JP6774629B2 - A sensitizer for immunochromatography using dengue virus as a measurement target substance and a measurement method using the sensitizer. - Google Patents
A sensitizer for immunochromatography using dengue virus as a measurement target substance and a measurement method using the sensitizer. Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F230/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal
- C08F230/02—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal containing phosphorus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
Description
本発明は、デング熱ウイルスを測定対象物質とするイムノクロマト測定法用増感剤、及び該増感剤の共存下で行うデング熱ウイルスを測定対象物質とするイムノクロマト測定法に関する。
本出願は、参照によりここに援用されるところの日本出願、特願2014-223503号優先権を請求する。The present invention relates to a sensitizer for immunochromatography using dengue virus as a substance to be measured, and an immunochromatography method using dengue virus as a substance to be measured in the presence of the sensitizer.
This application claims the priority of Japanese application, Japanese Patent Application No. 2014-223503, which is incorporated herein by reference.
イムノクロマト法は、メンブレンの毛細管現象を利用したクロマトグラフィーの手法と免疫学的手法を組み合わせた方法である。イムノクロマト法は、抗原抗体反応に起因する高い特異性を備え、操作が煩雑な重厚な設備や機器を必要とせず、軽便なキットを用いて簡易かつ迅速に測定を行え、かつ結果を目視で判定できることから、臨床診断法として広く普及している（参照：特許文献１）。 The immunochromatography method is a method that combines a chromatography method utilizing the capillary phenomenon of a membrane and an immunological method. The immunochromatography method has high specificity due to the antigen-antibody reaction, does not require heavy equipment or equipment that is complicated to operate, can perform simple and quick measurement using a convenient kit, and visually determines the result. Since it can be used, it is widely used as a clinical diagnostic method (see: Patent Document 1).
イムノクロマト法は、簡易に測定できる利点はあるが、検出感度の精度が低く、陽性検体を用いても偽陰性と判定される場合もあるので、より高感度のイムノクロマト測定法が望まれている。
これまでに、高感度のイムノクロマト測定法を確立する目的で種々の方法が検討されており、例えば、特許文献２では、イムノクロマトの試薬の添加位置を変える事で高感度化する手法が開示されている。また、特許文献３では、メンブレンの素材を改良することで高感度化する手法が開示されている。The immunochromatography method has an advantage that it can be easily measured, but the accuracy of detection sensitivity is low, and a false negative may be determined even if a positive sample is used. Therefore, a more sensitive immunochromatography method is desired.
So far, various methods have been studied for the purpose of establishing a highly sensitive immunochromatographic measurement method. For example, Patent Document 2 discloses a method for increasing the sensitivity by changing the addition position of the immunochromatographic reagent. There is. Further, Patent Document 3 discloses a method for increasing the sensitivity by improving the material of the membrane.
さらに、従来のイムノクロマト法に用いている検体希釈液に、添加剤を加えることで、高感度化を達成する方法が知られている。例えば、特許文献４、５及び６では、検体希釈液中に、ウシ血清アルブミン（ＢＳＡ）、ホスホリルコリン基を有する重合体、ヒアルロン酸を含有させることが開示されている。これらの開示では、試薬の添加位置を変える必要がなく、また従来のイムノクロマト用メンブレンを使用可能という利点がある。 Further, there is known a method of achieving high sensitivity by adding an additive to the sample diluent used in the conventional immunochromatography method. For example, Patent Documents 4, 5 and 6 disclose that bovine serum albumin (BSA), a polymer having a phosphorylcholine group, and hyaluronic acid are contained in a sample diluent. These disclosures have the advantages that it is not necessary to change the addition position of the reagent and that a conventional membrane for immunochromatography can be used.
しかしながら、上記特許文献では、フラビウイルス科フラビウイルス属の一つであるデング熱ウイルスの測定を高感度に行う方法に用いる増感剤については開示又は示唆をされていない。 However, the above patent document does not disclose or suggest a sensitizer used in a method for highly sensitive measurement of dengue virus, which is one of the flavivirus genus of the Flaviviridae family.
本発明では、デング熱ウイルスを高感度に検出できるイムノクロマト測定法用増感剤及び該増感剤を使用した測定法を提供することにある。 An object of the present invention is to provide a sensitizer for immunochromatography measurement method capable of detecting dengue virus with high sensitivity and a measurement method using the sensitizer.
本発明者らは、負電荷を有すグルコサミノグリカンが多くのフラビウイルスに対し接着因子として機能することを知見として得て、これまでのイムノクロマトグラフィー負電荷を有する免疫学的測定法用の公知の増感剤と比較して、デング熱ウイルスを高感度に検出できる化合物を見出すべく鋭意検討した結果、下記一般式［１］で示されるホスホリルコリン類似基構造を有する基を側鎖に有するポリマーが目的の性能を有していることを見出し、本発明を完成するに至った。
すなわち、本発明は以下よりなる。The present inventors have found that negatively charged glucosaminoglycans function as an adhesion factor for many flaviviruses, and have been used in conventional immunochromatography for immunological measurement methods having negative charges. As a result of diligent studies to find a compound capable of detecting dengue virus with high sensitivity as compared with known sensitizers, a polymer having a group having a phosphorylcholine-like group structure represented by the following general formula [1] in the side chain has been found. They have found that they have the desired performance and have completed the present invention.
That is, the present invention comprises the following.
下記一般式［１］で表されるポリマーを含んでなる、デング熱ウイルスを測定対象物質とするイムノクロマト測定法用増感剤。 A sensitizer for immunochromatography, which comprises a polymer represented by the following general formula [1] and contains a dengue virus as a measurement target substance.
