JP7627161B2 - Immunoassay reagent, immunoassay kit, and immunoassay method - Google Patents
Immunoassay reagent, immunoassay kit, and immunoassay method Download PDFInfo
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- JP7627161B2 JP7627161B2 JP2021059857A JP2021059857A JP7627161B2 JP 7627161 B2 JP7627161 B2 JP 7627161B2 JP 2021059857 A JP2021059857 A JP 2021059857A JP 2021059857 A JP2021059857 A JP 2021059857A JP 7627161 B2 JP7627161 B2 JP 7627161B2
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Description
本発明は、免疫測定用試薬、免疫測定用キット及び前記の試薬を使用する免疫測定方法に関する。 The present invention relates to an immunoassay reagent, an immunoassay kit, and an immunoassay method using the above-mentioned reagent.
免疫測定試薬においては、主要構成要素として、標識物質により標識されてなる物質がある（特許文献１）。免疫測定試薬においては、標識物質により標識されてなる物質が一定の活性を維持していることが望まれる。経時的に物質の活性が低下したり、保存状態によって物質の活性が変化したのでは正確な測定結果は期待できず、免疫測定試薬としては、大きな問題である。多数検体同時迅速検査を目的とした自動化免疫測定装置の開発において、さらなる保存安定性の改善が望まれている。 In immunoassay reagents, the main component is a substance labeled with a labeling substance (Patent Document 1). In immunoassay reagents, it is desirable that the substance labeled with the labeling substance maintain a certain level of activity. If the activity of the substance decreases over time or if the activity of the substance changes depending on the storage conditions, accurate measurement results cannot be expected, which is a major problem for immunoassay reagents. In the development of automated immunoassay devices aimed at rapid simultaneous testing of multiple samples, further improvements in storage stability are desired.
本発明の目的は、保存安定性に優れた免疫測定用試薬、該免疫測定用試薬を含む免疫測定用キット及び該キットを使用する免疫測定方法を提供することにある。 The object of the present invention is to provide an immunoassay reagent with excellent storage stability, an immunoassay kit including the immunoassay reagent, and an immunoassay method using the kit.
本発明者らは、上記目的を達成すべく鋭意検討した結果本発明に到達した。
即ち本発明は、物質（ＦＣ）と、ジギトニン、ククルビタシンＢ、ウアバイン及びサポニンからなる群から選ばれる少なくとも１種の化合物（Ｄ）であって、物質（ＦＣ）が物質（Ｆ）が標識物質（Ｃ）により標識されてなる物質であり、物質（Ｆ）が測定対象物質（Ｆ１）、測定対象物質の類似物質（Ｆ２）及び測定対象物質と特異的に結合する物質（Ｆ３）からなる群から選ばれる少なくとも１種の物質であり、標識物質（Ｃ）がペルオキシダーゼであり、（Ｘ）中の（Ｄ）の含有量が（Ｘ）の重量を基準として０．５１～１．５０重量％である免疫測定用試薬（Ｘ）；標識試薬（Ａ）及び固相担体試薬（Ｅ）を含む免疫測定用キットであって、標識試薬（Ａ）が該免疫測定用試薬（Ｘ）であり、固相担体試薬（Ｅ）が固相担体（Ｂ）の表面に物質（Ｆ）が固定化された固相担体（ＢＦ）を含有するものである免疫測定用キット；該免疫測定用キットを使用する免疫測定方法である。
The present inventors conducted extensive research to achieve the above object and arrived at the present invention.
That is, the present invention relates to a substance (FC) and at least one compound (D) selected from the group consisting of digitonin, cucurbitacin B, ouabain and saponin, wherein the substance (FC) is a substance obtained by labeling a substance (F) with a labeling substance (C), the substance (F) is at least one substance selected from the group consisting of a substance to be measured (F1), an analogue of the substance to be measured (F2) and a substance that specifically binds to the substance to be measured (F3), and the labeling substance (C) is peroxidase, An immunoassay reagent (X), in which the content of (D) in (X) is 0.51 to 1.50% by weight based on the weight of (X); an immunoassay kit comprising a labeled reagent (A) and a solid phase carrier reagent (E), wherein the labeled reagent (A) is the immunoassay reagent (X), and the solid phase carrier reagent (E) contains a solid phase carrier (BF) having a substance (F) immobilized on the surface of the solid phase carrier (B); and an immunoassay method using the immunoassay kit.
本発明の免疫測定用試薬及び免疫測定用キットを用いれば、長期間試薬を保管することができ、有効期間切れによる試薬ロスを低減することができる。 By using the immunoassay reagent and immunoassay kit of the present invention, the reagent can be stored for a long period of time, reducing reagent loss due to expiration.
＜免疫測定用試薬＞
本発明の免疫測定用試薬（Ｘ）は、物質（ＦＣ）と、ジギトニン、ククルビタシンＢ、ウアバイン及びサポニンからなる群から選ばれる少なくとも１種の化合物（Ｄ）とを含有する免疫測定用試薬（Ｘ）であって、物質（ＦＣ）が物質（Ｆ）が標識物質（Ｃ）により標識されてなる物質であり、物質（Ｆ）が測定対象物質（Ｆ１）、測定対象物質の類似物質（Ｆ２）及び測定対象物質と特異的に結合する物質（Ｆ３）からなる群から選ばれる少なくとも１種の物質であり、（Ｘ）中の（Ｄ）の含有量が（Ｘ）の重量を基準として０．５１～１．５０重量％である免疫測定用試薬である。
<Immunoassay Reagents>
The immunoassay reagent (X) of the present invention is an immunoassay reagent containing a substance (FC) and at least one compound (D) selected from the group consisting of digitonin, cucurbitacin B, ouabain and saponin, wherein the substance (FC) is a substance obtained by labeling a substance (F) with a labeling substance (C), the substance (F) is at least one substance selected from the group consisting of a substance to be measured (F1), an analogue of the substance to be measured (F2) and a substance that specifically binds to the substance to be measured (F3), and the content of (D) in (X) is 0.51 to 1.50% by weight based on the weight of (X).
本発明における物質（ＦＣ）は、上述の通り、物質（Ｆ）が標識物質（Ｃ）により標識されてなる物質である。物質（Ｆ）としては、測定対象物質（Ｆ１）、測定対象物質の類似物質（Ｆ２）及び測定対象物質と特異的に結合する物質（Ｆ３）からなる群から選ばれる少なくとも１種の物質が含まれる。
本発明における測定対象物質（Ｆ１）は、生物由来の試料［生体体液（血清、血液、リンパ液、腹水及び尿等）、各種細胞類及び培養液等）に含まれる物質等が挙げられる。
具体的には、タンパク質［アルブミン、ヘモグロビン、ミオグロビン、トランスフェリン、プロテインＡ、Ｃ反応性蛋白質（ＣＲＰ）、脂質蛋白質、酵素、免疫グロブリン、免疫グロブリンの断片、血液凝固関連因子、抗体、抗原及びホルモン等］、
核酸［デオキシリボ核酸（ＤＮＡ）及びリボ核酸（ＲＮＡ）等］、
薬物［抗てんかん薬、抗生物質、テオフィリン等］、
ウイルス［Ｃ型肝炎ウイルス、Ｂ型肝炎ウイルス、ヘパドナウイルス、アデノウイルス、ラブドウイルス、フラビウイルス、レトロウイルス、ヘルペスウイルス、オソミクソウイルス等］、
細菌［Ｏ－１５７、ピロリ菌、サルモネラ菌等］、
細胞［脂肪細胞、ＥＳ細胞、肝細胞、幹細胞、内皮細胞、上皮細胞、筋細胞、内分泌細胞、外分泌細胞、神経細胞、腫瘍細胞、ＩＰＳ細胞等］等が挙げられる。
前記の酵素としては、アルカリ性ホスファターゼ、アミラーゼ、酸性ホスファターゼ、γ－グルタミルトランスフェラーゼ（γ－ＧＴＰ）、リパーゼ、クレアチンキナーゼ（ＣＫ）、乳酸脱水素酵素（ＬＤＨ）、グルタミン酸オキザロ酢酸トランスアミナーゼ（ＧＯＴ）、グルタミン酸ピルビン酸トランスアミナーゼ（ＧＰＴ）、レニン、プロテインキナーゼ（ＰＫ）、チロシンキナーゼ等が挙げられる。
前記の脂質蛋白質としては、高比重リポ蛋白質（ＨＤＬ）、低比重リポ蛋白質（ＬＤＬ）、超低比重リポ蛋白質等が挙げられる。
前記の免疫グロブリンとしては、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＤ、ＩｇＥ等が挙げられる。
前記の免疫グロブリンの断片としては、Ｆｃ部、Ｆａｂ部、Ｆ（ａｂ）２部等が挙げられる。
前記の血液凝固関連因子としては、フィブリノーゲン、フィブリン分解産物（ＦＤＰ）、プロトロンビン、トロンビン等が挙げられる。
前記の抗体としては、抗ストレプトリジンＯ抗体、抗ヒトＢ型肝炎ウイルス表面抗原抗体（ＨＢｓ抗原）、抗ヒトＣ型肝炎ウイルス抗体、抗リュウマチ因子等が挙げられる。
前記の抗原としては、癌胎児性抗原（ＣＥＡ）等が挙げられる。
前記のホルモンとしては、甲状腺刺激ホルモン（ＴＳＨ）、甲状腺ホルモン（ＦＴ３、ＦＴ４、Ｔ３、Ｔ４）、副甲状腺ホルモン（ＰＴＨ）、ヒト絨毛性ゴナドトロピン（ｈＣＧ）、エストラジオール（Ｅ２）等が挙げられる。
なお、前記の抗原及び抗体としては、癌マーカー［ＰＩＶＫＡ－ＩＩ抗原、α－フェトプロテイン（ＡＦＰ）、癌胎児性抗原（ＣＥＡ）、ＣＡ１９－９、前立腺特異抗原（ＰＳＡ）等］及び心疾患マーカー［トロポニンＴ（ＴｎＴ）、ヒト脳性ナトリウム利尿ペプチド前駆体Ｎ端フラグメント（ＮＴ－ｐｒｏＢＮＰ）等］として知られている物質等も挙げられる。
上記に例示したものの中でも、商業的ニーズの観点から、抗体、ホルモン、癌マーカー及び心疾患マーカーからなる群より選ばれる少なくとも１種が好ましい。
As described above, the substance (FC) in the present invention is a substance obtained by labeling the substance (F) with a labeling substance (C). The substance (F) includes at least one substance selected from the group consisting of a substance to be measured (F1), a substance similar to the substance to be measured (F2), and a substance that specifically binds to the substance to be measured (F3).
The measurement target substance (F1) in the present invention may be a substance contained in a sample derived from a living organism [biological fluids (serum, blood, lymph, ascites, urine, etc.), various cells, culture fluids, etc.)].
Specifically, proteins [albumin, hemoglobin, myoglobin, transferrin, protein A, C-reactive protein (CRP), lipid proteins, enzymes, immunoglobulins, immunoglobulin fragments, blood coagulation-related factors, antibodies, antigens, hormones, etc.],
Nucleic acids [deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), etc.],
Drugs [antiepileptic drugs, antibiotics, theophylline, etc.]
Viruses [hepatitis C virus, hepatitis B virus, hepadnavirus, adenovirus, rhabdovirus, flavivirus, retrovirus, herpes virus, osomyxovirus, etc.],
Bacteria [O-157, Helicobacter pylori, Salmonella, etc.]
Examples of such cells include fat cells, ES cells, hepatic cells, stem cells, endothelial cells, epithelial cells, muscle cells, endocrine cells, exocrine cells, nerve cells, tumor cells, and IPS cells.
Examples of the enzymes include alkaline phosphatase, amylase, acid phosphatase, γ-glutamyltransferase (γ-GTP), lipase, creatine kinase (CK), lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), renin, protein kinase (PK), and tyrosine kinase.
Examples of the lipid protein include high density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein.
The immunoglobulins include IgG, IgM, IgA, IgD, IgE, and the like.
The immunoglobulin fragments include the Fc portion, the Fab portion, and the F(ab) 2 portion.
Examples of the blood coagulation-related factors include fibrinogen, fibrin degradation products (FDPs), prothrombin, thrombin, and the like.
Examples of the antibody include anti-streptolysin O antibody, anti-human hepatitis B virus surface antigen antibody (HBs antigen), anti-human hepatitis C virus antibody, and anti-rheumatoid factor.
The antigens include carcinoembryonic antigen (CEA) and the like.
Examples of the hormones include thyroid stimulating hormone (TSH), thyroid hormones (FT3, FT4, T3, T4), parathyroid hormone (PTH), human chorionic gonadotropin (hCG), estradiol (E2), and the like.
In addition, examples of the antigens and antibodies include substances known as cancer markers [PIVKA-II antigen, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA19-9, prostate-specific antigen (PSA), etc.] and heart disease markers [troponin T (TnT), N-terminal fragment of human brain natriuretic peptide precursor (NT-proBNP), etc.].
Among the above-listed examples, from the viewpoint of commercial needs, at least one selected from the group consisting of antibodies, hormones, cancer markers and heart disease markers is preferred.
