JP6931673B2 - Immunoassay methods and reagents - Google Patents
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本発明は、免疫分析方法及びそのための試薬に関する。 The present invention relates to immunoassay methods and reagents for them.
免疫分析方法は、血清、血漿、尿、便、髄液等の臨床検査で広く用いられ、近年、簡便・迅速に測定を行うことができることから、反応から測定までを一括して自動で行う自動分析装置が汎用されている。 The immunoassay method is widely used in clinical tests of serum, plasma, urine, stool, cerebrospinal fluid, etc., and in recent years, since it is possible to perform simple and rapid measurement, the reaction to the measurement are automatically performed collectively. Analyzers are widely used.
免疫分析方法は抗原抗体反応を利用した測定法で特異性が高い測定法であることが知られている。しかし試料によっては擬陽性または擬陰性などの非特異反応が起こる問題があった。例えば、抗体を認識して反応する因子が試料中に存在する場合があり、このような場合には試料中に測定しようとする抗原が存在しなくても陽性の測定値になる、また一方で試料中の抗原抗体反応を妨害する因子が存在する場合があり、このような場合は試料中に測定しようとする抗原が存在しても陰性の測定値になるなど、真値とは異なった測定値を示すという問題があった。 The immunoassay method is a measurement method using an antigen-antibody reaction and is known to be a highly specific measurement method. However, there is a problem that a non-specific reaction such as false positive or false negative occurs depending on the sample. For example, a factor that recognizes and reacts to an antibody may be present in the sample, in which case a positive measurement will be obtained even if the antigen to be measured is not present in the sample. There may be a factor that interferes with the antigen-antibody reaction in the sample. In such a case, even if the antigen to be measured is present in the sample, the measured value will be negative, which is different from the true value. There was a problem of showing the value.
非特異反応を抑制する手法としてヒトIgM自然抗体や、スルホン基又はその塩を有する芳香族モノマーが重合されたポリマーを添加することが知られていた（特許文献１、２参照）。しかし、これらの添加剤では不十分なことがあり、特に低濃度域での非特異反応抑制は困難な状態だった。更に高感度にした試薬においては、抗体の反応性を向上しているために非特異反応が起きやすい状況だった。 It has been known to add a human IgM natural antibody or a polymer obtained by polymerizing an aromatic monomer having a sulfone group or a salt thereof as a method for suppressing a non-specific reaction (see Patent Documents 1 and 2). However, these additives may be insufficient, and it has been difficult to suppress non-specific reactions, especially in the low concentration range. In the reagent with higher sensitivity, non-specific reaction was likely to occur because the reactivity of the antibody was improved.
本発明の目的は、免疫分析において、抗原を高感度でかつ正確に測定することができる免疫分析方法及びそのための試薬を提供することである。 An object of the present invention is to provide an immunoassay method capable of measuring an antigen with high sensitivity and accuracy in immunoassay, and a reagent for that purpose.
本願発明者は、鋭意研究の結果、免疫分析においてアルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤を存在させることにより、高感度な測定であっても、容易に非特異反応を抑制できることを見出した。 As a result of diligent research, the inventor of the present application facilitates even highly sensitive measurement by the presence of a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in the alkyl group in immunoassay. It was found that non-specific reactions can be suppressed.
すなわち、本発明は、以下の１）〜９）に係るものである。
１）アルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤（ポリオキシエチレンラウリルエーテルを除く）の存在下で抗原抗体反応及び／又は測定を行う、特異性が向上するラテックス凝集免疫分析方法。
２）前記ポリオキシエチレンアルキルエーテル界面活性剤のＨＬＢが１５〜１９の非イオン性界面活性剤である１）の方法。
３）前記ポリオキシエチレンアルキルエーテル界面活性剤が存在する反応系及び／又は測定系における該ポリオキシエチレンアルキルエーテル界面活性剤の濃度が０．０００１〜１％である１）又は２）の方法。
４）抗原抗体反応の開始から測定完了までを前記ポリオキシエチレンアルキルエーテル界面活性剤の存在下で行う１）〜３）のいずれかに記載の方法。
５）１）の方法に使用される、特異性が向上するラテックス凝集免疫分析試薬であって、アルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤（ポリオキシエチレンラウリルエーテルを除く）を含むことを特徴とする免疫分析試薬。
６）前記ポリオキシエチレンアルキルエーテル界面活性剤のＨＬＢが１５〜１９である５）の試薬。
That is, the present invention relates to the following 1) to 9).
