JP2015068764A - Method for measuring density of analysis object and test strip for lateral flow - Google Patents
Method for measuring density of analysis object and test strip for lateral flow Download PDFInfo
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Images
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Abstract
Description
本発明は、分析対象物の濃度測定方法及びラテラルフロー用テストストリップに関し、特に、試料中の分析対象物の濃度を定量的且つ迅速的に測定する方法及びその方法を用いたラテラルフロー用テストストリップに関する。 The present invention relates to a method for measuring the concentration of an analyte and a test strip for lateral flow, and more particularly to a method for quantitatively and rapidly measuring the concentration of an analyte in a sample and a test strip for lateral flow using the method. About.
基礎医学の目覚しい進歩により優れた治療薬や先進医療技術が開発されているものの、食生活・生活環境等の様々な要因により、がんや自己免疫疾患、感染症、循環器系疾患等の多種多様な疾患による不安は極めて大きい。こうした不安の解消には疾患の早期発見が重要である。近年、臨床検査においてベッドサイド検査やポイントオブケアテスティング(POCT)とよばれる、迅速且つ簡易なシステムでの検査が求められている。 Although excellent therapeutic drugs and advanced medical technologies have been developed due to remarkable advances in basic medicine, various factors such as cancer, autoimmune diseases, infectious diseases, cardiovascular diseases, etc., due to various factors such as eating habits and living environment Anxiety due to various diseases is extremely large. Early detection of the disease is important for eliminating such anxiety. In recent years, a rapid and simple system test called a bedside test or point-of-care testing (POCT) is required in clinical tests.
ラテラルフロー用テストストリップは、迅速且つ簡易に試料中の分析対象物を検出できるため、例えば、臨床分野、食品分野、環境検査等において広く応用されており、特に妊娠検査薬におけるホルモン検出、インフルエンザ検査におけるウイルス検出、ウイルス感染おける抗体検出等で利用されている。このラテラルフロー用テストストリップの一般的な構造は、滴下した試料を一定量保持可能なサンプルパッドと、その下流に配置され、試料に含まれる分析対象物である抗原に特異的な抗体が感作された粒子が乾燥状態で保持されている結合パッドと、その抗原を検出する抗体が固相化された部位(所謂、テストライン)及び粒子表面に備わる抗体を認識する抗体が固相化された部位(所謂、コントロールライン)を持つ展開膜(所謂、メンブレン)と、更にその下流に配置され、毛細管現象によって浸潤してきた試料を吸収する吸収パッドとの4種の特性を持った部材が一体化したものである。サンプルパッドに滴下された試料は、毛細管現象によりテストストリップ内を展開し、抗原抗体反応によりテストライン部分及びコントロールライン部分で粒子が検出される。一般的には、目視で確認できるようにテストストリップ幅は、5mmに設定されており、使用試料量は、100μl以上に設定されている。 Since the test strip for lateral flow can detect the analyte in the sample quickly and easily, it is widely applied in, for example, clinical field, food field, environmental test, etc., especially hormone detection in pregnancy test drugs, influenza test Is used for virus detection and antibody detection in virus infection. The general structure of this lateral flow test strip consists of a sample pad that can hold a fixed amount of the dropped sample, and an antibody specific to the antigen that is the analyte contained in the sample. An antibody that recognizes the antibody provided on the surface of the binding pad on which the particles are held in a dry state, a portion where the antibody for detecting the antigen is immobilized (a so-called test line), and the particle surface is immobilized. A member with four types of characteristics is integrated: a deployment membrane (so-called membrane) having a site (so-called control line) and an absorption pad that is arranged further downstream and absorbs a sample that has infiltrated by capillary action. It is a thing. The sample dropped on the sample pad develops in the test strip by capillary action, and particles are detected in the test line part and the control line part by antigen-antibody reaction. Generally, the test strip width is set to 5 mm so that it can be visually confirmed, and the amount of sample used is set to 100 μl or more.
通常用いられるラテラルフロー用テストストリップでは、試料滴下後、その試料を一定時間展開させた後、所定経過時間におけるシグナルを検出する。例えは、上記5mm幅のテストストリップであって、試料を100μl使用するものであれば、20分程度展開させた後にライン強度(シグナル強度)を測定する。このような技術としては、例えば、特許文献1に記載の技術がある。 In a commonly used lateral flow test strip, after dropping a sample, the sample is developed for a certain period of time, and then a signal at a predetermined elapsed time is detected. For example, if the test strip has a width of 5 mm and uses 100 μl of the sample, the line intensity (signal intensity) is measured after developing for about 20 minutes. As such a technique, there is a technique described in Patent Document 1, for example.
しかしながら、上記方法で試料中に含まれた分析対象物(例えば、抗原)の濃度を測定する際、シグナルを十分に検出するまで待機を要する等、実働における時間制限を要し、感染症等の迅速な診断においては大きな障害となることがある。このため、簡易であり、より迅速な方法で分析対象物の定量が可能な方法(つまり、分析対象物の濃度測定方法)が求められている。 However, when measuring the concentration of the analyte (for example, antigen) contained in the sample by the above method, it takes time until the signal is sufficiently detected, such as waiting for the actual operation, and infectious diseases, etc. A quick diagnosis can be a major obstacle. For this reason, there is a need for a simple method that can quantify the analyte by a more rapid method (that is, a method for measuring the concentration of the analyte).
そこで、本発明は、上記の問題点を鑑みて、従来技術と比較して、より迅速に分析対象物の濃度を測定することができる方法及びその方法を用いたラテラルフロー用テストストリップを提供することを目的とする。 Therefore, in view of the above problems, the present invention provides a method capable of measuring the concentration of an analyte more quickly than a conventional technique, and a lateral flow test strip using the method. For the purpose.
本発明の一態様は、分析対象物と第一物質の反応によって形成された複合物と第二物質とを反応させることで発生する検出可能なシグナルを測定し、測定した前記シグナルの強度から所定時間におけるシグナル増加率を算出し、算出した前記シグナル増加率を予め設定したシグナル増加率の基準値と比較することで前記分析対象物の濃度を判断することを特徴とする分析対象物の濃度測定方法である。 In one embodiment of the present invention, a detectable signal generated by reacting a complex formed by the reaction between an analyte and a first substance and a second substance is measured, and a predetermined value is determined from the measured intensity of the signal. Analyte concentration measurement characterized by calculating a signal increase rate over time and comparing the calculated signal increase rate with a preset reference value of the signal increase rate Is the method.
また、上記濃度測定方法において、前記所定時間は、前記複合物と前記第二物質との反応が飽和するまでの時間よりも短く、前記シグナル増加率は、前記分析対象物と前記第一物質との反応開始時から前記所定時間までの前記シグナル強度の時間変化率を利用して算出されることとしてもよい。
また、上記濃度測定方法において、前記シグナル増加率は、予め設定した単位時間での前記シグナル強度の時間変化量を利用して算出されることとしてもよい。
In the concentration measurement method, the predetermined time is shorter than a time until the reaction between the complex and the second substance is saturated, and the signal increase rate is determined by the analysis object, the first substance, and It is good also as calculating using the time change rate of the said signal intensity from the time of the reaction start to the said predetermined time.
In the concentration measurement method, the signal increase rate may be calculated using a time change amount of the signal intensity in a preset unit time.
また、上記濃度測定方法において、前記シグナルは、光学的、電気化学的、磁気化学的手法のうちいずれか1つによって検出可能なものであり、前記測定は、光学的、電気化学的、磁気化学的手法のうちいずれか1つを用いた測定であることとしてもよい。
また、上記濃度測定方法において、ラテラルフロー用テストストリップに用いることとしてもよい。
In the concentration measurement method, the signal can be detected by any one of optical, electrochemical, and magnetochemical methods, and the measurement can be performed by optical, electrochemical, or magnetic chemistry. The measurement may be performed using any one of the conventional methods.
Moreover, in the said density | concentration measuring method, it is good also as using for the test strip for lateral flow.
また、上記濃度測定方法において、前記第二物質は、前記ラテラルフロー用テストストリップを構成するメンブレンの所定の位置に固定化されていることとしてもよい。
また、上記濃度測定方法において、前記分析対象物と前記第一物質との反応は、前記分析対象物と前記第一物質とを混合する手段によって行われることとしてもよい。
また、上記濃度測定方法において、前記複合物と前記第二物質との反応は、前記分析対象物と前記第二物質とを混合する手段によって行われることとしてもよい。
In the concentration measurement method, the second substance may be fixed at a predetermined position of a membrane constituting the lateral flow test strip.
In the concentration measurement method, the reaction between the analysis object and the first substance may be performed by means for mixing the analysis object and the first substance.
In the concentration measurement method, the reaction between the composite and the second substance may be performed by means for mixing the analyte and the second substance.
