JP2016161329A - Method for forming a sample addition part of immuno-chromatography inspection device and immuno-chromatography inspection device - Google Patents
Method for forming a sample addition part of immuno-chromatography inspection device and immuno-chromatography inspection device Download PDFInfo
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本発明は、イムノクロマト法検査デバイスの試料添加部の形成方法及び該方法により形成された試料添加部を有するイムノクロマト法検査デバイスに関する。 The present invention relates to a method for forming a sample addition portion of an immunochromatography test device and an immunochromatography test device having a sample addition portion formed by the method.
近年、ウイルスや細菌等の病原体感染の有無、妊娠の有無などの様々な検査を短時間のうちに行う簡易検査試薬やキットが開発されている。病原体構成成分、ヒト絨毛性ゴナドトロピン等が検出あるいは定量の対象である。簡易検査試薬の多くは、特別な設備を必要とせず操作も簡単で安価であるという特徴を有しており、例えば、妊娠診断のための簡易検査試薬は一般薬局で販売されている。また、病原体の感染を検査する簡易検査試薬は、他の検査試薬と異なり、大病院や医療検査センター以外にも一般の病院や診療所で広く使用されている。これらの施設は患者が最初に訪れる医療機関である場合が多く、患者から採取した検体についてその場で感染の有無が判明すれば、早い段階で治療措置を施すことができるため、簡易検査試薬の医療における重要性は益々高まってきている。 In recent years, simple test reagents and kits have been developed that perform various tests such as the presence or absence of pathogens such as viruses and bacteria and the presence or absence of pregnancy within a short time. Pathogen components, human chorionic gonadotropins, and the like are targets for detection or quantification. Many of the simple test reagents have the feature that they do not require special equipment, are easy to operate and are inexpensive, and for example, simple test reagents for pregnancy diagnosis are sold at general pharmacies. Also, unlike other test reagents, simple test reagents for testing pathogen infection are widely used in general hospitals and clinics in addition to large hospitals and medical test centers. These facilities are often the first medical institutions visited by patients, and if the presence or absence of infection is identified on the spot for samples collected from patients, treatment measures can be taken at an early stage. The importance in medicine is increasing.
現在、簡易検査方法として、抗原抗体反応を利用した免疫測定法、特にイムノクロマト法が一般に知られている。イムノクロマト法は被検出物に特異的に結合する捕捉体(捕捉物質)、および被検出物に特異的に結合する標識体の複合体をメンブラン上に形成させて、標識を検出／定量することで、被検出物の検出（測定あるいは定量）を行う。イムノクロマト法は測定装置が簡単で、またコストの点でも優れているため多種多様の被検出物の検出に広く用いられている。 Currently, as a simple test method, an immunoassay method using an antigen-antibody reaction, particularly an immunochromatography method is generally known. Immunochromatography is a method of detecting / quantifying the label by forming a complex of a capture body (capture substance) that specifically binds to the target substance and a labeled body that specifically binds to the target object. Detecting (measuring or quantifying) an object to be detected. The immunochromatography method is widely used for detecting a wide variety of objects to be detected because of its simple measuring device and excellent cost.
イムノクロマト法の一つの形態においては、ニトロセルロース等のメンブランストリップ上に被検出物に特異的に結合する抗体を捕捉物質として固相化した検出部、及び被検出物に特異的に結合する標識体を含む標識体部を備えた検査デバイスに、被検出物を含む検体試料を滴下して、被検出物−標識体の複合体を形成させながら展開して検出部でこの複合体を捕捉することで標識を検出あるいは定量する（例えば特許文献１及び特許文献２）。 In one form of the immunochromatography method, a detection part in which an antibody that specifically binds to a detection target is immobilized on a membrane strip such as nitrocellulose as a capture substance, and a label that specifically binds to the detection target A specimen sample containing a detection object is dropped onto a test device having a label body portion containing, and developed while forming a complex of the detection object-label body, and the detection section captures this complex. The label is detected or quantified by using (for example, Patent Document 1 and Patent Document 2).
近年、イムノクロマト法検査デバイスの試料添加部に予め試薬を含浸させることにより、試料添加部上で検体試料を前処理する技術が開発されている。 In recent years, a technique has been developed for pretreating a specimen sample on a sample addition unit by impregnating a sample addition unit of an immunochromatography test device with a reagent in advance.
しかし、試料添加部に含浸させた試薬は乾燥ムラ等の原因により試料添加部内で濃度差が発生したりする。その結果、試料添加部に検体試料を添加すると、含浸させた試薬と検体が上手く混合されず、検体試料がメンブレン上にスムーズに展開されないことがあった。検体試料がメンブレン上に速やかに展開されないことで測定速度が遅くなったり、検体試料がメンブレン上に滞留することでバックグラウンドが高くなるという問題点があった。 However, the reagent impregnated in the sample addition part may cause a concentration difference in the sample addition part due to drying unevenness or the like. As a result, when the sample sample is added to the sample addition portion, the impregnated reagent and the sample are not mixed well, and the sample sample may not be smoothly spread on the membrane. There is a problem that the measurement speed becomes slow because the specimen sample is not rapidly developed on the membrane, and the background becomes high because the specimen sample stays on the membrane.