1) Antigen-antibody reaction and / or measurement is performed in the presence of a polyoxyethylene alkyl ether surfactant (excluding polyoxyethylene lauryl ether) having 8 to 14 carbon atoms in the alkyl group, which improves specificity. Latex agglutination immunoassay method.
2) The method of 1), wherein the polyoxyethylene alkyl ether surfactant has an HLB of 15 to 19 as a nonionic surfactant.
3) The method according to 1) or 2), wherein the concentration of the polyoxyethylene alkyl ether surfactant in the reaction system and / or the measurement system in which the polyoxyethylene alkyl ether surfactant is present is 0.0001 to 1%.
4) The method according to any one of 1) to 3), wherein the process from the start of the antigen-antibody reaction to the completion of the measurement is carried out in the presence of the polyoxyethylene alkyl ether surfactant.
5 ) A latex agglutination immunoassay reagent used in the method 1) with improved specificity, which is a polyoxyethylene alkyl ether surfactant (polyoxyethylene) having an alkyl group having 8 to 14 carbon atoms. An immunoassay reagent comprising (excluding lauryl ether).
6 ) The reagent of 5) in which the HLB of the polyoxyethylene alkyl ether surfactant is 15 to 19.
本発明の方法によれば、反応及び／又は測定系にアルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤を存在させるという簡易な手段により、高感度な免疫分析においても非特異反応を効果的に抑制することができ、免疫分析において、抗原を正確に測定でき特異性が向上する。 According to the method of the present invention, highly sensitive immunoassay is performed by a simple means of allowing a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in the alkyl group to be present in the reaction and / or measurement system. In addition, the non-specific reaction can be effectively suppressed, and the antigen can be accurately measured in the immunoassay, and the specificity is improved.
以下、本発明の方法について説明する。なお、本明細書中の「％」は特に断りがない限り質量基準（ｗ／ｖ％）を意味する。 Hereinafter, the method of the present invention will be described. In addition, "%" in this specification means mass-based (w / v%) unless otherwise specified.
本発明の免疫分析方法は、検体中の被測定物質に対し免疫的に反応する免疫分析試薬を用いて、抗原抗体反応を行い、得られた反応物を測定する免疫分析方法において、アルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤の存在下で反応及び／又は測定を行うことを特徴とするものである。 The immunoassay method of the present invention is an immunoassay method in which an antigen-antibody reaction is carried out using an immunoassay reagent that immunologically reacts with a substance to be measured in a sample, and the obtained reaction product is measured. It is characterized in that the reaction and / or measurement is carried out in the presence of a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms.
免疫分析の手法自体は周知である。本発明の方法が適用される免疫分析方法とは、公知のいかなる免疫分析方法であってもよいが、中でも免疫凝集法が好ましく、特に不溶性担体粒子としてラテックス粒子を用いるラテックス凝集法が好ましい。免疫凝集法において感作粒子の凝集を検出する方法は周知であり、本発明においても、感作粒子の凝集による吸光度又は光散乱等を検出する方法等の周知の方法が使用可能である。例えば、免疫比濁法（ＴＩＡ法、ラテックス凝集法）、比色法、ＲＰＬＡ法、ＣＬ法及びイムノクロマト法等が挙げられ、感度が高く定量精度が良い比濁法及び比色法が好適に用いられる。 The immunoassay method itself is well known. The immunoassay method to which the method of the present invention is applied may be any known immunoassay method, but the immunoagglutination method is preferable, and the latex agglutination method using latex particles as insoluble carrier particles is particularly preferable. A method for detecting agglutination of sensitized particles in an immunoaggregation method is well known, and in the present invention, a well-known method such as a method for detecting absorbance or light scattering due to agglutination of sensitized particles can be used. Examples thereof include an immunoturbidimetry method (TIA method, latex agglutination method), a colorimetric method, an RPLA method, a CL method and an immunochromatography method, and the turbidimetry method and the colorimetric method having high sensitivity and good quantification accuracy are preferably used. Be done.
