JP6740083B2 - Immunological measurement reagent, measurement method, and method for expanding measurement range - Google Patents
Immunological measurement reagent, measurement method, and method for expanding measurement range Download PDFInfo
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- JP6740083B2 JP6740083B2 JP2016208003A JP2016208003A JP6740083B2 JP 6740083 B2 JP6740083 B2 JP 6740083B2 JP 2016208003 A JP2016208003 A JP 2016208003A JP 2016208003 A JP2016208003 A JP 2016208003A JP 6740083 B2 JP6740083 B2 JP 6740083B2
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Description
本発明は、免疫学的測定方法、免疫学的測定試薬及び測定範囲の拡大方法に関する。 The present invention relates to an immunological measurement method, an immunological measurement reagent, and a method for expanding a measurement range.
臨床検査の分野では迅速かつ正確に測定を行うことが常に必要とされており、その要請に応じる形で、試薬組成や測定方法などについて、多くの改善努力がなされてきた。
臨床検査分野においては自動分析装置を使用することが多いが、高精度での測定という分析性能を左右する因子として、分析装置の性能と試薬の性能の両面からアプローチされる。特に複数の装置で使用されるような汎用性の高い測定試薬の場合には、その試薬性能として感度や特異性はもちろんのこと、試薬の安定性や測定範囲が十分に確保されていることが非常に重要である。
In the field of clinical testing, it is always necessary to perform quick and accurate measurement, and in response to the request, many efforts have been made to improve the reagent composition and the measurement method.
In the field of clinical testing, automatic analyzers are often used, but as a factor that influences the analytical performance of highly accurate measurement, the analytical instrument performance and the reagent performance are both approached. In particular, in the case of a highly versatile measuring reagent that is used in multiple devices, the reagent performance is not only sensitivity and specificity, but also the stability and measuring range of the reagent are sufficiently secured. Very important.
特に、分析対象物質を定量的に測定する場合、臨床検査等の分野では、正確な測定が行える分析対象物質の濃度範囲を測定範囲として設定している。測定範囲を超える高値の検体であった場合、希釈して再検査を行う必要がある。このような高値検体の再検率を減らすことにより、報告時間の短縮やコストダウンが見込めるため、臨床現場では、測定範囲をより広くすることが求められている。 In particular, in the case of quantitatively measuring a substance to be analyzed, in the field of clinical examination and the like, a concentration range of the substance to be analyzed that allows accurate measurement is set as a measurement range. If the sample has a high value that exceeds the measurement range, it is necessary to dilute and retest. By reducing the retesting rate of such high-value samples, it is possible to shorten the reporting time and reduce the cost. Therefore, it is required in clinical practice to broaden the measurement range.
正確な測定とは、分析対象物質の濃度を高い信頼性で決定できることであり、評価する指標の一つとして、希釈試験が挙げられる。希釈試験は、被検試料の希釈系列を作製・測定し、横軸に希釈倍率を、縦軸に検量線から求めた測定値をとったグラフを描いた時に、原点を通る直線となることを確認する試験である。このグラフが直線とならない場合は、被検試料中に含まれる分析対象物質と標準品の反応性が異なっていることになり、検量線から求めた測定値の信頼性は低くなる。なお、上述のグラフを描く代わりに、被検試料の希釈系列を測定して得られた吸光度をプロットした曲線を検量線に重ね、その形状が一致していることを確かめることでも、希釈直線性を確認することができる。 Accurate measurement means that the concentration of the substance to be analyzed can be determined with high reliability, and one of the indexes to be evaluated is a dilution test. In the dilution test, when a dilution series of the test sample is prepared and measured, a horizontal axis shows the dilution ratio and a vertical axis shows the measured value obtained from the calibration curve, a straight line that passes through the origin is drawn. This is a test to confirm. If this graph is not a straight line, it means that the analyte contained in the test sample and the standard product have different reactivities, and the reliability of the measured value obtained from the calibration curve is low. Incidentally, instead of drawing the above-mentioned graph, a curve plotting the absorbance obtained by measuring the dilution series of the test sample is superimposed on the calibration curve, and it is also possible to confirm that the shapes are the same, and the linearity of dilution is also confirmed. Can be confirmed.
