JP2019053060A - Immunoassay method for hepatitis B virus core antibody - Google Patents
Immunoassay method for hepatitis B virus core antibody Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
Description
本発明は、B型肝炎ウイルスコア抗体の免疫測定方法に関する。 The present invention relates to an immunoassay method for hepatitis B virus core antibody.
B型肝炎ウイルス(HBV)の本体は、直径42 nmの二重構造の球形粒子の形状をとっている「デーン(dane)粒子」と称される粒子である。デーン粒子は、その表面がHBs抗原と称される表面抗原で覆われており、さらに内部には、HBc抗原(コア抗原)、HBe抗原、及びウイルス遺伝子をコードする環状二重鎖DNAを含む直径27 nmのコア構造が存在することが知られている。 The main body of hepatitis B virus (HBV) is a particle called “dane particle” which takes the shape of a double-structured spherical particle having a diameter of 42 nm. The surface of the Dane particle is covered with a surface antigen called HBs antigen, and the inside contains a double-stranded DNA containing HBc antigen (core antigen), HBe antigen, and a viral gene. It is known that a 27 nm core structure exists.
これら抗原や抗原に対する自己抗体は、ウイルス感染の診断のための標的とされてきた。特に、HBc抗原に対する抗体(一般に「HBVコア抗体」、又は「抗HBc抗体」と呼ばれる)は、HBV感染の初期から陽性化し、感染後長期間にわたり陽性であり続けることから、HBV感染を広く検出できるマーカーとして用いられている。 These antigens and autoantibodies against the antigen have been targeted for the diagnosis of viral infection. In particular, antibodies against HBc antigen (generally called “HBV core antibody” or “anti-HBc antibody”) become positive from the early stage of HBV infection and remain positive for a long time after infection, thus widely detecting HBV infection It is used as a possible marker.
しかしながら、既存の抗HBc抗体測定試薬では陽性であるにもかかわらず検出できない場合もあり、高感度化が求められている。抗HBc抗体を高感度に測定するための手段として、反応系に添加する検体量を増やすことが考えられる。しかしながら、反応系に添加する検体量を増やすと、陽性検体のシグナル値は増加するが、同時に非特異的な反応によるバックグラウンドのシグナル値も増加してしまうため、感度を上げることができない。 However, there are cases where it cannot be detected despite the fact that the existing anti-HBc antibody measurement reagent is positive, and high sensitivity is required. As a means for measuring the anti-HBc antibody with high sensitivity, it is conceivable to increase the amount of the sample added to the reaction system. However, when the amount of the sample added to the reaction system is increased, the signal value of the positive sample increases, but at the same time, the background signal value due to the nonspecific reaction also increases, so the sensitivity cannot be increased.
抗HBc抗体の免疫測定系の非特異反応を抑制する方法として、例えば特許文献1には、前処理液又は一次反応液に2-メルカプトエタノールやジチオスレイトール等の還元剤を添加する方法が記載されている。特許文献1では偽陽性が排除できるとされており、非特異反応がある程度抑制されているものと推察されるが、還元剤を利用する手法では非特異反応の抑制効果が十分ではない。 As a method for suppressing the nonspecific reaction of the immunoassay system of anti-HBc antibody, for example, Patent Document 1 describes a method of adding a reducing agent such as 2-mercaptoethanol or dithiothreitol to a pretreatment solution or a primary reaction solution. Has been. In Patent Document 1, it is assumed that false positives can be eliminated, and it is surmised that the nonspecific reaction is suppressed to some extent. However, the method using a reducing agent is not sufficient for suppressing the nonspecific reaction.
特許文献2には、感染症急性期に生じる特異的IgM抗体の測定における問題点を解決する手段として、検体をIgM含有水溶液で希釈するという手法が開示されている。開示内容はIgM型抗体の測定に限定されており、主としてA型肝炎ウイルス(HAV)に対するIgM抗体の測定方法に関する。HAV抽出物をアニオン性界面活性剤で処理したものをHAV抗原試薬として用いることが記載されるほか、測定系内にアニオン性界面活性剤以外の界面活性剤を添加しても良いことが記載されており、実施例では0.1% Tween20を含む抗IgM抗体被覆微粒子試薬が用いられている。しかしながら、測定系内にアニオン性界面活性剤以外の界面活性剤を添加する目的やその効果は全く記載されていない。 Patent Document 2 discloses a technique of diluting a specimen with an IgM-containing aqueous solution as a means for solving the problems in the measurement of specific IgM antibodies that occur in the acute stage of infectious diseases. The disclosure is limited to the measurement of IgM antibodies, and mainly relates to a method for measuring IgM antibodies against hepatitis A virus (HAV). It is described that the HAV extract treated with an anionic surfactant is used as the HAV antigen reagent, and that a surfactant other than the anionic surfactant may be added to the measurement system. In the examples, an anti-IgM antibody-coated fine particle reagent containing 0.1% Tween20 is used. However, the purpose and effect of adding a surfactant other than the anionic surfactant in the measurement system are not described at all.
特許文献3には、HLB値が12〜19の非イオン性界面活性剤を抗原−抗体反応系に1.5〜6 w/w%の濃度で存在させて抗原抗体反応を行なうことにより、干渉物質による非特異的な妨害を減少させ、低値における免疫反応の測定感度及び測定精度が向上することが記載されている。実施例の記載によると、抗原−抗体反応系内の界面活性剤濃度が1.5%未満の低濃度では、バックグラウンドを下げる効果も免疫反応の促進効果も十分ではない。また実施例では、NCC−ST−439抗原の免疫測定における効果のみを検討しており、抗体の免疫測定に関しては全く検討していない。 In Patent Document 3, a nonionic surfactant having an HLB value of 12 to 19 is present in an antigen-antibody reaction system at a concentration of 1.5 to 6 w / w%, and an antigen-antibody reaction is performed. It is described that the non-specific interference is reduced and the measurement sensitivity and measurement accuracy of the immune reaction at low values are improved. According to the description of the examples, when the surfactant concentration in the antigen-antibody reaction system is a low concentration of less than 1.5%, neither the effect of lowering the background nor the effect of promoting the immune reaction is sufficient. In Examples, only the effect of NCC-ST-439 antigen in immunoassay is examined, and the immunoassay of antibody is not studied at all.
本発明は、抗HBc抗体の免疫測定において、従来法よりもさらに非特異反応を抑制し、検出感度をさらに高めることができる手段を提供することを目的とする。 An object of the present invention is to provide a means capable of further suppressing the nonspecific reaction and further enhancing the detection sensitivity in the immunoassay of an anti-HBc antibody.
本願発明者らは、鋭意研究の結果、一次反応系内に所定の非イオン性界面活性剤又は両性界面活性剤が共存する条件下で抗HBc抗体の免疫測定を行なうことにより、界面活性剤を利用した従来の類似技術よりもさらに非特異反応を抑制し、抗HBc抗体の検出感度を高めることができることを見出し、本願発明を完成した。 As a result of diligent research, the inventors of the present application conducted the immunoassay of the anti-HBc antibody under the condition that a predetermined nonionic surfactant or amphoteric surfactant coexists in the primary reaction system, thereby obtaining the surfactant. It was found that the non-specific reaction can be further suppressed and the detection sensitivity of the anti-HBc antibody can be enhanced as compared with the conventional similar technique used, and the present invention has been completed.
すなわち、本発明は、検体中の抗HBc抗体の免疫測定方法であって、下記(1)及び(2):
(1) 非イオン性界面活性剤
(2) 両性界面活性剤
から選択される少なくとも1種の界面活性剤が共存する条件下で、HBc抗原又は抗免疫グロブリン抗体若しくはその抗原結合性断片を含む試薬と検体を接触させて抗原抗体反応を行なうことを含む、方法を提供する。
また、本発明は、抗HBc抗体の免疫測定キットであって、上記(1)及び(2)から選択される少なくとも1種の界面活性剤を含有する溶液と、HBc抗原とを含む、キットを提供する。さらに、本発明は、抗HBc抗体の免疫測定キットであって、HBc抗原を含有する抗原試薬液を含み、該試薬液が、上記(1)及び(2)から選択される少なくとも1種の界面活性剤を含有する、キットを提供する。さらに、本発明は、抗HBc抗体の免疫測定キットであって、抗免疫グロブリン抗体又はその抗原結合性断片を含有する抗体試薬液と、HBc抗原とを含み、該抗体試薬液が、上記(1)及び(2)から選択される少なくとも1種の界面活性剤を含有する、キットを提供する。
That is, the present invention is an immunoassay method for an anti-HBc antibody in a specimen, which comprises the following (1) and (2):
(1) Nonionic surfactant
(2) An antigen-antibody reaction by contacting a specimen containing a reagent containing an HBc antigen or an anti-immunoglobulin antibody or an antigen-binding fragment thereof under conditions where at least one surfactant selected from amphoteric surfactants coexists. A method is provided that includes:
The present invention also provides an anti-HBc antibody immunoassay kit comprising a solution containing at least one surfactant selected from (1) and (2) above and an HBc antigen. provide. Furthermore, the present invention is an immunoassay kit for an anti-HBc antibody, comprising an antigen reagent solution containing an HBc antigen, wherein the reagent solution is at least one interface selected from (1) and (2) above. A kit is provided containing an active agent. Furthermore, the present invention is an anti-HBc antibody immunoassay kit, comprising an antibody reagent solution containing an anti-immunoglobulin antibody or an antigen-binding fragment thereof, and an HBc antigen, wherein the antibody reagent solution is the above (1) And a kit containing at least one surfactant selected from (2).
本発明によれば、抗HBc抗体の免疫測定時の非特異反応を良好に抑制し、バックグラウンドを従来法よりもさらに抑制することができる。従って、低値における検出感度が高まるほか、検体使用量を増加した場合でもバックグラウンドが抑えられるので、高感度な測定が可能になる。 According to the present invention, a non-specific reaction at the time of immunoassay of an anti-HBc antibody can be satisfactorily suppressed, and the background can be further suppressed as compared with the conventional method. Therefore, the detection sensitivity at a low value is increased, and the background can be suppressed even when the amount of sample used is increased, so that highly sensitive measurement is possible.
本発明は、検体中の抗HBc抗体を免疫測定により測定する方法であり、下記(1)及び(2)から選択される少なくとも1種の界面活性剤が共存する条件下で、HBc抗原又は抗免疫グロブリン抗体若しくはその抗原結合性断片を含む試薬と検体を接触させて抗原抗体反応を行なう。
(1) 非イオン性界面活性剤
(2) 両性界面活性剤
The present invention is a method for measuring an anti-HBc antibody in a specimen by immunoassay. Under the conditions in which at least one surfactant selected from the following (1) and (2) coexists, an HBc antigen or anti-antibody An antigen-antibody reaction is performed by contacting a specimen with a reagent containing an immunoglobulin antibody or an antigen-binding fragment thereof.
(1) Nonionic surfactant
(2) Amphoteric surfactant
HBc抗原又は抗免疫グロブリン抗体若しくはその抗原結合性断片を含む試薬(以下、便宜的に「抗原/抗体試薬」と呼ぶことがある)は、例えば、HBc抗原を含む試薬であってよく、この場合、上記の抗原抗体反応は、検体とHBc抗原を含む試薬との反応(検体中の抗HBc抗体とHBc抗原との反応)である。 The reagent containing the HBc antigen or anti-immunoglobulin antibody or antigen-binding fragment thereof (hereinafter sometimes referred to as “antigen / antibody reagent” for convenience) may be, for example, a reagent containing the HBc antigen. The antigen-antibody reaction described above is a reaction between a specimen and a reagent containing an HBc antigen (a reaction between an anti-HBc antibody in the specimen and an HBc antigen).
界面活性剤は、例えば、検体希釈液に添加されていてよい。すなわち、この態様では、上記少なくとも1種の界面活性剤を含む溶液が用いられ、検体と、当該溶液と、抗原/抗体試薬とを混合して抗原抗体反応を行なうことにより、界面活性剤共存下で抗原抗体反応が行われる(態様1)。抗原/抗体試薬には上記界面活性剤が含まれていなくてよい。抗原/抗体試薬にも上記界面活性剤が含まれる場合、下記の態様3に該当する。 The surfactant may be added to the specimen diluent, for example. That is, in this embodiment, a solution containing at least one surfactant is used, and an antigen-antibody reaction is performed by mixing a specimen, the solution, and an antigen / antibody reagent, thereby coexisting with the surfactant. The antigen-antibody reaction is carried out in (Aspect 1). The antigen / antibody reagent may not contain the surfactant. When the surfactant is also contained in the antigen / antibody reagent, it corresponds to the following embodiment 3.
界面活性剤は、抗原/抗体試薬に含まれていてもよい。すなわち、1つの態様において、検体と、上記少なくとも1種の界面活性剤を含む抗原/抗体試薬とを混合して抗原抗体反応を行なうことにより、界面活性剤共存下で抗原抗体反応が行われる(態様2)。検体希釈液が使用される場合、検体希釈液には上記界面活性剤が含まれていなくてよい。検体希釈液にも上記界面活性剤が含まれる場合、下記の態様3に該当する。 A surfactant may be included in the antigen / antibody reagent. That is, in one embodiment, an antigen-antibody reaction is carried out in the presence of a surfactant by mixing an analyte and an antigen / antibody reagent containing at least one surfactant as described above to perform an antigen-antibody reaction ( Aspect 2). When the sample diluent is used, the sample diluent may not contain the surfactant. When the above-mentioned surfactant is also contained in the specimen diluent, it corresponds to the following aspect 3.
