JP3350762B2 - Immunological diagnostics - Google Patents
Immunological diagnosticsInfo
- Publication number
- JP3350762B2 JP3350762B2 JP25346792A JP25346792A JP3350762B2 JP 3350762 B2 JP3350762 B2 JP 3350762B2 JP 25346792 A JP25346792 A JP 25346792A JP 25346792 A JP25346792 A JP 25346792A JP 3350762 B2 JP3350762 B2 JP 3350762B2
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- acid
- phosphate buffer
- reaction
- antigen
- 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.)
- Expired - Fee Related
Links
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Description
【0001】[0001]
【産業上の利用分野】本発明は、抗原抗体反応に基づく
高感度かつ再現性に優れ、またランニングコスト等にも
優れた新規な免疫学的診断薬に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel immunological diagnostic agent having high sensitivity and excellent reproducibility based on an antigen-antibody reaction, and also excellent in running costs and the like.
【0002】[0002]
【従来の技術】体外診断薬は、その検出・定量法によっ
てRI法、化学発光法、酵素法、ラテックス法などに分
類される。これらのうち、RI法は放射活性元素でラベ
ル化した抗体(抗原)を用いることにより、また化学発
光法とは、特開昭63− 57572号公報、特開昭63−101368
号公報などに開示されているようにアクリジン等の化学
発光物質によりラベル化した抗体(抗原) により、また
酵素法は特開昭63−141514号公報、特開昭60− 71958号
公報などに開示されているように酵素/基質による発光
により、検出・定量を行なうものである。これらはサン
ドイッチ法などの高度な免疫学的手法を組み合わすこと
により、ng/ml〜pg/mlの超微量定量が可能である。2. Description of the Related Art In vitro diagnostic agents are classified into RI method, chemiluminescent method, enzymatic method, latex method and the like according to their detection and quantification methods. Among them, the RI method uses an antibody (antigen) labeled with a radioactive element, and the chemiluminescence method is described in JP-A-63-57572 and JP-A-63-101368.
JP-A-63-141514 and JP-A-60-71958 disclose an antibody (antigen) labeled with a chemiluminescent substance such as acridine as disclosed in Japanese Unexamined Patent Publication No. As described above, detection and quantification are performed by luminescence from an enzyme / substrate. These can be combined with advanced immunological techniques such as a sandwich method to enable ultra-trace quantification of ng / ml to pg / ml.
【0003】一方、ラテックス法は、抗体(抗原)を固
定化した粒径サブミクロンオーダーのラテックスを用
い、これと検体中の抗原(抗体)との抗原抗体反応の結
果、生じるラテックスの凝集反応を検定するものであ
り、一般にmg/ml〜μg/mlの検出感度を有する。この
手法では、検定手段としてスライドガラス上で凝集反応
を起こし、これを目視により判定を行なうスライド法
や、また凝集反応の程度を透過光濁度、散乱光濁度、積
分級濁度として光学的に定量化することが可能であり、
簡便性、迅速性に優れた方法である。また、この際問題
となる非特異的凝集反応を抑制する手段として、特公昭
57− 29039号公報及び特公平2− 14661号公報には、抗
体を 2,4−ジニトロフェニル基あるいは疎水基により修
飾し、ラテックス微粒子への物理的吸着能を高める試み
が知られている。[0003] On the other hand, the latex method uses a latex having a submicron order particle size on which an antibody (antigen) is immobilized, and a latex agglutination reaction resulting from an antigen-antibody reaction between the latex and an antigen (antibody) in a sample is performed. And generally has a detection sensitivity of mg / ml to μg / ml. In this method, an agglutination reaction occurs on a slide glass as an assay means, and a slide method in which the agglutination reaction is visually determined, or the degree of the agglutination reaction is optically determined as transmitted light turbidity, scattered light turbidity, or integrated class turbidity. It is possible to quantify
This method is simple and quick. In addition, as a means for suppressing non-specific agglutination reactions,
JP-A-57-29039 and JP-B-2-14661 discloses an attempt to modify an antibody with a 2,4-dinitrophenyl group or a hydrophobic group to increase the physical adsorption ability to latex fine particles.