このような精度の高い測定が必要とされる試薬の一例として、Ｄダイマー測定試薬を例に説明する。Ｄダイマーとは、安定化フィブリン（cross-linked fibrin）のプラスミン分解産物（別称として、例えば、「Ｄ−Ｄダイマー」、「ＤＤ／Ｅ複合体」、「ＸＤＰ」と総称されることがある）のことであり、最小単位であるＤＤ／Ｅ（ＤＤ／Ｅモノマー）、並びにその高分子ＤＤ／Ｅ（ＤＤ／Ｅポリマー、例えば、ＤＸＤ／ＹＹ、ＹＸＹ／ＤＸＸＤ、ＤＸＸＹ／ＹＸＸＤなど）を含み、深部静脈血栓症（ＤＶＴ）の除外診断や、播種性血管内凝固症候群（ＤＩＣ）の診断マーカーとして広汎に使用されている。
Ｄダイマーの測定方法としては、Ｄダイマーを認識するモノクローナル抗体を、ラテックス粒子、プラスチックプレートなどの固相に固定してＤダイマーと結合させる抗原抗体反応に基づく方法、すなわちラテックス凝集法やＥＬＩＳＡ法などが知られている。
As an example of a reagent that requires such highly accurate measurement, a D-dimer measurement reagent will be described as an example. D-dimer is a plasmin-degradation product of stabilized cross-linked fibrin (may be collectively referred to as "DD-dimer", "DD/E complex", "XDP") That is, DD/E (DD/E monomer) which is the minimum unit, and its high molecular weight DD/E (DD/E polymer, for example, DXD/YY, YXY/DXXD, DXXY/YXXD, etc.), It is widely used as an exclusion diagnosis of deep vein thrombosis (DVT) and a diagnostic marker for disseminated intravascular coagulation (DIC).
As a method for measuring D-dimer, a method based on an antigen-antibody reaction in which a monoclonal antibody that recognizes D-dimer is immobilized on a solid phase such as latex particles or a plastic plate and bonded to D-dimer, that is, latex agglutination method or ELISA method, etc. It has been known.
血中での存在形式が多様で分子量が不均一であるＤダイマーを免疫学的に測定する場合、臨床検体に含まれるＤダイマーと標準品に含まれるＤダイマーでは含まれるＤダイマーの分子量の分布が異なるため、臨床検体によっては希釈系列を測定して得られた吸光度をプロットした曲線の形状が、検量線の形状から乖離することがある。その傾向は高濃度領域であればあるほどより顕著に表れるため、検量線から乖離しない（希釈直線性が得られる）濃度範囲を測定範囲として設定しようと試みると、特に、測定上限が制限され、実用上問題であった。 Distribution of molecular weight of D-dimer contained in clinical specimen and D-dimer contained in standard sample when immunologically measuring D-dimer having various molecular forms in blood and heterogeneous molecular weight Therefore, the shape of the curve plotting the absorbance obtained by measuring the dilution series may deviate from the shape of the calibration curve depending on the clinical sample. The tendency becomes more pronounced in higher concentration regions, so if you try to set a concentration range that does not deviate from the calibration curve (dilution linearity can be obtained) as the measurement range, the upper limit of measurement will be limited, It was a problem in practice.
これまで、Ｄダイマーの測定において分析結果の正確性を向上するために、希釈用緩衝液にＤモノマーを共存させる方法（特許文献１）や、血栓症患者の臨床挙動に合致したＤダイマー測定用の標準物質（特許文献２）、Ｄダイマーに特異的な抗体（特許文献３）などが提案されている。しかし、依然として、被検試料中のＤダイマーの反応性が検量線と一致する濃度範囲は限られており、低濃度域からより高濃度域まで、正確に測定可能なＤダイマー測定試薬が望まれていた。また、このことはＤダイマー測定試薬に限定された話ではなく、臨床検査で用いられる全ての試薬において求められるものである。 Up to now, in order to improve the accuracy of the analysis results in the measurement of D-dimer, a method in which a D-monomer coexists in a diluting buffer (Patent Document 1), and for D-dimer measurement that matches the clinical behavior of patients with thrombosis (Reference 2), an antibody specific to D dimer (Reference 3), and the like have been proposed. However, the concentration range in which the reactivity of the D-dimer in the test sample matches the calibration curve is still limited, and a D-dimer measuring reagent that can accurately measure from the low-concentration region to the higher-concentration region is desired. Was there. Further, this is not limited to the D-dimer measurement reagent, but is required for all reagents used in clinical tests.