本発明における測定対象物質の類似物質（アナログ）（Ｆ２）は、「測定対象物質と特異的に結合する物質（Ｆ３）」が有する「測定対象物質（Ｆ１）との結合部位」と結合し得るもの、言い換えれば、「測定対象物質（Ｆ１）」が有する「測定対象物質と特異的に結合する物質（Ｆ３）」との結合部位を有するもの、更に言い換えれば、「測定対象物質（Ｆ１）」と「測定対象物質と特異的に結合する物質（Ｆ３）」との反応時に共存させると、該反応と競合し得るものであれば何れでもよい。 In the present invention, the analogue (F2) of the substance to be measured may be any substance capable of binding to the "binding site for the substance to be measured (F1)" of the "substance (F3) that specifically binds to the substance to be measured," in other words, any substance that has a binding site for the "substance to be measured (F1)" of the "substance to be measured (F3) that specifically binds to the substance to be measured," or in other words, any substance that can compete with the reaction of the "substance to be measured (F1)" and the "substance that specifically binds to the substance to be measured (F3)" when present together.
本発明における「測定対象物質と特異的に結合する物質（Ｆ３）」としては、例えば「抗原」－「抗体」間反応、「糖鎖」－「タンパク質」間反応、「糖鎖」－「レクチン」間反応、「酵素」－「インヒビター」間反応、「タンパク質」－「ペプチド鎖」間反応、「染色体又はヌクレオチド鎖」－「ヌクレオチド鎖」間反応、「ヌクレオチド鎖」－「タンパク質」間反応等の相互反応によって、「測定対象物質又はその類似物質（Ｆ２）」と結合するもの等が挙げられる。
上記各組合せにおいて何れか一方が「測定対象物質（Ｆ１）又はその類似物質（Ｆ２）」である場合、他の一方がこの「測定対象物質と特異的に結合する物質（Ｆ３）」となる。
例えば、「測定対象物質（Ｆ１）又はその類似物質（Ｆ２）」が「抗原」であるときは、「測定対象物質と特異的に結合する物質（Ｆ３）」は「抗体」であり、「測定対象物質（Ｆ１）又はその類似物質（Ｆ２）」が「抗体」であるときは、「測定対象物質と特異的に結合する物質（Ｆ３）」は「抗原」である（以下、その他の上記各組合せにおいても同様である）。
In the present invention, the "substance (F3) that specifically binds to the measured substance" may be, for example, a substance that binds to the "measured substance or a substance similar thereto (F2)" through interactions such as an "antigen"-"antibody" reaction, a "sugar chain"-"protein" reaction, a "sugar chain"-"lectin" reaction, an "enzyme"-"inhibitor" reaction, a "protein"-"peptide chain" reaction, a "chromosome or nucleotide chain"-"nucleotide chain" reaction, and a "nucleotide chain"-"protein" reaction.
In each of the above combinations, when one of them is a "substance to be measured (F1) or an analogue thereof (F2)", the other is a "substance that specifically binds to the substance to be measured (F3)".
For example, when the "substance to be measured (F1) or its analogue (F2)" is an "antigen," the "substance that specifically binds to the substance to be measured (F3)" is an "antibody," and when the "substance to be measured (F1) or its analogue (F2)" is an "antibody," the "substance that specifically binds to the substance to be measured (F3)" is an "antigen" (the same applies to the other combinations mentioned below).
（Ｆ３）としては、前記の測定対象物質（Ｆ１）として例示した物質及びこれらに対する抗体等が挙げられる。
尚、本発明において用いられる抗体には、パパインやペプシン等の蛋白質分解酵素、或いは化学的分解により生じるＦａｂ、Ｆ（ａｂ’）２フラグメント等の分解産物も包含される。
また、測定対象物質（Ｆ１）がＰＩＶＫＡ－ＩＩ抗原である場合は、（Ｆ３）としては、抗プロトロンビンモノクローナル抗体（抗ＰＩＶＫＡ－ＩＩモノクローナル抗体等）であることが好ましい。
Examples of (F3) include the substances exemplified as the above-mentioned measurement target substance (F1) and antibodies against these substances.
The antibodies used in the present invention also include decomposition products such as Fab and F(ab') 2 fragments produced by proteolytic enzymes such as papain and pepsin, or by chemical decomposition.
When the substance to be measured (F1) is a PIVKA-II antigen, (F3) is preferably an anti-prothrombin monoclonal antibody (such as an anti-PIVKA-II monoclonal antibody).
上記の測定対象物質と特異的に結合する物質（Ｆ３）としては、「抗原」－「抗体」間反応或いは「糖鎖－タンパク質」間反応によって、測定対象物質（Ｆ１）又はその類似物質（Ｆ２）と結合するものが、入手が容易であることから好ましい。
具体的には、測定対象物質（Ｆ１）又はその類似物質（Ｆ２）に対する抗体、測定対象物質（Ｆ１）又はその類似物質（Ｆ２）が結合する抗原及び測定対象物質（Ｆ１）又はその類似物質（Ｆ２）に結合するタンパク質が好ましく、測定対象物質（Ｆ１）又はその類似物質（Ｆ２）に対する抗体及び測定対象物質（Ｆ１）又はその類似物質（Ｆ２）に結合するタンパク質が更に好ましい。
As the substance (F3) that specifically binds to the above-mentioned measured substance, a substance that binds to the measured substance (F1) or its analogue (F2) through an "antigen"-"antibody" reaction or a "glycan-protein" reaction is preferred because it is easily available.
Specifically, antibodies against the substance to be measured (F1) or its analogue (F2), antigens to which the substance to be measured (F1) or its analogue (F2) bind, and proteins that bind to the substance to be measured (F1) or its analogue (F2) are preferred, and antibodies against the substance to be measured (F1) or its analogue (F2) and proteins that bind to the substance to be measured (F1) or its analogue (F2) are even more preferred.
前記の物質（Ｆ）としては、保存安定性の観点から、抗プロトロンビンモノクローナル抗体が好ましい。 From the viewpoint of storage stability, anti-prothrombin monoclonal antibody is preferable as the substance (F).
本発明に用いられる標識物質（Ｃ）は、物質（Ｆ）を標識するために用いられるものであり、例えば酵素免疫測定法（ＥＩＡ）において用いられるアルカリホスファターゼ、β－ガラクトシダーゼ、ペルオキシダーゼ（以下においてＰＯＤと略記することがある）、マイクロペルオキシダーゼ、グルコースオキシダーゼ、グルコース－６－リン酸脱水素酵素、リンゴ酸脱水素酵素、ルシフェラーゼ、チロシナーゼ、酸性ホスファターゼ等の酵素類；
例えば放射免疫測定法（ＲＩＡ）において用いられる９９ｍＴｃ、１３１Ｉ、１２５Ｉ、１４Ｃ、３Ｈ、３２Ｐ等の放射性同位元素；
例えば蛍光免疫測定法（ＦＩＡ）において用いられるフルオレセイン、ダンシル、フルオレスカミン、クマリン、ナフチルアミン又はこれらの誘導体、グリーン蛍光タンパク質（ＧＦＰ）等の蛍光性物質；
例えばルシフェリン、イソルミノール、ルミノール、ビス（２，４，６－トリフロロフェニル）オキザレート等の発光性物質；
例えばフェノール、ナフトール、アントラセン又はこれらの誘導体等の紫外部に吸収を有する物質；
例えば４－アミノ－２，２，６，６－テトラメチルピペリジン－１－オキシル、３－アミノ－２，２，５，５－テトラメチルピロリジン－１－オキシル、２，６－ジ－ｔ－ブチル－α－（３，５－ジ－ｔ－ブチル－４－オキソ－２，５－シクロヘキサジエン－１－イリデン）－ｐ－トリオキシル等のオキシル基を有する化合物に代表されるスピンラベル化剤としての性質を有する物質等が挙げられる。
これらのうち、保存安定性の観点から、酵素及び蛍光性物質が好ましく、さらに好ましいのはアルカリホスファターゼ、ペルオキシダーゼ及びグルコースオキシダーゼであり、特に好ましいのはペルオキシダーゼである。
（Ｃ）は１種を用いてもよく、２種以上を併用してもよい。
The labeling substance (C) used in the present invention is used to label the substance (F), and examples of such labeling substances include enzymes used in enzyme immunoassay (EIA), such as alkaline phosphatase, β-galactosidase, peroxidase (hereinafter sometimes abbreviated as POD), microperoxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, luciferase, tyrosinase, and acid phosphatase;
Radioisotopes such as 99mTc, 131I, 125I, 14C, 3H, 32P, etc., used in radioimmunoassays (RIAs);
Fluorescent substances such as fluorescein, dansyl, fluorescamine, coumarin, naphthylamine or derivatives thereof, green fluorescent protein (GFP), etc., used in, for example, fluorescent immunoassay (FIA);
Luminescent substances such as luciferin, isoluminol, luminol, and bis(2,4,6-trifluorophenyl)oxalate;
Substances that absorb in the ultraviolet region, such as phenol, naphthol, anthracene, or derivatives thereof;
Examples of the compound include substances having properties as a spin labeling agent, such as compounds having an oxyl group, such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, 3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxyl, and 2,6-di-t-butyl-α-(3,5-di-t-butyl-4-oxo-2,5-cyclohexadiene-1-ylidene)-p-trioxyl.
Among these, from the viewpoint of storage stability, enzymes and fluorescent substances are preferred, alkaline phosphatase, peroxidase and glucose oxidase are more preferred, and peroxidase is particularly preferred.
(C) may be used alone or in combination of two or more kinds.
本発明において物質（ＦＣ）は、上記物質（Ｆ）が標識物質（Ｃ）により標識されてなる物質である。標識物質（Ｃ）を物質（Ｆ）に結合させて物質（ＦＣ）とする方法としては、一般的にこの分野で用いられる方法［例えば、医化学実験講座、第８巻、山村雄一監修、第１版、中山書店、１９７１；図説 蛍光抗体、川生明著、第１版、（株）ソフトサイエンス社、１９８３；酵素免疫測定法、石川栄治、河合忠、室井潔編、第２版、医学書院、１９８２等］等を利用すればよい。 In the present invention, the substance (FC) is a substance obtained by labeling the above-mentioned substance (F) with a labeling substance (C). The method for binding the labeling substance (C) to the substance (F) to obtain the substance (FC) may be a method generally used in this field [e.g., Medical Chemistry Experiment Lectures, Vol. 8, edited by Yamamura Yuichi, 1st Edition, Nakayama Shoten, 1971; Illustrated Fluorescent Antibodies, by Kawao Akira, 1st Edition, Soft Science Co., Ltd., 1983; Enzyme Immunoassay, edited by Ishikawa Eiji, Kawai Tadashi, and Muroi Kiyoshi, 2nd Edition, Igaku Shoin, 1982, etc.].
本発明において、免疫測定用試薬（Ｘ）中の物質（ＦＣ）の含有量は、免疫測定用試薬（Ｘ）の重量を基準として、保存安定性の観点から、０．０５～０．４重量％が好ましく、さらに好ましくは０．１～０．３重量％である。 In the present invention, the content of the substance (FC) in the immunoassay reagent (X) is preferably 0.05 to 0.4% by weight, and more preferably 0.1 to 0.3% by weight, based on the weight of the immunoassay reagent (X) from the viewpoint of storage stability.
本発明における化合物（Ｄ）は、前記の通り、ジギトニン、ククルビタシンＢ、ウアバイン及びサポニン（ステロイドサポニン及びトリテルペノイドサポニン等）からなる群から選ばれる少なくとも１種の化合物である。 As described above, compound (D) in the present invention is at least one compound selected from the group consisting of digitonin, cucurbitacin B, ouabain, and saponins (steroid saponins, triterpenoid saponins, etc.).
本発明において、免疫測定用試薬（Ｘ）中の化合物（Ｄ）の含有量は、保存安定性の観点から、免疫測定用試薬（Ｘ）の重量を基準として、０．５１～１．５０重量％であり、０．７５～１．５０重量％が好ましく、さらに好ましくは０．９５～１．５０重量％である。 In the present invention, the content of compound (D) in the immunoassay reagent (X) is 0.51 to 1.50% by weight, preferably 0.75 to 1.50% by weight, and more preferably 0.95 to 1.50% by weight, based on the weight of the immunoassay reagent (X), from the viewpoint of storage stability.
免疫測定用試薬（Ｘ）中には、さらにタンパク質を含有してもよい。タンパク質を含有すると、測定毎の測定値のぶれを抑制できる（同時再現性の向上）ため、好ましい。
タンパク質としては、同時再現性の観点から、カゼインが好ましい。
（Ｘ）中のタンパク質の含有量は、免疫測定用試薬（Ｘ）の重量を基準として、同時再現性の観点から、０．００５～１．０重量％が好ましく、さらに好ましくは０．０１～０．０３重量％である。
また、同時再現性を更に高める観点からは、カゼインの重量と前記の（Ｄ）の重量との比率［カゼインの重量／（化合物（Ｄ）の合計重量）］が、０．８～５．０であることが好ましい。
The immunoassay reagent (X) may further contain a protein. If a protein is contained, the fluctuation of the measured value between measurements can be suppressed (improvement of simultaneous reproducibility), which is preferable.
From the viewpoint of simultaneous reproducibility, casein is preferred as the protein.
The protein content in (X) is preferably 0.005 to 1.0% by weight, more preferably 0.01 to 0.03% by weight, based on the weight of the immunoassay reagent (X) from the viewpoint of simultaneous reproducibility.
From the viewpoint of further improving the simultaneous reproducibility, it is preferable that the ratio of the weight of casein to the weight of (D) [weight of casein/(total weight of compound (D)] is 0.8 to 5.0.
