JP2024002311A - Method of measuring c-peptide and reagent therefor - Google Patents
Method of measuring c-peptide and reagent therefor Download PDFInfo
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- JP2024002311A JP2024002311A JP2022101418A JP2022101418A JP2024002311A JP 2024002311 A JP2024002311 A JP 2024002311A JP 2022101418 A JP2022101418 A JP 2022101418A JP 2022101418 A JP2022101418 A JP 2022101418A JP 2024002311 A JP2024002311 A JP 2024002311A
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Abstract
Description
本発明は、Ｃ－ペプチドの測定方法及びそのための試薬に関する。 The present invention relates to a method for measuring C-peptide and a reagent therefor.
ヒトＣ－ペプチドは31個のアミノ酸からなるペプチドであり、インスリン前駆物質であるプロインスリンの構成成分である。Ｃ－ペプチドはプロインスリンが切断されインスリンが血中へ放出される際に、分解産物として同時に放出されるポリペプチドである。ヒトプロインスリンは、86アミノ酸からなるポリペプチドで、主として1～30番目のインスリンB鎖、33～63番目のＣ－ペプチド、66～86番目のインスリンA鎖で構成され、Ｃ－ペプチドとインスリンは31、32番目のArg、64番目Lys、65番目Argを経てそれぞれ結合している。Ｃ－ペプチドはインスリンが放出されると同時に血中に放出されるので、インスリンの分泌動態の指標としての役割を果たし、血中Ｃ－ペプチドの動態は糖尿病患者等の内因性インスリンの分泌能を調べるために重要な指標となり得る。 Human C-peptide is a peptide consisting of 31 amino acids and is a component of the insulin precursor proinsulin. C-peptide is a polypeptide that is simultaneously released as a degradation product when proinsulin is cleaved and insulin is released into the blood. Human proinsulin is a polypeptide consisting of 86 amino acids, mainly composed of insulin B chains 1 to 30, C-peptide 33 to 63, and insulin A chains 66 to 86. They are connected via the 31st and 32nd Arg, the 64th Lys, and the 65th Arg, respectively. Since C-peptide is released into the blood at the same time as insulin is released, it serves as an indicator of insulin secretion dynamics, and the dynamics of blood C-peptide influences the endogenous insulin secretion ability of diabetic patients. This can be an important indicator for investigation.
プロインスリンとの交叉反応性が低く、再現性が高く、かつ高感度でヒトＣ－ペプチドを測定し得る方法が報告されている（非特許文献１）。 A method capable of measuring human C-peptide with low cross-reactivity with proinsulin, high reproducibility, and high sensitivity has been reported (Non-Patent Document 1).
従来の方法でもヒトＣ－ペプチドを高感度に測定することが可能である。発明者らは、更に高感度なＣ－ペプチドの測定系を構築するため、従来の方法の定量下限（ＬＯＱ）よりも低濃度でＣ－ペプチドを含む検体を用いた測定系の構築を検討したところ、I型糖尿病患者の、特に低濃度のＣ－ペプチドを含む検体（低値検体）において、従来の方法では、Ｃ－ペプチドの濃度が正確に測定できない検体が存在することが判明した。 It is also possible to measure human C-peptide with high sensitivity using conventional methods. In order to construct an even more sensitive C-peptide measurement system, the inventors considered constructing a measurement system using a sample containing C-peptide at a concentration lower than the lower limit of quantification (LOQ) of conventional methods. However, it has been found that there are samples from type I diabetic patients that contain particularly low concentrations of C-peptide (low-value samples), in which the concentration of C-peptide cannot be accurately measured using conventional methods.
本発明は、低値検体においても、精度良くＣ－ペプチドを測定可能な、Ｃ－ペプチドの測定方法及び測定試薬を提供することを目的とする。 An object of the present invention is to provide a method for measuring C-peptide and a measuring reagent that can accurately measure C-peptide even in a sample with a low value.
本願発明者らは、鋭意研究の結果、試料中のＣ－ペプチドを測定する前に、アルカリ性物質又は酸性化剤のいずれかを含む前処理液と試料を混和することにより、低値検体においても、精度良くＣ－ペプチドを測定することが可能になることを見出し、本発明を完成した。 As a result of intensive research, the inventors of the present application have found that by mixing the sample with a pretreatment solution containing either an alkaline substance or an acidifying agent before measuring C-peptide in the sample, even low-value samples can be measured. They discovered that C-peptide can be measured with high accuracy, and completed the present invention.
すなわち、本発明は、以下のものを提供する。
(1) 生体から分離された試料と、アルカリ性物質又は酸性化剤のいずれかを含む前処理液とを混和する前処理工程を含む、生体から分離された試料中のＣ－ペプチドの測定方法。
(2) 前記試料中のＣ－ペプチドをイムノアッセイにより測定する、(1)に記載の方法。
(3) 前記前処理液がアルカリ性物質を含み、前処理工程が０．０１Ｎ以上１Ｎ以下のアルカリ濃度の条件下で行われる、(1)または(2)に記載の方法。
(4) 前記前処理液が酸性化剤を含み、前処理工程が０．０１Ｎ以上１Ｎ以下の酸濃度の条件下で行われる、(1)または(2)に記載の方法。
(5) 前記前処理液が界面活性剤を含む、(1)～(4)のいずれか１項に記載の方法。
(6) 前記前処理液がアルカリ性物質と界面活性剤を含み、該界面活性剤が陰イオン性界面活性剤である、(5)に記載の方法。
(7) 前記陰イオン性界面活性剤がＳＤＳ又はＮＬＳである、(6)に記載の方法。
(8) 前記前処理液が酸性化剤と界面活性剤を含み、該界面活性剤が陽イオン界面活性剤、非イオン性界面活性剤、又は両イオン性界面活性剤である、(5)に記載の方法。
(9) 前記前処理液が尿素を含む、(8)に記載の方法。
(10) 酸性化剤又はアルカリ性物質のいずれかを含む前処理液を備える、Ｃ－ペプチド測定用試薬。
That is, the present invention provides the following.
(1) A method for measuring C-peptide in a sample separated from a living body, including a pretreatment step of mixing the sample separated from the living body with a pretreatment liquid containing either an alkaline substance or an acidifying agent.
(2) The method according to (1), wherein the C-peptide in the sample is measured by immunoassay.
(3) The method according to (1) or (2), wherein the pretreatment liquid contains an alkaline substance, and the pretreatment step is performed at an alkaline concentration of 0.01N or more and 1N or less.
(4) The method according to (1) or (2), wherein the pretreatment liquid contains an acidifying agent, and the pretreatment step is performed at an acid concentration of 0.01N or more and 1N or less.
(5) The method according to any one of (1) to (4), wherein the pretreatment liquid contains a surfactant.
(6) The method according to (5), wherein the pretreatment liquid contains an alkaline substance and a surfactant, and the surfactant is an anionic surfactant.
(7) The method according to (6), wherein the anionic surfactant is SDS or NLS.
(8) According to (5), the pretreatment liquid contains an acidifying agent and a surfactant, and the surfactant is a cationic surfactant, a nonionic surfactant, or an amphoteric surfactant. Method described.
(9) The method according to (8), wherein the pretreatment liquid contains urea.
(10) A reagent for measuring C-peptide, comprising a pretreatment liquid containing either an acidifying agent or an alkaline substance.
本発明によれば、低値検体においても、精度良くＣ－ペプチドを測定することが可能になる。 According to the present invention, it is possible to accurately measure C-peptide even in a sample with a low value.
本明細書中で記載される「％」の濃度は、特に記載のない限り、重量／体積（ｗ／ｖ）の濃度表示である。 The "%" concentrations described herein are weight/volume (w/v) concentrations, unless otherwise specified.
＜Ｃ－ペプチドの測定方法＞
本発明で測定されるＣ－ペプチド（Ｃ－ペプチド）は、任意の動物由来のＣ－ペプチドであるが、好ましくは、哺乳動物（例、ヒト、サル、チンパンジー等の霊長類；マウス、ラット等の齧歯類、ウサギ等の重歯類；ウシ、ブタ、ヤギ、ウマ、ヒツジ等の有蹄類；イヌ、ネコ等の食肉類）；鳥類（例、ニワトリ））由来のＣ－ペプチドであり、より好ましくは霊長類由来のＣ－ペプチドであり、特に好ましくは、ヒト由来のＣ－ペプチドである。
<Method for measuring C-peptide>
The C-peptide (C-peptide) measured in the present invention is derived from any animal, but is preferably derived from mammals (e.g., humans, primates such as monkeys and chimpanzees; mice, rats, etc.). C-peptide derived from heavy teeth such as rodents and rabbits; ungulates such as cows, pigs, goats, horses, and sheep; carnivores such as dogs and cats; and birds (e.g., chickens)). , more preferably a C-peptide derived from a primate, and particularly preferably a C-peptide derived from a human.
１．前処理工程
本発明の方法は、生体から分離された生体試料中に存在するＣ－ペプチドを測定する方法である。Ｃ－ペプチドの測定方法としては、イムノアッセイが好ましいが、これに限定されるものではなく、例えば、質量分析等の他の方法によっても測定することができる。以下は、好ましい測定方法であるイムノアッセイでＣ－ペプチドを測定する場合について説明する。
1. Pretreatment Step The method of the present invention is a method for measuring C-peptide present in a biological sample separated from a living body. The preferred method for measuring C-peptide is immunoassay, but it is not limited thereto, and other methods such as mass spectrometry can also be used. The following describes the case where C-peptide is measured by immunoassay, which is a preferred measurement method.
本発明の方法は、イムノアッセイにおける免疫反応（反応工程）の前に、生体試料と前処理液とを混和することによる前処理工程を含むことを特徴とする。実施例に示すように、特に低濃度のＣ－ペプチドを含む検体（低値検体）において、従来の方法では、Ｃ－ペプチドの濃度が正確に測定できない検体が存在することが判明した。これは偽高値を引き起こす反応阻害物質によって引き起こされると考えられた。前処理工程により、低値検体においても、精度良くＣ－ペプチドを測定できるようになる。前処理液は、アルカリ性物質又は酸性化剤のいずれかを含む。 The method of the present invention is characterized by including a pretreatment step by mixing a biological sample and a pretreatment liquid before the immune reaction (reaction step) in the immunoassay. As shown in the Examples, it has been found that there are some samples in which the concentration of C-peptide cannot be accurately measured using conventional methods, especially in samples containing a low concentration of C-peptide (low-value samples). This was thought to be caused by reaction inhibitors that cause false highs. The pretreatment step makes it possible to accurately measure C-peptide even in low-value samples. The pretreatment liquid contains either an alkaline substance or an acidifying agent.
前記前処理工程において混和する生体試料と前処理液の体積比は、１：１０～１０：１、特に１：５～５：１、さらに１：３～３：１とすることが好ましい。本発明で用いられる生体試料は、Ｃ－ペプチドを含有し得る試料であれば特に限定されず、例えば、血清、血漿、全血、尿、便、口腔粘膜、咽頭粘膜、腸管粘膜、及び生検試料（例、甲状腺穿刺吸引細胞診（Ｆｉｎｅ ｎｅｅｄｌｅ ａｓｐｉｒａｔｉｏｎ：ＦＮＡ）試料、腸管試料、肝臓試料）が挙げられる。好ましくは、生体試料は、血清又は血漿である。 The volume ratio of the biological sample and the pretreatment liquid to be mixed in the pretreatment step is preferably 1:10 to 10:1, particularly 1:5 to 5:1, and more preferably 1:3 to 3:1. The biological sample used in the present invention is not particularly limited as long as it can contain C-peptide, and examples thereof include serum, plasma, whole blood, urine, stool, oral mucosa, pharyngeal mucosa, intestinal mucosa, and biopsy. Examples include samples (eg, fine needle aspiration (FNA) thyroid samples, intestinal samples, liver samples). Preferably, the biological sample is serum or plasma.
前記前処理液に含まれるアルカリ性物質としては、水酸化ナトリウム、水酸化カリウム等のアルカリ金属水酸化物、水酸化マグネシウム等のアルカリ土類水酸化物等を好適に使用できる。アルカリ性物質を使用する場合、前処理液のアルカリ性物質の規定度は、前処理時（混和後）の濃度として、０．０１Ｎ以上１Ｎ以下、特に０．１Ｎ以上０．４Ｎ以下とすることが好ましい。アルカリ性物質の規定度を０．０１Ｎ以上１Ｎ以下とすることで、前処理の効果が十分に得られ、かつ、後段の反応工程への影響を最小化することが可能である。 As the alkaline substance contained in the pretreatment liquid, alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkaline earth hydroxides such as magnesium hydroxide, and the like can be suitably used. When using an alkaline substance, the normality of the alkaline substance in the pretreatment liquid is preferably 0.01N or more and 1N or less, particularly 0.1N or more and 0.4N or less, as a concentration at the time of pretreatment (after mixing). . By setting the normality of the alkaline substance to 0.01N or more and 1N or less, the effect of the pretreatment can be sufficiently obtained, and the influence on the subsequent reaction process can be minimized.
