obtained latex sediment
1 was added and well stirred with a touch mixer, followed by centrifugal washing with a high speed cooling centrifuge at 19000 × g and 10 ° C. for 30 minutes. This washing operation was repeated 3 times. To the finally obtained precipitate, 2.0 ml of a latex storage buffer was added, and the mixture was thoroughly stirred with a touch mixer, and then ultrasonically disrupted (manufactured by Astrason, Microchip, output scale 3, 5).
0% cycle) and sonicate for 1 minute in an ice bath to disperse. After this, a further latex storage buffer of 6.0 m
1 was added and thoroughly mixed with a touch mixer, and then a latex suspension having a solid content of 0.125% (W / V) was prepared.
Stored at ° C.
【００２２】上記抗原液をフォスファチジルコリンエタ
ノール溶液０．１６７ｍｌ、コレステロールエタノール
溶液０．０５０ｍｌに代えた他は全く同じ条件でそれぞ
れの脂質固定化ラテックス懸濁液を調製した。このカル
ジオライピン、フォスファチジルコリン及びコレステロ
ール固定化ラテックス（固形分０．１２５％（Ｗ／
Ｖ））をそれぞれ２ｍｌ、１０ｍｌ及び３ｍｌとり、よ
く混合した。以上の操作を５回繰り返し行い、それぞれ
ロット１〜５の計５ロットの抗りん脂質抗体検出用ラテ
ックス試薬を得た。Each lipid-immobilized latex suspension was prepared under exactly the same conditions except that the antigen solution was replaced with 0.167 ml of a phosphatidylcholine ethanol solution and 0.050 ml of a cholesterol ethanol solution. This cardiolipin, phosphatidylcholine and cholesterol-immobilized latex (solid content 0.125% (W /
V)) was taken in 2 ml, 10 ml and 3 ml respectively and mixed well. The above operation was repeated 5 times to obtain a total of 5 lots of latex reagents for detecting antiphospholipid antibody, that is, lots 1 to 5, respectively.
【００２３】（３）検体の調製 （１）で得られた梅毒家兎血清のうち陽性検体として、
ＲＰＲ法で８倍を示したものを、生理食塩水（０．９％
塩化ナトリウム水溶液）にて、２倍及び８倍希釈し、４
倍及び１倍を示す血清を調製した。また、陰性検体とし
て正常家兎血清をそのまま用いた。(3) Preparation of specimen As a positive specimen of the syphilis rabbit serum obtained in (1),
What showed 8 times by RPR method was physiological saline (0.9%
2 times and 8 times diluted with an aqueous solution of sodium chloride, 4
Serums showing 2-fold and 1-fold were prepared. In addition, normal rabbit serum was used as it was as a negative sample.
【００２４】（４）自動分析装置による検体の測定 全自動生化学分析装置 日立７１５０形（日立製作所社
製）により、下記のようにして、各ロットのラテックス
試薬を用いて検体中の抗りん脂質抗体を測定した。 測定モード；Ｏｒｉｇｉｎａｌ Ａｂｓ パラメーター；検体量 ２０μｌ ラテックス試薬量 ５０μｌ 検体希釈用緩衝液量 ３５０μｌ 測定波長；５７０ｎｍ 測定時間；検体分注後、ただちに検体希釈用緩衝液量を
添加、混合し、その後、ラテックス試薬を添加、混合し
た。ラテックス試薬の添加後８０〜３２０秒後の吸光度
の変化量を求め、これを反応量とした。 検体；（３）で調製した梅毒陽性血清梅毒陰性血清をそ
れぞれｎ＝５で測定した。(4) Measurement of Specimen by Automatic Analyzer An anti-phospholipid in the specimen is measured by using a latex reagent of each lot by a fully automatic biochemical analyzer Hitachi 7150 (manufactured by Hitachi Ltd.) as follows. The antibody was measured. Measurement mode; Original Abs parameter; Specimen amount 20 μl Latex reagent amount 50 μl Specimen diluting buffer amount 350 μl Measurement wavelength; 570 nm Measurement time; Specimen diluting buffer amount is added and mixed immediately after sample dispensing, and then latex reagent Was added and mixed. The amount of change in the absorbance 80 to 320 seconds after the addition of the latex reagent was determined and used as the reaction amount. Specimen: Syphilis-positive serum prepared in (3) and syphilis-negative serum were measured at n = 5.
【００２５】実施例２ フォスファチジルコリンの代わりにレシチン１６ｍｇ／
ｍｌエタノール溶液を用いた以外は実施例１と同様に行
い抗りん脂質抗体検出用ラテックス試薬を得た。その測
定方法は実施例１と同様に行った。Example 2 Lecithin 16 mg / instead of phosphatidylcholine
A latex reagent for detecting antiphospholipid antibody was obtained in the same manner as in Example 1 except that a ml ethanol solution was used. The measurement method was the same as in Example 1.