また、上記濃度測定方法において、前記分析対象物と前記第一物質との反応は、物理的親和性、化学的親和性、化学結合、免疫学的方法のいずれか1つを利用して行われることとしてもよい。
また、上記濃度測定方法において、前記複合物と前記第二物質との反応は、物理的親和性、化学的親和性、化学結合、免疫学的方法のいずれか1つを利用して行われることとしてもよい。
In the concentration measurement method, the reaction between the analyte and the first substance is performed using any one of physical affinity, chemical affinity, chemical binding, and immunological methods. It is good as well.
In the concentration measurement method, the reaction between the complex and the second substance is performed using any one of physical affinity, chemical affinity, chemical binding, and immunological method. It is good.
また、上記濃度測定方法において、前記第一物質は、化学結合または物理結合によって第一支持体に固定化されていることとしてもよい。
また、上記濃度測定方法において、前記第二物質は、化学結合または物理結合によって第二支持体に固定化されることとしてもよい。
また、上記濃度測定方法において、前記第一物質と前記第二物質のそれぞれは、アミノ酸、核酸、ポリペプチド、ポリヌクレオチド、脂質、リン脂質、糖質、多糖、低分子化合物、無機物質及びこれらの融合体のいずれか1つであることとしてもよい。
In the concentration measurement method, the first substance may be immobilized on the first support by chemical bonding or physical bonding.
In the concentration measurement method, the second substance may be immobilized on the second support by chemical bonding or physical bonding.
In the concentration measurement method, each of the first substance and the second substance includes an amino acid, a nucleic acid, a polypeptide, a polynucleotide, a lipid, a phospholipid, a saccharide, a polysaccharide, a low molecular compound, an inorganic substance, and these substances. It may be any one of fusions.
また、上記濃度測定方法において、前記アミノ酸と前記ポリペプチドのそれぞれは、タンパク質、タンパク質断片、結合ドメイン、標的結合ドメイン、結合タンパク質、結合タンパク質断片、抗体、抗体断片、抗体重鎖、抗体軽鎖、1本鎖抗体、単一ドメイン抗体、Fab抗体断片、Fc抗体断片、Fv抗体断片、F(ab’)2抗体断片、Fab’抗体断片、1本鎖Fv(scFv)抗体断片、抗体結合ドメイン、抗原、抗原決定基、エピトープ、ハプテン、免疫原、免疫原断片、ビオチン、ビオチン誘導体、アビジン、ストレプトアビジン、基質、酵素、抗体酵素、補因子、受容体、受容体断片、受容体サブユニット、受容体サブユニット断片、ホルモン、レクチン、ポリヒスチジンのいずれか1つであることとしてもよい。 In the concentration measurement method, each of the amino acid and the polypeptide includes a protein, a protein fragment, a binding domain, a target binding domain, a binding protein, a binding protein fragment, an antibody, an antibody fragment, an antibody heavy chain, an antibody light chain, Single chain antibody, single domain antibody, Fab antibody fragment, Fc antibody fragment, Fv antibody fragment, F (ab ′) 2 antibody fragment, Fab ′ antibody fragment, single chain Fv (scFv) antibody fragment, antibody binding domain, Antigen, antigenic determinant, epitope, hapten, immunogen, immunogen fragment, biotin, biotin derivative, avidin, streptavidin, substrate, enzyme, antibody enzyme, cofactor, receptor, receptor fragment, receptor subunit, receptor It may be any one of a body subunit fragment, a hormone, a lectin, and a polyhistidine.
また、上記濃度測定方法において、前記核酸は、mRNA、総RNA、ゲノムDNA、プラスミドDNA、植物DNAのいずれか1つであることとしてもよい。
また、上記濃度測定方法において、前記分析対象物は、脂質、炭水化物、タンパク質、抗体、抗原、代謝産物、ホルモン、ウイルス、微生物、細胞、末梢血、血清、血漿、腹水、尿、脳脊髄液、痰、唾液、骨髄、滑液、眼房水、羊水、耳垢、母乳、気管支肺胞洗浄液、***、前立腺液、カウパー液若しくは***前液、汗、糞便、毛髪、涙、嚢胞液、胸水若しくは腹水、囲心腔液、リンパ液、糜粥、乳糜、胆汁、間質液、月経分泌物、膿、皮脂、嘔吐物、膣分泌物、粘膜からの分泌物、水便、膵液、鼻腔からの洗浄液、気管支肺吸引物、胚盤胞腔液、臍帯血のいずれか1つであることとしてもよい。
In the concentration measurement method, the nucleic acid may be any one of mRNA, total RNA, genomic DNA, plasmid DNA, and plant DNA.
In the concentration measurement method, the analysis object includes lipid, carbohydrate, protein, antibody, antigen, metabolite, hormone, virus, microorganism, cell, peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid, Sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostate fluid, cowper's fluid or pre-ejaculate fluid, sweat, feces, hair, tears, cyst fluid, pleural effusion or ascites , Pericardial fluid, lymph fluid, sputum, milk cake, bile, interstitial fluid, menstrual discharge, pus, sebum, vomiting, vaginal discharge, mucous secretion, water stool, pancreatic juice, nasal wash, It may be one of bronchopulmonary aspirate, blastocyst fluid, and umbilical cord blood.
また、上記濃度測定方法において、前記第一および第二支持体は、ポリメチルメタクリル酸メチル(PMMA)、ポリカーボネート、ポリプロピレン、ポリエチレン、ポリメチルペンテン、ポリスチレン、ポリテトラフルオロエチレン、ABS樹脂、ポリジメチルシロキサン、ニトロセルロース、シリコンの樹脂、それらの高分子化合物を含む共重合体あるいは複合体、石英ガラス、パイレックス(登録商標)ガラス、ソーダガラス、ホウ酸ガラス、ケイ酸ガラス、ホウケイ酸ガラス、セラミックス及びその複合体のいずれか1つによって構成されることとしてもよい。 In the above concentration measurement method, the first and second supports are polymethyl methyl methacrylate (PMMA), polycarbonate, polypropylene, polyethylene, polymethylpentene, polystyrene, polytetrafluoroethylene, ABS resin, polydimethylsiloxane. , Nitrocellulose, silicone resins, copolymers or composites containing these polymer compounds, quartz glass, Pyrex (registered trademark) glass, soda glass, borate glass, silicate glass, borosilicate glass, ceramics and the like It may be configured by any one of the complexes.
また、上記濃度測定方法において、前記第一支持体は、金コロイド、銀コロイド、鉄コロイド、アルミニウムコロイド、または白金コロイドを含む金属コロイド粒子、または高分子化合物を材料とする粒子であることとしてもよい。
また、上記濃度測定方法において、前記高分子化合物を材料とする粒子は、蛍光色素内包粒子または可視色素内包粒子であることとしてもよい。
In the concentration measurement method, the first support may be a metal colloid particle including a gold colloid, a silver colloid, an iron colloid, an aluminum colloid, or a platinum colloid, or a particle made of a polymer compound. Good.
In the concentration measurement method, the particles made of the polymer compound may be fluorescent dye-containing particles or visible dye-containing particles.
また、上記濃度測定方法において、前記蛍光色素内包粒子に内包された蛍光色素は、二価のマンガン、鉛、アンチモン、セリウム、三価のセリウム、三価のクロム、二価または三価の鉄、三価または四価のチタン、銅、銀、二価のサマリウム、二価または三価のユーロピウム、三価のテルビウム、三価のジスプロシウム、三価のホルミウム、三価のエルビウム、ウラニル化合物、ルテニウム化合物、錫化合物、タリウム化合物、ビスマス化合物、タングステン酸化合物、モリブデン酸化合物、硫黄、バナジウム化合物、ランタン化合物、プラセオジム化合物、ネオジム化合物、プロメチウム化合物、ガドリニウム化合物、ツリウム化合物、イッテルビウム化合物、ルテチウム化合物の群から選ばれる少なくとも1つの物質を含んだ色素であることとしてもよい。 In the concentration measurement method, the fluorescent dye encapsulated in the fluorescent dye-encapsulated particles may be divalent manganese, lead, antimony, cerium, trivalent cerium, trivalent chromium, divalent or trivalent iron, Trivalent or tetravalent titanium, copper, silver, divalent samarium, divalent or trivalent europium, trivalent terbium, trivalent dysprosium, trivalent holmium, trivalent erbium, uranyl compound, ruthenium compound , Tin compound, thallium compound, bismuth compound, tungstic acid compound, molybdate compound, sulfur, vanadium compound, lanthanum compound, praseodymium compound, neodymium compound, promethium compound, gadolinium compound, thulium compound, ytterbium compound, lutetium compound A pigment containing at least one substance It may be used as a.