ところで、非特異反応の抑制や感度の向上を目的として、反応液中の緩衝液濃度を検討したことが報告されている。特許文献４では、０．５ｍｏｌ／Ｌまたは０．１ｍｏｌ／ＬのＨＥＰＥＳ、ＰＩＰＥＳ、又はＭＯＰＳ緩衝液を凝集促進剤と共に用いた場合、０．１ｍｏｌ／Ｌリン酸緩衝液を凝集促進剤と共に用いる場合と比較して、抗梅毒トレポネーマ抗体を含まない検体における非特異反応が抑制されることが開示されている。
一方、特許文献５では、０．０５ｍｏｌ／Ｌ又は０．１ｍｏｌ／Ｌ（反応液中での濃度はそれぞれ０．０４ｍｏｌ／Ｌ又は０．０８ｍｏｌ／Ｌ）のＭＯＰＳ、ＰＩＰＥＳ、又はＨＥＰＥＳ緩衝液を用いた場合、高感度に標準梅毒トレポネーマ陽性血清を測定できるが、０．６ｍｏｌ／Ｌ（反応液中での濃度は０．５ｍｏｌ／Ｌ）の同緩衝液を用いた場合は、吸光度変化量の差が小さいため、感度の問題があることが開示されている。
しかし、これらの報告は、上記したような希釈直線性を改善して測定範囲を拡大することで正確な測定を達成できることを示唆するものはない。
By the way, it has been reported that the buffer concentration in the reaction solution was examined for the purpose of suppressing non-specific reaction and improving sensitivity. In Patent Document 4, when 0.5 mol/L or 0.1 mol/L HEPES, PIPES, or MOPS buffer is used with an aggregation promoter, when 0.1 mol/L phosphate buffer is used with an aggregation promoter. It is disclosed that the non-specific reaction is suppressed in a sample that does not contain the anti- Treponema pallidum antibody, as compared with.
On the other hand, in Patent Document 5, 0.05 mol/L or 0.1 mol/L (concentration in the reaction solution is 0.04 mol/L or 0.08 mol/L, respectively) of MOPS, PIPES, or HEPES buffer solution is used. When the standard syphilis Treponema pallidum serum can be measured with high sensitivity, the difference in absorbance changes when 0.6 mol/L (concentration in the reaction solution is 0.5 mol/L) of the same buffer solution is used. It is disclosed that there is a problem of sensitivity due to the small value.
However, these reports do not suggest that accurate measurement can be achieved by improving the dilution linearity as described above and expanding the measurement range.
このような状況に鑑み、免疫学的測定において、低濃度域からより高濃度域まで、正確に測定することができる、免疫学的測定方法および測定試薬を提供することが本発明の目的である。 In view of such a situation, it is an object of the present invention to provide an immunological measurement method and a measurement reagent capable of accurately measuring from a low concentration range to a higher concentration range in an immunological measurement. ..
本発明者らは、前記の問題点を解決すべく鋭意検討した結果、免疫学的測定において、免疫反応（抗原抗体反応）を行う反応液中の緩衝液濃度を０．１９ｍｏｌ／Ｌ以上にすることによって、被検試料の希釈直線性が良好となり、それにより測定範囲を拡大できることを見出し、本発明に至った。本発明はこの知見に基づいて、低濃度域からより高濃度域まで正確な測定を行うための測定方法及び測定試薬を完成させたものである。 As a result of intensive studies to solve the above-mentioned problems, the present inventors set the buffer concentration in the reaction solution in which the immunological reaction (antigen-antibody reaction) is 0.19 mol/L or more in the immunological measurement. As a result, it was found that the linearity of dilution of the sample to be tested becomes good, and thereby the measurement range can be expanded, and the present invention was completed. The present invention has completed a measurement method and a measurement reagent for performing accurate measurement from a low concentration range to a higher concentration range based on this finding.
