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JP3513075B2 - Immunoassay and reagent therefor - Google Patents

Immunoassay and reagent therefor

Info

Publication number
JP3513075B2
JP3513075B2 JP2000103600A JP2000103600A JP3513075B2 JP 3513075 B2 JP3513075 B2 JP 3513075B2 JP 2000103600 A JP2000103600 A JP 2000103600A JP 2000103600 A JP2000103600 A JP 2000103600A JP 3513075 B2 JP3513075 B2 JP 3513075B2
Authority
JP
Japan
Prior art keywords
antigen
antibody
insoluble carrier
reagent
particulate insoluble
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 - Lifetime
Application number
JP2000103600A
Other languages
Japanese (ja)
Other versions
JP2001289853A (en
Inventor
雅枝 佐藤
修一 目黒
忠 山▲崎▼
盛 関根
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Denka Seiken Co Ltd
Original Assignee
Denka Seiken Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Denka Seiken Co Ltd filed Critical Denka Seiken Co Ltd
Priority to JP2000103600A priority Critical patent/JP3513075B2/en
Publication of JP2001289853A publication Critical patent/JP2001289853A/en
Application granted granted Critical
Publication of JP3513075B2 publication Critical patent/JP3513075B2/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

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Description

【発明の詳細な説明】Detailed Description of the Invention

【0001】[0001]

【発明の属する技術分野】本発明は、凝集法による免疫
測定法及びそのための試薬に関する。
TECHNICAL FIELD The present invention relates to an immunoassay method by an agglutination method and a reagent therefor.

【0002】[0002]

【従来の技術】不溶性担体粒子を利用した免疫測定法
は、血清、血漿、尿、髄液などの体液に含まれる抗原性
物質の定量方法として臨床検査に応用され、現在は自動
化による簡便性・迅速性の理由から広く普及している。
近年は、更なる測定性能の向上を目的とした応用技術が
提案されている。例えば、少なくとも2つの異なった量
の同一抗体が負荷された2種の異ったサイズ範囲のラテ
ックス粒子を用いる試薬(特公昭63-14783号公報参照)や
平均粒径の異なる2種類の不溶性担体粒子に抗体を感作
して用いる測定法(特許第第2588174号公報参照)によれ
ば、広い濃度範囲におよぶ測定が可能となる。平均粒子
径が単一の不溶性担体粒子にポリクローナル抗体とモノ
クローナル抗体を併せて用いる方法(特開平10-90268公
報参照)によれば、同様な効果が得られる。また、平均
粒径の異なる2種類以上の不溶性担体粒子に特定の抗原
に対する2種以上のモノクローナル抗体を感作し、立体
障害による凝集阻害を回避することで正確な定量を可能
とする測定法(特開平10-123137公報参照)などが提案さ
れている。
2. Description of the Related Art Immunoassays using insoluble carrier particles have been applied to clinical tests as a method for quantifying antigenic substances contained in body fluids such as serum, plasma, urine, and cerebrospinal fluid. Widely used for reasons of speed.
In recent years, applied techniques have been proposed for the purpose of further improving measurement performance. For example, a reagent using two types of latex particles having different size ranges loaded with at least two different amounts of the same antibody (see JP-B-63-14783) or two types of insoluble carriers having different average particle sizes. According to the measuring method of sensitizing particles with an antibody (see Japanese Patent No. 2588174), it is possible to measure over a wide concentration range. The same effect can be obtained by the method of using both the polyclonal antibody and the monoclonal antibody in insoluble carrier particles having a single average particle size (see Japanese Patent Laid-Open No. 10-90268). In addition, two or more types of insoluble carrier particles having different average particle sizes are sensitized with two or more types of monoclonal antibodies against a specific antigen to avoid aggregation inhibition due to steric hindrance, thereby enabling accurate quantification ( Japanese Patent Laid-Open Publication No. 10-123137) has been proposed.

【0003】また、Journal of Clinical Laboratory A
nalysis 12:137-144 (1998)及び「医学と薬学」42巻
5号 1999年11月 42(5):781-788, 1999には、大小2
種類の担体粒子を用い、平均粒径の小さな担体粒子には
反応速度の小さなモノクローナル抗体を感作し、平均粒
径の大きな担体粒子には反応速度の大きなモノクローナ
ル抗体を感作した感作粒子混合物を用いて凝集法により
免疫測定を行うことが記載されている。しかしながら、
これらの文献に記載された方法では、測定可能な濃度範
囲が狭く、高濃度領域の測定が困難であるという問題を
有している。
The Journal of Clinical Laboratory A
nalysis 12: 137-144 (1998) and "Medical Science and Pharmacy" Vol. 42, No. 5, November 1999 42 (5): 781-788, 1999.
A mixture of sensitized particles in which different types of carrier particles are used, carrier particles having a small average particle size are sensitized with a monoclonal antibody having a low reaction rate, and carrier particles having a large average particle size are sensitized with a monoclonal antibody having a large reaction rate. It is described that the immunoassay is carried out by the agglutination method. However,
The methods described in these documents have a problem that the measurable concentration range is narrow and it is difficult to measure in a high concentration region.

【0004】[0004]

【発明が解決しようとする課題】臨床検査における血清
免疫検査項目では、低濃度域に臨床的な判定を下すため
の重要なポイントを持つものが多く、低濃度域の測定精
度が検査結果を左右する大きな要因になっていることは
明らかである。粒子径の異なる複数の不溶性担体を混合
して用いる技術は、特許第2588174号公報に示されると
おり、単一の粒子を用いる場合に比べて測定範囲は広く
することができ、低濃度域の測定も可能となるが、測定
精度の面では更なる向上が望まれる。測定精度の向上に
は測定系の高感度化が有効であるが、従来の技術におい
て測定系を高感度化することが測定範囲の縮小をもたら
してしまうことは、特許第2588174号公報に記載されて
いる通りである。また、Journal of Clinical Laborato
ry Analysis 12:137-144 (1998)及び「医学と薬学」4
2巻5号 1999年11月 42(5):781-788, 1999に記載さ
れた方法では高濃度域の測定が困難であり、測定範囲が
狭くなっている。
[Problems to be Solved by the Invention] Many serum immunoassay items in clinical tests have important points for making clinical judgments in the low concentration range, and the measurement accuracy in the low concentration range influences the test results. It is clear that this is a major factor that The technique of mixing a plurality of insoluble carriers having different particle diameters is, as shown in Japanese Patent No. 2588174, the measurement range can be made wider than in the case of using a single particle, and the measurement in a low concentration range can be performed. However, further improvement is desired in terms of measurement accuracy. Although it is effective to increase the sensitivity of the measurement system to improve the measurement accuracy, increasing the sensitivity of the measurement system in the conventional technique leads to a reduction in the measurement range, and it is described in Japanese Patent No. 2588174. It's just like that. Also, Journal of Clinical Laborato
ry Analysis 12: 137-144 (1998) and "Medical Science and Pharmacy" 4
Volume 2, No. 5, November 1999 42 (5): 781-788, 1999 is difficult to measure in the high-concentration range, and the measurement range is narrow.