界面活性剤は、抗原/抗体試薬と検体希釈液の両方に含まれていてもよい。すなわち、1つの態様において、検体と、上記少なくとも1種の界面活性剤を含む溶液と、上記少なくとも1種の界面活性剤を含む抗原/抗体試薬とを混合して抗原抗体反応を行なうことにより、界面活性剤共存下で抗原抗体反応が行われる(態様3)。 A surfactant may be included in both the antigen / antibody reagent and the sample diluent. That is, in one embodiment, by performing an antigen-antibody reaction by mixing a specimen, a solution containing the at least one surfactant, and an antigen / antibody reagent containing the at least one surfactant, Antigen-antibody reaction is performed in the presence of a surfactant (Aspect 3).
検体、検体希釈液、及び抗原/抗体試薬を混合する順番は限定されない。検体希釈液と検体を先に混合した後、希釈後検体の少なくとも一部を抗原/抗体試薬に添加してもよいし、検体希釈液と検体を別個に抗原/抗体試薬に添加してもよい。別個に添加する場合、検体希釈液と検体を同時に添加してもよいし、順次に添加してもよい。順次に添加する場合、どちらを先に抗原/抗体試薬に添加してもよい。検体希釈液を先に抗原/抗体試薬に添加した後、検体を添加することが一般的であり得るが、逆でも差し支えない。本明細書では便宜的に「検体希釈液」という語を用いているが、検体希釈液と検体を先に混合してから上記試薬に添加する態様に本発明の範囲を限定することを意図するものではない。 The order of mixing the specimen, the specimen diluent, and the antigen / antibody reagent is not limited. After first mixing the sample diluent and the sample, at least a part of the diluted sample may be added to the antigen / antibody reagent, or the sample diluent and the sample may be added separately to the antigen / antibody reagent. . When added separately, the sample diluent and the sample may be added simultaneously or sequentially. When adding sequentially, either may be added to the antigen / antibody reagent first. It is common to first add the specimen diluent to the antigen / antibody reagent and then add the specimen, but the reverse is also possible. In this specification, the term “specimen diluent” is used for convenience, but it is intended to limit the scope of the present invention to an embodiment in which the sample diluent and the specimen are first mixed and then added to the reagent. It is not a thing.
(1)の非イオン性界面活性剤としては、エーテル型又はエステルエーテル型の非イオン性界面活性剤を挙げることができる。HLB(Hydrophilic-Lipophilic Balance)値が10〜20、例えば11〜20、12〜19、又は17〜19の非イオン性界面活性剤を好ましく用いることができる。 Examples of the nonionic surfactant (1) include ether type or ester ether type nonionic surfactants. A nonionic surfactant having an HLB (Hydrophilic-Lipophilic Balance) value of 10 to 20, for example, 11 to 20, 12 to 19, or 17 to 19 can be preferably used.
エーテル型の非イオン性界面活性剤の好ましい例としては、ポリオキシエチレンアルキルフェニルエーテル系の界面活性剤、及びポリオキシエチレンアルキルエーテル系の界面活性剤を挙げることができる。 Preferable examples of the ether type nonionic surfactant include polyoxyethylene alkylphenyl ether surfactants and polyoxyethylene alkyl ether surfactants.
ポリオキシエチレンアルキルフェニルエーテル系界面活性剤の好ましい例としては、Triton(登録商標)の商品名で知られる各種の界面活性剤のうちでHLB値が上記範囲のもの、特にHLB値が17〜19のものを挙げることができる。具体例として、ポリオキシエチレン(40)オクチルフェニルエーテル(商品名Triton X-405、HLB=17.9)、ポリオキシエチレン(30)オクチルフェニルエーテル(商品名Triton X-305、HLB=17.3)、ポリオキシエチレン(8)オクチルフェニルエーテル(商品名Triton X-114、HLB=12.4)、ポリオキシエチレン(10)オクチルフェニルエーテル(商品名Triton X-100、HLB=13.5)、さらには、Triton X-705の商品名で知られるHLB=18.5のポリオキシエチレンアルキルフェニルエーテル系界面活性剤が挙げられるが、これらに限定されない。 Preferable examples of the polyoxyethylene alkylphenyl ether surfactant include various surfactants known under the trade name of Triton (registered trademark) having an HLB value in the above range, particularly an HLB value of 17 to 19 Can be mentioned. Specific examples include polyoxyethylene (40) octylphenyl ether (trade name Triton X-405, HLB = 17.9), polyoxyethylene (30) octylphenyl ether (trade name Triton X-305, HLB = 17.3), polyoxy Ethylene (8) octylphenyl ether (trade name Triton X-114, HLB = 12.4), polyoxyethylene (10) octylphenyl ether (trade names Triton X-100, HLB = 13.5), and Triton X-705 Examples thereof include, but are not limited to, polyoxyethylene alkylphenyl ether surfactants having an HLB of 18.5 known by trade names.
ポリオキシエチレンアルキルエーテル系界面活性剤の好ましい例としては、Brij(登録商標)の商品名で知られる各種の界面活性剤のうちでHLB値が上記範囲のものを挙げることができる。具体例として、ポリオキシエチレン(10)セチルエーテル(商品名Brij C10、HLB=13.0)、ポリオキシエチレン(20)セチルエーテル(商品名Brij 58、HLB=15.7)を挙げることができるが、これらに限定されない。 Preferable examples of the polyoxyethylene alkyl ether surfactant include those having an HLB value in the above range among various surfactants known under the trade name of Brij (registered trademark). Specific examples include polyoxyethylene (10) cetyl ether (trade name Brij C10, HLB = 13.0) and polyoxyethylene (20) cetyl ether (trade names Brij 58, HLB = 15.7). It is not limited.
エステルエーテル型の非イオン性界面活性剤の好ましい例としては、ポリオキシエチレンソルビタン脂肪酸エステル系の界面活性剤を挙げることができる。該界面活性剤の好ましい例としては、Tween(登録商標)の商品名で知られる各種の界面活性剤のうちでHLB値が上記範囲のものを挙げることができる。具体例として、ポリオキシエチレンソルビタンモノラウレート(商品名Tween 20、HLB=16.7)、ポリオキシエチレンソルビタンモノパルミタート(商品名Tween 40、HLB=15.6)、ポリオキシエチレンソルビタンモノステアラート(商品名Tween 60、HLB=14.9)、ポリオキシエチレンソルビタンモノオレエート(商品名Tween 80、HLB=15.0)を挙げることができるが、これらに限定されない。 Preferable examples of the ester ether type nonionic surfactant include polyoxyethylene sorbitan fatty acid ester surfactants. Preferable examples of the surfactant include those having an HLB value in the above range among various surfactants known under the trade name of Tween (registered trademark). Specific examples include polyoxyethylene sorbitan monolaurate (trade name Tween 20, HLB = 16.7), polyoxyethylene sorbitan monopalmitate (trade name Tween 40, HLB = 15.6), polyoxyethylene sorbitan monostearate (trade name) Examples include, but are not limited to, Tween 60, HLB = 14.9) and polyoxyethylene sorbitan monooleate (trade names Tween 80, HLB = 15.0).
(2)の両性界面活性剤としては、スルホベタイン型界面活性剤を好ましく用いることができる。中でも炭素数12以上の直鎖アルキル基を有するスルホベタイン型界面活性剤が好ましい。具体例として、N-ドデシル-N,N-ジメチル-3-アンモニオ-1-プロパンスルホネート(C12APS)、N-テトラデシル-N,N-ジメチル-3-アンモニオ-1-プロパンスルホネート(C14APS)、N-ヘキサデシル-N,N-ジメチル-3-アンモニオ-1-プロパンスルホネート(C16APS)などが挙げられるが、これらに限定されない。 As the amphoteric surfactant (2), a sulfobetaine surfactant can be preferably used. Of these, sulfobetaine surfactants having a linear alkyl group having 12 or more carbon atoms are preferred. Specific examples include N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (C12APS), N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (C14APS), N- Examples include, but are not limited to, hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (C16APS).
検体希釈液のベースとなる液体は特に限定されず、水性の液体であればよい。ヒトまたは動物の血液に由来する成分を含んでいる液体でもよい。例えば滅菌水、pH4.0〜11.0までの緩衝能を有するバッファー、血清を用いることができる。態様1及び態様3では、このような水性の液体に上記した所定の界面活性剤を所定濃度で含有する液体を検体希釈液として用いればよい。態様2では、このような水性の液体をそのまま検体希釈液として用いればよい。 The liquid used as the base of the specimen dilution liquid is not particularly limited as long as it is an aqueous liquid. It may be a liquid containing components derived from human or animal blood. For example, sterilized water, a buffer having a buffer capacity of pH 4.0 to 11.0, and serum can be used. In Aspect 1 and Aspect 3, a liquid containing such a predetermined surfactant in a predetermined concentration in such an aqueous liquid may be used as the specimen dilution liquid. In the aspect 2, such an aqueous liquid may be used as it is as the sample diluent.
上記した抗原抗体反応系内の上記界面活性剤の濃度(上記界面活性剤の2種以上を用いる場合には合計の濃度)は、好ましくは0.040 w/v%〜1.27 w/v%であり、例えば0.040 w/v%〜1.0 w/v%、0.040 w/v%〜0.80 w/v%、0.040 w/v%〜0.50 w/v%、又は0.040 w/v%〜0.40 w/v%であってよい。0.40 w/v%程度以下で用いた場合には、非特異反応を良好に抑制しつつ、陽性検体の反応性を特に高く維持することができる。 The concentration of the surfactant in the antigen-antibody reaction system (the total concentration when two or more of the surfactants are used) is preferably 0.040 w / v% to 1.27 w / v%, For example, 0.040 w / v% to 1.0 w / v%, 0.040 w / v% to 0.80 w / v%, 0.040 w / v% to 0.50 w / v%, or 0.040 w / v% to 0.40 w / v% It may be. When used at about 0.40 w / v% or less, the reactivity of positive specimens can be maintained particularly high while suppressing nonspecific reactions well.
従って、検体希釈液や抗原/抗体試薬は、上記した抗原抗体反応系内の界面活性剤濃度が0.040 w/v%〜1.27 w/v%となるように上記の界面活性剤を含んでいることが好ましい。下記実施例のように、検体10μLと検体希釈液190μLを混合し、そのうち10μLをHBc抗原試薬50μLと混合して一次反応を行なう免疫測定系を想定すると、一次反応系内の界面活性剤濃度が0.040 w/v%〜1.27 w/v%の場合、態様1における検体希釈液中の界面活性剤濃度は0.25 w/v%〜8.02 w/v%であり、態様2における抗原/抗体試薬中の界面活性剤濃度は0.048 w/v%〜1.52 w/v%であり、態様3では、一次反応系内の界面活性剤が0.040 w/v%〜1.27 w/v%となるように、検体希釈液及び抗原/抗体試薬の界面活性剤濃度を調整すればよい。 Therefore, the sample diluent and the antigen / antibody reagent should contain the above surfactant so that the surfactant concentration in the above antigen-antibody reaction system is 0.040 w / v% to 1.27 w / v%. Is preferred. As in the following examples, assuming an immunoassay system in which 10 μL of a sample is mixed with 190 μL of a sample diluent, 10 μL of which is mixed with 50 μL of HBc antigen reagent to perform a primary reaction, the surfactant concentration in the primary reaction system is In the case of 0.040 w / v% to 1.27 w / v%, the concentration of the surfactant in the specimen diluent in Embodiment 1 is 0.25 w / v% to 8.02 w / v%, and in the antigen / antibody reagent in Embodiment 2 The surfactant concentration is 0.048 w / v% to 1.52 w / v%, and in Embodiment 3, the sample dilution is performed so that the surfactant in the primary reaction system is 0.040 w / v% to 1.27 w / v%. The surfactant concentration of the solution and the antigen / antibody reagent may be adjusted.