また、上記濃度測定方法において、前記免疫学的方法は、酵素免疫測定法(EIA)、放射免疫測定法(RIA)、蛍光免疫検定法(FIA)、細胞免疫染色法、組織免疫染色法、免疫沈降法、フローサイトメトリー法、蛍光標示式細胞分取法(FACS)、イムノクロマトグラフィー法、水晶振動子マイクロバランス法(QCM)、表面プラズモン共鳴法(SPR)、二面偏波式干渉法(DPI)、エリプソメトリー法、ELISPOT法、ウェスタンブロッティング法のいずれか1つを用いて測定可能な方法であることとしてもよい。
また、本発明の別の態様は、上記記載の分析対象物の濃度測定方法を用いたことを特徴とするラテラルフロー用テストストリップである。
In the concentration measurement method, the immunological methods include enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), cell immunostaining, tissue immunostaining, Sedimentation method, flow cytometry method, fluorescence-labeled cell sorting method (FACS), immunochromatography method, quartz crystal microbalance method (QCM), surface plasmon resonance method (SPR), two-plane polarization interference method (DPI) , Ellipsometry method, ELISPOT method, and Western blotting method may be used.
Another aspect of the present invention is a lateral flow test strip characterized by using the above-described method for measuring an analyte concentration.
本発明の一態様であれば、試料の反応開始後、所定時間における検出シグナルの強度変化の傾き(つまり、時間変化率)を算出する。そして、その算出した値を、予め算出しておいた各設定濃度における分析対象物のシグナル強度変化の傾きと比較する。こうして、測定試料に含まれる分析対象物の濃度の決定を行う。
このため、本発明の一態様によれば、シグナルが十分に検出されるまで(シグナル強度が飽和するまで)待機する必要がないので、従来技術と比較して、より迅速に分析対象物の濃度を測定することができる。
According to one embodiment of the present invention, the slope of the change in the intensity of the detection signal at a predetermined time (that is, the time change rate) is calculated after the start of the sample reaction. Then, the calculated value is compared with the slope of the change in signal intensity of the analyte at each set concentration calculated in advance. In this way, the concentration of the analysis object contained in the measurement sample is determined.
For this reason, according to one embodiment of the present invention, it is not necessary to wait until a signal is sufficiently detected (until the signal intensity is saturated), so that the concentration of the analyte can be more quickly compared with the conventional technique. Can be measured.
まず、本実施形態で使用される試料と、本実施形態で測定される分析対象物について、具体例を挙げつつ説明する。
(試料について)
本発明の実施形態で使用される試料は、生体試料であって液状のものが好ましい。上記試料としては、例えば、末梢血、血清、血漿、腹水、尿、脳脊髄液、痰、唾液、骨髄、滑液、眼房水、羊水、耳垢、母乳、気管支肺胞洗浄液、***、前立腺液、カウパー液若しくは***前液、汗、糞便、毛髪、涙、嚢胞液、胸水若しくは腹水、囲心腔液、リンパ液、糜粥、乳糜、胆汁、間質液、月経分泌物、膿、皮脂、嘔吐物、膣分泌物、粘膜からの分泌物、水便、膵液、鼻腔からの洗浄液、気管支肺吸引物、胚盤胞腔液、臍帯血等である。
First, the sample used in this embodiment and the analyte to be measured in this embodiment will be described with specific examples.
(About the sample)
The sample used in the embodiment of the present invention is a biological sample and is preferably a liquid sample. Examples of the sample include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid, sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostate fluid , Cowper's fluid or pre-ejaculate fluid, sweat, feces, hair, tears, cyst fluid, pleural effusion or ascites, pericardial fluid, lymph fluid, sputum, milk fistula, bile, interstitial fluid, menstrual discharge, pus, sebum, vomiting Products, vaginal secretions, mucous secretions, water stool, pancreatic juice, nasal lavage fluid, bronchopulmonary aspirate, blastocyst fluid, umbilical cord blood and the like.
(分析対象物について)
本発明の実施形態における分析対象物は、例えば、脂質、炭水化物、タンパク質、抗体、抗原、代謝産物、ホルモン、ウイルス、微生物、細胞、末梢血、血清、血漿、腹水、尿、脳脊髄液、痰、唾液、骨髄、滑液、眼房水、羊水、耳垢、母乳、気管支肺胞洗浄液、***、前立腺液、カウパー液若しくは***前液、汗、糞便、毛髪、涙、嚢胞液、胸水若しくは腹水、囲心腔液、リンパ液、糜粥、乳糜、胆汁、間質液、月経分泌物、膿、皮脂、嘔吐物、膣分泌物、粘膜からの分泌物、水便、膵液、鼻腔からの洗浄液、気管支肺吸引物、胚盤胞腔液、臍帯血のいずれか1つである。より詳しくは、例えば、核酸、脂質、炭水化物、タンパク質等であって、特に、DNA、RNA、抗体、抗原、代謝産物、ホルモン、ウイルス、微生物、細胞、腫瘍マーカー、心疾患マーカーである。以下、これらの分析対象物の具体例について説明する。
(Analytical objects)
Analytes in the embodiments of the present invention include, for example, lipids, carbohydrates, proteins, antibodies, antigens, metabolites, hormones, viruses, microorganisms, cells, peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid, sputum , Saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostate fluid, cowper's fluid or pre-ejaculatory fluid, sweat, feces, hair, tears, cyst fluid, pleural effusion or ascites, Pericardial fluid, lymph fluid, sputum, milk cake, bile, interstitial fluid, menstrual discharge, pus, sebum, vomiting, vaginal discharge, mucous discharge, stool, pancreatic juice, nasal wash, bronchi Any one of lung aspirate, blastocyst fluid, and umbilical cord blood. More specifically, for example, nucleic acids, lipids, carbohydrates, proteins, etc., particularly DNA, RNA, antibodies, antigens, metabolites, hormones, viruses, microorganisms, cells, tumor markers, heart disease markers. Hereinafter, specific examples of these analysis objects will be described.
上述のタンパク質としては、例えば、血漿タンパク質、血清タンパク質、リポタンパク質、糖タンパク質、ポリペプチド、腫瘍マーカー、アポタンパク質、ウイルス抗原、自己抗体等が挙げられる。
また、上述の血漿タンパク質や血清タンパク質としては、例えば、免疫グロブリン(IgG、IgA、IgM、IgD、IgE)、補体成分(C3、C4、C5、C1q)、CRP、α1−アンチトリプシン、α1−マイクログロブリン、β2−マイクログロブリン、ハプトグロビン、トランスフェリン、セルロプラスミン、フェリチン等が挙げられる。
Examples of the protein include plasma protein, serum protein, lipoprotein, glycoprotein, polypeptide, tumor marker, apoprotein, viral antigen, autoantibody and the like.
Examples of the plasma protein and serum protein include immunoglobulins (IgG, IgA, IgM, IgD, IgE), complement components (C3, C4, C5, C1q), CRP, α1-antitrypsin, α1- Examples include microglobulin, β2-microglobulin, haptoglobin, transferrin, ceruloplasmin, ferritin and the like.
また、上述の腫瘍マーカーとしては、例えば、α−フェトプロテイン(AFP)、癌胎児性抗原(CEA)、CA19−9、CA125、CA15−3、SCC抗原、前立腺酸性ホスファターゼ(PAP)、PIVKA−II、γ−セミノプロテイン、TPA、エラスターゼI、神経特異エノラーゼ(NSE)、免疫抑制酸性タンパク(IAP)等が挙げられる。 Examples of the above-mentioned tumor marker include α-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA19-9, CA125, CA15-3, SCC antigen, prostate acid phosphatase (PAP), PIVKA-II, Examples include γ-seminoprotein, TPA, elastase I, nerve specific enolase (NSE), and immunosuppressive acidic protein (IAP).
また、上述のアポタンパク質としては、例えば、アポA−I、アポA−II、アポB、アポC−II、アポC−III、アポE等が挙げられる。
また、上述のウイルス抗原としては、例えば、B型肝炎ウイルス(HBV)関連抗原、C型肝炎ウイルス(HVC)関連抗原、HTLV−I、HIV、狂犬病ウイルス、インフルエンザウイルス、風疹ウイルス等が挙げられる。より詳しくは、HCV関連抗原としては、例えば、HCVc100−3リコビナント抗原、pHCV−31リコビナント抗原、pHCV−34リコビナント抗原等が挙げられ、それらの混合物が好ましく使用できる。また、HIV関連抗原としては、ウイルス表面抗原等が挙げられ、例えば、HIV−I env.gp41リコビナント抗原、HIV−I env.gp120リコビナント抗原、HIV−Igag.p24リコビナント抗原、HIV−II env.p36リコビナント抗原等が挙げられる。また、ウイルス以外の感染症としては、MRSA、ASO、トキソプラズマ、マイコプラズマ、STD等が挙げられる。
Examples of the apoprotein include apo AI, apo A-II, apo B, apo C-II, apo C-III, and apo E.
