JP2010237105A - Immunoassay reagent and immunoassay method - Google Patents
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Abstract
Description
本発明は、遊離型と複合型の２つの型を有する抗原の測定法に関し、さらに詳しくは補助抗体を用いた免疫学的凝集法および試薬に関する。 The present invention relates to a method for measuring an antigen having two types, a free type and a complex type, and more particularly to an immunological aggregation method and a reagent using an auxiliary antibody.
血液中などに存在する蛋白質には、遊離型として単独で存在するものと、別の蛋白質と結合して複合型蛋白質として存在するものがあり、その一例として前立腺特異抗原（以下「ＰＳＡ」と略）がある。 Proteins present in blood and the like include those that exist alone as a free form and those that bind to another protein and exist as a complex type protein. For example, prostate-specific antigen (hereinafter referred to as “PSA”). )
ＰＳＡは、１９７９年にＷａｎｇらによって最初に精製された抗原であり、前立腺上皮細胞より産生される分子量約３４,０００のセリンプロテアーゼである。アミノ酸配列と蛋白質分解酵素の機能に関する研究から、ＰＳＡは、１９６４年に原らにより見出されたγ−セミノプロテインと同一物質であることが明らかとなった。 PSA is an antigen that was first purified by Wang et al. In 1979 and is a serine protease having a molecular weight of about 34,000 produced from prostate epithelial cells. Studies on amino acid sequences and functions of proteolytic enzymes revealed that PSA is the same substance as the γ-seminoprotein found by Hara et al. In 1964.
ＰＳＡの分泌は、男性の前立腺に特異的ではあるが、前立腺癌に特異的というわけではない。たとえば、ＰＳＡの血中濃度は、前立腺肥大症や前立腺炎等の前立腺癌以外の前立腺疾患でも上昇することが報告されている。また、ＰＳＡは、正常な前立腺細胞でも産生され、血液中に浸出する場合がある。 The secretion of PSA is specific for the male prostate but not for prostate cancer. For example, it has been reported that the blood concentration of PSA increases even in prostate diseases other than prostate cancer such as prostatic hypertrophy and prostatitis. PSA is also produced by normal prostate cells and may leach into the blood.
ＰＳＡは、精液という比較的入手しやすい検体からも精製可能であるため、前立腺癌の各種治療の効果判定、治療後の再発・再燃の評価、さらには根治的前立腺全摘除術後の残存腫瘍の早期検出等の、前立腺癌に関連する幅広い用途に対し、非常に有用な指標となりうる。そのためＰＳＡは、従来利用されてきた前立腺酸性ホスファターゼに代わって、前立腺特異的な腫瘍マーカーとして位置付けられている。 Since PSA can be purified from semen samples that are relatively easily available, the effectiveness of various treatments for prostate cancer, evaluation of recurrence / relapse after treatment, and residual tumor after radical prostatectomy It can be a very useful indicator for a wide range of uses related to prostate cancer, such as early detection. For this reason, PSA is positioned as a prostate-specific tumor marker in place of conventionally used prostate acid phosphatase.
健常な成人男子では、血液中のＰＳＡの濃度は、０．１ｎｇ／ｍＬ以下と非常に低濃度である。一方、前立腺疾患患者では、悪性、良性を問わず、血液中のＰＳＡの濃度は、健常な成人男子の数１０倍の濃度となる。前立腺疾患において血清中に放出されたＰＳＡの大部分は、蛋白質分解酵素による組織障害を防ぐ作用のあるα１−アンチキモトリプシン（以下「ＡＣＴ」と略）やα２−マクログロブリン（以下「α２Ｍ」と略）等の蛋白質分解酵素阻害蛋白との複合型として存在している。しかし、ＰＳＡの一部は、非複合型である遊離型ＰＳＡ（以下「Ｆ−ＰＳＡ」と略）として存在しているため、ＡＣＴ複合型であるＰＳＡ(以下「ＰＳＡ−ＡＣＴ」と略)の濃度を測定しただけでは、ＰＳＡの濃度を正確に測定することはできない。 In healthy adult males, the concentration of PSA in the blood is as low as 0.1 ng / mL or less. On the other hand, in patients with prostate disease, the concentration of PSA in blood is several tens of times that of healthy adult males, regardless of whether it is malignant or benign. Most of the PSA released in serum in prostate diseases is abbreviated as α1-antichymotrypsin (hereinafter abbreviated as “ACT”) or α2-macroglobulin (hereinafter abbreviated as “α2M”), which has an effect of preventing tissue damage caused by proteolytic enzymes. ) Etc., and exists as a complex type with a protease inhibitor protein. However, a part of PSA exists as free PSA (hereinafter abbreviated as “F-PSA”) which is a non-complex type, and therefore PSA (hereinafter abbreviated as “PSA-ACT”) which is an ACT complex type. The concentration of PSA cannot be accurately measured only by measuring the concentration.
したがって、Ｆ−ＰＳＡとＰＳＡ−ＡＣＴの総ＰＳＡの濃度（以下「Ｔ−ＰＳＡ」と略）を正確に測定することが、前立腺疾患の検出等においては非常に重要である。なお、免疫学的に測定される複合型ＰＳＡは、通常ＰＳＡ−ＡＣＴである。α２Ｍと結合しているＰＳＡは、ＰＳＡ抗原全体がα２Ｍで覆われており、抗ＰＳＡ抗体では認識できず、ＰＳＡとして免疫学的に測定することはできないと考えられている。 Therefore, it is very important in the detection of prostate diseases and the like to accurately measure the concentration of total PSA of F-PSA and PSA-ACT (hereinafter abbreviated as “T-PSA”). In addition, the complex type PSA measured immunologically is usually PSA-ACT. PSA bound to α2M is thought to be unable to be immunologically measured as PSA because the entire PSA antigen is covered with α2M and cannot be recognized by an anti-PSA antibody.