検体の使用量を増やす場合は、一次反応系内の界面活性剤濃度が0.04 w/v%〜1.27 w/v%となるように、検体希釈液や抗原/抗体試薬の界面活性剤濃度を調整することが好ましい。検体の使用量を約10倍にした場合、例えば、検体10μLと検体希釈液190μLを混合し、希釈検体100μLをHBc抗原試薬50μLと混合して一次反応を行なう場合には、態様1における検体希釈液の界面活性剤濃度は0.063 w/v%〜2.01 w/v%、態様2における抗原/抗体試薬の界面活性剤濃度は0.12 w/v%〜3.81 w/v%とすればよく、態様3では、一次反応系内の界面活性剤が0.040 w/v%〜1.27 w/v%となるように、検体希釈液及び抗原/抗体試薬の界面活性剤濃度を調整すればよい。あるいは、検体100μLと検体希釈液100μLを混合し、そのうち10μLをHBc抗原試薬50μLと混合して一次反応を行なう場合には、態様1における検体希釈液中の界面活性剤濃度は0.48 w/v%〜15.2 w/v%、態様2における抗原/抗体試薬の界面活性剤濃度は0.048 w/v%〜1.52 w/v%とすればよく、態様3では、一次反応系内の界面活性剤が0.040 w/v%〜1.27 w/v%となるように、検体希釈液及び抗原/抗体試薬の界面活性剤濃度を調整すればよい。 When increasing the amount of sample used, adjust the surfactant concentration in the sample diluent and antigen / antibody reagent so that the surfactant concentration in the primary reaction system is 0.04 w / v% to 1.27 w / v%. It is preferable to do. When the amount of the sample used is about 10 times, for example, when 10 μL of the sample is mixed with 190 μL of the sample diluent, and 100 μL of the diluted sample is mixed with 50 μL of the HBc antigen reagent, the primary dilution is performed. The surfactant concentration of the solution may be 0.063 w / v% to 2.01 w / v%, and the surfactant concentration of the antigen / antibody reagent in embodiment 2 may be 0.12 w / v% to 3.81 w / v%. Then, the surfactant concentration of the sample diluent and the antigen / antibody reagent may be adjusted so that the surfactant in the primary reaction system is 0.040 w / v% to 1.27 w / v%. Alternatively, when 100 μL of the sample and 100 μL of the sample diluent are mixed and 10 μL of the sample is mixed with 50 μL of the HBc antigen reagent and the primary reaction is performed, the surfactant concentration in the sample diluent in Embodiment 1 is 0.48 w / v%. -15.2 w / v%, and the surfactant concentration of the antigen / antibody reagent in embodiment 2 may be 0.048 w / v%-1.52 w / v%. In embodiment 3, the surfactant in the primary reaction system is 0.040 w / v%. The concentration of the surfactant in the sample diluent and the antigen / antibody reagent may be adjusted so as to be w / v% to 1.27 w / v%.
本発明では、界面活性剤に組み合わせて塩を用いてもよい。例えば、検体希釈液が塩をさらに含んでいてもよい。また、抗原/抗体試薬は通常、100〜150mM程度の塩を含んでいるが、さらに高濃度の塩を含んでいてもよい。上記した所定の界面活性剤に塩を組み合わせることで、さらに高い非特異反応低減効果を得ることができる。塩は特に限定されず、無機塩でも有機塩でもよい。例えば、塩化ナトリウム、塩化カリウムなどのアルカリ金属塩を好ましく用いることができるが、これらに限定されない。複数種類の塩を組み合わせて用いても良い。 In the present invention, a salt may be used in combination with the surfactant. For example, the specimen diluent may further contain a salt. Further, the antigen / antibody reagent usually contains a salt of about 100 to 150 mM, but may contain a higher concentration of salt. A higher nonspecific reaction reducing effect can be obtained by combining a salt with the above-described predetermined surfactant. The salt is not particularly limited, and may be an inorganic salt or an organic salt. For example, alkali metal salts such as sodium chloride and potassium chloride can be preferably used, but are not limited thereto. A plurality of types of salts may be used in combination.
検体希釈液に塩を添加する場合、検体希釈液中の塩濃度(複数の塩を用いる場合は合計の濃度)は200 mM以上であればよく、例えば250 mM以上、300 mM以上、350 mM以上、400mM以上、500mM以上又は800 mM以上とすることができる。上限は特に限定されないが、通常は2000 mM程度以下であり、例えば1800 mM以下、又は1500 mM以下としてもよい。 When adding salt to the sample diluent, the salt concentration in the sample diluent (total concentration when multiple salts are used) may be 200 mM or more, for example, 250 mM or more, 300 mM or more, 350 mM or more 400 mM or more, 500 mM or more, or 800 mM or more. The upper limit is not particularly limited, but is usually about 2000 mM or less, for example, 1800 mM or less, or 1500 mM or less.
全血、血清、血漿などの血液由来の検体を用いる場合、検体由来の塩が一次反応液中に含まれる。血液由来の検体の場合、一般的に約150mM相当の塩を含む。下記実施例のように、血清検体10μLと検体希釈液190μLを混合し、そのうち10μLを塩濃度150mMのHBc抗原試薬50μLと混合して一次反応を行なう免疫測定系を想定すると、検体希釈液中の塩濃度が200mM〜2000mMの場合、一次反応系内の塩濃度は158mM〜443mMとなる。従って、本発明において塩を使用する場合の塩濃度を一次反応系内の濃度として表現すると、158mM〜443mMであってよく、例えば170mM〜443mM、180mM〜443mM、190mM〜443mM、又は200mM〜443mMであり得る。また、一次反応系内の塩濃度は、上記の範囲内で、例えば420mM以下、400mM以下、又は350mM以下としてもよい。また、本発明では、抗原/抗体試薬液の塩濃度を高めることによって、上記した一次反応系内の塩濃度を達成してもよい。つまり、本発明では、非特異反応低減のために、検体希釈液及び抗原/抗体試薬液のいずれか一方又は両者が、抗原抗体反応(一次反応)の反応系内の塩濃度が158 mM以上となるように塩を含んでいてもよい。 When using a blood-derived sample such as whole blood, serum, or plasma, a salt derived from the sample is contained in the primary reaction solution. In the case of a blood-derived specimen, it generally contains about 150 mM of salt. As in the examples below, assuming an immunoassay system in which 10 μL of serum sample and 190 μL of sample diluent are mixed, 10 μL of this is mixed with 50 μL of HBc antigen reagent with a salt concentration of 150 mM, and a primary reaction is performed. When the salt concentration is 200 mM to 2000 mM, the salt concentration in the primary reaction system is 158 mM to 443 mM. Therefore, when the salt concentration in the case of using the salt in the present invention is expressed as the concentration in the primary reaction system, it may be 158 mM to 443 mM, for example, 170 mM to 443 mM, 180 mM to 443 mM, 190 mM to 443 mM, or 200 mM to 443 mM. possible. The salt concentration in the primary reaction system may be, for example, 420 mM or less, 400 mM or less, or 350 mM or less within the above range. In the present invention, the salt concentration in the primary reaction system may be achieved by increasing the salt concentration of the antigen / antibody reagent solution. That is, in the present invention, in order to reduce the nonspecific reaction, either one or both of the sample diluent and the antigen / antibody reagent solution have a salt concentration of 158 mM or more in the reaction system of the antigen-antibody reaction (primary reaction). It may contain a salt.
本発明では、免疫測定の全てのステップに上記所定の界面活性剤を共存させる必要はない。例えば、後述するように2ステップのサンドイッチ免疫測定により抗HBc抗体の測定を行なう場合には、一次反応系内に界面活性剤が共存することになるが、B/F分離及び固相担体洗浄のステップで界面活性剤が反応系から除去される。それ以降の工程では、界面活性剤を反応系内に添加することは必須ではなく、添加してもしなくてもよい。例えば、第二の抗原抗体反応である二次反応は、上記した界面活性剤の存在下で実施してもよいし、非存在下で実施してもよい。 In the present invention, it is not necessary to allow the predetermined surfactant to coexist in all steps of immunoassay. For example, as described later, when anti-HBc antibody is measured by a two-step sandwich immunoassay, surfactants coexist in the primary reaction system, but B / F separation and solid phase carrier washing are performed. In step, the surfactant is removed from the reaction system. In the subsequent steps, it is not essential to add the surfactant into the reaction system, and it may or may not be added. For example, the secondary reaction that is the second antigen-antibody reaction may be performed in the presence or absence of the surfactant described above.
なお、免疫測定の分野で周知の通り、2ステップサンドイッチ法における一次反応とは、固相担体上に結合させた抗原又は抗体と検体との反応であり、二次反応とは、B/F分離及び洗浄後の固相と標識抗体又は標識抗原との反応である。本発明においては、HBc抗原を固相化する場合、担体上のHBc抗原と希釈検体との反応が一次反応、B/F分離及び洗浄後の固相と標識抗免疫グロブリン抗体との反応が二次反応であり、抗免疫グロブリン抗体を固相化する場合、担体上の抗免疫グロブリン抗体と希釈検体との反応が一次反応、B/F分離及び洗浄後の固相と標識HBc抗原との反応が二次反応である。 As is well known in the field of immunoassay, the primary reaction in the two-step sandwich method is a reaction between an antigen or antibody bound on a solid phase carrier and a specimen, and the secondary reaction is B / F separation. And a reaction between the solid phase after washing and the labeled antibody or labeled antigen. In the present invention, when the HBc antigen is immobilized, the reaction between the HBc antigen on the carrier and the diluted specimen is a primary reaction, and the reaction between the solid phase after B / F separation and washing and the labeled anti-immunoglobulin antibody is second. When the anti-immunoglobulin antibody is immobilized, the reaction between the anti-immunoglobulin antibody on the carrier and the diluted specimen is the primary reaction, and the reaction between the solid phase after B / F separation and washing and the labeled HBc antigen. Is a secondary reaction.
本発明で用いられる検体としては、全血、血漿、血清、尿、唾、脳脊髄液などの生物学的体液、および肝組織などが挙げられ、特に、全血、血清、血漿サンプルが好ましいが、これらに限定されない。 Examples of the specimen used in the present invention include whole body blood, plasma, serum, urine, saliva, cerebrospinal fluid and other biological body fluids, liver tissue and the like, and particularly, whole blood, serum and plasma samples are preferable. However, it is not limited to these.
被検個体は、哺乳動物であれば特に限定されないが、好ましくはヒトである。被検個体は、B型肝炎の疑いのある患者、又は肝炎(特にB型肝炎)を発症している患者であり得る。 The test individual is not particularly limited as long as it is a mammal, but is preferably a human. The test individual may be a patient suspected of having hepatitis B or a patient who has developed hepatitis (particularly hepatitis B).
測定対象の抗HBc抗体には、IgG型抗体とIgM型抗体が包含される。本発明においては、IgG型抗体及びIgM型抗体のいずれか一方(例えばIgG型抗体)のみを測定してもよいし、両者を測定してもよい。典型的には、測定対象の抗HBc抗体は、IgG型抗体を含む。 The anti-HBc antibodies to be measured include IgG type antibodies and IgM type antibodies. In the present invention, either one of IgG type antibody and IgM type antibody (for example, IgG type antibody) may be measured, or both may be measured. Typically, the anti-HBc antibody to be measured includes an IgG type antibody.
抗体の測定方法自体は周知の常法である。免疫測定を反応様式で分類すると、サンドイッチ法、競合法、凝集法、イムノクロマト法、ウエスタンブロット法等があり、標識で分類すると、放射免疫測定、蛍光免疫測定、酵素免疫測定(EIA)、ビオチン免疫測定等がある。また、抗原を用いた抗体検査法の具体例としては、これらに限定されないが、EIA法(ELISA、CLEIA(化学発光酵素免疫測定法)、ウエスタンブロット等)、化学発光免疫測定法(CLIA法)、凝集法(ラテックス凝集法等)、補体結合反応(CF)等が挙げられる。本発明においては、検体中の抗HBc抗体を特異的に検出できる免疫測定法であれば、いかなる手法を用いてもよい。特に限定されないが、本発明における免疫測定は、サンドイッチ法、特に2ステップサンドイッチ法による免疫測定であり得る。なお、本発明において、「測定」という語には、検出、定量及び半定量が包含される。 The antibody measurement method itself is a well-known conventional method. The immunoassay can be classified by reaction mode, such as sandwich method, competition method, aggregation method, immunochromatography method, Western blot method, etc. When classified by label, radioimmunoassay, fluorescent immunoassay, enzyme immunoassay (EIA), biotin immunity There are measurements. Specific examples of antibody testing methods using antigens include, but are not limited to, EIA methods (ELISA, CLEIA (chemiluminescent enzyme immunoassay), Western blot, etc.), chemiluminescent immunoassays (CLIA method) , Aggregation method (latex aggregation method, etc.), complement binding reaction (CF) and the like. In the present invention, any technique may be used as long as it is an immunoassay that can specifically detect an anti-HBc antibody in a specimen. Although not particularly limited, the immunoassay in the present invention can be an immunoassay by a sandwich method, particularly a two-step sandwich method. In the present invention, the term “measurement” includes detection, quantification, and semi-quantification.
以下、2ステップサンドイッチ法に基づくCLEIA法を例に、本発明の方法でヒト由来検体中の抗HBc抗体を測定する場合の各工程をより具体的に説明する。 Hereinafter, taking the CLEIA method based on the two-step sandwich method as an example, each step in the case of measuring an anti-HBc antibody in a human-derived specimen by the method of the present invention will be described more specifically.