当該免疫分析方法の態様としては、用いる不溶性担体粒子は特に限定されず、免疫分析試薬に従来用いられている周知のものであってよい。例えば、ポリエチレンやポリスチレン等のラテックス粒子、アルミナ粒子、シリカ粒子、金コロイド、磁性粒子等の粒子が挙げられる。これらの不溶性担体の中ではラテックス粒子、特にポリスチレンラテックス粒子が好適に用いられる。免疫凝集法は、抗原或いは抗体を感作した感作粒子の凝集を光学的に検出する方法として周知であり、検出には比濁法又は比色法が好適に用いられる。例えば、セル外部より可視光から近赤外域の光、例えば通常３００〜１０００ｎｍ、好ましくは５００〜９００ｎｍの光を照射し、吸光度変化又は散乱光の強度変化を検出することにより、当該感作粒子の凝集の程度が測定される。ラテックス粒子として特にポリスチレンラテックス粒子が好適に用いられる。ラテックス粒子のサイズは特に限定されないが、粒径は３０〜６００ｎｍであることが好ましい。 The mode of the immunoassay method is not particularly limited to the insoluble carrier particles used, and may be well-known ones conventionally used for immunoassay reagents. For example, latex particles such as polyethylene and polystyrene, alumina particles, silica particles, colloidal gold, magnetic particles and the like can be mentioned. Among these insoluble carriers, latex particles, particularly polystyrene latex particles, are preferably used. The immunoagglutination method is well known as a method for optically detecting the aggregation of sensitized particles sensitized with an antigen or antibody, and the turbidimetry method or the colorimetric method is preferably used for the detection. For example, by irradiating light in the visible to near-infrared region from the outside of the cell, for example, light of usually 300 to 1000 nm, preferably 500 to 900 nm, and detecting a change in absorbance or a change in intensity of scattered light, the sensitized particles The degree of agglomeration is measured. Polystyrene latex particles are particularly preferably used as the latex particles. The size of the latex particles is not particularly limited, but the particle size is preferably 30 to 600 nm.
上記したラテックス粒子に、測定すべき抗原と免疫的に反応する抗体若しくはその抗原結合性断片を固定化する。固定化の方法も周知であり、物理吸着又は共有結合等の周知の方法により行われる。得られた感作粒子の懸濁液と被検試料とを混合すると、被検試料中に含まれる被測定物質（抗原）によって感作粒子が凝集され、感作粒子懸濁液の吸光度が変化する。この吸光度の変化量（エンドポイント法）又は変化率（レート法）を測定する。測定すべき抗原を種々の既知濃度で含む複数の標準試料を準備し、それらについて上記方法により吸光度の変化量又は変化率を測定する。標準試料中の測定すべき抗原の濃度を横軸、測定された吸光度の変化量又は変化率を縦軸にプロットして検量線を描く。未知の被検試料についても同じ方法により吸光度の変化量又は変化率を測定し、測定結果を上記検量線に当てはめることにより、被検試料中の抗原を定量することができる。 An antibody or an antigen-binding fragment thereof that immunoreacts with the antigen to be measured is immobilized on the above-mentioned latex particles. The method of immobilization is also well known, and is carried out by a well-known method such as physical adsorption or covalent bond. When the obtained suspension of sensitized particles and the test sample are mixed, the sensitized particles are aggregated by the substance (antigen) to be measured contained in the test sample, and the absorbance of the sensitized particle suspension changes. do. The amount of change in absorbance (endpoint method) or rate of change (rate method) is measured. A plurality of standard samples containing the antigen to be measured at various known concentrations are prepared, and the amount of change or the rate of change in absorbance is measured for them by the above method. A calibration curve is drawn by plotting the concentration of the antigen to be measured in the standard sample on the horizontal axis and the amount of change or rate of change in the measured absorbance on the vertical axis. The amount of change or rate of change in absorbance of an unknown test sample is measured by the same method, and the measurement result is applied to the calibration curve to quantify the antigen in the test sample.