ＰＳＡの測定法はいくつか開発されているが、免疫放射定量法（以下「ＩＲＭＡ法」と略）や化学発光免疫測定法（以下「ＣＬＩＡ法」と略）等の免疫学的測定法が主流である。現在、Ｔ−ＰＳＡ測定用として、免疫学的測定法を利用した様々なキットが市販されている。しかし、血清中のＴ−ＰＳＡ濃度の測定値がキット間で大きく異なる点が問題とされている。これは、Ｆ−ＰＳＡとＰＳＡ−ＡＣＴのそれぞれに対する試薬の反応性が、キット毎に異なることが主な原因であると考えられている。特に、複合体であるＰＳＡ−ＡＣＴに対する各試薬の反応性の違いが、キット間の測定値のばらつきの原因であると考えられている。また、ＰＳＡ−ＡＣＴに比べてＦ−ＰＳＡの存在比が低くなると、キット間の測定値のばらつきがさらに顕著になると報告されている。 Several methods for measuring PSA have been developed, but immunoassays such as immunoradiometric assay (hereinafter abbreviated as “IRMA method”) and chemiluminescence immunoassay (hereinafter abbreviated as “CLIA method”) are mainly used. It is. Currently, various kits using immunological measurement methods are commercially available for T-PSA measurement. However, there is a problem in that the measured value of serum T-PSA concentration varies greatly between kits. It is thought that this is mainly due to the fact that the reactivity of the reagent with respect to each of F-PSA and PSA-ACT varies from kit to kit. In particular, the difference in reactivity of each reagent with PSA-ACT, which is a complex, is considered to be a cause of variation in measured values between kits. Further, it has been reported that when the abundance ratio of F-PSA is lower than that of PSA-ACT, the variation in measured values between kits becomes more remarkable.
１９９７年以降、血清Ｔ−ＰＳＡの標準化を目指した調査が、日本泌尿器科学会主催で行われている。Ｔ−ＰＳＡの標準化においては、臨床や検診の場で最も問題となる、２０ｎｇ／ｍＬ以下（特に４〜１０ｎｇ／ｍＬのグレーゾーン）の濃度でＴ−ＰＳＡを正確に測定できる試薬キットが要望されている。そのため、Ｔ−ＰＳＡを精度良く測定するために、血清中のＦ−ＰＳＡとＰＳＡ−ＡＣＴの存在比に関係なく、それぞれに対する反応性が等価（等モル反応）となるような測定法が求められている。 Since 1997, research aimed at standardizing serum T-PSA has been conducted by the Japanese Urological Association. In standardization of T-PSA, there is a demand for a reagent kit capable of accurately measuring T-PSA at a concentration of 20 ng / mL or less (especially, a gray zone of 4 to 10 ng / mL), which is the most problematic in clinical and screening settings. ing. Therefore, in order to measure T-PSA with high accuracy, a measurement method is required in which the reactivity to each is equivalent (equal molar reaction) regardless of the abundance ratio of F-PSA and PSA-ACT in serum. ing.
ＩＲＭＡ法やＣＬＩＡ法においては、Ｆ−ＰＳＡとＰＳＡ−ＡＣＴそれぞれに対するモノクローナル抗体を組み合わせることにより、Ｆ−ＰＳＡとＰＳＡ−ＡＣＴが等モルで反応することが報告されている（非特許文献１）。ＩＲＭＡ法やＣＬＩＡ法では、検出用抗体として使用される２つのモノクローナル抗体のうちの一方が、標識抗体として反応液中で自由に動き回れ（自由度が高い）、ＰＳＡ−ＡＣＴとの反応性が高いため、等モル反応が起きやすいと考えられる。 In the IRMA method and CLIA method, it has been reported that F-PSA and PSA-ACT react in equimolar amounts by combining monoclonal antibodies against F-PSA and PSA-ACT, respectively (Non-patent Document 1). In the IRMA method or CLIA method, one of two monoclonal antibodies used as detection antibodies can freely move around in the reaction solution as a labeled antibody (high degree of freedom), and has high reactivity with PSA-ACT. Therefore, it is considered that an equimolar reaction is likely to occur.
一方、ラテックス粒子等を使用する免疫学的凝集測定法では、Ｆ−ＰＳＡとＰＳＡ−ＡＣＴの反応性を等価にすることが困難であるとされてきた。その理由は、免疫学的凝集測定法では、使用される抗体が固相化されており、ＣＬＩＡ法等の場合とは異なり標識抗体の自由度が低いため、検出用抗体とＰＳＡ−ＡＣＴとの反応性が制限されるためであると考えられる。 On the other hand, it has been difficult to make the reactivity of F-PSA and PSA-ACT equivalent by immunological aggregation measurement methods using latex particles. The reason is that in the immunological agglutination measurement method, the antibody used is solid-phased, and unlike the CLIA method or the like, the degree of freedom of the labeled antibody is low, so the detection antibody and PSA-ACT This is thought to be because the reactivity is limited.
しかし、免疫学的凝集測定法は検出方法が簡便かつ迅速であるため、Ｆ−ＰＳＡとＰＳＡ−ＡＣＴを等モルで反応できるようにすることのメリットは大きい。そこで、免疫学的凝集測定法において、Ｆ−ＰＳＡとＰＳＡ−ＡＣＴの反応性を等価にする方法、またはＦ−ＰＳＡとＰＳＡ−ＡＣＴの両方の濃度を測定する方法として、幾つかの方法が提案されている。 However, since the immunological agglutination measurement method is simple and rapid, the merit of allowing F-PSA and PSA-ACT to react in equimolar amounts is great. Therefore, several methods have been proposed as methods for equalizing the reactivity of F-PSA and PSA-ACT, or for measuring the concentrations of both F-PSA and PSA-ACT in immunological aggregation measurement methods. Has been.
例えば、Ｆ−ＰＳＡに特異的な（ＰＳＡ−ＡＣＴとは反応しない）補助抗体を試料に添加し、次にＰＳＡに対する検出用抗体を感作した担体を添加する方法が提案されている（特許文献１）。本技術は、ＡＣＴと結合可能なＦ−ＰＳＡの領域に、ある程度分子量の大きい蛋白質（抗体）を結合させ、Ｆ−ＰＳＡとＰＳＡ−ＡＣＴの分子量を近づけ、エピトープの数を揃えることで、検出用抗体に対するＦ−ＰＳＡの反応性とＰＳＡ−ＡＣＴの反応性を近づけ、ＰＳＡに対する検出用抗体を感作した担体と、補助抗体が結合したＦ−ＰＳＡおよびＰＳＡ−ＡＣＴとを等モル反応で測定する方法であるとされている。 For example, a method has been proposed in which an auxiliary antibody specific to F-PSA (which does not react with PSA-ACT) is added to a sample, and then a carrier sensitized with a detection antibody against PSA is added (Patent Literature). 1). In this technology, a protein (antibody) with a certain degree of molecular weight is bound to the F-PSA region that can bind to ACT, the molecular weight of F-PSA and PSA-ACT are brought close to each other, and the number of epitopes is made uniform. The reactivity of F-PSA to the antibody and the reactivity of PSA-ACT are brought close to each other, and the carrier sensitized with the detection antibody to PSA and the F-PSA and PSA-ACT bound to the auxiliary antibody are measured by equimolar reaction. It is supposed to be a method.