【００２６】比較例１ 抗原液としてカルジオライピンエタノール溶液０．０３
３ｍｌ、フォスファチジルコリンエタノール溶液０．１
６７ｍｌ、コレステロールエタノール溶液０．０５０ｍ
ｌの混合液計０．２５ｍｌを用いた以外は実施例１と同
様に行い抗りん脂質抗体検出用ラテックス試薬を得た。
その測定方法は実施例１と同様に行った。Comparative Example 1 Ethanol solution of cardiolipin 0.03 as antigen solution
3 ml, phosphatidylcholine ethanol solution 0.1
67 ml, cholesterol ethanol solution 0.050 m
A latex reagent for detecting antiphospholipid antibody was obtained in the same manner as in Example 1 except that a total of 0.25 ml of the mixed solution of 1 was used.
The measurement method was the same as in Example 1.
【００２７】比較例２ 抗原液としてカルジオライピンエタノール溶液０．０３
３ｍｌ、レシチンエタノール溶液０．１６７ｍｌ、コレ
ステロールエタノール溶液０．０５０ｍｌの混合液計
０．２５ｍｌを用いた以外は実施例１と同様に行い抗り
ん脂質抗体検出用ラテックス試薬を得た。その測定方法
は実施例１と同様に行った。Comparative Example 2 Cardiolipin ethanol solution 0.03 as an antigen solution
A latex reagent for detecting antiphospholipid antibody was obtained in the same manner as in Example 1 except that a total of 0.25 ml of a mixed solution of 3 ml of lecithin ethanol solution 0.167 ml and cholesterol ethanol solution 0.050 ml was used. The measurement method was the same as in Example 1.
【００２８】比較例３ 使用する試薬をロットの異なるＲＰＲキット（化学及び
血清療法研究所社製）５キットを用いて同一の検体をｎ
＝１で測定した以外は実施例１と同様に行い抗りん脂質
抗体検出用ラテックス試薬を得た。その測定方法は実施
例１と同様に行った。評価１ 実施例１及び２並びに比較例１〜３で得られた各ロット
の抗りん脂質抗体検出用ラテックス試薬における検体の
吸光度変化量の平均値及びそのロット間の統計を表１及
び表２に示した。いずれもｎ＝５の平均値を示した。表
１及び表２中、ＣＶ値は、変動係数を表す。Comparative Example 3 The same sample was prepared by using 5 kits of RPR kits (Chemical and Serum Therapy Research Institute) of different lots.
A latex reagent for detecting antiphospholipid antibody was obtained in the same manner as in Example 1 except that the measurement was performed at = 1. The measurement method was the same as in Example 1. Evaluation 1 Tables 1 and 2 show the average value of the amount of change in absorbance of the samples in the latex reagents for detecting antiphospholipid antibody of each lot obtained in Examples 1 and 2 and Comparative Examples 1 to 3 and the statistics between the lots. Indicated. All showed the average value of n = 5. In Tables 1 and 2, the CV value represents a coefficient of variation.
【００２９】実施例３ 平均粒径０．１９１μｍのポリスチレンラテックス（積
水化学工業社製、固形分１０％（Ｗ／Ｖ））を使用し、
抗原液の容量をそれぞれ実施例１の４倍とした以外は実
施例１と同様に行い抗りん脂質抗体検出用ラテックス試
薬を得た。その測定方法は実施例１と同様に行った。Example 3 Polystyrene latex having an average particle size of 0.191 μm (Sekisui Chemical Co., Ltd., solid content 10% (W / V)) was used,
A latex reagent for detecting antiphospholipid antibody was obtained in the same manner as in Example 1 except that the volume of the antigen solution was set to 4 times that in Example 1. The measurement method was the same as in Example 1.
【００３０】実施例４ 平均粒径０．１９１μｍのポリスチレンラテックス（積
水化学工業社製、固形分１０％（Ｗ／Ｖ））を使用し、
抗原液の容量をそれぞれ実施例２の４倍とした以外は実
施例２と同様に行い抗りん脂質抗体検出用ラテックス試
薬を得た。その測定方法は実施例１と同様に行った。 比較例４ 添加する抗原液の順番をコレステロール、フォスファチ
ジルコリン、カルジオライピンにした以外は実施例３と
同様に行い抗りん脂質抗体検出用ラテックス試薬を得
た。その測定方法は実施例１と同様に行った。Example 4 Polystyrene latex having an average particle size of 0.191 μm (Sekisui Chemical Co., Ltd., solid content 10% (W / V)) was used.