Examples of the viral antigen include hepatitis B virus (HBV) -related antigen, hepatitis C virus (HVC) -related antigen, HTLV-I, HIV, rabies virus, influenza virus, rubella virus and the like. More specifically, examples of the HCV-related antigen include HCVc100-3 recombinant antigen, pHCV-31 recombinant antigen, pHCV-34 recombinant antigen, and a mixture thereof can be preferably used. Examples of HIV-related antigens include virus surface antigens and the like, for example, HIV-I env. gp41 recombinant antigen, HIV-I env. gp120 recombinant antigen, HIV-Igag. p24 recombinant antigen, HIV-II env. Examples include p36 recombinant antigen. Examples of infectious diseases other than viruses include MRSA, ASO, toxoplasma, mycoplasma, STD and the like.
また、上述の自己抗体としては、例えば、抗マイクロゾーム抗体、抗サイログロブリン抗体、抗核抗体、リュウマチ因子、抗ミトコンドリア抗体、ミエリン抗体等が挙げられる。
また、上述のホルモンとしては、例えば、下垂体ホルモン(LH、FSH、GH、ACTH、TSH、プロラクチン)、甲状腺ホルモン(T3、T4、サイログロブリン)、カルシトニン、副甲状腺ホルモン(PTH)、副腎皮質ホルモン(アルドステロン、コルチゾール)、性腺ホルモン(hCG、エストロゲン、テストステロン、hPL)、膵・消化管ホルモン(インスリン、C−ペプチド、グルカゴン、ガストリン)、その他(レニン、アンジオテンシンI,II、エンケファリン、エリスロポエチン)等が挙げられる。
Examples of the autoantibodies include anti-microsomal antibodies, anti-thyroglobulin antibodies, antinuclear antibodies, rheumatoid factors, anti-mitochondrial antibodies, and myelin antibodies.
Examples of the hormones described above include pituitary hormones (LH, FSH, GH, ACTH, TSH, prolactin), thyroid hormones (T3, T4, thyroglobulin), calcitonin, parathyroid hormone (PTH), adrenal cortex hormone ( Aldosterone, cortisol), gonadal hormones (hCG, estrogen, testosterone, hPL), pancreatic / gastrointestinal hormones (insulin, C-peptide, glucagon, gastrin), others (renin, angiotensin I, II, enkephalin, erythropoietin) It is done.
また、上述の心疾患マーカーとしては、例えば、クレアチニンキナーゼ(CK)、CKのアイソザイムであるCK−MB、ミオグロビン、心臓型脂肪酸結合蛋白(H−FABP)、ミオシン軽鎖(MLC)、心筋トロポニンT(TnT)、心筋トロポニンI(TnI)、心房性ナトリウム利尿ペプチド(ANP)、脳性ナトリウム利尿ペプチド(BNP)等が挙げられる。 Examples of the above-mentioned heart disease markers include creatinine kinase (CK), CK isozyme CK-MB, myoglobin, cardiac fatty acid binding protein (H-FABP), myosin light chain (MLC), cardiac troponin T (TnT), cardiac troponin I (TnI), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and the like.
以下、本実施形態に係る分析対象物の濃度測定方法で用いられるラテラルフロー用テストストリップについて説明する。
本実施形態に係るラテラルフロー用テストストリップは、試料に含まれる分析対象物を検出して定量するものであって、試料滴下部材であるサンプルパッド、試料が流れる方向に分析対象物に特異的親和性のある第一物質が標識された粒子が乾燥固化されている結合パッド、そして分析対象物に特異的親和性のある第二物質が固相化された部位(テストライン)と粒子表面の第一物質を認識する第二物質が固相化された部位(コントロールライン)を持つ展開膜(メンブレン)、更にその下流に毛細管現象により浸潤してきた試料を吸収する吸収パッドの4種の部材が直列に連結されたものである。なお、上述の分析対象物に特異的親和性のある第一物質が標識された粒子は、例えば、平均粒径20〜100nmの金コロイド粒子である。また、分析対象物を検出するための第二物質は、例えば、メンブレンにおいては線状に固定化されている。また、ラテラルフロー用テストストリップの幅は、例えば、1.5mm〜5mmであり、サンプルパッドに滴下する分析対象物を含む試料の体積は、例えば25μl〜100μlである。
Hereinafter, a lateral flow test strip used in the method for measuring the concentration of an analyte according to this embodiment will be described.
The test strip for lateral flow according to the present embodiment detects and quantifies an analyte contained in a sample, and has a specific affinity for the analyte in the direction of sample flow, a sample pad that is a sample dropping member. A binding pad on which particles labeled with a sexual first substance are dried and solidified, and a site (test line) on which a second substance having specific affinity for an analyte is immobilized and a first surface on the particle surface. There are four types of members in series: a development membrane (membrane) having a part (control line) in which a second substance recognizing one substance is solidified, and an absorption pad that absorbs a sample that has infiltrated downstream by capillary action. It is connected to. In addition, the particle | grains by which the 1st substance with specific affinity to the above-mentioned to-be-analyzed object was labeled are gold colloid particles with an average particle diameter of 20-100 nm, for example. In addition, the second substance for detecting the analysis target is immobilized in a linear shape on the membrane, for example. Moreover, the width | variety of the test strip for lateral flows is 1.5 mm-5 mm, for example, and the volume of the sample containing the analysis object dripped at a sample pad is 25 microliters-100 microliters, for example.
本実施形態に係るラテラルフロー用テストストリップは、テストラインにおいては分析対象物と第一物質と第二物質との複合体が形成されることにより、検出強度が分析対象物の濃度依存的に上昇し、コントロールラインにおいては第一物質標識粒子が毛細管現象にて流れてきたものを検出するものである。より詳しくは、サンプルパッドに試料を滴下した時点から、光学的手法を用いて、第二物質が固定化された部位のシグナルを経時的に検出するものである。 In the test strip for lateral flow according to the present embodiment, the detection intensity increases depending on the concentration of the analyte by forming a complex of the analyte, the first substance, and the second substance in the test line. In the control line, the first substance-labeled particles are detected by the capillary phenomenon. More specifically, the signal at the site where the second substance is immobilized is detected over time using an optical technique from the time when the sample is dropped onto the sample pad.
以下、本実施形態に係るラテラルフロー用テストストリップを構成する部材の材料及び構成について、詳細に説明する。
(部材の材料について)
上述の第一物質と第二物質のそれぞれは、例えば、アミノ酸、核酸、ポリペプチド、ポリヌクレオチド、脂質、リン脂質、糖質、多糖、低分子化合物、無機物質及びこれらの融合体のいずれか1つである。
Hereinafter, materials and structures of members constituting the lateral flow test strip according to the present embodiment will be described in detail.
(About material of parts)
Each of the first substance and the second substance is, for example, any one of amino acids, nucleic acids, polypeptides, polynucleotides, lipids, phospholipids, carbohydrates, polysaccharides, low molecular compounds, inorganic substances, and fusions thereof. One.
また、上述のアミノ酸とポリペプチドのそれぞれは、例えば、タンパク質、タンパク質断片、結合ドメイン、標的結合ドメイン、結合タンパク質、結合タンパク質断片、抗体、抗体断片、抗体重鎖、抗体軽鎖、1本鎖抗体、単一ドメイン抗体、Fab抗体断片、Fc抗体断片、Fv抗体断片、F(ab’)2抗体断片、Fab’抗体断片、1本鎖Fv(scFv)抗体断片、抗体結合ドメイン、抗原、抗原決定基、エピトープ、ハプテン、免疫原、免疫原断片、ビオチン、ビオチン誘導体、アビジン、ストレプトアビジン、基質、酵素、抗体酵素、補因子、受容体、受容体断片、受容体サブユニット、受容体サブユニット断片、ホルモン、レクチン、ポリヒスチジンのいずれか1つである。 Each of the above-mentioned amino acids and polypeptides includes, for example, a protein, a protein fragment, a binding domain, a target binding domain, a binding protein, a binding protein fragment, an antibody, an antibody fragment, an antibody heavy chain, an antibody light chain, and a single chain antibody. Single domain antibody, Fab antibody fragment, Fc antibody fragment, Fv antibody fragment, F (ab ′) 2 antibody fragment, Fab ′ antibody fragment, single chain Fv (scFv) antibody fragment, antibody binding domain, antigen, antigen determination Group, epitope, hapten, immunogen, immunogenic fragment, biotin, biotin derivative, avidin, streptavidin, substrate, enzyme, antibody enzyme, cofactor, receptor, receptor fragment, receptor subunit, receptor subunit fragment , Any one of hormone, lectin, and polyhistidine.