本発明の免疫測定用試薬（Ｘ）は、保存安定性が高いので、免疫測定における標識試薬（Ａ）として好ましく用いることができる。 The immunoassay reagent (X) of the present invention has high storage stability and can therefore be preferably used as a labeling reagent (A) in immunoassays.
本発明の免疫測定用キットは、標識試薬（Ａ）及び固相担体試薬（Ｅ）を含む免疫測定用キットであって、標識試薬（Ａ）が上記本発明の免疫測定用試薬（Ｘ）であり、固相担体試薬（Ｅ）が固相担体（Ｂ）の表面上に物質（Ｆ）が固定化された固相担体（ＢＦ）を含有するものである免疫測定用キットである。 The immunoassay kit of the present invention is an immunoassay kit that includes a labeled reagent (A) and a solid phase carrier reagent (E), in which the labeled reagent (A) is the above-mentioned immunoassay reagent (X) of the present invention, and the solid phase carrier reagent (E) contains a solid phase carrier (BF) having a substance (F) immobilized on the surface of the solid phase carrier (B).
本発明において、固相担体（Ｂ）は、一般的にこの分野で使用されるものであれば特に限定されないが、例えばガラスビーズ、ポリスチレンビーズ、磁性シリカ粒子（Ｂ１）、マイクロプレート、ラテックス等が代表的なものとして挙げられる。
磁性シリカ粒子（Ｂ１）としては、特開２０１４－２１０６８０号公報及び特開２０１３－０１９８８９号公報等に記載の公知の磁性シリカ粒子等が挙げられる。
これらのうち、短時間で測定できる観点から、磁性シリカ粒子（Ｂ１）が好ましい。
In the present invention, the solid phase carrier (B) is not particularly limited as long as it is one generally used in this field, and representative examples thereof include glass beads, polystyrene beads, magnetic silica particles (B1), microplates, latex, etc.
Examples of the magnetic silica particles (B1) include known magnetic silica particles described in JP-A-2014-210680 and JP-A-2013-019889.
Of these, the magnetic silica particles (B1) are preferred from the viewpoint of enabling measurement in a short period of time.
前記の磁性シリカ粒子（Ｂ１）は、シリカのマトリックス中に体積平均粒子径が１～１５ｎｍで超常磁性を有する金属酸化物を分散されているものである。超常磁性とは、外部磁場の存在下で物質の個々の原子磁気モーメントが整列し誘発された一時的な磁場を示し、外部磁場を取り除くと、部分的な整列が損なわれ磁場を示さなくなることをいう。
なお、金属酸化物の体積平均粒子径は、任意の２００個の粒子について走査型電子顕微鏡（日本電子株式会社製「ＪＳＭ－７０００Ｆ」等）で観察して測定された粒子径の平均値である。
The magnetic silica particles (B1) have a volume average particle size of 1 to 15 nm and a metal oxide having superparamagnetism dispersed in a silica matrix. Superparamagnetism refers to a temporary magnetic field induced by the alignment of individual atomic magnetic moments of a substance in the presence of an external magnetic field, and when the external magnetic field is removed, the partial alignment is lost and the magnetic field is no longer exhibited.
The volume average particle size of the metal oxide is the average value of particle sizes measured by observing 200 random particles with a scanning electron microscope (such as "JSM-7000F" manufactured by JEOL Ltd.).
体積平均粒子径が１～１５ｎｍで超常磁性を示す超常磁性金属酸化物としては、鉄、コバルト、ニッケル及びこれらの合金等の酸化物が挙げられるが、磁界に対する感応性が優れていることから、酸化鉄が特に好ましい。超常磁性金属酸化物は、１種を単独で用いても２種以上を併用してもよい。 Superparamagnetic metal oxides that have a volume average particle size of 1 to 15 nm and exhibit superparamagnetism include oxides of iron, cobalt, nickel, and alloys thereof, among which iron oxide is particularly preferred due to its excellent sensitivity to magnetic fields. Superparamagnetic metal oxides may be used alone or in combination of two or more types.
酸化鉄としては、公知の種々の酸化鉄を用いることができる。酸化鉄の内、特に化学的
な安定性に優れることから、マグネタイト、γ－ヘマタイト、マグネタイト－α－ヘマタイト中間酸化鉄及びγ－ヘマタイト－α－ヘマタイト中間酸化鉄が好ましく、大きな飽和磁化を有し、外部磁場に対する感応性が優れていることから、マグネタイトが更に好ましい。
As the iron oxide, various known iron oxides can be used. Among the iron oxides, magnetite, γ-hematite, magnetite-α-hematite intermediate iron oxide, and γ-hematite-α-hematite intermediate iron oxide are preferred because of their excellent chemical stability, and magnetite is more preferred because of its large saturation magnetization and excellent sensitivity to an external magnetic field.
前記の磁性シリカ粒子（Ｂ１）中の超常磁性金属酸化物の含有量の下限は、６０重量％が好ましく、さらに好ましくは６５重量％であり、上限は９５重量％が好ましく、さらに好ましくは８０重量％である。
超常磁性金属酸化物の含有量が６０重量％以上であると、得られた磁性シリカ粒子（Ｂ１）の磁性が十分であるため、実際の用途面における分離操作に時間がかからないので好ましい。また９５重量％以下であると、合成が容易であるので好ましい。
The lower limit of the content of the superparamagnetic metal oxide in the magnetic silica particles (B1) is preferably 60% by weight, more preferably 65% by weight, and the upper limit is preferably 95% by weight, more preferably 80% by weight.
If the content of the superparamagnetic metal oxide is 60% by weight or more, the magnetic properties of the magnetic silica particles (B1) obtained are sufficient, so that the separation operation in practical applications does not take much time, which is preferable. Also, if the content is 95% by weight or less, the synthesis is easy, which is preferable.
超常磁性金属酸化物の製造方法は、特に限定されないが、Ｍａｓｓａｒｔにより報告されたものをベースとして水溶性鉄塩及びアンモニアを用いる共沈殿法（Ｒ．Ｍａｓｓａｒｔ，ＩＥＥＥ Ｔｒａｎｓ．Ｍａｇｎ．１９８１，１７，１２４７）や水溶性鉄塩の水溶液中の酸化反応を用いた方法により合成することができる。 The method for producing the superparamagnetic metal oxide is not particularly limited, but it can be synthesized by a coprecipitation method using a water-soluble iron salt and ammonia based on the method reported by Massart (R. Massart, IEEE Trans. Magn. 1981, 17, 1247) or a method using an oxidation reaction in an aqueous solution of a water-soluble iron salt.
前記の磁性シリカ粒子（Ｂ１）の体積平均粒子径は、好ましくは１～５μｍ、更に好ましくは１～３μｍである。体積平均粒子径が１μｍ以上であると、分離回収の際に時間がかからない傾向にある。また、５μｍ以下であると、表面積が比較的大きくなる結果、固定化する物質（測定対象物質、測定対象物質の類似物質又は測定対象物質と特異的に結合する物質）の結合量も多くなり、結合効率が上昇する傾向にある。 The volume average particle diameter of the magnetic silica particles (B1) is preferably 1 to 5 μm, and more preferably 1 to 3 μm. If the volume average particle diameter is 1 μm or more, separation and recovery tend not to take much time. Furthermore, if it is 5 μm or less, the surface area becomes relatively large, and as a result, the amount of the immobilized substance (the substance to be measured, a substance similar to the substance to be measured, or a substance that specifically binds to the substance to be measured) that is bound also increases, and the binding efficiency tends to increase.
前記の磁性シリカ粒子（Ｂ１）の体積平均粒子径は、任意の２００個の磁性シリカ粒子について走査型電子顕微鏡（日本電子株式会社製「ＪＳＭ－７０００Ｆ」）で観察して測定された粒子径の平均値である。 The volume average particle diameter of the magnetic silica particles (B1) is the average particle diameter measured by observing 200 random magnetic silica particles with a scanning electron microscope (JSM-7000F manufactured by JEOL Ltd.).
前記の磁性シリカ粒子（Ｂ１）の体積平均粒子径は、後述の水中油型エマルションを作製する際の混合条件（せん断力等）を調節して水中油型エマルションの粒子径を調整することにより制御することができる。また、磁性シリカ粒子製造時の水洗工程の条件変更や一般的な分級等の方法によっても平均粒子径を所望の値とすることができる。 The volume average particle size of the magnetic silica particles (B1) can be controlled by adjusting the mixing conditions (shear force, etc.) when preparing the oil-in-water emulsion described below to adjust the particle size of the oil-in-water emulsion. The average particle size can also be adjusted to a desired value by changing the conditions of the water washing process during the production of magnetic silica particles or by general classification methods.
前記の磁性シリカ粒子（Ｂ１）は、例えば、体積平均粒子径が１～１５ｎｍの超常磁性金属酸化物粒子の重量に基づいて、３０～５００重量％の（アルキル）アルコキシシラン及び分散剤を含有する分散液と、水、水溶性有機溶媒、非イオン性界面活性剤及び（アルキル）アルコキシシランの加水分解用触媒を含有する溶液とを混合して水中油型エマルションを形成後、（アルキル）アルコキシシランの加水分解反応及び縮合反応を行い、超常磁性金属酸化物がシリカに包含された磁性シリカ粒子の水性分散体が得た後、磁性シリカ粒子の水性分散体を遠心分離及び／又は集磁により固液分離し、水及びメタノール等で洗浄することにより得られる。
上記及び以下において、（アルキル）アルコキシシランとは、アルキルアルコキシシラン及び／又はアルコキシシランを意味する。
The magnetic silica particles (B1) can be obtained, for example, by mixing a dispersion containing 30 to 500% by weight of (alkyl)alkoxysilane and a dispersant based on the weight of superparamagnetic metal oxide particles having a volume average particle diameter of 1 to 15 nm with a solution containing water, a water-soluble organic solvent, a nonionic surfactant, and a catalyst for hydrolysis of (alkyl)alkoxysilane to form an oil-in-water emulsion, then carrying out a hydrolysis reaction and a condensation reaction of the (alkyl)alkoxysilane to obtain an aqueous dispersion of magnetic silica particles in which the superparamagnetic metal oxide is encapsulated in silica, and then subjecting the aqueous dispersion of magnetic silica particles to solid-liquid separation by centrifugation and/or magnetic collection, and washing with water, methanol, or the like.
Above and below, (alkyl)alkoxysilane means alkylalkoxysilane and/or alkoxysilane.
前記の磁性シリカ粒子（Ｂ１）は、超常磁性金属酸化物がシリカに包含され、粒子表面に存在しないことから、多くの測定対象物質、測定対象物質の類似物質、又は測定対象物質と特異的に結合する物質をその表面に固定化することができる。 The magnetic silica particles (B1) have superparamagnetic metal oxides encapsulated in silica and not present on the particle surface, so that many target substances, substances similar to the target substances, or substances that specifically bind to the target substances can be immobilized on their surfaces.
本発明において、固相担体（Ｂ）｛好ましくは磁性シリカ粒子（Ｂ１）｝に、測定対象物質（Ｆ１）、測定対象物質の類似物質（Ｆ２）及び測定対象物質と特異的に結合する物質（Ｆ３）からなる群より選ばれる少なくとも１種を固定化し、固相担体（Ｂ）の表面に
物質（Ｆ）が固定化された固相担体（ＢＦ）を製造する方法としては、上述の（Ｂ）に測定対象物質（Ｆ１）等を物理吸着させる方法が挙げられるが、より効率良く測定対象物質（Ｆ１）等を固定化させる観点から、グルタルアルデヒド、アルブミン、カルボジイミド、ストレプトアビジン、ビオチン及び官能基を有するアルキルアルコキシシランからなる群から選ばれる少なくとも１種の有機化合物を（Ｂ）表面に結合させ、それらを介して測定対象物質（Ｆ１）等を（Ｂ）に固定化させる方法が好ましい。
（Ｂ）表面に結合させる有機化合物の内、特定の測定対象物質（Ｆ１）等を結合させる観点から、官能基（エチレン性不飽和基、エポキシ基、アミノ基、メルカプト基及びイソシアネート基等）を有するアルキルアルコキシシランが更に好ましい。
このような官能基を有するアルキルアルコキシシランとしては、ビニルトリメトキシシラン、ビニルトリエトキシシラン、２－（３，４－エポキシシクロヘキシル）エチルトリメトキシシラン、３－グリシドキシプロピルメチルジメトキシシラン、３－グリシドキシプロピルトリメトキシシラン、３－グリシドキシプロピルメチルジエトキシシラン、３－グリシドキシプロピルトリエトキシシラン、ｐ－スチリルトリメトキシシラン、３－メタクリロキシプロピルメチルジメトキシシラン、３－メタクリロキシプロピルトリメトキシシラン、３－メタクリロキシプロピルメチルジエトキシシラン、３－メタクリロキシプロピルトリエトキシシラン、３－アクリロキシプロピルトリメトキシシラン、Ｎ－２－（アミノエチル）－３－アミノプロピルメチルジメトキシシラン、Ｎ－２－（アミノエチル）－３－アミノプロピルトリメトキシシラン、３－アミノプロピルトリメトキシシラン、３－アミノプロピルトリエトキシシラン、３－トリエトキシシリル－Ｎ－（１，３－ジメチル－ブチリデン）プロピルアミン、Ｎ－フェニル－３－アミノプロピルトリメトキシシラン、トリス－（トリメトキシシリルプロピル）イソシアヌレート、３－ウレイドプロピルトリアルコキシシラン、３－メルカプトプロピルメチルジメトキシシラン、３－メルカプトプロピルトリメトキシシラン、３－イソシアネートプロピルトリエトキシシラン等が挙げられる。
In the present invention, a method for producing a solid phase carrier (BF) in which at least one selected from the group consisting of a substance to be measured (F1), a substance similar to the substance to be measured (F2), and a substance that specifically binds to the substance to be measured (F3) is immobilized on a solid phase carrier (B) {preferably magnetic silica particles (B1)} and a substance (F) is immobilized on the surface of the solid phase carrier (B) includes a method in which the substance to be measured (F1) or the like is physically adsorbed on the above-mentioned (B). However, from the viewpoint of more efficiently immobilizing the substance to be measured (F1) or the like, a method in which at least one organic compound selected from the group consisting of glutaraldehyde, albumin, carbodiimide, streptavidin, biotin, and alkylalkoxysilanes having a functional group is bound to the surface of (B) and the substance to be measured (F1) or the like is immobilized on (B) via them is preferred.