すなわち、本発明は、
［１］測定対象物質に対する抗体若しくはそのフラグメント又は抗原を固定化した不溶性担体を用いる、被検試料中の前記測定対象物質を免疫学的に測定する方法において、０．１９ｍｏｌ／Ｌ以上の濃度の緩衝液の存在下で抗原抗体反応を行う、測定方法、
［２］前記不溶性担体が、ラテックス粒子である、［１］の測定方法、
［３］前記緩衝液が、ＨＥＰＥＳ緩衝液、Ｔｒｉｓ緩衝液、又はＢｉｓ−Ｔｒｉｓ緩衝液から選ばれる、［１］又は［２］の測定方法、
［４］前記測定対象物質が、ＣＲＰ、プロテインＣ、ヒアルロン酸、ＡＦＰ（α−フェトプロテイン）、ＩｇＥ、ＲＦ（リウマチ因子）、ＩＲＥ（エラスターゼ１）、ＡＳＯ（抗ストレプトリジンＯ）、アディポネクチン（ＡＤＮ）、ＡＴIII（アンチトロンビンIII）、β２Ｍ（β ２ マイクログロブリン）、ＣＥＡ（癌胎児性抗原）、第ＸIII因子（１３因子、Ｆ１３）、ＦＤＰ（フィブリン・フィブリノゲン分解産物）、ＦＲＴＮ（フェリチン）、ＨＣＧ（ヒト絨毛性ゴナドトロピン）、ＨＰＬ（ヒト胎盤性ラクトゲン）、ｔＰＡＩ（組織プラスミノゲンアクチベータ・プラスミノゲンアクチベータインヒビター１複合体）、Ｄダイマー、ＳＦ（可溶性フィブリン）、ＰＰＩ（ＰＩＣ、プラスミン・プラスミンインヒビター複合体）、ＴＡＴ（トロンビン・アンチトロンビン複合体）、Ｃｙｓ Ｃ（シスタチンＣ）、ＰＳＡ（前立腺特異抗原）、ＰＧ（ペプシノゲンＩ・II）、ヘモグロビン、Ｔｆ（尿中トランスフェリン、便中トランスフェリン）、尿中ＢＴＡ（膀胱腫瘍抗原）、ＩＲＩ（インスリン）、Ｅ２（エストラジオール、エストロゲン）、Ｅ３（エストリオール）、ＭＭＰ−３（マトリックスメタロプロティナーゼ−３）、Ｍｂ（ミオグロビン）、Ｈ−ＦＡＢＰ（ヒト心臓由来脂肪酸結合蛋白）、Ｌ−ＦＡＢＰ（尿中Ｌ型脂肪酸結合蛋白）、ＫＬ−６（シアル化糖鎖抗原）、ＲＢＰ（レチノール結合蛋白）、ＩＶ型コラーゲン、ＨＣＶ抗体、ＨＢｓ抗原、ＨＢｅ抗原・抗体、ＨＢｃ抗体、ＨＩＶ抗体、ＨＴＬＶ−１抗体である、［１］〜［３］のいずれかに記載の測定方法、
［５］測定対象物質に対する抗体若しくはそのフラグメント又は抗原、及び緩衝液を含む、免疫学的測定試薬であって、前記緩衝液の濃度が、抗原抗体反応時の反応液中の濃度として０．１９ｍｏｌ／Ｌ以上となる濃度である、免疫学的測定試薬、
［６］前記測定対象物質が、ＣＲＰ、プロテインＣ、ヒアルロン酸、ＡＦＰ（α−フェトプロテイン）、ＩｇＥ、ＲＦ（リウマチ因子）、ＩＲＥ（エラスターゼ１）、ＡＳＯ（抗ストレプトリジンＯ）、アディポネクチン（ＡＤＮ）、ＡＴIII（アンチトロンビンIII）、β２Ｍ（β ２ マイクログロブリン）、ＣＥＡ（癌胎児性抗原）、第ＸIII因子（１３因子、Ｆ１３）、ＦＤＰ（フィブリン・フィブリノゲン分解産物）、ＦＲＴＮ（フェリチン）、ＨＣＧ（ヒト絨毛性ゴナドトロピン）、ＨＰＬ（ヒト胎盤性ラクトゲン）、ｔＰＡＩ（組織プラスミノゲンアクチベータ・プラスミノゲンアクチベータインヒビター１複合体）、Ｄダイマー、ＳＦ（可溶性フィブリン）、ＰＰＩ（ＰＩＣ、プラスミン・プラスミンインヒビター複合体）、ＴＡＴ（トロンビン・アンチトロンビン複合体）、Ｃｙｓ Ｃ（シスタチンＣ）、ＰＳＡ（前立腺特異抗原）、ＰＧ（ペプシノゲンＩ・II）、ヘモグロビン、Ｔｆ（尿中トランスフェリン、便中トランスフェリン）、尿中ＢＴＡ（膀胱腫瘍抗原）、ＩＲＩ（インスリン）、Ｅ２（エストラジオール、エストロゲン）、Ｅ３（エストリオール）、ＭＭＰ−３（マトリックスメタロプロティナーゼ−３）、Ｍｂ（ミオグロビン）、Ｈ−ＦＡＢＰ（ヒト心臓由来脂肪酸結合蛋白）、Ｌ−ＦＡＢＰ（尿中Ｌ型脂肪酸結合蛋白）、ＫＬ−６（シアル化糖鎖抗原）、ＲＢＰ（レチノール結合蛋白）、ＩＶ型コラーゲン、ＨＣＶ抗体、ＨＢｓ抗原、ＨＢｅ抗原・抗体、ＨＢｃ抗体、ＨＩＶ抗体、ＨＴＬＶ−１抗体である、［５］の免疫学的測定試薬、