従って、本発明の目的は、前記の問題点を改善し、従来の測定方法よりも更に迅速且つ高感度に被検出物の存在を検出又は定量することを可能にするイムノクロマト法検査デバイス並びにその試料添加部の形成方法を提供することである。 Accordingly, an object of the present invention is to improve the above-mentioned problems, and to detect or quantify the presence of an object to be detected more quickly and with higher sensitivity than conventional measurement methods, and an immunochromatographic test device and a sample thereof It is to provide a method for forming an addition portion.
本発明者らは、鋭意研究の結果、ニトロセルロースメンブラン等の多孔性基材を用いるイムノクロマト法において、試料添加部に含浸させた試薬の復元性を改良した試料添加部を備えた検査デバイスを用いることにより、迅速且つ高感度に測定が可能になることを見出し、本発明を完成させた。 As a result of diligent research, the present inventors use a test device having a sample addition part that improves the recovery of the reagent impregnated in the sample addition part in an immunochromatography method using a porous substrate such as a nitrocellulose membrane. Thus, it was found that measurement can be performed quickly and with high sensitivity, and the present invention was completed.
すなわち、本発明は、以下のものを提供する。
(1) 試料添加部に常態で白色粉末状である界面活性剤を施し乾燥させることを含む、イムノクロマト法検査デバイスの試料添加部の形成方法。
(2) 前記界面活性剤が、分子量２００〜９００の陰イオン性、非イオン性又は両性界面活性剤である(1)記載の方法。
(3) 前記界面活性剤が、デオキシコール酸及びその塩並びにコール酸及びその塩から成る群より選ばれる少なくとも１種である(1)記載の方法。
(4) 前記界面活性剤が、デオキシコール酸ナトリウムである(3)記載の方法。
(5) (1)〜(4)のいずれか１項に記載の方法により形成された試料添加部を含むイムノクロマト法検査デバイス。
That is, the present invention provides the following.
(1) A method for forming a sample addition part of an immunochromatography test device, which comprises applying a normal white powdery surfactant to the sample addition part and drying it.
(2) The method according to (1), wherein the surfactant is an anionic, nonionic or amphoteric surfactant having a molecular weight of 200 to 900.
(3) The method according to (1), wherein the surfactant is at least one selected from the group consisting of deoxycholic acid and salts thereof and cholic acid and salts thereof.
(4) The method according to (3), wherein the surfactant is sodium deoxycholate.
(5) An immunochromatographic test device including a sample addition part formed by the method according to any one of (1) to (4).
本発明の方法を用いて形成された試料添加部を備えた検査デバイスを用いることにより、検体試料と試料添加部に含浸させた試薬が上手く混合され、検体試料がメンブレン上に速やかに展開されるため、バックグラウンドが低く、偽陽性が少なく、迅速で高感度に測定することができる。 By using a test device having a sample addition portion formed by using the method of the present invention, the sample sample and the reagent impregnated in the sample addition portion are mixed well, and the sample sample is rapidly developed on the membrane. Therefore, the background is low, there are few false positives, and the measurement can be performed quickly and with high sensitivity.
本発明における試料添加部とは、イムノクロマト法検査デバイスにおいて検体試料を添加する部位である。試料添加部は、吸水性のある材質、例えばスポンジ、ガラス繊維などの不織布等の多孔性基材からなるパッドをデバイス本体を構成するニトロセルロースメンブラン等の多孔性基材上に載置することで形成される。パッドを用いる場合、該パッドを試料添加パッドと呼ぶことができる。また、試料添加部をパッド状とせず、デバイス本体を構成するニトロセルロースメンブラン等の多孔性基材の一部領域を試料添加部とすることも可能である。試料添加部は試験片上の一端に設けるのが好ましい。 The sample addition part in the present invention is a part where a specimen sample is added in an immunochromatography test device. The sample addition part is made by placing a pad made of a porous substrate such as a nonwoven material such as sponge or glass fiber on a porous substrate such as a nitrocellulose membrane constituting the device body. It is formed. When a pad is used, the pad can be called a sample addition pad. Moreover, it is also possible to make a sample addition part into a part area | region of porous base materials, such as a nitrocellulose membrane which comprises a device main body, without making a sample addition part into pad shape. The sample addition part is preferably provided at one end on the test piece.
試料添加部には、予め抗原抗体反応に必要な試薬を施すことができる。例えば検体試料中の非特異成分が抗原抗体反応に関与しないように界面活性剤を施したり、血液検体を測定する際は溶血剤を施すこともできる。イムノクロマト法は検体試料を予め浮遊液で浮遊させてから試料添加部に添加することが多いが、その場合、検体試料が希釈されることで被検出物もまた希釈され、検出感度に支障をきたすことになる。そこで、予め検体浮遊液の成分である緩衝剤、界面活性剤、各種蛋白、塩類、糖類を施しておくこともできる。 Reagents necessary for the antigen-antibody reaction can be applied in advance to the sample addition part. For example, a surfactant can be applied so that non-specific components in the specimen sample do not participate in the antigen-antibody reaction, or a hemolytic agent can be applied when measuring a blood specimen. In the immunochromatography method, the specimen sample is often suspended in the suspension in advance and then added to the sample addition part. In this case, the analyte is diluted by diluting the specimen sample, which impairs the detection sensitivity. It will be. Therefore, a buffer, a surfactant, various proteins, salts, and saccharides that are components of the sample suspension can be applied in advance.