【0004】[0004]
【発明が解決しようとする課題】RI法、化学発光法、
酵素法では高感度が得られるものの、他方では、大がか
りな設備あるいは装置が必要であり、またランニングコ
ストが高く、さらに前処理から測定までに長時間を要す
るなどの問題点がある。またラテックス法では、簡便か
つ迅速な診断が可能ではあるが、ラテックスが高価であ
り、ランニングコストが高いという問題点がある。SUMMARY OF THE INVENTION The RI method, the chemiluminescence method,
Although high sensitivity can be obtained with the enzymatic method, on the other hand, there are problems such as the necessity of large-scale equipment or equipment, high running cost, and a long time from pretreatment to measurement. The latex method allows simple and quick diagnosis, but latex is expensive.
And the running cost is high .
【0005】本発明の目的は、濁度の発生を検定するシ
ステムにおいて、従来のラテックス等の担体を使用する
ことなく、高感度で、簡便かつ迅速な検定が可能な免疫
学的診断薬を提供することである。[0005] An object of the present invention is to provide an immunological diagnostic agent capable of performing a highly sensitive, simple and rapid assay without using a conventional carrier such as latex in a system for assaying the generation of turbidity. It is to be.
【0006】[0006]
【課題を解決するための手段】本発明者らは鋭意研究の
結果、ラテックス等の担体は使用せず、抗体自身を化学
的に疎水化修飾することにより、抗体の親水/疎水バラ
ンスを最適化し、抗体抗原反応時に検出に好都合な濁度
を起こし易い物質を調製することによって、より高感度
かつ再現性に優れた診断薬となしうることを見出し、本
発明を完成させるに至った。Means for Solving the Problems As a result of intensive studies, the present inventors have optimized the hydrophilic / hydrophobic balance of an antibody by chemically modifying the antibody itself without using a carrier such as latex. The present inventors have found that by preparing a substance which easily causes turbidity which is convenient for detection during an antibody-antigen reaction, a diagnostic agent having higher sensitivity and excellent reproducibility can be obtained, and the present invention has been completed.
【0007】以下本発明をさらに詳細に説明する。Hereinafter, the present invention will be described in more detail.
【0008】すなわち、本発明の課題を解決するための
手段は、炭素原子数2〜24個のアシル基で修飾された抗
体を、濁度発生成分として含有することを特徴とする免
疫学的診断薬を提供することである。[0008] That is, a means for solving the problem of the present invention is to provide an immunodiagnostic method characterized by containing an antibody modified with an acyl group having 2 to 24 carbon atoms as a turbidity generating component. To provide medicine.
【0009】本発明において、抗体を修飾させて疎水性
を付与する化合物(以下、「疎水性化合物」と略称す
る)としては、脂肪酸、特に炭素原子数が2〜24個の脂
肪酸が好ましい。しかして、特に好ましいものは、炭素
原子数2〜18個の脂肪酸である。炭素原子数が25個以上
の脂肪酸を使用すると、修飾された抗体同士の相互間の
凝集が顕著に強くなるため好ましくない。In the present invention, as the compound that imparts hydrophobicity by modifying an antibody (hereinafter abbreviated as “hydrophobic compound”), a fatty acid, particularly a fatty acid having 2 to 24 carbon atoms is preferable. Thus, particularly preferred are fatty acids having 2 to 18 carbon atoms. It is not preferable to use a fatty acid having 25 or more carbon atoms, since the aggregation between the modified antibodies becomes remarkably strong.
【0010】脂肪酸のうち、例えば、飽和直鎖脂肪酸
(脂肪族モノカルボン酸)として具体的には、酢酸、プ
ロピオン酸、酪酸、吉草酸、カプロン酸、エナント酸、
カプリル酸、ペラルゴン酸、カプリン酸、ウンデカン
酸、ラウリン酸、トリデカン酸、ミリスチン酸、ペンタ
デカン酸、パルミチン酸、マルガリン酸、ステアリン
酸、ノンデカン酸、アラキン酸などを挙げることができ
る。これらの脂肪酸を使用することにより、一価飽和直
鎖アシル基として、例えば、アセチル、プロピニオニ
ル、ブチリル、バレリル、ヘキサノイル、ヘプタノイ
ル、オクタノイル、ノナノイル、デカノイルなどが抗体
に修飾される。Among the fatty acids, specific examples of the saturated linear fatty acid (aliphatic monocarboxylic acid) include acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, and the like.
