JP2007155624A - Anti-Treponema / Paridum antibody measurement reagent - Google Patents
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Abstract
【課題】血清干渉の発生を防止して、梅毒の感染の診断を精度良く行うことが可能な抗トレポネーマ・パリダム抗体測定試薬を提供することを目的とする。
【解決手段】梅毒感染の診断に用いられる抗トレポネーマ・パリダム抗体測定試薬であって、抗トレポネーマ・パリダム抗原を担持した不溶性担体と、２−メタクリロイルオキシエチルホスホリルコリンに由来するセグメント及び親水性モノマーに由来するセグメントを有する共重合体とを含有する抗トレポネーマ・パリダム抗体測定試薬。
【選択図】 なしAn object of the present invention is to provide a reagent for measuring anti-treponema and paridum antibodies capable of preventing the occurrence of serum interference and accurately diagnosing syphilis infection.
A reagent for measuring anti-treponema pallidum antibody used for diagnosis of syphilis infection, derived from an insoluble carrier carrying an anti-treponema pallidum antigen, a segment derived from 2-methacryloyloxyethyl phosphorylcholine, and a hydrophilic monomer And an anti-treponema pallidum antibody measuring reagent containing a copolymer having a segment to form.
[Selection figure] None
Description
本発明は、血清干渉の発生を防止して、梅毒の感染の診断を精度良く行うことが可能な抗トレポネーマ・パリダム抗体測定試薬に関する。 The present invention relates to a reagent for measuring an anti-treponema paridum antibody capable of preventing the occurrence of serum interference and accurately diagnosing syphilis infection.
梅毒の病原体であるトレポネーマ・パリダム（Ｔｒｅｐｏｎｅｍａ Ｐａｌｌｉｄｕｍ）が生体に感染すると、該病原体に対する抗体が産生される。抗トレポネーマ・パリダム抗体測定試薬は血中の抗トレポネーマ・パリダム抗体の有無を測定することにより、梅毒の感染を判定する試薬である。 When a syphilis pathogen, Treponema Pallidum, infects a living body, antibodies against the pathogen are produced. The anti-treponema / paridum antibody measurement reagent is a reagent for determining the infection of syphilis by measuring the presence or absence of anti-treponema / paridum antibody in the blood.
従来、抗トレポネーマ・パリダム抗体測定は血球凝集を利用したＴＰＨＡ等の用手法で行われていたが、近年、生化学自動分析装置に適用可能な試薬（自動化試薬）が発売されている。この自動化試薬においては、患者の採血負担を軽減する目的で、用手法と比べて少量の血清で測定されることが多い。そのため、試薬と血中の抗体の反応により起こる抗原抗体反応量も少量となる。よって、自動化試薬においては、抗原抗体反応を促進する増感剤を添加する場合がある。一般に用いられる増感剤としては、ポリエチレングリコール、デキストラン等がある。また、抗トレポネーマ・パリダム抗体測定試薬における増感剤としては、特許文献１にグルコシド誘導体をモノマー単位として含む水溶性重合体及び／又は溶性共重合体が効果を示すことが開示されている。 Conventionally, anti-treponema and paridum antibody measurement has been performed by a technique such as TPHA using hemagglutination, but in recent years, a reagent (automated reagent) applicable to a biochemical automatic analyzer has been released. This automated reagent is often measured with a small amount of serum compared to the conventional method for the purpose of reducing the burden of blood collection on the patient. Therefore, the amount of antigen-antibody reaction caused by the reaction between the reagent and the antibody in the blood is also small. Therefore, in an automated reagent, a sensitizer that promotes an antigen-antibody reaction may be added. Commonly used sensitizers include polyethylene glycol and dextran. Moreover, as a sensitizer in an anti- treponema pallidum antibody measuring reagent, it is disclosed by patent document 1 that the water-soluble polymer and / or soluble copolymer which contain a glucoside derivative as a monomer unit show an effect.
しかしながら、このような増感剤は、抗原抗体反応を促進する効果には優れているが、一部の検体で正確な測定結果が得られないという問題があった。具体的には、検体が血清である場合に、血清中に含まれている成分が変動することにより、測定に悪影響を与える血清干渉と呼ばれる現象が発生し、正確な測定結果が得られないという問題があった。
本発明は、上記現状に鑑み、血清干渉の発生を防止して、梅毒の感染の診断を精度良く行うことが可能な抗トレポネーマ・パリダム抗体測定試薬を提供することを目的とする。 In view of the above situation, an object of the present invention is to provide an anti-treponema paridum antibody measuring reagent capable of preventing the occurrence of serum interference and accurately diagnosing syphilis infection.