本発明の方法では、試料添加部に常態で白色粉末状である界面活性剤を施すことが必要である。本発明者等は、鋭意検討を行った結果、このことによって、イムノクロマト法において、検体試料が速やかにメンブレン上に展開され、高感度に被検出物を検出することができることを見出し、本発明に至った。なお、「常態で白色粉末状」とは、その界面活性剤が、常温、常圧下に単独で存在した場合に白色粉末状という意味である。これ以外の界面活性剤として、液状、ペースト状、又はワックス状である界面活性剤を用いた場合は、乾燥が不十分であったり、試料添加部の安定性に問題があったり、試料が添加された際の迅速な浸透効果が十分でなく、本発明には適さない。界面活性剤は、通常、水系媒体に溶解して多孔性基材に施されるので、水溶解性に優れたものが好ましい。また、白色粉末状の界面活性剤の中でも、分子量２００〜９００の界面活性剤は、水溶解性、安定性に優れ、本発明の効果が得られる点で好ましい。 In the method of the present invention, it is necessary to apply a surfactant that is normally in the form of a white powder to the sample addition part. As a result of intensive studies, the present inventors have found that in this immunochromatography method, the specimen sample can be rapidly developed on the membrane, and the detection object can be detected with high sensitivity. It came. In addition, “normally white powder” means that the surfactant is in the form of white powder when present alone at normal temperature and normal pressure. If a surfactant in the form of liquid, paste, or wax is used as the other surfactant, drying may be insufficient, there may be a problem with the stability of the sample addition section, or the sample may be added. When used, the rapid penetration effect is not sufficient and is not suitable for the present invention. Since the surfactant is usually dissolved in an aqueous medium and applied to the porous substrate, one having excellent water solubility is preferable. Among white powdery surfactants, surfactants having a molecular weight of 200 to 900 are preferable because they are excellent in water solubility and stability, and the effects of the present invention can be obtained.
このような常態において白色粉末状であり、水溶解性に優れた界面活性剤としては、以下のものが挙げられる。これらの界面活性剤は、単独で用いることもできるし２種以上を組み合わせて用いることもできる。
コール酸ナトリウム、
デオキシコール酸ナトリウム、
ラウリル硫酸ナトリウム、
スクロースモノコレート、
β-D-フラクトピラノシル-α-D-グルコピラノシドモノドデカノエート、
β-D-フラクトピラノシル-α-D-グルコピラノシドモノデカノエート、
n-オクタノイル-N-メチルグルカミド、
n-ノナノイル-N -メチルグルカミド、
n-デカノイル-N-メチルグルカミド、
n-オクチル-β-D-チオグルコピラノシド、
n-オクチル-β-D-マルトピラノシド、
n-オクチル-β-D-グルコピラノシド、
n-ノニル-β-D-チオマルトピラノシド、
n-ドデシル-β-D-マルトピラノシド、
n-デシル-β-D-マルトピラノシド、
N,N-ビス(3-D-グルコンアミドプロピル)コラミド、
N,N-ビス(3-D-グルコンアミドプロピル) デオキシコラミド、
3-[(3-コラミドプロピル)ジメチルアンモニオ]プロパンスルホン酸、
3-[(3-コラミドプロピル)ジメチルアンモニオ]-2-ヒドロキシプロパンスルホン酸、
ツビッタージェント (ZWITTERGENT )3-10デタージェント (商品名) カルビオケム製、
ツビッタージェント 3-12デタージェント (商品名) カルビオケム製、
ツビッタージェント 3-14デタージェント (商品名) カルビオケム製。
これらの界面活性剤の中でも、陰イオン性である、デオキシコール酸及びその塩並びにコール酸及びその塩から成る群より選ばれる少なくとも１種が好ましく、特に、コール酸ナトリウム、デオキシコール酸ナトリウムは、水溶解性が良く、安価であるのでより好ましい。
Examples of the surfactant that is white powder in such a normal state and excellent in water solubility include the following. These surfactants can be used alone or in combination of two or more.