Examples thereof include caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nondecanoic acid, and arachiic acid. By using these fatty acids, for example, acetyl, propionyl, butyryl, valeryl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl and the like are modified into monovalent saturated linear acyl groups.
【0011】上記疎水性化合物を抗体中に導入して疏水
性修飾抗体を製造するのには、疎水性化合物と抗体とを
直接反応させる方法と、疎水性化合物をいったん活性化
してから抗体と反応させる方法とが用いられる。In order to produce a hydrophobically modified antibody by introducing the above-mentioned hydrophobic compound into an antibody, a method of directly reacting the antibody with the hydrophobic compound or a method of reacting the antibody with the antibody after activating the hydrophobic compound once is used. And a method of causing it to be used.
【0012】 前者では、脱水縮合剤として、BOP試
薬(和光純薬社製)、1,1’−カルボニルジイミダゾ
ール、N−シクロヘキシル−N’−(2−モルホルノエ
チル)カルボジイミド・メソ−p−トルエンスルホネー
ト、ジシクロヘキシルカルボジイミド、シアノリン酸ジ
エチル、N−エトキシカルボニル−2−エトキシ−1,
2−ジヒドロキノリン、1−エチル−3−(3−ジメチ
ルアミノプロピル)カルボジイミド塩酸塩などが好まし
く使用される。In the former, a BOP reagent (manufactured by Wako Pure Chemical Industries, Ltd.), 1,1′-carbonyldiimidazole, N-cyclohexyl-N ′-(2-morphformoethyl) carbodiimide.meso - p-toluenesulfonate are used as a dehydrating condensing agent. , Dicyclohexylcarbodiimide, diethyl cyanophosphate, N-ethoxycarbonyl-2-ethoxy-1,
2-Dihydroquinoline, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and the like are preferably used.
【0013】また後者では、疎水性基中のカルボキシル
基を酸クロライド化または酸無水物化したもの、あるい
は、活性化試薬として、炭酸N,N'−ジスクシンイミジ
ル、1−ヒドロキシベンゾトリアゾール、N−ヒドロキ
シ−5−ノルボルネン−2,3 −ジカルボキシイミド、N
−ヒドロキシコハク酸イミド、N−ヒドロキシフタルイ
ミド、クロロギ酸イソブチル、4−ヒドロキシフェニル
ジメチルスルホニウムメチルサルフェート等を用い、活
性化エステル化したものが好ましく使用される。また、
ホルムアルデヒドやアセトアルデヒド等の脂肪族アルデ
ヒド類を抗体中のアミノ基に作用させ、さらに水素化ホ
ウ素ナトリウム等により還元することによりN−アルキ
ル化する方法なども用いられる。In the latter, a carboxyl group in a hydrophobic group is converted to an acid chloride or an acid anhydride, or N, N'-disuccinimidyl carbonate, 1-hydroxybenzotriazole, -Hydroxy-5-norbornene-2,3-dicarboximide, N
Activated esterification using -hydroxysuccinimide, N-hydroxyphthalimide, isobutyl chloroformate, 4-hydroxyphenyldimethylsulfonium methyl sulfate or the like is preferably used. Also,
A method in which an aliphatic aldehyde such as formaldehyde or acetaldehyde is allowed to act on an amino group in the antibody, and further reduced with sodium borohydride or the like to perform N-alkylation is also used.
【0014】本発明において使用する抗体は、任意のも
のを使用すべく、しかして、例えばウサギに免疫して得
られた抗ヒトCRP抗体、ヤギに免疫して得られた抗ヒ
トCRP抗体を挙げることができる。As the antibody used in the present invention, any antibody may be used, and examples thereof include an anti-human CRP antibody obtained by immunizing a rabbit and an anti-human CRP antibody obtained by immunizing a goat. be able to.
【0015】抗体1モルに対する疎水性化合物の反応仕
込み時のモル比は、特に限定はされないが、1:0.01〜
1:10000 、好ましくは1:1〜1:1000である。この
場合、0.01以下では期待される効果が得られず、また10
000 以上では修飾抗体自身のみで沈殿を生じてしまうた
め好ましくない。The molar ratio of the hydrophobic compound to 1 mol of the antibody at the time of preparing the reaction is not particularly limited.