また、上述の核酸は、例えば、mRNA、総RNA、ゲノムDNA、プラスミドDNA、植物DNAのいずれか1つである。
また、上述の第一物質が標識された粒子(第一支持体)は、例えば、金コロイド、銀コロイド、鉄コロイド、アルミニウムコロイド、または白金コロイドを含む金属コロイド粒子、または高分子化合物を材料とする粒子である。より詳しくは、高分子化合物を材料とする粒子は、蛍光色素内包粒子または可視色素内包粒子である。また、蛍光色素内包粒子に内包される蛍光色素は、例えば、二価のマンガン、鉛、アンチモン、セリウム、三価のセリウム、三価のクロム、二価または三価の鉄、三価または四価のチタン、銅、銀、二価のサマリウム、二価または三価のユーロピウム、三価のテルビウム、三価のジスプロシウム、三価のホルミウム、三価のエルビウム、ウラニル化合物、ルテニウム化合物、錫化合物、タリウム化合物、ビスマス化合物、タングステン酸化合物、モリブデン酸化合物、硫黄、バナジウム化合物、ランタン化合物、プラセオジム化合物、ネオジム化合物、プロメチウム化合物、ガドリニウム化合物、ツリウム化合物、イッテルビウム化合物、ルテチウム化合物の群から選ばれる少なくとも1つの物質を含んだ色素である。
The above-mentioned nucleic acid is, for example, any one of mRNA, total RNA, genomic DNA, plasmid DNA, and plant DNA.
The particles (first support) labeled with the above-mentioned first substance are, for example, metal colloid particles including gold colloid, silver colloid, iron colloid, aluminum colloid, or platinum colloid, or a polymer compound as a material. Particles. More specifically, the particles made of a polymer compound are fluorescent dye-containing particles or visible dye-containing particles. The fluorescent dyes encapsulated in the fluorescent dye-encapsulated particles are, for example, divalent manganese, lead, antimony, cerium, trivalent cerium, trivalent chromium, divalent or trivalent iron, trivalent or tetravalent. Titanium, copper, silver, divalent samarium, divalent or trivalent europium, trivalent terbium, trivalent dysprosium, trivalent holmium, trivalent erbium, uranyl compound, ruthenium compound, tin compound, thallium Compound, bismuth compound, tungstic acid compound, molybdate compound, sulfur, vanadium compound, lanthanum compound, praseodymium compound, neodymium compound, promethium compound, gadolinium compound, thulium compound, ytterbium compound, lutetium compound It is a pigment containing.
また、上述の第二物質が固定化されたメンブレン(第二支持体)は、例えば、ポリメチルメタクリル酸メチル(PMMA)、ポリカーボネート、ポリプロピレン、ポリエチレン、ポリメチルペンテン、ポリスチレン、ポリテトラフルオロエチレン、ABS樹脂、ポリジメチルシロキサン、ニトロセルロース、シリコンの樹脂、それらの高分子化合物を含む共重合体あるいは複合体、石英ガラス、パイレックス(登録商標)ガラス、ソーダガラス、ホウ酸ガラス、ケイ酸ガラス、ホウケイ酸ガラス、セラミックス及びその複合体のいずれか1つによって構成される。
なお、上述の第一物質は、化学結合または物理結合によって第一支持体に固定化されている。また、上述の第二物質は、化学結合または物理結合によって第二支持体に固定化されている。
Moreover, the membrane (second support) on which the second substance is immobilized is, for example, polymethyl methyl methacrylate (PMMA), polycarbonate, polypropylene, polyethylene, polymethylpentene, polystyrene, polytetrafluoroethylene, ABS. Resin, Polydimethylsiloxane, Nitrocellulose, Silicone resin, Copolymer or complex containing these high molecular compounds, Quartz glass, Pyrex (registered trademark) glass, Soda glass, Borate glass, Silicate glass, Borosilicate It is composed of any one of glass, ceramics, and composites thereof.
The first substance described above is immobilized on the first support by a chemical bond or a physical bond. Moreover, the above-mentioned second substance is immobilized on the second support by chemical bonding or physical bonding.
(構造について)
ラテラルフロー用テストストリップの形状は、滴下する試料がテストストリップ中を毛細管現象により移動することができる限り、特に制限を受けない。例えば、サンプルパッド、結合パッド、メンブレン、吸水パッドの4部材が、基板となるバッキングシート上にこの順番で一方向に直列連結して固定化された構造が代表的である。各部材をバッキングシートに固定化した後、そのバッキングシートを裁断機で2mm〜5mm幅に裁断してテストストリップとするのが好ましい。
(About structure)
The shape of the test strip for lateral flow is not particularly limited as long as the dropped sample can move through the test strip by capillary action. For example, a structure in which four members, a sample pad, a bonding pad, a membrane, and a water absorbing pad, are connected in series in one order in this order on a backing sheet serving as a substrate, and is representative. After each member is fixed to the backing sheet, the backing sheet is preferably cut into a width of 2 mm to 5 mm by a cutting machine to form a test strip.
以下、ラテラルフロー用テストストリップを構成する上記4部材の詳細について説明する。
(1)サンプルパッド
サンプルパッドは、滴下された、分析対象物を含む液体試料を一定時間保持する部材である。サンプルパッドとしては、セルロースを素材としたものを使用するのが好ましく、より詳しくは、例えばCellulose Absorbent Pad(Pall社製)等が挙げられる。また、滴下された試料によりpHが異なることを鑑み、サンプルパッドに緩衝液を浸潤させて乾燥させた後に、そのサンプルパッドを使用するのが好ましい。
Hereinafter, the details of the four members constituting the lateral flow test strip will be described.
(1) Sample pad The sample pad is a member that holds a dropped liquid sample containing an analysis object for a certain period of time. As the sample pad, it is preferable to use a cellulose material, and more specifically, for example, Cellulose Absorbent Pad (manufactured by Pall). In view of the difference in pH depending on the dropped sample, it is preferable to use the sample pad after the sample pad is infiltrated with a buffer solution and dried.
(2)結合パッド
結合パッドは、分析対象物に対する標識粒子が乾燥固化された部材であり、サンプルパッドから毛細管現象により移動してきた試料に含まれる分析対象物が抗原抗体反応等の特異的認識反応で上記標識粒子と結合される部分である。結合パッドの材質は、ガラス繊維で不織状のものが好ましく、より詳しくは、例えばGlass Fiber Pad(Millipore社製)等が挙げられる。なお、上述の分析対象物と標識粒子に備わる抗体(第一物質)との反応は、物理的親和性、化学的親和性、化学結合、免疫学的方法のいずれか1つを利用して行われるものである。
(2) Binding pad The binding pad is a member obtained by drying and solidifying the labeled particles for the analyte, and the analyte contained in the sample that has moved from the sample pad by capillary action is a specific recognition reaction such as an antigen-antibody reaction. It is a part couple | bonded with the said labeling particle | grains. The material of the bonding pad is preferably a non-woven glass fiber, and more specifically, for example, Glass Fiber Pad (Millipore). The reaction between the analyte and the antibody (first substance) provided on the labeled particle is performed using any one of physical affinity, chemical affinity, chemical binding, and immunological methods. It is what is said.
結合パッドにおける単位面積(cm2)当たりの上記標識粒子の含有量は、特に制限はないが、1μg〜4μgであることが好ましい。また、結合パッドへの上記標識粒子の浸潤方法としては、例えば、標識粒子を懸濁させ分散させた液体を、結合パッドに塗布、滴下又は噴霧した後に、その結合パッドを乾燥させる方法が好ましい。
また、上記標識粒子は、上述の材料の中でも、金属コロイド粒子、蛍光色素粒子、着色ラテックス粒子、アップコンバージョン燐光体粒子、量子ドット粒子等が好ましい。また、上記標識粒子の粒子径は、20nm〜1000nmであることが好ましい。
また、上記標識粒子に備わる抗体としては、上述の抗体の中でも、マウスIgG、ヤギIgGの他、例えば、ウサギIgG、ニワトリIgY、マウスIgE、ヒトIgG、ヒトIgE等が好ましい。
The content of the labeling particles per unit area (cm 2 ) in the bonding pad is not particularly limited, but is preferably 1 μg to 4 μg. Further, as a method for infiltrating the labeled particles into the bonding pad, for example, a method of drying the bonding pad after applying, dripping or spraying a liquid in which the labeled particles are suspended and dispersed to the bonding pad is preferable.
The labeling particles are preferably metal colloid particles, fluorescent dye particles, colored latex particles, up-conversion phosphor particles, quantum dot particles, etc. among the above materials. The particle size of the labeling particles is preferably 20 nm to 1000 nm.
As the antibody provided in the labeled particles, among the above-mentioned antibodies, in addition to mouse IgG and goat IgG, for example, rabbit IgG, chicken IgY, mouse IgE, human IgG, human IgE and the like are preferable.