Sodium cholate,
Sodium deoxycholate,
Sodium lauryl sulfate,
Sucrose monocholate,
β-D-fructopyranosyl-α-D-glucopyranoside monododecanoate,
β-D-fructopyranosyl-α-D-glucopyranoside monodecanoate,
n-octanoyl-N-methylglucamide,
n-nonanoyl-N-methylglucamide,
n-decanoyl-N-methylglucamide,
n-octyl-β-D-thioglucopyranoside,
n-octyl-β-D-maltopyranoside,
n-octyl-β-D-glucopyranoside,
n-nonyl-β-D-thiomaltopyranoside,
n-dodecyl-β-D-maltopyranoside,
n-decyl-β-D-maltopyranoside,
N, N-bis (3-D-gluconamidopropyl) colamide,
N, N-bis (3-D-gluconamidopropyl) deoxycolamide,
3-[(3-Colamidopropyl) dimethylammonio] propanesulfonic acid,
3-[(3-colamidopropyl) dimethylammonio] -2-hydroxypropanesulfonic acid,
Zwittergent (ZWITTERGENT) 3-10 Detergent (trade name) Made by Calbiochem,
Zubittergent 3-12 Detergent (trade name) Made by Calbiochem,
Zubittergent 3-14 Detergent (trade name) Made by Calbiochem.
Among these surfactants, at least one member selected from the group consisting of deoxycholic acid and its salt and cholic acid and its salt, which are anionic, is preferred. In particular, sodium cholate and sodium deoxycholate are: It is more preferable because it has good water solubility and is inexpensive.
試料添加部に上記界面活性剤を施す方法としては、界面活性剤の溶液、好ましくは、水又は水系緩衝液を溶媒とする水溶液を、含浸や滴下等により添加する方法が好ましい。この場合、界面活性剤溶液中の界面活性剤の濃度は、通常、0.005質量％〜5質量％、好ましくは、0.05質量％〜 2質量％である。また、試料添加部への界面活性剤の添加量は、通常、0.0013mg〜10mg程度、好ましくは、0.005mg〜4mg程度である。 As a method for applying the surfactant to the sample addition part, a method of adding a surfactant solution, preferably an aqueous solution containing water or an aqueous buffer as a solvent, by impregnation or dropping is preferable. In this case, the concentration of the surfactant in the surfactant solution is usually 0.005% to 5% by mass, preferably 0.05% to 2% by mass. Moreover, the addition amount of the surfactant to the sample addition part is usually about 0.0013 mg to 10 mg, preferably about 0.005 mg to 4 mg.
界面活性剤を含有させることの効果は、前記の浮遊液を多孔性基材に施し乾燥する際に、水分子の蒸発にともない界面活性剤が微細な結晶状態として試料添加部に存在することにより、検体試料がムラなく試料添加部に浸透することでメンブレン上に速やかに展開されるためと考えられる。また、予め試料添加部に抗原抗体反応に必要な試薬を施した際は、抗原抗体反応に必要な試薬の乾燥時の濃度ムラや自己凝集が著しく防止されることにより、検体試料と抗原抗体反応に必要な試薬がムラなく混合されることにより、メンブレン上に速やかに展開され、しかも自己凝集によるバックグラウンド並びに偽陽性もなく、Ｓ／Ｎ比を大幅に上げることができる。また、界面活性剤は、常態で粉末状であるので乾燥状態で保持することができ、保存安定性が良く、取扱いも容易である。さらに、白色であるので、目視による検査結果の判定の妨げにならない。 The effect of containing the surfactant is that when the suspension is applied to the porous substrate and dried, the surfactant is present in the sample addition portion as a fine crystalline state as the water molecules evaporate. This is probably because the specimen sample penetrates into the sample addition part without unevenness and is rapidly developed on the membrane. In addition, when the reagent necessary for the antigen-antibody reaction is applied to the sample addition part in advance, the concentration unevenness and self-aggregation during drying of the reagent necessary for the antigen-antibody reaction are remarkably prevented. By mixing the necessary reagents without any unevenness, the S / N ratio can be greatly increased without any rapid development on the membrane and without background and false positives due to self-aggregation. Further, since the surfactant is normally powdery, it can be kept in a dry state, has good storage stability, and is easy to handle. Furthermore, since it is white, it does not interfere with the determination of the inspection result by visual inspection.
本発明のイムノクロマト法試験デバイスを用いて検出する被検出物や検体試料は限定されない。例えば、検体試料は、咽頭あるいは鼻腔拭い液、鼻腔吸引液、咽頭あるいは鼻腔洗浄液、唾液、血清、血漿、全血、便懸濁液、尿、培養液およびそれらを緩衝液で希釈したものや希釈せずにそのまま用いることができる。被検出物も何ら限定されず、検出しようとするいかなる物質であってもよい。具体例として、インフルエンザウイルス、アデノウイルス、ＲＳウイルス、ＨＡＶ、ＨＢｓ、ＨＩＶ、ノロウイルス等のウイルス抗原、ＭＲＳＡ、Ａ群溶連菌、Ｂ群溶連菌、レジオネラ属菌等の細菌抗原、細菌等が産生する毒素、マイコプラズマ、クラミジア・トラコマティス、ヒト絨毛性ゴナドトロピン等のホルモン、Ｃ反応性タンパク質、ミオグロビン、心筋トロポニン、各種腫瘍マーカー、農薬、環境ホルモン等の抗原を挙げることができ、さらに、上記細菌、ウイルス等に対する抗体を挙げることができる。 An object to be detected and a specimen sample to be detected using the immunochromatographic test device of the present invention are not limited. For example, specimen samples include pharyngeal or nasal wipes, nasal aspirate, pharyngeal or nasal wash, saliva, serum, plasma, whole blood, stool suspension, urine, culture fluid and those diluted with buffer or diluted Can be used as is. An object to be detected is not limited at all, and may be any substance to be detected. Specific examples include virus antigens such as influenza virus, adenovirus, RS virus, HAV, HBs, HIV, norovirus, MRSA, group A streptococcus, group B streptococcus, bacterial antigens such as Legionella, toxins produced by bacteria, Examples include hormones such as mycoplasma, chlamydia trachomatis, human chorionic gonadotropin, C-reactive protein, myoglobin, cardiac troponin, various tumor markers, pesticides, environmental hormones, and other antigens. Mention may be made of antibodies.