式中、Ｒは、水素原子又は陽イオンから選ばれる１種以上を示し、ｍとｎはモル比であり、ｍは９９〜５５、ｎは１〜４５である。 In the formula, R represents one or more selected from hydrogen atom or cation, m and n are molar ratios, m is 99 to 55, and n is 1 to 45.
前記ポリマーは、下記一般式［２］で表されるモノマーに基づく構成単位と、メタクリル酸に基づく構成単位を有するコポリマーである。 The polymer is a copolymer having a structural unit based on a monomer represented by the following general formula [2] and a structural unit based on methacrylic acid.
前記ポリマーは、２−(メタクリロイルオキシ)エチル−２'−(トリメチルアンモニオ)エチルホスフェートとメタクリル酸の構成比が９９〜５５：１〜４５である。
前記ポリマーは、２−(メタクリロイルオキシ)エチル−２'−(トリメチルアンモニオ)エチルホスフェートとメタクリル酸の構成比が９９〜７０：１〜３０である。
前記ポリマーは、重量平均分子量１０，０００〜１，０００，０００の水溶性のポリマーである。The polymer has a composition ratio of 2- (methacryloyloxy) ethyl-2'-(trimethylammonio) ethyl phosphate to methacrylic acid of 99 to 55: 1-45.
The polymer has a composition ratio of 2- (methacryloyloxy) ethyl-2'-(trimethylammonio) ethyl phosphate to methacrylic acid of 99 to 70: 1-30.
The polymer is a water-soluble polymer having a weight average molecular weight of 10,000 to 1,000,000.
前項に記載のイムノクロマト測定法用増感剤を含む、デング熱ウイルスを測定対象物質とするイムノクロマト測定法用検体希釈液。 A sample diluent for immunochromatography, which contains the sensitizer for immunochromatography described in the preceding section and uses dengue virus as the measurement target substance.
前項に記載のイムノクロマト測定法用増感剤の存在下で抗原抗体反応を行わせることを特徴とする、デング熱ウイルスを測定対象物質とするイムノクロマト測定法。 An immunochromatographic measurement method using dengue virus as a measurement target substance, which comprises causing an antigen-antibody reaction in the presence of the sensitizer for immunochromatographic measurement described in the preceding section.
前項に記載のイムノクロマト測定法用増感剤を含む、デング熱ウイルスを測定対象物質とするイムノクロマト測定用器具。 An immunochromatographic measurement device containing a dengue virus as a measurement target substance, which comprises the sensitizer for the immunochromatographic measurement method described in the preceding section.
以下の（１）（２）の工程を含むデング熱の治療方法。
（１）下記一般式［１］で表されるポリマーを含んでなるイムノクロマト測定法用増感剤の存在下で抗原抗体反応を行うデング熱ウイルスの測定工程。
（２）上記（１）の測定結果に基づき、デング熱ウイルス治療を患者に施す工程。A method for treating dengue fever, which comprises the following steps (1) and (2).
(1) A step for measuring a dengue virus in which an antigen-antibody reaction is carried out in the presence of a sensitizer for immunochromatography, which comprises a polymer represented by the following general formula [1].
(2) A step of applying dengue virus treatment to a patient based on the measurement result of (1) above.
上記式中、Ｒは、水素原子又は陽イオンから選ばれる１種以上を示し、ｍとｎはモル比であり、ｍは９９〜５５、ｎは１〜４５である。 In the above formula, R represents one or more selected from hydrogen atom or cation, m and n are molar ratios, m is 99 to 55, and n is 1 to 45.
上記ポリマーは、下記一般式［２］で表されるモノマーに基づく構成単位と、メタクリル酸に基づく構成単位を有するコポリマーである。 The polymer is a copolymer having a structural unit based on a monomer represented by the following general formula [2] and a structural unit based on methacrylic acid.
前記ポリマーは、２−(メタクリロイルオキシ)エチル−２'−(トリメチルアンモニオ)エチルホスフェートとメタクリル酸の構成比が９９〜５５：１〜４５である。
前記ポリマーは、２−(メタクリロイルオキシ)エチル−２'−(トリメチルアンモニオ)エチルホスフェートとメタクリル酸の構成比が９９〜７０：１〜３０である。
前記ポリマーは、重量平均分子量１０，０００〜１，０００，０００の水溶性のポリマーである。The polymer has a composition ratio of 2- (methacryloyloxy) ethyl-2'-(trimethylammonio) ethyl phosphate to methacrylic acid of 99 to 55: 1-45.
The polymer has a composition ratio of 2- (methacryloyloxy) ethyl-2'-(trimethylammonio) ethyl phosphate to methacrylic acid of 99 to 70: 1-30.
The polymer is a water-soluble polymer having a weight average molecular weight of 10,000 to 1,000,000.
本発明のデング熱ウイルスを測定対象物質とするイムノクロマト測定法用増感剤を使用した測定方法では、従来のデング熱ウイルスを検出するイムノクロマト測定法用と比較して、高感度でデング熱ウイルスを検出することができる。
さらに、本発明のデング熱の治療方法は、高感度でデング熱ウイルスを検出することができるので、デング熱の患者に迅速にデング熱治療を施すことができる。In the measurement method using the sensitizer for immunochromatography using the dengue virus of the present invention as the measurement target substance, the dengue virus is detected with higher sensitivity than the conventional immunochromatography method for detecting dengue virus. Can be done.