なお、このような免疫凝集法を行う自動装置が種々市販されており、市販の免疫凝集法用自動装置を用いて、容易、簡便に行うことができる。 Various automatic devices for performing such an immuno-aggregation method are commercially available, and can be easily and easily performed by using a commercially available automatic device for the immuno-aggregation method.
本発明における免疫分析による被測定物質としては、免疫分析により測定可能な物質であれば何ら限定されないが、被測定物質が抗原の場合、例えばＣＲＰ（Ｃ−反応性蛋白質）、前立腺特異抗原、フェリチン、β−２マイクログロブリン、ミオグロビン、ヘモグロビン、アルブミン、クレアチニン等のタンパク質マーカー、ＩｇＧ、ＩｇＡ、ＩｇＭ等の免疫グロブリン、各種腫瘍マーカー、ＬＤＬ、ＨＤＬ、ＴＧ等のリポ蛋白、Ａ型インフルエンザウイルス、Ｂ型インフルエンザウイルス、ＲＳウイルス（ＲＳＶ）、ライノウイルス、ロタウイルス、ノロウイルス、アデノウイルス、アストロウイルス、ＨＡＶ、ＨＢs、ＨＣＶ、ＨＩＶ、ＥＢＶ等のウイルス抗原、クラミジア・トラコマティス、溶連菌、百日咳菌、ヘリコバクター・ピロリ、レプトスピラ、トレポネーマ・パリダム、トキソプラズマ・ゴンディ、ボレリア、レジオネラ属菌、炭疽菌、MRSA等の細菌抗原、細菌等が産生する毒素、マイコプラズマ脂質抗原、ヒト絨毛製ゴナドトロピン等のペプチドホルモン、ステロイドホルモン等のステロイド、エピネフリンやモルヒネ等の生理活性アミン類、ビタミンＢ類等のビタミン類、プロスタグランジン類、テトラサイクリン等の抗生物質、農薬、環境ホルモン等が挙げられるがこれらに限定されるものではない。好ましい例として、ＣＲＰ、前立腺特異抗原、フェリチン、β−２マイクログロブリン及びヘモグロビン等の抗原が挙げられる。 The substance to be measured by immunoassay in the present invention is not limited as long as it is a substance that can be measured by immunoassay, but when the substance to be measured is an antigen, for example, CRP (C-reactive protein), prostate-specific antigen, ferritin. , Β-2 microglobulin, myoglobin, hemoglobin, albumin, creatinine and other protein markers, IgG, IgA, IgM and other immunoglobulins, various tumor markers, LDL, HDL, TG and other lipoproteins, type A influenza virus, type B Influenza virus, RS virus (RSV), rhinovirus, rotavirus, norovirus, adenovirus, astrovirus, HAV, HBs, HCV, HIV, EBV and other viral antigens, Chlamydia trachomatis, lytic bacteria, pertussis, helicobacter pylori , Leptspira, Treponema paridam, Toxoplasma gondii, Borrelia, Regionella spp., Charcoal antigen, MRSA and other bacterial antigens, Bacterial toxins, Mycoplasma lipid antigens, Human villus gonadotropin and other peptide hormones, steroid hormones, etc. Examples thereof include, but are not limited to, steroids, physiologically active amines such as epinephrine and morphine, vitamins such as vitamin B, antibiotics such as prostaglandins and tetracyclines, pesticides, and environmental hormones. Preferred examples include antigens such as CRP, prostate-specific antigen, ferritin, β-2 microglobulin and hemoglobin.
被測定物質が抗体の場合、上記のタンパク質マーカー、各種腫瘍マーカー、リポ蛋白、ウイルス抗原、細菌抗原、細菌等が産生する毒素、ペプチドホルモン、ステロイド、生理活性アミン類、ビタミン類、抗生物質、農薬、環境ホルモン等の抗原と特異的に反応する抗体等が挙げられる。 When the substance to be measured is an antibody, the above protein markers, various tumor markers, lipoproteins, viral antigens, bacterial antigens, toxins produced by bacteria, peptide hormones, steroids, physiologically active amines, vitamins, antibiotics, pesticides , Antibodies that specifically react with antigens such as environmental hormones.