本発明において、アルカリ性物質を含む前処理液と試料とを混和した際のｐＨは、添加するアルカリ性物質にもよるが、例えばｐＨ１０．０以上、好ましくはｐＨ１１．０以上、より好ましくはｐＨ１２．０以上である。また、本発明において、アルカリ性物質を含む前処理液と試料とを混和した際のｐＨは、添加するアルカリ性物質にもよるが、例えばｐＨ１３．７以下、好ましくはｐＨ１３．５以下、より好ましくはｐＨ１３．３以下である。具体的には、アルカリ性物質を含む前処理液と試料とを混和した際のｐＨは、例えばｐＨ１０．０～１３．７、好ましくはｐＨ１１．０～１３．５、より好ましくはｐＨ１２．０～１３．３である。前処理工程におけるｐＨをこれらの範囲とすることで、前処理の効果が十分に得られ、かつ、後段の反応工程への影響を最小化することが可能である。 In the present invention, the pH when the pretreatment liquid containing an alkaline substance and the sample are mixed depends on the alkaline substance added, but is, for example, pH 10.0 or higher, preferably pH 11.0 or higher, and more preferably pH 12.0. That's all. Further, in the present invention, the pH when the sample is mixed with the pretreatment liquid containing an alkaline substance depends on the alkaline substance added, but is, for example, pH 13.7 or lower, preferably pH 13.5 or lower, more preferably pH 13. .3 or less. Specifically, the pH when the pretreatment liquid containing an alkaline substance and the sample are mixed is, for example, pH 10.0 to 13.7, preferably pH 11.0 to 13.5, more preferably pH 12.0 to 13. .3. By setting the pH in the pretreatment step within these ranges, the effect of the pretreatment can be sufficiently obtained, and the influence on the subsequent reaction step can be minimized.
前記前処理液に含まれる酸性化剤としては、塩酸、硫酸、酢酸等を好適に使用できる。酸性化剤を使用する場合、前処理液の酸の規定度は、前処理時の濃度として、０．０１Ｎ以上１Ｎ以下、特に０．１Ｎ以上０．４Ｎ以下とすることが好ましい。酸の規定度を０．０１Ｎ以上１Ｎ以下とすることで、前処理の効果が十分に得られ、かつ、後段の反応工程への影響を最小化することが可能である。 As the acidifying agent contained in the pretreatment liquid, hydrochloric acid, sulfuric acid, acetic acid, etc. can be suitably used. When using an acidifying agent, the normality of the acid in the pretreatment liquid is preferably 0.01N or more and 1N or less, particularly 0.1N or more and 0.4N or less, as a concentration during pretreatment. By setting the normality of the acid to 0.01N or more and 1N or less, the effect of the pretreatment can be sufficiently obtained, and the influence on the subsequent reaction process can be minimized.
本発明において、酸性物質を含む前処理液と試料とを混和した際のｐＨは、添加する酸性物質にもよるが、例えばｐＨ４．８以下、好ましくはｐＨ４．５以下、より好ましくはｐＨ４．２以下である。また、本発明において、酸性物質を含む前処理液と試料とを混和した際のｐＨは、添加する酸性物質にもよるが、例えばｐＨ０．３以上、好ましくはｐＨ０．４以上、より好ましくはｐＨ０．５以上である。具体的には、酸性物質を含む前処理液と試料とを混和した際のｐＨは、例えばｐＨ０．３～４．８、好ましくはｐＨ０．４～４．５、より好ましくはｐＨ０．５～４．２である。前処理工程におけるｐＨをこれらの範囲とすることで、前処理の効果が十分に得られ、かつ、後段の反応工程への影響を最小化することが可能である。 In the present invention, the pH when the pretreatment liquid containing an acidic substance and the sample are mixed depends on the acidic substance added, but is, for example, pH 4.8 or lower, preferably pH 4.5 or lower, more preferably pH 4.2. It is as follows. In addition, in the present invention, the pH when the pretreatment liquid containing an acidic substance and the sample are mixed depends on the acidic substance added, but is, for example, pH 0.3 or higher, preferably pH 0.4 or higher, and more preferably pH 0. .5 or more. Specifically, the pH when mixing the pretreatment liquid containing an acidic substance and the sample is, for example, pH 0.3 to 4.8, preferably pH 0.4 to 4.5, more preferably pH 0.5 to 4. .2. By setting the pH in the pretreatment step within these ranges, the effect of the pretreatment can be sufficiently obtained, and the influence on the subsequent reaction step can be minimized.
本発明において、酸性物質を含む前処理液と試料とを混和した際のｐＨは、添加する界面活性剤や変性剤の種類や濃度によって、上記の例示の範囲で、より至適なｐＨを設定することができる。 In the present invention, the pH when the sample is mixed with the pretreatment liquid containing an acidic substance is set to a more optimal pH within the above-mentioned range depending on the type and concentration of the surfactant and denaturant to be added. can do.
前処理液は、界面活性剤を含んでいてもよい。前処理液がアルカリ性物質を含む場合、界面活性剤としては、陰イオン性界面活性剤が好ましい。陰イオン性界面活性剤としては、ドデシル硫酸ナトリウム（ＳＤＳ）、ドデシル硫酸リチウム等の硫酸エステル型界面活性剤、Ｎ-ラウロイルサルコシンナトリウム（ＮＬＳ）等のカルボン酸型界面活性剤、ドデシルベンゼンスルホン酸ナトリウム等のスルホン酸型界面活性剤、デオキシコール酸、コール酸等の胆汁酸若しくはその誘導体、又はそれらの塩等を好適に使用でき、特にＳＤＳ及びＮＬＳを好適に使用できる。界面活性剤の濃度は、特に限定されず、適宜設定できるが、例えばＳＤＳ又はＮＬＳを使用する場合は、生体試料と混和した混和液の前処理時の濃度として、０．０１～１２．５％、特に０．０５～１０％、さらに０．１～７．５％とすることが好ましい。ＳＤＳを使用する場合は、ＳＤＳの濃度を０．０５～１０％とすることで、本発明の効果をさらに高めることができる。 The pretreatment liquid may contain a surfactant. When the pretreatment liquid contains an alkaline substance, the surfactant is preferably an anionic surfactant. Examples of anionic surfactants include sulfate ester type surfactants such as sodium dodecyl sulfate (SDS) and lithium dodecyl sulfate, carboxylic acid type surfactants such as sodium N-lauroylsarcosine (NLS), and sodium dodecylbenzenesulfonate. Sulfonic acid type surfactants such as deoxycholic acid, bile acids such as cholic acid or derivatives thereof, or salts thereof can be preferably used, and SDS and NLS can be particularly preferably used. The concentration of the surfactant is not particularly limited and can be set as appropriate, but for example, when using SDS or NLS, the concentration at the time of pretreatment of the mixture mixed with the biological sample is 0.01 to 12.5%. , particularly preferably from 0.05 to 10%, more preferably from 0.1 to 7.5%. When using SDS, the effect of the present invention can be further enhanced by setting the concentration of SDS to 0.05 to 10%.
前処理液が陰イオン性界面活性剤を含む場合、前処理後に、反応系に持ち込まれる陰イオン性界面活性剤の影響を軽減するために、陽イオン性界面活性剤、両イオン性界面活性剤、非イオン性界面活性剤を単独又は複数を添加してもよい。前処理後に添加する陽イオン性界面活性剤、両イオン性界面活性剤、及び非イオン性界面活性剤は、下記の中和液に添加してもよい。 When the pretreatment solution contains an anionic surfactant, a cationic surfactant or amphoteric surfactant is added to reduce the effect of the anionic surfactant brought into the reaction system after the pretreatment. , one or more nonionic surfactants may be added. The cationic surfactant, amphoteric surfactant, and nonionic surfactant added after the pretreatment may be added to the neutralization solution described below.
前処理液が酸性化剤と界面活性剤を含む場合、該界面活性剤が陽イオン性界面活性剤、非イオン性界面活性剤、両イオン性界面活性剤、又はそれらの組合せであることが好ましい。陽イオン性界面活性剤としては、炭素数１０個以上の一本鎖アルキル基と、第３級アミン又は第４級アンモニウム塩を同分子中に有している陽イオン性界面活性剤が好ましい。このような陽イオン性界面活性剤の例としては、デシルトリメチルアンモニウムクロライド、ドデシルトリメチルアンモニウムクロライド、テトラデシルトリメチルアンモニウムクロライド、ヘキサデシルトリメチルアンモニウムクロライド、デシルトリメチルアンモニウムブロマイド、ドデシルトリメチルアンモニウムブロマイド、テトラデシルトリメチルアンモニウムブロマイド、ヘキサデシルトリメチルアンモニウムブロマイド、ラウリルピリジニウムクロライド、テトラデシルピリジニウムクロライド、セチルピリジニウムクロライド等が挙げられる。非イオン性界面活性剤としては、ポリオキシエチレンソルビタンモノラウレート（Ｔｗｅｅｎ２０）、ポリオキシエチレンソルビタンモノオレエート（Ｔｗｅｅｎ８０）等のポリオキシエチレンソルビタン脂肪酸エステル（例えば、Ｔｗｅｅｎ（商品名・登録商標）シリーズ）、ポリオキシエチレンオクチルフェニルエーテル等のポリオキシエチレンアルキルフェニルエーテル（例えば、Ｔｒｉｔｏｎ（商品名・登録商標）シリーズ）等が挙げられる。両イオン性界面活性剤としては、ＣＨＡＰＳ、ＣＨＡＰＳＯ、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）等のスルホベタイン型界面活性剤等が挙げられる。これらの界面活性剤の添加量は、検体との混和時の濃度で０．１％以上１５%以下が好ましく、さらに、０．５％～１０％が好ましい。 When the pretreatment liquid includes an acidifying agent and a surfactant, the surfactant is preferably a cationic surfactant, a nonionic surfactant, a zwitterionic surfactant, or a combination thereof. . As the cationic surfactant, a cationic surfactant having a single-chain alkyl group having 10 or more carbon atoms and a tertiary amine or a quaternary ammonium salt in the same molecule is preferable. Examples of such cationic surfactants include decyltrimethylammonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride, decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium Bromide, hexadecyltrimethylammonium bromide, laurylpyridinium chloride, tetradecylpyridinium chloride, cetylpyridinium chloride and the like. Examples of nonionic surfactants include polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monolaurate (Tween 20) and polyoxyethylene sorbitan monooleate (Tween 80) (for example, Tween (trade name/registered trademark) series). ), polyoxyethylene alkyl phenyl ethers such as polyoxyethylene octylphenyl ether (eg, Triton (trade name, registered trademark) series), and the like. Examples of amphoteric surfactants include CHAPS, CHAPSO, N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C12APS), N-tetradecyl-N,N-dimethyl-3-ammonio-1 Examples include sulfobetaine type surfactants such as -propanesulfonate (C14APS) and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C16APS). The amount of these surfactants added is preferably 0.1% or more and 15% or less, and more preferably 0.5% to 10% in concentration when mixed with the specimen.
前処理液には、必要に応じて、尿素、チオ尿素等、他のタンパク変性剤が含まれていてもよい。変性剤の濃度は、処理時濃度で０．１Ｍ以上が好ましく、さらに０．５Ｍ以上４Ｍ未満が好ましい。また、前処理液には、処理効果を増強させるために、単糖類、二糖類、クエン酸、及びクエン酸塩類のいずれか、又はこれらを組合せて添加してもよい。さらに、前処理液には、ＥＤＴＡ等のキレート剤が含まれていてもよい。 The pretreatment liquid may contain other protein denaturing agents such as urea and thiourea as necessary. The concentration of the modifier during treatment is preferably 0.1M or more, more preferably 0.5M or more and less than 4M. Furthermore, in order to enhance the treatment effect, any one of monosaccharides, disaccharides, citric acid, and citrates, or a combination of these may be added to the pretreatment liquid. Furthermore, the pretreatment liquid may contain a chelating agent such as EDTA.
前処理工程は、生体試料と前処理液を混和し、混合液を室温で放置することにより行うことができる。前処理時間は、精度良くＣ－ペプチドを測定できるように検体を処理可能な時間であればよく、特に限定されないが、例えば、1秒以上、10秒以上、30秒以上、１分以上、３分以上、５分以上とすることができる。前処理時間の上限は特に存在しないが、６０分以下、３０分以下、特には１５分以下でよい。 The pretreatment step can be performed by mixing the biological sample and the pretreatment liquid and leaving the mixed liquid at room temperature. The pretreatment time is not particularly limited, as long as the sample can be processed so that C-peptide can be measured with high precision, but examples include 1 second or more, 10 seconds or more, 30 seconds or more, 1 minute or more, or 30 seconds or more. The duration may be longer than 5 minutes, or longer than 5 minutes. There is no particular upper limit to the pretreatment time, but it may be 60 minutes or less, 30 minutes or less, particularly 15 minutes or less.