The ratio is 1: 10000, preferably 1: 1 to 1: 1000. In this case, if the value is less than 0.01, the expected effect cannot be obtained.
A molecular weight of 000 or more is not preferred because precipitation occurs only with the modified antibody itself.
【0016】これらの疎水性化合物の抗体中への導入反
応は、抗体の失活を最低限に抑えるために、水あるいは
緩衝液中での反応が最も好ましく、0〜80℃、好ましく
は4〜40℃で10分〜72時間、好ましくは1時間〜24時間
反応させることにより行なわれる。The reaction for introducing these hydrophobic compounds into the antibody is most preferably performed in water or a buffer solution to minimize the inactivation of the antibody, and is carried out at 0 to 80 ° C., preferably at 4 to 80 ° C. The reaction is carried out at 40 ° C. for 10 minutes to 72 hours, preferably for 1 hour to 24 hours.
【0017】また水または緩衝液以外の溶媒としてジメ
チルホルムアミド、ジメチルアセトアミド、ジメチルス
ルホキシド、テトラヒドロフラン、1,4 −ジオキサン等
の有機溶媒およびこれらと水あるいは緩衝液との混合溶
媒なども使用できる。Organic solvents such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, tetrahydrofuran and 1,4-dioxane, and mixed solvents of these with water or a buffer can also be used as solvents other than water or the buffer.
【0018】得られた疎水性修飾抗体は、そのままで、
あるいはさらに透析、限外濾過、ゲル濾過、吸着剤処
理、抽出等により精製した後に、本発明の診断薬の組成
分として用いられる。この際、疎水性修飾抗体は、pH4.
5 〜8.5 の水溶液あるいは緩衝液、またはジメチルホル
ムアミド、ジメチルアセトアミド、ジメチルスルホキシ
ド、テトラヒドロフラン、1,4 −ジオキサン等の有機溶
媒およびこれらと水あるいは緩衝液との混合溶媒中に均
一に溶解、あるいは分散させて使用される。また添加物
としてポリエチレングリコール、ポリビニルアルコー
ル、デキストラン等の水溶性高分子およびドデシルベン
ゼンスルホン酸ソーダ、トライトンX等のアニオン系、
カチオン系、ノニオン系の種々の界面活性剤等を使用す
ることもできる。The resulting hydrophobically modified antibody is
Alternatively, after further purification by dialysis, ultrafiltration, gel filtration, adsorbent treatment, extraction, etc., it is used as a component of the diagnostic agent of the present invention. At this time, the hydrophobic modified antibody has a pH of 4.
5 to 8.5 aqueous solution or buffer, or an organic solvent such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, tetrahydrofuran, 1,4-dioxane, or a mixed solvent of these and water or a buffer. Used. As additives, water-soluble polymers such as polyethylene glycol, polyvinyl alcohol and dextran and anionics such as sodium dodecylbenzenesulfonate and Triton X;
Various cationic and nonionic surfactants can also be used.
【0019】免疫学的診断薬における修飾抗体の濃度範
囲については、使用する抗体により異なるため特に限定
されないが、測定系の最終濃度として10 −4 〜10 4
mg/ml、特に好ましくは10 −1 〜10 2 mg/m
lである。10 −4 mg/ml未満では十分な感度が得
られず、また10 4 mg/mlより高い濃度では、修飾
抗体同士の相互間の凝集が起こり易くなるため好ましく
ない。The concentration range of the modified antibody in the immunological diagnostic agent is not particularly limited because it varies depending on the antibody used, but the final concentration of the measurement system is 10 -4 to 10 4.
mg / ml, particularly preferably 10 -1 ~10 2 mg / m
l. If the concentration is less than 10 −4 mg / ml, sufficient sensitivity cannot be obtained, and if the concentration is higher than 10 4 mg / ml, the modified antibodies are likely to aggregate with each other, which is not preferable.