本発明は、上記のようにして形成される検体添加部位を含むイムノクロマト法検査デバイスをも提供する。試料添加部に常態で白色粉末状である界面活性剤を施す点を除き、検査デバイスの構成自体は、例えば特許文献１及び特許文献２に記載されるような、周知のものであってよい。本発明のイムノクロマト法検査デバイスの具体例の模式図を図１に示すが、本発明の検査デバイスはこれに限定されるものではない。 The present invention also provides an immunochromatographic test device including the specimen addition site formed as described above. The configuration of the inspection device itself may be a well-known one as described in, for example, Patent Document 1 and Patent Document 2, except that a surfactant that is in the form of a white powder is applied to the sample adding portion. Although the schematic diagram of the specific example of the immunochromatography method testing device of this invention is shown in FIG. 1, the testing device of this invention is not limited to this.
図１の上が上面図、下が切断断面図である。図示の具体例では、プラスチック板（ヘ）上に、２個の検出部（ハ）が形成されたニトロセルロースメンブラン（イ）、濾紙で形成された吸収パッド部（ホ）、上記方法により形成された標識体部（ロ）、及びガラス繊維フィルターで形成された試料添加部（ニ）がそれぞれ積層されている。そして、図示のように、吸収パッド部（ホ）の一方の端部領域と、ニトロセルロースメンブラン（イ）の一方の端部領域、ニトロセルロースメンブラン（イ）の他方の端部領域と標識体部（ロ）の一方の端部領域、標識体部（ロ）の他方の端部領域と試料添加部（ニ）の一方の端部領域がそれぞれ重ね合わされており、これにより、連続したラテラルフローの流路が形成されている。 The top of FIG. 1 is a top view and the bottom is a cut cross-sectional view. In the illustrated example, a nitrocellulose membrane (ii) in which two detection parts (c) are formed on a plastic plate (f), an absorption pad part (e) made of filter paper, and formed by the above method. The labeled part (b) and the sample addition part (d) formed of a glass fiber filter are laminated. And, as shown in the figure, one end region of the absorbent pad portion (e), one end region of the nitrocellulose membrane (A), the other end region of the nitrocellulose membrane (A), and the labeling body portion One end region of (b), the other end region of the marker part (b) and one end region of the sample addition part (d) are overlapped, respectively. A flow path is formed.
次にこの検査デバイスを用いた免疫測定法について説明する。先ず、検体を検体浮遊／抽出用緩衝液に浮遊／抽出させた検体試料を調製する。プラスチック板（ヘ）上に積層されたニトロセルロースメンブラン（イ）上に、被検出物と抗原抗体反応する抗体を着色ラテックス粒子で標識した、上記方法により形成される安定化乾燥標識体パッドを含む標識体部（ロ）を備え、更に被検出物と抗原抗体反応する抗体を捕捉物質としてライン状に固相化された検出部（ハ）を備えた検査デバイスの試料添加部（ニ）に前記検体試料を滴下する。被検出物を含む検体試料は、メンブラン上を水平方向に移動しながら標識体を展開するので、被検出物が存在すれば、被検出物−標識体の複合体を形成し、更に検出部（ハ）に到達するとそのライン上に、捕捉抗体−被検出物−標識体の複合体が形成され、この複合体中の着色ラテックス粒子により、複合体の存在を検出することで検体中の被検出物の有無を判定する。なお、検出部（ハ）は、被検出物質と抗原抗体反応し、且つ、着色ラテックス粒子上の抗体又はその抗原結合性断片と同時に被検出物に結合することが可能な、抗体又はその抗原結合性断片をライン状に固相化した領域である。反応に関与しなかった他の成分等は、吸収パッド部（ホ）に吸収される。なお、図１に示す例では、検出部（ハ）が２個存在するが、これは、例えば下記実施例に記載するように、Ａ型インフルエンザウイルスとＢ型インフルエンザウイルスのような２種類の被検出物をそれぞれ捕捉するためのものである。このような検出部（ハ）を複数設けることにより、複数種類の被検出物を同時に免疫測定することが可能である。 Next, an immunoassay method using this test device will be described. First, a specimen sample in which a specimen is suspended / extracted in a specimen suspension / extraction buffer is prepared. Including a stabilized dry label pad formed by the above-described method in which an antibody that reacts with an object to be antigen-antibody is labeled with colored latex particles on a nitrocellulose membrane (b) laminated on a plastic plate (f). In addition to the sample addition part (d) of the inspection device comprising a labeling part (b), and further comprising a detection part (c) solidified in a line using an antibody that undergoes an antigen-antibody reaction with the detected substance as a capture substance Drop the specimen sample. The specimen sample containing the detection object develops the label while moving on the membrane in the horizontal direction. Therefore, if the detection object exists, a complex of the detection object and the label is formed, and the detection unit ( C), a complex of a capture antibody, an object to be detected, and a label is formed on the line, and the presence of the complex is detected by the colored latex particles in the complex, thereby detecting the object in the sample. Determine the presence or absence of objects. The detection unit (c) is an antibody or antigen binding thereof that reacts with the detected substance by antigen-antibody and can bind to the detected substance simultaneously with the antibody on the colored latex particles or antigen-binding fragment thereof. This is a region in which sex fragments are immobilized in a line. Other components that are not involved in the reaction are absorbed by the absorbent pad portion (e). In the example shown in FIG. 1, there are two detection units (c). As shown in the following examples, for example, two detection units (type A influenza virus and type B influenza virus) are detected. This is for capturing each detected object. By providing a plurality of such detection units (c), it is possible to perform immunoassay on a plurality of types of detection objects at the same time.