Furthermore, the method for treating dengue fever of the present invention can detect dengue virus with high sensitivity, so that a patient with dengue fever can be rapidly treated with dengue fever.
本発明のデング熱ウイルスを測定対象物質とするイムノクロマト測定法用増感剤、該増感剤を含むデング熱ウイルスを測定対象物質とするイムノクロマト測定法用検体希釈液、該増感剤の存在下で抗原抗体反応を行わせるデング熱ウイルスを測定対象物質とするイムノクロマト測定法、該増感剤を含むデング熱ウイルスを測定対象物質とするイムノクロマト測定用器具、及び、該増感剤の存在下で抗原抗体反応を行わせる工程を含むデング熱の治療方法を、以下で説明する。
なお、本発明のイムノクロマト測定法用増感剤を使用する測定法では、測定対象物質としてはデング熱ウイルスであるが、他の測定対象物質も検出できる。しかし、本発明のイムノクロマト測定法用増感剤は、デング熱ウイルスを高感度に検出できることを特徴とする。A sensitizer for immunochromatography using the dengue virus of the present invention as a substance to be measured, a sample diluent for immunochromatography using the dengue virus containing the sensitizer as a substance to be measured, and an antigen in the presence of the sensitizer. An immunochromatographic measurement method using a dengue virus as a measurement target substance to cause an antibody reaction, an immunochromatographic measurement device containing a dengue fever virus as a measurement target substance containing the sensitizer, and an antigen-antibody reaction in the presence of the sensitizer. A method of treating dengue fever, including the steps to be performed, will be described below.
In the measurement method using the sensitizer for immunochromatography measurement method of the present invention, the substance to be measured is dengue virus, but other substances to be measured can also be detected. However, the sensitizer for immunochromatography measurement of the present invention is characterized in that it can detect dengue virus with high sensitivity.
本発明のデング熱ウイルスを測定対象物質とするイムノクロマト測定法用増感剤として用いられるポリマーは、下記一般式［１］で表される。 The polymer used as a sensitizer for immunochromatography using the dengue virus of the present invention as a measurement target substance is represented by the following general formula [1].
上記式中、Ｒは、水素原子又は陽イオンから選ばれる１種以上を示す。ここで、陽イオンは、ナトリウムイオン、カリウムイオン、アンモニウムイオン等が挙げられ、これらの組み合わせであっても良い。
これらの組み合わせの例としては、メタクリル酸、メタクリル酸ナトリウム、メタクリル酸カリウム、メタクリル酸アンモニウム等を例示することができる。In the above formula, R represents one or more selected from hydrogen atom or cation. Here, examples of the cation include sodium ion, potassium ion, ammonium ion and the like, and a combination thereof may be used.
Examples of these combinations include methacrylic acid, sodium methacrylate, potassium methacrylate, ammonium methacrylate and the like.
上記式中、ｍとｎはモル比であり、ｍとｎの割合は、９９〜５５：１〜４５、好ましくは９０〜６０：１０〜４０、より好ましくは８０〜６５：２０〜３５、最も好ましくは７０：３０である。 In the above formula, m and n are molar ratios, and the ratio of m and n is 99 to 55: 1 to 45, preferably 90 to 60: 10 to 40, more preferably 80 to 65: 20 to 35, most. It is preferably 70:30.
上記式で表されるポリマーは、重量平均分子量１０，０００〜１，０００，０００、好ましくは５０，０００〜５００，０００の水溶性ポリマーである。 The polymer represented by the above formula is a water-soluble polymer having a weight average molecular weight of 10,000 to 1,000,000, preferably 50,000 to 500,000.
本発明の一般式［１］で示される水溶性ポリマーは、下記一般式［２］で表されるモノマー｛MPC：２−(メタクリロイルオキシ)エチル−２'−(トリメチルアンモニオ)エチルホスフェート｝に基づく構成単位と、メタクリル酸に基づく構成単位を有するコポリマーにより成る。 The water-soluble polymer represented by the general formula [1] of the present invention is a monomer represented by the following general formula [2] {MPC: 2- (methacrylicloyloxy) ethyl-2'-(trimethylammonio) ethyl phosphate}. It consists of a copolymer having a constituent unit based on and a constituent unit based on methacrylic acid.
イムノクロマト測定法用増感剤として用いられるポリマーは、好ましくは、以下を例示することができるが特に限定されない。
２−(メタクリロイルオキシ)エチル−２'−(トリメチルアンモニオ)エチルホスフェートとメタクリル酸の構成比が９９〜５５：１〜４５である。
２−(メタクリロイルオキシ)エチル−２'−(トリメチルアンモニオ)エチルホスフェートとメタクリル酸の構成比が９９〜７０：１〜３０である。
２−(メタクリロイルオキシ)エチル−２'−(トリメチルアンモニオ)エチルホスフェートとメタクリル酸の構成比が９０〜６０：１〜４０である。
２−(メタクリロイルオキシ)エチル−２'−(トリメチルアンモニオ)エチルホスフェートとメタクリル酸の構成比が８０〜６５：２０〜３５である。
２−(メタクリロイルオキシ)エチル−２'−(トリメチルアンモニオ)エチルホスフェートとメタクリル酸の構成比が約７０：約３０である。
前記ポリマーは、重量平均分子量１０，０００〜１，０００，０００の水溶性のポリマーである。The polymer used as a sensitizer for immunochromatography can preferably exemplify the following, but is not particularly limited.
The composition ratio of 2- (methacryloyloxy) ethyl-2'-(trimethylammonio) ethyl phosphate to methacrylic acid is 99 to 55: 1-45.