まず、検体希釈液でヒト由来検体を希釈した後、固相担体上に固定化されたHBc抗原と希釈検体を接触させるか、あるいは、検体希釈液及びヒト由来検体を別個に順次にHBc抗原と接触させる(一般的には検体希釈液を先に接触させるが、検体が先でも良い)ことにより、担体上のHBc抗原と希釈検体中の抗HBc抗体とを反応させる(一次反応)。この一次反応により、検体中の抗HBc抗体がHBc抗原を介して担体上に結合する。態様1では検体希釈液中に、態様2では固相化HBc抗原を含む試薬中に、態様3では検体希釈液と固相化HBc抗原試薬の両者に、界面活性剤が含まれており、一次反応は界面活性剤の共存下で進行する。反応後、B/F分離を行ない、分離した担体を洗浄する。このB/F分離及び洗浄ステップにより、検体希釈液又は固相化HBc抗原試薬に含まれていた上記所定の界面活性剤は反応系から除去される。 First, after diluting the human-derived sample with the sample diluent, the HBc antigen immobilized on the solid support is brought into contact with the diluted sample, or the sample diluent and the human-derived sample are separately and sequentially separated from the HBc antigen. By contacting (generally, the sample dilution solution is contacted first, but the sample may be first), the HBc antigen on the carrier is reacted with the anti-HBc antibody in the diluted sample (primary reaction). By this primary reaction, the anti-HBc antibody in the specimen is bound on the carrier via the HBc antigen. In aspect 1, the specimen diluent contains a surfactant, in aspect 2, the reagent containing the immobilized HBc antigen, and in aspect 3, both the specimen diluent and the immobilized HBc antigen reagent contain a surfactant. The reaction proceeds in the presence of a surfactant. After the reaction, B / F separation is performed and the separated carrier is washed. By the B / F separation and washing step, the predetermined surfactant contained in the specimen diluent or the immobilized HBc antigen reagent is removed from the reaction system.
次いで、標識された抗ヒト免疫グロブリン抗体を系内に添加し、HBc抗原に結合した抗HBc抗体と、標識された抗ヒト免疫グロブリン抗体を反応させる(二次反応)。この二次反応により、固相担体上にHBc抗原−抗HBc抗体−標識抗ヒト免疫グロブリン抗体の複合体が形成される。次いで、B/F分離を行い、担体を洗浄することで、未反応の標識抗ヒト免疫グロブリン抗体を除去する。未反応の標識抗ヒト免疫グロブリン抗体を除去した後、HBc抗原タンパク質及び抗HBc抗体を介して担体に結合した標識抗ヒト免疫グロブリン抗体からのシグナルを適当な方法で検出することにより、検体中に含まれる抗HBc抗体を特異的に測定することが可能となる。 Next, a labeled anti-human immunoglobulin antibody is added to the system, and the anti-HBc antibody bound to the HBc antigen is reacted with the labeled anti-human immunoglobulin antibody (secondary reaction). By this secondary reaction, a complex of HBc antigen-anti-HBc antibody-labeled anti-human immunoglobulin antibody is formed on the solid phase carrier. Subsequently, B / F separation is performed, and the carrier is washed to remove unreacted labeled anti-human immunoglobulin antibody. After removing the unreacted labeled anti-human immunoglobulin antibody, the signal from the labeled anti-human immunoglobulin antibody bound to the carrier via the HBc antigen protein and the anti-HBc antibody is detected by an appropriate method in the sample. It becomes possible to specifically measure the contained anti-HBc antibody.
二次反応ステップにおいて、標識抗ヒト免疫グロブリン抗体として、標識抗ヒトIgG抗体を用いれば、IgG型の抗HBc抗体を測定することができ、標識抗ヒトIgM抗体を用いれば、IgM型の抗HBc抗体を測定することができる。IgG型抗HBc抗体とIgM型抗HBc抗体の両者を測定したい場合には、標識抗ヒトIgG抗体と標識抗ヒトIgM抗体の両者を用いればよい。両者を区別する必要がなければ、同じ標識物質を結合させた標識抗ヒトIgG抗体と標識抗ヒトIgM抗体を混合して二次反応を行えばよい。IgG型抗HBc抗体とIgM型抗HBc抗体を区別して測定したい場合には、異なる標識物質を結合させた標識抗ヒトIgG抗体及び標識抗ヒトIgM抗体を同時に用いて二次反応を行ない、後述する検出工程で両者を区別して検出するか、あるいは、二次反応ステップを別々に実施すればよい。また、後述する通り、抗ヒト免疫グロブリン抗体に代えて抗原結合性断片を用いてもよい。 In the secondary reaction step, if a labeled anti-human IgG antibody is used as a labeled anti-human immunoglobulin antibody, an IgG-type anti-HBc antibody can be measured, and if a labeled anti-human IgM antibody is used, an IgM-type anti-HBc antibody can be measured. Antibodies can be measured. When it is desired to measure both IgG anti-HBc antibody and IgM anti-HBc antibody, both labeled anti-human IgG antibody and labeled anti-human IgM antibody may be used. If it is not necessary to distinguish between the two, a labeled anti-human IgG antibody bound with the same labeling substance and a labeled anti-human IgM antibody may be mixed and subjected to a secondary reaction. If you want to distinguish between IgG-type anti-HBc antibody and IgM-type anti-HBc antibody, perform a secondary reaction using a labeled anti-human IgG antibody and labeled anti-human IgM antibody combined with different labeling substances at the same time, as described later. The detection process may be performed by distinguishing both, or the secondary reaction step may be performed separately. Further, as described later, an antigen-binding fragment may be used in place of the anti-human immunoglobulin antibody.
以上、2ステップ法を例に説明したが、1ステップ法で免疫測定を実施してもよい。また、固相担体に抗ヒト免疫グロブリン抗体を結合させ、標識されたHBc抗原タンパク質を用いて測定を行なうこともできる。この場合、固相抗体と希釈検体との反応が一次反応であり、洗浄後の固相と標識HBc抗原との反応が二次反応である。また、上述したように本発明の免疫測定法はサンドイッチ法に限定されず、競合法によって抗HBc抗体を検出してもよい。 Although the two-step method has been described above as an example, immunoassay may be performed by the one-step method. Alternatively, an anti-human immunoglobulin antibody can be bound to a solid support and measurement can be performed using a labeled HBc antigen protein. In this case, the reaction between the solid phase antibody and the diluted specimen is a primary reaction, and the reaction between the washed solid phase and the labeled HBc antigen is a secondary reaction. Further, as described above, the immunoassay method of the present invention is not limited to the sandwich method, and the anti-HBc antibody may be detected by a competition method.
抗HBc抗体の測定に使用するHBc抗原としては、抗HBc抗体と特異的に結合するものであればいかなるものであってもよい。市販のリコンビナントHBc抗原を用いることができるし、また、周知の遺伝子工学的手法によりリコンビナントHBc抗原を調製して用いることもできる。B型肝炎ウイルス感染細胞からHBc抗原を抽出して得ることもできる。 The HBc antigen used for measurement of the anti-HBc antibody may be any as long as it specifically binds to the anti-HBc antibody. Commercially available recombinant HBc antigens can be used, and recombinant HBc antigens can also be prepared and used by well-known genetic engineering techniques. It can also be obtained by extracting HBc antigen from hepatitis B virus-infected cells.
HBcタンパク質のアミノ酸配列およびこれをコードするヌクレオチド配列は、NCBIのGenBank等のデータベースから入手することができるので、当業者であれば、周知の遺伝子工学的手法を用いてリコンビナントHBc抗原を容易に調製することができる。配列表の配列番号1および2に示した配列は、公知のHBc遺伝子およびこれにコードされるHBcタンパク質の配列の一例であり、GenBankにaccession No. AF324125.1で登録されている配列である。これまでに公知となっているHBcタンパク質のアミノ酸配列の大部分は、配列番号2のアミノ酸と95%程度以上の同一性を有するか、あるいは1〜数個のアミノ酸が置換、欠失、挿入及び/又は付加されたアミノ酸配列を有しており、そのようなアミノ酸配列のHBcタンパク質であれば抗HBc抗体測定のためのHBc抗原として好ましく使用可能である。もっとも、HBc抗原の配列はこれに限定されるものではなく、配列番号2のアミノ酸配列と同一性が95%未満のものや、N末端あるいはC末端にタグ配列等の余分なアミノ酸配列が付加されたものであっても、HBVに感染した個体の体内で誘導される抗HBc抗体との反応性を有する限り、HBc抗原として使用可能である。本発明において、「HBc抗原」といった場合には、上記した各種のHBcタンパク質が包含される。 The amino acid sequence of the HBc protein and the nucleotide sequence that encodes it can be obtained from databases such as NCBI's GenBank. Therefore, those skilled in the art can easily prepare recombinant HBc antigens using well-known genetic engineering techniques. can do. The sequences shown in SEQ ID Nos. 1 and 2 in the sequence listing are examples of known HBc genes and HBc protein sequences encoded thereby, and are registered in GenBank with accession No. AF324125.1. Most of the amino acid sequences of HBc proteins known so far have about 95% or more identity with the amino acid of SEQ ID NO: 2, or 1 to several amino acids are substituted, deleted, inserted and An HBc protein having an added amino acid sequence and having such an amino acid sequence can be preferably used as an HBc antigen for anti-HBc antibody measurement. However, the sequence of the HBc antigen is not limited to this, and the amino acid sequence of SEQ ID NO: 2 is less than 95% identical or an extra amino acid sequence such as a tag sequence is added to the N-terminal or C-terminal. Can be used as an HBc antigen as long as it has reactivity with an anti-HBc antibody induced in the body of an individual infected with HBV. In the present invention, “HBc antigen” includes the various HBc proteins described above.
なお、アミノ酸配列の同一性とは、比較すべき2つのアミノ酸配列のアミノ酸残基ができるだけ多く一致するように両アミノ酸配列を整列させ、一致したアミノ酸残基数を全アミノ酸残基数で除したものを百分率で表したものである。上記整列の際には、必要に応じ、比較する2つの配列の一方又は双方に適宜ギャップを挿入する。このような配列の整列化は、例えばBLAST、FASTA、CLUSTAL W等の周知のプログラムを用いて行なうことができる。ギャップが挿入される場合、上記全アミノ酸残基数は、1つのギャップを1つのアミノ酸残基として数えた残基数となる。このようにして数えた全アミノ酸残基数が、比較する2つの配列間で異なる場合には、同一性(%)は、長い方の配列の全アミノ酸残基数で、一致したアミノ酸残基数を除して算出される。ただし、比較すべき配列が他の任意の配列(例えばタグ配列、リンカー配列、他のタンパク質の配列等)と連結された状態にある場合には、そのような任意の配列は除外して同一性が算出される。 The amino acid sequence identity means that both amino acid sequences are aligned so that the amino acid residues of the two amino acid sequences to be compared match as much as possible, and the number of matching amino acid residues is divided by the total number of amino acid residues. The thing is expressed as a percentage. In the above alignment, a gap is appropriately inserted in one or both of the two sequences to be compared as necessary. Such sequence alignment can be performed using a known program such as BLAST, FASTA, CLUSTAL W, and the like. When gaps are inserted, the total number of amino acid residues is the number of residues obtained by counting one gap as one amino acid residue. When the total number of amino acid residues counted in this way is different between the two sequences to be compared, the identity (%) is the total number of amino acid residues in the longer sequence, and the number of amino acid residues matched. Is calculated by dividing. However, if the sequence to be compared is linked to any other sequence (for example, a tag sequence, a linker sequence, another protein sequence, etc.), such an arbitrary sequence is excluded and the identity is excluded. Is calculated.
天然のタンパク質を構成する20種類のアミノ酸は、低極性側鎖を有する中性アミノ酸(Gly, Ile, Val, Leu, Ala, Met, Pro)、親水性側鎖を有する中性アミノ酸(Asn, Gln, Thr, Ser, Tyr, Cys)、酸性アミノ酸(Asp, Glu)、塩基性アミノ酸(Arg, Lys, His)、芳香族アミノ酸(Phe, Tyr, Trp)のように類似の性質を有するものにグループ分けできる。これらグループ内での置換であればタンパク質の性質が変化しないことが多いことが知られている。従って、配列番号2に例示した公知のHBcタンパク質とアミノ酸配列が一部相違するタンパク質であっても、これらグループ内での置換であれば、抗HBc抗体との結合性も維持される可能性が高い。 Twenty kinds of amino acids constituting natural proteins are neutral amino acids having low polarity side chains (Gly, Ile, Val, Leu, Ala, Met, Pro) and neutral amino acids having hydrophilic side chains (Asn, Gln). , Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp) Can be divided. It is known that substitution within these groups often does not change the properties of the protein. Therefore, even if the protein is partially different in amino acid sequence from the known HBc protein exemplified in SEQ ID NO: 2, it is possible that the binding to the anti-HBc antibody is maintained if the substitution is within these groups. high.
固相用担体は特に限定されず、免疫測定用に従来用いられているものであってよい。例えば、プラスチックプレート、微粒子、繊維状物質等を用いることができる。これら担体の材質としては、従来公知のものを用いることができ、特に限定されないが、微粒子であれば、例えば、ガラスビーズ、ポリスチレン等の各種プラスチックビーズ、ラテックス粒子および各種フェライト粒子(例えば、特開平3−115862号公報参照)等を挙げることができる。また、繊維状物質としては、例えば、セルロース、ニトロセルロース、キトサン、ポリエチレングリコール重合体、シラン重合体等を構成成分とするものを挙げることができる。 The solid phase carrier is not particularly limited, and may be one conventionally used for immunoassay. For example, a plastic plate, fine particles, a fibrous substance, or the like can be used. Conventionally known materials can be used as the material of these carriers, and are not particularly limited. However, if the particles are fine particles, for example, various kinds of plastic beads such as glass beads and polystyrene, latex particles, and various types of ferrite particles (for example, JP-A-HEI 3-115862 publication). Examples of the fibrous substance include those containing cellulose, nitrocellulose, chitosan, polyethylene glycol polymer, silane polymer and the like as constituent components.