被検出物と抗原抗体反応する抗体又はその抗原結合性断片を結合させたラテックス粒子を安定化乾燥標識体として用いる本発明の検査デバイスを用いることにより、迅速且つ簡便に被検出物を測定できる。 By using the test device of the present invention that uses latex particles bound with an antibody or antigen-binding fragment thereof that reacts with an object for antigen-antibody reaction as a stabilized dry label, the object to be detected can be measured quickly and easily.
以下、本発明を実施例及び比較例に基づきより具体的に説明する。但し、本発明は下記実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples and comparative examples. However, the present invention is not limited to the following examples.
イムノクロマト法によるインフルエンザウイルス抗原の検出
１．抗インフルエンザウイルスモノクローナル抗体の作製
（１）抗Ａ型インフルエンザウイルスＮＰ抗体
Ａ型インフルエンザウイルス抗原をＢＡＬＢ／ｃマウスに免疫し、一定期間飼育したマウスから脾臓を摘出し、ケラーらの方法（Kohler et al., Nature, vol, 256, p495-497(1975)）によりマウスミエローマ細胞（Ｐ３×６３）と融合した。得られた融合細胞（ハイブリドーマ）を、３７℃インキュベーター中で維持し、Ａ型インフルエンザウイルスＮＰ抗原を固相したプレートを用いたＥＬＩＳＡにより上清の抗体活性を確認しながら細胞の純化（単クローン化）を行った。取得した該細胞２株をそれぞれプリスタン処理したＢＡＬＢ／ｃマウスに腹腔投与し、約２週間後、抗体含有腹水を採取した。得られた腹水からプロティンＡカラムを用いたアフィニティークロマトグラフィーにより、それぞれＩｇＧを精製し、２種類の精製抗Ａ型インフルエンザウイルスＮＰ抗体を得た。
Detection of influenza virus antigens by immunochromatography Preparation of anti-influenza virus monoclonal antibody (1) Anti-influenza A virus NP antibody Influenza A virus antigens were immunized to BALB / c mice, and the spleen was removed from mice bred for a certain period of time, and the method of Keller et al. , Nature, vol, 256, p495-497 (1975)) and fused with mouse myeloma cells (P3 × 63). The obtained fused cells (hybridoma) were maintained in a 37 ° C. incubator, and cell purification was performed while confirming the antibody activity of the supernatant by ELISA using a plate on which influenza A virus NP antigen was immobilized (monocloning) ) The obtained two cell lines were each intraperitoneally administered to pristane-treated BALB / c mice, and about 2 weeks later, antibody-containing ascites was collected. IgG was purified from the obtained ascites by affinity chromatography using a protein A column to obtain two types of purified anti-influenza A virus NP antibodies.
（２）抗Ｂ型インフルエンザウイルスＮＰ抗体
Ｂ型インフルエンザウイルス抗原を用い、（１）と同様の方法で、２種類の精製抗Ｂ型インフルエンザウイルスＮＰ抗体を得た。
(2) Anti-B influenza virus NP antibody Two types of purified anti-B influenza virus NP antibodies were obtained in the same manner as in (1) using influenza B virus antigen.