【0005】従って、本発明の課題は、測定範囲を広範
囲に維持したまま、低濃度域の測定をより高感度化する
測定方法を提供することである。
Therefore, an object of the present invention is to provide a measuring method for increasing the sensitivity of measurement in a low concentration range while maintaining a wide measuring range.

【0006】[0006]

【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、大小2種類の担体粒子を用い、平均粒径の小
さな担体粒子には反応速度の小さなポリクローナル抗体
を感作し、平均粒径の大きな担体粒子には反応速度の大
きなポリクローナル抗体を感作した感作粒子混合物であ
って、大小の粒子の平均粒径の比率、それらの重量比率
及びそれらの平均粒径の値を最適化することにより、測
定範囲を広範囲に維持したまま、低濃度域の測定を従来
法よりも高感度化することができることを見出し、本発
明を完成した。
Means for Solving the Problems As a result of earnest research, the inventors of the present invention used two types of carrier particles, large and small, and sensitized a carrier antibody having a small average particle size with a polyclonal antibody having a small reaction rate, It is a mixture of sensitized particles sensitized with a polyclonal antibody with a large reaction rate for carrier particles with a large particle size, and the ratio of the average particle size of large and small particles, their weight ratio and their average particle size values are optimized. It was found that the sensitivity of the measurement in the low concentration range can be made higher than that of the conventional method while maintaining the measurement range in a wide range, and the present invention has been completed.

【0007】すなわち、本発明は、第1の粒子状不溶性
担体上に、測定すべき抗原と抗原抗体反応する第1のポ
リクローナル抗体又はその抗原結合性断片を担持した第
1の感作粒子と、第2の粒子状不溶性担体上に、測定す
べき抗原と抗原抗体反応する第2のポリクローナル抗体
又はその抗原結合性断片を担持した第2の感作粒子との
混合物であって、前記第1の粒子状不溶性担体の平均粒
子径が0.05〜0.10μmであり、前記第2の粒子状不溶性
担体の平均粒子径は、前記第1の粒子状不溶性担体の平
均粒子径の1.5〜5.0倍であり、前記第1の粒子状不溶性
担体の重量が前記第2の粒子状不溶性担体の総重量の
2.5〜10.0倍であり、前記第2のポリクローナル抗体の
抗原抗体反応速度は、前記第1のポリクローナル抗体の
抗原抗体反応速度よりも大きい、第1及び第2の感作粒
子混合物と、測定すべき抗原を含む検体とを反応させ、
凝集の生成速度を光学的に測定することを含む、凝集法
による免疫測定方法を提供する。また、本発明は、第1
の粒子状不溶性担体上に、測定すべき抗原と抗原抗体反
応する第1のポリクローナル抗体又はその抗原結合性断
片を担持した第1の感作粒子と、第2の粒子状不溶性担
体上に、測定すべき抗原と抗原抗体反応する第2のポリ
クローナル抗体又はその抗原結合性断片を担持した第2
の感作粒子との混合物であって、前記第1の粒子状不溶
性担体の平均粒子径が0.05〜0.10μmであり、前記第2
の粒子状不溶性担体の平均粒子径は、前記第1の粒子状
不溶性担体の平均粒子径の1.5〜5.0倍であり、前記第1
の粒子状不溶性担体の重量が前記第2の粒子状不溶性
担体の総重量の2.5〜10.0倍であり、前記第2のポリク
ローナル抗体の抗原抗体反応速度は、前記第1のポリク
ローナル抗体の抗原抗体反応速度よりも大きい、第1及
び第2の感作粒子混合物から成る、凝集法による免疫測
定用試薬を提供する。
That is, the present invention provides a first sensitized particle in which a first polyclonal antibody or an antigen-binding fragment thereof that reacts with an antigen to be measured reacts on a first particulate insoluble carrier. A mixture of second sensitized particles carrying a second polyclonal antibody or an antigen-binding fragment thereof which reacts with an antigen to be measured on a second particulate insoluble carrier, the mixture comprising: The average particle size of the particulate insoluble carrier is 0.05 to 0.10 μm, the average particle size of the second particulate insoluble carrier is 1.5 to 5.0 times the average particle size of the first particulate insoluble carrier, The total weight of the first particulate insoluble carrier is greater than the total weight of the second particulate insoluble carrier .
2.5 to 10.0 times, and the antigen-antibody reaction rate of the second polyclonal antibody is higher than the antigen-antibody reaction rate of the first polyclonal antibody, which should be measured with the mixture of the first and second sensitized particles. React with a sample containing antigen,
Provided is an immunoassay method by an agglutination method, which comprises optically measuring an agglutination production rate. The present invention also provides a first
The first sensitized particles carrying a first polyclonal antibody or an antigen-binding fragment thereof that reacts with the antigen to be measured on the particulate insoluble carrier, and the second particulate insoluble carrier A second polyclonal antibody or an antigen-binding fragment thereof carrying a second polyclonal antibody that reacts with the antigen to be reacted.
The sensitized particles of 1., wherein the first particulate insoluble carrier has an average particle size of 0.05 to 0.10 μm,
The average particle size of the particulate insoluble carrier is 1.5 to 5.0 times the average particle size of the first particulate insoluble carrier.
The total weight of the particulate insoluble carrier is 2.5 to 10.0 times the total weight of the second particulate insoluble carrier, and the antigen-antibody reaction rate of the second polyclonal antibody is the antigen antibody of the first polyclonal antibody. Provided is an agglutination immunoassay reagent comprising a mixture of first and second sensitized particles having a rate higher than a reaction rate.