The composition ratio of 2- (methacryloyloxy) ethyl-2'-(trimethylammonio) ethyl phosphate to methacrylic acid is 99 to 70: 1-30.
The composition ratio of 2- (methacryloyloxy) ethyl-2'-(trimethylammonio) ethyl phosphate to methacrylic acid is 90 to 60: 1-40.
The composition ratio of 2- (methacryloyloxy) ethyl-2'-(trimethylammonio) ethyl phosphate to methacrylic acid is 80 to 65:20 to 35.
The composition ratio of 2- (methacryloyloxy) ethyl-2'-(trimethylammonio) ethyl phosphate to methacrylic acid is about 70: about 30.
The polymer is a water-soluble polymer having a weight average molecular weight of 10,000 to 1,000,000.
一般式［１］で示される水溶性ポリマーの具体的な製造例は、特開平０８−３３３４２１号、特許文献６に記載の方法を例示することができるが特に限定されない。 Specific production examples of the water-soluble polymer represented by the general formula [1] can be exemplified by the methods described in Japanese Patent Application Laid-Open No. 08-333421 and Patent Document 6, but are not particularly limited.
本発明のイムノクロマト測定法用増感剤として用いられるポリマーは、イムノクロマト測定法に用いられる各種試薬や試料に溶解させて用いてもよく、また、イムノクロマト測定法用キットに担持させて用いてもよいが、好ましくは、検体希釈液として用いることが好ましい。
なお、本発明のイムノクロマト測定法とは、用いられるイムノクロマト測定法用キットとして一般に、検体希釈液とテストストリップからなる。The polymer used as the sensitizer for the immunochromatography method of the present invention may be used by being dissolved in various reagents and samples used in the immunochromatography measurement method, or may be supported on a kit for the immunochromatography measurement method. However, it is preferable to use it as a sample diluent.
The immunochromatographic measurement method of the present invention generally comprises a sample diluent and a test strip as a kit for the immunochromatographic measurement method used.
検体希釈液とは、イムノクロマトグラフィー法で測定するに際しての、適当な希釈剤であり、トリス緩衝剤、リン酸緩衝剤、ベロナール緩衝剤、ホウ酸緩衝剤、グッド緩衝剤等の緩衝成分、アルブミン、グロブリン、カゼイン、血清、水溶性ゼラチン、界面活性剤、糖類、キレート剤等の安定化成分、サリチル酸、安息香酸、アジ化ナトリウム等の防腐成分を含むことができる。 The sample diluent is an appropriate diluent for measurement by the immunochromatography method, and is a buffer component such as Tris buffer, phosphate buffer, veronal buffer, boric acid buffer, Good buffer, albumin, etc. It can contain stabilizing components such as globulin, casein, serum, water-soluble gelatin, surfactants, sugars and buffers, and antiseptic components such as salicylic acid, benzoic acid and sodium azide.
本発明の増感剤を、検体希釈液に溶解させて用いる場合、その濃度は、抗原抗体反応がなされる際の濃度として、特に限定されないが、通常0.1〜5.0w/v％、好ましくは0.5〜1.0w/v％となるように用いられる。 When the sensitizer of the present invention is used by dissolving it in a sample diluent, the concentration thereof is not particularly limited as the concentration at which the antigen-antibody reaction is carried out, but is usually 0.1 to 5.0 w / v%, preferably 0.5. It is used so that it becomes ~ 1.0w / v%.
テストストリップとは、一般に、検体の第一の抗原決定基にて抗原抗体反応可能な第一の物質と、前記検体の第二の抗原決定基にて抗原抗体反応可能で且つ標識された第二の物質と、膜担体とを少なくとも備え、前記第一の物質は前記膜担体の所定位置に予め固定されて捕捉部位を形成し、前記第二の物質は前記捕捉部位から隔離した位置で前記担体にてクロマト展開可能なように配置されて構成される。 A test strip generally refers to a first substance capable of an antigen-antibody reaction with a first antigenic determinant of a sample and a second substance capable of an antigen-antibody reaction with a second antigen-antibody group of the sample and labeled. The substance and the membrane carrier are provided, the first substance is fixed in advance at a predetermined position of the membrane carrier to form a trapping site, and the second substance is isolated from the trapping site of the carrier. It is arranged and configured so that it can be chromatographed at.
イムノクロマト測定用テストストリップは、市販品を用いても、公知の方法により作製したものを用いてもよい。公知の方法により作製する場合、上記展開膜で用いられる基材としては、通常この分野で用いられるものを用いればよく、例えばセルロース、ニトロセルロース、ナイロン等が好ましいものとして挙げられる。 As the test strip for immunochromatography measurement, a commercially available product or a test strip prepared by a known method may be used. When produced by a known method, the base material used in the developing film may be one usually used in this field, and for example, cellulose, nitrocellulose, nylon and the like are preferable.