前処理工程は、生体試料と前処理液を混和した後、さらに加熱してもよい。加熱温度は３０～５０℃とすることが好ましい。また、加熱時間は、前処理時間に含まれ、精度良くＣ－ペプチドを測定できるように検体を処理可能な時間であればよく、特に限定されないが、例えば、1秒以上、10秒以上、30秒以上、１分以上、３分以上、５分以上とすることができる。加熱時間の上限は特に存在しないが、通常、６０分以下、３０分以下、特には１５分以下でよい。 In the pretreatment step, the biological sample and the pretreatment liquid may be mixed and then further heated. The heating temperature is preferably 30 to 50°C. The heating time is not particularly limited, as long as it is included in the pretreatment time and allows the sample to be processed so that C-peptide can be measured with high accuracy. The time can be set to be longer than seconds, longer than 1 minute, longer than 3 minutes, or longer than 5 minutes. There is no particular upper limit to the heating time, but it may generally be 60 minutes or less, 30 minutes or less, particularly 15 minutes or less.
前処理液がアルカリ性物質を含む場合、前処理後に、ｐＨを中和するために酸性化剤を含む中和液を添加する工程を更に含んでもよい。酸性化剤としては上記したものが挙げられる。また、中和液を添加する工程を含まない場合、例えば、下記の反応工程において、混合する緩衝液の緩衝能及びｐＨ等を調整することで、前処理液のアルカリ性物質を中和し、反応工程における影響を緩和してもよい。反応工程におけるｐＨは、反応工程に含まれる成分によって適宜設定することができるが、例えば、ｐＨ５．５～９．５になるように反応工程の液中に含まれる酸性化剤又は緩衝液等の添加量を適宜設定することができる。また、中和液を添加後のｐＨが、上記の反応工程におけるｐＨよりも高い場合又は低い場合は、反応工程において、混合する緩衝液の緩衝能及びｐＨ等を調整することで、前処理液のアルカリ性物質を中和し、反応工程における影響を緩和してもよい。 When the pretreatment liquid contains an alkaline substance, the method may further include a step of adding a neutralizing liquid containing an acidifying agent to neutralize the pH after the pretreatment. Examples of the acidifying agent include those mentioned above. In addition, if the step of adding a neutralizing solution is not included, for example, in the reaction step below, by adjusting the buffer capacity and pH of the buffer solution to be mixed, the alkaline substance in the pretreatment solution is neutralized and the reaction is performed. The influence on the process may be alleviated. The pH in the reaction process can be set appropriately depending on the components included in the reaction process, but for example, the pH in the reaction process may be adjusted using an acidifying agent or a buffer solution contained in the reaction process solution to adjust the pH to 5.5 to 9.5. The amount added can be set appropriately. In addition, if the pH after adding the neutralizing solution is higher or lower than the pH in the above reaction step, in the reaction step, by adjusting the buffer capacity and pH of the buffer solution to be mixed, The alkaline substance may be neutralized to reduce its influence on the reaction process.
前処理液が酸性化剤を含む場合、前処理後に、ｐＨを中和するためにアルカリ性物質を含む中和液を添加する工程を更に含んでもよい。アルカリ性物質としては上記したものが挙げられる。また、中和液を添加する工程を含まない場合、例えば、下記の反応工程において、混合する緩衝液の緩衝能及びｐＨ等を調整することで、前処理液の酸性化剤を中和し、反応工程における影響を緩和してもよい。反応工程におけるｐＨは、反応工程に含まれる成分によって適宜設定することができるが、例えば、ｐＨ５．５～９．５になるように反応工程の液中に含まれる酸性化剤又は緩衝液等の添加量を適宜設定することができる。また、中和液を添加後のｐＨが、上記の反応工程におけるｐＨよりも高い場合又は低い場合は、反応工程において、混合する緩衝液の緩衝能及びｐＨ等を調整することで、前処理液の酸性化剤を中和し、反応工程における影響を緩和してもよい。 When the pretreatment liquid contains an acidifying agent, the method may further include a step of adding a neutralizing liquid containing an alkaline substance to neutralize the pH after the pretreatment. Examples of the alkaline substance include those mentioned above. In addition, if the step of adding a neutralizing solution is not included, for example, in the following reaction step, the acidifying agent in the pretreatment solution is neutralized by adjusting the buffer capacity and pH of the buffer solution to be mixed, The influence on the reaction process may be alleviated. The pH in the reaction process can be set appropriately depending on the components included in the reaction process, but for example, the pH in the reaction process may be adjusted using an acidifying agent or a buffer solution contained in the reaction process solution to adjust the pH to 5.5 to 9.5. The amount added can be set appropriately. In addition, if the pH after adding the neutralizing solution is higher or lower than the pH in the above reaction step, in the reaction step, by adjusting the buffer capacity and pH of the buffer solution to be mixed, The acidifying agent may be neutralized to reduce its effect on the reaction process.
２．反応工程
本発明の方法の上記前処理工程で得られた生体試料混和液（生体試料と前処理液とを混和し前処理して得られた試料、又は混和液を中和して得られた試料）は、次いでイムノアッセイの反応工程に供される。反応工程においては、生体試料混和液を緩衝液と混合させ、混合液中の抗原をＣ－ペプチドに対する抗体と反応させる。なお、Ｃ－ペプチドのイムノアッセイ自体は種々の方法が周知であり、Ｃ－ペプチドを定量可能ないずれのイムノアッセイをも採用することができる。
2. Reaction step: The biological sample mixture obtained in the above pretreatment step of the method of the present invention (a sample obtained by mixing and pretreating a biological sample and a pretreatment liquid, or a sample obtained by neutralizing the mixture) The sample) is then subjected to an immunoassay reaction step. In the reaction step, the biological sample mixture is mixed with a buffer solution, and the antigen in the mixture is reacted with an antibody against C-peptide. Note that various methods for C-peptide immunoassay are well known, and any immunoassay capable of quantifying C-peptide can be employed.
前記緩衝液としては、例えば、ＭＥＳ緩衝液、リン酸緩衝液、Ｔｒｉｓ緩衝液、炭酸緩衝液をベースとしたものが挙げられ、特にＴｒｉｓ緩衝液をベースとしたものを好適に使用できる。前処理液として界面活性剤を含有するものを使用した場合には、未反応の界面活性剤を吸収するために、例えば、ＢＳＡ、ポリビニルピロリドン（ＰＶＰ）、ポリビニルアルコール（ＰＶＡ）デキストラン硫酸ナトリウム等の水溶性高分子を、前処理後の混和液と混合した際の終濃度で０．０１～１０．０％、特に０．０５～５．０％程度含む緩衝液を使用することが好ましい。また、前処理工程において、アルカリ性物質を含有する前処理液を使用した場合、前記緩衝液はアルカリ性物質の影響を緩和し得る緩衝能を有する緩衝液を使用してもよい。前記の通り、中和工程を含まない場合、又は中和後のｐＨが高い場合は、アルカリ性物質の影響を緩和し得る緩衝能を有する緩衝液を使用することが好ましい。また、酸性化剤を含有する前処理液を使用した場合は、前処理液の酸の影響を緩和し得る緩衝能を有する緩衝液を使用してもよい。前記の通り、中和工程を含まない場合、又は中和後のｐＨが低い場合は、酸性化剤の影響を緩和し得る緩衝能を有する緩衝液を使用することが好ましい。前処理工程の混和液と緩衝液との混合は、体積比で、例えば、１：１０～１０：１、特に１：５～５：１、さらに１：３～３：１としてもよい。 Examples of the buffer include those based on MES buffer, phosphate buffer, Tris buffer, and carbonate buffer, and those based on Tris buffer can be particularly preferably used. When using a pretreatment liquid containing a surfactant, for example, BSA, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), dextran sodium sulfate, etc. are used to absorb unreacted surfactant. It is preferable to use a buffer solution containing the water-soluble polymer at a final concentration of about 0.01 to 10.0%, especially about 0.05 to 5.0% when mixed with the mixed solution after pretreatment. Further, in the pretreatment step, when a pretreatment liquid containing an alkaline substance is used, the buffer may be a buffer having a buffering capacity capable of alleviating the influence of the alkaline substance. As mentioned above, when the neutralization step is not included or when the pH after neutralization is high, it is preferable to use a buffer solution having a buffering capacity capable of alleviating the influence of alkaline substances. Furthermore, when a pretreatment liquid containing an acidifying agent is used, a buffer having a buffering capacity capable of alleviating the influence of the acid in the pretreatment liquid may be used. As mentioned above, when the neutralization step is not included or when the pH after neutralization is low, it is preferable to use a buffer solution having a buffering capacity that can alleviate the influence of the acidifying agent. The mixture and buffer solution in the pretreatment step may be mixed at a volume ratio of, for example, 1:10 to 10:1, particularly 1:5 to 5:1, and further 1:3 to 3:1.
本発明の方法で使用されるＣ－ペプチドに対する抗体は、Ｃ－ペプチドのアミノ酸配列の少なくとも一部をエピトープとして認識する抗体である。Ｃ－ペプチドに対する抗体は、特に限定されず、既知のエピトープを認識する抗体をいずれも使用することができるが、好ましくは、Ｃ－ペプチドに対する抗体は、Ｃ－ペプチド特異的エピトープ（特に、ヒトＣ－ペプチド特異的エピトープ）を認識する抗体である。 The antibody against C-peptide used in the method of the present invention is an antibody that recognizes at least part of the amino acid sequence of C-peptide as an epitope. The antibody against C-peptide is not particularly limited, and any antibody that recognizes a known epitope can be used. Preferably, the antibody against C-peptide is a C-peptide-specific epitope (particularly human C-peptide). - an antibody that recognizes a peptide-specific epitope).
Ｃ－ペプチドに対する抗体は、ポリクローナル抗体又はモノクローナル抗体のいずれであってもよい。Ｃ－ペプチドに対する抗体は、免疫グロブリン（例、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＤ、ＩｇＥ、ＩｇＹ）のいずれのアイソタイプであってもよい。Ｃ－ペプチドに対する抗体はまた、全長抗体であってもよい。全長抗体とは、可変領域及び定常領域を各々含む重鎖及び軽鎖を含む抗体（例、２つのＦａｂ部分及びＦｃ部分を含む抗体）をいう。Ｃ－ペプチドに対する抗体はまた、このような全長抗体に由来する抗体断片であってもよい。抗体断片は、全長抗体の一部であり、例えば、定常領域欠失抗体（例、Ｆ（ａｂ’）２、Ｆａｂ’、Ｆａｂ、Ｆｖ）が挙げられる。Ｃ－ペプチドに対する抗体はまた、単鎖抗体等の改変抗体であってもよい。 Antibodies against C-peptide may be either polyclonal or monoclonal antibodies. Antibodies against C-peptide may be of any isotype of immunoglobulin (eg, IgG, IgM, IgA, IgD, IgE, IgY). Antibodies against C-peptide may also be full-length antibodies. A full-length antibody refers to an antibody that includes a heavy chain and a light chain, each containing a variable region and a constant region (eg, an antibody that includes two Fab portions and an Fc portion). Antibodies against C-peptide may also be antibody fragments derived from such full-length antibodies. Antibody fragments are parts of full-length antibodies, and include, for example, constant region deleted antibodies (eg, F(ab')2, Fab', Fab, Fv). Antibodies against C-peptide may also be modified antibodies such as single chain antibodies.
Ｃ－ペプチドに対する抗体は、従前公知の方法を用いて作製することができる。例えば、Ｃ－ペプチドに対する抗体は、上記のエピトープを抗原として用いて作製することができる。また、上述したようなエピトープを認識するＣ－ペプチドに対する多数の抗体が市販されているので、このような市販品を使用することもできる。 Antibodies against C-peptide can be produced using previously known methods. For example, antibodies against C-peptide can be generated using the epitopes described above as antigens. Furthermore, since many antibodies against C-peptide that recognize the above-mentioned epitopes are commercially available, such commercially available products can also be used.