本発明は、梅毒感染の診断に用いられる抗トレポネーマ・パリダム抗体測定試薬であって、抗トレポネーマ・パリダム抗原を担持した不溶性担体と、２−メタクリロイルオキシエチルホスホリルコリンに由来するセグメント及び親水性モノマーに由来するセグメントを有する共重合体とを含有する抗トレポネーマ・パリダム抗体測定試薬である。
以下に、本発明を詳述する。
The present invention is an anti-treponema paridum antibody measurement reagent used for diagnosis of syphilis infection, derived from an insoluble carrier carrying an anti-treponema paridum antigen, a segment derived from 2-methacryloyloxyethyl phosphorylcholine, and a hydrophilic monomer And an anti-treponema pallidum antibody measuring reagent containing a copolymer having a segment.
The present invention is described in detail below.
本発明者らは、鋭意検討の結果、抗トレポネーマ・パリダム抗体測定試薬中に、増感剤として２−メタクリロイルオキシエチルホスホリルコリンに由来するセグメント及び親水性モノマーに由来するセグメントを有する共重合体を含有させることにより、血清中の抗トレポネーマ・パリダム抗体を測定する場合に、血清干渉の発生を防止して、抗トレポネーマ・パリダム抗体の測定を精度良く行うことが可能となることを見出し、本発明を完成させるに至った。 As a result of intensive studies, the present inventors have included a copolymer having a segment derived from 2-methacryloyloxyethyl phosphorylcholine and a segment derived from a hydrophilic monomer as a sensitizer in an anti-treponema / paridum antibody measurement reagent. Thus, when measuring anti-treponema paridum antibody in serum, it has been found that it is possible to prevent the occurrence of serum interference and accurately measure anti-treponema paridum antibody. It came to complete.
本発明の抗トレポネーマ・パリダム抗体測定試薬は、梅毒に対する抗原を担持した不溶性担体と、２−メタクリロイルオキシエチルホスホリルコリンに由来するセグメント及び親水性モノマーに由来するセグメントを有する共重合体（以下、単に共重合体ともいう）とを含有する。
上記親水性モノマーとして、メタクリル酸を用いた場合における上記共重合体の構造を下記一般式（１）に示す。
The reagent for measuring anti-treponema pallidum antibody of the present invention is a copolymer (hereinafter simply referred to as copolymer) having an insoluble carrier carrying an antigen against syphilis, a segment derived from 2-methacryloyloxyethyl phosphorylcholine, and a segment derived from a hydrophilic monomer. (Also referred to as a polymer).
The structure of the copolymer when methacrylic acid is used as the hydrophilic monomer is shown in the following general formula (1).
本発明の抗トレポネーマ・パリダム抗体測定試薬は、上記共重合体を含有することで、検体として血清を使用する場合であっても、血清干渉を引き起こしてしまうことがなく、正確な測定結果を得ることができる。
なお、上記血清干渉を確認する方法としては、例えば、添加回収試験という方法を用いることができる。上記添加回収試験は、被測定物質である抗原又は抗体が高濃度で含まれる標準物質を、０．１〜５％程度になるように生理食塩水（変動成分がない血清のモデル）に添加して添加前後での測定値の差を求めた後、同様にして標準物質を被測定物質である抗原又は抗体を含む血清に添加して添加前後での測定値の差を求め、標準物質を生理食塩水に添加した場合での測定値の差を１００％としたときの割合を回収率として算出することにより、血清干渉の発生を確認する試験である。
The anti-treponema and paridum antibody measuring reagent of the present invention contains the above-mentioned copolymer, so that even when serum is used as a specimen, serum interference is not caused and an accurate measurement result is obtained. be able to.
As a method for confirming the serum interference, for example, a method called addition recovery test can be used. In the above addition / recovery test, a standard substance containing a high concentration of antigen or antibody, which is a substance to be measured, is added to physiological saline (a model of serum without fluctuation components) so that the concentration is about 0.1 to 5%. Then, after obtaining the difference between the measured values before and after the addition, add the standard substance to the serum containing the antigen or antibody to be measured in the same manner to obtain the difference between the measured values before and after the addition. This is a test for confirming the occurrence of serum interference by calculating the ratio when the difference in measured value when added to saline is 100% as the recovery rate.
上記２−メタクリロイルオキシエチルホスホリルコリンは、メタクリロイル基を有することから、他の重合性モノマーと共重合することが可能である。共重合可能な親水性モノマーとしては、例えば、２−ヒドロキシエチルメタクリレート、（メタ）アクリレート、（メタ）アクリルアミド等の（メタ）アクリル酸系単量体等が挙げられる。 Since the 2-methacryloyloxyethyl phosphorylcholine has a methacryloyl group, it can be copolymerized with other polymerizable monomers. Examples of the copolymerizable hydrophilic monomer include (meth) acrylic acid monomers such as 2-hydroxyethyl methacrylate, (meth) acrylate, and (meth) acrylamide.