本発明の増感剤を含むデング熱ウイルスを測定対象物質とするイムノクロマト測定用器具としては、例えば（１）展開膜からなるもの、（２）検体標識部並びに展開膜からなり、且つ該検体標識部と該展開膜とが毛管現象により移動可能なように形成されたもの、（３）検体標識部、展開膜並びに液体吸収部からなり、且つ該検体標識部と該展開膜と該液体吸収部とが、この順に毛管現象により移動可能なように形成されたもの、（４）検体滴下部、検体標識部、展開膜並びに液体吸収部からなり、且つ該検体滴下部と該検体標識部と該展開膜と該液体吸収部とが、この順に毛管現象により移動可能なように形成されたもの等が挙げられるが、特に限定されない。 Examples of the immunochromatographic measurement device for measuring the dengue fever virus containing the sensitizer of the present invention include (1) a developing film, (2) a sample labeling section and a developing film, and the sample labeling section. And the developing film formed so as to be movable by capillary action, (3) a sample labeling part, a developing film and a liquid absorbing part, and the sample labeling part, the developing film and the liquid absorbing part. Is formed in this order so as to be movable by capillarity, (4) a sample dropping part, a sample labeling part, a developing film and a liquid absorbing part, and the sample dropping part, the sample labeling part and the developing part. Examples thereof include those in which the membrane and the liquid absorbing portion are formed so as to be movable by a capillary phenomenon in this order, but the present invention is not particularly limited.
本発明の増強剤を、前記イムノクロマト測定法用器具に担持させて用いる場合、その量は、担持される場所、使用する感度増強剤の種類、使用する測定対象物質の種類、使用する標識物質等により変動するが、例えばイムノクロマト測定法用器具の展開膜、検体標識部、検体滴下部等に担持させる場合、単位面積（cm2）当たりに含まれる感度増強剤の量として、通常0.01μg〜10ｍｇ、好ましくは0.1μg〜4ｍｇ、より好ましくは1〜800μgである。また、担持される面積は、用いられるイムノクロマト測定法用器具の種類及び大きさ、測定用試料の量により変動するが、イムノクロマト測定法用器具の展開膜の場合には、通常総面積の10〜60％、好ましくは20〜30％、検体標識部の場合には、通常総面積の1〜30％、好ましくは5〜15％、検体滴下部の場合には、通常総面積の5〜40％、好ましくは10〜20％である。When the enhancer of the present invention is supported on the immunochromatographic measurement instrument and used, the amount thereof includes the place where the enhancer is supported, the type of the sensitivity enhancer used, the type of the substance to be measured, the labeling substance used, and the like. The amount of the sensitivity enhancer contained per unit area (cm 2 ) is usually 0.01 μg to 10 mg when supported on a developing film, a sample labeling part, a sample dropping part, etc. of an immunochromatographic measurement instrument, for example. , Preferably 0.1 μg to 4 mg, more preferably 1 to 800 μg. The area to be supported varies depending on the type and size of the immunochromatography instrument used and the amount of the measurement sample, but in the case of the developing film of the immunochromatography instrument, it is usually 10 to 10 to the total area. 60%, preferably 20 to 30%, usually 1 to 30% of the total area in the case of the sample labeling part, preferably 5 to 15%, and usually 5 to 40% of the total area in the case of the sample dropping part. , Preferably 10 to 20%.
本発明のデング熱の治療方法は、少なくとも以下の（１）及び（２）の工程を含む。
（１）前記ポリマーを含んでなるイムノクロマト測定法用増感剤の存在下で抗原抗体反応を行うデング熱ウイルスの測定工程。
（２）上記（１）の測定結果に基づき、デング熱ウイルス治療を患者に施す工程。
なお、患者は、デング熱に感染しているおそれのある人も含む。なお、測定とは、患者由来の検体中にデング熱ウイルスが存在するかどうかを検出することを意味する。
また、デング熱ウイルス治療を患者に施す工程とは、公知治療薬、今後上市される治療薬、臨床段階の治療薬候補を患者に投与するだけでなく、対症療法（アスピリン投与、補液・輸液投与等）も含む。
本発明のデング熱の治療方法は、高感度でデング熱ウイルスを検出することができるので、デング熱の患者に迅速にデング熱治療を施すことができる。The method for treating dengue fever of the present invention includes at least the following steps (1) and (2).
(1) A step of measuring a dengue virus in which an antigen-antibody reaction is carried out in the presence of a sensitizer for immunochromatography, which comprises the polymer.
(2) A step of applying dengue virus treatment to a patient based on the measurement result of (1) above.
Patients also include those who may be infected with dengue fever. The measurement means detecting whether or not the dengue virus is present in the sample derived from the patient.
In addition, the process of applying dengue virus treatment to patients is not only to administer known therapeutic agents, therapeutic agents to be launched in the future, and clinical stage therapeutic agent candidates to patients, but also symptomatic treatment (administration of aspirin, replacement fluid / infusion, etc.) ) Is also included.
Since the method for treating dengue fever of the present invention can detect a dengue virus with high sensitivity, it is possible to rapidly treat a patient with dengue fever.
以下、本実施例により本発明を更に詳細に説明するが、本発明はこれらに限定されない。 Hereinafter, the present invention will be described in more detail with reference to the present examples, but the present invention is not limited thereto.
（本発明のポリマー及び比較例のポリマーの作成）
本発明のイムノクロマト測定法用増感剤として用いられるポリマー（実施態様例１、２）及び比較例のポリマーを作成した。詳細は、以下の通りである。(Preparation of Polymer of the Present Invention and Polymer of Comparative Example)
Polymers (Examples 1 and 2) used as a sensitizer for the immunochromatography method of the present invention and polymers of Comparative Examples were prepared. The details are as follows.
本発明のポリマー及び比較例のポリマーは、２−(メタクリロイルオキシ)エチル−２'−(トリメチルアンモニオ)エチルホスフェート（ＭＰＣ）と１種又は２種のモノマーから合成した（参照：下記表１）。 The polymers of the present invention and the polymers of Comparative Examples were synthesized from 2- (methacryloyloxy) ethyl-2'-(trimethylammonio) ethyl phosphate (MPC) and one or two monomers (see Table 1 below). ..