A latex reagent for detecting antiphospholipid antibody was obtained in the same manner as in Example 2 except that the volume of the antigen solution was set to 4 times that in Example 2. The measurement method was the same as in Example 1. Comparative Example 4 A latex reagent for detecting antiphospholipid antibody was obtained in the same manner as in Example 3, except that the order of the antigen solution to be added was cholesterol, phosphatidylcholine, and cardiolipin. The measurement method was the same as in Example 1.
【００３１】比較例５ 添加する抗原液の順番をコレステロール、カルジオライ
ピン、フォスファチジルコリンにした以外は実施例４と
同様に行い抗りん脂質抗体検出用ラテックス試薬を得
た。その測定方法は実施例１と同様に行った。 比較例６ 使用する試薬をロットの異なるＲＰＲキット（化学及び
血清療法研究所社製）５キットを用いて同一の検体をｎ
＝１で測定した以外は実施例３と同様に行い抗りん脂質
抗体検出用ラテックス試薬を得た。その測定方法は実施
例１と同様に行った。Comparative Example 5 A latex reagent for detecting antiphospholipid antibody was obtained in the same manner as in Example 4, except that the order of the antigen solution to be added was cholesterol, cardiolipin and phosphatidylcholine. The measurement method was the same as in Example 1. Comparative Example 6 Using the RPR kits (manufactured by Chemistry and Serum Therapy Research Institute) 5 kits of different lots as reagents, the same sample was used.
A latex reagent for detecting antiphospholipid antibody was obtained in the same manner as in Example 3 except that the measurement was performed at = 1. The measurement method was the same as in Example 1.
【００３２】評価２ 実施例３及び４並びに比較例４〜６で得られた各ロット
の抗りん脂質抗体検出用ラテックス試薬における検体の
吸光度変化量の平均値及びそのロット間の統計を表３及
び表４に示した。いずれもｎ＝５の平均値を示した。表
３及び表４中、ＣＶ値は、変動係数を表す。 Evaluation 2 The average value of the amount of change in absorbance of the sample in the latex reagents for detecting antiphospholipid antibody of each lot obtained in Examples 3 and 4 and Comparative Examples 4 to 6 and the statistics between the lots are shown in Table 3 and. The results are shown in Table 4. All showed the average value of n = 5. In Tables 3 and 4, the CV value represents a coefficient of variation.
【００３３】[0033]
【表１】 [Table 1]
【００３４】[0034]
【表２】 [Table 2]
【００３５】[0035]
【表３】 [Table 3]
【００３６】[0036]
【表４】 [Table 4]
【００３７】[0037]
【発明の効果】本発明は、りん脂質をそれぞれ個別にラ
テックス粒子に固定化させて、それぞれのりん脂質固定
化ラテックス水性懸濁液を調製し、これらを混合するこ
とにより、ロット間差が小さく、安定した再現性を示す
抗りん脂質抗体検出用ラテックス試薬を得ることができ
る。INDUSTRIAL APPLICABILITY According to the present invention, phospholipids are individually immobilized on latex particles to prepare respective phospholipid-immobilized latex aqueous suspensions, and by mixing these, the difference between lots is reduced. Thus, it is possible to obtain a latex reagent for detecting antiphospholipid antibody which shows stable reproducibility.
Claims (1)
リン及びコレステロールをそれぞれ個別にラテックス粒
子に固定化させてそれぞれのりん脂質固定化ラテックス
水性懸濁液を調製し、その後、これらの少なくとも１種
を混合してラテックス試薬を得ることを特徴とする抗り
ん脂質抗体検出用ラテックス試薬の製造方法。1. Cardiolipin, phosphatidylcholine and cholesterol are individually immobilized on latex particles to prepare respective phospholipid-immobilized latex aqueous suspensions, and at least one of these is then mixed. A method for producing a latex reagent for detecting an antiphospholipid antibody, which comprises obtaining a latex reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP24152494A JP3439542B2 (en) | 1994-10-05 | 1994-10-05 | Method for producing reagent for measuring antiphospholipid antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP24152494A JP3439542B2 (en) | 1994-10-05 | 1994-10-05 | Method for producing reagent for measuring antiphospholipid antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH08105890A true JPH08105890A (en) | 1996-04-23 |
| JP3439542B2 JP3439542B2 (en) | 2003-08-25 |
Family
ID=17075632
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP24152494A Expired - Lifetime JP3439542B2 (en) | 1994-10-05 | 1994-10-05 | Method for producing reagent for measuring antiphospholipid antibody |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3439542B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002365297A (en) * | 2001-06-08 | 2002-12-18 | Sekisui Chem Co Ltd | Reagent for measuring anti-phosphatide antibody |
| JP2010249846A (en) * | 2008-11-12 | 2010-11-04 | Sekisui Medical Co Ltd | Insoluble carrier for use in anti-phospholipid antibody measurement reagent, anti-phospholipid antibody measurement reagent, and method for measuring anti-phospholipid antibody |
-
1994
- 1994-10-05 JP JP24152494A patent/JP3439542B2/en not_active Expired - Lifetime
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002365297A (en) * | 2001-06-08 | 2002-12-18 | Sekisui Chem Co Ltd | Reagent for measuring anti-phosphatide antibody |