また、Ｆ−ＰＳＡにもＰＳＡ−ＡＣＴにも反応するが互いに競合しない２種のモノクローナル抗体と、Ｆ−ＰＳＡには反応せずＰＳＡ−ＡＣＴには反応するモノクローナル抗体とを別々に不溶性担体に感作し、Ｔ−ＰＳＡとＰＳＡ−ＡＣＴを測定する免疫凝集測定法が開示されている（特許文献２）。本技術は、３種類のモノクローナル抗体を感作したラテックスについて、それぞれ特定の２種類を組み合わせて（混合させて）用いることで、Ｆ−ＰＳＡとＰＳＡ−ＡＣＴの存在比に影響されることなく測定できる試薬と、複合体のみに特異的な試薬であるとされている。 In addition, two types of monoclonal antibodies that react with F-PSA and PSA-ACT but do not compete with each other and monoclonal antibodies that do not react with F-PSA but react with PSA-ACT are separately sensed by an insoluble carrier. An immunoagglutination measurement method for measuring T-PSA and PSA-ACT is disclosed (Patent Document 2). This technology uses latex that has been sensitized with 3 types of monoclonal antibodies in combination (mixed) with each of the 2 types, so that measurement is not affected by the abundance ratio of F-PSA and PSA-ACT. It is said that it is a reagent specific to only the complex and the complex that can be produced.
また、ラテックス等の不溶性担体粒子を使用した免疫学的凝集測定法において、Ｆ−ＰＳＡとＰＳＡ−ＡＣＴに対し、反応性の異なるモノクローナル抗体を利用した測定法およびそれに使用する測定試薬が開示されている（特許文献３）。本技術は、Ｆ−ＰＳＡとＰＳＡ−ＡＣＴの両方に反応するが、Ｆ−ＰＳＡより反応が遅いＰＳＡ−ＡＣＴにより強く結合することによって凝集速度を早め、Ｆ−ＰＳＡとＰＳＡ−ＡＣＴが等モル反応となるようにした測定法であるとされている。 In addition, in immunological agglutination measurement methods using insoluble carrier particles such as latex, a measurement method using monoclonal antibodies having different reactivities to F-PSA and PSA-ACT and a measurement reagent used therefor are disclosed. (Patent Document 3). This technology reacts to both F-PSA and PSA-ACT, but accelerates the aggregation rate by binding strongly to PSA-ACT, which is slower than F-PSA, and F-PSA and PSA-ACT react equimolarly. It is said that this is a measurement method designed to be
しかし、たとえば、特許文献１に係る方法では、必ずしもＦ−ＰＳＡとＰＳＡ−ＡＣＴが等モルにならないという問題があった。つまり、特許文献１に習い、Ｆ−ＰＳＡ特異的なモノクローナル抗体を補助抗体として使用しても、検出用抗体に対するＦ−ＰＳＡおよびＰＳＡ−ＡＣＴの反応が等価にならない場合があるという問題があった。 However, for example, the method according to Patent Document 1 has a problem that F-PSA and PSA-ACT are not always equimolar. In other words, even if F-PSA-specific monoclonal antibody is used as an auxiliary antibody according to Patent Document 1, the reaction of F-PSA and PSA-ACT to the detection antibody may not be equivalent. .
また、特許文献２に係る方法では、Ｆ−ＰＳＡに対してもＰＳＡ−ＡＣＴに対しても結合力に違いがない補助抗体が使用されていた。その結果、検出用抗体との反応性が、Ｆ−ＰＳＡの１００％に対し、ＰＳＡ−ＡＣＴでは約８０％しかなく、検出用抗体とＦ−ＰＳＡおよびＰＳＡ−ＡＣＴとを、等価に反応させることができないという問題があった。 Further, in the method according to Patent Document 2, an auxiliary antibody that has no difference in binding force to F-PSA and PSA-ACT has been used. As a result, the reactivity with the detection antibody is only about 80% with PSA-ACT compared to 100% with F-PSA, and the detection antibody, F-PSA and PSA-ACT are allowed to react equivalently. There was a problem that could not.
本発明はこうした状況に鑑みてなされたものであり、検出用抗体が遊離型抗原および複合型抗原と等価に反応するような、補助抗体を用いた測定方法を提供することを目的としている。 The present invention has been made in view of such circumstances, and an object of the present invention is to provide a measurement method using an auxiliary antibody such that a detection antibody reacts equivalently to a free antigen and a complex antigen.
上記課題を解決するために、本発明のある態様の免疫学的測定試薬は、遊離型抗原に対する反応性が、遊離型抗原と遊離型抗原に結合する別の抗原とで形成される複合型抗原に対する反応性より高い検出用抗体の反応性を等価に補正する補助抗体を含む免疫学的測定試薬であって、補助抗体は遊離型抗原と複合型抗原の両方に結合可能であり、かつ遊離型抗原と補助抗体との反応性が複合型抗原と補助抗体との反応性よりも高く、検出用抗体が加えられた場合に、遊離型抗原と検出用抗体との反応が複合型抗原と検出用抗体との反応よりも強く抑制される。 In order to solve the above-mentioned problems, the immunoassay reagent according to an aspect of the present invention is a complex antigen formed by reacting a free antigen with another antigen that binds to the free antigen. An immunoassay reagent comprising an auxiliary antibody that equivalently corrects the reactivity of a detection antibody higher than the reactivity to the antibody, wherein the auxiliary antibody can bind to both free and complex antigens and is free The reactivity between the antigen and the auxiliary antibody is higher than the reactivity between the complex antigen and the auxiliary antibody, and when the detection antibody is added, the reaction between the free antigen and the detection antibody reacts with the complex antigen and the detection antibody. It is suppressed more strongly than the reaction with the antibody.
本発明の別の態様は、免疫学的測定方法である。この方法は、遊離型抗原に対する反応性が、遊離型抗原と遊離型抗原に結合する別の抗原とで形成される複合型抗原に対する反応性より高い検出用抗体の反応性を等価に補正する補助抗体を用いた免疫学的測定方法であって、遊離型抗原とも複合型抗原とも結合し、遊離型抗原との反応性が複合型抗原との反応性よりも高い補助抗体を添加し、続いて検出用抗体を用いて補助抗体を添加後の遊離型抗原と複合型抗原を含む溶液の濃度を測定する場合に、遊離型抗原の濃度の低下率を複合型抗原の濃度の低下率よりも高くした。 Another embodiment of the present invention is an immunological measurement method. This method assists in equivalently correcting the reactivity of the detection antibody with a higher reactivity to the free antigen than to the complex antigen formed by the free antigen and another antigen that binds to the free antigen. An immunological measurement method using an antibody, which binds to a free antigen or a complex antigen, and adds a co-antibody that has a higher reactivity with the free antigen than the reactivity with the complex antigen, followed by When measuring the concentration of a solution containing free antigen and complex antigen after adding an auxiliary antibody using a detection antibody, the decrease rate of the concentration of free antigen is higher than the decrease rate of the concentration of complex antigen. did.