Of the organic compounds to be bonded to the surface of (B), from the viewpoint of bonding a specific substance to be measured (F1) or the like, alkylalkoxysilanes having functional groups (ethylenically unsaturated groups, epoxy groups, amino groups, mercapto groups, isocyanate groups, and the like) are more preferred.
Examples of alkylalkoxysilanes having such functional groups include vinyltrimethoxysilane, vinyltriethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyl trimethoxysilane, N-2-(aminoethyl)-3-aminopropyl methyl dimethoxysilane, N-2-(aminoethyl)-3-aminopropyl trimethoxysilane, 3-aminopropyl trimethoxysilane, 3-aminopropyl triethoxysilane, 3-triethoxysilyl-N-(1,3-dimethyl-butylidene)propylamine, N-phenyl-3-aminopropyl trimethoxysilane, tris-(trimethoxysilylpropyl)isocyanurate, 3-ureidopropyl trialkoxysilane, 3-mercaptopropyl methyl dimethoxysilane, 3-mercaptopropyl trimethoxysilane, 3-isocyanatopropyl triethoxysilane, and the like.
固相担体試薬（Ｅ）は、上記固相担体（Ｂ）の表面上に物質（Ｆ）が固定化された固相担体（ＢＦ）を含有するものである。
前記の固相担体（Ｂ）の表面上に固定化する物質（Ｆ）としては、物質（ＦＣ）の説明の際に例示した物質（Ｆ）が挙げられる。
前記の固相担体（Ｂ）の表面上に固定化する物質（Ｆ）として好ましいものは、標識試薬（Ａ）における物質（ＦＣ）の種類にもよるが、標識試薬（Ａ）における物質（ＦＣ）がＰＯＤ標識抗プロトロンビンモノクローナル抗体である場合は、抗ＰＩＶＫＡ－ＩＩモノクローナル抗体が好ましい。
The solid phase carrier reagent (E) contains a solid phase carrier (BF) having a substance (F) immobilized on the surface of the solid phase carrier (B).
The substance (F) to be immobilized on the surface of the solid phase carrier (B) includes the substance (F) exemplified in the explanation of the substance (FC).
The substance (F) to be immobilized on the surface of the solid phase carrier (B) is preferably an anti-PIVKA-II monoclonal antibody, although it depends on the type of the substance (FC) in the labeled reagent (A). When the substance (FC) in the labeled reagent (A) is a POD-labeled anti-prothrombin monoclonal antibody, an anti-PIVKA-II monoclonal antibody is preferable.
本発明の免疫測定用キットは、上記固相担体試薬（Ｅ）及び標識試薬（Ａ）以外に、さらに緩衝液、ルミノール発光試薬（Ｋ）及び過酸化物水溶液（Ｌ）を含んでいてもよい。
ルミノール発光試薬（Ｋ）は、上記標識試薬（Ａ）中に含まれる物質（ＦＣ）を標識するものとして用いられている標識物質（Ｃ）に基づいて選択され、例えば、標識物質（Ｃ）がペルオキシダーゼである場合、２，３－ジヒドロ－１，４－フタラジンジオン化合物及び化学発光増強剤を必須構成成分としてなるルミノール発光試薬等が挙げられる。
過酸化物水溶液（Ｌ）は、過酸化物及び水を必須構成成分としてなる過酸化物水溶液である。
The immunoassay kit of the present invention may further contain a buffer solution, a luminol luminescence reagent (K) and an aqueous peroxide solution (L) in addition to the above-mentioned solid phase carrier reagent (E) and labeling reagent (A).
The luminol luminescence reagent (K) is selected based on the labeling substance (C) used to label the substance (FC) contained in the labeling reagent (A). For example, when the labeling substance (C) is peroxidase, the luminol luminescence reagent may include a luminol luminescence reagent containing a 2,3-dihydro-1,4-phthalazinedione compound and a chemiluminescence enhancer as essential components.
The aqueous peroxide solution (L) is an aqueous peroxide solution containing a peroxide and water as essential components.
本発明の免疫測定用キットに用いる緩衝液としては、一般的に免疫測定の分野で用いられている、トリス緩衝液、リン酸緩衝液、ベロナール緩衝液、ホウ酸緩衝液及びグッド緩衝液等が挙げられ、そのｐＨは、本発明の効果を阻害しない範囲であればよく、５～９が好ましい。
また、このような緩衝液中には、本発明の効果を阻害しない範囲であれば、塩（塩化ナトリウム等）、アルブミン、グロブリン、水溶性ゼラチン、ポリエチレングリコール等の
安定化剤、界面活性剤及び糖類等を含有させておいてもよい。
Buffer solutions used in the immunoassay kit of the present invention include Tris buffer, phosphate buffer, veronal buffer, borate buffer, Good's buffer, etc., which are commonly used in the field of immunoassays. The pH of the solution may be within a range that does not inhibit the effects of the present invention, and a range of 5 to 9 is preferable.
In addition, such a buffer solution may contain salts (e.g., sodium chloride), albumin, globulin, water-soluble gelatin, stabilizers such as polyethylene glycol, surfactants, sugars, etc., as long as the effects of the present invention are not impaired.
ルミノール発光試薬（Ｋ）において、２，３－ジヒドロ－１，４－フタラジンジオン化合物としては、例えば、特開平２－２９１２９９号公報、特開平１０－３１９０１５号公報及び特開２０００－２７９１９６号公報等に記載の公知の２，３－ジヒドロ－１，４－フタラジンジオン化合物並びにこれらの混合物等が使用できる。
これらの内、ルミノール、イソルミノール、Ｎ－アミノヘキシル－Ｎ－エチルイソルミノール（ＡＨＥＩ）、Ｎ－アミノブチル－Ｎ－エチルイソルミノール（ＡＢＥＩ）及びこれらの金属塩（アルカリ金属塩等）が好ましく、更に好ましいのはルミノール及びその金属塩、特に好ましいのはルミノールのナトリウム塩である。
In the luminol luminescence reagent (K), the 2,3-dihydro-1,4-phthalazinedione compound may be, for example, the known 2,3-dihydro-1,4-phthalazinedione compounds described in JP-A-2-291299, JP-A-10-319015, JP-A-2000-279196, or mixtures thereof.
Of these, luminol, isoluminol, N-aminohexyl-N-ethylisoluminol (AHEI), N-aminobutyl-N-ethylisoluminol (ABEI) and metal salts thereof (such as alkali metal salts) are preferred, luminol and metal salts thereof are more preferred, and sodium salt of luminol is particularly preferred.
化学発光増強剤としては、例えば、特開昭５９－５００２５２号公報、特開昭５９－１７１８３９号公報及び特開平２－２９１２９９号公報等に記載の公知の化学発光増強剤並びにこれらの混合物等が使用できる。
これらの内、化学発光増強効果等の観点から、フェノール（フェノール誘導体を含む）が好ましく、更に好ましいのはＰ－ヨードフェノール、４－（シアノメチルチオ）フェノール及び４－シアノメチルチオ－２－クロロフェノールであり、特に好ましいのは４－（シアノメチルチオ）フェノールである。
As the chemiluminescence enhancer, for example, known chemiluminescence enhancers described in JP-A-59-500252, JP-A-59-171839, JP-A-2-291299, etc., and mixtures thereof can be used.
Among these, from the viewpoint of chemiluminescence enhancing effect, etc., phenol (including phenol derivatives) is preferred, more preferred are p-iodophenol, 4-(cyanomethylthio)phenol and 4-cyanomethylthio-2-chlorophenol, and particularly preferred is 4-(cyanomethylthio)phenol.
ルミノール発光試薬（Ｋ）には、２，３－ジヒドロ－１，４－フタラジンジオン化合物及び化学発光増強剤以外に、緩衝液及び／又はキレート剤等を含むことができる。 The luminol luminescence reagent (K) may contain a buffer solution and/or a chelating agent in addition to the 2,3-dihydro-1,4-phthalazinedione compound and the chemiluminescence enhancer.
ルミノール発光試薬（Ｋ）が含有する緩衝液としては、例えば、特開平１０－３１９０１５号公報及び特開２００３－２７９４８９号公報等に記載の公知の緩衝液並びにこれらの混合物等が使用できる。
これらの内、化学発光増強効果及び保存安定性等の観点から、３－［４－（２－ヒドロキシエチル）－１－ピペラジニル］プロパンスルホン酸／水酸化ナトリウム緩衝液、２－ヒドロキシ－３－［４－（２－ヒドロキシエチル）－１－ピペラジニル］プロパンスルホン酸・１水和物／水酸化ナトリウム緩衝液及びピペラジニル－１，４－ビス（２－ヒドロキシ－３－プロパンスルホン酸）・２水和物／水酸化ナトリウム緩衝液が好ましく、更に好ましいのは３－［４－（２－ヒドロキシエチル）－１－ピペラジニル］プロパンスルホン酸／水酸化ナトリウム緩衝液及び２－ヒドロキシ－３－［４－（２－ヒドロキシエチル）－１－ピペラジニル］プロパンスルホン酸・１水和物／水酸化ナトリウム緩衝液であり、特に好ましいのは３－［４－（２－ヒドロキシエチル）－１－ピペラジニル］プロパンスルホン酸／水酸化ナトリウム緩衝液である。
As the buffer solution contained in the luminol luminescence reagent (K), for example, known buffer solutions as described in JP-A-10-319015 and JP-A-2003-279489, and mixtures thereof, can be used.
Among these, from the viewpoints of chemiluminescence enhancing effect and storage stability, etc., 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid/sodium hydroxide buffer solution, 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid.monohydrate/sodium hydroxide buffer solution, and piperazinyl-1,4-bis(2-hydroxy-3-propanesulfonic acid).dihydrate/sodium hydroxide buffer solution are preferred, more preferred are 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid/sodium hydroxide buffer solution, and 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid.monohydrate/sodium hydroxide buffer solution, and particularly preferred is 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid/sodium hydroxide buffer solution.
ルミノール発光試薬（Ｋ）が含有するキレート剤としては、例えば、特開平９－７５０９９号公報及び特開２００３－２７９４８９号公報等に記載の公知のキレート剤並びにこれらの混合物等が使用できる。
これらの内、化学発光増強効果及び保存安定性等の観点から、４配位キレート剤が好ましく、更に好ましいのはエチレンジアミン四酢酸（ＥＤＴＡ）及びその塩（エチレンジアミン四酢酸二ナトリウム、エチレンジアミン四酢酸三ナトリウム、エチレンジアミン四酢酸四ナトリウム、エチレンジアミン四酢酸二カリウム及びエチレンジアミン四酢酸三カリウム等）並びにトランス－１，２ジアミノシクロヘキサン－Ｎ，Ｎ，Ｎ’，Ｎ’－四酢酸（ＣｙＤＴＡ）であり、特に好ましいのはエチレンジアミン四酢酸（ＥＤＴＡ）及びその塩である。
As the chelating agent contained in the luminol luminescence reagent (K), for example, known chelating agents described in JP-A-9-75099 and JP-A-2003-279489 and mixtures thereof can be used.
Among these, from the viewpoints of chemiluminescence enhancing effect and storage stability, etc., tetracoordinate chelating agents are preferred, more preferred are ethylenediaminetetraacetic acid (EDTA) and salts thereof (disodium ethylenediaminetetraacetate, trisodium ethylenediaminetetraacetate, tetrasodium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, tripotassium ethylenediaminetetraacetate, etc.) and trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CyDTA), and particularly preferred are ethylenediaminetetraacetic acid (EDTA) and salts thereof.
ルミノール発光試薬（Ｋ）は、液体であることが好ましく、また、酵素の蛍光強度の観点からはアルカリ性であることが好ましい。
ルミノール発光試薬（Ｋ）のｐＨは、７～１１が好ましく、更に好ましくは８～１０である。尚、ｐＨは、ＪＩＳ Ｋ０４００－１２－１０：２０００に準拠して測定される（測定温度２５℃）。
The luminol luminescent reagent (K) is preferably a liquid, and is preferably alkaline from the viewpoint of the fluorescence intensity of the enzyme.
The pH of the luminol luminescence reagent (K) is preferably 7 to 11, and more preferably 8 to 10. The pH is measured in accordance with JIS K0400-12-10:2000 (measurement temperature: 25° C.).