上記親水性モノマーとしては特に限定されないが、例えば、（メタ）アクリル酸、（メタ）アクリルアミド等が挙げられる。なかでも、血液成分中のタンパク質等と静電反発することが期待される理由から、カチオン性である（メタ）アクリル酸が好適である。
ここで、（メタ）アクリル酸とは、アクリル酸又はメタクリル酸を意味する。
Although it does not specifically limit as said hydrophilic monomer, For example, (meth) acrylic acid, (meth) acrylamide, etc. are mentioned. Of these, cationic (meth) acrylic acid is preferred because it is expected to have electrostatic repulsion with proteins and the like in blood components.
Here, (meth) acrylic acid means acrylic acid or methacrylic acid.
上記共重合体における上記２−メタクリロイルオキシエチルホスホリルコリンに由来するセグメントと、親水性モノマーに由来するセグメントとの比としては特に限定されず、必要に応じて適宜選択されればよいが、モル比で５：５〜３：７であることが好ましい。上記２−メタクリロイルオキシエチルホスホリルコリンに由来するセグメントの比がこれ未満であると、抗トレポネーマ・パリダム抗体測定における血清干渉の発生を効果的に防ぐことができないことがある。 The ratio of the segment derived from the 2-methacryloyloxyethyl phosphorylcholine and the segment derived from the hydrophilic monomer in the copolymer is not particularly limited, and may be appropriately selected as necessary. It is preferably 5: 5 to 3: 7. If the ratio of the segment derived from 2-methacryloyloxyethyl phosphorylcholine is less than this, it may be impossible to effectively prevent the occurrence of serum interference in the measurement of anti-treponema / paridum antibody.
上記共重合体の重量平均分子量としては特に限定されないが、好ましい下限は５万、好ましい上限は５００万である。５万未満であると、凝集促進効果がなくなることがあり、５００万を超えると、試薬中に添加した場合に粘性が上がりすぎ、再現性等が悪化してしまうことがある。 Although it does not specifically limit as a weight average molecular weight of the said copolymer, A preferable minimum is 50,000 and a preferable upper limit is 5 million. If it is less than 50,000, the effect of promoting aggregation may be lost, and if it exceeds 5,000,000, when added to the reagent, the viscosity is excessively increased and the reproducibility may be deteriorated.
本発明の抗トレポネーマ・パリダム抗体測定試薬における上記共重合体の含有量の好ましい下限は０．１（ｗ／ｖ）％、好ましい上限は１．２（ｗ／ｖ）％である。０．１（ｗ／ｖ）％未満であると、血清干渉の発生を効果的に防止できないことがあり、１．２（ｗ／ｖ）％を超えると、試薬の粘性が上がりすぎ、再現性が低下することがある。
より好ましい下限は０．２（ｗ／ｖ）％、より好ましい上限は０．８（ｗ／ｖ）％である。
The preferable lower limit of the content of the copolymer in the anti-treponema pallidum antibody measuring reagent of the present invention is 0.1 (w / v)%, and the preferable upper limit is 1.2 (w / v)%. If it is less than 0.1 (w / v)%, it may not be possible to effectively prevent the occurrence of serum interference. If it exceeds 1.2 (w / v)%, the reagent viscosity becomes too high and reproducibility. May decrease.
A more preferred lower limit is 0.2 (w / v)%, and a more preferred upper limit is 0.8 (w / v)%.
本発明の抗トレポネーマ・パリダム抗体測定試薬は、上記共重合体に加えて、抗トレポネーマ・パリダム抗原を担持した不溶性担体を含有する。 The reagent for measuring anti-treponema pallidum antibody of the present invention contains an insoluble carrier carrying an anti-treponema pallidum antigen in addition to the above copolymer.
上記抗トレポネーマ・パリダム抗原としては、例えば、トレポネーマ・パリダム菌体由来抗原を用いることが好ましい。 As the anti-treponema pallidum antigen, for example, an antigen derived from Treponema pallidum is preferably used.
上記不溶性担体としては特に限定されないが、例えば、ポリスチレン、スチレン−スチレンスルホン酸塩重合体、アクリロニトリル−ブタジエン−スチレン共重合体、塩化ビニル−アクリル酸エステル共重合体、ポリ酢酸ビニルアクリレート等からなるものが挙げられる。 Although it does not specifically limit as said insoluble support | carrier, For example, what consists of polystyrene, a styrene styrene sulfonate polymer, an acrylonitrile butadiene styrene copolymer, a vinyl chloride acrylate copolymer, a polyvinyl acetate acrylate etc. Is mentioned.