【0020】本発明の診断薬は、該疎水性修飾抗体が均
一に溶解あるいは分散した溶液中に検体を加えた際に起
こる抗原抗体反応により生起される濁度発生により検定
を行なうものである。この際、凝集反応の程度を凝集比
濁法、積分級濁度法、粒度分布法、比ろう法などにより
光学的に定量化することが可能である。また、光源とし
てキセノンランプ、水素・重水素ランプ、水銀ランプ、
タングステンランプ、タングステン−ハロゲンランプ、
ヘリウムランプ、クリプトンランプ、各種レーザー、発
光ダイオードなどを使用し、340nm 以上、特に好ましく
は、340 〜700nm あるいは 800〜2400nmの波長を用いて
測定される。The diagnostic agent of the present invention performs an assay by generating turbidity caused by an antigen-antibody reaction that occurs when a sample is added to a solution in which the hydrophobic modified antibody is uniformly dissolved or dispersed. At this time, the extent of the agglutination reaction can be optically quantified by an agglutination turbidimetric method, an integral class turbidity method, a particle size distribution method, a turbidimetric method, or the like. Xenon lamps, hydrogen / deuterium lamps, mercury lamps,
Tungsten lamp, tungsten-halogen lamp,
It is measured using a helium lamp, a krypton lamp, various lasers, a light emitting diode, or the like, and a wavelength of 340 nm or more, particularly preferably 340 to 700 nm or 800 to 2400 nm.
【0021】[0021]
【発明による効果】本発明により、高感度かつ再現性に
優れ、さらに迅速な検定が可能な新規な診断薬が提供さ
れる。According to the present invention, a novel diagnostic agent having high sensitivity and excellent reproducibility and capable of performing a rapid assay is provided.
【0022】[0022]
【実施例】以下、実施例によりさらに具体的に説明を行
う。ただし、本発明は、これらの実施例のみに限定され
るものではない。The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to only these examples.
【0023】[実施例1]ウサギに免疫して得られた抗
ヒトCRP抗体を0.1Mのリン酸緩衝液(pH=8.0)にて50
μg /mlに調整した。このうちの1mlに、酢酸ナトリウ
ム1gを添加し溶解させた後、さらに無水酢酸2μlを
加え、4℃で一晩反応させた。反応終了後、0.01M りん
酸緩衝液(pH=7.4)中、4℃で一晩透析を行い、酢酸を
除去し、さらにフィルター(0.45μm)により不溶物を
濾過除去し、アセチル化抗体を得た。得られたアセチル
化抗体はベッカー法にて力価を測定し、0.05M NaCl−0.
01Mりん酸緩衝液(pH=8.2)にて 0.5mgAg/mlに調整し
以下の評価に用いた。Example 1 An anti-human CRP antibody obtained by immunizing a rabbit was treated with 0.1 M phosphate buffer (pH = 8.0) for 50 days.
Adjusted to μg / ml. To 1 ml of the mixture, 1 g of sodium acetate was added and dissolved, and then 2 μl of acetic anhydride was further added and reacted at 4 ° C. overnight. After completion of the reaction, dialysis was performed overnight at 4 ° C. in a 0.01 M phosphate buffer (pH = 7.4) to remove acetic acid, and then insolubles were removed by filtration with a filter (0.45 μm) to obtain an acetylated antibody. Was. The obtained acetylated antibody was measured for titer by the Becker method, and 0.05 M NaCl-0.
It was adjusted to 0.5 mg Ag / ml with a 01 M phosphate buffer (pH = 8.2) and used for the following evaluation.
【0024】 上記により得られた試薬の50μlと、
1〜100mg/dlのヒトCRPの20μlと、0.
5%ポリエチレングリコール/0.01Mリン酸緩衝液
(pH=7.4)の300μlとを混合した。5分間の
後、吸光度(使用機器;日立7150型自動分析機、主
波長340nm、副波長700nm)を測定した。表1
に測定値(× 10−4)を示す。50 μl of the reagent obtained above,
20 μl of 1-100 mg / dl human CRP;
300 μl of 5% polyethylene glycol / 0.01 M phosphate buffer (pH = 7.4) was mixed. After 5 minutes, the absorbance (device used: Hitachi 7150 automatic analyzer, main wavelength 340 nm, auxiliary wavelength 700 nm) was measured. Table 1
Shows measured values (× 10 −4 ).