これら担体へのHBc抗原の固相化は、物理吸着、架橋剤を用いた化学結合等公知の方法を適宜用いることができる。 For immobilization of the HBc antigen on these carriers, known methods such as physical adsorption and chemical bonding using a crosslinking agent can be used as appropriate.
本発明に使用する抗免疫グロブリン抗体としては、測定したいクラスの抗HBc抗体に特異的に結合するものであればよく、抗IgG抗体又はその抗原結合性断片、及び抗IgM抗体又はその抗原結合性断片の少なくともいずれかを用いることができる。検体の由来の適した抗免疫グロブリン抗体が好ましく使用され、例えば、ヒト由来の検体を用いる場合は、上述したように抗ヒト免疫グロブリン抗体又はその抗原結合性断片を用いることが好ましい。抗免疫グロブリン抗体又はその抗原結合性断片の具体例としては、マウス、ウサギ、ニワトリ、ヤギ、ヒツジ、ウシなどの実験動物を免疫して得られるポリクローナル抗体;免疫した実験動物から脾臓細胞を分離し、ミエローマ細胞と融合させることによって得られるハイブリドーマが産生するモノクローナル抗体;免疫した実験動物から分離した脾臓細胞または血中白血球をEBウイルスによって不死化させた細胞が産生するモノクローナル抗体;組換え抗体;免疫グロブリンへの結合性を有する上記抗体の断片(例えば、Fab、Fab'、F(ab')2、Fv、scFv)など、測定したいクラスの抗HBc抗体に高い特異性、親和性を示す分子であればいかなるものでも用いることができる。抗免疫グロブリン抗体又はその抗原結合性断片は、市販品を用いてもよいし、周知の常法により抗免疫グロブリン抗体又はその抗原結合性断片を作製して用いることもできる。 The anti-immunoglobulin antibody used in the present invention may be any antibody that specifically binds to the class of anti-HBc antibody to be measured, such as an anti-IgG antibody or an antigen-binding fragment thereof, and an anti-IgM antibody or an antigen-binding property thereof. At least one of the fragments can be used. A suitable anti-immunoglobulin antibody derived from a specimen is preferably used. For example, when a specimen derived from a human is used, it is preferable to use an anti-human immunoglobulin antibody or an antigen-binding fragment thereof as described above. Specific examples of anti-immunoglobulin antibodies or antigen-binding fragments thereof include polyclonal antibodies obtained by immunizing laboratory animals such as mice, rabbits, chickens, goats, sheep and cows; spleen cells are isolated from the immunized experimental animals Monoclonal antibodies produced by hybridomas obtained by fusing with myeloma cells; Monoclonal antibodies produced by cells immortalized by EB virus from spleen cells or blood leukocytes isolated from immunized experimental animals; Recombinant antibodies; A molecule that exhibits high specificity and affinity for the anti-HBc antibody of the class to be measured, such as fragments of the above-mentioned antibodies having binding properties to globulin (eg, Fab, Fab ′, F (ab ′) 2 , Fv, scFv). Anything can be used. As the anti-immunoglobulin antibody or antigen-binding fragment thereof, a commercially available product may be used, or an anti-immunoglobulin antibody or antigen-binding fragment thereof may be prepared and used by a known conventional method.
抗免疫グロブリン抗体若しくはその抗原結合性断片又はHBc抗原の標識に用いる標識物質としては、例えば酵素、蛍光物質、化学発光物質、染色物質、放射性物質などが挙げられる。酵素としては、アルカリホスファターゼ(ALP)、パーオキシダーゼ、βガラクトシダーゼ等、公知のものを用いることができるが、これに限定されるものではない。高い検出感度の測定系を提供するためには、ALPを用いることが望ましい。 Examples of the labeling substance used for labeling the anti-immunoglobulin antibody or antigen-binding fragment thereof or HBc antigen include enzymes, fluorescent substances, chemiluminescent substances, staining substances, radioactive substances and the like. As the enzyme, known ones such as alkaline phosphatase (ALP), peroxidase, β-galactosidase can be used, but the enzyme is not limited thereto. In order to provide a measurement system with high detection sensitivity, it is desirable to use ALP.
標識物質として酵素を用いる場合、該酵素に対応した発色基質、蛍光基質又は発光基質等の基質を測定系内に添加し、酵素反応により生じる発色や発光等のシグナルを吸光光度計やルミノメーター等を用いて測定すればよい。例えば、標識物質としてALPを用いる場合、3−(4−メトキシスピロ(1,2−ジオキセタン−3,2'−トリシクロ[3.3.1.13,7]デカン)−4−イル)フェニルホスフェート2ナトリウム(例えば商品名AMPPD(登録商標))などの発光基質を用いることができる。標識物質としてビオチン又はハプテンが用いられる場合には、酵素、蛍光物質、化学発光物質、染色物質、又は放射性物質などを結合したストレプトアビジン又はハプテン抗体などを用いればよい。 When an enzyme is used as a labeling substance, a chromogenic substrate, a fluorescent substrate or a luminescent substrate corresponding to the enzyme is added to the measurement system, and signals such as chromogenic or luminescent generated by the enzyme reaction are absorptiometers, luminometers, etc. What is necessary is just to measure using. For example, when ALP is used as a labeling substance, 3- (4-methoxyspiro (1,2-dioxetane-3,2′-tricyclo [3.3.1.1 3,7 ] decane) -4-yl) phenyl A luminescent substrate such as phosphate disodium (for example, trade name AMPPD (registered trademark)) can be used. When biotin or hapten is used as the labeling substance, streptavidin or a hapten antibody to which an enzyme, a fluorescent substance, a chemiluminescent substance, a staining substance, a radioactive substance or the like is bound may be used.
抗免疫グロブリン抗体の標識は、公知の共有結合法等により直接標識してもよいし、ビオチン−アビジン等他の結合対を用いて間接的に標識してもよい。 The anti-immunoglobulin antibody may be labeled directly by a known covalent bond method or the like, or may be indirectly labeled using another binding pair such as biotin-avidin.
抗HBc抗体を定量する場合には、抗HBc抗体を種々の濃度で含む濃度既知の標準試料について、HBc抗原を用いて抗HBc抗体の測定を行ない、標識からのシグナルの量と標準試料中の抗HBc抗体濃度との相関関係をプロットして標準曲線を作成すればよい。抗HBc抗体濃度が未知の対象検体について同じ操作を行ない、標識からのシグナル量を測定し、測定されたシグナル量をこの標準曲線に当てはめることにより、検体中の抗HBc抗体を定量することができる。抗HBc抗体用のWHO第1国際標準品(WHO International Standard First International Standard for anti-Hepatitis B core antigen(anti-HBc), plasma, human. NIBSC code: 95/522)を用いて標準曲線を作成し、この標準曲線に基づいて被検個体由来の検体中の抗HBc抗体量を算出すれば、国際単位による濃度(IU/mL)で抗体量を表すことができる。あるいは、上記の国際標準品に対して較正された他の標準品を用いても、国際単位による濃度(IU/mL)で抗体量を求めることができる。 When quantifying the anti-HBc antibody, the anti-HBc antibody is measured using the HBc antigen for a standard sample of known concentration containing the anti-HBc antibody at various concentrations, and the amount of the signal from the label and the amount in the standard sample are measured. A standard curve may be created by plotting the correlation with the anti-HBc antibody concentration. By performing the same operation on the target sample whose anti-HBc antibody concentration is unknown, measuring the amount of signal from the label, and applying the measured amount of signal to this standard curve, the anti-HBc antibody in the sample can be quantified. . Create a standard curve using WHO International Standard First International Standard for anti-Hepatitis B core antigen (anti-HBc), plasma, human. NIBSC code: 95/522 If the amount of anti-HBc antibody in the specimen derived from the test individual is calculated based on this standard curve, the amount of antibody can be expressed by the concentration (IU / mL) in international units. Alternatively, the amount of antibody can be determined at a concentration in international units (IU / mL) using another standard calibrated against the above international standard.
抗HBc抗体を測定する場合、検体に加えて、陰性コントロール及び陽性コントロールを測定してもよい。陰性コントロールとしては、抗HBc抗体が陰性であることを確認された血液試料、抗HBc抗体を含まない各種緩衝液などを用いることができる。陽性コントロールとしては、抗HBc抗体が陰性であることを確認された血液試料又は緩衝液に、上記の抗HBc抗体用のWHO第1国際標準品を所定の濃度で添加した溶液、または該血液試料又は緩衝液に、上記の国際標準品に対して較正された他の標準品を所定の濃度で添加した溶液、抗HBc抗体陽性検体、ヒトより精製した抗HBc抗体を所定の濃度で添加した溶液などを用いることができる。 When measuring an anti-HBc antibody, a negative control and a positive control may be measured in addition to the specimen. As a negative control, a blood sample confirmed to be negative for anti-HBc antibody, various buffers not containing anti-HBc antibody, and the like can be used. As a positive control, a solution obtained by adding the above-mentioned WHO first international standard for anti-HBc antibody at a predetermined concentration to a blood sample or buffer solution confirmed to be negative for anti-HBc antibody, or the blood sample Or a solution in which another standard calibrated against the above international standard is added to the buffer solution at a predetermined concentration, a solution in which an anti-HBc antibody positive specimen, or an anti-HBc antibody purified from humans is added at a predetermined concentration Etc. can be used.
本発明はまた、上記本発明の方法にて検体中の抗HBc抗体を測定するための免疫測定キットも提供する。 The present invention also provides an immunoassay kit for measuring an anti-HBc antibody in a specimen by the method of the present invention.
1つの態様において、該キットは、上記した検体希釈液、すなわち上記(1)及び(2)から選択される少なくとも1種の界面活性剤を含有する溶液と、HBc抗原とを含む(態様A)。この態様Aは、後述するように、上記した本発明の態様のうちの態様1及び態様3を包含する。検体希釈液は、先に説明した通り、抗原抗体反応系内における濃度が0.040 w/v%〜1.27 w/v%となる濃度で上記界面活性剤を含有することが好ましい。検体希釈液中の界面活性剤濃度は、上述したように、検体や検体希釈液の使用量に応じて、0.25 w/v%〜8.02 w/v%、0.063 w/v%〜2.01 w/v%、0.48 w/v%〜15.2 w/v%等の値を取り得る。抗原抗体反応系内(2ステップの場合は特に一次反応系内)の界面活性剤濃度が0.040 w/v%〜1.27 w/v%となるように、検体希釈液中の界面活性剤を適宜に設定することができる。 In one embodiment, the kit comprises the above-described specimen diluent, that is, a solution containing at least one surfactant selected from (1) and (2) above, and an HBc antigen (Aspect A). . This aspect A includes aspects 1 and 3 of the aspects of the present invention described above, as will be described later. As described above, the specimen diluent preferably contains the surfactant at a concentration of 0.040 w / v% to 1.27 w / v% in the antigen-antibody reaction system. As described above, the surfactant concentration in the sample diluent is 0.25 w / v% to 8.02 w / v%, 0.063 w / v% to 2.01 w / v, depending on the amount of sample and sample diluent used. %, Values from 0.48 w / v% to 15.2 w / v% can be taken. The surfactant in the sample diluent is appropriately adjusted so that the surfactant concentration in the antigen-antibody reaction system (especially in the primary reaction system in the case of two steps) is 0.040 w / v% to 1.27 w / v%. Can be set.
本発明の免疫測定キットは、サンドイッチ法による免疫測定キットであってもよく、抗免疫グロブリン抗体又はその抗原結合性断片をさらに含み得る。検体希釈液、免疫測定方法、HBc抗原、抗免疫グロブリン抗体又はその抗原結合性断片等についての好ましい条件は上記と同様である。例えば、検体希釈液は、上記所定の界面活性剤に加え、上記した塩をさらに含んでいてよい。また、抗免疫グロブリン抗体又はその抗原結合性断片は、抗IgG抗体でも抗IgM抗体でもよく、典型的には抗IgG抗体、特に抗ヒトIgG抗体を含み得る。 The immunoassay kit of the present invention may be an immunoassay kit by a sandwich method, and may further contain an anti-immunoglobulin antibody or an antigen-binding fragment thereof. Preferred conditions for the sample diluent, immunoassay method, HBc antigen, anti-immunoglobulin antibody, or antigen-binding fragment thereof are the same as described above. For example, the sample dilution liquid may further contain the above-described salt in addition to the predetermined surfactant. In addition, the anti-immunoglobulin antibody or antigen-binding fragment thereof may be an anti-IgG antibody or an anti-IgM antibody, and typically may include an anti-IgG antibody, particularly an anti-human IgG antibody.
該キットにおいて、HBc抗原は、プレートや粒子(例えば磁性粒子)などの固相担体に結合された形態であってもよい。この場合、抗免疫グロブリン抗体又はその抗原結合性断片は、適当な媒体中に溶解した抗体液の形態でキットに含まれていてよく、典型的には、抗免疫グロブリン抗体又はその抗原結合性断片は、標識物質で標識された形態で標識抗体液としてキットに含まれ得る。この態様において、抗体液は、上記した所定の界面活性剤を含んでいてもよいし、含んでいなくてもよい。 In the kit, the HBc antigen may be in a form bound to a solid phase carrier such as a plate or particle (eg, magnetic particle). In this case, the anti-immunoglobulin antibody or antigen-binding fragment thereof may be included in the kit in the form of an antibody solution dissolved in an appropriate medium, and typically, the anti-immunoglobulin antibody or antigen-binding fragment thereof. Can be included in the kit as a labeled antibody solution in a form labeled with a labeling substance. In this embodiment, the antibody solution may or may not contain the predetermined surfactant described above.