上記不溶性担体の粒子径としては、用いる測定方法や測定機器にもよるが、好ましい下限が０．０５μｍ、好ましい上限は１．０μｍである。０．０５μｍ未満であると、凝集による光学的変化量が小さく、測定に必要な高い感度が得られないことがあり、１．０μｍを超えると、ラテックス粒子の凝集による光学的変化量が測定可能域を超えてしまい、測定範囲が小さくなることがある。 The particle size of the insoluble carrier depends on the measurement method and measuring instrument used, but the preferred lower limit is 0.05 μm and the preferred upper limit is 1.0 μm. If it is less than 0.05 μm, the amount of optical change due to aggregation is small, and high sensitivity required for measurement may not be obtained. If it exceeds 1.0 μm, the amount of optical change due to aggregation of latex particles can be measured. The measurement range may be reduced.
上記不溶性担体に抗トレポネーマ・パリダム抗原を担持させる方法としては特に限定されず、公知の方法により、物理的、化学的結合により担持させる方法等が挙げられる。なお、このときに用いる抗トレポネーマ・パリダム抗原は、菌体破砕物であってもよく、精製物であってもよい。また、遺伝子組み換え技術により人工的に合成されたものを１種又はそれ以上組み合わせたものでもよい。 The method for supporting the anti-treponema / paridum antigen on the insoluble carrier is not particularly limited, and examples thereof include a method for supporting it by a physical or chemical bond by a known method. In addition, the anti-treponema pallidum antigen used at this time may be a crushed cell or a purified product. Moreover, what was synthesize | combined artificially by the gene recombination technique and what combined 1 or more types may be used.
本発明の抗トレポネーマ・パリダム抗体測定試薬は、上記抗トレポネーマ・パリダム抗原を担持した不溶性担体と、上記共重合体とを同一の媒体に分散及び溶解させることにより１液系のラテックス試薬として使用してもよく、また、上記抗トレポネーマ・パリダム抗原を担持した不溶性担体を含有する第１試薬と、上記共重合体を媒体に添加した緩衝液からなる第２試薬とで構成された２液系の試薬として使用してもよい。 The reagent for measuring anti-treponema pallidum antibody of the present invention is used as a one-component latex reagent by dispersing and dissolving the insoluble carrier carrying the anti-treponema pallidum antigen and the copolymer in the same medium. Alternatively, a two-component system comprising a first reagent containing an insoluble carrier carrying the anti-treponema pallidum antigen and a second reagent comprising a buffer solution in which the copolymer is added to a medium. It may be used as a reagent.
上記媒体としては特に限定されず、例えば、リン酸緩衝液、グリシン緩衝液、トリス塩緩衝液等が挙げられる。 The medium is not particularly limited, and examples thereof include a phosphate buffer, a glycine buffer, and a tris salt buffer.
上記１液系のラテックス試薬中には、更に、ウシ血清アルブミン、ショ糖、塩化ナトリウム、ＥＤＴＡ・２Ｎａ、界面活性剤等を適宜溶解させもよい。
また、２液系の試薬として使用する場合にも、それぞれにウシ血清アルブミン、ショ糖、塩化ナトリウム、ＥＤＴＡ・２Ｎａ、界面活性剤等を適宜溶解させてもよい。
In the one-component latex reagent, bovine serum albumin, sucrose, sodium chloride, EDTA · 2Na, a surfactant and the like may be appropriately dissolved.
In addition, when used as a two-component reagent, bovine serum albumin, sucrose, sodium chloride, EDTA · 2Na, a surfactant and the like may be appropriately dissolved in each.
本発明の抗トレポネーマ・パリダム抗体測定試薬を用い、検体中の抗トレポネーマ・パリダム抗体との抗原抗体反応により生じる凝集の度合いを光学的に測定又は目視にて観察することにより、検体中の抗トレポネーマ・パリダム抗体を測定することができる。 By using the anti-treponema pallidum antibody measuring reagent of the present invention, the degree of aggregation caused by the antigen-antibody reaction with the anti-treponema pallidum antibody in the sample is optically measured or visually observed, thereby anti-treponema in the sample.・ Paridum antibody can be measured.
本発明の抗トレポネーマ・パリダム抗体測定試薬を用い、抗原抗体反応を行う際のｐＨの好ましい上限は４．５、好ましい下限は１０．０であり、より好ましい下限は６．０、より好ましい上限は８．０である。
また、反応温度の好ましい下限は０℃、好ましい上限は５０℃であり、反応時間は適宜選択される。
The preferable upper limit of the pH when performing the antigen-antibody reaction using the anti-treponema / paridum antibody measurement reagent of the present invention is 4.5, the preferable lower limit is 10.0, the more preferable lower limit is 6.0, and the more preferable upper limit is 8.0.
Moreover, the preferable minimum of reaction temperature is 0 degreeC, and a preferable upper limit is 50 degreeC, and reaction time is selected suitably.