本発明によれば、検出用抗体が遊離型抗原および複合型抗原と等価に反応することができる。 According to the present invention, the detection antibody can react equivalently to free antigen and complex antigen.
以下、実施の形態に係る免疫学的測定試薬および免疫学的測定方法について説明する。 Hereinafter, the immunological measurement reagent and the immunological measurement method according to the embodiment will be described.
本実施形態は、抗原が、遊離型と複合型の２つの型を有する場合に、（１）まず補助抗体を含む第１試薬を抗原に加えることによって、遊離型抗原と複合型抗原の検出用抗体に対する反応性を等価にし、（２）次いで検出用抗体を含む第２試薬を加え、免疫学的測定法により抗原の総量を測定する技術に関する発明である。つまり、本実施形態の免疫学的測定試薬は、測定対象物である抗原と結合する抗体として、遊離型抗原と複合型抗原の反応性を等価に補正する抗体である補助抗体と、その後に抗原の濃度の測定のために用いられる検出用抗体の２種類の抗体とを含む。 In this embodiment, when the antigen has two types, a free type and a complex type, (1) First, a first reagent containing an auxiliary antibody is added to the antigen to detect the free type antigen and the complex type antigen. This invention relates to a technique for equalizing the reactivity to an antibody, (2) adding a second reagent containing a detection antibody, and measuring the total amount of the antigen by an immunoassay. That is, the immunological measurement reagent of the present embodiment includes an auxiliary antibody that is an antibody that equally corrects the reactivity of a free antigen and a complex antigen as an antibody that binds to an antigen that is a measurement target, and then an antigen. And two types of antibodies for detection used for the measurement of the concentration of
本実施形態の補助抗体は、遊離型抗原に対する反応性が、遊離型抗原と遊離型抗原に結合する別の抗原とで形成される複合型抗原に対する反応性より高い検出用抗体の反応性を等価に補正する抗体である。また、補助抗体は、遊離型抗原と複合型抗原の両方に結合可能であり、かつ遊離型抗原と補助抗体との反応性が複合型抗原と補助抗体との反応性よりも高い。その結果、検出用抗体が加えられた場合に、遊離型抗原と検出用抗体との反応が複合型抗原と検出用抗体との反応よりも強く抑制される。これにより、遊離型抗原および複合型抗原が、検出用抗体と等モルで反応するように補正することができる。補助抗体はモノクローナル抗体であることが望ましい。 The auxiliary antibody of this embodiment is equivalent in reactivity to the detection antibody in that the reactivity to the free antigen is higher than the reactivity to the complex antigen formed by the free antigen and another antigen that binds to the free antigen. It is an antibody which correct | amends to. The auxiliary antibody can bind to both the free antigen and the complex antigen, and the reactivity between the free antigen and the auxiliary antibody is higher than the reactivity between the complex antigen and the auxiliary antibody. As a result, when the detection antibody is added, the reaction between the free antigen and the detection antibody is suppressed more strongly than the reaction between the complex antigen and the detection antibody. Thereby, it can correct | amend so that a free type antigen and a complex type | mold antigen may react with an antibody for a detection by equimolarity. The auxiliary antibody is preferably a monoclonal antibody.
本実施形態の免疫学的測定試薬は、上記の補助抗体を含む試薬であり、補助抗体を含む第１試薬に限られず、第１試薬および第２試薬を含む試薬もしくは免疫学的測定用キットであってもよい。 The immunological measurement reagent of this embodiment is a reagent containing the above-mentioned auxiliary antibody, and is not limited to the first reagent containing the auxiliary antibody, but is a reagent containing the first reagent and the second reagent or an immunological measurement kit. There may be.
検出用抗体は、抗原の濃度測定のために用いられる、第２試薬に含まれる抗体である。検出用抗体は、不溶性担体粒子に感作された状態で使用される。検出用抗体は、複合型抗原と反応するポリクローナル抗体であるか、または異なるエピトープを認識し、かつ遊離型抗原および複合型抗原の両方に結合する２種類以上のモノクローナル抗体である。 The detection antibody is an antibody contained in the second reagent used for measuring the antigen concentration. The detection antibody is used in a state sensitized to insoluble carrier particles. The detection antibody is a polyclonal antibody that reacts with the complex antigen, or two or more monoclonal antibodies that recognize different epitopes and bind to both the free antigen and the complex antigen.
本実施形態では、より具体的には、測定対象物である抗原は、前立腺特異抗原（ＰＳＡ）である。ＰＳＡは、遊離型（Ｆ−ＰＳＡ）および複合型（ＰＳＡ−ＡＣＴ）として生体中に存在する。 In the present embodiment, more specifically, the antigen that is the measurement target is a prostate specific antigen (PSA). PSA exists in the living body as a free form (F-PSA) and a composite form (PSA-ACT).
本実施形態に係るモノクローナル抗体は、公知の方法によって得ることができる。本実施形態に係るモノクローナル抗体は、公知の方法によって得ることができる。すなわち、マウスをポリクローナル抗体の免疫操作と同様に免疫し、抗体価の上昇を確認したところで、抗体産生細胞を回収する。回収した抗体産生細胞とミエローマとの細胞融合を行った後に、抗体を産生する不死化した細胞を選択培地によって選択する。こうして得られたハイブリドーマから、目的の抗原と反応する抗体を産生している細胞をスクリーニングし、クローニングして抗体産生株として樹立する。得られた抗体産生株をマウスの腹腔に接種して培養し、腹水としてモノクローナル抗体を得る。なお、マウスにかえて、ラット等の免疫動物を用いてもよい。また、ミエローマとの細胞融合でハイブリドーマを作製するのにかえて、Ｂ細胞をＥＢＶトランスフォーム等により形質転換させ不死化してもよい。また、モノクローナル抗体の作製は、ｉｎ ｖｉｔｒｏで抗体産生株を大量に培養することによって行ってもよい。 The monoclonal antibody according to this embodiment can be obtained by a known method. The monoclonal antibody according to this embodiment can be obtained by a known method. That is, mice are immunized in the same manner as polyclonal antibody immunization, and when an increase in antibody titer is confirmed, antibody-producing cells are collected. After the collected antibody-producing cells and myeloma are fused, immortalized cells that produce antibodies are selected using a selective medium. From the thus obtained hybridoma, cells producing an antibody that reacts with the target antigen are screened and cloned to establish an antibody producing strain. The obtained antibody-producing strain is inoculated into the peritoneal cavity of a mouse and cultured to obtain a monoclonal antibody as ascites. Instead of mice, immunized animals such as rats may be used. Further, instead of producing a hybridoma by cell fusion with myeloma, B cells may be transformed and immortalized by EBV transform or the like. Monoclonal antibodies may be produced by culturing a large amount of antibody-producing strains in vitro.