免疫分析に用いられる検体は、被測定物質を含むものであれば特に限定されないが、血液、血清、血漿、尿、便、唾液、組織液、髄液、ぬぐい液等の体液等又はその希釈物が挙げられ、血液、血清、血漿、尿、便、髄液又はこれらの希釈物が好ましい。 The sample used for immunoanalysis is not particularly limited as long as it contains the substance to be measured, but blood, serum, plasma, urine, stool, saliva, tissue fluid, spinal fluid, body fluid such as swab, or a dilution thereof. Blood, serum, plasma, urine, stool, spinal fluid or dilutions thereof are preferred.
上記の通り、本発明の方法では、反応系及び／又は測定系において、アルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤が存在する状態下で抗原抗体反応及び／又は測定を行うことを特徴としている。ポリオキシエチレンアルキルエーテル界面活性剤のＨＬＢは特に限定されないが、１５〜１９が好ましい。 As described above, in the method of the present invention, in the reaction system and / or the measurement system, the antigen-antibody reaction and the antigen-antibody reaction are carried out in the presence of a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in the alkyl group. / Or it is characterized by making measurements. The HLB of the polyoxyethylene alkyl ether surfactant is not particularly limited, but 15 to 19 is preferable.
本発明の方法において、当該アルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤は、抗原抗体反応の開始から抗原抗体反応量の検出・定量が終了するまでのいずれかの段階でアルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤が反応及び／又は測定系（「反応・測定系」とも称する）内に含まれていればよいが、抗原抗体反応の開始から検出・定量までの間に亘って含まれていることが好ましい。従って、アルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤は、抗原抗体反応の開始前又は開始と同時に反応系内に添加するのが好ましい。具体的には、検体を希釈する際に添加してもよいし、抗体又は抗原と検体とを混合する際に添加してもよい。 In the method of the present invention, the polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in the alkyl group can be used from the start of the antigen-antibody reaction to the end of detection and quantification of the amount of the antigen-antibody reaction. At this stage, a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in the alkyl group may be contained in the reaction and / or measurement system (also referred to as “reaction / measurement system”). Is preferably contained from the start of the antigen-antibody reaction to the detection and quantification. Therefore, the polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in the alkyl group is preferably added into the reaction system before or at the same time as the start of the antigen-antibody reaction. Specifically, it may be added when the sample is diluted, or when the antibody or antigen is mixed with the sample.
また、免疫分析に用いる各種試薬に予めアルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤を含有させることでもよく、本発明は斯かるアルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤を含有した免疫分析試薬を提供するものでもある。ここで、免疫分析に用いる各種試薬としては、例えば、検体希釈液、抗体／抗原希釈液、固相化抗体／抗原、感作粒子懸濁液、洗浄液、酵素液、基質液、検量線作成用の被検物質標準液等が挙げられ、アルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤を含有した免疫分析試薬としては、これらの試薬にアルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤を添加したもの、例えば、検体を希釈する緩衝液や、抗体又は抗原を含む試薬等にアルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤を含有させたものが挙げられる。 Further, various reagents used for immunoanalysis may contain a polyoxyethylene alkyl ether surfactant having an alkyl group having 8 to 14 carbon atoms in advance, and the present invention has such an alkyl group having 8 to 14 carbon atoms. It also provides an immunoassay reagent containing 8 to 14 polyoxyethylene alkyl ether surfactants. Here, as various reagents used for immunoanalysis, for example, a sample diluent, an antibody / antigen diluent, an immobilized antibody / antigen, a sensitized particle suspension, a washing solution, an enzyme solution, a substrate solution, and a calibration line preparation are used. Examples of the immunoassay reagent containing a polyoxyethylene alkyl ether surfactant having an alkyl group having 8 to 14 carbon atoms include the standard solution of the test substance in the above, and these reagents have an alkyl group carbon atom. A reagent containing a polyoxyethylene alkyl ether surfactant having a number of 8 to 14, for example, a buffer for diluting a sample, a reagent containing an antibody or an antigen, etc., has an alkyl group having 8 to 14 carbon atoms. Examples thereof include those containing a polyoxyethylene alkyl ether surfactant.