上記凝集の度合いを光学的に測定する方法としては、公知の方法が用いられ、例えば、用いる不溶性担体の粒子の大きさ、濃度の選択、反応時間の設定により、散乱光強度、吸収光度、透過光強度の増減を測定する。また、これらの方法を併用することも可能である。また、上記測定を行う際の光の波長としては、３００〜９００ｎｍが好ましい。 As a method for optically measuring the degree of aggregation, a known method is used. For example, the size, concentration, and reaction time of insoluble carrier particles to be used are selected according to the scattered light intensity, absorbed light intensity, and transmission. Measure the increase or decrease of light intensity. Also, these methods can be used in combination. Moreover, as a wavelength of the light at the time of performing the said measurement, 300-900 nm is preferable.
上記の光学的測定方法に用いる装置としては、散乱光強度、透過光強度、吸光度等を検出できる光学機器が挙げられ、一般的に使用されている生化学自動分析機であればいずれのものであっても使用することができる。 Examples of the apparatus used for the above optical measurement method include optical instruments capable of detecting scattered light intensity, transmitted light intensity, absorbance, etc., and any commonly used biochemical automatic analyzer can be used. It can be used even if it exists.
上記凝集の度合いを目視にて観察する方法としては、通常、検体と本発明の抗トレポネーマ・パリダム抗体測定試薬とを含む溶液を判定板上で混合し、混合液を揺り動かした後、凝集の有無を判定する方法等を用いることができる。なお、凝集の度合いの観察には、目視による方法以外に、凝集状態をビデオカメラで撮影し、画像処理を施す方法を用いることも可能である。 As a method of visually observing the degree of aggregation, usually, a solution containing a specimen and the anti-treponema / paridum antibody measurement reagent of the present invention is mixed on a judgment plate, the mixture is shaken, and then the presence or absence of aggregation. Or the like can be used. For observation of the degree of aggregation, in addition to the visual method, a method of photographing the aggregation state with a video camera and performing image processing can be used.
本発明によれば、血清干渉の発生を防止して、梅毒の感染の診断を精度良く行うことが可能な抗トレポネーマ・パリダム抗体測定試薬を提供することができる。 ADVANTAGE OF THE INVENTION According to this invention, generation | occurrence | production of serum interference can be prevented and the anti- treponema paridum antibody measuring reagent which can diagnose the infection of syphilis accurately can be provided.
以下に実施例を掲げて本発明を更に詳しく説明するが、本発明はこれら実施例のみに限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
（実施例１）
（１）抗トレポネーマ・パリダム抗体測定試薬の調製
以下の手順に従い抗トレポネーマ・パリダム抗原担持ラテックス液からなる第１試薬と、検体希釈液からなる第２試薬とから構成される２液系の抗トレポネーマ・パリダム抗体測定試薬を調製した。
Example 1
(1) Preparation of reagent for measuring anti-treponema / paridum antibody According to the following procedure, a two-component anti-treponema composed of a first reagent composed of an anti-treponema / paridum antigen-carrying latex and a second reagent composed of a sample diluent -A paridum antibody measurement reagent was prepared.
（１−１）抗トレポネーマ・パリダム抗原担持ラテックス液の調製
１００ｍＭリン酸緩衝液（ｐＨ７．４）に１５０μｇ／ｍｌの蛋白濃度で溶解した抗トレポネーマ・パリダム抗原液４００μｌを平均粒径０．４００μｍのポリスチレンラテックス（固形分１０（ｗ／ｖ）％、積水化学工業社製）１００μｌに添加し、４℃にて１時間攪拌した。次いで、ウシ血清アルブミン（セロロジカル製；ＢＳＡ）を１（ｗ／ｖ）％含有する１００ｍＭリン酸緩衝液（ｐＨ７．４）２ｍｌを添加し、１．５時間攪拌した。
得られた液体を１０℃にて３０分間、１８，０００ｒｐｍで遠心分離し、得られた沈殿物を、ＢＳＡを０．２５（ｗ／ｖ）％含有する１００ｍＭリン酸緩衝液（ｐＨ７．４）４ｍｌに添加し、ラテックスを懸濁させることにより抗トレポネーマ・パリダム抗原担持ラテックス液を調製した。
(1-1) Preparation of anti-treponema paridum antigen-supporting latex solution 400 μl of anti-treponema paridum antigen solution dissolved in 100 mM phosphate buffer (pH 7.4) at a protein concentration of 150 μg / ml has an average particle size of 0.400 μm. Polystyrene latex (solid content 10 (w / v)%, manufactured by Sekisui Chemical Co., Ltd.) 100 μl was added and stirred at 4 ° C. for 1 hour. Subsequently, 2 ml of 100 mM phosphate buffer (pH 7.4) containing 1 (w / v)% of bovine serum albumin (Cerological, BSA) was added and stirred for 1.5 hours.