【0008】[0008]

【発明の実施の形態】本発明の凝集法による免疫測定法
では、平均粒子径の異なる大小2種類の粒子状不溶性担
体が用いられる。平均粒子径(直径)が小さい方の、第
1の粒子状不溶性担体の平均粒子径は0.05〜0.10μm、
好ましくは0.06〜0.08μmである。第1の粒子状不溶性
担体の平均粒子径がこの範囲内にあると、高濃度域での
正確な測定が可能になり、測定可能な濃度範囲が高濃度
側に広がる。また、平均粒子径が大きい方の、第2の粒
子状不溶性担体の平均粒子径は、前記第1の粒子状不溶
性担体の平均粒子径の1.5〜5.0倍、好ましくは2.0〜4.0
倍である。第2の粒子状不溶性担体の平均粒子径が、第
1の粒子状不溶性担体の平均粒子径の1.5〜5.0倍の範囲
内にあると、低濃度域での測定感度が高くなる。
BEST MODE FOR CARRYING OUT THE INVENTION In the immunoassay method by the agglutination method of the present invention, two types of particulate insoluble carriers having different average particle sizes are used. The average particle diameter of the first particulate insoluble carrier having a smaller average particle diameter (diameter) is 0.05 to 0.10 μm,
It is preferably 0.06 to 0.08 μm. When the average particle size of the first particulate insoluble carrier is within this range, accurate measurement in the high concentration range is possible, and the measurable concentration range is widened to the high concentration side. The average particle size of the second particulate insoluble carrier having the larger average particle size is 1.5 to 5.0 times, preferably 2.0 to 4.0 times the average particle size of the first particulate insoluble carrier.
Double. When the average particle size of the second particulate insoluble carrier is within the range of 1.5 to 5.0 times the average particle size of the first particulate insoluble carrier, the measurement sensitivity in the low concentration range becomes high.

【0009】なお、粒子状不溶性担体自体は、従来から
凝集法に広く用いられているいずれの担体粒子であって
もよく、例えばラテックス粒子や赤血球粒子等を挙げる
ことができる。ポリスチレンラテックス等のラテックス
粒子を特に好ましく用いることができる。
The particulate insoluble carrier itself may be any carrier particle that has been widely used in the agglutination method, and examples thereof include latex particles and red blood cell particles. Latex particles such as polystyrene latex can be particularly preferably used.

【0010】前記第1及び第2の粒子状不溶性担体に
は、反応速度が異なるポリクローナル抗体がそれぞれ感
作される。平均粒子径が小さな第1の粒子状不溶性担体
には、反応速度が小さな第1のポリクローナル抗体が感
作され、平均粒径が大きな第2の粒子状不溶性担体には
反応速度が大きな第2のポリクローナル抗体が感作され
る。ここで、反応速度の大小は、各ポリクローナル抗体
を対応抗原と反応させることにより生じる凝集を吸光度
の変化により測定する場合の、反応初期(反応開始から
1分以内、好ましくは20秒以内)の吸光度の変化率の
大小により表される。抗原抗体反応開始時点からの時間
を横軸に取り、吸光度を縦軸に取り、抗原抗体反応開始
前の吸光度を同じにして抗原抗体反応を行わせ、反応開
始から20秒後の吸光度の変化を比較することにより反
応速度の大小を比較した場合、第2のポリクローナル抗
体の反応速度は、第1のポリクローナル抗体の反応速度
の1.3〜2.0倍程度が好ましく、特に好ましくは1.5〜1.7
倍程度である。なお、反応速度の大小は、力価の大小と
は必ずしも相関しない。下記実施例に示すように、力価
がほぼ同じでも反応速度はかなり異なる場合がある。抗
体を担体粒子に感作する方法は周知の常法により行うこ
とができる。すなわち、抗体溶液に粒子を浮遊させて放
置することにより物理的に吸着させることができる。
Polyclonal antibodies having different reaction rates are sensitized to the first and second particulate insoluble carriers, respectively. The first particulate insoluble carrier having a small average particle size is sensitized with the first polyclonal antibody having a small reaction rate, and the second particulate insoluble carrier having a large average particle size has a large reaction rate. The polyclonal antibody is sensitized. Here, the magnitude of the reaction rate is the absorbance at the initial stage of the reaction (within 1 minute from the start of the reaction, preferably within 20 seconds) when the aggregation caused by reacting each polyclonal antibody with the corresponding antigen is measured by the change in the absorbance. It is represented by the magnitude of the change rate of The time from the start of the antigen-antibody reaction is plotted on the horizontal axis, the absorbance is plotted on the vertical axis, and the antigen-antibody reaction is carried out with the same absorbance before the initiation of the antigen-antibody reaction. When the reaction rates are compared by comparison, the reaction rate of the second polyclonal antibody is preferably about 1.3 to 2.0 times the reaction rate of the first polyclonal antibody, and particularly preferably 1.5 to 1.7.
It is about double. The magnitude of the reaction rate does not necessarily correlate with the magnitude of the titer. As shown in the examples below, the reaction rates may differ considerably even if the titers are almost the same. The carrier particles can be sensitized by a known method. That is, the particles can be physically adsorbed by suspending the particles in the antibody solution and allowing the particles to stand.

【0011】なお、粒子状不溶性担体には、ポリクロー
ナル抗体に代えて、Fabフラグメントや、F(ab')2フラグ
メントのような、そのポリクローナル抗体の抗原結合性
断片を感作することもできる。
The particulate insoluble carrier may be sensitized with an antigen-binding fragment of the polyclonal antibody such as Fab fragment or F (ab ') 2 fragment instead of the polyclonal antibody.

【0012】反応に供される前記第1の粒子状不溶性担
体の重量が前記第2の粒子状不溶性担体の重量の2.
5〜10.0倍であり、特に3.0〜7.0倍であることが好まし
い。第1及び第2の粒子状不溶性担体の重量比率が上記
の範囲内にあると、高濃度域での正確な測定が可能にな
り、測定可能な濃度範囲が高濃度側に広がる。
The total weight of the first particulate insoluble carrier subjected to the reaction is 2. The total weight of the second particulate insoluble carrier.
It is 5 to 10.0 times, and particularly preferably 3.0 to 7.0 times. When the weight ratio of the first and second particulate insoluble carriers is within the above range, accurate measurement in the high concentration range becomes possible, and the measurable concentration range expands to the high concentration side.

【0013】第1及び第2の感作粒子の反応系中での濃
度は特に限定されないが、測定感度の観点から、反応系
中の前記第2の感作粒子の濃度が0.02〜0.07 W/V%程度
が好ましい。
The concentration of the first and second sensitized particles in the reaction system is not particularly limited, but from the viewpoint of measurement sensitivity, the concentration of the second sensitized particles in the reaction system is 0.02 to 0.07 W / About V% is preferable.