Ｃ－ペプチドに対する抗体は、固相に固相化されていてもよい。またＣ－ペプチドに対する抗体は、反応工程において固相に固相化され得る抗体であってもよい。本明細書において、固相に固相化された抗体、及び反応工程において固相に固相化され得る抗体を、単に固相化抗体ということがある。固相としては、例えば、液相を収容又は搭載可能な固相（例、プレート、メンブレン、試験管等の支持体、及びウェルプレート、マイクロ流路、ガラスキャピラリー、ナノピラー、モノリスカラム等の容器）、ならびに液相中に懸濁又は分散可能な固相（例、粒子等の固相担体）が挙げられる。固相の材料としては、例えば、ガラス、プラスチック、金属、及びカーボンが挙げられる。固相の材料としてはまた、非磁性材料、又は磁性材料を用いることができるが、操作の簡便性等の観点から、磁性材料が好ましい。固相は、好ましくは固相担体であり、より好ましくは磁性固相担体であり、さらにより好ましくは磁性粒子である。抗体の固相化方法としては、従前公知の方法を利用することができる。このような方法としては、例えば、物理的吸着法、共有結合法、親和性物質（例、ビオチン、ストレプトアビジン）を利用する方法、及びイオン結合法が挙げられる。特定の実施形態では、Ｃ－ペプチドに対する抗体は、固相に固相化された抗体であり、好ましくは、磁性の固相に固相化された抗体であり、より好ましくは、磁性粒子に固相化された抗体である。 The antibody against C-peptide may be immobilized on a solid phase. Furthermore, the antibody against C-peptide may be an antibody that can be immobilized on a solid phase in the reaction step. In this specification, an antibody immobilized on a solid phase and an antibody that can be immobilized on a solid phase in a reaction step are sometimes simply referred to as immobilized antibodies. Examples of the solid phase include solid phases that can accommodate or load a liquid phase (e.g., supports such as plates, membranes, and test tubes, and containers such as well plates, microchannels, glass capillaries, nanopillars, and monolith columns). , as well as solid phases (eg, solid phase supports such as particles) that can be suspended or dispersed in a liquid phase. Examples of solid phase materials include glass, plastic, metal, and carbon. As the material of the solid phase, a non-magnetic material or a magnetic material can be used, but a magnetic material is preferable from the viewpoint of ease of operation. The solid phase is preferably a solid phase carrier, more preferably a magnetic solid phase carrier, and even more preferably magnetic particles. As a method for immobilizing antibodies, previously known methods can be used. Examples of such methods include physical adsorption methods, covalent bonding methods, methods using affinity substances (eg, biotin, streptavidin), and ionic bonding methods. In certain embodiments, the antibody against C-peptide is an antibody immobilized on a solid phase, preferably an antibody immobilized on a magnetic solid phase, more preferably an antibody immobilized on a magnetic particle. It is a phased antibody.
反応工程は、前処理工程の混和液と緩衝液とを混合した後、固相化抗体に接触させてもよく、前記混和液と、固相化抗体を含む緩衝液とを混合してもよい。例えば、緩衝液中に、粒子上に固相化した抗体を予め入れて粒子液とし、前記混和液と粒子液とを混合させてもよい。反応工程は、例えば免疫凝集法や競合法のように一次反応工程のみで実施してもよいが、サンドイッチ法のように二次反応工程を設けてもよい。なお、二次反応工程を設ける場合、一次反応工程と二次反応工程の間に、未反応成分を除去するための洗浄工程を設けてもよい。またサンドイッチ１ステップ法のように、一次反応と二次反応を同時に行ってもよい。 In the reaction step, the mixed solution of the pretreatment step and a buffer solution may be mixed and then brought into contact with the immobilized antibody, or the mixed solution and a buffer solution containing the immobilized antibody may be mixed. . For example, an antibody immobilized on particles may be added to a buffer solution in advance to form a particle liquid, and the mixed liquid and the particle liquid may be mixed. The reaction step may be carried out using only a primary reaction step, such as an immunoagglutination method or a competitive method, or may include a secondary reaction step, such as a sandwich method. In addition, when providing a secondary reaction step, a washing step for removing unreacted components may be provided between the primary reaction step and the secondary reaction step. Moreover, the primary reaction and the secondary reaction may be performed simultaneously as in the sandwich one-step method.
Ｃ－ペプチドに対する抗体は、標識物質で標識化されていてもよい。本明細書において、標識物質で標識化された抗体を、単に標識化抗体ということがある。標識物質としては、例えば、酵素（例、ペルオキシダーゼ、アルカリフォスファターゼ、ルシフェラーゼ、βガラクトシダーゼ）、親和性物質（例、ストレプトアビジン、ビオチン）、蛍光物質又は蛍光タンパク質（例、フルオレセイン、フルオレセインイソチオシアネート、ローダミン、緑色蛍光タンパク質、赤色蛍光タンパク質）、発光物質又は吸光物質（例、ルシフェリン、エクオリン、アクリジニウム、ルテニウム）、放射性物質（例、３Ｈ、１４Ｃ、３２Ｐ、３５Ｓ、１２５Ｉ）が挙げられる。また、本発明の方法では二次反応を設ける場合、二次反応に用いる抗体は、このような標識物質で標識化されていてもよい。 The antibody against C-peptide may be labeled with a labeling substance. In this specification, an antibody labeled with a labeling substance may be simply referred to as a labeled antibody. Examples of labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin), fluorescent substances or fluorescent proteins (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent or light-absorbing substances (eg, luciferin, aequorin, acridinium, ruthenium), and radioactive substances (eg, 3 H, 14 C, 32 P, 35 S, 125 I). Furthermore, when a secondary reaction is provided in the method of the present invention, the antibody used in the secondary reaction may be labeled with such a labeling substance.
特定の実施形態では、本発明の方法は、二次反応に用いる抗体として、一次反応に用いるＣ－ペプチドに対する抗体と異なるエピトープを認識するＣ－ペプチドに対する別の抗体を含む。このような別の抗体が認識するエピトープの詳細は、上述したＣ－ペプチドに対する抗体について詳述したエピトープと同様である（但し、併用される場合、エピトープの種類は異なる）。Ｃ－ペプチドに対する抗体により認識されるエピトープと、Ｃ－ペプチドに対する別の抗体により認識されるエピトープとの組合せは、特に限定されない。このような別の抗体の使用は、例えば、サンドイッチ法が利用される場合に好ましい。 In certain embodiments, the methods of the invention include as the antibody used in the secondary reaction another antibody to C-peptide that recognizes a different epitope than the antibody to C-peptide used in the primary reaction. The details of the epitope recognized by such another antibody are the same as the epitope detailed above for the antibody against C-peptide (however, when used in combination, the types of epitopes are different). The combination of an epitope recognized by an antibody against C-peptide and an epitope recognized by another antibody against C-peptide is not particularly limited. The use of such further antibodies is preferred, for example, when a sandwich method is utilized.
３． 検出工程
一次抗体又は二次抗体に標識を用いた場合、使用する標識に適した方法、例えば酵素標識を用いた場合は酵素の基質を添加することによって、検出することができる。例えば、アルカリフォスファターゼ（ＡＬＰ）を標識抗体として用いた場合は、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）を酵素基質として用いた化学発光酵素イムノアッセイ法（ＣＬＥＩＡ）の系とすることができる。
3. Detection Step When a label is used for the primary antibody or secondary antibody, detection can be performed by a method suitable for the label used, for example, when an enzyme label is used, by adding a substrate for the enzyme. For example, when alkaline phosphatase (ALP) is used as a labeled antibody, 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane disodium salt It can be used as a chemiluminescent enzyme immunoassay (CLEIA) system using (AMPPD) as an enzyme substrate.
本発明の方法は、Ｃ－ペプチドに対する抗体を使用するイムノアッセイである。このようなイムノアッセイとしては、例えば、直接競合法、間接競合法、及びサンドイッチ法が挙げられる。また、このようなイムノアッセイとしては、例えば、化学発光酵素イムノアッセイ法（ＣＬＥＩＡ）、化学発光イムノアッセイ（ＣＬＩＡ）、免疫比濁法（ＴＩＡ）、酵素イムノアッセイ法（ＥＩＡ）（例、直接競合ＥＬＩＳＡ、間接競合ＥＬＩＳＡ、及びサンドイッチＥＬＩＳＡ）、ラジオイムノアッセイ（ＲＩＡ）、ラテックス凝集反応法、蛍光イムノアッセイ（ＦＩＡ）、及びイムノクロマトグラフィー法が挙げられる。これらのイムノアッセイ自体は周知であり、ここで詳しく述べる必要はないが、それぞれ簡単に説明する。 The method of the invention is an immunoassay using antibodies against C-peptide. Such immunoassays include, for example, direct competitive methods, indirect competitive methods, and sandwich methods. In addition, such immunoassays include, for example, chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), immunoturbidimetry (TIA), enzyme immunoassay (EIA) (e.g., direct competitive ELISA, indirect competitive ELISA). ELISA, and sandwich ELISA), radioimmunoassay (RIA), latex agglutination, fluorescence immunoassay (FIA), and immunochromatography. These immunoassays themselves are well known and need not be described in detail here, but each will be briefly explained.
直接競合法は、例えば、測定すべき標的抗原（本発明ではＣ－ペプチド）に対する抗体を固相に固相化し（固相及び固相化については上記のとおり）、非特異吸着を防ぐためのブロッキング処理（血清アルブミン等のタンパク質溶液で固相を処理）後、この抗体と、前記標的抗原を含む被検試料（本発明では、上記のとおり前処理工程を行った生体試料）と、一定量の標識した抗原（標識は上記のとおり）とを反応させ、洗浄後、固相に結合した標識を定量する方法である。被検試料中の抗原と標識抗原とが、抗体に対して競合的に結合するので、被検試料中の抗原量が多いほど、固相に結合する標識の量が少なくなる。種々の既知濃度の抗原標準液を作製し、それぞれについて固相に固定化された標識量（標識の性質に応じて、吸光度、発光強度、蛍光強度等、以下同じ）を測定して、抗原濃度を横軸、標識量を縦軸にとった検量線を作成する。未知の被検試料について、標識量を測定し、測定された標識量を検量線に当てはめることにより、未知の被検試料中の抗原量を測定することができる。直接競合法自体はこの分野において周知であり、例えば、US 20150166678Aに記載されている。 In the direct competition method, for example, an antibody against the target antigen to be measured (C-peptide in the present invention) is immobilized on a solid phase (the solid phase and immobilization are as described above), and a After blocking treatment (treatment of the solid phase with a protein solution such as serum albumin), this antibody and a test sample containing the target antigen (in the present invention, a biological sample that has undergone the pretreatment step as described above) are combined in a certain amount. This method involves reacting with a labeled antigen (the label is as described above) and, after washing, quantifying the label bound to the solid phase. Since the antigen in the test sample and the labeled antigen competitively bind to the antibody, the larger the amount of antigen in the test sample, the smaller the amount of label bound to the solid phase. Prepare antigen standard solutions with various known concentrations, measure the amount of label immobilized on the solid phase for each (absorbance, luminescence intensity, fluorescence intensity, etc., depending on the nature of the label, the same applies hereinafter), and determine the antigen concentration. Create a calibration curve with the amount of label on the horizontal axis and the amount of label on the vertical axis. The amount of antigen in the unknown test sample can be determined by measuring the amount of label in the unknown test sample and applying the measured amount of label to a calibration curve. The direct competition method itself is well known in the field and is described, for example, in US 20150166678A.
間接競合法では、例えば、標的抗原（本発明ではＣ－ペプチド）を固相に固相化する（固相及び固相化については上記のとおり）。次いで、固相のブロッキング処理後、標的抗原を含む被検試料（本発明では、上記のとおり前処理工程を行った生体試料）と、一定量の抗標的抗原抗体とを混合し、前記固相化抗原と反応させる。洗浄後、固相に結合された前記抗標的抗原抗体を定量する。これは、前記抗標的抗原抗体に対する標識した二次抗体（標識は上記のとおり）を反応させ、洗浄後、標識量を測定することにより行うことができる。種々の既知濃度の抗原標準液を作製し、それぞれについて固相に固定化されて標識量を測定して、検量線を作成する。未知の被検試料について、標識量を測定し、測定された標識量を検量線に当てはめることにより、未知の被検試料中の抗原量を測定することができる。なお、標識二次抗体を用いずに、標識した一次抗体を用いることも可能である。間接競合法自体はこの分野において周知であり、例えば、上記したUS 20150166678Aに記載されている。 In the indirect competition method, for example, the target antigen (C-peptide in the present invention) is immobilized on a solid phase (the solid phase and immobilization are as described above). Next, after blocking the solid phase, a test sample containing the target antigen (in the present invention, a biological sample that has been subjected to the pretreatment step as described above) and a certain amount of anti-target antigen antibody are mixed, and the solid phase is react with the chemical antigen. After washing, the anti-target antigen antibody bound to the solid phase is quantified. This can be done by reacting a labeled secondary antibody against the anti-target antigen antibody (the label is as described above), and after washing, measuring the amount of label. Antigen standard solutions of various known concentrations are prepared, each is immobilized on a solid phase, and the amount of label is measured to create a calibration curve. The amount of antigen in the unknown test sample can be determined by measuring the amount of label in the unknown test sample and applying the measured amount of label to a calibration curve. Note that it is also possible to use a labeled primary antibody without using a labeled secondary antibody. The indirect competition method itself is well known in the field and is described, for example, in the above-mentioned US 20150166678A.