The obtained liquid was centrifuged at 18,000 rpm for 30 minutes at 10 ° C., and the resulting precipitate was added to a 100 mM phosphate buffer (pH 7.4) containing 0.25 (w / v)% BSA. An anti-treponema / paridum antigen carrying latex solution was prepared by adding to 4 ml and suspending the latex.
（１−２）検体希釈液の調製
ＢＳＡを１％含有する１００ｍＭリン酸緩衝液（ｐＨ７．４）にＬｉｐｉｄｕｒｅ（２−メタクリロイルオキシエチルホスホリルコリンとメタクリル酸との共重合体：分子量１００万；日本油脂社製）を０．２（ｗ／ｖ）％添加した。
(1-2) Preparation of specimen dilution liquid Lipidure (2-methacryloyloxyethyl phosphorylcholine and methacrylic acid copolymer: molecular weight 1 million; Nippon Oil & Fats) in 100 mM phosphate buffer (pH 7.4) containing 1% BSA 0.2 (w / v)% was added.
（実施例２）
実施例１の（１−１）抗トレポネーマ・パリダム抗原担持ラテックス液の調製において、得られた沈殿物にＢＳＡを０．２５（ｗ／ｖ）％含有する１００ｍＭリン酸緩衝液（ｐＨ７．４）５ｍｌを添加するとともに、（１−２）検体希釈液の調製において、Ｌｉｐｉｄｕｒｅを０．６（ｗ／ｖ）％添加した以外は、実施例１と同様にして、抗トレポネーマ・パリダム抗体測定試薬を調製した。
(Example 2)
In the preparation of the latex liquid carrying the anti-treponema pallidum antigen (1-1) in Example 1, a 100 mM phosphate buffer (pH 7.4) containing 0.25 (w / v)% BSA in the resulting precipitate. In addition to adding 5 ml, (1-2) In the preparation of the sample diluent, except that 0.6 (w / v) Lipidure was added, the anti-treponema pallidum antibody measurement reagent was prepared in the same manner as in Example 1. Prepared.
（実施例３）
実施例１の（１−１）抗トレポネーマ・パリダム抗原担持ラテックス液の調製において、得られた沈殿物にＢＳＡを０．２５（ｗ／ｖ）％含有する１００ｍＭリン酸緩衝液（ｐＨ７．４）１２ｍｌを添加するとともに、（１−２）検体希釈液の調製において、Ｌｉｐｉｄｕｒｅを１．０（ｗ／ｖ）％添加した以外は、実施例１と同様にして、抗トレポネーマ・パリダム抗体測定試薬を調製した。
(Example 3)
In the preparation of the latex liquid carrying the anti-treponema pallidum antigen (1-1) in Example 1, a 100 mM phosphate buffer (pH 7.4) containing 0.25 (w / v)% BSA in the resulting precipitate. In addition to the addition of 12 ml, (1-2) In the preparation of the sample diluent, 1.0 (w / v)% of Lipidure was added in the same manner as in Example 1 except that the anti-Treponema paridum antibody measurement reagent was used. Prepared.
（比較例１）
実施例１の（１−２）検体希釈液の調製において、ＢＳＡを１％含有する１００ｍＭリン酸緩衝液（ｐＨ７．４）に、Ｌｉｐｉｄｕｒｅの代わりにｐＧＥＭＡ（グリコシルエチルメタクリレートのホモポリマー、平均分子量１１４万、日本精化社製）を１（ｗ／ｖ）％添加した以外は実施例１と同様にして、抗トレポネーマ・パリダム抗体測定試薬を調製した。
(Comparative Example 1)
In the preparation of the sample diluent (1-2) in Example 1, a 100 mM phosphate buffer solution (pH 7.4) containing 1% BSA was replaced with pGEMA (a homopolymer of glycosylethyl methacrylate, average molecular weight of 114) instead of Lipidure. An anti-treponema paridum antibody measuring reagent was prepared in the same manner as in Example 1 except that 1 (w / v)% was added.