HBc抗原が液体中に含まれた抗原試薬液の形態で(例えば、粒子担体上に固定化されたHBc抗原を含有する抗原試薬液の形態で)キットに含まれる場合には、上記した所定の界面活性剤が抗原試薬液にも含まれていてよい。検体希釈液のみに界面活性剤が含有されるキットは、上記した本発明の態様のうちの態様1に該当し、検体希釈液及び抗原試薬液の両方に界面活性剤が含有されるキットは、本発明の態様のうちの態様3に該当する。 When the HBc antigen is contained in a kit in the form of an antigen reagent solution contained in a liquid (for example, in the form of an antigen reagent solution containing an HBc antigen immobilized on a particle carrier), A surfactant may also be included in the antigen reagent solution. The kit containing the surfactant only in the sample diluent corresponds to the embodiment 1 of the above-described embodiments of the present invention, and the kit containing the surfactant in both the sample diluent and the antigen reagent solution is This corresponds to aspect 3 of the aspects of the present invention.
非特異反応のさらなる低減のために、検体希釈液が塩を含んでいてもよいし、検体希釈液に塩を添加する代わりに、あるいは検体希釈液への塩の添加と組み合わせて、抗原試薬液中の塩濃度を高めてもよい。すなわち、本発明のキットでは、検体希釈液及び抗原試薬液のいずれか一方若しくは両者が、抗原抗体反応系内における塩濃度が158 mM以上となるように塩を含有していてよい。 For further reduction of non-specific reaction, the sample diluent may contain a salt, and instead of adding salt to the sample diluent, or in combination with the addition of salt to the sample diluent, the antigen reagent solution The salt concentration inside may be increased. That is, in the kit of the present invention, either one or both of the sample diluent and the antigen reagent solution may contain a salt so that the salt concentration in the antigen-antibody reaction system is 158 mM or more.
あるいは、HBc抗原は、標識物質で標識された形態で標識抗原液としてキットに含まれていてもよい。この場合、抗免疫グロブリン抗体又はその抗原結合性断片が、固相担体に結合された形態で該キットに含まれ得る。この態様において、標識HBc抗原液は、上記した所定の界面活性剤を含んでいてもよいし、含んでいなくてもよい。 Alternatively, the HBc antigen may be included in the kit as a labeled antigen solution in a form labeled with a labeling substance. In this case, the anti-immunoglobulin antibody or antigen-binding fragment thereof can be included in the kit in a form bound to a solid phase carrier. In this embodiment, the labeled HBc antigen solution may or may not contain the predetermined surfactant described above.
抗免疫グロブリン抗体又はその抗原結合性断片が液体中に含まれた抗体試薬液の形態で(例えば、粒子担体上に固定化された抗免疫グロブリン抗体又はその抗原結合性断片を含有する抗体試薬液の形態で)キットに含まれる場合には、上記した所定の界面活性剤が抗体試薬液にも含まれていてよい。検体希釈液のみに界面活性剤が含有されるキットは、上記した本発明の態様のうちの態様1に該当し、検体希釈液及び抗体試薬液の両方に界面活性剤が含有されるキットは、本発明の態様のうちの態様3に該当する。 An antibody reagent solution containing an anti-immunoglobulin antibody or antigen-binding fragment thereof in a liquid form (for example, an antibody reagent solution containing an anti-immunoglobulin antibody or antigen-binding fragment thereof immobilized on a particle carrier) In the case of being included in the kit), the above-mentioned predetermined surfactant may be included in the antibody reagent solution. The kit containing the surfactant only in the sample diluent corresponds to the above-described embodiment 1 of the embodiments of the present invention, and the kit containing the surfactant in both the sample diluent and the antibody reagent solution is This corresponds to aspect 3 of the aspects of the present invention.
非特異反応のさらなる低減のために、検体希釈液が塩を含んでいてもよいし、検体希釈液に塩を添加する代わりに、あるいは検体希釈液への塩の添加と組み合わせて、抗体試薬液中の塩濃度を高めてもよい。すなわち、本発明のキットでは、検体希釈液及び抗体試薬液のいずれか一方若しくは両者が、抗原抗体反応系内における塩濃度が158 mM以上となるように塩を含有していてよい。 In order to further reduce non-specific reactions, the sample diluent may contain a salt. Instead of adding salt to the sample diluent, or in combination with the addition of salt to the sample diluent, the antibody reagent solution The salt concentration inside may be increased. That is, in the kit of the present invention, either one or both of the sample diluent and the antibody reagent solution may contain a salt so that the salt concentration in the antigen-antibody reaction system is 158 mM or more.
別の態様において、本発明の免疫測定キットは、HBc抗原を含有する抗原試薬液を含み、該抗原試薬液が、上記した(1)及び(2)から選択される少なくとも1種の界面活性剤を含有する(態様B)。この態様Bのキットは、上記した本発明の態様のうちの態様2に該当する。抗原試薬液は、HBc抗原が粒子担体上に固定化された形態で該抗原を含有する試薬であり得る。 In another embodiment, the immunoassay kit of the present invention includes an antigen reagent solution containing an HBc antigen, and the antigen reagent solution is at least one surfactant selected from (1) and (2) above. (Aspect B). This kit of aspect B corresponds to aspect 2 of the aspects of the present invention described above. The antigen reagent solution may be a reagent containing the antigen in a form in which the HBc antigen is immobilized on a particle carrier.
非特異反応のさらなる低減のために、抗原試薬液中の塩濃度を高めてもよく、その場合の塩濃度は、抗原抗体反応系内の塩濃度が158 mM以上となる濃度であればよい。あるいは、該キットは、検体希釈液を含んでいてよく、該検体希釈液及び抗原試薬液のいずれか一方又は両者が、抗原抗体反応系内における塩濃度が158 mM以上となるように塩を含有していてもよい。 In order to further reduce the non-specific reaction, the salt concentration in the antigen reagent solution may be increased, and the salt concentration in that case may be a concentration at which the salt concentration in the antigen-antibody reaction system is 158 mM or more. Alternatively, the kit may contain a sample diluent, and either or both of the sample diluent and the antigen reagent solution contain a salt such that the salt concentration in the antigen-antibody reaction system is 158 mM or more. You may do it.
態様Bのキットについてのその他の好ましい条件は、態様2による免疫測定方法と同様である。 Other preferred conditions for the kit of aspect B are the same as in the immunoassay method according to aspect 2.
さらに別の態様において、本発明の免疫測定キットは、抗免疫グロブリン抗体又はその抗原結合性断片を含有する抗体試薬液と、HBc抗原とを含み、該抗体試薬液が、上記した(1)及び(2)から選択される少なくとも1種の界面活性剤を含有する(態様C)。この態様Cのキットは、上記した本発明の態様のうちの態様2に該当する。抗体試薬液は、抗免疫グロブリン抗体又はその抗原結合性断片が粒子担体上に固定化された形態で、該抗体又はその抗原結合性断片を含有する試薬であり得る。 In yet another embodiment, the immunoassay kit of the present invention comprises an antibody reagent solution containing an anti-immunoglobulin antibody or an antigen-binding fragment thereof, and an HBc antigen, wherein the antibody reagent solution comprises the above (1) and It contains at least one surfactant selected from (2) (Aspect C). This kit of embodiment C corresponds to embodiment 2 of the embodiments of the present invention described above. The antibody reagent solution can be a reagent containing the antibody or antigen-binding fragment thereof in a form in which an anti-immunoglobulin antibody or antigen-binding fragment thereof is immobilized on a particle carrier.
非特異反応のさらなる低減のために、抗体試薬液中の塩濃度を高めてもよく、その場合の塩濃度は、抗原抗体反応系内の塩濃度が158 mM以上となる濃度であればよい。あるいは、該キットは、検体希釈液を含んでいてよく、該検体希釈液及び抗体試薬液のいずれか一方又は両者が、抗原抗体反応系内における塩濃度が158 mM以上となるように塩を含有していてもよい。 In order to further reduce the non-specific reaction, the salt concentration in the antibody reagent solution may be increased, and the salt concentration in that case may be a concentration at which the salt concentration in the antigen-antibody reaction system is 158 mM or more. Alternatively, the kit may contain a sample diluent, and either or both of the sample diluent and the antibody reagent solution contain a salt so that the salt concentration in the antigen-antibody reaction system is 158 mM or more. You may do it.
態様Cのキットについてのその他の好ましい条件も、態様2による免疫測定方法と同様である。 Other preferable conditions for the kit of aspect C are the same as those of the immunoassay method according to aspect 2.
本発明の抗HBc抗体の免疫測定キットは、さらに、使用する標識物質に応じて適当な基質液や、洗浄液等の、免疫測定キットに一般的に含まれているその他の試薬類等も含んでいてよい。 The anti-HBc antibody immunoassay kit of the present invention further includes other reagents generally contained in the immunoassay kit, such as an appropriate substrate solution and washing solution depending on the labeling substance to be used. May be.
以下、本発明を実施例に基づきより具体的に説明する。もっとも、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. However, the present invention is not limited to the following examples.
参考例1.測定方法について
(1) HBc抗原結合粒子の作製
リコンビナントHBc抗原結合粒子を、次の通り調製した。
磁性フェライト粒子とリコンビナントHBc抗原とを重量比500:1の割合で、0.1Mリン酸緩衝液中で混合し、25℃で1時間ゆるやかに攪拌しながらインキュベートした。反応後、フェライト粒子を磁石で集めて反応液から分離し、粒子を50 mM Tris緩衝液(150mM NaCl、0.1%アジ化ナトリウム、2%BSA、pH7.2)にて洗浄し、HBc抗原結合粒子を得た。
Reference Example 1 Measurement method
(1) Production of HBc antigen-binding particles Recombinant HBc antigen-binding particles were prepared as follows.
Magnetic ferrite particles and recombinant HBc antigen were mixed at a weight ratio of 500: 1 in 0.1 M phosphate buffer and incubated at 25 ° C. for 1 hour with gentle stirring. After the reaction, ferrite particles are collected with a magnet and separated from the reaction solution. The particles are washed with 50 mM Tris buffer (150 mM NaCl, 0.1% sodium azide, 2% BSA, pH 7.2), and HBc antigen-binding particles. Got.
(2) アルカリホスファターゼ標識抗IgG抗体の作製
マウス抗ヒトIgGモノクローナル抗体とウシ小腸由来アルカリホスファターゼ(オリエンタル酵母社製)とをヨシタケらの方法(Yoshitakeet al., J.Biochem. 1982, 92(5), p1413-1424)により結合させ、アルカリホスファターゼ標識抗体を調製した。通例行われる通り、脱塩処理したマウス抗ヒトIgGモノクローナル抗体とペプシンを0.1Mクエン酸緩衝液(pH3.5)中で混合し、37℃で1時間静置してペプシン消化を行った。反応を停止させた後にゲルろ過精製を行い、Fc領域を除去した抗ヒトIgG抗体を得た。次に2−メルカプトエチルアミン塩酸塩を添加して、チオール化を行った。更に脱塩処理し、抗ヒトIgG抗体のFabを得た。
(2) Preparation of alkaline phosphatase-labeled anti-IgG antibody Mouse anti-human IgG monoclonal antibody and bovine small intestine-derived alkaline phosphatase (produced by Oriental Yeast Co., Ltd.) were used by the method of Yoshitake et al. , p1413-1424) to prepare an alkaline phosphatase-labeled antibody. As usual, desalted mouse anti-human IgG monoclonal antibody and pepsin were mixed in 0.1 M citrate buffer (pH 3.5) and left at 37 ° C. for 1 hour for pepsin digestion. After stopping the reaction, gel filtration purification was performed to obtain an anti-human IgG antibody from which the Fc region was removed. Next, 2-mercaptoethylamine hydrochloride was added to effect thiolation. Further, desalting treatment was performed to obtain an anti-human IgG antibody Fab.
一方、脱塩したアルカリホスファターゼとN-(4-マレイミドブチリロキシ)-スクシンイミド(GMBS)(終濃度0.3 mg/mL)を混合し、30℃で1時間静置してマレイミド化を行った。脱塩した後、Fabとマレイミド化アルカリホスファターゼをモル比1:1の割合で混合し、25℃にて30分静置してカップリングを行った。カップリング液を15℃にて1時間静置し、2−メルカプトエチルアミン塩酸塩を加え、反応を停止させた。濃縮および脱塩後、50 mM Tris緩衝液(150mM NaCl、0.1%アジ化ナトリウム、2%BSA、1mM MgCl2、0.1mM ZnCl2、pH7.2)を加え、アルカリホスファターゼ標識抗IgG抗体溶液とした。 On the other hand, desalted alkaline phosphatase and N- (4-maleimidobutyryloxy) -succinimide (GMBS) (final concentration 0.3 mg / mL) were mixed and allowed to stand at 30 ° C. for 1 hour for maleimidation. After desalting, Fab and maleimidated alkaline phosphatase were mixed at a molar ratio of 1: 1 and allowed to stand at 25 ° C. for 30 minutes for coupling. The coupling solution was allowed to stand at 15 ° C. for 1 hour, and 2-mercaptoethylamine hydrochloride was added to stop the reaction. After concentration and desalting, 50 mM Tris buffer (150 mM NaCl, 0.1% sodium azide, 2% BSA, 1 mM MgCl 2 , 0.1 mM ZnCl 2 , pH 7.2) was added to obtain an alkaline phosphatase-labeled anti-IgG antibody solution .