【0025】[0025]
【表1】 [Table 1]
【0026】[実施例2]ヤギに免疫して得られた抗ヒ
トCRP抗体を0.1Mりん酸緩衝液(pH=8.0)で10mg/ml
に調整した。このうちの1mlに、アセトアルデヒド10μ
lを加え、4℃で30分間反応させ、さらに水素化ホウ素
ナトリウムの1mgを加え、4℃で30分間反応させた。反
応終了後、0.01M りん酸緩衝液(pH=7.4)中、4℃で一
晩透析を行ない、さらにフィルター(0.45μm)により
不溶物を濾過除去し、エチル化抗体を得た。得られたエ
チル化抗体は、ベッカー法にて力価を測定し、0.05M Na
Cl−0.01M りん酸緩衝液(pH=8.2)にて0.5 mgAg/mlに
調整し、以下実施例1と同様の評価を行なった。表1に
測定値を併せて示す。Example 2 An anti-human CRP antibody obtained by immunizing goats was immunized with a 0.1 M phosphate buffer (pH = 8.0) at 10 mg / ml.
Was adjusted. In 1 ml of this, acetaldehyde 10μ
1 was added and reacted at 4 ° C. for 30 minutes, and 1 mg of sodium borohydride was further added and reacted at 4 ° C. for 30 minutes. After completion of the reaction, dialysis was performed overnight at 4 ° C. in a 0.01 M phosphate buffer (pH = 7.4), and insolubles were removed by filtration with a filter (0.45 μm) to obtain an ethylated antibody. The obtained ethylated antibody was measured for titer by the Becker method, and 0.05 M Na
It was adjusted to 0.5 mgAg / ml with a Cl-0.01M phosphate buffer (pH = 8.2), and the same evaluation as in Example 1 was performed. Table 1 also shows the measured values.
【0027】[実施例3]ヤギに免疫して得られた抗ヒ
トCRP抗体を0.1Mりん酸緩衝液(pH=7.4)にて10mg/
mlに調整した。このうちの1mlに500 mg/mlの N−デカ
ノイルオキシこはく酸イミド/ジメチルホルムアミド溶
液1mlを添加し、40℃で6時間反応させた。反応終了
後、0.01M りん酸緩衝液(pH=7.4)中、4℃で一晩透析
を行ない、さらにフィルター(0.45μm )により不溶物
を濾過除去し、デカノイル化抗体を得た。得られたデカ
ノイル化抗体はベッカー法にて力価を測定し、0.05M Na
Cl−0.01M りん酸緩衝液(pH=8.2)にて0.5 mgAg/mlに
調整し、以下実施例1と同様の評価を行なった。表1に
測定値を併せて示す。[Example 3] An anti-human CRP antibody obtained by immunizing a goat was treated with a 0.1 M phosphate buffer (pH = 7.4) at 10 mg / mg.
Adjusted to ml. To 1 ml of the solution, 1 ml of a 500 mg / ml N-decanoyloxysuccinimide / dimethylformamide solution was added and reacted at 40 ° C. for 6 hours. After completion of the reaction, dialysis was performed overnight at 4 ° C. in a 0.01 M phosphate buffer (pH = 7.4), and insolubles were removed by filtration with a filter (0.45 μm) to obtain a decanoylated antibody. The obtained decanoylated antibody was measured for titer by the Becker method, and 0.05 M Na
It was adjusted to 0.5 mgAg / ml with a Cl-0.01M phosphate buffer (pH = 8.2), and the same evaluation as in Example 1 was performed. Table 1 also shows the measured values.
【0028】[ 実施例4]ヤギに免疫して得られた抗ヒ
トCRP抗体を0.1Mクエン酸緩衝液(pH=4.5)にて10mg
/mlに調整した。このうちの1mlに、500 mg/mlの4−
(ラウロイルオキシ)フェニルジメチルスルホニウムメ
チルサルフェートの0.1Mクエン酸緩衝液(pH=4.5)溶液
1mlを添加し、40℃で4時間反応させた。反応終了後、
0.01M りん酸緩衝液(pH=7.4)中、4℃で一晩透析を行
ない、さらにフィルター(0.45μm)により不溶物を濾
過除去し、ラウロイル化抗体を得た。得られたラウロイ
ル化抗体は、ベッカー法にて力価を測定し、0.05M NaCl
−0.01M りん酸緩衝液(pH=8.2 )にて0.5 mgAg/mlに
調整し、以下実施例1と同様の評価を行なった。表1に
測定値を併せて示す。Example 4 10 mg of an anti-human CRP antibody obtained by immunizing a goat in 0.1 M citrate buffer (pH = 4.5)
/ Ml. 1 ml of this contains 500 mg / ml 4-
1 ml of a 0.1 M citrate buffer (pH = 4.5) solution of (lauroyloxy) phenyldimethylsulfonium methyl sulfate was added, and the mixture was reacted at 40 ° C. for 4 hours. After the reaction,
Dialysis was performed overnight at 4 ° C. in a 0.01 M phosphate buffer (pH = 7.4), and insolubles were removed by filtration with a filter (0.45 μm) to obtain a lauroylated antibody. The obtained lauroylated antibody was measured for titer by the Becker method, and 0.05 M NaCl
It was adjusted to 0.5 mgAg / ml with a -0.01 M phosphate buffer (pH = 8.2), and the same evaluation as in Example 1 was performed. Table 1 also shows the measured values.