【0014】免疫測定自体は、従来の凝集法と全く同様
にして行うことができる。すなわち、上記第1及び第2
の感作粒子を緩衝液に懸濁し、これに検体を加え、凝集
の生成速度を光学的に測定することにより行うことがで
きる。凝集の生成速度は、反応液の吸光度、濁度又は光
透過率等を連続的に又は時間間隔をあけて断続的に測定
することにより容易に測定することができる。凝集速度
は、例えば抗原抗体反応開始60秒後から90秒後の吸
光度の変化を測定することにより測定することができ
る。もっとも、測定時間はこれに限定されるものではな
く、反応の比較的初期(好ましくは反応開始から2分間
以内)の任意の時間における吸光度等の変化を測定する
ことにより測定することができる。凝集速度は、検体中
の抗原濃度と相関するので、既知の抗原濃度の標準試料
をいくつか作製して反応を行い、凝集速度を測定して検
量線を作成しておき、検体中の抗原濃度は、測定された
凝集速度をその検量線にあてはめることにより知ること
ができる。反応温度は、特に限定されないが、通常、3
7℃程度が好ましい。
The immunoassay itself can be carried out in exactly the same manner as the conventional agglutination method. That is, the first and second
It can be carried out by suspending the sensitized particles of 1. in a buffer solution, adding a sample thereto, and optically measuring the rate of aggregation formation. The generation rate of aggregation can be easily measured by measuring the absorbance, turbidity, light transmittance, etc. of the reaction solution continuously or intermittently at intervals. The aggregation rate can be measured, for example, by measuring the change in absorbance 60 seconds to 90 seconds after the start of the antigen-antibody reaction. However, the measurement time is not limited to this, and it can be measured by measuring a change in absorbance or the like at an arbitrary time relatively early in the reaction (preferably within 2 minutes from the start of the reaction). The agglutination rate correlates with the antigen concentration in the sample, so some standard samples with known antigen concentrations are prepared and reacted, and the agglutination rate is measured to prepare a calibration curve. Can be determined by fitting the measured aggregation rate to the calibration curve. The reaction temperature is not particularly limited, but is usually 3
About 7 ° C is preferable.

【0015】本発明の方法により測定することができる
抗原は何ら限定されるものではなく、それに対応するポ
リクローナル抗体を作製することができるあらゆる抗原
が本発明の方法により測定可能である。例えば、C反応
性タンパク質(CRP)のような疾病のマーカーとなる
各種タンパク質、細菌やウイルスのような病原体等を挙
げることができるがこれらに限定されるものではない。
また、検体も何ら限定されるものではなく、血清、血
漿、尿、髄液、痰等の体液や、飲食物又はその抽出液な
どを挙げることができるがこれらに限定されるものでは
ない。なお、体液等をそのまま用いた場合に抗原濃度が
高すぎて正確な測定が困難な場合には、体液等を希釈し
たものを検体として用いることができることは言うまで
もない。
The antigen that can be measured by the method of the present invention is not limited at all, and any antigen that can produce a corresponding polyclonal antibody can be measured by the method of the present invention. Examples include, but are not limited to, various proteins such as C-reactive protein (CRP), which are markers for diseases, and pathogens such as bacteria and viruses.
Further, the sample is not limited at all, and examples thereof include body fluids such as serum, plasma, urine, spinal fluid, and sputum, and foods and drinks or extracts thereof, but are not limited thereto. Needless to say, a diluted body fluid or the like can be used as a sample when the antigen concentration is too high and accurate measurement is difficult when the body fluid or the like is used as it is.

【0016】本発明は、抗原性物質に対する抗体の反応
速度の違いを利用することにより、被検試料中の抗原性
物質をその含有濃度に応じて効果的に捉えることを可能
とし、測定の精度を向上させるものである。つまり、被
検試料中の抗原性物質の濃度が非常に希薄な場合には、
反応速度の速い抗体が優先的に抗原性物質を捉えること
で該抗体が感作された粒子径の大きい不溶性担体が優先
的に凝集を起こし、反応は僅かでも大きい光学的変化と
して捉えられる。一方、被検試料中の抗原性物質の濃度
が高い場合には、反応速度の速い抗体は急速に消費され
るため粒子径の大きい不溶性担体は瞬時に凝集してしま
うが、反応速度の遅い抗体がゆっくりと反応を持続する
ことで該抗体が感作された粒子径の小さい不溶性担体の
凝集反応が維持され、光学的変化を抑えながら継続させ
る事ができる。本発明は、これらの効果により測定範囲
を広範囲に維持したまま低濃度域の測定をより高感度化
することを可能とする測定方法である。さらに、本発明
では、大小の粒子の平均粒径の比率、それらの重量比率
及びそれらの平均粒径の値を最適化することにより、測
定範囲を広範囲に維持したまま低濃度域の測定をより高
感度化することが特に良好に達成される。
The present invention makes it possible to effectively capture an antigenic substance in a test sample according to its content concentration by utilizing the difference in the reaction rate of the antibody to the antigenic substance, and the accuracy of the measurement. Is to improve. In other words, when the concentration of the antigenic substance in the test sample is extremely dilute,
When the antibody having a fast reaction rate preferentially captures the antigenic substance, the insoluble carrier having a large particle size, to which the antibody has been sensitized, preferentially aggregates, and the reaction is captured as a slight optical change. On the other hand, when the concentration of the antigenic substance in the test sample is high, the antibody having a fast reaction rate is rapidly consumed, and the insoluble carrier having a large particle size is instantaneously aggregated, but the antibody having a slow reaction rate is used. By slowly continuing the reaction, the agglutination reaction of the insoluble carrier having a small particle size sensitized with the antibody can be maintained, and the optical change can be continued while being suppressed. The present invention is a measurement method that enables measurement in a low concentration range with higher sensitivity while maintaining a wide measurement range due to these effects. Further, in the present invention, by optimizing the ratio of the average particle size of large and small particles, the weight ratio thereof and the value of the average particle size thereof, it is possible to further improve the measurement in the low concentration range while maintaining the measurement range in a wide range. Higher sensitivity is achieved particularly well.

【0017】[0017]

【実施例】以下、本発明を実施例に基づきより具体的に
説明する。もっとも、本発明は下記実施例に限定される
ものではない。
EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

【0018】参考例1 抗ヒトCRPウサギポリクロー
ナル抗体の反応速度 抗体の反応速度について、市販されている抗ヒトC反応
性タンパク質(C-Reactive Protein;CRP)ウサギポリ
クローナル抗体による実例を示す。抗ヒトCRPウサギ
ポリクローナル抗体A、および抗ヒトCRPウサギポリ
クローナル抗体Bを、抗原性物質であるヒトCRPに対
する凝集力価が等しくなるようにグリシン緩衝液で希釈
したものをそれぞれ反応液とし、抗原性物質との抗原抗
体反応により生成される凝集を光学的に測定した。測定
試料には、ヒトCRP精製抗原を正常ヒト血清で希釈し
てCRP濃度が約10mg/dLとなるように調製した
ものを用いた。
Reference Example 1 Reaction Rate of Anti-Human CRP Rabbit Polyclonal Antibody Regarding the reaction rate of the antibody, an example using a commercially available anti-human C-reactive protein (CRP) rabbit polyclonal antibody is shown. Anti-human CRP rabbit polyclonal antibody A and anti-human CRP rabbit polyclonal antibody B diluted with glycine buffer so that the agglutination titers against human CRP, which is an antigenic substance, are equal to each other and used as reaction solutions. The aggregation produced by the antigen-antibody reaction with was measured optically. The measurement sample used was one prepared by diluting human CRP purified antigen with normal human serum so as to have a CRP concentration of about 10 mg / dL.