本実施形態に係る検出用の抗体を感作する不溶性担体粒子としては、ラテックス粒子が好適である。ラテックス粒子は、様々な免疫化学項目の測定ができる専用型自動分析装置、および生化学活性等も測定が可能な汎用型自動分析装置で使用するのに適している。ラテックス粒子の材質としては、ポリスチレン、スチレン―ブタジエン重合体等が挙げられる。免疫学的凝集測定法で汎用されているポリスチレン製が材質として特に好ましいが、抗体感作に適していればポリスチレン製のラテックス粒子に限定されない。また、ラテックス粒子の粒子径は、不溶性担体粒子として通常使用されている５０〜７００ｎｍが好ましい。ラテックス粒子の粒子径は均一であってもよく、また異なる粒子径を有するラテックス粒子が混合されていてもよい。 Latex particles are suitable as the insoluble carrier particles for sensitizing the detection antibody according to this embodiment. Latex particles are suitable for use in a dedicated automatic analyzer capable of measuring various immunochemical items and a general-purpose automatic analyzer capable of measuring biochemical activity and the like. Examples of the material of the latex particles include polystyrene and styrene-butadiene polymer. Polystyrene, which is widely used in immunological aggregation measurement methods, is particularly preferable as a material, but is not limited to polystyrene latex particles as long as it is suitable for antibody sensitization. Moreover, the particle diameter of latex particles is preferably 50 to 700 nm, which is usually used as insoluble carrier particles. The particle diameter of the latex particles may be uniform, or latex particles having different particle diameters may be mixed.
ラテックス粒子への抗体の感作は、公知の技術を利用して実施できる。通常は、ポリスチレンの表面は疎水性であるため、ラテックス表面への物理的吸着で抗体の感作が可能である。また、表面にアミノ基やカルボキシル基を有するラテックス粒子を用いる場合、グルタルアルデヒド、カルボジイミド試薬を使用した化学的結合によって、ラテックス粒子表面に抗体を結合させてもよい。 Sensitization of antibodies to latex particles can be performed using known techniques. Usually, since the surface of polystyrene is hydrophobic, the antibody can be sensitized by physical adsorption to the latex surface. When latex particles having an amino group or a carboxyl group on the surface are used, the antibody may be bound to the latex particle surface by chemical bonding using glutaraldehyde or a carbodiimide reagent.
（測定方法）
ここで、本実施形態の免疫学的測定方法について説明する。本実施形態の免疫学的測定方法は、遊離型抗原に対する反応性が、遊離型抗原と遊離型抗原に結合する別の抗原とで形成される複合型抗原に対する反応性より高い検出用抗体の反応性を等価に補正する補助抗体を用い、遊離型抗原とも複合型抗原とも結合し、遊離型抗原との反応性が複合型抗原との反応性よりも高い補助抗体を添加し、続いて検出用抗体を用いて補助抗体を添加後の遊離型抗原と複合型抗原を含む溶液の濃度を測定する方法である。本実施形態の免疫学的測定方法では、遊離型抗原の濃度の低下率は、複合型抗原の濃度の低下率よりも高くなっている。
(Measuring method)
Here, the immunological measurement method of the present embodiment will be described. In the immunological measurement method of this embodiment, the reactivity of the detection antibody is higher than the reactivity to the complex antigen formed by the free antigen and another antigen that binds to the free antigen. Using an auxiliary antibody that corrects for gender, add an auxiliary antibody that binds to both free and complex antigens, and has a higher reactivity with the free antigen than with the complex antigen, followed by detection. This is a method for measuring the concentration of a solution containing a free antigen and a complex antigen after adding an auxiliary antibody using an antibody. In the immunological measurement method of this embodiment, the rate of decrease in the concentration of free antigen is higher than the rate of decrease in the concentration of complex antigen.
本実施形態では、測定対象物である抗原は、ＰＳＡである。ＰＳＡは、遊離型（Ｆ−ＰＳＡ）および複合型（ＰＳＡ−ＡＣＴ）として生体中に存在する。また、補助抗体はモノクローナル抗体であることが望ましい。 In this embodiment, the antigen that is the measurement object is PSA. PSA exists in the living body as a free form (F-PSA) and a composite form (PSA-ACT). The auxiliary antibody is preferably a monoclonal antibody.
本実施形態では、抗原の濃度測定方法として免疫学的凝集測定法を用いたが、抗原の濃度測定は、公知の操作方法により行うことができる。例えば、抗原の濃度測定に光学的測定法を用いる場合、血清等の生体試料と、担体粒子に感作させた抗体とを反応させ、エンドポイント法またはレート法により、透過光や散乱光による吸光度を測定し、濃度を算出することができる。 In the present embodiment, an immunological agglutination measurement method is used as the antigen concentration measurement method, but the antigen concentration measurement can be performed by a known operation method. For example, when an optical measurement method is used for measuring the concentration of an antigen, a biological sample such as serum is reacted with an antibody sensitized to carrier particles, and the absorbance by transmitted light or scattered light is determined by the endpoint method or the rate method. And the concentration can be calculated.
本実施形態では、補助抗体を用いた補正により、抗原−補助抗体の複合体と検出用抗体との反応性が、遊離型抗原と検出用抗体との反応性と等価になることが特徴である。抗原がＰＳＡである場合、本反応のメカニズムとしては、例えば以下のものが想定される。 This embodiment is characterized in that the reactivity between the antigen-auxiliary antibody complex and the detection antibody becomes equivalent to the reactivity between the free antigen and the detection antibody by correction using the auxiliary antibody. . When the antigen is PSA, as the mechanism of this reaction, for example, the following can be assumed.
Ｆ−ＰＳＡの分子量は約３４，０００であるが、ＩｇＧである補助抗体および検出用抗体の分子量は共に約１５０，０００であり、Ｆ−ＰＳＡの分子量よりもＩｇＧの分子量は４倍以上と非常に大きい。そのため、補助抗体がＦ−ＰＳＡに結合すると、Ｆ−ＰＳＡのエピトープ部位の大部分が補助抗体との結合により覆われてしまうと考えられる。また、補助抗体との結合に関与していないエピトープ部位についても、立体障害により、検出用抗体がＦ−ＰＳＡと補助抗体の複合体（以下「補正後Ｆ−ＰＳＡ」と略）に接近しにくくなる可能性がある。以上の理由により、補助抗体を加えない場合に比べ、検出用抗体と補正後Ｆ−ＰＳＡとの反応性が大幅に低下するものと考えられる。 Although the molecular weight of F-PSA is about 34,000, the molecular weight of both the auxiliary antibody and detection antibody, which are IgG, is about 150,000, and the molecular weight of IgG is 4 times or more than the molecular weight of F-PSA. Big. Therefore, it is considered that when the auxiliary antibody binds to F-PSA, most of the epitope site of F-PSA is covered by the binding with the auxiliary antibody. In addition, even for epitope sites that are not involved in binding to the auxiliary antibody, the detection antibody is difficult to approach the complex of F-PSA and the auxiliary antibody (hereinafter abbreviated as “corrected F-PSA”) due to steric hindrance. There is a possibility. For the above reasons, it is considered that the reactivity between the detection antibody and the corrected F-PSA is greatly reduced as compared with the case where no auxiliary antibody is added.