(3)メンブレン
メンブレンは、上記標識粒子と分析対象物との複合体、上記標識粒子及び試料が毛細管現象によって移動する部材であり、固定化抗体−分析対象物−標識粒子からなる免疫反応が行われる抗体固定化部(テストライン)と、固定化抗体−標識粒子からなる免疫反応が行われる抗体固定化部(コントロールライン)とを有している。上述の分析対象物と固定化抗体(第二物質)との反応は、物理的親和性、化学的親和性、化学結合、免疫学的方法のいずれか1つを利用して行われるものである。
(3) Membrane The membrane is a member in which the labeled particle / analyte complex, the labeled particle, and the sample move by capillary action, and an immune reaction consisting of immobilized antibody / analyte / labeled particle is performed. An antibody immobilization part (test line), and an antibody immobilization part (control line) in which an immune reaction comprising immobilized antibody-labeled particles is carried out. The reaction between the analyte and the immobilized antibody (second substance) is performed using any one of physical affinity, chemical affinity, chemical binding, and immunological method. .
メンブレンは、タンパク質を物理吸着可能な素材で構成されたもの、特にニトロセルロースで構成されたものが好ましく、より詳しくは、例えばHi−Flow Plus120メンブレン(Millipore社製)等が挙げられる。
上記メンブレンにおける抗体固定化部(判定部)の形状としては、局所的に捕捉用抗体が固定化されている限り特に制限はなく、例えば、ライン状、円状、帯状等が挙げられる。その形状の中でも、幅0.5〜2.0mmのライン状であることが好ましく、また、1cmあたりの抗体量が0.1〜2μgであることが好ましい。
The membrane is preferably composed of a material capable of physically adsorbing protein, particularly composed of nitrocellulose, and more specifically, for example, Hi-Flow Plus 120 membrane (manufactured by Millipore).
The shape of the antibody immobilization part (determination part) in the membrane is not particularly limited as long as the capture antibody is locally immobilized, and examples thereof include a line shape, a circular shape, and a belt shape. Among these shapes, a line shape having a width of 0.5 to 2.0 mm is preferable, and an antibody amount per 1 cm is preferably 0.1 to 2 μg.
メンブレンに備わる抗体としては、上述の抗体の中でも、マウスIgG、ヤギIgGの他、例えば、ウサギIgG、ニワトリIgY、マウスIgE、ヒトIgG、ヒトIgE等が好ましい。例えば、標識粒子にマウスIgGが表面修飾されている場合は、ヤギ抗マウスIgG抗体が固定化された抗体固定化部をコントロールラインとして用いることができる。また、抗体をメンブレンに固定化した後、タンパク質の非特異的吸着を防ぐために、例えば、アルブミン、カゼイン、ポリビニルアルコール等のブロッキング剤の溶液でそのメンブレンを浸潤させた後に乾燥させるのが好ましい。 As the antibody provided in the membrane, among the above-mentioned antibodies, mouse IgG, goat IgG, for example, rabbit IgG, chicken IgY, mouse IgE, human IgG, human IgE and the like are preferable. For example, when mouse IgG is surface-modified on the labeled particles, an antibody immobilization part on which a goat anti-mouse IgG antibody is immobilized can be used as a control line. Moreover, after immobilizing the antibody on the membrane, in order to prevent non-specific adsorption of the protein, for example, the membrane is preferably infiltrated with a blocking agent solution such as albumin, casein, or polyvinyl alcohol and then dried.
テストラインにおいては、固定化抗体−分析対象物−標識粒子からなる免疫反応により、形成された複合体(分析対象物−標識粒子)が捕捉される。そして、このテストラインで、捕捉された標識粒子に由来する発色又は蛍光等(具体的には、蛍光、発光、呈色、着色等)の強度(シグナル)の程度により分析対象物の有無を判定、すなわち陽性・陰性を判定する。一方、コントロールラインにおいては、固定化抗体−標識粒子からなる免疫反応により、試料の移動を確認できる。こうして、抗体固定化部に標識粒子が濃縮され、シグナルを目視的に又は検出機器を用いて検出、判定できる。 In the test line, the formed complex (analyte-labeled particle) is captured by an immune reaction consisting of immobilized antibody-analyte-labeled particle. In this test line, the presence or absence of the analyte is determined based on the intensity (signal) of color development or fluorescence derived from the captured labeled particles (specifically, fluorescence, luminescence, coloration, coloring, etc.). That is, positive / negative is determined. On the other hand, in the control line, the movement of the sample can be confirmed by an immune reaction consisting of immobilized antibody-labeled particles. Thus, the labeled particles are concentrated in the antibody immobilization part, and the signal can be detected and judged visually or using a detection device.
(4)吸水パッド
吸水パッドは、毛細管現象でメンブレンを移動してきた試料及び標識粒子を吸収し、常に一定の流れを生じさせるための部材である。吸水パッドの材質としては、セルロース素材の繊維状のものが好ましく、より詳しくは、例えばCellulose Fiber Sample Pad(Millipore社製)等が挙げられる。
(5)バッキングシート
バッキングシートは、上記のサンプルパッド、結合パッド、メンブレン、吸収パッドの4部材を固定化させるための粘着性を有したシートである。バッキングシートとしては、例えばバッキングシートAR9020(Adhesives Research社製)等が挙げられる。
(4) Water-absorbing pad The water-absorbing pad is a member that absorbs the sample and marker particles that have moved through the membrane by capillary action and always generates a constant flow. The material of the water absorbing pad is preferably a fibrous material of cellulose material, and more specifically, for example, Cellulose Fiber Sample Pad (manufactured by Millipore).
(5) Backing sheet The backing sheet is a sheet having adhesiveness for fixing the four members of the sample pad, the bonding pad, the membrane, and the absorption pad. Examples of the backing sheet include backing sheet AR9020 (manufactured by Adhesives Research).
以下、上記テストラインで、捕捉された標識粒子に由来するシグナルの検出方法について説明する。
上記テストストリップにおいて、分析対象物と、対象物特異的物質が標識された粒子との反応で生じるシグナル(検出シグナル)は、光学的な方法で検出するのが好ましい。光学的な方法としては、例えば反射光度又は蛍光検出、発光検出、あるいは電気化学発光等が挙げられる。より詳しくは、赤色系/青色系を経時的に検出できる装置としては、イムノクロマトリーダC10066−10(浜松ホトニクス社製)等が挙げられる。
Hereinafter, a method for detecting a signal derived from the captured labeled particles in the test line will be described.
In the test strip, it is preferable to detect a signal (detection signal) generated by the reaction between the analyte and the particles labeled with the object-specific substance by an optical method. Examples of the optical method include reflected light intensity or fluorescence detection, luminescence detection, or electrochemiluminescence. More specifically, as an apparatus capable of detecting red / blue system over time, an immunochromatography reader C10066-10 (manufactured by Hamamatsu Photonics) and the like can be mentioned.
次に、シグナルに対する時間変化率である傾きの算出方法について説明する。
上記の通り、分析対象物と分析対象物特異的物質とは、免疫学的反応を利用したテストストリップで検出される。本実施形態に係る算出方法は、テストストリップにて分析対象物が既知濃度の試料を滴下し、シグナル検出装置を用いてシグナル強度の測定を開始する。既知濃度の種類は、既知濃度がゼロを含め5段階以上あるのが好ましい。シグナルは、10秒〜60秒の間隔で検出(測定)し、経過時間に対し各時間におけるシグナル強度を記録する。各既知濃度試料に対するシグナル強度変化の傾きを算出するために、各既知濃度試料を30分程度テストストリップにて展開させ、30分間のデータを解析するのが好ましい。
Next, a method for calculating the slope, which is the rate of change with respect to the signal, will be described.
As described above, the analyte and the analyte-specific substance are detected by the test strip using an immunological reaction. In the calculation method according to this embodiment, a sample having a known concentration of an analyte is dropped on a test strip, and signal intensity measurement is started using a signal detection device. It is preferable that there are five or more types of known concentrations including zero known concentrations. The signal is detected (measured) at intervals of 10 to 60 seconds, and the signal intensity at each time is recorded with respect to the elapsed time. In order to calculate the slope of the change in signal intensity with respect to each known concentration sample, it is preferable to develop each known concentration sample on a test strip for about 30 minutes and analyze the data for 30 minutes.