【0019】測定装置には、生化学検査用自動分析装置
TBA−30Rを用い、測定試料25μLに対して、凝
集促進剤として3%のポリエチレングリコールを含むグ
リシン緩衝液250μLを添加し、その5分後に抗体A
およびBのそれぞれから調製された反応液を50μL加
える設定とし、抗原抗体反応により生成される凝集を光
波長340nm(主波長)における吸光度の経時的変化
として20秒毎に5分間測定し、その結果を図1のグラ
フに示した。なお、バックグランドの吸光度を消去する
ために、波長804nm(副波長)における吸光度を測
定し、主波長における吸光度から差し引いて吸光度を算
出した。
An automatic analyzer for biochemical examination TBA-30R was used as a measuring device, and 250 μL of a glycine buffer containing 3% polyethylene glycol as an aggregation accelerator was added to 25 μL of the measurement sample, and 5 minutes after that. Later antibody A
The reaction solution prepared from each of B and B was set to add 50 μL, and the aggregation generated by the antigen-antibody reaction was measured every 20 seconds for 5 minutes as the change with time in the absorbance at the light wavelength of 340 nm (main wavelength). Is shown in the graph of FIG. In order to eliminate the background absorbance, the absorbance at a wavelength of 804 nm (sub wavelength) was measured and subtracted from the absorbance at the main wavelength to calculate the absorbance.

【0020】抗原抗体反応が開始される5分の点以降、
抗体Aでは吸光度が急激に上昇しているのに対し、抗体
Bでは比較的緩やかに吸光度が上昇する。このことは、
抗体Aが抗体Bに比べ反応速度が速いことを意味してい
る。また、測定の最終点である10分で抗体Aと抗体B
の吸光度は、ほぼ同じレベルにあり凝集力価としては同
等であることを示している。
From the point 5 minutes after the antigen-antibody reaction is started,
The absorbance of antibody A rapidly increases, while the absorbance of antibody B relatively slowly increases. This is
This means that antibody A has a faster reaction rate than antibody B. In addition, at the final point of measurement, 10 minutes, antibody A and antibody B
The absorbances of 1 and 2 are almost at the same level, indicating that they are equivalent in agglutination titer.

【0021】実施例1、比較例1及び2 ヒトCRPの
測定 抗ヒトCRPウサギポリクローナル抗体A、および抗ヒ
トCRPウサギポリクローナル抗体Bをポリスチレンラ
テックスC(粒子径0.21μm)、およびポリスチレンラ
テックスD(粒子径0.06μm)との組み合わせでそれぞ
れ感作し、グリシン緩衝液中に分散浮遊液状とし、計4
種類の試薬を調製した。これら4種類の試薬をそれぞれ
混合して使用した。なお、試薬はCambiasoらの方法(Me
thods inEnzymology Vol.74; Section I, B [6]:106-13
9)に従い、グリシン緩衝液中で抗体とポリスチレンラ
テックスの規定量を混合し、室温で60分間放置するこ
とにより感作をおこない、遠心操作により剰余分の抗体
を除去した後、ウシ血清アルブミンを含むグリシン緩衝
液によりポリスチレンラテックス粒子表面の抗体未感作
部分のブロッキングを施し、再び遠心操作により抗体感
作ポリスチレンラテックス粒子を集め、グリシン緩衝液
中に再浮遊することにより調製した。
Example 1, Comparative Examples 1 and 2 Measurement of Human CRP Anti-human CRP rabbit polyclonal antibody A and anti-human CRP rabbit polyclonal antibody B were added to polystyrene latex C (particle diameter 0.21 μm) and polystyrene latex D (particle diameter). 0.06 μm) and sensitized each to form a suspension liquid in glycine buffer, total 4
Different types of reagents were prepared. These four types of reagents were mixed and used. The reagent is the method of Cambiaso et al. (Me
thods inEnzymology Vol.74; Section I, B [6]: 106-13
According to 9), the antibody and polystyrene latex were mixed in a specified amount in glycine buffer, and the mixture was left at room temperature for 60 minutes for sensitization, and the excess antibody was removed by centrifugation to contain bovine serum albumin. It was prepared by blocking the antibody-unsensitized portion on the surface of the polystyrene latex particles with a glycine buffer, collecting the antibody-sensitized polystyrene latex particles by centrifugation again, and resuspending in the glycine buffer.

【0022】測定用試料として、ヒトCRP精製抗原を
正常ヒト血清で希釈して調製したCRP標準品を用い
た。CRP標準品は血漿蛋白国際標準品CRM470に
準拠して値付けされたもので、1,5,10,20,3
0,50mg/dLを用意した。0mg/dLとして生理
的食塩液を使用した。
As a measurement sample, a CRP standard product prepared by diluting a human CRP purified antigen with normal human serum was used. CRP standard products are priced in accordance with plasma protein international standard product CRM470, 1, 5, 10, 20, 3
0,50 mg / dL was prepared. Physiological saline was used as 0 mg / dL.

【0023】生化学検査用自動分析装置TBA−80F
Rを用い、次の条件パラメータにて測定をおこなった。
測定検体量4μLに対し、第1試薬としてグリシン緩衝
液を200μL、調製した試薬を第2試薬として200
μL、反応開始(第2試薬添加後)60秒後から90秒間
の平均反応速度を光波長572nmの吸光度変化により
測定する設定とした。
Automatic analyzer for biochemical examination TBA-80F
The measurement was performed using R under the following condition parameters.
For a measurement sample volume of 4 μL, 200 μL of glycine buffer solution as the first reagent and 200 μL of the prepared reagent as the second reagent
μL, and the setting was made so that the average reaction rate from 60 seconds after the start of the reaction (after addition of the second reagent) to 90 seconds was measured by the change in absorbance at a light wavelength of 572 nm.

【0024】本発明の実施例として、抗体Aが感作され
たラテックスCと抗体Bが感作されたラテックスDをそ
れぞれの粒子濃度が0.03%(W/V)、および0.17%
(W/V)となるようにグリシン緩衝液中で混合したものを
調製し、試薬[1](実施例1)とした。
As an example of the present invention, latex C sensitized with antibody A and latex D sensitized with antibody B have a particle concentration of 0.03% (W / V) and 0.17%, respectively.
A mixture was prepared in a glycine buffer solution to give (W / V), and was designated as reagent [1] (Example 1).