サンドイッチ法は、例えば、固相に抗標的抗原抗体を固相化し（固相及び固相化については上記のとおり）、ブロッキング処理後、標的抗原を含む被検試料（本発明では、上記のとおり前処理工程を行った生体試料）を反応させ、洗浄後、標的抗原に対する標識した二次抗体（標識は上記のとおり）を反応させ、洗浄後、固相に結合した標識を定量する方法である。種々の既知濃度の抗原標準液を作製し、それぞれについて固相に固定化された標識量を測定して、検量線を作成する。未知の被検試料について、標識量を測定し、測定された標識量を検量線に当てはめることにより、未知の被検試料中の抗原量を測定することができる。サンドイッチ法自体はこの分野において周知であり、例えば、US 20150309016Aに記載されている。 In the sandwich method, for example, an anti-target antigen antibody is immobilized on a solid phase (the solid phase and immobilization are as described above), and after a blocking treatment, a test sample containing the target antigen (in the present invention, as described above) is immobilized on a solid phase. After washing, a labeled secondary antibody against the target antigen (the label is as described above) is reacted, and after washing, the amount of the label bound to the solid phase is quantified. . Antigen standard solutions of various known concentrations are prepared, and the amount of label immobilized on the solid phase is measured for each to create a calibration curve. The amount of antigen in the unknown test sample can be determined by measuring the amount of label in the unknown test sample and applying the measured amount of label to a calibration curve. The sandwich method itself is well known in the art and is described, for example, in US 20150309016A.
上記した各種イムノアッセイのうち、化学発光酵素イムノアッセイ法（ＣＬＥＩＡ）、化学発光イムノアッセイ（ＣＬＩＡ）、酵素イムノアッセイ法（ＥＩＡ）、ラジオイムノアッセイ（ＲＩＡ）、蛍光イムノアッセイ（ＦＩＡ）は、上記した直接競合法、間接競合法、サンドイッチ法等を行う際に用いる標識の種類に基づいて分類したイムノアッセイである。化学発光酵素イムノアッセイ法（ＣＬＥＩＡ）は、標識として酵素（例えば、上記したアルカリフォスファターゼ）を用い、基質として化学発光性化合物を生じる基質（例えば、上記したＡＭＰＰＤ）を用いる、イムノアッセイである。酵素イムノアッセイ法（ＥＩＡ）は、標識として酵素（例えば、上記したペルオキシダーゼ、アルカリフォスファターゼ、ルシフェラーゼ、βガラクトシダーゼ等）を用いるイムノアッセイである。各酵素の基質としては、吸光度測定等により定量可能な化合物が用いられる。例えば、ペルオキシダーゼの場合には、1,2-フェニレンジアミン(OPD)や3,3'5,5'-テトラメチルベンチジン(TMB)等、アルカリフォスファターゼの場合には、p-ニトロフェニルフォスフェート(pNPP)等、β－ガラクトシダーゼの場合には、MG：4-メチルウンベリフェリルガラクトシド、NG：ニトロフェニルガラクトシド等、ルシフェラーゼの場合には、ルシフェリン等が用いられる。ラジオイムノアッセイ（ＲＩＡ）は、標識として放射性物質を用いる方法であり、放射性物質としては、上記のとおり３Ｈ、１４Ｃ、３２Ｐ、３５Ｓ、１２５Ｉ等の放射性元素が挙げられる。蛍光イムノアッセイ（ＦＩＡ）は、標識として蛍光物質又は蛍光タンパク質を用いる方法であり、蛍光物質又は蛍光タンパク質としては、上記のとおりフルオレセイン、フルオレセインイソチオシアネート、ローダミン、緑色蛍光タンパク質、赤色蛍光タンパク質等が挙げられる。これらの標識を用いるイムノアッセイ自体はこの分野において周知であり、例えば、US 8039223BやUS 20150309016Aに記載されている。 Among the various immunoassays described above, chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and fluorescent immunoassay (FIA) are the direct competitive method and indirect method described above. Immunoassays are classified based on the type of label used when conducting competitive methods, sandwich methods, etc. Chemiluminescent enzyme immunoassay (CLEIA) is an immunoassay that uses an enzyme as a label (eg, alkaline phosphatase, described above) and a substrate that produces a chemiluminescent compound (eg, AMPPD, described above) as a substrate. Enzyme immunoassay (EIA) is an immunoassay that uses enzymes (eg, peroxidase, alkaline phosphatase, luciferase, β-galactosidase, etc. as described above) as a label. As a substrate for each enzyme, a compound that can be quantified by absorbance measurement or the like is used. For example, in the case of peroxidase, 1,2-phenylenediamine (OPD) and 3,3'5,5'-tetramethylbenzidine (TMB), etc., and in the case of alkaline phosphatase, p-nitrophenyl phosphate ( pNPP), etc., in the case of β-galactosidase, MG: 4-methylumbelliferyl galactoside, NG: nitrophenyl galactoside, etc., and in the case of luciferase, luciferin, etc. are used. Radioimmunoassay (RIA) is a method using a radioactive substance as a label, and examples of the radioactive substance include radioactive elements such as 3 H, 14 C, 32 P, 35 S, and 125 I as described above. Fluorescence immunoassay (FIA) is a method that uses a fluorescent substance or fluorescent protein as a label, and examples of the fluorescent substance or fluorescent protein include fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein, etc. as described above. . Immunoassays using these labels are themselves well known in the art and are described, for example, in US 8039223B and US 20150309016A.
免疫比濁法（ＴＩＡ）は、測定すべき標的抗原（本発明ではＣ－ペプチド）と、該抗原に対する抗体との抗原抗体反応により生成された抗原抗体複合物により濁度が増大する現象を利用したイムノアッセイである。抗標的抗原抗体溶液に、種々の既知濃度の抗原を添加し、それぞれ濁度を測定し、検量線を作成する。未知の被検試料について、同様に濁度を測定し、測定された濁度を検量線に当てはめることにより、未知の被検試料中の抗原量を測定することができる。免疫比濁法自体は周知であり、例えば、US 20140186238Aに記載されている。ラテックス凝集法は、免疫比濁法と類似しているが、免疫比濁法における抗体溶液に代えて、表面に抗標的抗原抗体を固定化したラテックス粒子の浮遊液を用いる方法である。免疫比濁法及びラテックス凝集法自体はこの分野において周知であり、例えば、US7820398Bに記載されている。 Immune turbidimetry (TIA) utilizes the phenomenon in which turbidity increases due to an antigen-antibody complex generated by an antigen-antibody reaction between a target antigen to be measured (in the present invention, C-peptide) and an antibody against the antigen. This is an immunoassay. Various known concentrations of antigen are added to the anti-target antigen antibody solution, the turbidity of each is measured, and a calibration curve is created. By similarly measuring the turbidity of an unknown test sample and applying the measured turbidity to a calibration curve, the amount of antigen in the unknown test sample can be determined. Immunoturbidimetry itself is well known and is described, for example, in US 20140186238A. The latex agglutination method is similar to the immunoturbidimetric method, but instead of the antibody solution used in the immunoturbidimetric method, a suspension of latex particles having anti-target antigen antibodies immobilized on the surface is used. Immunoturbidimetry and latex agglutination methods themselves are well known in the art and are described, for example, in US7820398B.
イムノクロマトグラフィー法は、ろ紙、セルロースメンブレン、ガラス繊維、不織布等の多孔性材料で形成された基体（マトリックスやストリップとも呼ばれる）上で上記したサンドイッチ法や競合法を行う方法である。例えば、サンドイッチ法によるイムノクロマトグラフィー法の場合、抗標的抗原抗体を固定化した検出ゾーンを上記基体上に設け、標的抗原を含む被検試料（本発明では、上記のとおり前処理工程を行った生体試料）を基体に添加し、上流側から展開液を流して標的抗原を検出ゾーンまで移動させ、検出ゾーンに固定化させる。固定化された標的抗原を、標識した二次抗体でサンドイッチして、検出ゾーンに固定化された標識を検出することにより、被検試料中の標的抗原を検出する。標識二次抗体を含む標識ゾーンを検出ゾーンよりも上流側に形成しておくことにより、標的抗原と標識二次抗体との結合体が検出ゾーンに固定化される。標識が酵素の場合には、酵素の基質を含めた基質ゾーンも検出ゾーンよりも上流側に設けられる。競合法の場合には、例えば、検出ゾーンに標的抗原を固定化しておき、被検試料中の標的抗原と、検出ゾーンに固定化された標的抗原とを競合させることができる。検出ゾーンよりも上流側に標識抗体ゾーンを設けておき、被検試料中の標的抗原と標識抗体を反応させ、未反応の標識抗体を検出ゾーンに固定化して標識を検出又は定量することにより、被検試料中の標的抗原を検出又は定量することができる。イムノクロマトグラフィー法自体は、この分野において周知であり、例えばUS6210898Bに記載されている。 The immunochromatography method is a method in which the above-described sandwich method or competitive method is performed on a substrate (also called a matrix or strip) formed of a porous material such as filter paper, cellulose membrane, glass fiber, or nonwoven fabric. For example, in the case of immunochromatography using a sandwich method, a detection zone in which an anti-target antigen antibody is immobilized is provided on the above-mentioned substrate, and a test sample containing the target antigen (in the present invention, a detection zone in which an anti-target antigen antibody is immobilized is prepared) A sample) is added to the substrate, and a developing solution is flowed from the upstream side to move the target antigen to the detection zone, where it is immobilized. The target antigen in the test sample is detected by sandwiching the immobilized target antigen with a labeled secondary antibody and detecting the immobilized label in the detection zone. By forming a labeled zone containing a labeled secondary antibody upstream of the detection zone, a conjugate of the target antigen and labeled secondary antibody is immobilized in the detection zone. When the label is an enzyme, a substrate zone containing a substrate for the enzyme is also provided upstream of the detection zone. In the case of the competition method, for example, a target antigen can be immobilized on the detection zone, and the target antigen in the test sample can compete with the target antigen immobilized on the detection zone. A labeled antibody zone is provided upstream of the detection zone, the target antigen in the test sample is reacted with the labeled antibody, and the unreacted labeled antibody is immobilized in the detection zone to detect or quantify the label. A target antigen in a test sample can be detected or quantified. Immunochromatography methods themselves are well known in the art and are described, for example, in US6210898B.
＜Ｃ－ペプチドの測定試薬＞
本発明のＣ－ペプチドの測定試薬は、上述のＣ－ペプチドの測定方法を実現し得る測定試薬である。本発明の測定試薬は、通常のイムノアッセイに使用される構成に加え、アルカリ性物質又は酸性化剤のいずれかを含む前処理液を構成成分として含むことを特徴とする。
<C-peptide measurement reagent>
The C-peptide measurement reagent of the present invention is a measurement reagent that can implement the above-described C-peptide measurement method. The measurement reagent of the present invention is characterized by containing a pretreatment liquid containing either an alkaline substance or an acidifying agent as a component in addition to the configuration used in ordinary immunoassays.
本発明の試薬は、互いに隔離された形態又は組成物の形態において各構成成分を含む。具体的には、各構成成分はそれぞれ異なる容器（例、チューブ、プレート）に収容された形態で提供されてもよいが、一部の構成成分が組成物の形態（例、同一溶液中）で提供されてもよい。あるいは、本発明の試薬は、デバイスの形態で提供されてもよい。具体的には、構成成分の全部がデバイス中に収容された形態で提供されてもよい。あるいは、構成成分の一部がデバイス中に収容された形態で提供され、残りのものがデバイス中に収容されない形態（例、異なる容器に収容された形態）で提供されてもよい。この場合、デバイス中に収容されない構成成分は、標的物質の測定の際に、デバイス中に注入されることにより使用されてもよい。 The reagents of the present invention include each component in isolated form from each other or in the form of a composition. Specifically, each component may be provided in a form housed in a different container (e.g., tube, plate), but some components may be provided in the form of a composition (e.g., in the same solution). may be provided. Alternatively, the reagents of the invention may be provided in the form of a device. In particular, all of the components may be provided in the form contained within the device. Alternatively, some of the components may be provided in a form contained within the device, and the remaining components may be provided in a form not contained in the device (eg, contained in a different container). In this case, components not contained in the device may be used by being injected into the device during measurement of the target substance.
好ましい実施形態では、本発明の試薬は、採用されるべきイムノアッセイの種類に応じた構成を有していてもよい。例えば、サンドイッチ法が採用される場合、本発明の試薬は、必須の構成成分として、ｉ）前処理液、ｉｉ）Ｃ－ペプチドに対する抗体、ｉｉｉ）緩衝液、並びに任意の構成成分として、ｉｖ）Ｃ－ペプチドに対する別の抗体、ｖ）標識物質、ｖｉ）希釈液、及び、必要に応じて、ｖｉｉ）標識物質と反応する基質を含んでいてもよい。ｉｉ）及びｉｉｉ）の構成成分は、同一溶液に含まれていてもよい。ｉｖ）の構成成分は、ｖ）標識物質で標識化されていてもよい。好ましくは、Ｃ－ペプチドに対する抗体は、磁性粒子に固相化されていてもよい。 In a preferred embodiment, the reagent of the invention may have a configuration depending on the type of immunoassay to be employed. For example, when a sandwich method is employed, the reagent of the present invention comprises, as essential components, i) a pretreatment solution, ii) an antibody against C-peptide, iii) a buffer, and as an optional component, iv) It may also contain another antibody against C-peptide, v) a labeling substance, vi) a diluent, and, if necessary, vii) a substrate that reacts with the labeling substance. Components ii) and iii) may be contained in the same solution. The component of iv) may be labeled with v) a labeling substance. Preferably, the antibody against C-peptide may be immobilized on magnetic particles.