測定対象により傾きが異なるため、最初に定量値が求められる傾きを決定する必要がある。各既知濃度におけるシグナル強度変化の傾きを測定開始後から1分間隔で算出する。各経過時間での傾きに対し、測定開始30分後における各既知濃度のシグナル強度との相関を求める。相関が最も高い経過時間での傾きが最短で測定可能な時間と決定され、反応が飽和に達する30分の反応を待たず、迅速測定が可能となる(図1)。この時の分析対象物の濃度と傾きとで散布図を作成し、検量線も算出する。
Since the slope varies depending on the measurement object, it is necessary to first determine the slope at which the quantitative value is obtained. The slope of the change in signal intensity at each known concentration is calculated at 1 minute intervals from the start of measurement. The correlation with the signal intensity of each known
この決定した経過時間を利用し、分析対象物が未知濃度の試料をテストストリップで測定する。分析対象物濃度未知試料をテストストリップに滴下し、経時的な測定を開始する。次に、上記で決定された経過時間における傾きを算出し、検量線を利用し分析対象物の濃度を算出する。
換言すると、本実施形態に係る分析対象物の濃度測定方法は、分析対象物と第一物質の反応によって形成された複合物と第二物質とを反応させることで発生する検出可能なシグナルを測定し、測定した前記シグナルの強度から所定時間におけるシグナル増加率を算出し、算出した前記シグナル増加率を予め設定したシグナル増加率の基準値と比較することで前記分析対象物の濃度を判断するものである。
Using this determined elapsed time, a sample having an unknown analyte concentration is measured with a test strip. A sample with unknown analyte concentration is dropped on the test strip and measurement over time is started. Next, the slope at the elapsed time determined above is calculated, and the concentration of the analyte is calculated using the calibration curve.
In other words, the analyte concentration measurement method according to the present embodiment measures a detectable signal generated by reacting the complex formed by the reaction between the analyte and the first substance with the second substance. And calculating the signal increase rate at a predetermined time from the measured signal intensity, and comparing the calculated signal increase rate with a preset reference value of the signal increase rate to determine the concentration of the analyte. It is.
また、前記所定時間は、前記複合物と前記第二物質との反応が飽和するまでの時間よりも短く、前記シグナル増加率は、前記分析対象物と前記第一物質との反応開始時から前記所定時間までの前記シグナル強度の時間変化率を利用して算出されるものである。または、前記シグナル増加率は、予め設定した単位時間での前記シグナル強度の時間変化率を利用して算出されるものである。 Further, the predetermined time is shorter than the time until the reaction between the complex and the second substance is saturated, and the signal increase rate is determined from the start of the reaction between the analyte and the first substance. It is calculated using the time change rate of the signal intensity up to a predetermined time. Alternatively, the signal increase rate is calculated using a time change rate of the signal intensity in a preset unit time.
(変形例)
上述の実施形態では、分析対象物と第一物質との反応は、結合パッドで開始される場合について説明したが、本発明はこれに限定されるものではない。また、上述の実施形態では、複合物と第二物質との反応は、メンブレンで開始される場合について説明したが、本発明はこれに限定されるものではない。
(Modification)
In the above-described embodiment, the case where the reaction between the analyte and the first substance is initiated by the bond pad is described, but the present invention is not limited to this. In the above-described embodiment, the case where the reaction between the composite and the second substance is initiated by the membrane has been described, but the present invention is not limited to this.
以下、本発明を実施例に基づいてさらに詳細に説明する。
(1)抗体調製
分析対象物をPSA(Prostate Specific Antigen:前立腺特異抗原)とし、このPSAを検出するため、メンブレン固相化抗体にはマウスモノクローナル抗PSA抗体1H12(HyTest社製)を使用した。また、コントロールラインには、抗マウスイムノグロブリン抗体(Dako社製)を使用した。この2種の抗体を50mMリン酸緩衝液(pH7.2)で透析カートリッジSlide−A−Lyzer 10,000MWCO(Pierce社製)を用いて4℃で透析した。透析後の抗体濃度を測定し、抗PSA抗体は1mg/ml、抗マウスイムノグロブリンは0.5mg/mlにそれぞれ調製した。
Hereinafter, the present invention will be described in more detail based on examples.
(1) Antibody preparation The analysis object was PSA (Prostate Specific Antigen), and in order to detect this PSA, mouse monoclonal anti-PSA antibody 1H12 (manufactured by HyTest) was used as the membrane-immobilized antibody. In addition, an anti-mouse immunoglobulin antibody (manufactured by Dako) was used as a control line. The two types of antibodies were dialyzed at 4 ° C. with a 50 mM phosphate buffer (pH 7.2) using a dialysis cartridge Slide-A-Lyzer 10,000 MWCO (Pierce). The antibody concentration after dialysis was measured, and anti-PSA antibody was prepared at 1 mg / ml, and anti-mouse immunoglobulin was prepared at 0.5 mg / ml.
(2)金コロイド溶液の調製
マウスモノクローナル抗PSA抗体(5A6)を透析し、リン酸緩衝液(pH7.4)で60μg/mlに調製した。次に、1ODになるよう調製した金コロイド(粒子径40nm、田中貴金属工業社製)溶液9mlとリン酸二水素カリウム溶液(pH8.0)1mlとの混合液に本抗体溶液を加えピペッティング後、室温で15分間静置し、1%PEG20,000溶液及び10%BSA溶液を加えて混合した。この混合溶液を8000×gで15分遠心分離した後に上清を廃棄し、結合パッド塗布液を加えて攪拌・超音波処理し、再び遠心分離した後に上清を廃棄した。この操作を計2回繰り返し金コロイド溶液とした。
(2) Preparation of colloidal gold solution Mouse monoclonal anti-PSA antibody (5A6) was dialyzed and adjusted to 60 μg / ml with phosphate buffer (pH 7.4). Next, this antibody solution is added to a mixed solution of 9 ml of a gold colloid (particle size 40 nm, manufactured by Tanaka Kikinzoku Kogyo Co., Ltd.) solution prepared to 1 OD and 1 ml of potassium dihydrogen phosphate solution (pH 8.0), and pipetting is performed. The mixture was allowed to stand at room temperature for 15 minutes, and 1% PEG 20,000 solution and 10% BSA solution were added and mixed. After centrifuging this mixed solution at 8000 × g for 15 minutes, the supernatant was discarded, and the binding pad coating solution was added, stirred and sonicated, centrifuged again, and the supernatant was discarded. This operation was repeated twice in total to obtain a gold colloid solution.
(3)サンプルパッドの作製
セルロースを素材としたサンプルパッド(Cellulose Absorbent Pad #111、Pall社製)を1.8mm×150mmに裁断し、50mM Tris−HCl(pH7.4)を1ml滴下して、50℃で1時間乾燥させた。
(4)結合パッドの作製
1.5mlエッペンドルフチューブに塗布液と金コロイド溶液とを加え、28kHzで超音波処理した後、計900μlの金コロイド塗布溶液を10mm×150mmのガラスファイバーパッド(glass fiber Pad、Millipore社製)に滴下し、そのガラスファイバーパッドを真空減圧ポンプで減圧した環境下で18時間乾燥させた。
(3) Preparation of sample pad A sample pad made of cellulose (Cellulose Absorbent Pad # 111, manufactured by Pall) was cut into 1.8 mm × 150 mm, and 1 ml of 50 mM Tris-HCl (pH 7.4) was dropped. Dry at 50 ° C. for 1 hour.
(4) Preparation of Bond Pad After adding the coating solution and colloidal gold solution to a 1.5 ml Eppendorf tube and sonicating at 28 kHz, a total of 900 μl of colloidal gold coating solution was added to a 10 mm × 150 mm glass fiber pad (glass fiber pad). , Manufactured by Millipore), and the glass fiber pad was dried for 18 hours in an environment reduced in pressure by a vacuum vacuum pump.
(5)メンブレンの作製
メンブレン(HiFlow Plus HF120、Millipore社製)に抗体塗布機XYZ3060(BioDot社製)で、上記(1)で調整した抗体(固相化抗体)を1μl/cmで塗布した。塗布後、0.5%乳性カゼイン溶液でブロッキングし、スクロースや界面活性剤を含む洗浄液で洗浄・安定化して、一晩室温で乾燥させた。
(5) Production of membrane The antibody (solid-phase antibody) prepared in (1) above was applied at 1 μl / cm to the membrane (HiFlow Plus HF120, manufactured by Millipore) with an antibody coating machine XYZ3060 (BioDot). After the application, it was blocked with a 0.5% dairy casein solution, washed and stabilized with a washing solution containing sucrose and a surfactant, and dried overnight at room temperature.
(6)テストストリップの作製
乾燥させたメンブレン、吸収パッド(CFSP203000、Millipore社製)、結合パッド、サンプルパッドのそれぞれをバッキングシート(Adhesive Research社製)に貼り付け、そのバッキングシートをカッティング機器CM4000(BioDot社製)にて5mm幅で裁断した後、ハウジングケースに収めてテストストリップキットとした。
(6) Preparation of test strip Each of the dried membrane, the absorption pad (CFSP203000, manufactured by Millipore), the bonding pad, and the sample pad was attached to a backing sheet (manufactured by Adhesive Research), and the backing sheet was cut into a cutting device CM4000 ( (Product of BioDot) was cut to a width of 5 mm, and then housed in a housing case to form a test strip kit.