ルミノール発光試薬（Ｋ）は、２，３－ジヒドロ－１，４－フタラジンジオン化合物、化学発光増強剤並びに必要により緩衝液及び／又はキレート剤を均一混合することにより容易に得ることができる。 The luminol luminescence reagent (K) can be easily obtained by homogeneously mixing a 2,3-dihydro-1,4-phthalazinedione compound, a chemiluminescence enhancer, and, if necessary, a buffer solution and/or a chelating agent.
過酸化物水溶液（Ｌ）が含有する過酸化物としては、例えば、特開平８－２６１９４３号公報及び特開２０００－２７９１９６号公報等に記載の公知の酸化剤等［無機の過酸化物（過酸化水素、過ホウ酸ナトリウム及び過ホウ酸カリウム等）、有機過酸化物（過酸化ジアルキル及び過酸化アシル等）、ペルオキソ酸化合物（ペルオキソ硫酸及びペルオキソリン酸等）等］の水溶液が挙げられる。
これらの内、保存安定性等の観点から、過酸化水素水溶液、過ホウ酸ナトリウム水溶液及び過ホウ酸カリウム水溶液が好ましく、更に好ましいのは過酸化水素水溶液である。
Examples of the peroxide contained in the aqueous peroxide solution (L) include aqueous solutions of known oxidizing agents such as inorganic peroxides (hydrogen peroxide, sodium perborate, potassium perborate, etc.), organic peroxides (dialkyl peroxides, acyl peroxides, etc.), and peroxoacid compounds (peroxosulfuric acid, peroxolinic acid, etc.), as described in JP-A-8-261943 and JP-A-2000-279196.
Among these, from the viewpoint of storage stability, etc., an aqueous solution of hydrogen peroxide, an aqueous solution of sodium perborate and an aqueous solution of potassium perborate are preferred, and an aqueous solution of hydrogen peroxide is more preferred.
過酸化物水溶液（Ｌ）が含有する水としては、蒸留水、逆浸透水及び脱イオン水等が挙げられる。これらの内、化学発光増強効果及び保存安定性等の観点から、蒸留水及び脱イオン水が好ましく、更に好ましいのは脱イオン水である。 Examples of the water contained in the aqueous peroxide solution (L) include distilled water, reverse osmosis water, and deionized water. Of these, from the viewpoints of chemiluminescence enhancement effect and storage stability, distilled water and deionized water are preferred, and deionized water is more preferred.
過酸化物水溶液（Ｌ）は、過酸化物及び水以外にキレート剤等を含むことができる。
キレート剤としては、上述のルミノール発光試薬（Ｋ）に含むことができるキレート剤として例示したものと同様のものが挙げられ、好ましいものも同様である。
過酸化物水溶液（Ｌ）は、過酸化物、水及び必要によりキレート剤を均一混合することにより容易に得られる。
The aqueous peroxide solution (L) may contain a chelating agent and the like in addition to the peroxide and water.
Examples of the chelating agent include the same as those exemplified as the chelating agent that can be contained in the above-mentioned luminol luminescence reagent (K), and the preferred ones are also the same.
The aqueous peroxide solution (L) can be easily obtained by uniformly mixing a peroxide, water and, if necessary, a chelating agent.
本発明の免疫測定用キットは、試料中の測定対象物質（Ｆ１）を定量する免疫測定方法としてこの分野で一般的に行われる方法に用いることができ、例えば、文献［例えば、酵素免疫測定法第２版（石川栄治ら編集、医学書院）１９８２年］記載のサンドイッチ法、競合法及び特開平６－１３００６３号公報記載の測定法に準じて行えばよい。 The immunoassay kit of the present invention can be used in immunoassay methods commonly used in this field to quantify the substance to be measured (F1) in a sample, and may be performed, for example, in accordance with the sandwich method and competitive method described in the literature [e.g., Enzyme Immunoassay Methods, 2nd Edition (edited by Ishikawa Eiji et al., Igaku Shoin, 1982)] and the measurement method described in JP-A-6-130063.
サンドイッチ法は、例えば、「測定対象物質（Ｆ１）」を含む試料と、「測定対象物質と特異的に結合する物質（Ｆ３）」を磁性シリカ粒子（Ｂ１）の表面に固定化した磁性シリカ粒子（Ｂ１Ｆ３）と、「標識物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）」とを接触させて、磁性シリカ粒子（Ｂ１）表面に担持された「測定対象物質と特異的に結合する物質（Ｆ３）」と、「測定対象物質（Ｆ１）」と、「標識物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）」との標識複合体｛（Ｆ３）／（Ｆ１）／（Ｆ３Ｃ）｝を形成させ、該標識複合体をＢ／Ｆ分離して、複合体中の標識物質量を測定し、その結果に基づいて試料中の測定対象物質（Ｆ１）を定量することによりなされる。 The sandwich method is carried out, for example, by contacting a sample containing a "substance to be measured (F1)," magnetic silica particles (B1F3) having a "substance that specifically binds to the substance to be measured (F3)" immobilized on the surface of the magnetic silica particles (B1), and a "substance that specifically binds to the substance to be measured labeled with a labeling substance (F3C)," forming a labeled complex {(F3)/(F1)/(F3C)} of the "substance that specifically binds to the substance to be measured (F3)" supported on the surface of the magnetic silica particles (B1), the "substance to be measured (F1)," and the "substance that specifically binds to the substance to be measured labeled with a labeling substance (F3C)," B/F separating the labeled complex, measuring the amount of the labeled substance in the complex, and quantifying the substance to be measured (F1) in the sample based on the results.
具体的には例えば、「測定対象物質（Ｆ１）を含む試料」と、「測定対象物質と特異的に結合する物質（Ｆ３）を磁性シリカ粒子（Ｂ１）の表面に固定化した磁性シリカ粒子（Ｂ１Ｆ３）」とを接触させて、磁性シリカ粒子（Ｂ１）表面に担持された「測定対象物質と特異的に結合する物質（Ｆ３）」と「測定対象物質（Ｆ１）」との複合体を形成させ、更に該複合体に「標識物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）」を接触させて、磁性シリカ粒子（Ｂ１）に担持された「測定対象物質と特異的に結合する物質（Ｆ３）」と、「測定対象物質（Ｆ１）」と、「標識物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）」との標識複合体｛（Ｆ３）／（Ｆ１）／（Ｆ３Ｃ）｝を形成させ、該標識複合体をＢ／Ｆ分離して、標識複合体中の標識物質量を測定し、その結果に基づいて試料中の測定対象物質を定量すればよい。
該方法においては、「測定対象物質（Ｆ１）」と「測定対象物質と特異的に結合する物質（Ｆ３）」が固定化された磁性シリカ粒子（Ｂ１Ｆ３）とを反応させた後、「標識物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）」を反応させているが、「標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）」と「測定対象物質（Ｆ１）」とを反応させた後に「測定対象物質と特異的に結合する物質（Ｆ３）」が固定化された磁性シリカ粒子（Ｂ１Ｆ３）とを反応させても、これら３つを同時に反応させても構わない。
Specifically, for example, a "sample containing a measured substance (F1)" is contacted with "magnetic silica particles (B1F3) having a substance (F3) that specifically binds to the measured substance immobilized on the surface of the magnetic silica particles (B1)" to form a complex between the "substance (F3) that specifically binds to the measured substance" carried on the surface of the magnetic silica particles (B1) and the "measured substance (F1)". The complex is then contacted with a "substance (F3C) that specifically binds to the measured substance, labeled with a labeling substance", to form a labeled complex {(F3)/(F1)/(F3C)} between the "substance (F3) that specifically binds to the measured substance" carried on the magnetic silica particles (B1), the "measured substance (F1)", and the "substance (F3C) that specifically binds to the measured substance, labeled with a labeling substance", and the labeled complex is subjected to B/F separation to measure the amount of the labeled substance in the labeled complex. The measured substance in the sample can be quantified based on the result.
In this method, the "substance to be measured (F1)" is reacted with magnetic silica particles (B1F3) having immobilized thereon a "substance that specifically binds to the substance to be measured (F3)" and then reacted with a "substance that specifically binds to the substance to be measured labeled with a labeling substance (F3C)". However, it is also possible to react the "substance that specifically binds to the labeled substance to be measured (F3C)" with the "substance to be measured (F1)" and then react it with the magnetic silica particles (B1F3) having immobilized thereon a "substance that specifically binds to the substance to be measured (F3)", or to react these three simultaneously.
上記サンドイッチ法におけるＢ／Ｆ分離とは、上記標識複合体｛（Ｆ３）／（Ｆ１）／（Ｆ３Ｃ）｝と、標識複合体の形成に関与しなかった「標識物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）」との分離を意味し、具体的には、「磁性シリカ粒子（Ｂ１）に担持された測定対象物質と特異的に結合する物質（Ｆ３）」、「磁性シリカ粒子（Ｂ１）に担持された測定対象物質と特異的に結合する物質（Ｆ３）と測定対象物質（Ｆ１）との複合体｛（Ｆ３）／（Ｆ１）｝」及び上記の「標識複合体｛（Ｆ３）／（Ｆ１）／（Ｆ３Ｃ）｝」と、他の成分（試料中の測定対象物質（Ｆ１）以外の成分、標識複合体の形成に関与しなかった標識物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）等）との分離を意味する。
また、Ｂ／Ｆ分離工程は標識複合体｛（Ｆ３）／（Ｆ１）／（Ｆ３Ｃ）｝の形成後には必須の工程であるが、磁性シリカ粒子（Ｂ１）表面に担持された「測定対象物質と特異的に結合する物質（Ｆ３）」と「測定対象物質（Ｆ１）」との複合体｛（Ｆ３）／（Ｆ１）｝を形成させた後においても実施することができる。
The B/F separation in the sandwich method means separation of the labeled complex {(F3)/(F1)/(F3C)} and the "substance (F3C) that specifically binds to the analyte labeled with a labeling substance" that was not involved in the formation of the labeled complex, specifically means separation of the "substance (F3) that specifically binds to the analyte supported on the magnetic silica particles (B1)", the "complex {(F3)/(F1)}" of the substance (F3) that specifically binds to the analyte supported on the magnetic silica particles (B1) and the analyte (F1), and the above "labeled complex {(F3)/(F1)/(F3C)}" from other components (components other than the analyte (F1) in the sample, the substance (F3C) that specifically binds to the analyte labeled with a labeling substance that was not involved in the formation of the labeled complex, etc.).
In addition, the B/F separation step is an essential step after the formation of the labeled complex {(F3)/(F1)/(F3C)}, but it can also be performed after the formation of a complex {(F3)/(F1)} between the "substance (F3) that specifically binds to the substance to be measured" supported on the surface of the magnetic silica particles (B1) and the "substance to be measured (F1)."
競合法としては、「測定対象物質（Ｆ１）」を含む試料、「標識物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）」、及び、「測定対象物質（Ｆ１）及び／又は測定対象物質の類似物質（Ｆ２）」をその表面に固定化している磁性シリカ粒子（Ｂ１Ｆ１／Ｆ２）とを接触させて、磁性シリカ粒子（Ｂ１）に担持された「測定対象物質（Ｆ１）及び／又はその類似物質（Ｆ２）」と「標識物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）」との標識複合体｛（Ｆ１）／（Ｆ３Ｃ）及び／又は（Ｆ２）／（Ｆ３Ｃ）｝を形成させ、該標識複合体を担持した磁性シリカ粒子をＢ／Ｆ分離して、標識複合体中の標識物質量を測定し、その結果に基づいて試料中の測定対象物質を定量することによりなされる。 The competitive method is carried out by contacting a sample containing the "measurement target substance (F1)," a "substance (F3C) that specifically binds to the measurement target substance labeled with a labeling substance," and magnetic silica particles (B1F1/F2) on whose surfaces the "measurement target substance (F1) and/or an analogue of the measurement target substance (F2)" are immobilized, forming a labeled complex {(F1)/(F3C) and/or (F2)/(F3C)} between the "measurement target substance (F1) and/or its analogue (F2)" supported on the magnetic silica particles (B1) and the "substance (F3C) that specifically binds to the measurement target substance labeled with a labeling substance," B/F separation of the magnetic silica particles supporting the labeled complex, measuring the amount of the labeled substance in the labeled complex, and quantifying the measurement target substance in the sample based on the results.