| JP2010249846A (en) * | 2008-11-12 | 2010-11-04 | Sekisui Medical Co Ltd | Insoluble carrier for use in anti-phospholipid antibody measurement reagent, anti-phospholipid antibody measurement reagent, and method for measuring anti-phospholipid antibody |
| EP2352029A1 (en) * | 2008-11-12 | 2011-08-03 | Sekisui Medical Co., Ltd. | Insoluble carrier for use in anti-phospholipid antibody measurement reagent, anti-phospholipid antibody measurement reagent, and method for measuring anti-phospholipid antibody |
| KR20110093763A (en) * | 2008-11-12 | 2011-08-18 | 세키스이 메디칼 가부시키가이샤 | Insoluble carrier for use in anti-phospholipid antibody measurement reagent, anti-phospholipid antibody measurement reagent, and method for measuring anti-phospholipid antibody |
| CN102203612A (en) * | 2008-11-12 | 2011-09-28 | 积水医疗株式会社 | Insoluble carrier for use in anti-phospholipid antibody measurement reagent, anti-phospholipid antibody measurement reagent, and method for measuring anti-phospholipid antibody |
| EP2352029A4 (en) * | 2008-11-12 | 2012-05-16 | Sekisui Medical Co Ltd | Insoluble carrier for use in anti-phospholipid antibody measurement reagent, anti-phospholipid antibody measurement reagent, and method for measuring anti-phospholipid antibody |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3439542B2 (en) | 2003-08-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0070527B1 (en) | Method of assaying biologically active substances and labelling agents therefor | |
| US20140363899A1 (en) | Method for avoiding influence of endogenous lipoprotein and reagent | |
| TWI757328B (en) | Antibody assay method using antigen-immobilized insoluble-carrying particles with antigen immobilized in different ways, reagent for antibody assay | |
| JPS5838862A (en) | Method of examining auto-blocking antibody and reagent kit used for said method | |
| JP3439542B2 (en) | Method for producing reagent for measuring antiphospholipid antibody | |
| JP3786543B2 (en) | Immunological reagent | |
| JP3328058B2 (en) | Method for producing immunodiagnostic drug | |
| US20180074052A1 (en) | Method for stabilizing glycerophospholipids and reagents using same | |
| JP3444649B2 (en) | Immunoassay reagent and method for producing the same | |
| JP3618797B2 (en) | Immunoassay | |
| JPH10282096A (en) | Method and reagent for measuring anti-phospholipid antibody | |
| JP3290520B2 (en) | Antiphospholipid antibody assay | |
| JPH09304386A (en) | Manufacture of immunity diagnostic drug and immunity diagnostic drug obtained | |
| JP3359411B2 (en) | Method for producing reagent for measuring antiphospholipid antibody | |
| JPH11258236A (en) | Reagent for measuring anti phospholipid antibody | |
| JP3403520B2 (en) | Method for producing reagent for measuring antiphospholipid antibody | |
| JP3487651B2 (en) | Method for producing immunodiagnostic reagent | |
| JP2004117068A (en) | Immunoassay reagent and immunoassay method | |
| JPH07113641B2 (en) | Method for manufacturing syphilis diagnostic reagent | |
| JPH11118800A (en) | Production of reagent for measuring antiphospholipid antibody | |
| JPH09304388A (en) | Quantitative method for human immunoglobulin e and measuring kit | |
| JPH05312808A (en) | Immunity examination method using lipidal antigen | |
| JPH10132821A (en) | Antiphosphorus lipid antibody measuring immune agglutination reagent and measuring method for antiphosphorus lipid antibody | |
| JPH10239315A (en) | Method for manufacturing anti-phospholipid antibody measuring reagent and reagent | |
| JP2000009731A (en) | Anti-phospholipid antibody measuring reagent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FPAY | Renewal fee payment (prs date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20090613 Year of fee payment: 6 |
|
| FPAY | Renewal fee payment (prs date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20100613 Year of fee payment: 7 |
|
| FPAY | Renewal fee payment (prs date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20100613 Year of fee payment: 7 |
|
| FPAY | Renewal fee payment (prs date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20110613 Year of fee payment: 8 |
|
| FPAY | Renewal fee payment (prs date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20110613 Year of fee payment: 8 |
|
| FPAY | Renewal fee payment (prs date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20120613 Year of fee payment: 9 |
|
| FPAY | Renewal fee payment (prs date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20120613 Year of fee payment: 9 |
|
| FPAY | Renewal fee payment (prs date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20130613 Year of fee payment: 10 |
|
| EXPY | Cancellation because of completion of term |