また、例えば、抗体又は抗原を固定化（感作）したラテックス粒子（感作粒子）を含む免疫凝集試薬にアルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤を含有させておくことができる。この場合、免疫分析試薬中の感作粒子の濃度は、特に限定されないが、０．０１〜０．５％であることが好ましい。感作粒子浮遊液中の抗体量及び抗原量は常法どおりであってよく、特に限定されないが、例えば抗体感作ラテックスの場合、抗体量はラテックス浮遊液中に０．０１〜２．０ｍｇ／ｍＬとするのが好ましい。 Further, for example, a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms of an alkyl group is added to an immunoaggregating reagent containing latex particles (sensitized particles) on which an antibody or antigen is immobilized (sensitized). It can be contained. In this case, the concentration of the sensitized particles in the immunoassay reagent is not particularly limited, but is preferably 0.01 to 0.5%. The amount of antibody and the amount of antigen in the sensitized particle suspension may be as usual and are not particularly limited. For example, in the case of antibody-sensitized latex, the amount of antibody is 0.01 to 2.0 mg / in the latex suspension. It is preferably mL.
ポリカルボン酸型界面活性剤の反応・測定系内における濃度は、非特異反応抑制の点から、好ましくは０．０００１〜１％であり、さらに好ましくは０．００１〜０．５％である。したがって、免疫分析試薬に予めアルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤を含有させる場合には、反応及び／又は測定系内での濃度が上記の濃度になるように免疫分析試薬に含有させればよい。 The concentration of the polycarboxylic acid-type surfactant in the reaction / measurement system is preferably 0.0001 to 1%, more preferably 0.001 to 0.5%, from the viewpoint of suppressing non-specific reactions. Therefore, when the immunoassay reagent contains a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in the alkyl group in advance, the concentration in the reaction and / or measurement system becomes the above concentration. It may be contained in the immunoassay reagent so as to be.
免疫分析に用いられるブランク試料は、被測定物質を含み得ないものであれば特に限定されないが、精製水、生理食塩液、緩衝液、陰性検体又はその希釈物が好ましい。 The blank sample used for immunoassay is not particularly limited as long as it does not contain the substance to be measured, but purified water, physiological saline, buffer solution, negative sample or a dilution thereof is preferable.
後記実施例に記載されるように、アルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤を反応及び／又は測定系内に存在させた場合、非特異反応が抑制される。そして、特異性は、アルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤を存在させない場合に比べて、優位に向上する。したがって、本発明の方法を用いることにより、特に非特異反応が起きやすい高感度化した試薬においては、従来よりも試薬性能を向上することが可能になる。 As described in the examples below, when a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in the alkyl group is present in the reaction and / or measurement system, the non-specific reaction is suppressed. Will be done. Then, the specificity is significantly improved as compared with the case where the polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms of the alkyl group is not present. Therefore, by using the method of the present invention, it is possible to improve the reagent performance as compared with the conventional one, especially in the reagent with high sensitivity in which a non-specific reaction is likely to occur.
以下、本発明を実施例及び比較例に基づきより具体的に説明する。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 in more detail based on Examples and Comparative Examples. However, the present invention is not limited to the following examples.
実施例１〜６、比較例１
（１）試薬の調製
フェリチンに対する抗体を用いて、以下の通りに免疫凝集法による測定試薬を調製した。
ｉ）抗フェリチン抗体を平均粒径３００ｎｍのポリスチレンラテックス浮遊液１ｍＬに対し０．０３ｍｇ担持させてなる感作粒子を、緩衝液（トリス、ｐＨ８．０）に０．０４％となるように懸濁し、ラテックス浮遊液を調製した。
ii）緩衝液（トリス、ｐＨ８．５）に、アルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤(「エマルゲン１１１８Ｓ−７０」（商品名、花王株式会社から市販）、ＨＬＢ１６．４)を添加し、下記の試薬Ａ〜Ｆを調製した（実施例１〜６）。比較例１として、何も添加しない試薬Ｇを調製した。
Examples 1-6, Comparative Example 1
(1) Preparation of Reagents Using an antibody against ferritin, a reagent for measurement by the immunoagglutination method was prepared as follows.