具体的には例えば、「測定対象物質（Ｆ１）」を含む試料と、「標識物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）」と、「測定対象物質（Ｆ１）及び／又はその類似物質（Ｆ２）」を担持した磁性シリカ粒子（Ｂ１Ｆ１／Ｆ２）とを接触させて、「標識物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）」に、試料中の「測定対象物質（Ｆ１）」と磁性シリカ粒子（Ｂ１）表面に担持された「測定対象物質（Ｆ１）及び／又はその類似物質（Ｆ２）」とを競合反応させて、磁性シリカ粒子（Ｂ１）表面に担持された「測定対象物質（Ｆ１）及び／又はその類似物質（Ｆ２）」と「標識物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）」との標識複合体｛（Ｆ１）／（Ｆ３Ｃ）及び／又は（Ｆ２）／（Ｆ３Ｃ）｝を形成させ、該標識複合体を担持した磁性シリカ粒子をＢ／Ｆ分離して、標識複合体中の標識物質量を測定し、その結果に基づいて試料中の測定対象物質（Ｆ１）を定量すればよい。
該方法においては、「測定対象物質（Ｆ１）」、「標識物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）」、並びに磁性シリカ粒子（Ｂ１）表面に固定化された「測定対象物質（Ｆ１）及び／又はその類似物質（Ｆ２）」を同時に競合反応させているが、「測定対象物質（Ｆ１）」と磁性シリカ粒子（Ｂ１）表面に固定化された「測定対象物質（Ｆ１）及び／又はその類似物質（Ｆ２）」とを接触させた後に、「標識対象物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）」を加えて競合反応させても、「測定対象物質（Ｆ１）」と「標識物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）」を接触させた後に、「測定対象物質（Ｆ１）及び／又はその類似物質（Ｆ２）」を担持した磁性シリカ粒子（Ｂ１Ｆ１／Ｆ２）を加えて競合反応させてもよい。
Specifically, for example, a sample containing a "measurement target substance (F1)" is contacted with a "substance (F3C) that specifically binds to the measurement target substance labeled with a labeling substance," and magnetic silica particles (B1F1/F2) carrying the "measurement target substance (F1) and/or its analogue (F2)," and the "substance (F3C) that specifically binds to the measurement target substance labeled with a labeling substance" reacts with the "measurement target substance (F1)" in the sample and the "measurement target substance (F1) and/or its analogue (F2)" carried on the surface of the magnetic silica particles (B1). The magnetic silica particles (B1) are subjected to a competitive reaction with the "substance (F1) and/or its analog (F2)" carried on the surface of the magnetic silica particles (B1) to form a labeled complex {(F1)/(F3C) and/or (F2)/(F3C)} between the "substance (F1) to be measured and/or its analog (F2)" carried on the surface of the magnetic silica particles (B1) and the "substance (F3C) that specifically binds to the substance to be measured and that is labeled with a labeling substance," the magnetic silica particles carrying the labeled complex are then subjected to B/F separation to measure the amount of the labeled substance in the labeled complex, and the substance to be measured (F1) in the sample is quantified based on the result.
In this method, the "measurement substance (F1)", the "substance (F3C) that specifically binds to the measurement substance labeled with a labeling substance", and the "measurement substance (F1) and/or its analogue (F2)" immobilized on the surface of the magnetic silica particles (B1) are simultaneously subjected to a competitive reaction. Alternatively, the "measurement substance (F1)" and the "substance (F1) and/or its analogue (F2)" immobilized on the surface of the magnetic silica particles (B1) may be brought into contact with each other, and then the "substance (F3C) that specifically binds to the measurement substance labeled with a labeling substance" may be added to carry out the competitive reaction. Alternatively, the "measurement substance (F1)" and the "substance (F3C) that specifically binds to the measurement substance labeled with a labeling substance" may be brought into contact with each other, and then the magnetic silica particles (B1F1/F2) carrying the "measurement substance (F1) and/or its analogue (F2)" may be added to carry out the competitive reaction.
上記競合法におけるＢ／Ｆ分離とは、上記標識複合体｛（Ｆ１）／（Ｆ３Ｃ）及び／又は（Ｆ２）／（Ｆ３Ｃ）｝と、標識複合体の形成に関与しなかった、「標識物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）」、及び「標識物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）と測定対象物質（Ｆ１）との複合体｛（Ｆ１）／（Ｆ３Ｃ）｝」との分離を意味し、具体的には、「測定対象物質（Ｆ１）及び／又はその類似物質（Ｆ２）を担持した磁性シリカ粒子」、並びに、「測定対象物質（Ｆ１）及び／又はその類似物質（Ｆ２）を担持した磁性シリカ粒子と標識物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）との複合体｛（Ｂ１Ｆ１／Ｆ２）／（Ｆ３Ｃ）｝」と、他の成分（試料中の測定対象物質（Ｆ１）以外の成分、「標識物質により標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）」、複合体｛（Ｆ１）／（Ｆ３Ｃ）｝等）との分離を意味する。 The B/F separation in the competitive method refers to the separation of the labeled complex {(F1)/(F3C) and/or (F2)/(F3C)} from the "substance (F3C) that specifically binds to the analyte labeled with a labeling substance" that was not involved in the formation of the labeled complex, and the "complex of the substance (F3C) that specifically binds to the analyte labeled with a labeling substance and the analyte (F1) {(F1)/(F3C)}" and specifically refers to the "analyte (F1) and/or its analogues." This means separation of "magnetic silica particles carrying a substance (F2)" and "complexes {(B1F1/F2)/(F3C)} of magnetic silica particles carrying a substance to be measured (F1) and/or its analogue (F2) and a substance (F3C) that specifically binds to the substance to be measured labeled with a labeling substance" from other components (components other than the substance to be measured (F1) in the sample, "substance (F3C) that specifically binds to the substance to be measured labeled with a labeling substance," complexes {(F1)/(F3C)}, etc.).
また、競合法の別の態様としては、「測定対象物質（Ｆ１）」を含む試料と、「標識物質により標識された測定対象物質及び／又はその類似物質（Ｆ１／Ｆ２Ｃ）」と、「測定対象物質と特異的に結合する物質（Ｆ３）」を担持した磁性シリカ粒子（Ｂ１Ｆ３）とを接触させて、磁性シリカ粒子（Ｂ１）に担持された「測定対象物質と特異的に結合する物質（Ｆ３）」と「標識物質により標識された測定対象物質及び／又はその類似物質（Ｆ１／Ｆ２Ｃ）」との標識複合体｛（Ｆ３）／（Ｆ１／Ｆ２Ｃ）｝とを形成させ、該標識複合体を担持した磁性シリカ粒子をＢ／Ｆ分離して、標識複合体中の標識物質量を測定し、その結果に基づいて試料中の測定対象物質を測定することによりなされる。 In another embodiment of the competitive method, a sample containing the "measurement target substance (F1)" is contacted with magnetic silica particles (B1F3) carrying the "measurement target substance and/or its analogue labeled with a labeling substance (F1/F2C)" and the "substance that specifically binds to the measurement target substance (F3)" to form a labeled complex {(F3)/(F1/F2C)} between the "substance that specifically binds to the measurement target substance (F3)" carried on the magnetic silica particles (B1) and the "measurement target substance and/or its analogue labeled with a labeling substance (F1/F2C)", the magnetic silica particles carrying the labeled complex are subjected to B/F separation, the amount of the labeled substance in the labeled complex is measured, and the measurement target substance in the sample is measured based on the result.
具体的には例えば、「測定対象物質（Ｆ１）」を含む試料と、「標識物質により標識された測定対象物質及び／又はその類似物質（Ｆ１／Ｆ２Ｃ）」と、「測定対象物質と特異的に結合する物質（Ｆ３）」を担持させた磁性シリカ粒子（Ｂ１Ｆ３）とを接触させて、磁性シリカ粒子（Ｂ１）に担持された「測定対象物質と特異的に結合する物質（Ｆ３）」に、「試料中の測定対象物質（Ｆ１）」と「標識物質により標識された測定対象物質及び／又はその類似物質（Ｆ１／Ｆ２Ｃ）」とを競合反応させて、磁性シリカ粒子（Ｂ１）に担持された「測定対象物質と特異的に結合する物質（Ｆ３）」と試料中の「測定対象物質（Ｆ１）」との複合体｛（Ｆ３）／（Ｆ１）｝、並びに、「標識物質により標識された測定対象物質及び／又はその類似物質（Ｆ１／Ｆ２Ｃ）」との標識複合体｛（Ｆ３）／（Ｆ１／Ｆ２Ｃ）｝とを形成させ、該複合体又は標識複合体を担持した磁性シリカ粒子をＢ／Ｆ分離して、標識複合体中の標識物質量を測定し、その結果に基づいて試料中の測定対象物質（Ｆ１）を測定すればよい。
該競合法においては、「測定対象物質（Ｆ１）」、「標識物質により標識された測定対象物質及び／又はその類似物質（Ｆ１／Ｆ２Ｃ）」、並びに「測定対象物質と特異的に結合する物質（Ｆ３）」を担持させた磁性シリカ粒子（Ｂ１Ｆ３）を同時に競合反応させているが、「測定対象物質（Ｆ１）」と「測定対象物質と特異的に結合する物質（Ｆ３）」を担持させた磁性シリカ粒子とを接触させた後｛複合体（Ｆ３）／（Ｆ１）を形成させた後｝に、「標識物質により標識された測定対象物質及び／又はその類似物質（Ｆ１／Ｆ２Ｃ）」を加えて競合反応｛磁性シリカ粒子（Ｂ１Ｆ３）において、試料中の測定対象物質（Ｆ１）が反応しなかった（Ｆ３）に、（Ｆ１／Ｆ２Ｃ）を反応｝させても、「標識物質により標識された測定対象物質及び／又はその類似物質（Ｆ１／Ｆ２Ｃ）」と「測定対象物質と特異的に結合する物質（Ｆ３）」を担持させた磁性シリカ粒子（Ｂ１Ｆ３）とを接触させた後｛複合体（Ｆ３）／（Ｆ１／Ｆ２Ｃ）を形成させた後｝に、「測定対象物質（Ｆ１）」を加えて競合反応｛磁性シリカ粒子（Ｂ１Ｆ３）において、（Ｆ１／Ｆ２Ｃ）が反応しなかった（Ｆ３）に、試料中の測定対象物質（Ｆ１）を反応｝させてもよい。
Specifically, for example, a sample containing a "measurement target substance (F1)" is contacted with magnetic silica particles (B1F3) carrying a "measurement target substance and/or its analogue labeled with a labeling substance (F1/F2C)" and a "substance that specifically binds to the measurement target substance (F3)," and the "measurement target substance (F1) in the sample" and the "measurement target substance and/or its analogue labeled with a labeling substance (F1/F2C)" undergo a competitive reaction with the "substance that specifically binds to the measurement target substance (F3)" carried on the magnetic silica particles (B1), A complex {(F3)/(F1)} is formed between the "substance (F3) that specifically binds to the measured substance" supported on the magnetic silica particles (B1) and the "measured substance (F1)" in the sample, as well as a labeled complex {(F3)/(F1/F2C)} between the "measured substance and/or its analogue labeled with a labeling substance (F1/F2C)". The complex or the magnetic silica particles supporting the labeled complex are then subjected to B/F separation to measure the amount of the labeled substance in the labeled complex, and the measured substance (F1) in the sample is measured based on the results.
In this competitive method, the "measurement target substance (F1)", the "measurement target substance labeled with a labeling substance and/or its analogue (F1/F2C)", and the magnetic silica particles (B1F3) carrying the "substance that specifically binds to the measurement target substance (F3)" are simultaneously subjected to a competitive reaction. After the "measurement target substance (F1)" is contacted with the magnetic silica particles carrying the "substance that specifically binds to the measurement target substance (F3)" {after the complex (F3)/(F1) is formed}, the "measurement target substance labeled with a labeling substance and/or its analogue (F1/F2C)" is added to carry out the competitive reaction {magnetic silica particles In (B1F3), (F1/F2C) may be reacted with (F3) to which the measured substance (F1) in the sample has not reacted}, or the measured substance (F1) may be contacted with magnetic silica particles (B1F3) carrying a "measured substance and/or its analogue (F1/F2C) labeled with a labeling substance" {after forming a complex (F3)/(F1/F2C)}, and then the "measured substance (F1)" may be added to cause a competitive reaction {the measured substance (F1) in the sample may be reacted with (F3) to which (F1/F2C) has not reacted in the magnetic silica particles (B1F3)}.
上記競合法におけるＢ／Ｆ分離とは、上記標識複合体と、標識複合体の形成に関与しなかった「標識物質により標識された測定対象物質及び／又はその類似物質（Ｆ１／Ｆ２Ｃ）」との分離を意味し、具体的には、「測定対象物質と特異的に結合する物質（Ｆ３）を担持させた磁性シリカ粒子（Ｂ１Ｆ３）」、「磁性シリカ粒子（Ｂ１）上の測定対象物質と特異的に結合する物質（Ｆ３）と、測定対象物質（Ｆ１）との複合体｛（Ｆ３）／（Ｆ１）｝」、及び「磁性シリカ粒子（Ｂ１）上の測定対象物質と特異的に結合する物質（Ｆ３）と、標識物質により標識された測定対象物質及び／又はその類似物質（Ｆ１／Ｆ２Ｃ）との複合体｛（Ｆ３）／（Ｆ１／Ｆ２Ｃ）｝」と、他の成分（試料中の測定対象物質（Ｆ１）以外の成分、標識複合体の形成に関与しなかった標識物質により標識された測定対象物質及び／又はその類似物質（Ｆ１／Ｆ２Ｃ）等）との分離を意味する。
また、Ｂ／Ｆ分離工程は標識複合体の形成後には必須の工程であるが、「測定対象物質（Ｆ１）」と「測定対象物質と特異的に結合する物質（Ｆ３）」を担持させた磁性シリカ粒子とを接触させた後においても実施することができる。
The B/F separation in the competitive method means separation of the labeled complex from the "measurement substance labeled with a labeling substance and/or its analogue (F1/F2C)" that was not involved in the formation of the labeled complex, and specifically means separation of "magnetic silica particles (B1F3) carrying a substance (F3) that specifically binds to the measurement substance", "a complex {(F3)/(F1)}" of the measurement substance (F3) on the magnetic silica particles (B1) that specifically binds to the measurement substance on the magnetic silica particles (B1) and the measurement substance (F1)," and "a complex {(F3)/(F1/F2C)}" of the substance (F3) that specifically binds to the measurement substance on the magnetic silica particles (B1) and the measurement substance labeled with a labeling substance and/or its analogue (F1/F2C)" from other components (components other than the measurement substance (F1) in the sample, the measurement substance labeled with a labeling substance that was not involved in the formation of the labeled complex and/or its analogue (F1/F2C), etc.).