（実施態様例のポリマー）
○実施態様例１ ＰＭＡ９９
ＭＰＣモノマーとメタクリル酸とからなるコポリマー、分子量；100×103、ＭＰＣモノマーとメタクリル酸の構成比；９９：１。
○実施態様例２ ＰＭＡ７０
ＭＰＣモノマーとメタクリル酸とからなるコポリマー、分子量；230×103、ＭＰＣモノマーとメタクリル酸の構成比；７０：３０。(Polymer of Embodiment)
Example Example 1 PMA99
Copolymer of the MPC monomer and methacrylic acid, the molecular weight; 100 × 10 3, composition ratio of MPC monomer and methacrylic acid; 99: 1.
○ Example 2 PMA70
Copolymer consisting of MPC monomer and methacrylic acid, molecular weight; 230 × 10 3 , composition ratio of MPC monomer and methacrylic acid; 70:30.
（比較例のポリマー）
○比較例１ ＰＭＰＣ
ＭＰＣモノマーのホモポリマー、分子量；1,000×103。
○比較例２ ＰＭＡ５０
ＭＰＣモノマーとメタクリル酸とからなるコポリマー、分子量；1100×103、ＭＰＣモノマーとメタクリル酸の構成比；５０：５０。
○比較例３ ＰＭＡ３０
ＭＰＣモノマーとメタクリル酸とからなるコポリマー、分子量；700×103、ＭＰＣモノマーとメタクリル酸の構成比；３０：７０。
○比較例４ ＰＭＢ８０
ＭＰＣモノマーとメタクリル酸ブチルとからなるコポリマー、分子量；600×103、ＭＰＣモノマーとメタクリル酸ブチルの構成比；８０：２０。
○比較例５ ＰＭＢ３０
ＭＰＣモノマーとメタクリル酸ブチルとからなるコポリマー、分子量；90×103、ＭＰＣモノマーとメタクリル酸ブチルの構成比；３０：７０。
○比較例６ ＰＭＥ７０
ＭＰＣモノマーとモノメチルエーテルポリエチレングリコールメタクリレートとからなるコポリマー、分子量；140×103、ＭＰＣモノマーとモノメチルエーテルポリエチレングリコールメタクリレートの構成比；７０：３０。
○比較例７ ＰＭＳ８０
ＭＰＣモノマーとメタクリル酸オクタデシルとからなるポリマー、分子量；40×103、ＭＰＣモノマーとメタクリル酸オクタデシルの構成比；８０：２０。
○比較例８ ＰＭＺ８０
ＭＰＣモノマーとメタクリル酸ベンジルとからなるポリマー、分子量；240×103、ＭＰＣモノマーとメタクリル酸ベンジルの構成比；８０：２０。
○比較例９ ＰＭＩ８０
ＭＰＣモノマーとメタクリル酸イソボルニルとからなるポリマー、分子量；260×103、ＭＰＣモノマーとメタクリル酸イソボルニルの構成比；８０：２０。
○比較例１０ ＰＭＳ５０
ＭＰＣモノマーとメタクリル酸オクタデシルとからなるポリマー、分子量；200×103、ＭＰＣモノマーとメタクリル酸オクタデシルの構成比；５０：５０。
○比較例１１ ＰＭＳ３０
ＭＰＣモノマーとメタクリル酸オクタデシルとからなるポリマー、分子量；200×103、ＭＰＣモノマーとメタクリル酸オクタデシルの構成比；３０：７０。(Comparative example polymer)
○ Comparative example 1 PMPC
Homopolymer of MPC monomer, molecular weight; 1,000 × 10 3 .
○ Comparative example 2 PMA50
Copolymer of the MPC monomer and methacrylic acid, the molecular weight; 1100 × 10 3, composition ratio of MPC monomer and methacrylic acid; 50: 50.
○ Comparative example 3 PMA30
Copolymer of the MPC monomer and methacrylic acid, the molecular weight; 700 × 10 3, composition ratio of MPC monomer and methacrylic acid; 30: 70.
○ Comparative example 4 PMB80
Copolymer consisting of MPC monomer and butyl methacrylate, molecular weight; 600 × 10 3 , composition ratio of MPC monomer and butyl methacrylate; 80:20.
○ Comparative example 5 PMB30
Copolymer consisting of MPC monomer and butyl methacrylate, molecular weight; 90 × 10 3 , composition ratio of MPC monomer and butyl methacrylate; 30:70.
○ Comparative example 6 PME70
Copolymer of the MPC monomer and monomethyl ether of polyethylene glycol methacrylate, molecular weight; 140 × 10 3, composition ratio of MPC monomer and monomethyl ether of polyethylene glycol methacrylate; 70: 30.
○ Comparative Example 7 PMS80
Polymer consisting of MPC monomer and octadecyl methacrylate; molecular weight; 40 × 10 3 , composition ratio of MPC monomer and octadecyl methacrylate; 80:20.
○ Comparative example 8 PMZ80
Polymer consisting of MPC monomer and benzyl methacrylate; molecular weight; 240 × 10 3 , composition ratio of MPC monomer and benzyl methacrylate; 80:20.
○ Comparative example 9 PMI80
Polymer consisting of MPC monomer and isobornyl methacrylate; molecular weight; 260 × 10 3 , composition ratio of MPC monomer and isobornyl methacrylate; 80:20.
○ Comparative Example 10 PMS50
Polymer consisting of MPC monomer and octadecyl methacrylate; molecular weight; 200 × 10 3 , composition ratio of MPC monomer and octadecyl methacrylate; 50:50.