【0025】本発明の比較例として、抗体Aが感作され
たラテックスC、およびラテックスDをそれぞれの粒子
濃度が0.03%(W/V)、および0.17%(W/V)となるよ
うにグリシン緩衝液中で混合したものを調製し、試薬
[2](比較例1)とした。また、抗体Bが感作された
ラテックスC、およびラテックスDについても同様の濃
度で混合し、試薬[3](比較例2)とした。
As a comparative example of the present invention, latex C and latex D sensitized with antibody A had particle concentrations of 0.03% (W / V) and 0.17% (W / V), respectively. A mixture was prepared in a glycine buffer solution as described above to prepare a reagent [2] (Comparative Example 1). Further, latex C and latex D sensitized with antibody B were also mixed at the same concentration to obtain reagent [3] (Comparative Example 2).

【0026】試薬[1](実施例1)、試薬[2](比
較例1)、試薬[3](比較例3)について、試薬毎に
CRP濃度と一分間当たりの吸光度変化量との関係を示
す検量線を作成した。これを表1、および図2に示す。
For the reagent [1] (Example 1), the reagent [2] (Comparative example 1), and the reagent [3] (Comparative example 3), the relationship between the CRP concentration and the amount of change in absorbance per minute for each reagent. Was prepared. This is shown in Table 1 and FIG.

【0027】[0027]

【表1】 [Table 1]

【0028】その結果、試薬[2](比較例1)におい
ては吸光度変化量の濃度依存性が得られる範囲が10m
g/dLまでと非常に短く、一方試薬[1](実施例
1)、および試薬[3](比較例2)については50m
g/dLまでとなった。試薬[1](実施例1)と試薬
[3](比較例2)を比較すると測定範囲はほぼ同等と
見られるが、低濃度域の吸光度変化量において試薬
[1](実施例1)が優位にあり、数値的にもCRP濃
度1mg/dLにおいて3倍以上の差が見られた。
As a result, in the reagent [2] (Comparative Example 1), the range in which the concentration dependence of the amount of change in absorbance was obtained was 10 m.
Very short up to g / dL, while 50 m for reagent [1] (Example 1) and reagent [3] (Comparative Example 2).
Up to g / dL. When the reagent [1] (Example 1) and the reagent [3] (Comparative example 2) are compared, the measurement ranges seem to be almost the same, but the reagent [1] (Example 1) shows a similar change in the absorbance change amount in the low concentration range. It was superior, and a difference of 3 times or more was observed numerically at a CRP concentration of 1 mg / dL.

【0029】試薬[2](比較例1)では、反応速度の
速い抗体Aのみを用いていることにより、抗原抗体反応
を介する不溶性担体粒子の凝集生成が速やかに進行する
ため、CRP濃度の低い試料でも非常に高感度な測定が
実現される。しかし、CRP濃度の高い試料では反応が
瞬時に起こり、測定が開始される前に凝集の生成は進行
し、測定時間内には飽和状態に達してしまうため、平均
反応速度は見掛け上少ないものとして測定されてしま
う。
In the reagent [2] (Comparative Example 1), since only the antibody A having a fast reaction rate is used, the aggregate formation of the insoluble carrier particles rapidly proceeds through the antigen-antibody reaction, so that the CRP concentration is low. Very sensitive measurement can be realized even with a sample. However, in a sample having a high CRP concentration, the reaction occurs instantaneously, the formation of aggregation proceeds before the measurement is started, and the saturated state is reached within the measurement time. Therefore, the average reaction rate is apparently low. It will be measured.

【0030】試薬[3](比較例2)では、反応速度の
遅い抗体Bのみを用いていることにより、抗原抗体反応
を介する不溶性担体粒子の凝集生成は緩やかに進行する
ため、 CRP濃度の高い試料に対して試薬[2](比
較例1)で見られるような現象はCRP濃度50mg/
dLまで見られず測定範囲は広いが、 CRP濃度1m
g/dLでの吸光度変化量は試薬[2](比較例1)の
半分程度となってしまう。これは、特開昭63-65369号公
報に示される技術であり、不溶性担体粒子を混合して用
いることで得られる性能である。
In the reagent [3] (Comparative Example 2), since only the antibody B having a slow reaction rate is used, the aggregate formation of the insoluble carrier particles through the antigen-antibody reaction proceeds slowly, so that the CRP concentration is high. The phenomenon observed with the reagent [2] (Comparative Example 1) on the sample showed a CRP concentration of 50 mg /
The measurement range is wide because it is not seen up to dL, but the CRP concentration is 1 m.
The amount of change in absorbance at g / dL is about half that of reagent [2] (Comparative Example 1). This is the technique disclosed in JP-A-63-65369, and is the performance obtained by mixing insoluble carrier particles.

【0031】本発明の実施例である試薬[1]では、低
濃度域において試薬[2](比較例1)と同等の吸光度
変化量を有し、測定範囲において試薬[3](比較例
2)と同等の性能を有している。これは、抗体A,Bの
反応速度の違いが試料中のCRP濃度に応じて、粒子径
が互いに異なるラテックスC,Dの持つ性能を効果的に
発揮させていることを示している。
The reagent [1], which is an example of the present invention, has the same absorbance change amount as the reagent [2] (Comparative Example 1) in the low concentration range, and the reagent [3] (Comparative Example 2) in the measurement range. ) Has the same performance as. This indicates that the difference in the reaction rate of the antibodies A and B effectively exerts the performance of the latexes C and D having different particle sizes depending on the CRP concentration in the sample.

【0032】実施例2、比較例3及び4 ヒトβ2-ミク
ログロブリン(Beta-2-Microglobulin;BMG)の測定 参考例2に記載した、抗ヒトBMGウサギポリクローナ
ル抗体E、および抗ヒトBMGウサギポリクローナル抗
体FをポリスチレンラテックスG(粒子径0.14μm)、
およびポリスチレンラテックスH(粒子径0.07μm)と
の組み合わせでそれぞれ感作し、グリシン緩衝液中に分
散浮遊液状とし、計4種類の試薬を調製した。これら4
種類の試薬をそれぞれ混合して使用した。感作は実施例
1と同様に行った。
[0032] Example 2, Comparative Examples 3 and 4 human beta 2 - microglobulin; described in measured Reference Example 2 (B eta-2- M icro g lobulin BMG), anti-human BMG rabbit polyclonal antibodies E, and anti Human BMG rabbit polyclonal antibody F was added to polystyrene latex G (particle diameter 0.14 μm),
And a combination of polystyrene latex H (particle diameter 0.07 μm) were sensitized and dispersed in a glycine buffer to prepare a suspension liquid. These 4
Each type of reagent was mixed and used. The sensitization was performed in the same manner as in Example 1.