一方、ＰＳＡ−ＡＣＴでは、ＰＳＡのエピトープ部位の多くは、ＡＣＴとの結合により覆われている。また、ＡＣＴにより覆われていないエピトープ部位であっても、立体障害により、ＰＳＡ−ＡＣＴと補助抗体が結合できないか、または弱い結合しか形成できない可能性がある。そのため補助抗体は、ＡＣＴに覆われておらず、かつ立体障害も起こらないエピトープ部位でしかＰＳＡ−ＡＣＴとの複合体（以下「補正後ＰＳＡ−ＡＣＴ」と略）を形成できないと考えられる。以上の理由により、検出用抗体と補正後ＰＳＡ−ＡＣＴとの反応性は、補助抗体を加えない場合と比べて、それほど大きく低下しないと考えられる。 On the other hand, in PSA-ACT, many of the epitope sites of PSA are covered by binding to ACT. Moreover, even if the epitope site is not covered by ACT, there is a possibility that PSA-ACT and the auxiliary antibody cannot be bound or only weak bonds can be formed due to steric hindrance. Therefore, it is considered that the auxiliary antibody can form a complex with PSA-ACT (hereinafter abbreviated as “corrected PSA-ACT”) only at an epitope site that is not covered with ACT and does not cause steric hindrance. For the above reasons, it is considered that the reactivity between the detection antibody and the corrected PSA-ACT does not decrease so much as compared with the case where no auxiliary antibody is added.
以上のように、本実施形態の補助抗体を用いれば、検出用抗体が補正後Ｆ−ＰＳＡおよび補正後ＰＳＡ−ＡＣＴと結合できる部位を、補正後Ｆ−ＰＳＡおよび補正後ＰＳＡ−ＡＣＴの両方において検出用抗体が立体障害を受けることなく結合可能な部位のみに制限することができる。これにより、検出用抗体が、補正後Ｆ−ＰＳＡとも補正後ＰＳＡ−ＡＣＴとも等価に反応するようになると考えられる。 As described above, when the auxiliary antibody of the present embodiment is used, the site where the detection antibody can bind to the corrected F-PSA and the corrected PSA-ACT in both the corrected F-PSA and the corrected PSA-ACT. The detection antibody can be limited only to a site that can be bound without steric hindrance. Thereby, it is considered that the detection antibody reacts equivalently to both the corrected F-PSA and the corrected PSA-ACT.
本実施形態は、遊離型抗原と複合型抗原の反応性を、立体障害も考慮しつつ補正することにより、遊離型抗原と複合型抗原の反応性を等価にする技術を開示するものである。つまり、本発明の技術思想は、抗原がＰＳＡである場合に限定されず、抗原が遊離型と複合型として存在する場合であれば、あらゆる免疫学的測定に応用可能である。 The present embodiment discloses a technique for making the reactivity of the free antigen and the complex antigen equivalent by correcting the reactivity of the free antigen and the complex antigen in consideration of steric hindrance. That is, the technical idea of the present invention is not limited to the case where the antigen is PSA, and can be applied to any immunological measurement as long as the antigen exists as a free type and a complex type.
なお、例えば引用文献１に係る発明では、遊離型に特異的な補助抗体を使用しているが、検出用抗体と補正後ＰＳＡ−ＡＣＴの反応において、立体障害による影響を考慮していなかった。したがって、補助抗体として用いるモノクローナル抗体のロットや実験条件によっては、検出用抗体と、補正後Ｆ−ＰＳＡおよび補正後ＰＳＡ−ＡＣＴの反応性を等価にできない場合があった。 For example, in the invention according to the cited document 1, an auxiliary antibody specific for the free form is used, but the influence of steric hindrance was not considered in the reaction between the detection antibody and the corrected PSA-ACT. Therefore, depending on the lot of monoclonal antibody used as an auxiliary antibody and experimental conditions, the reactivity of the detection antibody, the corrected F-PSA and the corrected PSA-ACT may not be equivalent.
一方、本実施形態では、補助抗体を用い、立体障害も考慮しつつ、遊離型抗原だけではなく複合型抗原のエピトープ部位の反応性をも補正するものである。つまり、本実施形態は、引用文献１等に係る発明とは技術思想を異にするものである。本実施形態では、遊離型抗原と複合型抗原の反応性が、条件に左右されず等価になる。さらに、両ＰＳＡ抗原の存在比や濃度に左右されることなく、等モル反応が起きるものと考えられる。 On the other hand, in this embodiment, an auxiliary antibody is used to correct not only the free antigen but also the reactivity of the epitope site of the complex antigen while taking into consideration steric hindrance. That is, the present embodiment is different in technical idea from the invention according to the cited document 1 and the like. In the present embodiment, the reactivity of the free antigen and the complex antigen is equivalent regardless of the conditions. Furthermore, it is considered that an equimolar reaction occurs regardless of the abundance ratio and concentration of both PSA antigens.
以下に実施例を挙げて本発明を具体的に説明するが、本発明はこれらにより何ら限定されるものではなく、当業者の知識に基づいて各種の設計変更等の変形を加えることも可能である。 Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to these examples, and various modifications such as design changes can be added based on the knowledge of those skilled in the art. is there.
（実験１．補助抗体の取得）
Ｆ−ＰＳＡとの反応性がＰＳＡ−ＡＣＴとの反応性よりも強い補助抗体の取得を行った。本実施例では、まず以下の手順により、Ｆ−ＰＳＡに対するモノクローナル抗体（補助抗体）を得た。
(Experiment 1. Acquisition of auxiliary antibody)
An auxiliary antibody having a higher reactivity with F-PSA than that with PSA-ACT was obtained. In this example, a monoclonal antibody (auxiliary antibody) against F-PSA was first obtained by the following procedure.