In addition, although the B/F separation step is an essential step after the formation of the labeled complex, it can also be performed after contacting the "substance to be measured (F1)" with magnetic silica particles carrying the "substance that specifically binds to the substance to be measured (F3)."
また、「測定対象物質（Ｆ１）」が酵素の場合には、上記サンドイッチ法や競合法以外の酵素活性方法を用いる方法、例えば、「測定対象物質（Ｆ１）」を含む試料と、「測定対象物質と特異的に結合する物質（Ｆ３）（例えば、抗体等の酵素と結合し得る物質）」を表面に固定化した磁性シリカ粒子（Ｂ１Ｆ３）とを接触させて、磁性シリカ粒子（Ｂ１）に担持された「測定対象物質と特異的に結合する物質（Ｆ３）」との複合体｛（Ｆ３）／（Ｆ１）｝を形成させ、複合体を担持した磁性シリカ粒子をＢ／Ｆ分離した後、酵素の種類に応じた基質及び発色剤、要すれば更に共役酵素を添加し、その基質の変化又は発色剤の発色結果に基づいて試料中の酵素量を測定する方法により、測定してもよい。
尚、基質、発色剤、共役酵素は、公知のものを用いればよく、例えば酵素がペルオキシダーゼの場合には、過酸化水素とルミノール発光試薬等を用いればよい。これらの使用量も一般的にこの分野で用いられる範囲であればよい。上記方法におけるＢ／Ｆ分離とは、「測定対象物質（Ｆ１）」と、磁性シリカ粒子上に固定化された「測定対象物質と特異的に結合する物質（Ｆ３）」との複合体｛（Ｆ３）／（Ｆ１）｝と、その他の成分（試料中の測定対象物質（Ｆ１）以外の成分等）との分離を意味する。
Furthermore, when the "substance to be measured (F1)" is an enzyme, the measurement may be performed by a method using an enzyme activity method other than the sandwich method or competitive method, for example, by contacting a sample containing the "substance to be measured (F1)" with magnetic silica particles (B1F3) having immobilized on their surfaces a "substance (F3) that specifically binds to the substance to be measured (e.g., a substance that can bind to an enzyme such as an antibody)" to form a complex {(F3)/(F1)} with the "substance (F3) that specifically binds to the substance to be measured" supported on the magnetic silica particles (B1), and after B/F separation of the magnetic silica particles supporting the complex, a substrate and a color developer according to the type of enzyme, and if necessary, a conjugated enzyme, are added, and the amount of the enzyme in the sample is measured based on the change in the substrate or the color development result of the color developer.
The substrate, coloring agent, and conjugated enzyme may be known. For example, when the enzyme is peroxidase, hydrogen peroxide and luminol luminescence reagent may be used. The amounts of these may be within the range generally used in this field. The B/F separation in the above method means separation of a complex {(F3)/(F1)} of the "substance to be measured (F1)" and the "substance (F3) that specifically binds to the substance to be measured" immobilized on magnetic silica particles, from other components (components other than the substance to be measured (F1) in the sample, etc.).
本発明の免疫測定方法において、試料、物質（Ｆ）が固定化された磁性シリカ粒子（Ｂ１Ｆ）、標識された測定対象物質と特異的に結合する物質（Ｆ３Ｃ）、標識された測定対象物質（Ｆ１Ｃ）又はその類似物質（Ｆ２Ｃ）等を接触させる方法としては、一般的になされる撹拌、混合等の処理により、磁性シリカ粒子が分散されればよい。反応時間は、測定対象物質（Ｆ１）、用いられる測定対象物質と特異的に結合する物質（Ｆ３）、サンドイッチ法、競合法等の違いに応じて適宜設定されればよいが、１～１０分が好ましく、さらに好ましくは１～５分である。 In the immunoassay method of the present invention, the sample, the magnetic silica particles (B1F) having the immobilized substance (F), the substance (F3C) that specifically binds to the labeled analyte, the labeled analyte (F1C) or its analogue (F2C), etc. can be brought into contact with each other by a general process such as stirring or mixing to disperse the magnetic silica particles. The reaction time can be set appropriately depending on the analyte (F1), the substance (F3) that specifically binds to the analyte used, the sandwich method, the competitive method, etc., but is preferably 1 to 10 minutes, more preferably 1 to 5 minutes.
本発明の免疫測定方法におけるＢ／Ｆ分離は、例えば、磁性シリカ粒子の磁性を利用し、反応槽の外側等から磁石等により磁性シリカ粒子を集めて、反応液を排出し、洗浄液を加えた後、磁石を取り除き、磁性シリカ粒子を混合して分散させ、洗浄することによりなされる。上記操作を１～３回繰り返してもよい。洗浄液としては、一般的にこの分野で用いられるもの（生理食塩水等）等を用いることができる。 In the immunoassay method of the present invention, B/F separation is performed, for example, by utilizing the magnetism of the magnetic silica particles, collecting the magnetic silica particles from the outside of the reaction vessel with a magnet or the like, discharging the reaction liquid, adding a washing liquid, removing the magnet, mixing and dispersing the magnetic silica particles, and washing them. The above operation may be repeated 1 to 3 times. As the washing liquid, a liquid generally used in this field (such as physiological saline) can be used.
以下、実施例により、本発明を更に説明するが、本発明はこれに限定されるものではない。なお実施例２、５、８、１１は、それぞれ、参考例１～４である。
The present invention will be further described below with reference to examples, but the present invention is not limited thereto. Examples 2, 5, 8, and 11 are Reference Examples 1 to 4, respectively.
＜実施例１＞
以下に示す方法により、固相担体試薬（Ｅ）［抗ＰＩＶＫＡ－ＩＩモノクローナル抗体
を固定化した磁性シリカ粒子（ＢＦ）を含有］、標識試薬（Ａ１）（ＰＯＤ標識抗プロトロンビンモノクローナル抗体試薬）、免疫反応緩衝液、ルミノール発光試薬（Ｋ）及び過酸化水素液（Ｌ）から構成される本発明の免疫測定用キット（Ｓ１）を得た。
Example 1
By the method described below, an immunoassay kit (S1) of the present invention was obtained, which is composed of a solid phase carrier reagent (E) [containing magnetic silica particles (BF) to which anti-PIVKA-II monoclonal antibody is immobilized], a labeled reagent (A1) (POD-labeled anti-prothrombin monoclonal antibody reagent), an immune reaction buffer, a luminol luminescence reagent (K) and a hydrogen peroxide solution (L).
固相担体試薬（Ｅ）の作製：
１重量％γ－アミノプロピルトリエトキシシラン含有水溶液４０ｍＬの入った蓋付きポリスチレン瓶に製造した磁性シリカ粒子（商品名：マグラピッド、三洋化成工業（株）製）４０ｍｇを加え、２５℃で１時間反応させ、ネオジウム磁石で磁性シリカ粒子を集磁後、液をアスピレーターで吸引除去した。次いで脱イオン水４０ｍＬを加えて蓋をし、ポリスチレン瓶をゆっくりと２回倒置攪拌した後、ネオジウム磁石で磁性シリカ粒子を集磁後、液をアスピレーターで吸引除去して磁性シリカ粒子を洗浄した。この洗浄操作を５回行った。次いで、この洗浄後の磁性シリカ粒子を２重量％グルタルアルデヒド含有水溶液４０ｍＬの入った蓋付きポリスチレン瓶に加え、２５℃で１時間反応させた。そして、脱イオン水４０ｍＬを加えて蓋をし、ポリスチレン瓶をゆっくりと２回倒置攪拌したのち、ネオジウム磁石で磁性シリカ粒子を集磁後、液をアスピレーターで吸引除去して磁性シリカ粒子を洗浄した。この洗浄操作を１０回行った。更にこの洗浄後の磁性シリカ粒子を、物質（Ｆ）として抗ＰＩＶＫＡ－ＩＩモノクローナル抗体（コスモ・バイオ（株）製）１０μｇ／ｍＬの濃度で含む０．０２Ｍリン酸緩衝液（ｐＨ８．７）１２０ｍＬの入った蓋付きポリスチレン瓶に加え、２５℃で１時間反応させた。反応後、ネオジウム磁石で物質（Ｆ）が固定化された磁性シリカ粒子（ＢＦ）を集磁後、抗ＰＩＶＫＡ－ＩＩモノクローナル抗体（Ｆ）含有リン酸緩衝液を除去した。次いで、得られた磁性シリカ粒子（ＢＦ）を１重量％のブロックエース（ＤＳファーマバイオメディカル（株）製）含有の０．０２Ｍリン酸緩衝液（ｐＨ７．０）４０ｍＬの入った蓋付きポリエチレン瓶に４０ｍｇ加え、２５℃で１２時間浸漬させた。これにより、抗ＰＩＶＫＡ－ＩＩモノクローナル抗体（Ｆ）を固定化した磁性シリカ粒子（ＢＦ）を含有する固相担体試薬（Ｅ）を得て、冷蔵（２～１０℃）で保存した。
Preparation of solid support reagent (E):
40 mg of the magnetic silica particles (product name: Magrapid, manufactured by Sanyo Chemical Industries, Ltd.) was added to a lidded polystyrene bottle containing 40 mL of an aqueous solution containing 1 wt % γ-aminopropyltriethoxysilane, and the mixture was reacted at 25 ° C. for 1 hour. The magnetic silica particles were collected with a neodymium magnet, and the liquid was removed by aspirating with an aspirator. Next, 40 mL of deionized water was added, the lid was placed, and the polystyrene bottle was slowly inverted and stirred twice, after which the magnetic silica particles were collected with a neodymium magnet, and the liquid was removed by aspirating with an aspirator to wash the magnetic silica particles. This washing operation was performed five times. Next, the washed magnetic silica particles were added to a lidded polystyrene bottle containing 40 mL of an aqueous solution containing 2 wt % glutaraldehyde, and the mixture was reacted at 25 ° C. for 1 hour. Then, 40 mL of deionized water was added, the lid was placed, and the polystyrene bottle was slowly inverted and stirred twice, after which the magnetic silica particles were collected with a neodymium magnet, and the liquid was removed by aspirating with an aspirator to wash the magnetic silica particles. This washing operation was performed 10 times. Further, the washed magnetic silica particles were added to a lidded polystyrene bottle containing 120 mL of 0.02 M phosphate buffer (pH 8.7) containing anti-PIVKA-II monoclonal antibody (manufactured by Cosmo Bio Co., Ltd.) at a concentration of 10 μg/mL as substance (F), and reacted at 25 ° C. for 1 hour. After the reaction, the magnetic silica particles (BF) to which the substance (F) was immobilized were collected with a neodymium magnet, and the phosphate buffer containing the anti-PIVKA-II monoclonal antibody (F) was removed. Next, 40 mg of the obtained magnetic silica particles (BF) were added to a lidded polyethylene bottle containing 40 mL of 0.02 M phosphate buffer (pH 7.0) containing 1 wt % Block Ace (manufactured by DS Pharma Biomedical Co., Ltd.), and immersed at 25 ° C. for 12 hours. As a result, a solid phase carrier reagent (E) containing magnetic silica particles (BF) having anti-PIVKA-II monoclonal antibody (F) immobilized thereon was obtained, and the solid phase carrier reagent was stored in a refrigerator (2 to 10° C.).
標識試薬（Ａ１）の作製：
抗プロトロンビンモノクローナル抗体（コスモ・バイオ（株）製）、西洋ワサビ由来ＰＯＤ（東洋紡（株）製）を用い、文献（エス・ヨシタケ、エム・イマガワ、イー・イシカワ、エトール；ジェイ．バイオケム，Ｖｏｌ．９２，１９８２，１４１３－１４２４）に記載の方法を用いて、ＰＯＤで標識された抗プロトロンビンモノクローナル抗体（ＦＣ）を調製した。これを、（Ｄ）として０．９５重量％ジギトニン（ナカライテスク（株）製）含有の０．０２Ｍリン酸緩衝液で、ＰＯＤ標識抗プロトロンビンモノクローナル抗体（ＦＣ）濃度として２．０μｇ／ｍＬとなるように希釈し、標識試薬（Ａ１）を調製した。
標識試薬（Ａ１）は、４℃で７日間保存したものと、３５℃で７日間保存したものをそれぞれ準備した。
なお、標識試薬（Ａ１）中のジギトニンの含有量は、標識試薬（Ａ１）の重量を基準として、０．９５重量％である。
Preparation of labeling reagent (A1):
Using anti-prothrombin monoclonal antibody (manufactured by Cosmo Bio Co., Ltd.) and horseradish-derived POD (manufactured by Toyobo Co., Ltd.), a POD-labeled anti-prothrombin monoclonal antibody (FC) was prepared according to the method described in the literature (S. Yoshitake, M. Imagawa, E. Ishikawa, Ethol; J. Biochem, Vol. 92, 1982, 1413-1424). This was diluted with 0.02 M phosphate buffer containing 0.95 wt. % digitonin (manufactured by Nacalai Tesque Co., Ltd.) as (D) so that the concentration of the POD-labeled anti-prothrombin monoclonal antibody (FC) was 2.0 μg/mL, to prepare a labeled reagent (A1).