【0033】測定用試料として、ヒトBMG精製抗原を
緩衝液で希釈して調製したBMG標準品を用いた。BM
G標準品はWHO国際標準品に準拠して値付けされたも
ので、50mg/Lを生理的食塩液にて2倍階段希釈し
たものを1/28まで用意した。0mg/Lとして生理的
食塩液を使用した。
As a sample for measurement, a BMG standard product prepared by diluting a human BMG purified antigen with a buffer was used. BM
G standard is one that is priced in compliance with WHO international standard was prepared which was 2-fold serial dilution with a physiological saline solution 50 mg / L to 1/2 8. A physiological saline solution was used as 0 mg / L.

【0034】生化学検査用自動分析装置TBA−30R
を用い、次の条件パラメータにて測定をおこなった。測
定検体量3μLに対し、第1試薬としてグリシン緩衝液
を150μL、調製した試薬を第2試薬として150μ
L、反応開始(第2試薬添加後)60秒後から90秒間の
平均反応速度を光波長572nmの吸光度変化により測
定する設定とした。
Automatic analyzer for biochemical examination TBA-30R
Was measured under the following condition parameters. 150 μL of glycine buffer solution as the first reagent and 150 μL of the prepared reagent as the second reagent for the measurement sample volume of 3 μL
L, the average reaction rate from 90 seconds after the start of the reaction (after addition of the second reagent) to 90 seconds was set to be measured by the change in absorbance at a light wavelength of 572 nm.

【0035】本発明の実施例として、参考例2に記載し
た抗体Eが感作されたラテックスGと抗体Fが感作され
たラテックスHをそれぞれの粒子濃度が0.06%(W/
V)、および0.17%(W/V)となるようにグリシン緩衝液
中で混合したものを調製し、試薬[4](実施例2)と
した。
As an example of the present invention, the particle concentration of latex G sensitized with antibody E and latex H sensitized with antibody F described in Reference Example 2 was 0.06% (W /
V) and 0.17% (W / V) were mixed in a glycine buffer to prepare a reagent [4] (Example 2).

【0036】本発明の比較例として、抗体Eが感作され
たラテックスG、およびラテックスHをそれぞれの粒子
濃度が0.06%(W/V)、および0.17%(W/V)となるよ
うにグリシン緩衝液中で混合したものを調製し、試薬
[5](比較例3)とした。また、抗体Fが感作された
ラテックスG、およびラテックスHについても同様の濃
度で混合し、試薬[6](比較例4)とした。
As a comparative example of the present invention, latex G and latex H sensitized with antibody E had a particle concentration of 0.06% (W / V) and 0.17% (W / V), respectively. A mixture was prepared in a glycine buffer solution as described above, and was designated as a reagent [5] (Comparative Example 3). Further, the latex G and the latex H sensitized with the antibody F were also mixed at the same concentration to obtain a reagent [6] (Comparative Example 4).

【0037】試薬[4](実施例2)、試薬[5](比
較例3)、試薬[6](比較例4)について測定をおこ
ない、試薬毎にBMG濃度と一分間当たりの吸光度変化
量の関係を示す検量線を作成した。これを表2、および
図3に示す。
The reagent [4] (Example 2), the reagent [5] (Comparative Example 3) and the reagent [6] (Comparative Example 4) were measured, and the BMG concentration and the change in absorbance per minute for each reagent were measured. A calibration curve showing the relationship of was prepared. This is shown in Table 2 and FIG.

【0038】[0038]

【表2】 [Table 2]

【0039】その結果、試薬[5](比較例3)におい
て吸光度変化量の濃度依存性が得られる範囲が25mg
/Lまでであり、試薬[4](実施例2)、および試薬
[6](比較例4)については50mg/Lまでとなっ
た。試薬[6](比較例4)で測定範囲は広範に確保さ
れるが低濃度域での吸光度変化量は試薬[5]に比べて
非常に低い。試薬[4](実施例2)において、低濃度
域の吸光度変化量は試薬[5](比較例3)と同等であ
り、測定範囲は50mg/Lまでの性能を示した。
As a result, in the reagent [5] (Comparative Example 3), the concentration dependence of the amount of change in absorbance was 25 mg.
/ L and up to 50 mg / L for reagent [4] (Example 2) and reagent [6] (Comparative Example 4). The reagent [6] (Comparative Example 4) ensures a wide measurement range, but the amount of change in absorbance in the low concentration range is much lower than that of the reagent [5]. In the reagent [4] (Example 2), the amount of change in absorbance in the low concentration range was equivalent to that of the reagent [5] (Comparative example 3), and the measurement range showed performance up to 50 mg / L.

【0040】本発明の実施例である試薬[4](実施例
2)では、低濃度域において試薬[5](比較例3)と
同等の吸光度変化量、および試薬[6](比較例4)と
同等の測定範囲を有している。これは実施例1と同様の
結果であり、本発明が特定の抗原性物質、およびそれに
対する特定の抗体で成立するものではないことを示して
いる。
The reagent [4] (Example 2), which is an example of the present invention, has an absorbance change equivalent to that of the reagent [5] (Comparative Example 3) in the low concentration range, and the reagent [6] (Comparative Example 4). ) Has a measurement range equivalent to. This is the same result as in Example 1, and shows that the present invention does not consist of a specific antigenic substance and a specific antibody against it.

【0041】[0041]

【発明の効果】本発明による免疫学的測定法によれば、
低濃度域の高感度化と広範囲な測定範囲の維持が可能と
なり、血清免疫検査等において臨床的な判定を下すため
の重要である低濃度域の測定精度を向上させ、より正確
な判定が可能となる。
According to the immunological assay method of the present invention,
High sensitivity in the low concentration range and maintenance of a wide measurement range are possible, and the measurement accuracy in the low concentration range, which is important for making clinical judgments in serum immunoassays, etc., is improved, enabling more accurate judgments. Becomes

【図面の簡単な説明】[Brief description of drawings]

【図1】参考例1における各抗体の抗原抗体反応による
凝集生成速度を比較した図である。
FIG. 1 is a diagram comparing the rate of aggregate formation by the antigen-antibody reaction of each antibody in Reference Example 1.

【図2】実施例1での各試薬におけるCRP濃度と1分
間当たりの吸光度変化量の関係をグラフに示し、比較し
た図である。
FIG. 2 is a graph showing the relationship between the CRP concentration of each reagent and the amount of change in absorbance per minute in Example 1, and is a diagram for comparison.

【図3】実施例2での各試薬におけるBMG濃度と1分
間当たりの吸光度変化量の関係をグラフに示し、比較し
た図である。
FIG. 3 is a graph showing the relationship between the BMG concentration of each reagent and the amount of change in absorbance per minute in Example 2 and compared them.