i) Sensitive particles in which 0.03 mg of an anti-ferritin antibody is carried in 1 mL of a polystyrene latex suspension having an average particle size of 300 nm are suspended in a buffer solution (Tris, pH 8.0) so as to be 0.04%. , Latex buffer was prepared.
ii) In a buffer solution (Tris, pH 8.5), a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms of an alkyl group (“Emargen 1118S-70” (trade name, commercially available from Kao Corporation) ), HLB16.4) was added to prepare the following reagents A to F (Examples 1 to 6). As Comparative Example 1, a reagent G to which nothing was added was prepared.
（２）自動分析装置による測定
自動分析装置は日立社７１８０型自動分析装置によりエンドポイント法で自動測定を行った。
前述の試薬Ａ〜Ｇを用いて、非特異反応を起こすことで知られている偽陽性２検体及び正常１検体の測定を行った。検体溶液１５．０μＬに試薬Ａ〜Ｇの上記で調製した緩衝液１００μＬを添加し、この混合液を３７℃で撹拌混合した。５分間放置後、ラテックス浮遊液１００μＬを添加し、更に３７℃で撹拌混合した。約５分間の凝集反応を吸光度変化量として測定し、検量線より各検体のフェリチン濃度を算出した。
(2) Measurement by automatic analyzer The automatic analyzer used the Hitachi 7180 automatic analyzer to perform automatic measurement by the endpoint method.
Using the above-mentioned reagents A to G, two false positive samples and one normal sample known to cause a non-specific reaction were measured. To 15.0 μL of the sample solution, 100 μL of the buffer solution prepared above for Reagents A to G was added, and the mixed solution was stirred and mixed at 37 ° C. After standing for 5 minutes, 100 μL of the latex suspension was added, and the mixture was further stirred and mixed at 37 ° C. The agglutination reaction for about 5 minutes was measured as the amount of change in absorbance, and the ferritin concentration of each sample was calculated from the calibration curve.
（３）既存試薬との感度比較
上記実施例１〜６及び比較例１の測定感度と、非特異反応が抑制されていることがわかっている既存の市販ラテックス試薬であるＦＥＲ−ラテックスＸ２「生研」ＣＮ（デンカ生研）（以下、「既存試薬」）との感度の比較を行った。結果を図１示す。
(3) Comparison of Sensitivity with Existing Reagents The measurement sensitivities of Examples 1 to 6 and Comparative Example 1 and the existing commercially available latex reagent FER-Latex X2 "Seiken", which is known to suppress non-specific reactions. We compared the sensitivity with CN (Denka Seiken) (hereinafter, "existing reagent"). The results are shown in FIG.
図１に示されるように、実施例１〜６及び比較例１の方法では、既存試薬を用いた方法と比較して、同じフェリチン濃度であれば吸光度変化量が大きくなっており、実施例１〜５及び比較例１の方法の方が測定感度が高いことがわかる。 As shown in FIG. 1, in the methods of Examples 1 to 6 and Comparative Example 1, the amount of change in absorbance was large at the same ferritin concentration as compared with the method using the existing reagent, and Example 1 It can be seen that the methods of ~ 5 and Comparative Example 1 have higher measurement sensitivities.
（４）偽陽性検体の測定値皮革
上記実施例１〜６と比較例１の測定結果を比較した。結果を表２に示す。
(4) Measured value of false positive sample Leather The measurement results of Examples 1 to 6 and Comparative Example 1 were compared. The results are shown in Table 2.
既実施例１〜６の測定結果と、比較例１の測定結果を比べると、高感度化した免疫分析方法において、アルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤を添加することで、非特異反応が抑制され、正常検体の測定結果は変動せずに偽陽性検体の測定値が低下することが示された。 Comparing the measurement results of Examples 1 to 6 with the measurement results of Comparative Example 1, the polyoxyethylene alkyl ether surface activity in which the number of carbon atoms of the alkyl group is 8 to 14 in the highly sensitive immunoanalytical method. It was shown that the addition of the agent suppressed the non-specific reaction, and the measurement results of the normal specimens did not fluctuate and the measured values of the false positive specimens decreased.