ポリクローナル抗体の免疫操作と同様にマウスを免疫し、抗体価の上昇を確認したところで、その抗体産生細胞を回収した。回収した抗体産生細胞を、マウスミエローマ細胞との細胞融合により、ハイブリドーマとした。これを抗体産生能に基づきスクリーニングし、更に必要な活性を持つ抗体を産生する株をクローニングし、抗体産生株を樹立した。抗体産生株をマウスの腹腔に接種して培養し、腹水としてモノクローナル抗体を得た。 Mice were immunized in the same manner as polyclonal antibody immunization, and when antibody titer increase was confirmed, the antibody-producing cells were recovered. The collected antibody-producing cells were used as hybridomas by cell fusion with mouse myeloma cells. This was screened based on antibody production ability, and a strain producing an antibody having the necessary activity was further cloned to establish an antibody producing strain. The antibody-producing strain was inoculated into the abdominal cavity of the mouse and cultured to obtain a monoclonal antibody as ascites.
次に、得られたＰＳＡに対するモノクローナル抗体（補助抗体）から、Ｆ−ＰＳＡとの反応性がＰＳＡ−ＡＣＴとの反応性よりも強い補助抗体を選別した。本実施形態では、二抗体法（ＲＩＡ）により抗体力価の測定を行うことにより、抗ＰＳＡモノクローナル抗体の抗原に対する結合力を測定した。具体的には、以下の手順により、測定を行った。 Next, an auxiliary antibody having a higher reactivity with F-PSA than that with PSA-ACT was selected from the obtained monoclonal antibody (auxiliary antibody) against PSA. In this embodiment, the antibody titer was measured by the two-antibody method (RIA) to measure the binding force of the anti-PSA monoclonal antibody to the antigen. Specifically, the measurement was performed according to the following procedure.
試験管に、得られた補助抗体であるモノクローナル抗体を含む第１試薬（１ｍｇ／Ｌ）０．０１ｍＬと、１２５Ｉで標識した各抗原（Ｆ−ＰＳＡ、ＰＳＡ−ＡＣＴ）０．２ｍＬとを加えて混和した後、静置（２時間、室温）した。次に、検出用抗体として抗マウスＩｇＧ抗体を含む第２試薬を０．１ｍＬ加えて混和した後、静置（３０分間、４℃）した。遠心（３，０００ｒｐｍ、３０分、４℃）後、上清を吸引し、沈殿の放射能量を測定した。沈殿の放射能量の、試験管に加えた標識体の総放射能量に対する比率（％）を算出し、抗体力価とした。 To a test tube, add 0.01 mL of the first reagent (1 mg / L) containing the obtained monoclonal antibody as an auxiliary antibody and 0.2 mL of each antigen labeled with 125 I (F-PSA, PSA-ACT). After mixing, the mixture was allowed to stand (2 hours, room temperature). Next, 0.1 mL of a second reagent containing an anti-mouse IgG antibody as a detection antibody was added and mixed, and then allowed to stand (30 minutes, 4 ° C.). After centrifugation (3,000 rpm, 30 minutes, 4 ° C.), the supernatant was aspirated and the radioactivity of the precipitate was measured. The ratio (%) of the radioactivity of the precipitate to the total radioactivity of the labeled body added to the test tube was calculated and used as the antibody titer.
本実施例の実験結果を表１に示す。Ｆ−ＰＳＡとの反応性は強いがＰＳＡ−ＡＣＴとも弱く反応する補助抗体として、抗体Ｌｏｔ Ｎｏ．ＰＡ０５（以下「実施例」とする）が得られた。一方、Ｆ−ＰＳＡに特異的な補助抗体として得られた抗体Ｌｏｔ Ｎｏ．ＰＡ０８（以下「比較例１」とする）も、比較のために示す。 The experimental results of this example are shown in Table 1. As an auxiliary antibody that reacts strongly with F-PSA but weakly reacts with PSA-ACT, the antibody Lot No. PA05 (hereinafter referred to as “Example”) was obtained. On the other hand, antibody Lot No. obtained as an auxiliary antibody specific for F-PSA was used. PA08 (hereinafter referred to as “Comparative Example 1”) is also shown for comparison.
本実験においては、抗体（補助抗体）の抗原に対する反応性を、抗体力価の値（％）に基づき、以下の４段階に分類した。
４０％以上（強く反応する場合）： ＋＋＋
２０〜４０％（中程度に反応する場合）：＋＋
５〜２０％（弱く反応する場合）： ＋
５％未満（全く反応しない場合）： −
In this experiment, the reactivity of the antibody (auxiliary antibody) to the antigen was classified into the following four stages based on the value (%) of the antibody titer.
40% or more (when reacting strongly): ++
20-40% (when reacting moderately): ++
5-20% (when reacting weakly): +
Less than 5% (when there is no reaction): −
Ｆ−ＰＳＡとの反応においては、実施例、比較例１の抗体とも反応性が「＋＋＋」であり、非常に高い抗体力価を示した。一方、ＰＳＡ-ＡＣＴに対する比較例１の抗体の反応性は「−」であり、抗体を加えない対照実験の抗体力価と同程度であった。また、実施例の抗体のＰＳＡ−ＡＣＴに対する反応性は「＋」であり、バックグランドよりは３倍以上高かったが、実施例の抗体のＦ−ＰＳＡに対する反応性の１／５程度であった。 In the reaction with F-PSA, the reactivity of both the antibody of Example and Comparative Example 1 was “++++”, indicating a very high antibody titer. On the other hand, the reactivity of the antibody of Comparative Example 1 to PSA-ACT was “−”, which was comparable to the antibody titer of the control experiment in which no antibody was added. In addition, the reactivity of the antibody of the example to PSA-ACT was “+”, which was 3 times higher than the background, but about 1/5 of the reactivity of the antibody of the example to F-PSA. .
（実験２．補助抗体を用いた免疫学的測定）
実験１で得られた、Ｆ−ＰＳＡと強く反応しＰＳＡ−ＡＣＴとも弱く反応する実施例の抗体（ＰＡ０５）を補助抗体として使用した。また、Ｆ−ＰＳＡに特異的な補助抗体である比較例１の抗体（ＰＡ０８）も、比較のために使用した。これらの補助抗体を補正用の第１試薬に添加し、Ｆ−ＰＳＡ（１０ｎｇ／ｍＬ）及びＰＳＡ−ＡＣＴ（１０ｎｇ／ｍＬ）の各抗原と反応させた。次に、２つの抗ＰＳＡモノクローナル抗体（検出用抗体）を感作したラテックス試薬である測定用の第２試薬を用いて、生化学自動分析装置でＦ−ＰＳＡ（１０ｎｇ／ｍＬ）及びＰＳＡ−ＡＣＴ（１０ｎｇ／ｍＬ）の濃度を測定した。対照として、補助抗体が添加されていない第１試薬に関しても測定を行い（比較例２）、それらの測定値を比較した。
(Experiment 2. Immunological measurement using auxiliary antibody)
The antibody of Example (PA05) obtained in Experiment 1 that strongly reacts with F-PSA and weakly reacts with PSA-ACT was used as an auxiliary antibody. The antibody of Comparative Example 1 (PA08), which is an auxiliary antibody specific for F-PSA, was also used for comparison. These auxiliary antibodies were added to the first reagent for correction, and reacted with each antigen of F-PSA (10 ng / mL) and PSA-ACT (10 ng / mL). Next, using a second reagent for measurement, which is a latex reagent sensitized with two anti-PSA monoclonal antibodies (detection antibodies), F-PSA (10 ng / mL) and PSA-ACT are analyzed using a biochemical automatic analyzer. The concentration of (10 ng / mL) was measured. As a control, measurement was also performed for the first reagent to which no auxiliary antibody was added (Comparative Example 2), and the measured values were compared.