２．標識体パッドの作製
精製抗Ａ型インフルエンザウイルスＮＰ抗体及び精製抗Ｂ型インフルエンザウイルスＮＰ抗体のうちそれぞれ1種類ずつを使用した。青色ラテックス粒子に抗Ａ型インフルエンザウイルス抗体を共有結合させ、浮遊液に懸濁し、ソニケーションを行って充分に分散浮遊させた抗Ａ型ラテックス浮遊液を調製した。また、同様に抗Ｂ型インフルエンザウイルス抗体を共有結合させた抗Ｂ型ラテックス浮遊液を調製した。抗Ａ型ラテックス浮遊液と抗Ｂ型ラテックス浮遊液とを混合し、大きさが20cm×1cmのガラス繊維に塗布し、温風下で良く乾燥させて、乾燥混合物を形成した標識体パッドを作製した。
2. Preparation of labeled body pad One of each of the purified anti-influenza A virus NP antibody and the purified anti-influenza B virus NP antibody was used. Anti-A influenza virus antibody was covalently bound to the blue latex particles, suspended in the suspension, and sonicated to prepare an anti-A latex suspension that was sufficiently dispersed and suspended. Similarly, an anti-B type latex suspension liquid to which an anti-type B influenza virus antibody was covalently bound was prepared. Anti-A-type latex suspension and anti-B-type latex suspension were mixed, applied to glass fiber with a size of 20cm x 1cm, and dried well under warm air to produce a marker pad that formed a dry mixture. .
３．試料添加パッドの作製
大きさが2.0cm×20cmのガラス繊維を使用した。
3. Preparation of sample addition pad Glass fiber having a size of 2.0 cm × 20 cm was used.
４．検査デバイスの作製
検査デバイスは、図１に示すものと同様の構成のものを用いた。ニトロセルロースメンブランを2cm×20cmの大きさに裁断し接着剤がついたプラスチック板でバッキングした、下端から 0.6cmと1.0cmの位置に約1mm幅になる量の抗Ａ型インフルエンザウイルス抗体（上記と別の抗体）液、並びに抗Ｂ型インフルエンザウイルス抗体（上記と別の抗体）液を各々20cm塗布し、温風下で良く乾燥させて抗体を固相化した（検出部）。次に、3cm×20cmの大きさの濾紙をニトロセルロースメンブランの上端に5mm重ねて吸収パッド部を設けた。更に、標識体パッドをニトロセルロースメンブランの下端に2mm重ねて標識体部を設け、更に、試料添加パッドを標識体パッドの上端から7mm離れた位置に合わせて重ね、試料添加部を設けた。次いで、カッターで幅5mmの短冊に裁断して一体化された検査デバイスを作製した。
4). Production of Inspection Device An inspection device having the same configuration as that shown in FIG. 1 was used. A nitrocellulose membrane is cut into a 2cm x 20cm size and backed with a plastic plate with an adhesive, and an anti-influenza A virus antibody (approx. 20 cm each of another antibody) solution and anti-type B influenza virus antibody (another antibody) solution were applied and dried well under warm air to immobilize the antibody (detector). Next, a filter paper having a size of 3 cm × 20 cm was stacked 5 mm on the upper end of the nitrocellulose membrane to provide an absorbent pad portion. Further, a label body part was provided by overlapping the label body pad by 2 mm on the lower end of the nitrocellulose membrane, and the sample addition pad was overlapped at a position 7 mm away from the upper end of the label body pad to provide a sample addition section. Next, an inspection device integrated by cutting with a cutter into a strip having a width of 5 mm was produced.
５．試験
実施例１及び比較例１
まず、上記のとおり作製した検査デバイスの試料添加部に、検体展開成分（100mM Tris-HCl（pH8.0）、1%Tween20、 0.5%BSA）および食用赤色色素（濃度0.1%）を含浸した（比較例１）。また、これらに0.2質量%のデオキシコール酸ナトリウム(DOC)をさらに含む溶液を試料添加部に含浸させた（実施例１）。試料として生理食塩水を試料添加部に滴下し、試料滴下直後の食用色素の放出の様子と、試験終了後の試料添加部の様子を観察した。その結果、試験直後の色素の放出は、デオキシコール酸ナトリウム含有の試料添加部を持つ検査デバイスで明らかに放出量が多かった（図２）。また試験終了後の試料添加部においても、デオキシコール酸ナトリウム含有サンプルでは色素の色残りが少なかった（図３）。以上からデオキシコール酸ナトリウムが検体添加部に含有されることで検体展開成分の放出が改善し、それに伴い食用色素の放出も改善したものと推察される。なお、図面は白黒なので差がわかりにくいかもしれないが、カラー写真であれば、図２及び図３における実施例１と比較例１の差異は肉眼で明瞭に理解できる。
5. Test Example 1 and Comparative Example 1
First, the sample addition part of the test device produced as described above was impregnated with a sample developing component (100 mM Tris-HCl (pH 8.0), 1% Tween20, 0.5% BSA) and an edible red pigment (concentration 0.1%) ( Comparative Example 1). Further, the sample addition part was impregnated with a solution further containing 0.2% by mass of sodium deoxycholate (DOC) (Example 1). As a sample, physiological saline was dropped on the sample addition part, and the state of release of the food dye immediately after the sample was dropped and the state of the sample addition part after the test was observed. As a result, the release of the dye immediately after the test was obviously large in the test device having the sample addition part containing sodium deoxycholate (FIG. 2). Further, in the sample addition part after the test was completed, the color residue of the dye was small in the sample containing sodium deoxycholate (FIG. 3). From the above, it can be inferred that sodium deoxycholate is contained in the specimen addition part, so that the release of the specimen developing component is improved, and the release of the food dye is also improved accordingly. Since the drawings are black and white, the difference may be difficult to understand. However, in the case of a color photograph, the difference between Example 1 and Comparative Example 1 in FIGS. 2 and 3 can be clearly understood with the naked eye.