(7)抗原濃度既知溶液の調製
検出標的であるPSA(抗原)は、Recombinant PSA(HyTest社製)を使用した。この抗原溶液を1×PBS、5%BSA溶液にて、抗原濃度0、0.16、0.8、4、20ng/mlに希釈して測定用抗原溶液とした。
(8)テストストリップでの対象物既知濃度試料の測定
上記(6)で作製したテストストリップを水平に置き、上記(7)の抗原濃度既知溶液を1枚のテストストリップあたり100μl滴下した。滴下開始後、イムノクロマトリーダC10066−10(浜松ホトニクス社製)内の測定位置に設置し、1分間隔で30分間継続して測定した。こうして測定して結果を図2に示す。測定データは、テストストリップの展開方向にスキャニングし、テストライン、コントロールラインにおけるピーク中心(Height:ピークトップ)におけるシグナル強度を測定データとした。また、30分間の測定において傾きがゼロになる時間を反応が飽和に達した時間とした。
(7) Preparation of antigen concentration known solution Recombinant PSA (manufactured by HyTest) was used as PSA (antigen) as a detection target. This antigen solution was diluted with 1 × PBS, 5% BSA solution to an antigen concentration of 0, 0.16, 0.8, 4, 20 ng / ml to obtain an antigen solution for measurement.
(8) Measurement of sample with known concentration of target on test strip The test strip prepared in (6) above was placed horizontally, and 100 μl of the antigen concentration known solution of (7) was dropped per test strip. After the start of dropping, it was placed at a measurement position in Immunochromato Reader C10066-10 (manufactured by Hamamatsu Photonics), and measurement was continued for 30 minutes at 1 minute intervals. The measurement results are shown in FIG. The measurement data was scanned in the development direction of the test strip, and the signal intensity at the peak center (Height: peak top) in the test line and control line was used as the measurement data. In addition, the time when the slope reached zero in the measurement for 30 minutes was defined as the time when the reaction reached saturation.
(9)対象物既知濃度による反応最適時間の決定
上記(8)の結果から、傾きがゼロとなる30分後でのシグナル強度を各抗原濃度での反応飽和におけるシグナル強度とした。測定開始(0分)から1分間隔で経過時間でのシグナル強度を経過時間におけるシグナル強度とし、反応が飽和に達するまでの時間にて、一定時間でのシグナル強度を所定時間におけるシグナル強度とした。0〜20ng/mlの各既知濃度の反応飽和におけるシグナル強度に対し、経過時間におけるシグナル強度、所定時間におけるシグナル強度の相関を算出し、相関が最も高い経過時間又は所定時間を傾きによる定量測定に最適な時間とした。測定開始(0分)から所定時間までの相関係数を表1に、また、1分間隔での所定時間の相関係数を表2に示した。
(9) Determination of optimum reaction time based on the known concentration of the object From the result of (8) above, the signal intensity at 30 minutes after the slope becomes zero was defined as the signal intensity at reaction saturation at each antigen concentration. The signal intensity at the elapsed time from the start of measurement (0 minutes) is defined as the signal intensity at the elapsed time, and the signal intensity at a predetermined time is defined as the signal intensity at the predetermined time until the reaction reaches saturation. . Calculates the correlation between the signal intensity at the elapsed time and the signal intensity at the predetermined time for the signal intensity at the reaction saturation of each known concentration of 0 to 20 ng / ml, and the elapsed time with the highest correlation or the predetermined time is used for quantitative measurement by the slope. The time was optimal. Table 1 shows the correlation coefficient from the start of measurement (0 minutes) to the predetermined time, and Table 2 shows the correlation coefficient for the predetermined time at 1 minute intervals.
表1は、測定開始時間(0分)から、2分、3分、4分、5分経過におけるシグナル強度の傾きと、30分後のシグナル強度との相関係数を算出した結果である。なお、傾きは、0分におけるシグナル強度と算出終点時間におけるシグナル強度の2点から算出したものである。 Table 1 shows the results of calculating the correlation coefficient between the slope of the signal intensity after 2 minutes, 3 minutes, 4 minutes, and 5 minutes from the measurement start time (0 minutes) and the signal intensity after 30 minutes. The slope is calculated from two points: the signal intensity at 0 minutes and the signal intensity at the calculation end point time.
表2は、測定開始時間(0分)からの所定経過時間を経過算出始点時間とし、1分間隔で算出終点時間を設定し、シグナル強度の傾きと30分後のシグナル強度との相関係数を算出した結果である。なお、傾きは、各算出始点時間から1分後の算出終点時間までの2点間におけるシグナル強度から算出したものである。 Table 2 shows the correlation coefficient between the slope of the signal intensity and the signal intensity after 30 minutes, with the predetermined elapsed time from the measurement start time (0 minute) as the elapsed calculation start time and the calculated end time set at 1 minute intervals. It is the result of having calculated. Note that the slope is calculated from the signal intensity between two points from each calculation start point time to the calculation end point time one minute later.
図2に示すように、反応開始後5分で、抗原濃度4ng/mlと20ng/mlで反応増加率が低下する傾向が確認された。このため、反応開始後5分までを傾き算出時間の目安とした。また、表1に示すように、測定開始(0分)から5分まで所定時間の傾きと30分後のシグナル強度との相関は高く、30分後のシグナル強度を利用した場合と同等の検量線作成が可能である。ただし、テストストリップの特性上、毛細管現象の影響により測定開始時は展開のムラがあるため、測定開始後1〜2分は傾き算出に利用しないのが好ましい。また、表2に示すように、測定開始(0分)からの所定時間における算出始点時間から1分間隔での傾きと30分後のシグナル強度の相関は高く、30分後のシグナル強度を利用した場合と同等の検量線作成が可能である。こうして、本実施例における最適な時間は、4分から6分の時間と算出した。 As shown in FIG. 2, it was confirmed that the reaction increase rate decreased at antigen concentrations of 4 ng / ml and 20 ng / ml 5 minutes after the start of the reaction. For this reason, up to 5 minutes after the start of the reaction was used as a measure of the slope calculation time. In addition, as shown in Table 1, the correlation between the slope of the predetermined time from the start of measurement (0 minutes) to 5 minutes and the signal intensity after 30 minutes is high, and the same calibration as when using the signal intensity after 30 minutes Line creation is possible. However, because of the characteristics of the test strip, there is uneven development at the start of measurement due to the influence of the capillary phenomenon. Therefore, it is preferable not to use the calculation for the inclination for 1-2 minutes after the start of measurement. Moreover, as shown in Table 2, the correlation between the slope at the 1-minute interval from the calculation start point time at the predetermined time from the start of measurement (0 minutes) and the signal intensity after 30 minutes is high, and the signal intensity after 30 minutes is used. It is possible to create a calibration curve equivalent to this. Thus, the optimum time in this example was calculated as 4 to 6 minutes.
分析対象物の濃度が未知である試料に対しては、上記のようにして決定された経過時間におけるシグナル強度変化の傾きを算出し、予め作製しておいた検量線を利用して、分析対象物の濃度を判断する。
以上のように、本実施例であれば、一般的なラテラルフロー用テストストリップでの測定時間である20分よりも時間短縮ができ、5分程度で定量的な測定が可能となった。よって、本発明の実施形態に係る測定方法は、より緊急を要す疾患に関し、迅速な診断を可能とすることが明らかとなった。
なお、本実施例は本発明の一実施例であり、本発明はこれらの実施例に何ら限定されるものではない。
For samples where the concentration of the analyte is unknown, calculate the slope of the change in signal intensity over the elapsed time determined as described above, and use the calibration curve prepared in advance to analyze the analyte. Determine the concentration of the object.
As described above, in this embodiment, the time can be shortened from 20 minutes, which is the measurement time of a general lateral flow test strip, and quantitative measurement is possible in about 5 minutes. Therefore, it has been clarified that the measurement method according to the embodiment of the present invention enables a quick diagnosis regarding a more urgent disease.
In addition, a present Example is one Example of this invention, and this invention is not limited to these Examples at all.
Claims (22)
前記シグナル増加率は、前記分析対象物と前記第一物質との反応開始時から前記所定時間までの前記シグナル強度の時間変化率を利用して算出されることを特徴とする請求項1に記載の分析対象物の濃度測定方法。 The predetermined time is shorter than the time until the reaction between the composite and the second substance is saturated,
The signal increase rate is calculated using a time change rate of the signal intensity from the start of the reaction between the analyte and the first substance to the predetermined time. Method for measuring the concentration of the analyte.
前記測定は、光学的、電気化学的、磁気化学的手法のうちいずれか1つを用いた測定であることを特徴とする請求項1から請求項3のいずれか一項に記載の分析対象物の濃度測定方法。 The signal is detectable by any one of optical, electrochemical and magnetochemical techniques;
The analysis object according to any one of claims 1 to 3, wherein the measurement is measurement using any one of optical, electrochemical, and magnetochemical methods. Concentration measurement method.
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