実施例７、８、比較例２、３
（１）試薬の調製
フェリチンに対する抗体を用いて、以下の通りに免疫凝集法による測定試薬を調製した。
ｉ）抗フェリチン抗体を平均粒径３００ｎｍのポリスチレンラテックス浮遊液１ｍＬに対し０．０３ｍｇ担持させてなる感作粒子を、緩衝液（トリス、ｐＨ８．０）に０．０４％となるように懸濁し、ラテックス浮遊液を調製した。
ii）緩衝液（トリス、ｐＨ８．５）に、アルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤(「エマルゲン１１１８Ｓ−７０」（商品名、花王株式会社から市販）、ＨＬＢ１６．４)及びヒトIgM自然抗体を添加し、下記の試薬Ｈ（実施例７）、試薬Ｉ（実施例８）を調製した。比較例２として何も添加しない試薬Ｊ、ヒトIgM自然抗体のみを添加した試薬Ｋを調製した。
Examples 7 and 8, Comparative Examples 2 and 3
(1) Preparation of Reagents Using an antibody against ferritin, a reagent for measurement by the immunoagglutination method was prepared as follows.
i) Sensitive particles in which 0.03 mg of an anti-ferritin antibody is carried in 1 mL of a polystyrene latex suspension having an average particle size of 300 nm are suspended in a buffer solution (Tris, pH 8.0) so as to be 0.04%. , Latex buffer was prepared.
ii) A polyoxyethylene alkyl ether surfactant (“Emargen 1118S-70” (trade name, commercially available from Kao Corporation) in which the number of carbon atoms of the alkyl group is 8 to 14 in a buffer solution (Tris, pH 8.5). ), HLB16.4) and human IgM natural antibody were added to prepare the following Reagent H (Example 7) and Reagent I (Example 8). As Comparative Example 2, Reagent J to which nothing was added and Reagent K to which only human IgM natural antibody was added were prepared.
（２）自動分析装置による測定
前述の試薬Ｈ〜Ｋを用いて、非特異反応を起こすことで知られている偽陽性２検体の測定を行った。検体溶液１５．０μＬに試薬Ｈ〜Ｋの上記で調製した緩衝液１００μＬを添加し、この混合液を３７℃で撹拌混合した。５分間放置後、ラテックス浮遊液１００μＬを添加し、更に３７℃で撹拌混合した。約５分間の凝集反応を吸光度変化量として測定し、検量線より各検体のフェリチン濃度を算出した。
(2) Measurement by automatic analyzer Using the reagents HK described above, two false positive samples known to cause a non-specific reaction were measured. To 15.0 μL of the sample solution, 100 μL of the buffer solution prepared above for Reagents HK was added, and the mixed solution was stirred and mixed at 37 ° C. After standing for 5 minutes, 100 μL of the latex suspension was added, and the mixture was further stirred and mixed at 37 ° C. The agglutination reaction for about 5 minutes was measured as the amount of change in absorbance, and the ferritin concentration of each sample was calculated from the calibration curve.
（４）既存試薬との比較
上記実施例７、８と比較例２、３の測定結果を比較した。結果を表４に示す。
(4) Comparison with existing reagents The measurement results of Examples 7 and 8 and Comparative Examples 2 and 3 were compared. The results are shown in Table 4.
実施例７、８の測定結果と、比較例２、３の測定結果を比べると、高感度化した免疫分析方法において、非特異反応抑制することで知られているヒト自然IgM抗体では非特異反応が抑制できず、アルキル基の炭素原子数が８〜１４個であるポリオキシエチレンアルキルエーテル界面活性剤を添加することで、非特異反応が抑制され、偽陽性検体の測定結果が低下することが示された。 Comparing the measurement results of Examples 7 and 8 with the measurement results of Comparative Examples 2 and 3, the non-specific reaction of the human natural IgM antibody known to suppress the non-specific reaction in the highly sensitive immunoassay method However, by adding a polyoxyethylene alkyl ether surfactant having 8 to 14 carbon atoms in the alkyl group, the non-specific reaction can be suppressed and the measurement result of the false positive sample can be deteriorated. Shown.
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