フロントページの続き (72)発明者 関根 盛 新潟県五泉市大字木越字鏡田1359番1 デンカ生研株式会社生産本部内 (58)調査した分野(Int.Cl.7,DB名) G01N 33/543 G01N 21/27 G01N 21/59 Front page continuation (72) Inventor Mori Sekine 1359-1 Kagamida, Koshigoshi, Gosen City, Niigata Prefecture Denka Seiken Co., Ltd. Production Headquarters (58) Fields investigated (Int.Cl. 7 , DB name) G01N 33/543 G01N 21/27 G01N 21/59

Claims (3)

(57)【特許請求の範囲】(57) [Claims] 【請求項1】 第1の粒子状不溶性担体上に、測定すべ
き抗原と抗原抗体反応する第1のポリクローナル抗体又
はその抗原結合性断片を担持した第1の感作粒子と、第
2の粒子状不溶性担体上に、測定すべき抗原と抗原抗体
反応する第2のポリクローナル抗体又はその抗原結合性
断片を担持した第2の感作粒子との混合物であって、前
記第1の粒子状不溶性担体の平均粒子径が0.05〜0.10μ
mであり、前記第2の粒子状不溶性担体の平均粒子径
は、前記第1の粒子状不溶性担体の平均粒子径の1.5〜
5.0倍であり、前記第1の粒子状不溶性担体の重量が
前記第2の粒子状不溶性担体の総重量の2.5〜10.0倍で
あり、前記第2のポリクローナル抗体の抗原抗体反応速
度は、前記第1のポリクローナル抗体の抗原抗体反応速
度よりも大きい、第1及び第2の感作粒子混合物と、測
定すべき抗原を含む検体とを反応させ、凝集の生成速度
を光学的に測定することを含む、凝集法による免疫測定
法。
1. A first sensitizing particle carrying a first polyclonal antibody or an antigen-binding fragment thereof which reacts with an antigen to be measured and an antigen-antibody, on a first particulate insoluble carrier, and a second particle. Which is a mixture of second sensitized particles carrying a second polyclonal antibody or an antigen-binding fragment thereof that reacts with an antigen to be measured on the particulate insoluble carrier, the first particulate insoluble carrier Has an average particle size of 0.05 to 0.10μ
m, and the average particle size of the second particulate insoluble carrier is 1.5 to 1.5 times the average particle size of the first particulate insoluble carrier.
5.0 times, the total weight of the first particulate insoluble carrier is 2.5 to 10.0 times the total weight of the second particulate insoluble carrier, and the antigen-antibody reaction rate of the second polyclonal antibody is It is possible to react the mixture of the first and second sensitized particles, which is larger than the antigen-antibody reaction rate of the first polyclonal antibody, with the sample containing the antigen to be measured, and optically measure the rate of aggregation formation. Immunoassay by agglutination, including.
【請求項2】 反応系中の前記第2の感作粒子の濃度が
0.02〜0.07 W/V%である請求項1記載の免疫測定法。
2. The concentration of the second sensitized particles in the reaction system is
The immunoassay method according to claim 1, wherein the immunoassay is 0.02 to 0.07 W / V%.
【請求項3】 第1の粒子状不溶性担体上に、測定すべ
き抗原と抗原抗体反応する第1のポリクローナル抗体又
はその抗原結合性断片を担持した第1の感作粒子と、第
2の粒子状不溶性担体上に、測定すべき抗原と抗原抗体
反応する第2のポリクローナル抗体又はその抗原結合性
断片を担持した第2の感作粒子との混合物であって、前
記第1の粒子状不溶性担体の平均粒子径が0.05〜0.10μ
mであり、前記第2の粒子状不溶性担体の平均粒子径
は、前記第1の粒子状不溶性担体の平均粒子径の1.5〜
5.0倍であり、前記第1の粒子状不溶性担体の重量が
前記第2の粒子状不溶性担体の総重量の2.5〜10.0倍で
あり、前記第2のポリクローナル抗体の抗原抗体反応速
度は、前記第1のポリクローナル抗体の抗原抗体反応速
度よりも大きい、第1及び第2の感作粒子混合物から成
る、凝集法による免疫測定用試薬。
3. A first sensitizing particle carrying a first polyclonal antibody or an antigen-binding fragment thereof which reacts with the antigen to be measured on the first particulate insoluble carrier, and a second particle. Which is a mixture of second sensitized particles carrying a second polyclonal antibody or an antigen-binding fragment thereof that reacts with an antigen to be measured on the particulate insoluble carrier, the first particulate insoluble carrier Has an average particle size of 0.05 to 0.10μ
m, and the average particle size of the second particulate insoluble carrier is 1.5 to 1.5 times the average particle size of the first particulate insoluble carrier.
5.0 times, the total weight of the first particulate insoluble carrier is 2.5 to 10.0 times the total weight of the second particulate insoluble carrier, and the antigen-antibody reaction rate of the second polyclonal antibody is A reagent for immunoassay by an agglutination method, which comprises a mixture of first and second sensitized particles, which is larger than the antigen-antibody reaction rate of the first polyclonal antibody.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006025401A1 (en) 2004-08-31 2006-03-09 Denka Seiken Co., Ltd. Method of assaying antigen and reagent therefor
US10994271B2 (en) 2016-06-14 2021-05-04 Denka Company Limited Membrane carrier for liquid sample test kit, liquid sample test kit, and method for producing liquid sample test kit
US11162938B2 (en) 2017-03-28 2021-11-02 Denka Company Limited Membrane carrier, kit for testing liquid sample using same, and manufacturing method thereof
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Publication number Priority date Publication date Assignee Title
US20080044926A1 (en) * 2004-12-24 2008-02-21 Daiichi Pure Chemicals Co., Reagent for Assaying Antigen and Method of Assaying Antigen
US20080081379A1 (en) * 2006-07-13 2008-04-03 Sigler Gerald F Homogeneous double receptor agglutination assay for immunosuppressant drugs
JP4853666B2 (en) * 2007-05-30 2012-01-11 Jsr株式会社 Target substance detection method, mixed particles, and target substance detection reagent
EP3006922B1 (en) 2013-05-31 2018-08-01 Sekisui Medical Co., Ltd. Method of agglutination immunoassay

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006025401A1 (en) 2004-08-31 2006-03-09 Denka Seiken Co., Ltd. Method of assaying antigen and reagent therefor
US10994271B2 (en) 2016-06-14 2021-05-04 Denka Company Limited Membrane carrier for liquid sample test kit, liquid sample test kit, and method for producing liquid sample test kit
US11162938B2 (en) 2017-03-28 2021-11-02 Denka Company Limited Membrane carrier, kit for testing liquid sample using same, and manufacturing method thereof
US11385227B2 (en) 2017-03-28 2022-07-12 Denka Company Limited Membrane carrier and kit for testing liquid sample using same

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