以下、本発明を実施例に基づき具体的に説明する。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 specifically explained based on Examples. However, the present invention is not limited to the following examples.
（参考例１）抗Ｃ－ペプチド抗体固相化磁性粒子液の調製
１０ｍＭ ＭＥＳ緩衝液（ｐＨ５．０）中で磁性粒子に抗Ｃ－ペプチド抗体を添加して、０．０４ｍｇ／ｍＬ 抗Ｃ－ペプチド抗体及び５ｍｇ／ｍＬ磁性粒子を含む懸濁液を得た。この懸濁液をゆるやかに攪拌しながら２５℃で１時間インキュベートして抗Ｃ－ペプチド抗体を磁性粒子に固相化した。その後、磁性粒子を磁石で集磁し、磁性粒子を洗浄液（５０ｍＭ トリス緩衝液、１５０ｍＭ ＮａＣｌ、２．０％ＢＳＡ、ｐＨ７．２）にて洗浄し、抗Ｃ－ペプチド抗体固相化粒子を得た。測定では、抗Ｃ－ペプチド抗体固相化粒子を、粒子希釈液（５０ｍＭ Ｔｒｉｓ緩衝液、１ｍＭ ＥＤＴＡ２Ｎａ、０．１％ ＮａＮ３、２．０％ＢＳＡ、ｐＨ７．２）中に懸濁した。これを抗体結合粒子液とした。
(Reference Example 1) Preparation of anti-C-peptide antibody immobilized magnetic particle solution Anti-C-peptide antibody was added to magnetic particles in 10 mM MES buffer (pH 5.0), and 0.04 mg/mL anti-C- A suspension containing the peptide antibody and 5 mg/mL magnetic particles was obtained. This suspension was incubated at 25° C. for 1 hour with gentle stirring to immobilize the anti-C-peptide antibody on the magnetic particles. Thereafter, the magnetic particles were collected with a magnet, and the magnetic particles were washed with a washing solution (50mM Tris buffer, 150mM NaCl, 2.0% BSA, pH 7.2) to obtain anti-C-peptide antibody immobilized particles. Ta. In the measurement, anti-C-peptide antibody immobilized particles were suspended in a particle diluent (50 mM Tris buffer, 1 mM EDTA2Na, 0.1% NaN 3 , 2.0% BSA, pH 7.2). This was used as an antibody-bound particle solution.
実施例１ アルカリ処理の希釈直線性確認試験
(1) 希釈検体の調製
Ｉ型糖尿病の購入検体（１）と検体（２）をルミパルス（登録商標）検体希釈液（富士レビオ社製）を用いて２倍希釈、４倍希釈、８倍希釈になるように調製した。１倍希釈検体は、未希釈の検体とし、この１～８倍希釈の検体を、段階的に希釈された検体という。
Example 1 Dilution linearity confirmation test of alkali treatment
(1) Preparation of diluted samples Dilute purchased specimens (1) and (2) of type I diabetes 2 times, 4 times, and 8 times using Lumipulse (registered trademark) sample diluent (manufactured by Fujirebio). It was prepared so that A 1-fold diluted specimen is an undiluted specimen, and a 1- to 8-fold diluted specimen is referred to as a stepwise diluted specimen.
(2) 検体及びキャリブレータのアルカリ処理
各検体４５μＬにアルカリ処理液（条件３：０．２Ｍ ＮａＯＨ、０．８％ Ｎ－ラウロイルサルコシンナトリウム（ＮＬＳ）、前処理時ｐＨ１２．８、 条件４：０．２Ｍ ＮａＯＨ、０．１６％ ＳＤＳ、前処理時ｐＨ１２．７）７５μＬを混合し、３７℃で７分間インキュベートした後、７５μＬの中和液（０．２Ｍ ＨＣｌ）を添加して速やかに撹拌し、アルカリ処理検体を得た（条件３：中和時ｐＨ７．２、条件４：中和時ｐＨ９．８）。
(2) Alkaline treatment of specimen and calibrator 45 μL of each specimen was treated with alkaline treatment solution (condition 3: 0.2M NaOH, 0.8% N-lauroylsarcosinate sodium (NLS), pH 12.8 during pretreatment, condition 4: 0.2M NaOH, 0.8% N-lauroylsarcosinate sodium (NLS), pH 12.8 during pretreatment, condition 4: 0.2M NaOH, 0.8% N-lauroylsarcosinate sodium (NLS), pH 12.8 during pretreatment. After mixing 75 μL of 2M NaOH, 0.16% SDS, pH 12.7 during pretreatment and incubating at 37°C for 7 minutes, 75 μL of neutralization solution (0.2M HCl) was added and quickly stirred. Alkali-treated specimens were obtained (condition 3: pH 7.2 during neutralization, condition 4: pH 9.8 during neutralization).
測定のキャリブレータとして、ルミパルスプレスト（登録商標）Ｃ－ペプチドキャリブレータ（富士レビオ社製、濃度：０、０．０３、０．３、３、３０ｎｇ／ｍＬ）を使用した。このキャリブレータについても、条件３と４は上記と同様の方法で処理して、アルカリ処理キャリブレータを得た。 Lumipulse Presto (registered trademark) C-peptide calibrator (manufactured by Fujirebio Co., Ltd., concentration: 0, 0.03, 0.3, 3, 30 ng/mL) was used as a calibrator for the measurement. This calibrator was also treated under conditions 3 and 4 in the same manner as above to obtain an alkali-treated calibrator.
(3) 検体中のＣ－ペプチド測定
得られたアルカリ処理検体及びキャリブレータを、自動分析装置ルミパルスＬ２４００を用いてＣ－ペプチド濃度の測定を行った。
(3) C-peptide measurement in specimen The C-peptide concentration of the obtained alkali-treated specimen and calibrator was measured using an automatic analyzer Lumipulse L2400.
抗体結合粒子液５０μＬとサンプル（段階的に希釈された検体又はキャリブレータ）１０μＬとをキュベットに分注した。撹拌後、３７℃で８分間インキュベートした（条件３：１次反応時ｐＨ７．１５、条件４：１次反応時ｐＨ７．４７）。キュベット内の粒子を磁石で集磁し、キュベット内を洗浄液（０．０５％ Ｔｗｅｅｎ２０）／ＰＢＳ）にて洗浄した。洗浄後のキュベットに、ルミパルスプレストＣ－ペプチドに付属する酵素標識抗体液（アルカリフォスファターゼ（ＡＬＰ）標識抗Ｃ－ペプチドモノクローナル抗体）５０μＬを加え、３７℃で８分間インキュベートした。キュベット内の粒子を磁石で集磁し、キュベット内を洗浄液にて洗浄した後、基質としてＡＭＰＰＤを含む基質液（ルミパルスプレスト基質液(共通試薬)）２００μＬを添加し、３７℃で４分間反応させた。波長４６３ｎｍに極大吸収波長を持つ光の発光量（カウント）を測定した。キャリブレータのカウントを用いてそれぞれの検量線を作成し、希釈系列検体中のＣ－ペプチド濃度を算出した。 50 μL of the antibody-bound particle solution and 10 μL of the sample (stepwise diluted specimen or calibrator) were dispensed into a cuvette. After stirring, the mixture was incubated at 37° C. for 8 minutes (condition 3: pH 7.15 during the first reaction, condition 4: pH 7.47 during the first reaction). The particles in the cuvette were collected with a magnet, and the inside of the cuvette was washed with a cleaning solution (0.05% Tween 20/PBS). 50 μL of an enzyme-labeled antibody solution (alkaline phosphatase (ALP) labeled anti-C-peptide monoclonal antibody) attached to Lumipulse Presto C-peptide was added to the washed cuvette, and incubated at 37° C. for 8 minutes. After collecting the particles in the cuvette with a magnet and washing the inside of the cuvette with a washing solution, 200 μL of a substrate solution containing AMPPD as a substrate (Lumipulse Presto substrate solution (common reagent)) was added, and the mixture was allowed to react at 37°C for 4 minutes. Ta. The amount of light emitted (count) having a maximum absorption wavelength at 463 nm was measured. Each calibration curve was created using the counts of the calibrator, and the C-peptide concentration in the diluted series samples was calculated.
対照として未処理の検体及びキャリブレータについて、同様にＣ－ペプチド濃度の測定を行った（条件１）。 As a control, the C-peptide concentration was similarly measured for the untreated specimen and the calibrator (condition 1).
条件２は、一次反応時に界面活性剤（ＮＬＳ）のみ存在した場合の測定系への影響を確認するため、参考例１に記載する粒子希釈液にＮＬＳを終濃度０．０６１５％となるように更に添加して、段階的に希釈された検体とキャリブレータを条件１と同様に測定した。 Condition 2 is to confirm the effect on the measurement system when only the surfactant (NLS) is present during the primary reaction, so NLS was added to the particle dilution solution described in Reference Example 1 to a final concentration of 0.0615%. Furthermore, the samples and calibrators that were diluted in stages were measured in the same manner as in Condition 1.
条件３は、上記のＮＬＳを含むアルカリ処理液によってアルカリ処理された段階的に希釈された検体と、アルカリ処理されたキャリブレータを、条件１と同様に測定した。 Under condition 3, a stepwise diluted sample treated with an alkali treatment solution containing the above-mentioned NLS and a calibrator treated with an alkali were measured in the same manner as in condition 1.
条件４は、上記のＳＤＳを含むアルカリ処理液によってアルカリ処理された段階的に希釈された検体と、アルカリ処理されたキャリブレータを、粒子液と混合させるサンプル量が３０μＬである点を除き、条件１と同様に測定した。 Condition 4 is the same as Condition 1 except that the sample volume in which the stepwise diluted specimen treated with the above-mentioned SDS-containing alkaline treatment solution and the alkali-treated calibrator are mixed with the particle liquid is 30 μL. Measured in the same manner.
段階的に希釈された検体の、実測定値、実測定値に希釈倍率を乗して算出した換算値、各希釈倍率の換算値を１倍の換算値で割り、その割合を百分率で表した値（回収率）を表１に示す。回収率は、１００％に近いほど希釈直線性が良好で、検体中の対象物を適切に定量できていることを示す。一方、低い回収率は、検体中の対象物の実際の濃度よりも高い値（偽高値）となっていることを示し、高い回収率は、実際の濃度よりも低い値（偽低値）となっていることを示す。条件１と条件２では、検体（１）と検体（２）のいずれも、２倍希釈において、回収率が５０％を下回った。一方で、アルカリ処理を行った条件３及び条件４では、回収率の改善が確認された。ＮＬＳを含むアルカリ処理液で処理した条件３では、検体（１）では４倍希釈まで、検体（２）では８倍希釈まで、１００±５０％の回収率を示した。ＳＤＳを含むアルカリ処理液で処理した条件４では、検体（１）と検体（２）のどちらも８倍希釈まで１００±４０％の回収率を示した。これらの結果から、アルカリ性物質を含む前処理液で処理を行うことで、偽高値を起こす反応が低減され、検体中のＣ－ペプチドが低値であっても正確に測定できることが示された。 The actual measured value of the sample diluted in stages, the converted value calculated by multiplying the actual measured value by the dilution factor, the converted value of each dilution factor divided by the 1x converted value, and the ratio expressed as a percentage ( Table 1 shows the recovery rate). The closer the recovery rate is to 100%, the better the dilution linearity is, indicating that the target substance in the sample can be appropriately quantified. On the other hand, a low recovery rate indicates that the concentration of the target substance in the sample is higher than the actual concentration (false high value), and a high recovery rate indicates that the concentration is lower than the actual concentration (false low value). Indicates that Under conditions 1 and 2, the recovery rate was less than 50% for both sample (1) and sample (2) when diluted 2 times. On the other hand, under conditions 3 and 4 where alkali treatment was performed, improvement in recovery rate was confirmed. Under condition 3, in which the sample was treated with an alkaline treatment solution containing NLS, a recovery rate of 100±50% was shown for sample (1) up to 4-fold dilution and sample (2) up to 8-fold dilution. Under condition 4, in which samples were treated with an alkaline treatment solution containing SDS, both sample (1) and sample (2) showed a recovery rate of 100±40% up to 8-fold dilution. These results showed that by treating with a pretreatment solution containing an alkaline substance, reactions that cause falsely high values were reduced, and even low values of C-peptide in the sample could be measured accurately.