(3) 抗HBc抗体の測定
検体、陰性コントロール又は陽性コントロール10μLと検体希釈液190μLを混合した。
検体としては、ProMedDx社から購入した陰性血清検体(HBs抗原陰性、HBcAb陰性)を使用した。以下の実施例で用いた検体1〜6は、詳細検討を実施し、何れも陰性検体であることを確認している。陰性コントロールと陽性コントロールは、B型肝炎ウイルスコア抗体測定キット「ルミパルスプレスト(登録商標)HBcAb-III」(富士レビオ社製)に付属された「HBcAb-III用標準陰性溶液」と「HBcAb-III用標準陽性溶液」を用いた。検体希釈液としては、50mM Tris緩衝液(150mM NaCl、1mM EDTA・2Na、pH7.2)に所定濃度となるように各界面活性剤を添加したものを使用した。
(3) Measurement of anti-HBc antibody 10 μL of the sample, negative control or positive control and 190 μL of the sample diluent were mixed.
As a sample, a negative serum sample (HBs antigen negative, HBcAb negative) purchased from ProMedDx was used. Samples 1 to 6 used in the following examples have been examined in detail and confirmed to be negative samples. The negative control and the positive control are the “standard negative solution for HBcAb-III” and “HBcAb-III” included in the “Lumipulse Presto (registered trademark) HBcAb-III” (Fujirebio Inc.) Standard positive solution "was used. As the sample dilution solution, a 50 mM Tris buffer solution (150 mM NaCl, 1 mM EDTA · 2Na, pH 7.2) to which each surfactant was added to a predetermined concentration was used.
50 mM Tris緩衝液(150mM NaCl、0.1%アジ化ナトリウム、2%BSA、pH7.2)に懸濁したHBc抗原結合粒子50μLと、希釈した検体10μLを反応槽に分注し、攪拌した。その後37℃で8分間インキュベーションし、B/F分離・洗浄を行った。その後、アルカリホスファターゼ標識抗IgG抗体溶液50μLを反応槽に分注し、攪拌後37℃で8分間インキュベーションし、B/F分離・洗浄を行った。その後、化学発光基質である3-(2'-スピロアダマンタン)-4-メトキシ-4-(3''-ホスホリルオキシ)フェニル-1,2-ジオキセタン・2ナトリウム塩(AMPPD)を含むルミパルス基質液200μLを反応槽に分注し、攪拌後37℃で4分間インキュベーションした後、発光量をルミノメーターで測定し、カウント数を得た。実際の測定は全自動化学発光酵素免疫測定システム「ルミパルスプレストII」(富士レビオ社製)にて行った。なお、洗浄液とルミパルス基質液は「ルミパルスプレストHBcAb-III」に付属されているものを使用した。 50 μL of HBc antigen-binding particles suspended in 50 mM Tris buffer (150 mM NaCl, 0.1% sodium azide, 2% BSA, pH 7.2) and 10 μL of diluted specimen were dispensed into a reaction vessel and stirred. Thereafter, incubation was performed at 37 ° C. for 8 minutes, and B / F separation and washing were performed. Thereafter, 50 μL of alkaline phosphatase-labeled anti-IgG antibody solution was dispensed into the reaction vessel, and after stirring, incubated at 37 ° C. for 8 minutes to separate and wash B / F. Then, Lumipulse substrate solution containing chemiluminescent substrate 3- (2'-spiroadamantane) -4-methoxy-4- (3 ''-phosphoryloxy) phenyl-1,2-dioxetane disodium salt (AMPPD) 200 μL was dispensed into a reaction vessel, stirred and incubated at 37 ° C. for 4 minutes, and then the luminescence was measured with a luminometer to obtain the count number. The actual measurement was performed with a fully automatic chemiluminescent enzyme immunoassay system “Lumipulse Presto II” (Fujirebio Inc.). The cleaning solution and the Lumipulse substrate solution used were those attached to “Lumipulse Presto HBcAb-III”.
各測定で得られたカウント数について、界面活性剤を添加していない検体希釈液を用いた場合のカウント数に対する、界面活性剤を添加した検体希釈液を用いた場合のカウント数の比率から、非特異反応低減率を算出し、各界面活性剤の添加効果を評価した。
非特異反応低減率(%)=X/Y×100
X:界面活性剤を添加した検体希釈液を用いた場合のカウント数
Y:界面活性剤を添加していない検体希釈液を用いた場合のカウント数
For the number of counts obtained in each measurement, from the ratio of the number of counts when using a sample diluent with a surfactant to the number of counts when using a sample diluent without adding a surfactant, The reduction rate of non-specific reaction was calculated and the effect of adding each surfactant was evaluated.
Nonspecific reaction reduction rate (%) = X / Y x 100
X: Count when using sample dilution with surfactant added
Y: Count when using sample dilution without surfactant
界面活性剤の添加が「効果あり」と評価する基準として、以下を設定した。
(1) 陽性コントロールの非特異反応低減率が85%以上
(2) 2以上の検体で非特異反応低減率が90%未満
The following was set as a standard for evaluating that the addition of a surfactant was “effective”.
(1) Non-specific reaction reduction rate of positive control is 85% or more
(2) Reduction rate of non-specific reaction is less than 90% for 2 or more samples
実施例1.各種濃度の界面活性剤の添加
参考例1に記載の測定方法に従って、検体1〜6の抗HBc抗体を測定した。検体希釈液としては、非イオン性界面活性剤であるTritonX-405(ポリオキシエチレンオクチルフェニルエーテル、HLB値17.9)を各濃度で添加した希釈液を用いた。
Example 1. Addition of various concentrations of surfactant According to the measurement method described in Reference Example 1, anti-HBc antibodies in Samples 1 to 6 were measured. As the sample diluent, a diluent obtained by adding Triton X-405 (polyoxyethylene octylphenyl ether, HLB value 17.9), which is a nonionic surfactant, at each concentration was used.
結果を表1に示す。表1には、各種濃度の界面活性剤を添加した検体希釈液を用いた場合のカウント数と、非特異反応低減率(%)を示す。一次反応液中の濃度が0.04w/v%以上となるように、0.25%w/v以上の濃度で検体希釈液に界面活性剤を添加した場合、陰性検体のカウント数が低下し、複数の検体で非特異反応低減率が90%未満に低下した。一方、陽性コントロールのカウント数はほとんど低下せず、その非特異反応低減率は85%以上であった。これらの結果から、一次反応液中の濃度が0.04%以上の濃度になるように界面活性剤を検体希釈液に添加することにより、非特異反応が抑制されることが明らかとなった。 The results are shown in Table 1. Table 1 shows the number of counts and the nonspecific reaction reduction rate (%) in the case of using a sample dilution solution to which various concentrations of a surfactant were added. When a surfactant is added to the sample diluent at a concentration of 0.25% w / v or more so that the concentration in the primary reaction solution is 0.04 w / v% or more, the number of negative sample counts is reduced. Non-specific reaction reduction rate decreased to less than 90% in the sample. On the other hand, the count of the positive control hardly decreased, and the nonspecific reaction reduction rate was 85% or more. From these results, it has been clarified that non-specific reactions are suppressed by adding a surfactant to the sample diluent so that the concentration in the primary reaction solution is 0.04% or more.
実施例2.各種非イオン性界面活性剤の添加
他の非イオン性界面活性剤についても検討するため、各種非イオン性界面活性剤を添加した検体希釈液を用いて、実施例1と同様に抗HBc抗体を測定した。
Example 2 Addition of various nonionic surfactants In order to investigate other nonionic surfactants, anti-HBc antibodies were prepared in the same manner as in Example 1 using specimen diluents to which various nonionic surfactants were added. It was measured.
結果を表2に示す。表2には、各種界面活性剤を1.5w/v%の濃度で添加した検体希釈液を用いた場合(一次反応液中の各種界面活性剤の濃度は0.238w/v%)のカウント数と、非特異反応低減率(%)を示す。TritonX-100、TritonX-114、TritonX-305、TritonX-705、Brij58、BrijC10、Tween20、Tween40、Tween80の非イオン性界面活性剤を検体希釈液に添加することにより、複数の検体で非特異反応低減率が90%未満に低下した。一方、陽性コントロールの非特異反応低減率は85%以上であった。これらの結果から、非イオン性界面活性剤を検体希釈液に添加することにより非特異反応が抑制されることが明らかとなった。特にTriton類(ポリオキシエチレンオクチルフェニルエーテル)を添加した場合は、多くの検体で非特異反応が抑制され、非特異反応低減率も大きく低下した。 The results are shown in Table 2. Table 2 shows the number of counts when using a sample diluent to which various surfactants were added at a concentration of 1.5 w / v% (the concentration of various surfactants in the primary reaction solution was 0.238 w / v%). The non-specific reaction reduction rate (%) is shown. Reduce non-specific reaction in multiple samples by adding nonionic surfactants of TritonX-100, TritonX-114, TritonX-305, TritonX-705, Brij58, BrijC10, Tween20, Tween40, Tween80 to the sample diluent The rate dropped to less than 90%. On the other hand, the nonspecific reaction reduction rate of the positive control was 85% or more. From these results, it was revealed that non-specific reaction is suppressed by adding a nonionic surfactant to the sample diluent. In particular, when Tritons (polyoxyethylene octylphenyl ether) was added, the nonspecific reaction was suppressed in many specimens, and the nonspecific reaction reduction rate was greatly reduced.
実施例3.両性界面活性の添加
さらに、両性界面活性剤についても検討するため、両性界面活性剤を添加した検体希釈液を用いて、実施例1と同様に抗HBc抗体を測定した。
Example 3 Addition of Amphoteric Surfactant Further, in order to examine amphoteric surfactant, anti-HBc antibody was measured in the same manner as in Example 1 using a sample dilution solution to which amphoteric surfactant was added.
結果を表3に示す。表3には、各種界面活性剤を1.5w/v%の濃度で検体希釈液に添加した場合(一次反応液中の各種界面活性剤の濃度は0.238w/v%)のカウント数と、非特異反応低減率(%)を示す。C12APS(N-ドデシル-N,N-ジメチル-3-アンモニオ-1-プロパンスルホナート)、C14APS(N-テトラデシル-N,N-ジメチル-3-アンモニオ-1-プロパンスルホナート)、C16APS(N-ヘキサデシル-N,N-ジメチル-3-アンモニオ-1-プロパンスルホナート)を検体希釈液に添加することにより、複数の検体に対する非特異反応低減率が90%未満に低下した。一方、陽性コントロールに対する非特異反応低減率は85%以上だった。これらの結果から、両性界面活性剤を検体希釈液に添加することにより、非特異反応が抑制されることが明らかとなった。 The results are shown in Table 3. Table 3 shows the number of counts when various surfactants are added to the specimen diluent at a concentration of 1.5 w / v% (the concentration of various surfactants in the primary reaction solution is 0.238 w / v%). Specific reaction reduction rate (%) is shown. C12APS (N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate), C14APS (N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate), C16APS (N- By adding hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate) to the sample diluent, the non-specific reaction reduction rate for a plurality of samples decreased to less than 90%. On the other hand, the reduction rate of non-specific reaction relative to the positive control was 85% or more. From these results, it became clear that non-specific reactions were suppressed by adding amphoteric surfactants to the sample diluent.
実施例4.塩濃度との組み合わせ効果
塩濃度と界面活性剤の組合せについて検討した。50mM Tris緩衝液(1mM EDTA・2Na、0.5% TritonX-405、pH7.2)に各濃度になるように塩を添加した検体希釈液を用い、実施例1と同様に抗HBc抗体を測定した。塩としては、塩化ナトリウムを用いた。また、実施例1と同様に非特異反応低減率を求めるため、界面活性剤を添加していない検体希釈液(150mM NaCl、1mM EDTA・2Na、pH7.2)を用いた場合についても同様に抗HBc抗体を測定した。
Example 4 Combination effect with salt concentration The combination of salt concentration and surfactant was investigated. The anti-HBc antibody was measured in the same manner as in Example 1 using a sample dilution solution in which salt was added to each concentration in 50 mM Tris buffer (1 mM EDTA · 2Na, 0.5% TritonX-405, pH 7.2). Sodium chloride was used as the salt. In addition, in order to determine the nonspecific reaction reduction rate as in Example 1, the anti-reaction rate was similarly used in the case of using a sample diluent (150 mM NaCl, 1 mM EDTA · 2Na, pH 7.2) to which no surfactant was added. HBc antibody was measured.