The labeling reagent (A1) was prepared by storing at 4° C. for 7 days and at 35° C. for 7 days.
The content of digitonin in the labeling reagent (A1) was 0.95% by weight based on the weight of the labeling reagent (A1).
免疫反応緩衝液の作製：
ウシ血清アルブミン（Ｂｏｖａｌ Ｃａｍｐａｎｙ製）を０．１重量％、エマルミンＬ－９０－Ｓ（三洋化成工業（株）製）を１重量％、塩化ナトリウム（富士フィルム和光純薬（株）製）を０．８５重量％含有した０．０２Ｍリン酸緩衝液（ｐＨ７．０）を調製し、冷蔵（２～１０℃）で保管した。
Preparation of immunoreaction buffer:
A 0.02 M phosphate buffer solution (pH 7.0) containing 0.1 wt % bovine serum albumin (Bovale Company), 1 wt % Emulmin L-90-S (Sanyo Chemical Industries, Ltd.), and 0.85 wt % sodium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.) was prepared and stored refrigerated (2 to 10° C.).
ルミノール発光試薬（Ｋ）の作製：
ルミノールのナトリウム塩［シグマ アルドリッチ ジャパン（株）製］０．７ｇ及び４－（シアノメチルチオ）フェノール０．１ｇを１，０００ｍＬメスフラスコに仕込んだ。３－［４－（２－ヒドロキシエチル）－１－ピペラジニル］プロパンスルホン酸／水酸化ナトリウム緩衝液（１０ｍＭ、ｐＨ８．６）を溶液の容量が１，０００ｍＬになるように仕込み、２５℃で均一混合してルミノール発光試薬（Ｋ）を調製した。測定に用いるまで冷蔵（２～１０℃）保存した。
Preparation of luminol luminescence reagent (K):
0.7 g of luminol sodium salt [Sigma-Aldrich Japan Co., Ltd.] and 0.1 g of 4-(cyanomethylthio)phenol were placed in a 1,000 mL measuring flask. 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid/sodium hydroxide buffer (10 mM, pH 8.6) was added so that the volume of the solution became 1,000 mL, and the solution was mixed uniformly at 25°C to prepare luminol luminescence reagent (K). The solution was stored in a refrigerator (2 to 10°C) until it was used for measurement.
過酸化水素液（Ｌ）の作製：
過酸化水素［富士フィルム和光純薬（株）製、試薬特級、濃度３０重量％］６．６ｇを１，０００ｍＬメスフラスコに仕込んだ。脱イオン水を溶液の容量が１，０００ｍＬになるように仕込み、２５℃で均一混合して過酸化水素液（Ｌ）を調製した。測定に用いるまで冷蔵（２～１０℃）保存した。
Preparation of hydrogen peroxide solution (L):
6.6 g of hydrogen peroxide [Fujifilm Wako Pure Chemical Industries, Ltd., special grade reagent, concentration 30% by weight] was placed in a 1,000 mL measuring flask. Deionized water was added to make the solution volume 1,000 mL, and the solution was mixed uniformly at 25°C to prepare hydrogen peroxide solution (L). The solution was stored refrigerated (2 to 10°C) until it was used for measurement.
＜実施例２～１２＞
実施例１の「標識試薬（Ａ１）の作製」において、０．０２Ｍリン酸緩衝液中の化合物（Ｄ）の種類及び濃度を表１に記載のものとする以外は実施例１と同様にして標識試薬（Ａ２）～（Ａ１２）を作製し、実施例１と同じ固相担体試薬（Ｅ）、免疫反応緩衝液、ルミノール発光試薬（Ｋ）及び過酸化水素液（Ｌ）と組み合わせて免疫測定用キット（Ｓ２）～（Ｓ１２）とした。
<Examples 2 to 12>
In "Preparation of labeled reagent (A1)" of Example 1, labeled reagents (A2) to (A12) were prepared in the same manner as in Example 1, except that the type and concentration of compound (D) in 0.02 M phosphate buffer were those shown in Table 1. These were combined with the same solid phase carrier reagent (E), immune reaction buffer, luminol luminescence reagent (K), and hydrogen peroxide solution (L) as in Example 1 to prepare immunoassay kits (S2) to (S12).
＜比較例１～３＞
実施例１の「標識試薬（Ａ１）の作製」において、０．０２Ｍリン酸緩衝液中の化合物（Ｄ）の種類及び濃度を表１に記載のものとする以外は実施例１と同様にして標識試薬（Ａ’１）～（Ａ’３）を作製し、実施例１と同じ固相担体試薬（Ｅ）、免疫反応緩衝液、ルミノール発光試薬（Ｋ）及び過酸化水素液（Ｌ）と組み合わせて免疫測定用キット（Ｓ’１）～（Ｓ’３）とした。
<Comparative Examples 1 to 3>
In "Preparation of labeled reagent (A1)" of Example 1, labeled reagents (A'1) to (A'3) were prepared in the same manner as in Example 1, except that the type and concentration of compound (D) in 0.02 M phosphate buffer was as shown in Table 1. These were combined with the same solid phase carrier reagent (E), immune reaction buffer, luminol luminescence reagent (K) and hydrogen peroxide solution (L) as in Example 1 to prepare immunoassay kits (S'1) to (S'3).
なお、表１中、各成分は下記を用いた。
ジギトニン：富士フイルム和光純薬（株）製
ククルビタシンＢ：東京化成工業（株）製
ウアバイン：富士フイルム和光純薬（株）製
サポニン：富士フイルム和光純薬（株）製
In Table 1, the following components were used.
Digitonin: Manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Cucurbitacin B: Manufactured by Tokyo Chemical Industry Co., Ltd. Ouabain: Manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Saponin: Manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
以下の方法により免疫測定用試薬（Ｓ１）～（Ｓ１２）及び（Ｓ’１）～（Ｓ’３）を用いて、免疫測定を行い、免疫測定用キットの保存安定性を評価した。結果を表１に示す。 Immunoassays were performed using the immunoassay reagents (S1) to (S12) and (S'1) to (S'3) according to the following method, and the storage stability of the immunoassay kits was evaluated. The results are shown in Table 1.
＜実施例１３～２４及び比較例４～６＞
＜免疫測定方法＞
○工程（１）
実施例及び比較例で得た免疫測定用キットの固相担体試薬（Ｅ）をそれぞれ０．０２５ｍＬ、試験管に入れ、試験管の外側からネオジウム磁石で磁性シリカ粒子を１０秒間集め、試験管中の液をアスピレーターで除き、ネオジウム磁石を側面から十分に離した。その後、免疫反応緩衝液０．２ｍＬと、測定対象物質（Ｆ１）であるＰＩＶＫＡ－ＩＩ抗原の濃度が３０００ｍＡＵ／ｍＬになるように調製したプール血清０．０２５ｍＬとを試験管に入れて混合、試験管中で３７℃３分間反応させ、抗ＰＩＶＫＡ－ＩＩモノクローナル抗体固定化磁性シリカ粒子上に抗ＰＩＶＫＡ－ＩＩモノクローナル抗体（Ｆ）／ＰＩＶＫＡ―ＩＩ抗原（Ｆ１）の複合体を形成させた。反応後、試験管の外側からネオジウム磁石で磁性シリカ粒子を１０秒間集め、試験管中の液をアスピレーターで除き、ネオジウム磁石を側面から十分に離した。その後、生理食塩水０．５ｍＬを加えて磁性シリカ粒子を分散させて集磁後、アスピレーターで液を除く洗浄操作を３回行った。
<Examples 13 to 24 and Comparative Examples 4 to 6>
<Immunoassay method>
Step (1)
0.025 mL of each solid phase carrier reagent (E) of the immunoassay kit obtained in the Examples and Comparative Examples was placed in a test tube, magnetic silica particles were collected from the outside of the test tube with a neodymium magnet for 10 seconds, the liquid in the test tube was removed with an aspirator, and the neodymium magnet was sufficiently removed from the side. Then, 0.2 mL of immune reaction buffer and 0.025 mL of pooled serum prepared so that the concentration of the PIVKA-II antigen, which is the measurement target substance (F1), was 3000 mAU/mL, were placed in the test tube and mixed, and reacted in the test tube at 37°C for 3 minutes to form a complex of anti-PIVKA-II monoclonal antibody (F)/PIVKA-II antigen (F1) on the magnetic silica particles with the anti-PIVKA-II monoclonal antibody immobilized thereon. After the reaction, magnetic silica particles were collected from the outside of the test tube with a neodymium magnet for 10 seconds, the liquid in the test tube was removed with an aspirator, and the neodymium magnet was sufficiently removed from the side. Thereafter, 0.5 mL of physiological saline was added to disperse the magnetic silica particles and collect the magnetic particles, after which a washing operation of removing the liquid with an aspirator was performed three times.
○工程（２）
続いて、それぞれの免疫測定用キットの標識試薬０．１ｍＬ［４℃で７日間保存した標識試薬又は３５℃で７日間保存した標識試薬をそれぞれ使用］をそれぞれ試験管に注入し、試験管中で３７℃３分間反応させ、抗ＰＩＶＫＡ－ＩＩモノクローナル抗体固定化磁性シリカ粒子上に抗ＰＩＶＫＡ－ＩＩ抗体（Ｆ）／ＰＩＶＫＡ―ＩＩ抗原（Ｆ１）／ＰＯＤ標識抗プロトロンビンモノクローナル抗体（ＦＣ）の複合体を形成させた。反応後、試験管の外側からネオジウム磁石で磁性シリカ粒子を１０秒間集め、試験管中の液をアスピレーターで除き、ネオジウム磁石を側面から十分に離した。その後、生理食塩水０．５ｍＬを加えて磁性シリカ粒子を分散させて集磁後、アスピレーターで液を除く洗浄操作を２回行った。
Step (2)
Subsequently, 0.1 mL of the labeled reagent of each immunoassay kit [the labeled reagent stored at 4°C for 7 days or the labeled reagent stored at 35°C for 7 days was used] was injected into each test tube, and reacted in the test tube at 37°C for 3 minutes to form a complex of anti-PIVKA-II antibody (F) / PIVKA-II antigen (F1) / POD-labeled anti-prothrombin monoclonal antibody (FC) on the anti-PIVKA-II monoclonal antibody-immobilized magnetic silica particles. After the reaction, the magnetic silica particles were collected from the outside of the test tube with a neodymium magnet for 10 seconds, the liquid in the test tube was removed with an aspirator, and the neodymium magnet was sufficiently separated from the side. Then, 0.5 mL of physiological saline was added to disperse the magnetic silica particles and collect them, and a washing operation of removing the liquid with an aspirator was performed twice.
○化学発光・検出工程
最後に、ルミノール発光試薬（Ｋ）０．１ｍＬと過酸化水素液（Ｌ）０．１ｍＬとを同時に加え、３７℃で発光反応させ、ルミノール発光試薬（Ｋ）及び過酸化水素液（Ｌ）を添加後４０～４５秒の一秒当たりの平均発光量をルミノメーター［ベルトールドジャパン社製「Ｌｕｍａｔ ＬＢ９５０７」］で測定した。結果を表１に示す。
Finally, 0.1 mL of luminol luminescence reagent (K) and 0.1 mL of hydrogen peroxide solution (L) were added at the same time, and the luminescence reaction was allowed to proceed at 37°C. The average amount of luminescence per second from 40 to 45 seconds after the addition of the luminol luminescence reagent (K) and hydrogen peroxide solution (L) was measured using a luminometer [Berthold Japan "Lumat LB9507"]. The results are shown in Table 1.
＜保存安定性の評価方法＞
保存安定性については、以下の基準で判定した。
○：（３５℃で７日間保存した標識試薬（Ａ）を用いた場合の平均発光量／４℃で７日間保存した標識試薬（Ａ）を用いた場合の平均発光量）×１００＝９０％～１１０％
×：（３５℃で７日間保存した標識試薬（Ａ）を用いた場合の平均発光量／４℃で７日間保存した標識試薬（Ａ）を用いた場合の平均発光量）×１００＜９０％、又は＞１１０％
なお、測定濃度／実際の測定濃度が１００％に近いほど、保存安定性が高いことを意味する。
<Evaluation method for storage stability>
The storage stability was evaluated according to the following criteria.
○: (average luminescence amount when using labeling reagent (A) stored at 35° C. for 7 days/average luminescence amount when using labeling reagent (A) stored at 4° C. for 7 days)×100=90% to 110%
×: (average luminescence amount when labeling reagent (A) stored at 35° C. for 7 days was used/average luminescence amount when labeling reagent (A) stored at 4° C. for 7 days was used)×100<90%, or >110%
It should be noted that the closer the measured concentration/actual measured concentration is to 100%, the higher the storage stability is.
実施例１３～２４及び比較例４～６の免疫測定方法で評価した保存安定性の結果を表１に示す。 The results of storage stability evaluated by the immunoassay method for Examples 13 to 24 and Comparative Examples 4 to 6 are shown in Table 1.
本発明の免疫測定用キット及び免疫測定方法は、保存安定性に優れることから、放射免疫測定法、酵素免疫測定法、蛍光免疫測定法及び化学発光免疫測定法等の臨床検査に幅広く適用できる。 The immunoassay kit and immunoassay method of the present invention have excellent storage stability and can be widely applied to clinical tests such as radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, and chemiluminescent immunoassay.
Claims (6)
An immunoassay method, which uses the immunoassay kit according to any one of claims 3 to 5.
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