［７］測定対象物質に対する抗体若しくはそのフラグメント又は抗原を固定化した不溶性担体を用いる、免疫学的測定において、０．１９ｍｏｌ／Ｌ以上の濃度の緩衝液の存在下で抗原抗体反応を行う、前記測定における測定範囲の拡大方法、
［８］前記測定対象物質が、ＣＲＰ、プロテインＣ、ヒアルロン酸、ＡＦＰ（α−フェトプロテイン）、ＩｇＥ、ＲＦ（リウマチ因子）、ＩＲＥ（エラスターゼ１）、ＡＳＯ（抗ストレプトリジンＯ）、アディポネクチン（ＡＤＮ）、ＡＴIII（アンチトロンビンIII）、β２Ｍ（β ２ マイクログロブリン）、ＣＥＡ（癌胎児性抗原）、第ＸIII因子（１３因子、Ｆ１３）、ＦＤＰ（フィブリン・フィブリノゲン分解産物）、ＦＲＴＮ（フェリチン）、ＨＣＧ（ヒト絨毛性ゴナドトロピン）、ＨＰＬ（ヒト胎盤性ラクトゲン）、ｔＰＡＩ（組織プラスミノゲンアクチベータ・プラスミノゲンアクチベータインヒビター１複合体）、Ｄダイマー、ＳＦ（可溶性フィブリン）、ＰＰＩ（ＰＩＣ、プラスミン・プラスミンインヒビター複合体）、ＴＡＴ（トロンビン・アンチトロンビン複合体）、Ｃｙｓ Ｃ（シスタチンＣ）、ＰＳＡ（前立腺特異抗原）、ＰＧ（ペプシノゲンＩ・II）、ヘモグロビン、Ｔｆ（尿中トランスフェリン、便中トランスフェリン）、尿中ＢＴＡ（膀胱腫瘍抗原）、ＩＲＩ（インスリン）、Ｅ２（エストラジオール、エストロゲン）、Ｅ３（エストリオール）、ＭＭＰ−３（マトリックスメタロプロティナーゼ−３）、Ｍｂ（ミオグロビン）、Ｈ−ＦＡＢＰ（ヒト心臓由来脂肪酸結合蛋白）、Ｌ−ＦＡＢＰ（尿中Ｌ型脂肪酸結合蛋白）、ＫＬ−６（シアル化糖鎖抗原）、ＲＢＰ（レチノール結合蛋白）、ＩＶ型コラーゲン、ＨＣＶ抗体、ＨＢｓ抗原、ＨＢｅ抗原・抗体、ＨＢｃ抗体、ＨＩＶ抗体、ＨＴＬＶ−１抗体である、［７］の拡大方法
に関する。
That is, the present invention is
[1] In a method for immunologically measuring the above-mentioned substance to be measured in a test sample using an insoluble carrier on which an antibody or fragment thereof against the substance to be measured or an antigen is immobilized, a concentration of 0.19 mol/L or more is used. A measuring method, which comprises performing an antigen-antibody reaction in the presence of a buffer,
[2] The measuring method according to [1], wherein the insoluble carrier is latex particles,
[3] The measuring method according to [1] or [2], wherein the buffer is selected from HEPES buffer, Tris buffer, and Bis-Tris buffer.