○ Comparative Example 11 PMS30
Polymer consisting of MPC monomer and octadecyl methacrylate; molecular weight; 200 × 10 3 , composition ratio of MPC monomer and octadecyl methacrylate; 30:70.
（本発明のイムノクロマト測定法用増感剤として用いられるポリマーの検出感度の確認）
本実施例では、本発明のイムノクロマト測定法用増感剤として用いられるポリマーのデング熱ウイルスの検出感度を確認するために、イムノクロマト用キットを反映したＥＬＩＳＡ法による定量的な比較（比較例のポリマーとの比較）を行った。詳細は、以下の通りである。(Confirmation of detection sensitivity of polymer used as sensitizer for immunochromatography measurement method of the present invention)
In this example, in order to confirm the detection sensitivity of the dengue virus of the polymer used as the sensitizer for the immunochromatography method of the present invention, a quantitative comparison by the ELISA method reflecting the immunochromatography kit (with the polymer of the comparative example). Comparison) was performed. The details are as follows.
（ポリマーの検出感度の測定方法）
抗DVｍAB（抗デング熱ウイルスモノクローナル抗体）の１０μg/mL溶液を５０μLずつマイクロチューブに入れ、さらに、終濃度が０．５、１．０重量％となるよう各実施態様例又は比較例のポリマーを含むイムノクロマト用ブロッキング試薬を該チューブに１００μＬずつ添加した。次に、イムノクロマト用ブロッキング剤（０．５重量％のカゼインを含むホウ酸緩衝液）を適量の血清で希釈したものを５０μＬと混合して、３７℃、６０分間静置した。
次に、該イムノクロマト用ブロッキング試薬を、さらに、該イムノクロマト用ブロッキング剤で前処理したＥＬＩＳＡプレートに添加し、３７℃、６０分間静置した。静置後、プレートを３００μＬの洗浄液（０．１重量％のＴｗｅｅｎを含むＰＢＳ溶液）で３回洗浄した。
次に、抗抗原２次抗体（１重量％のＢＳＡを含むリン酸緩衝液にて２０００倍に希釈したＰＯＤ標識の抗抗原２次抗体溶液）５０μＬを各ウェルに添加し３７℃、２時間静置した。各ウェルを３００μＬの洗浄液で３回洗浄し、過剰な抗抗原２次抗体を除去後、５０μＬのＴＭＢ（３，３'，５，５'−ｔｅｔｒａｍｅｔｈｙｌｂｅｎｚｉｄｉｎｅ）を各ウェルに添加し室温にて５分間静置した。その後、１Ｎ−硫酸５０μＬを添加して反応停止し、４５０ｎｍの吸光度を測定した。なお、ポリマー非添加系の吸光度の平均は、０．２１０であり、これに対する増感度比を評価した。ここで、増感度比が２．０以上であれば増感効果があると判断した。(Measuring method of polymer detection sensitivity)
50 μL of a 10 μg / mL solution of anti-DVmAB (anti-Dengue virus monoclonal antibody) is placed in a microtube, and further contains a polymer of each embodiment or comparative example so that the final concentration is 0.5 or 1.0% by weight. 100 μL of a blocking reagent for immunochromatography was added to the tube. Next, a blocking agent for immunochromatography (boric acid buffer containing 0.5% by weight of casein) diluted with an appropriate amount of serum was mixed with 50 μL and allowed to stand at 37 ° C. for 60 minutes.
Next, the immunochromatographic blocking reagent was further added to an ELISA plate pretreated with the immunochromatographic blocking agent, and allowed to stand at 37 ° C. for 60 minutes. After standing, the plate was washed 3 times with 300 μL of wash solution (PBS solution containing 0.1 wt% Tween).
Next, 50 μL of anti-antigen secondary antibody (POD-labeled anti-antigen secondary antibody solution diluted 2000-fold with phosphate buffer containing 1 wt% BSA) was added to each well and allowed to stand at 37 ° C. for 2 hours. Placed. Each well was washed 3 times with 300 μL of wash solution to remove excess anti-antigen secondary antibody, and then 50 μL of TMB (3,3', 5,5'-tetramethylbenzine) was added to each well for 5 minutes at room temperature. It was left still. Then, 50 μL of 1N-sulfuric acid was added to terminate the reaction, and the absorbance at 450 nm was measured. The average absorbance of the polymer-free system was 0.210, and the sensitivity ratio to this was evaluated. Here, it was determined that if the sensitization ratio is 2.0 or more, there is a sensitization effect.
（ポリマーの検出感度の向上の結果）
各ポリマーでの増感度比の結果を図１に示す。
実施態様例１及び実施態様例２のポリマーのみが増感度比２．０を超えた。すなわち、カルボキシル基を有するＭＰＣポリマーを用いた場合には、顕著な増感度を確認した。さらに、ＰＭＡ９９（実施態様例１）、ＰＭＡ７０（実施態様例２）において、より高い増感効果示し、同じカルボキシル基を有するポリマーであるＰＭＡ５０（比較例２）、ＰＭＡ３０（比較例３）では増感効果が２倍未満であることを確認した。
これにより、一般式［１］で示される水溶性ポリマーのｍとｎの割合は、９９〜５５：１〜４５、好ましくは９０〜６０：１０〜４０、より好ましくは８０〜６５：２０〜３５、最も好ましくは７０：３０であることを確認した。(Result of improved detection sensitivity of polymer)
The results of the sensitivity ratio for each polymer are shown in FIG.