（評価）
（１）抗トレポネーマ・パリダム抗体標準液の測定
抗トレポネーマ・パリダム抗体標準液として、梅毒陽性標準血清（積水化学工業社製、５濃度）１６μＬを採取し、これに検体希釈液１７５μＬを混和し、３７℃で適時保持した後、抗トレポネーマ・パリダム抗原担持ラテックス液２５μＬを添加、撹拌した後、約８０秒から３００秒までの間の波長７００ｎｍでの吸光度の変化量を測定し、吸光度変化量（△ａｂｓ）とした。測定は自動分析装置日立７１７０型を使用した。結果を表１に示した。なお、Ｔ．Ｕ．は抗トレポネーマ・パリダム抗体測定試薬であるメディエースＴＰＬＡ（積水化学工業社製）で血清を測定した場合の抗トレポネーマ・パリダム抗体の抗体価を表す単位であり、１０Ｔ．Ｕ．以上を陽性とする。
(Evaluation)
(1) Measurement of anti-treponema / paridum antibody standard solution As anti-treponema / paridum antibody standard solution, 16 μL of syphilis positive standard serum (Sekisui Chemical Co., Ltd., 5 concentration) was collected, and 175 μL of sample dilution solution was mixed with this. After holding at 37 ° C for a timely time, 25 μL of anti-treponema / paridum antigen-supporting latex solution was added and stirred, and the amount of change in absorbance at a wavelength of 700 nm from about 80 seconds to 300 seconds was measured. Δabs). The automatic analyzer Hitachi 7170 type was used for the measurement. The results are shown in Table 1. T. T. U. Is a unit representing the antibody titer of anti-treponema paridum antibody when serum is measured with Mediace TPLA (manufactured by Sekisui Chemical Co., Ltd.) which is a reagent for measuring anti-treponema paridum antibody. U. The above is positive.
（２）添加回収試験
生理食塩水２４５μＬに、２，０００Ｔ．Ｕ．の抗トレポネーマ・パリダム抗体標準液５μＬを添加し、評価（１）と同様の方法で添加前後の吸光度変化量を測定することにより、添加前後の抗体価の測定値の差を求めた。同様にして、５種類の梅毒陽性血清２４５μＬのそれぞれに２，０００Ｔ．Ｕ．の抗トレポネーマ・パリダム抗体標準液５μＬを添加し、添加前後の抗体価の測定値の差を求めた後、生理食塩水に抗トレポネーマ・パリダム抗体標準液を添加した場合における添加前後の抗体価の測定値の差を１００％として回収率を算出した。結果を表２に示した。
(2) Addition / recovery test To 245 μL of physiological saline, 2,000 T.P. U. Was added, and the amount of change in the antibody titer before and after the addition was measured by the same method as in Evaluation (1) to determine the difference in the measured antibody titer before and after the addition. Similarly, each of five types of syphilis positive 245 μL of 2,000 T.P. U. After adding 5 μL of the anti-treponema / paridum antibody standard solution and determining the difference between the measured antibody titers before and after the addition, the antibody titer before and after the addition when the anti-treponema / paridum antibody standard solution was added to the physiological saline. The recovery rate was calculated with the difference between the measured values as 100%. The results are shown in Table 2.
表２に示すように、Ｌｉｐｉｄｕｒｅを反応促進剤として使用した実施例１〜３では、各検体ともｐＧＥＭＡを反応促進剤として使用した比較例１よりも添加回収率が大幅に改善していることがわかる。 As shown in Table 2, in Examples 1 to 3 in which Lipidure was used as a reaction accelerator, the addition recovery rate was greatly improved over Comparative Example 1 in which each sample used pGEMA as a reaction accelerator. Recognize.
本発明によれば、血清干渉の発生を防止して、梅毒の感染の診断を精度良く行うことが可能な抗トレポネーマ・パリダム抗体測定試薬を提供することができる。 ADVANTAGE OF THE INVENTION According to this invention, generation | occurrence | production of serum interference can be prevented and the anti- treponema paridum antibody measuring reagent which can diagnose the infection of syphilis accurately can be provided.
Claims (2)
抗トレポネーマ・パリダム抗原を担持した不溶性担体と、２−メタクリロイルオキシエチルホスホリルコリンに由来するセグメント及び親水性モノマーに由来するセグメントを有する共重合体とを含有する
ことを特徴とする抗トレポネーマ・パリダム抗体測定試薬。 An anti-treponema and paridum antibody measuring reagent used for diagnosis of syphilis infection,
An anti-treponema pallidum antibody measurement comprising an insoluble carrier carrying an anti-treponema pallidum antigen, and a copolymer having a segment derived from 2-methacryloyloxyethyl phosphorylcholine and a segment derived from a hydrophilic monomer reagent.
The reagent for measuring anti-Treponema pallidum antibody according to claim 1, wherein the anti-Treponema pallidum antigen is an antigen derived from Treponema pallidum cells.