実施例および比較例１、２の測定結果を表２に示す。補助抗体を未添加の場合（比較例２）、検出用抗体のＦ−ＰＳＡに対する反応性（以下「反応性（１）」と略）が、検出用抗体のＰＳＡ―ＡＣＴに対する反応性（以下「反応性（２）」と略）より高く、等価な反応性を示さなかった。 The measurement results of Examples and Comparative Examples 1 and 2 are shown in Table 2. When the auxiliary antibody is not added (Comparative Example 2), the reactivity of the detection antibody to F-PSA (hereinafter abbreviated as “reactivity (1)”) is the reactivity of the detection antibody to PSA-ACT (hereinafter “ Reactivity (abbreviated as “(2)”) and did not show equivalent reactivity.
また、第１試薬に比較例１の補助抗体（ＰＡ０８）を添加した場合、反応性（１）、反応性（２）の値とも、補助抗体を未添加の場合よりいずれもわずかに高く、その結果、反応性（１）／反応性（２）比は補助抗体を未添加の場合（比較例２）とほぼ同程度となった。 In addition, when the auxiliary antibody (PA08) of Comparative Example 1 was added to the first reagent, both the reactivity (1) and reactivity (2) values were slightly higher than when no auxiliary antibody was added. As a result, the ratio of reactivity (1) / reactivity (2) was almost the same as that when the auxiliary antibody was not added (Comparative Example 2).
一方、第１試薬に実施例の補助抗体（ＰＡ０５）を添加した場合、反応性（２）は補助抗体を未添加の場合（比較例２）および比較例１の補助抗体を添加した場合よりわずかに高かったが、補助抗体を未添加の場合（比較例２）および比較例１の補助抗体を添加した場合よりも反応性（１）が低い値を示した。その結果、反応性（１）／反応性（２）比は１．００となった。 On the other hand, when the auxiliary antibody (PA05) of Example was added to the first reagent, the reactivity (2) was slightly lower than when the auxiliary antibody was not added (Comparative Example 2) and when the auxiliary antibody of Comparative Example 1 was added. The reactivity (1) was lower than when the auxiliary antibody was not added (Comparative Example 2) and when the auxiliary antibody of Comparative Example 1 was added. As a result, the reactivity (1) / reactivity (2) ratio was 1.00.
第１試薬に補助抗体を未添加の場合（比較例２）との比較から、Ｆ−ＰＳＡを特異的に補正する補助抗体（ＰＡ０８）を補助抗体として用いた場合（比較例１）、検出用抗原に対するＦ−ＰＳＡおよびＰＳＡ−ＡＣＴの反応性を等価にできなかった。一方、補助抗体を未添加の場合（比較例２）および補助抗体を添加した場合（比較例１）との比較から、実施例の補助抗体（ＰＡ０５）を用いた場合（実施例）、Ｆ−ＰＳＡと検出用抗体の反応性を抑制することにより、ＰＡ０５およびＦ−ＰＳＡと、ＰＡ０５およびＰＳＡ−ＡＣＴとの反応性が等価になることが確認された。 From the comparison with the case where no auxiliary antibody was added to the first reagent (Comparative Example 2), when the auxiliary antibody (PA08) that specifically corrects F-PSA was used as the auxiliary antibody (Comparative Example 1), for detection The reactivity of F-PSA and PSA-ACT to the antigen could not be equivalent. On the other hand, in comparison with the case where no auxiliary antibody was added (Comparative Example 2) and the case where the auxiliary antibody was added (Comparative Example 1), when the auxiliary antibody (PA05) of Example was used (Example), F- It was confirmed that the reactivity of PA05 and F-PSA with PA05 and PSA-ACT becomes equivalent by suppressing the reactivity of PSA and the antibody for detection.
Claims (7)
前記補助抗体は前記遊離型抗原と前記複合型抗原の両方に結合可能であり、かつ
前記遊離型抗原と前記補助抗体との反応性が前記複合型抗原と前記補助抗体との反応性よりも高く、
前記検出用抗体が加えられた場合に、前記遊離型抗原と前記検出用抗体との反応が前記複合型抗原と前記検出用抗体との反応よりも強く抑制されることを特徴とする免疫学的測定試薬。 Auxiliary antibody that equivalently corrects the reactivity of the antibody for detection higher than the reactivity to the complex antigen formed by the free antigen and another antigen that binds to the free antigen. An immunoassay reagent comprising:
The auxiliary antibody can bind to both the free antigen and the complex antigen, and the reactivity between the free antigen and the auxiliary antibody is higher than the reactivity between the complex antigen and the auxiliary antibody. ,
An immunological feature characterized in that when the detection antibody is added, the reaction between the free antigen and the detection antibody is suppressed more strongly than the reaction between the complex antigen and the detection antibody. Measuring reagent.
前記遊離型抗原とも前記複合型抗原とも結合し、前記遊離型抗原との反応性が前記複合型抗原との反応性よりも高い前記補助抗体を添加し、
続いて検出用抗体を用いて前記補助抗体を添加後の前記遊離型抗原と前記複合型抗原を含む溶液の濃度を測定する場合に、前記遊離型抗原の濃度の低下率を前記複合型抗原の濃度の低下率よりも高くしたことを特徴とする免疫学的測定方法。 Auxiliary antibody that equivalently corrects the reactivity of the antibody for detection higher than the reactivity to the complex antigen formed by the free antigen and another antigen that binds to the free antigen. In the immunological measurement method used,
Adding the auxiliary antibody that binds to both the free antigen and the complex antigen, the reactivity with the free antigen is higher than the reactivity with the complex antigen,
Subsequently, when measuring the concentration of the solution containing the free antigen and the complex antigen after adding the auxiliary antibody using a detection antibody, the rate of decrease in the concentration of the free antigen is determined by An immunological measurement method characterized by being higher than the rate of decrease in concentration.
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