実施例２ 偽高値を示す検体について、アルカリ処理による吸収試験を用いた特異性確認試験
Ｉ型糖尿病の購入検体（購入先ＩＩＣ－Ｊａｐａｎ）について検体未処理の条件（条件１）により、実施例１と同様にＣ－ペプチドの濃度を測定したところ、０．００２ ｎｇ／ｍＬ以上の測定値を示す、５検体（検体（３）～（７））が抽出された。これら５検体について、実施例１の条件１と条件４の方法でそれぞれ測定したところ、アルカリ前処理を行う条件４の測定では、２検体（検体（３）、検体（４））はアルカリ未処理の方法より２倍以上高い値を示し、３検体（検体（５）、検体（６）、検体（７））は１／２以下の低値を示した（実施例１の表２中の抗体未添加測定値参照）。これらの検体について、特異性を確認するために以下に記載する吸収試験を行った。
Example 2 Specificity confirmation test using an absorption test with alkali treatment for a sample showing a falsely high value Example 1 A purchased sample for type I diabetes (purchased from IIC-Japan) was tested under the condition that the sample was not treated (condition 1). When the concentration of C-peptide was measured in the same manner as above, 5 samples (specimens (3) to (7)) showing a measured value of 0.002 ng/mL or more were extracted. These five samples were measured using the methods of Condition 1 and Condition 4 in Example 1, and two samples (Sample (3) and Sample (4)) were not treated with alkali. 3 samples (sample (5), sample (6), sample (7)) showed a value less than half that of the antibody method in Table 2 of Example 1. (See measurements without addition). Absorption tests described below were conducted on these samples to confirm specificity.
吸収試験は、吸収剤として、測定に用いる固相用抗体（抗体結合粒子に使用する抗体）と標識用抗体（アルカリフォスファターゼ標識抗体に使用する抗体）を各１００μｇ／ｍＬとなるように抗体結合粒子液に添加した吸収試験用粒子液を、抗体結合粒子液の代わりに用いる以外は、実施例１と同様の方法でＣ－ペプチドの濃度を測定することにより行った。条件５は実施例１の条件１と同様に、条件６は実施例１の条件４と同様にキャリブレータ及び上記５検体（３）～（７）を測定した。 In the absorption test, the solid-phase antibody (antibody used for antibody-bound particles) used for measurement and the labeling antibody (antibody used for alkaline phosphatase-labeled antibody) are used as absorbents in antibody-bound particles at a concentration of 100 μg/mL each. The concentration of C-peptide was measured in the same manner as in Example 1, except that the absorption test particle solution added to the solution was used instead of the antibody-bound particle solution. Condition 5 was the same as Condition 1 of Example 1, and Condition 6 was the same as Condition 4 of Example 1, in which the calibrator and the five samples (3) to (7) were measured.
検体（３）～（７）の、抗体結合粒子液に吸収用抗体が添加されていない試薬で測定した測定値（抗体未添加測定値）、抗体結合粒子液に吸収用抗体を添加した試薬（吸収試験用粒子液）で測定した測定値（抗体添加測定値）、及び「１－（抗体添加測定値／抗体未添加測定値）」の百分率で表した値（吸収率）を表２に示す。 Measured values of samples (3) to (7) measured with a reagent in which an absorption antibody was not added to the antibody-bound particle solution (measured value without antibody addition), and a reagent in which an absorption antibody was added to the antibody-bound particle solution ( Table 2 shows the measured value (measured value with antibody addition) measured with the particle liquid for absorption test) and the value (absorption rate) expressed as a percentage of "1 - (measured value with antibody added/measured value without antibody addition)". .
アルカリ未処理の方法よりもアルカリ処理法により高値を示した２検体（検体（３）、（４））について、吸収試験を行った結果、アルカリ処理を行った条件６では全ての検体において、吸収率９０％以上を示した。この結果はアルカリ処理によって特異的にＣ－ペプチドを検出できていることを示す。アルカリ前処理法よりもアルカリ未処理の方法で高値を示した３検体（検体（５）、（６）、（７））について、吸収試験を行った結果、アルカリ処理を行わない条件５では、測定用抗体による吸収が起こらなかった。つまり、この３検体は、アルカリ処理を行わない場合、偽高値を示す検体であり、このような検体であっても、アルカリ処理を行うことで非特異的な反応を回避し、より正確にＣ－ペプチドを検出できていることを示す。 Absorption tests were conducted on two samples (sample (3) and (4)) that showed higher values with the alkali treatment method than with the alkali untreated method. As a result, absorption The rate was over 90%. This result shows that C-peptide can be specifically detected by alkali treatment. As a result of conducting an absorption test on three samples (specimens (5), (6), and (7)) that showed higher values with the method without alkali treatment than with the alkali pretreatment method, it was found that under condition 5 without alkali treatment, No absorption by the measuring antibody occurred. In other words, these three samples show falsely high values without alkali treatment. Even with such samples, alkali treatment can avoid non-specific reactions and more accurately detect C. - Indicates that the peptide has been detected.
実施例３ アルカリ処理した場合の検出限界（ＬＯＤ）と定量限界（ＬＯＱ）の算出
(1) 希釈試料の調製
Ｃ－ペプチドが０、０．００１、０．００２、０．００３、０．００４、０．００５、０．００６、０．００８、０．０１ｎｇ／ｍＬの濃度で含まれる希釈試料を使用した。
Example 3 Calculation of limit of detection (LOD) and limit of quantification (LOQ) when treated with alkali
(1) Preparation of diluted samples Contains C-peptide at concentrations of 0, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.008, and 0.01 ng/mL. A diluted sample was used.
(2) 試料及びキャリブレータのアルカリ処理
各希釈試料３０μＬにアルカリ処理液（０．２Ｍ ＮａＯＨ、０．８％ Ｎ－ラウロイルサルコシンナトリウム（ＮＬＳ））７５μＬを混合し、３７℃で７分間インキュベートした後、５０μＬの中和液（０．２Ｍ ＨＣｌ）を添加して速やかに撹拌し、アルカリ処理希釈試料を得た。
(2) Alkaline treatment of samples and calibrators 75 μL of alkaline treatment solution (0.2 M NaOH, 0.8% N-lauroylsarcosine sodium (NLS)) was mixed with 30 μL of each diluted sample, and after incubation at 37°C for 7 minutes, 50 μL of neutralizing solution (0.2 M HCl) was added and stirred rapidly to obtain an alkali-treated diluted sample.
測定のキャリブレータとして、ルミパルスプレスト（登録商標）Ｃ－ペプチドキャリブレータ（富士レビオ社製、濃度：０、０．０３、０．３、３、３０ｎｇ／ｍＬ）を使用した。このキャリブレータについても、上記と同様の方法で処理して、アルカリ処理キャリブレータを得た。 Lumipulse Presto (registered trademark) C-peptide calibrator (manufactured by Fujirebio Co., Ltd., concentration: 0, 0.03, 0.3, 3, 30 ng/mL) was used as a calibrator for the measurement. This calibrator was also treated in the same manner as above to obtain an alkali-treated calibrator.
(3) 試料中のＣ－ペプチド測定
得られたアルカリ処理希釈試料及びキャリブレータを、自動分析装置ルミパルスＬ２４００を用いて、実施例１条件３と同様に、Ｃ－ペプチド濃度の測定を行った。
(3) C-peptide measurement in sample The C-peptide concentration of the obtained alkali-treated diluted sample and calibrator was measured using the automatic analyzer Lumipulse L2400 in the same manner as in Example 1, Condition 3.
アルカリ処理希釈試料は１試料につき１０回測定（Ｎ＝１０）し、キャリブレータは１濃度につき２回測定（Ｎ＝２）した。 The alkali-treated diluted sample was measured 10 times per sample (N=10), and the calibrator was measured twice per concentration (N=2).
Ｎ＝１０で測定したアルカリ処理希釈試料のうち、０ｎｇ／ｍＬの試料の平均値＋３ＳＤの値（検出限界（ＬＯＤ））を求めた。算出された検出限界（ＬＯＤ）は０．００１１ｎｇ／ｍＬ であった。さらに、アルカリ処理希釈試料の標準偏差を平均値で割った数の百分率（変動係数（ＣＶ））を求めた。各濃度のＣＶを求め、ＣＶが１０%以下となる最小の濃度（定量限界（ＬＯＱ））を求めた。算出された定量限界は、０．００８ｎｇ／ｍＬであった。これらの結果から、アルカリ性物質を含む前処理液で処理を行うことで、例えば試料中に含まれるＣ－ペプチドの濃度が０．０２ｎｇ／ｍＬ未満であっても精度良くＣ－ペプチドを測定又は定量可能であることが示された。さらに、試料中に含まれるＣ－ペプチドの濃度が０．００８ｎｇ／ｍＬ～０．０１５ｎｇ／ｍＬであっても精度良くＣ－ペプチドを測定又は定量可能であることが示された。 Among the alkali-treated diluted samples measured at N=10, the average value + 3SD value (limit of detection (LOD)) of the 0 ng/mL sample was determined. The calculated limit of detection (LOD) was 0.0011 ng/mL. Furthermore, the percentage of the standard deviation of the alkali-treated diluted sample divided by the average value (coefficient of variation (CV)) was determined. The CV of each concentration was determined, and the minimum concentration (limit of quantification (LOQ)) at which the CV was 10% or less was determined. The calculated limit of quantification was 0.008 ng/mL. From these results, by treating with a pretreatment solution containing an alkaline substance, it is possible to accurately measure or quantify C-peptide even if the concentration of C-peptide contained in the sample is less than 0.02 ng/mL. It has been shown that it is possible. Furthermore, it was shown that C-peptide can be accurately measured or quantified even if the concentration of C-peptide contained in the sample is 0.008 ng/mL to 0.015 ng/mL.
実施例４ 偽高値を示す検体における、酸処理による希釈直線性確認試験
（１）希釈検体の調製
希釈検体は、実施例１（１）に記載した、段階的に希釈された検体と同じものを使用した。
Example 4 Dilution linearity confirmation test using acid treatment for a sample showing a false high value (1) Preparation of diluted sample The diluted sample was the same as the stepwise diluted sample described in Example 1 (1). used.
（２）検体の酸処理及び検体中のＣ－ペプチドの検出
条件７と条件８と条件９の酸処理及び検体中のＣ－ペプチドの検出は以下の通りに行った。条件７は、実施例１の条件１と同様の方法で希釈系列検体とキャリブレータを測定した。条件８は、各検体４５μＬに前処理液（０．５Ｍ ＨＣｌ、２Ｍ Ｕｒｅａ、４％ ＴｒｉｔｏｎＸ－１００）７５μＬを混合し（前処理時ｐＨ１．０２）、３７℃で７分間インキュベートした後、７５μＬの中和液（０．５Ｍ ＮａＯＨ)を添加して速やかに撹拌し（中和時ｐＨ１０．１４）、酸処理検体を得た。
(2) Acid treatment of specimen and detection of C-peptide in the specimen Acid treatment under conditions 7, 8, and 9 and detection of C-peptide in the specimen were performed as follows. Under condition 7, the diluted series specimen and calibrator were measured in the same manner as in condition 1 of Example 1. Condition 8 is to mix 45 μL of each sample with 75 μL of pretreatment solution (0.5M HCl, 2M Urea, 4% Triton A neutralizing solution (0.5 M NaOH) was added and rapidly stirred (pH 10.14 at the time of neutralization) to obtain an acid-treated specimen.
得られた酸処理検体３０μＬ のＣ－ペプチドの濃度を、実施例１と同様に測定した（１次反応時ｐＨ７．４４）。 The concentration of C-peptide in 30 μL of the acid-treated sample obtained was measured in the same manner as in Example 1 (pH 7.44 during the first reaction).
ルミパルスＣ－ペプチドキャリブレータ（富士レビオ社製、濃度：０、０．０３、０．３、３、３０ｎｇ／ｍＬ）についても、上記と同様の方法で酸処理キャリブレータを調製し、実施例１と同様に測定した。 Regarding Lumipulse C-peptide calibrator (manufactured by Fujirebio, concentration: 0, 0.03, 0.3, 3, 30 ng/mL), acid-treated calibrators were prepared in the same manner as above, and the same as in Example 1. was measured.
条件９は、検体の酸処理及び中和をルミパルスＬ２４００の装置上で行った。 In Condition 9, acid treatment and neutralization of the specimen were performed on a Lumipulse L2400 device.