[4] The substance to be measured is CRP, protein C, hyaluronic acid, AFP (α-fetoprotein), IgE, RF (rheumatoid factor), IRE (elastase 1), ASO (antistreptolysin O), adiponectin (ADN) , ATIII (antithrombin III), β2M (β 2 microglobulin), CEA (carcinoembryonic antigen), factor XIII (factor 13, F13), FDP (fibrin/fibrinogen degradation product), FRTN (ferritin), HCG ( Human chorionic gonadotropin), HPL (human placental lactogen), tPAI (tissue plasminogen activator/plasminogen activator inhibitor 1 complex), D dimer, SF (soluble fibrin), PPI (PIC, plasmin/plasmin inhibitor complex), TAT (Thrombin/antithrombin complex), Cys C (cystatin C), PSA (prostate specific antigen), PG (pepsinogen I/II), hemoglobin, Tf (urinary transferrin, fecal transferrin), urinary BTA (bladder tumor) Antigen), IRI (insulin), E2 (estradiol, estrogen), E3 (estriol), MMP-3 (matrix metalloproteinase-3), Mb (myoglobin), H-FABP (human heart-derived fatty acid binding protein), L -FABP (urinary L-type fatty acid binding protein), KL-6 (sialylated sugar chain antigen), RBP (retinol binding protein), type IV collagen, HCV antibody, HBs antigen, HBe antigen/antibody, HBc antibody, HIV antibody The measuring method according to any one of [1] to [3], which is an HTLV-1 antibody,
[5] An immunological measurement reagent containing an antibody or fragment thereof or an antigen against a substance to be measured, and a buffer solution, wherein the concentration of the buffer solution is 0.19 mol as the concentration in the reaction solution at the time of the antigen-antibody reaction. /L or more, an immunological measurement reagent,
[6] The substance to be measured is CRP, protein C, hyaluronic acid, AFP (α-fetoprotein), IgE, RF (rheumatoid factor), IRE (elastase 1), ASO (antistreptolysin O), adiponectin (ADN) , ATIII (antithrombin III), β2M (β 2 microglobulin), CEA (carcinoembryonic antigen), factor XIII (factor 13, F13), FDP (fibrin/fibrinogen degradation product), FRTN (ferritin), HCG ( Human chorionic gonadotropin), HPL (human placental lactogen), tPAI (tissue plasminogen activator/plasminogen activator inhibitor 1 complex), D dimer, SF (soluble fibrin), PPI (PIC, plasmin/plasmin inhibitor complex), TAT (Thrombin/antithrombin complex), Cys C (cystatin C), PSA (prostate specific antigen), PG (pepsinogen I/II), hemoglobin, Tf (urinary transferrin, fecal transferrin), urinary BTA (bladder tumor) Antigen), IRI (insulin), E2 (estradiol, estrogen), E3 (estriol), MMP-3 (matrix metalloproteinase-3), Mb (myoglobin), H-FABP (human heart-derived fatty acid binding protein), L -FABP (urinary L-type fatty acid binding protein), KL-6 (sialylated sugar chain antigen), RBP (retinol binding protein), type IV collagen, HCV antibody, HBs antigen, HBe antigen/antibody, HBc antibody, HIV antibody An immunological assay reagent of [5], which is an HTLV-1 antibody,