【0029】[実施例5]ヤギに免疫して得られた抗ヒ
トCRP抗体を0.1Mクエン酸緩衝液(pH=4.5)にて10mg
/mlに調整した。このうちの1mlに、50mg/mlの4−
(ミリストイルオキシ)フェニルジメチルスルホニウム
メチルサルフェート/ジメチルホルムアミド溶液1mlを
添加し、40℃で4時間反応させた。反応終了後、0.01M
りん酸緩衝液(pH=7.4)中4℃で一晩透析を行ない、さ
らにフィルター(0.45μm)により不溶物を濾過除去
し、ミリストイル化抗体を得た。得られたミリストイル
化抗体は、ベッカー法にて力価を測定し、0.05M NaCl−
0.01M りん酸緩衝液(pH=8.2)にて0.5 mgAg/mlに調整
し、以下実施例1と同様の評価を行なった。表1に測定
値を併せて示す。Example 5 10 mg of an anti-human CRP antibody obtained by immunizing a goat in 0.1 M citrate buffer (pH = 4.5)
/ Ml. 1 ml of this contains 50 mg / ml 4-
1 ml of a solution of (myristoyloxy) phenyldimethylsulfonium methyl sulfate / dimethylformamide was added and reacted at 40 ° C. for 4 hours. After the reaction is completed, 0.01M
Dialysis was performed overnight at 4 ° C. in a phosphate buffer (pH = 7.4), and insolubles were removed by filtration with a filter (0.45 μm) to obtain a myristoylated antibody. The obtained myristoylated antibody was measured for titer by the Becker method, and 0.05 M NaCl-
It was adjusted to 0.5 mgAg / ml with a 0.01 M phosphate buffer (pH = 8.2), and the same evaluation as in Example 1 was performed. Table 1 also shows the measured values.
【0030】[比較例] 未修飾のヤギ由来抗ヒトCRP抗体をベッカー法にて力
価を測定し、0.05MNaCl−0.01Mりん酸緩
衝液(pH=8.2)にて1.0mgAg/mlに調整
した。このうちの50μlと、1〜100mg/dlの
ヒトCRPを含有する0.01Mりん酸緩衝液(pH=
7.4)300μlとを混合した。5分間後の吸光度
(使用機器;日立7150型自動分折機、主波長340
nm、副波長700nm)を測定した。表1に測定値を
併せて示す。[Comparative Example] The titer of an unmodified goat-derived anti-human CRP antibody was measured by the Becker method, and 1.0 mg Ag in 0.05 M NaCl-0.01 M phosphate buffer (pH = 8.2). / Ml. Of these, 50 μl and 0.01 M phosphate buffer containing 1 to 100 mg / dl human CRP (pH =
7.4) 300 μl were mixed. Absorbance after 5 minutes (device used: Hitachi 7150 automatic analyzer, dominant wavelength 340
nm, subwavelength 700 nm). Table 1 also shows the measured values.
【0031】 以上の結果より、本発明による疎水化修
飾抗体は、未修飾抗体に比べてCRP濃度1〜100m
g/dlの広い範囲に亘って、有意に高い感度を有する
ことがわかる。From the above results, the hydrophobized modified antibody according to the present invention has a CRP concentration of 1 to 100 m
It can be seen that it has significantly higher sensitivity over a wide range of g / dl .