Priority Applications (11)
| Application Number | Priority Date | Filing Date | Title |
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| JP2005354081A JP4629564B2 (en) | 2005-12-07 | 2005-12-07 | Anti-Treponema / Paridum antibody measurement reagent |
| US12/086,082 US20090246884A1 (en) | 2005-12-07 | 2006-12-07 | Reagent for Assaying Antiphospholipid Antibody and Reagent for Assaying Anti-Treponema Pallidum Antibody |
| KR1020087012187A KR101440938B1 (en) | 2005-12-07 | 2006-12-07 | Reagent for assaying antiphospholipid antibody and reagent for assaying anti-treponema pallidum antibody |
| PCT/JP2006/324473 WO2007066731A1 (en) | 2005-12-07 | 2006-12-07 | Reagent for assaying antiphospholipid antibody and reagent for assaying anti-treponema pallidum antibody |
| DK06834228.6T DK1956373T3 (en) | 2005-12-07 | 2006-12-07 | PROCEDURE FOR ANALYTICAL ANTI-TREPONEMA PALLIDUM ANTIBODY ANALYSIS |
| CA2631636A CA2631636C (en) | 2005-12-07 | 2006-12-07 | Reagent for assaying antiphospholipid antibody and reagent for assaying anti-treponema pallidum antibody |
| ES06834228.6T ES2615524T3 (en) | 2005-12-07 | 2006-12-07 | Method to analyze anti-Treponema pallidum antibodies |
| EP06834228.6A EP1956373B1 (en) | 2005-12-07 | 2006-12-07 | Method for assaying anti-treponema pallidum antibody |
| CN201110427687.3A CN102565397B (en) | 2005-12-07 | 2006-12-07 | Method for preventing serum interference and improving storage stability of reagent of anti-phospholipid antibody |
| CN2006800455547A CN101341408B (en) | 2005-12-07 | 2006-12-07 | Reagent for assaying antiphospholipid antibody and reagent for assaying anti-treponema pallidum antibody |
| AU2006323739A AU2006323739B2 (en) | 2005-12-07 | 2006-12-07 | Reagent for assaying antiphospholipid antibody and reagent for assaying anti-treponema pallidum antibody |
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| JP2007155623A (en) * | 2005-12-07 | 2007-06-21 | Sekisui Chem Co Ltd | Antiphospholipid antibody measurement reagent |
| JP2009031061A (en) * | 2007-07-25 | 2009-02-12 | Jsr Corp | Target substance detection method and reagent for latex agglutination reaction |
| WO2009054538A1 (en) | 2007-10-22 | 2009-04-30 | Alfresa Pharma Corporation | Method and kit for measurement of acrolein adduct in sample utilizing agglutination reaction of immunological microparticle |
| WO2009113158A1 (en) * | 2008-03-11 | 2009-09-17 | 住友ベークライト株式会社 | Method for immobilizing biologically active substance |
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| EP4600649A1 (en) | 2022-10-06 | 2025-08-13 | Sekisui Medical Co., Ltd. | Non-specific reaction inhibitor, method for using non-specific reaction inhibitor, method for inhibiting non-specific reaction, biochemical measurement reagent, specimen pretreatment solution, and biochemical measurement reagent kit |
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| JP2001242171A (en) * | 2000-02-29 | 2001-09-07 | Sysmex Corp | Reagent for measuring anti-phospholipid antibody, its preparation method and method for measuring anti phospholipid antibody |
| JP2003344413A (en) * | 2002-05-24 | 2003-12-03 | Wako Pure Chem Ind Ltd | Sensitivity enhancer for immunochromatography measurement method, measurement method and instrument for measurement method |
| JP2007155623A (en) * | 2005-12-07 | 2007-06-21 | Sekisui Chem Co Ltd | Antiphospholipid antibody measurement reagent |
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| JP2001242171A (en) * | 2000-02-29 | 2001-09-07 | Sysmex Corp | Reagent for measuring anti-phospholipid antibody, its preparation method and method for measuring anti phospholipid antibody |
| JP2003344413A (en) * | 2002-05-24 | 2003-12-03 | Wako Pure Chem Ind Ltd | Sensitivity enhancer for immunochromatography measurement method, measurement method and instrument for measurement method |
| JP2007155623A (en) * | 2005-12-07 | 2007-06-21 | Sekisui Chem Co Ltd | Antiphospholipid antibody measurement reagent |
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| JP2007155623A (en) * | 2005-12-07 | 2007-06-21 | Sekisui Chem Co Ltd | Antiphospholipid antibody measurement reagent |
| JP2009031061A (en) * | 2007-07-25 | 2009-02-12 | Jsr Corp | Target substance detection method and reagent for latex agglutination reaction |
| WO2009054538A1 (en) | 2007-10-22 | 2009-04-30 | Alfresa Pharma Corporation | Method and kit for measurement of acrolein adduct in sample utilizing agglutination reaction of immunological microparticle |
| WO2009113158A1 (en) * | 2008-03-11 | 2009-09-17 | 住友ベークライト株式会社 | Method for immobilizing biologically active substance |
| JP2015148558A (en) * | 2014-02-07 | 2015-08-20 | 国立大学法人京都工芸繊維大学 | Protein adsorption inhibitor and protein adsorption inhibition method |
| JP2021067492A (en) * | 2019-10-18 | 2021-04-30 | キヤノンメディカルシステムズ株式会社 | Specimen suspension, method of preparing specimen suspension, and detection method |
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