[7] In an immunological assay using an antibody against the substance to be measured or a fragment thereof or an insoluble carrier on which an antigen is immobilized, an antigen-antibody reaction is performed in the presence of a buffer solution having a concentration of 0.19 mol/L or more, Method of expanding measurement range in measurement,
[8] The substance to be measured is CRP, protein C, hyaluronic acid, AFP (α-fetoprotein), IgE, RF (rheumatoid factor), IRE (elastase 1), ASO (antistreptolysin O), adiponectin (ADN) , ATIII (antithrombin III), β2M (β 2 microglobulin), CEA (carcinoembryonic antigen), factor XIII (factor 13, F13), FDP (fibrin/fibrinogen degradation product), FRTN (ferritin), HCG ( Human chorionic gonadotropin), HPL (human placental lactogen), tPAI (tissue plasminogen activator/plasminogen activator inhibitor 1 complex), D dimer, SF (soluble fibrin), PPI (PIC, plasmin/plasmin inhibitor complex), TAT (Thrombin/antithrombin complex), Cys C (cystatin C), PSA (prostate specific antigen), PG (pepsinogen I/II), hemoglobin, Tf (urinary transferrin, fecal transferrin), urinary BTA (bladder tumor) Antigen), IRI (insulin), E2 (estradiol, estrogen), E3 (estriol), MMP-3 (matrix metalloproteinase-3), Mb (myoglobin), H-FABP (human heart-derived fatty acid binding protein), L -FABP (urinary L-type fatty acid binding protein), KL-6 (sialylated sugar chain antigen), RBP (retinol binding protein), type IV collagen, HCV antibody, HBs antigen, HBe antigen/antibody, HBc antibody, HIV antibody , HTLV-1 antibody, [7].
本発明の測定方法を用いることにより、低濃度域の感度を維持したまま、より高濃度域まで高精度に測定することができる。また、本発明の測定試薬を用いることにより、被検試料の希釈直線性が向上し、より広い測定範囲で正確に測定することができる。 By using the measuring method of the present invention, it is possible to measure with high accuracy in a higher concentration range while maintaining the sensitivity in the low concentration range. Further, by using the measuring reagent of the present invention, the dilution linearity of the test sample is improved, and it is possible to perform accurate measurement in a wider measurement range.
本明細書における以下の項において、測定方法、測定試薬、測定範囲の拡大方法について実施の一態様としてＤダイマー測定を例に記載するが、本発明はこの範囲に限定されるものではない。
また、本明細書において、単に「Ｄダイマー」と称する場合には、前記「Ｄダイマー」は、安定化フィブリン（特にはヒト安定化フィブリン）のプラスミン分解産物であるＤダイマーを意味し、ＤＤ／Ｅモノマー及びＤＤ／Ｅポリマーを含む。
In the following sections of the present specification, the measurement method, the measurement reagent, and the method for expanding the measurement range will be described by taking D-dimer measurement as an example of an embodiment, but the present invention is not limited to this range.
Further, in the present specification, when simply referred to as “D dimer”, the “D dimer” means a D dimer which is a plasmin degradation product of stabilized fibrin (particularly human stabilized fibrin), and DD/ Includes E monomer and DD/E polymer.