Only the polymers of Embodiment 1 and Embodiment 2 exceeded the sensitivity ratio of 2.0. That is, when an MPC polymer having a carboxyl group was used, a remarkable desensitization was confirmed. Further, PMA99 (Example 1) and PMA70 (Example 2) show a higher sensitizing effect, and PMA50 (Comparative Example 2) and PMA30 (Comparative Example 3), which are polymers having the same carboxyl group, are sensitized. It was confirmed that the effect was less than double.
As a result, the ratio of m to n of the water-soluble polymer represented by the general formula [1] is 99 to 55: 1 to 45, preferably 90 to 60: 10 to 40, and more preferably 80 to 65: 20 to 35. , Most preferably 70:30.
（公知のイムノクロマト測定用器具を使用しての検出感度の確認）
本実施例では、本発明のポリマーが、公知のデング熱ウイルスの検出キットの検出感度を向上させることができるか（増感効果があるか）どうかを確認した。詳細は、以下の通りである。(Confirmation of detection sensitivity using a known immunochromatography measuring instrument)
In this example, it was confirmed whether the polymer of the present invention can improve the detection sensitivity of a known dengue virus detection kit (whether it has a sensitizing effect). The details are as follows.
市販のRapiDeng Agキット（株式会社バイオメディカル研究所社製品）であるＩＣＡキットで評価した。より詳しくは、血清型が４種類（DV1、DV2、DV3及びDV4）のデング熱ウイルスを検出するために、DenV検出ICAキットの展開剤（５重量％EDTA、５μg／mLマウス血清、０．１重量％Tween／ＰＢＳ）に終濃度０．２重量％となるようＰＭＡ７０を添加し、２×１０６pfu/mLのDV溶液から１０倍、１００倍に希釈した溶液を加えICAを展開した。Evaluation was performed using the ICA kit, which is a commercially available RapiDeng Ag kit (product of Biomedical Research Institute Co., Ltd.). More specifically, to detect dengue virus of four serotypes (DV1, DV2, DV3 and DV4), a developing agent of the DenV detection ICA kit (5 wt% EDTA, 5 μg / mL mouse serum, 0.1 wt) PMA70 was added to% Tween / PBS) to a final concentration of 0.2% by weight, and a solution diluted 10-fold and 100-fold from a 2 × 10 6 pfu / mL DV solution was added to develop ICA.
（イムノクロマト測定用器具を使用しての検出感度の確認の結果）
公知のデング熱ウイルス検出用イムノクロマト測定用器具を使用しての検出感度向上の結果を図２に示す。
０．２重量％のＰＭＡ７０を添加した場合は、１００倍希釈したウイルス溶液を加えたICAキットでも増感により検出可能であった。一方、コントロールの無添加系では１０倍希釈したDV溶液では検出できず、原液のみにしか検出できなかった。(Result of confirmation of detection sensitivity using immunochromatography measuring instrument)
FIG. 2 shows the results of improving the detection sensitivity using a known immunochromatographic measuring instrument for detecting dengue virus.
When 0.2% by weight of PMA70 was added, it could be detected by sensitization even with an ICA kit containing a virus solution diluted 100-fold. On the other hand, in the control-free system, it could not be detected in the DV solution diluted 10 times, but only in the stock solution.
（総論）
以上の実施例より、本発明は、公知のデング熱ウイルスを測定対象物質とするイムノクロマト測定法と比較して、著しく増感効果が優れているイムノクロマト測定法を提供することができた。
さらに、本発明のデング熱の治療方法は、高感度でデング熱ウイルスを検出することができるので、デング熱の患者又はデング熱に感染しているおそれの患者に迅速にデング熱治療を施すことができるので治療効果が高い。(General)
From the above examples, the present invention has been able to provide an immunochromatographic measurement method having a remarkably excellent sensitizing effect as compared with an immunochromatographic measurement method using a known dengue virus as a measurement target substance.
Further, the method for treating dengue fever of the present invention can detect dengue fever virus with high sensitivity, so that dengue fever treatment can be rapidly performed on a patient with dengue fever or a patient who may be infected with dengue fever. Is high.
本発明は、デング熱ウイルスを測定対象物質とするイムノクロマト測定法用増感剤を提供することができる。 The present invention can provide a sensitizer for immunochromatography, which uses dengue virus as a measurement target substance.
Claims (8)
A sensitizer for immunochromatography, which comprises a polymer represented by the following general formula [1] and contains a dengue virus as a measurement target substance.
The dengue virus according to claim 1 or 2, wherein the polymer has a composition ratio of 2- (methacryloyloxy) ethyl-2'-(trimethylammonio) ethyl phosphate to methacrylic acid of 99 to 55: 1-45. A sensitizer for immunochromatography as a target substance.
The dengue virus according to claim 1 or 2, wherein the polymer has a composition ratio of 2- (methacryloyloxy) ethyl-2'-(trimethylammonio) ethyl phosphate and methacrylic acid of 99 to 70: 1 to 30. A sensitizer for immunochromatography as a target substance.
The sensitizer for immunochromatography, which comprises the dengue virus according to any one of claims 1 to 4, which is a water-soluble polymer having a weight average molecular weight of 10,000 to 1,000,000. ..
A sample diluent for immunochromatography, which comprises the sensitizer for immunochromatography according to any one of claims 1 to 5, and which contains dengue virus as a substance to be measured.
An immunochromatographic measurement method using a dengue virus as a measurement target substance, which comprises causing an antigen-antibody reaction in the presence of the sensitizer for the immunochromatographic measurement method according to any one of claims 1 to 5.
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