実施例２及び比較例２
試料添加部に含浸させた溶液が、赤色食用色素を含まないことを除き、実施例１及び比較例１と同様にして、上記検体展開成分とデオキシコール酸ナトリウムを含む試料添加部を有する検査デバイス（実施例２）と、検体展開成分のみを含む試料添加部を有する検査デバイス（比較例２）を作製した。これらのそれぞれにて、インフルエンザウイルスB型の抗原を使用してイムノクロマト試験を行い、シグナル強度のばらつきをイムノクロマトリーダー（浜松フォトニクス社製）で測定した（各n=10）。
Example 2 and Comparative Example 2
A test device having a sample addition portion containing the sample developing component and sodium deoxycholate in the same manner as in Example 1 and Comparative Example 1 except that the solution impregnated in the sample addition portion does not contain a red food dye. A test device (Comparative Example 2) having (Example 2) and a sample addition portion containing only the sample developing component was produced. In each of these, an immunochromatographic test was performed using an influenza virus type B antigen, and variations in signal intensity were measured with an immunochromatographic reader (manufactured by Hamamatsu Photonics) (each n = 10).
結果を図４に示す。図４に示されるように、デオキシコール酸ナトリウムを含む試料添加部を有する検査デバイス（実施例２）で、明らかにシグナル強度のばらつきが少なかった（図４．デオキシコール酸ナトリウムあり：CV＝7.48%、デオキシコール酸ナトリウムなし：CV=15.02%、）。以上からデオキシコール酸ナトリウムが含まれる試料添加部を用いることで、試料添加部からの検体展開成分の放出が改善し、安定した試験結果を得られたものと考えられる。 The results are shown in FIG. As shown in FIG. 4, in the test device (Example 2) having the sample addition portion containing sodium deoxycholate, there was clearly little variation in signal intensity (FIG. 4. With sodium deoxycholate: CV = 7.48) %, No sodium deoxycholate: CV = 15.02%). From the above, it is considered that by using the sample addition part containing sodium deoxycholate, the release of the sample developing component from the sample addition part was improved, and a stable test result was obtained.
イ ニトロセルロースメンブラン
ロ 標識体部
ハ 検出部
ニ 試料添加部
ホ 吸収パッド部
ヘ プラスチック板
A Nitrocellulose membrane Marker part C Detection part D Sample addition part E Absorption pad part Plastic plate
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|---|---|---|---|---|
| WO2018168905A1 (en) * | 2017-03-14 | 2018-09-20 | デンカ生研株式会社 | Immunochromatographic device for extracting and measuring carbohydrate antigens |
| WO2019117275A1 (en) * | 2017-12-15 | 2019-06-20 | 積水メディカル株式会社 | Immunochromatographic test device |
| JP2019109091A (en) * | 2017-12-15 | 2019-07-04 | 積水メディカル株式会社 | Device for immunochromatographic testing |
| WO2019138899A1 (en) | 2018-01-11 | 2019-07-18 | 東洋紡株式会社 | Measurement sample dilution liquid, kit, and measurement method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07325085A (en) * | 1994-05-30 | 1995-12-12 | Teikoku Hormone Mfg Co Ltd | Method and device for obtaining semi-quantitative determination of hapten using immuno-chromatography |
| JP2008268043A (en) * | 2007-04-23 | 2008-11-06 | Denka Seiken Co Ltd | Method for forming detection part of examination device, and examination device for measuring lateral flow immunity |
| JP2009052945A (en) * | 2007-08-24 | 2009-03-12 | Sunstar Inc | Treatment method of specimen originated in oral cavity inside in immunochromatographic examination |
| JP2009210505A (en) * | 2008-03-06 | 2009-09-17 | Tanaka Kikinzoku Kogyo Kk | Immunoassay method and kit and developing solution therefor |
-
2015
- 2015-02-27 JP JP2015038499A patent/JP6452491B2/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07325085A (en) * | 1994-05-30 | 1995-12-12 | Teikoku Hormone Mfg Co Ltd | Method and device for obtaining semi-quantitative determination of hapten using immuno-chromatography |
| JP2008268043A (en) * | 2007-04-23 | 2008-11-06 | Denka Seiken Co Ltd | Method for forming detection part of examination device, and examination device for measuring lateral flow immunity |
| JP2009052945A (en) * | 2007-08-24 | 2009-03-12 | Sunstar Inc | Treatment method of specimen originated in oral cavity inside in immunochromatographic examination |
| JP2009210505A (en) * | 2008-03-06 | 2009-09-17 | Tanaka Kikinzoku Kogyo Kk | Immunoassay method and kit and developing solution therefor |
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