───────────────────────────────────────────────────── フロントページの続き (72)発明者 首藤 昇一 佐賀県佐賀市日の出1−18−18 (56)参考文献 特開 昭61−100660(JP,A) 特開 昭59−224565(JP,A) (58)調査した分野(Int.Cl.7,DB名) G01N 33/536 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Shoichi Shuto 1-18-18 Hinode, Saga City, Saga Prefecture (56) References JP-A-61-100660 (JP, A) JP-A-59-224565 (JP, A) (58) Fields surveyed (Int. Cl. 7 , DB name) G01N 33/536
Claims (2)
子数2〜24個のアシル基で修飾された抗体を、濁度発
生成分として含有することを特徴とする免疫学的診断
薬。1. An immunological diagnostic agent comprising, as a turbidity-generating component, an antibody modified with an acyl group having 2 to 24 carbon atoms without using an insoluble carrier .
を含む液とを混合し、発生した濁度を定量することを特
徴とする抗原の測定方法。2. A method for measuring an antigen, comprising mixing the immunological diagnostic agent according to claim 1 and a liquid containing an antigen, and quantifying the generated turbidity.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP25346792A JP3350762B2 (en) | 1992-08-31 | 1992-08-31 | Immunological diagnostics |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP25346792A JP3350762B2 (en) | 1992-08-31 | 1992-08-31 | Immunological diagnostics |
Publications (2)
Publication Number | Publication Date |
---|---|
JPH0682449A JPH0682449A (en) | 1994-03-22 |
JP3350762B2 true JP3350762B2 (en) | 2002-11-25 |
Family
ID=17251794
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP25346792A Expired - Fee Related JP3350762B2 (en) | 1992-08-31 | 1992-08-31 | Immunological diagnostics |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP3350762B2 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0640836A3 (en) * | 1993-08-24 | 1995-12-20 | Wako Pure Chem Ind Ltd | Immunoassay method. |
-
1992
- 1992-08-31 JP JP25346792A patent/JP3350762B2/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
JPH0682449A (en) | 1994-03-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN101310186B (en) | Method of assaying antigen and kit to be used therein | |
JP2022188271A (en) | Anti-drug antibody assay with suppressed target interference | |
JP2007127438A (en) | Non-specific reaction inhibitor, immunoassay of immunological measuring reagent and method of manufacturing non-specific reaction inhibitor | |
JP4704662B2 (en) | Immunological latex turbidimetric analysis method and reagent | |
JP2816414B2 (en) | Submicron particles, preparation and use in immunological diagnostics | |
JP3413415B2 (en) | Immunoassay reagent and immunoassay using the same | |
JP3350762B2 (en) | Immunological diagnostics | |
CN112255421B (en) | Lipoprotein a detection kit and detection method | |
CN110672836B (en) | Magnetic bead coating, preparation method and application thereof, and detection kit | |
US7736910B2 (en) | One-step production of gold sols | |
JPH0376422B2 (en) | ||
CN108267576B (en) | Modified CCP antigen and use thereof, anti-CCP antibody detection kit and manufacturing method thereof | |
JPH11248706A (en) | Non-specific adsorption inhibitor | |
US6777246B2 (en) | Tertiary amine compounds for use in immunoassays | |
JP2019028050A (en) | Means for preventing deterioration of immunoassay reagent containing insoluble carrier particles | |
CN112881675A (en) | IgM antibody detection diluent | |
JP3396231B2 (en) | Immune reaction measurement reagent and immunoreactivity measurement method | |
CN115792207B (en) | Single-molecule detection method based on fulvene compounds | |
JP3438962B2 (en) | Immunological agglutination reagent | |
JPS62138502A (en) | Polymer particle | |
JP3858277B2 (en) | Method for producing an immunological diagnostic agent | |
JPH0792455B2 (en) | Aqueous solvent for immunological test | |
JP4121432B2 (en) | Casein for immunoassay reagents | |
CN116003663A (en) | Long-chain carboxyl microsphere, preparation method thereof and kit | |
CN118409094A (en) | Magnetic bead-collectin complex and collectin antibody detection kit comprising same |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20070920 Year of fee payment: 5 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20080920 Year of fee payment: 6 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20080920 Year of fee payment: 6 |
|
S533 | Written request for registration of change of name |
Free format text: JAPANESE INTERMEDIATE CODE: R313533 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20080920 Year of fee payment: 6 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20080920 Year of fee payment: 6 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20090920 Year of fee payment: 7 |
|
LAPS | Cancellation because of no payment of annual fees |