条件９は、具体的には、酸処理液（２．４Ｍ Ｕｒｅａ、０．１６Ｍ ＨＣｌ、４％ Ｔｗｅｅｎ ８０、６．４％ 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）、ｐＨ３．４３）９０μＬと検体又はキャリブレータ３０μＬを混合し、３７℃で６．５分間インキュベートした後（前処理時ｐＨ４．１３）、Ｃ－ペプチド抗体固相化磁性粒子を粒子希釈液（１Ｍ Ｔｒｉｓ緩衝液、２０ｍＭ ＥＤＴＡ、３％ ＢＳＡ、０．１％ Ｔｗｅｅｎ ８０、ｐＨ ７．９）で希釈した粒子液８０μＬを加えて、３７℃で８分間反応させた（1次反応時ｐＨ７．６７）。０．０５％ Ｔｗｅｅｎ２０／ＰＢＳで洗浄後、ルミパルスプレストＣ－ペプチドに付属する酵素標識抗体液（アルカリフォスファターゼ（ＡＬＰ）標識抗Ｃ－ペプチドモノクローナル抗体）５０μＬを加え、３７℃で８分間反応させた。０．０５％ Ｔｗｅｅｎ２０／ＰＢＳで洗浄後、基質液２００μＬを添加し、３７℃で４分間反応させた。波長４６３ｎｍに極大吸収波長を持つ光の発光量（カウント）を測定した。キャリブレータのカウントを用いてそれぞれの検量線を作成し、段階的に希釈された検体中のＣ－ペプチド濃度を算出した。 Condition 9 specifically includes an acid treatment solution (2.4M Urea, 0.16M HCl, 4% Tween 80, 6.4% N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate). (C16APS), pH 3.43) and 30 μL of the sample or calibrator, and after incubating at 37°C for 6.5 minutes (pH 4.13 during pretreatment), add the C-peptide antibody immobilized magnetic particles to the particle dilution solution. 80 μL of particle solution diluted with (1M Tris buffer, 20 mM EDTA, 3% BSA, 0.1% Tween 80, pH 7.9) was added and reacted at 37°C for 8 minutes (first reaction pH 7.9). 67). After washing with 0.05% Tween 20/PBS, 50 μL of an enzyme-labeled antibody solution (alkaline phosphatase (ALP) labeled anti-C-peptide monoclonal antibody) attached to Lumipulse Presto C-peptide was added and reacted at 37° C. for 8 minutes. After washing with 0.05% Tween 20/PBS, 200 μL of substrate solution was added and reacted at 37° C. for 4 minutes. The amount of light emitted (count) having a maximum absorption wavelength at 463 nm was measured. Each calibration curve was created using the counts of the calibrator, and the C-peptide concentration in the serially diluted samples was calculated.
段階的に希釈された検体の、実測定値、実測定値に希釈倍率を乗して算出した換算値、各希釈倍率の換算値を１倍の換算値で割り、その割合を百分率で表した値（回収率）を表３に示す。 The actual measured value of the sample diluted in stages, the converted value calculated by multiplying the actual measured value by the dilution factor, the converted value of each dilution factor divided by the 1x converted value, and the ratio expressed as a percentage ( Table 3 shows the recovery rate).
条件７では、検体（１）と検体（２）のいずれも、２倍希釈において、回収率が５０％を下回った。一方で、酸処理を行った条件８及び条件９では、回収率の向上が確認された。条件８では、検体（１）では４倍希釈まで１００±４０％、検体（２）では８倍希釈まで１００±３０％の回収率を示した。陽イオン性界面活性剤を含む酸処理液で処理し、Ｔｒｉｓ緩衝液で中和を行った条件９では、検体（１）と検体（２）のどちらも８倍希釈まで１００±１０％の回収率を示した。これらの結果から、酸性化剤を含む前処理液で処理を行うことで、偽高値を起こす反応が低減され、検体中のＣ－ペプチドが低値であっても正確に測定できることが示された。 Under condition 7, the recovery rate was less than 50% for both sample (1) and sample (2) at 2-fold dilution. On the other hand, under conditions 8 and 9 in which acid treatment was performed, an improvement in the recovery rate was confirmed. Under condition 8, sample (1) showed a recovery rate of 100±40% up to 4-fold dilution, and sample (2) showed a recovery rate of 100±30% up to 8-fold dilution. Under condition 9, which was treated with an acid treatment solution containing a cationic surfactant and neutralized with Tris buffer, recovery of both specimen (1) and specimen (2) was 100 ± 10% up to 8-fold dilution. The rate was shown. These results indicate that by treating with a pretreatment solution containing an acidifying agent, reactions that cause falsely high values can be reduced and accurate measurements can be made even if the C-peptide in the sample is low. .
実施例５ 偽高値を示す検体について、酸処理による吸収試験を用いた特異性の確認
実施例２と同様に、実施例２に記載したＩ型糖尿病の購入検体について、実施例４の条件７と条件８の方法でそれぞれ測定したところ、酸処理を行う条件８では、２検体（検体（３）、検体（４））は検体を処理しない条件７より１．７倍以上高い値を示し、３検体（検体（５）、検体（６）、検体（７））は１／２以下の低値を示した（実施例４の表４中の抗体未添加測定値参照）。これらの検体について、特異性を確認するために粒子液に測定用抗体を添加した吸収試験を行った。
Example 5 Confirmation of specificity using absorption test by acid treatment for specimens showing falsely high values Similarly to Example 2, condition 7 of Example 4 was applied to purchased specimens for type I diabetes described in Example 2. When measured using the method of condition 8, two samples (sample (3) and sample (4)) showed values 1.7 times or more higher in condition 8, where acid treatment was performed, than in condition 7, where no specimen was treated, and 3 The samples (specimen (5), sample (6), and sample (7)) showed a low value of 1/2 or less (see the measured values without antibody addition in Table 4 of Example 4). For these samples, an absorption test was conducted in which a measurement antibody was added to the particle solution to confirm specificity.
吸収試験は、吸収剤として、測定に用いる固相用抗体（抗体結合粒子に使用する抗体）と標識用抗体（アルカリフォスファターゼ標識抗体に使用する抗体）を各１００μｇ／ｍＬとなるように抗体結合粒子液に添加した吸収試験用粒子液を、抗体結合粒子液の代わりに用いる以外は、実施例４と同様の方法でＣ－ペプチドの濃度を測定することにより行った。条件１０は実施例４の条件７と同様に、条件１１は実施例４の条件８と同様に、条件１２は実施例４の条件９と同様にキャリブレータ及び上記５検体（３）～（７）を測定した。 In the absorption test, the solid-phase antibody (antibody used for antibody-bound particles) used for measurement and the labeling antibody (antibody used for alkaline phosphatase-labeled antibody) are used as absorbents in antibody-bound particles at a concentration of 100 μg/mL each. The concentration of C-peptide was measured in the same manner as in Example 4, except that the absorption test particle liquid added to the liquid was used instead of the antibody-bound particle liquid. Condition 10 is the same as condition 7 of Example 4, condition 11 is the same as condition 8 of Example 4, and condition 12 is the same as condition 9 of Example 4, including the calibrator and the above five samples (3) to (7). was measured.
検体（３）～（７）の、抗体結合粒子液に吸収用抗体が添加されていない試薬で測定した測定値（抗体未添加測定値）、抗体結合粒子液に吸収用抗体を添加した試薬（吸収試験用粒子液）で測定した測定値（抗体添加測定値）、及び「１－（抗体添加測定値／抗体未添加測定値）」の百分率で表した値（吸収率）を表４に示す。 Measured values of samples (3) to (7) measured with a reagent in which an absorption antibody was not added to the antibody-bound particle solution (measured value without antibody addition), and a reagent in which an absorption antibody was added to the antibody-bound particle solution ( Table 4 shows the measured value (measured value with addition of antibody) measured with particle liquid for absorption test) and the value (absorption rate) expressed as a percentage of "1 - (measured value with addition of antibody / measured value without addition of antibody)". .
検体を処理しない方法よりも酸処理法により高値を示した２検体について、吸収試験の結果、酸処理を行った条件１１及び条件１２は、全検体で吸収率９５%以上を示した。この結果は酸処理によって特異的にＣ－ペプチドを検出できていることを示す。酸前処理法よりも酸未処理の方法で高値を示した３検体（検体（５）、（６）、（７））について、吸収試験を行った結果、酸処理を行わない条件１０では、測定用抗体による吸収が起こらなかった。つまり、この３検体は、酸処理を行わない場合、偽高値を示す検体であり、このような検体であっても、酸処理を行うことで非特異的な反応を回避し、より正確にＣ－ペプチドを検出できていることを示す。 Regarding the two samples that showed higher values with the acid treatment method than with the method without sample treatment, the results of the absorption test showed that under conditions 11 and 12 where acid treatment was performed, all the samples showed an absorption rate of 95% or more. This result shows that C-peptide can be specifically detected by acid treatment. As a result of conducting an absorption test on the three samples (specimens (5), (6), and (7)) that showed higher values with the non-acid treatment method than with the acid pretreatment method, it was found that under condition 10 without acid treatment, No absorption by the measuring antibody occurred. In other words, these three samples show falsely high values without acid treatment. Even with such samples, acid treatment can avoid non-specific reactions and more accurately detect C. - Indicates that the peptide has been detected.
実施例６ 酸処理した場合の検出限界（ＬＯＤ）と定量限界（ＬＯＱ）の算出
(1)希釈試料の調製
Ｃ－ペプチドが０、０．００１、０．００２、０．００３、０．００４、０．００５、０．００６、０．００８、０．０１ｎｇ／ｍＬの濃度で含まれる希釈試料を使用した。
Example 6 Calculation of limit of detection (LOD) and limit of quantification (LOQ) in the case of acid treatment
(1) Preparation of diluted sample Contains C-peptide at concentrations of 0, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.008, and 0.01 ng/mL. A diluted sample was used.
(2)試料及びキャリブレータの酸処理及びＣ－ペプチド測定
自動分析装置ルミパルスＬ２４００を用いて、実施例４条件９と同様に、試料中のＣ－ペプチド濃度の測定を行った。即ち、各希釈試料３０μＬに酸処理液（２．４Ｍ Ｕｒｅａ、０．１６Ｍ ＨＣｌ、４％ Ｔｗｅｅｎ ８０、６．４％ 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）、ｐＨ３．４３）９０μＬを混合し、３７℃で６．５分間インキュベートした後、８０μＬのＣ－ペプチド抗体固相化磁性粒子を粒子希釈液（１Ｍ Ｔｒｉｓ緩衝液、２０ｍＭ ＥＤＴＡ、３％ ＢＳＡ、０．１％ Ｔｗｅｅｎ ８０、ｐＨ ７．９）で希釈した粒子液を加えて、３７℃で８分間反応させた。
(2) Acid treatment of sample and calibrator and C-peptide measurement The C-peptide concentration in the sample was measured using the automatic analyzer Lumipulse L2400 in the same manner as in Example 4, Condition 9. That is, 30 μL of each diluted sample was mixed with an acid treatment solution (2.4 M Urea, 0.16 M HCl, 4% Tween 80, 6.4% N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C16APS). ), pH 3.43) and incubated at 37°C for 6.5 minutes, then 80 μL of C-peptide antibody immobilized magnetic particles were mixed with particle dilution solution (1M Tris buffer, 20mM EDTA, 3% BSA, A particle solution diluted with 0.1% Tween 80 (pH 7.9) was added and reacted at 37°C for 8 minutes.
測定のキャリブレータとして、ルミパルスプレスト（登録商標）Ｃ－ペプチドキャリブレータ（富士レビオ社製、濃度：０、０．０３、０．３、３、３０ｎｇ／ｍＬ）を使用した。このキャリブレータについても、上記と同様の方法でＣ－ペプチドを測定した。 Lumipulse Presto (registered trademark) C-peptide calibrator (manufactured by Fujirebio Co., Ltd., concentration: 0, 0.03, 0.3, 3, 30 ng/mL) was used as a calibrator for the measurement. C-peptide was also measured for this calibrator in the same manner as above.
酸処理希釈試料は１試料につき１０回測定（Ｎ＝１０）し、キャリブレータは１濃度につき２回測定（Ｎ＝２）した。 The acid-treated diluted sample was measured 10 times per sample (N=10), and the calibrator was measured twice per concentration (N=2).
Ｎ＝１０で測定した酸処理希釈試料のうち、０ｎｇ／ｍＬの試料の平均値＋３ＳＤの値（検出限界（ＬＯＤ））を求めた。算出された検出限界（ＬＯＤ）は０．０００２ｎｇ／ｍＬ であった。さらに、アルカリ処理希釈試料の標準偏差を平均値で割った数の百分率（変動係数（ＣＶ））を求めた。各濃度のＣＶを求め、ＣＶが１０%以下となる最小の濃度（定量限界（ＬＯＱ））を求めた。算出された定量限界は、０．００１ｎｇ／ｍＬであった。これらの結果から、アルカリ性物質を含む前処理液で処理を行うことで、例えば試料中に含まれるＣ－ペプチドの濃度が０．０２ｎｇ／ｍＬ未満であっても精度良くＣ－ペプチドを測定又は定量可能であることが示された。さらに、試料中に含まれるＣ－ペプチドの濃度が０．００１ｎｇ／ｍＬ～０．０１５ｎｇ／ｍＬであっても精度良くＣ－ペプチドを測定又は定量可能であることが示された。 Among the acid-treated diluted samples measured at N=10, the average value + 3SD value (limit of detection (LOD)) of the 0 ng/mL sample was determined. The calculated limit of detection (LOD) was 0.0002 ng/mL. Furthermore, the percentage of the standard deviation of the alkali-treated diluted sample divided by the average value (coefficient of variation (CV)) was determined. The CV of each concentration was determined, and the minimum concentration (limit of quantification (LOQ)) at which the CV was 10% or less was determined. The calculated limit of quantification was 0.001 ng/mL. From these results, by treating with a pretreatment solution containing an alkaline substance, it is possible to accurately measure or quantify C-peptide even if the concentration of C-peptide contained in the sample is less than 0.02 ng/mL. It has been shown that it is possible. Furthermore, it was shown that C-peptide can be accurately measured or quantified even if the concentration of C-peptide contained in the sample is 0.001 ng/mL to 0.015 ng/mL.
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