結果を表4に示す。検体希釈液に200 mM、500 mM、800 mMの塩を添加した場合(一次反応液中の塩濃度が158 mM、205 mM、253mMとなる場合)、陰性検体の非特異反応低減率が、150 mMの塩濃度の場合に比べて、さらに低下していた。この結果から、界面活性剤に加えて、一次反応液中の塩濃度が158 mM以上になるように塩を検体希釈液に添加することによって、非特異反応をさらに抑制できることが明らかとなった。 The results are shown in Table 4. When 200 mM, 500 mM, or 800 mM salt is added to the sample dilution (when the salt concentration in the primary reaction solution is 158 mM, 205 mM, or 253 mM), the nonspecific reaction reduction rate of the negative sample is 150 Compared with the salt concentration of mM, it was further reduced. From this result, it became clear that non-specific reaction can be further suppressed by adding salt to the specimen diluent so that the salt concentration in the primary reaction solution becomes 158 mM or more in addition to the surfactant.
実施例5 塩濃度との組み合わせ効果
高濃度の塩と界面活性剤の組み合わせについて検討した。検体としては、ProMedDx社から購入した陰性血清検体(HBs抗原陰性、HBcAb陰性)である検体7〜12を用いた。検体7〜12は参考例1と同様に詳細検討を実施し、何れも陰性検体であることを確認している。検体希釈液としては、445mM塩化ナトリウムと0.24% TritonX-705を含む50 mM Tris緩衝液(445mM NaCl、1mM EDTA・2Na、0.24% TritonX-705、pH7.2)を用いた。また、HBc抗原結合粒子としては、445mM塩化ナトリウムと0.24% TritonX-705を含む50 mM Tris緩衝液(445mM NaCl、0.1%アジ化ナトリウム、2%BSA、0.24% TritonX-705、pH7.2)に懸濁したHBc抗原結合粒子を用いて、実施例1と同様に抗HBc抗体を測定した。さらに、実施例1と同様に非特異反応低減率を求めるため、検体希釈液として界面活性剤を添加していない50 mM Tris緩衝液(150mM NaCl、1mM EDTA・2Na、pH7.2)と、50 mM Tris緩衝液(150mM NaCl、0.1%アジ化ナトリウム、2%BSA、pH7.2)に懸濁したHBc抗原結合粒子を用いた場合についても同様に抗HBc抗体を測定した。
Example 5 Combination Effect with Salt Concentration A combination of a high concentration salt and a surfactant was examined. As specimens, specimens 7 to 12 which were negative serum specimens (HBs antigen negative, HBcAb negative) purchased from ProMedDx were used. Samples 7 to 12 were examined in detail as in Reference Example 1 and confirmed to be negative samples. As a specimen dilution solution, a 50 mM Tris buffer solution (445 mM NaCl, 1 mM EDTA · 2Na, 0.24% TritonX-705, pH 7.2) containing 445 mM sodium chloride and 0.24% TritonX-705 was used. As HBc antigen-binding particles, 50 mM Tris buffer containing 445 mM sodium chloride and 0.24% TritonX-705 (445 mM NaCl, 0.1% sodium azide, 2% BSA, 0.24% TritonX-705, pH 7.2) Using the suspended HBc antigen-binding particles, anti-HBc antibody was measured in the same manner as in Example 1. Further, in order to obtain the nonspecific reaction reduction rate as in Example 1, 50 mM Tris buffer solution (150 mM NaCl, 1 mM EDTA · 2Na, pH 7.2) to which a surfactant was not added as a specimen dilution solution, Anti-HBc antibodies were similarly measured when HBc antigen-binding particles suspended in mM Tris buffer (150 mM NaCl, 0.1% sodium azide, 2% BSA, pH 7.2) were used.
結果を表5に示す。検体希釈液とHBc抗原結合粒子懸濁液に445mM塩化ナトリウムと0.24% TritonX-705を添加した場合(一次反応液中の塩濃度が443 mM、TritonX-705濃度が0.238%となる場合)、陰性である検体7〜12のカウント数が大幅に低下することにより、非特異反応低減率が大幅に低下していた。陽性コントロールは非特異反応低減率の数値が高く維持され、陽性のカウント数への悪影響は認められなかった。この結果から、界面活性剤に加えて、一次反応液中の塩濃度を443 mMにした場合において、非特異反応をさらに抑制できることが明らかとなった。 The results are shown in Table 5. Negative when 445 mM sodium chloride and 0.24% TritonX-705 are added to the sample diluent and HBc antigen-binding particle suspension (when the salt concentration in the primary reaction solution is 443 mM and the TritonX-705 concentration is 0.238%) As a result, the non-specific reaction reduction rate was greatly reduced. The positive control maintained a high nonspecific reaction reduction rate, and no adverse effect on the positive count was observed. From this result, it was revealed that nonspecific reaction can be further suppressed when the salt concentration in the primary reaction solution is 443 mM in addition to the surfactant.
実施例6 検体量を増やした場合の、塩および界面活性剤の添加効果
検体量を増やした場合の、塩および界面活性剤の添加効果について検討した。検体、陰性コントロール又は陽性コントロール10μLと検体希釈液190μLを混合した。検体としては、ProMedDx社から購入した陰性血清検体(HBs抗原陰性、HBcAb陰性)である検体13〜18を用いた。検体13〜18は参考例1と同様に詳細検討を実施し、何れも陰性検体であることを確認している。界面活性剤の添加効果を検討するため、検体希釈液として、0.25% TritonX-705を含む50 mM Tris緩衝液(150mM NaCl、1 mM EDTA・2Na、0.25% TritonX-705、pH7.2)を用いた。次に、0.25% TritonX-705を含む50 mM Tris緩衝液(150mM NaCl、0.1%アジ化ナトリウム、2%BSA、0.25% TritonX-705、pH7.2)に懸濁したHBc抗原結合粒子50μLと、希釈した検体20μLを反応槽に分注し、実施例1と同様に抗HBc抗体を測定した。このとき、一次反応液中のTritonX-705濃度は0.246%となる。また、塩および界面活性剤の添加効果を検討するため、検体希釈液として、200mM塩化ナトリウムと0.25% TritonX-705を含む50 mM Tris緩衝液(200mM NaCl、1 mM EDTA・2Na、0.25% TritonX-705、pH7.2)を用い、HBc抗原結合粒子の懸濁液として、200mM塩化ナトリウムと0.25% TritonX-705を含む50 mM Tris緩衝液(200mM NaCl、0.1%アジ化ナトリウム、2%BSA、0.25% TritonX-705、pH7.2)に懸濁したHBc抗原結合粒子50μLを用い、同様に抗HBc抗体を測定した。このとき、一次反応液中の塩濃度は199 mM、TritonX-705濃度は0.246%となる。
Example 6 Effect of addition of salt and surfactant when the amount of sample was increased The effect of addition of salt and surfactant when the amount of sample was increased was examined. The sample, negative control or positive control 10 μL and sample dilution liquid 190 μL were mixed. As samples, samples 13 to 18 which were negative serum samples (HBs antigen negative, HBcAb negative) purchased from ProMedDx were used. Samples 13 to 18 were examined in detail as in Reference Example 1 and confirmed to be negative samples. Use 50 mM Tris buffer solution (150 mM NaCl, 1 mM EDTA · 2Na, 0.25% TritonX-705, pH 7.2) containing 0.25% TritonX-705 as a sample diluent to examine the effect of adding surfactants. It was. Next, 50 μL of HBc antigen-binding particles suspended in 50 mM Tris buffer (150 mM NaCl, 0.1% sodium azide, 2% BSA, 0.25% TritonX-705, pH 7.2) containing 0.25% TritonX-705, 20 μL of diluted specimen was dispensed into a reaction vessel, and anti-HBc antibody was measured in the same manner as in Example 1. At this time, the concentration of TritonX-705 in the primary reaction solution is 0.246%. In addition, in order to examine the effects of addition of salt and surfactant, the specimen dilution solution was 50 mM Tris buffer containing 200 mM sodium chloride and 0.25% TritonX-705 (200 mM NaCl, 1 mM EDTA · 2Na, 0.25% TritonX- 705, pH 7.2), and as a suspension of HBc antigen-binding particles, 50 mM Tris buffer (200 mM NaCl, 0.1% sodium azide, 2% BSA, 0.25) containing 200 mM sodium chloride and 0.25% TritonX-705 Anti-HBc antibody was similarly measured using 50 μL of HBc antigen-binding particles suspended in% TritonX-705, pH 7.2). At this time, the salt concentration in the primary reaction solution is 199 mM, and the Triton X-705 concentration is 0.246%.
結果を表6に示す。検体量を増やした場合においても、一次反応時に界面活性剤を添加することにより、非特異反応低減率が低下し、非特異反応を抑制できることが示された。さらに、高濃度の塩と界面活性剤を組み合わせることにより、非特異反応低減率が大幅に低下した。このことにより、塩と界面活性剤の組み合わせは、検体量に依存せず非特異反応減少に効果的であることが示された。 The results are shown in Table 6. Even when the amount of the sample was increased, it was shown that by adding a surfactant during the primary reaction, the reduction rate of the non-specific reaction is reduced and the non-specific reaction can be suppressed. Furthermore, the nonspecific reaction reduction rate was significantly reduced by combining a high concentration salt and a surfactant. This indicates that the combination of the salt and the surfactant is effective in reducing the nonspecific reaction regardless of the amount of the sample.
実施例7 一次反応液への界面活性剤の添加効果
一次反応液への界面活性剤の添加効果について検討した。検体としては、ProMedDx社から購入した陰性血清検体(HBs抗原陰性、HBcAb陰性)である検体19〜24を用いた。検体19〜24は参考例1と同様に詳細検討を実施し、何れも陰性検体であることを確認している。検体希釈液としては、50 mM Tris緩衝液(150mM NaCl、1mM EDTA・2Na、pH7.2)を用いた。また、HBc抗原結合粒子としては、TritonX-305、TritonX-405およびTrironX-705をそれぞれ0.5%の濃度で含む50 mM Tris緩衝液(150mM NaCl、0.1%アジ化ナトリウム、2%BSA、pH7.2)に懸濁したHBc抗原結合粒子を用いて、実施例1と同様に抗HBc抗体を測定し、非特異反応低減率を求めた。
Example 7 Effect of addition of surfactant to primary reaction solution The effect of addition of surfactant to the primary reaction solution was examined. As samples, samples 19 to 24, which were negative serum samples (HBs antigen negative, HBcAb negative) purchased from ProMedDx, were used. Samples 19 to 24 were examined in detail in the same manner as in Reference Example 1 and confirmed to be negative samples. As the sample dilution, 50 mM Tris buffer (150 mM NaCl, 1 mM EDTA · 2Na, pH 7.2) was used. As HBc antigen-binding particles, 50 mM Tris buffer (150 mM NaCl, 0.1% sodium azide, 2% BSA, pH 7.2, each containing 0.5% TritonX-305, TritonX-405 and TrironX-705) The anti-HBc antibody was measured in the same manner as in Example 1 using the HBc antigen-binding particles suspended in (1), and the nonspecific reaction reduction rate was determined.
結果を表7に示す。HBc抗原結合粒子懸濁液に0.5%の界面活性剤を添加した場合(一次反応液中の界面活性剤の濃度が0.417%となる場合)、非特異反応低減率が大幅に低下していた。この結果から、一次反応液中に界面活性剤を含有させることにより、非特異反応が抑制できることが確認された。 The results are shown in Table 7. When 0.5% of a surfactant was added to the HBc antigen-binding particle suspension (when the concentration of the surfactant in the primary reaction solution was 0.417%), the nonspecific reaction reduction rate was greatly reduced. From this result, it was confirmed that a non-specific reaction can be suppressed by including a surfactant in the primary reaction solution.
Claims (20)
(1) 非イオン性界面活性剤
(2) 両性界面活性剤
から選択される少なくとも1種の界面活性剤が共存する条件下で、HBc抗原又は抗免疫グロブリン抗体若しくはその抗原結合性断片を含む試薬と検体を接触させて抗原抗体反応を行なうことを含む、方法。 An immunoassay method for anti-HBc antibody in a specimen, comprising the following (1) and (2):
(1) Nonionic surfactant
(2) An antigen-antibody reaction by contacting a specimen containing a reagent containing an HBc antigen or an anti-immunoglobulin antibody or an antigen-binding fragment thereof under conditions where at least one surfactant selected from amphoteric surfactants coexists. Performing the method.
(1) 非イオン性界面活性剤
(2) 両性界面活性剤
から選択される少なくとも1種の界面活性剤を含有する溶液と、HBc抗原とを含む、キット。 An anti-HBc antibody immunoassay kit comprising the following (1) and (2):
(1) Nonionic surfactant
(2) A kit comprising a solution containing at least one surfactant selected from amphoteric surfactants and an HBc antigen.
(1) 非イオン性界面活性剤
(2) 両性界面活性剤
から選択される少なくとも1種の界面活性剤を含有する、キット。 An anti-HBc antibody immunoassay kit comprising an antigen reagent solution containing an HBc antigen, the reagent solution comprising the following (1) and (2):
(1) Nonionic surfactant
(2) A kit containing at least one surfactant selected from amphoteric surfactants.
(1) 非イオン性界面活性剤
(2) 両性界面活性剤
から選択される少なくとも1種の界面活性剤を含有する、キット。 An anti-HBc antibody immunoassay kit comprising an antibody reagent solution containing an anti-immunoglobulin antibody or an antigen-binding fragment thereof and an HBc antigen, wherein the antibody reagent solution comprises the following (1) and (2):
(1) Nonionic surfactant
(2) A kit containing at least one surfactant selected from amphoteric surfactants.
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