（１）測定項目
本発明は、抗体またはその抗体に由来するフラグメント（以下、併せて、単に抗体と称することがあります）、あるいは、抗原を不溶性担体に固定化した免疫学的反応を利用した測定方法、測定試薬において使用することができる。後述する不溶性担体に抗体若しくはそのフラグメント又は抗原を固定化して免疫学的反応を行うものであれば、本発明を利用して希釈直線性の改善、測定範囲の拡大ができるため、測定項目は特に限定されない。特に、後述するように抗体を固定化するラテックス粒子の粒径が比較的大きい場合に好ましく用いることができる。
(1) Measurement items The present invention uses an antibody or a fragment derived from the antibody (hereinafter, also simply referred to as an antibody) or an immunological reaction in which an antigen is immobilized on an insoluble carrier. It can be used in the method and the measuring reagent. As long as an antibody or a fragment thereof or an antigen is immobilized on an insoluble carrier described below to perform an immunological reaction, the present invention can be used to improve the dilution linearity and expand the measurement range. Not limited. In particular, it can be preferably used when the particle size of the latex particles for immobilizing the antibody is relatively large as described later.
本発明を好ましく使用できる項目は特に限定されるものではないが、血中濃度が低い等の理由から高感度を求められる項目において、特に有用である。ここで高感度とは、例えば数十μｇ／ｍＬ以下を意味し、前記感度が必要とされる高感度が求められる項目では、測定の際に粒径の大きなラテックス粒子を使用する可能性が高いことが想定されるため、本発明を好ましく使用することができる。
具体的測定項目としては、例えば、ＣＲＰ、プロテインＣ、ヒアルロン酸、ＡＦＰ（α−フェトプロテイン）、ＩｇＥ、ＲＦ（リウマチ因子）、ＩＲＥ（エラスターゼ１）、ＡＳＯ（抗ストレプトリジンＯ）、アディポネクチン（ＡＤＮ）、ＡＴIII（アンチトロンビンIII）、β２Ｍ（β ２ マイクログロブリン）、ＣＥＡ（癌胎児性抗原）、第ＸIII因子（１３因子、Ｆ１３）、ＦＤＰ（フィブリン・フィブリノゲン分解産物）、ＦＲＴＮ（フェリチン）、ＨＣＧ（ヒト絨毛性ゴナドトロピン）、ＨＰＬ（ヒト胎盤性ラクトゲン）、ｔＰＡＩ（組織プラスミノゲンアクチベータ・プラスミノゲンアクチベータインヒビター１複合体）、Ｄダイマー、ＳＦ（可溶性フィブリン）、ＰＰＩ（ＰＩＣ、プラスミン・プラスミンインヒビター複合体）、ＴＡＴ（トロンビン・アンチトロンビン複合体）、Ｃｙｓ Ｃ（シスタチンＣ）、ＰＳＡ（前立腺特異抗原）、ＰＧ（ペプシノゲンＩ・II）、ヘモグロビン、Ｔｆ（尿中トランスフェリン、便中トランスフェリン）、尿中ＢＴＡ（膀胱腫瘍抗原）、ＩＲＩ（インスリン）、Ｅ２（エストラジオール、エストロゲン）、Ｅ３（エストリオール）、ＭＭＰ−３（マトリックスメタロプロティナーゼ−３）、Ｍｂ（ミオグロビン）、Ｈ−ＦＡＢＰ（ヒト心臓由来脂肪酸結合蛋白）、Ｌ−ＦＡＢＰ（尿中Ｌ型脂肪酸結合蛋白）、ＫＬ−６（シアル化糖鎖抗原）、ＲＢＰ（レチノール結合蛋白）、ＩＶ型コラーゲン、ＨＣＶ抗体、ＨＢｓ抗原、ＨＢｅ抗原・抗体、ＨＢｃ抗体、ＨＩＶ抗体、ＨＴＬＶ−１抗体などが挙げられるが、これに限定されるものではない。
The item for which the present invention can be preferably used is not particularly limited, but it is particularly useful for items for which high sensitivity is required because of low blood concentration and the like. Here, high sensitivity means, for example, several tens of μg/mL or less, and in the item requiring high sensitivity requiring the above-mentioned sensitivity, there is a high possibility that latex particles having a large particle size are used in the measurement. Therefore, the present invention can be preferably used.
Specific measurement items include, for example, CRP, protein C, hyaluronic acid, AFP (α-fetoprotein), IgE, RF (rheumatoid factor), IRE (elastase 1), ASO (antistreptolysin O), adiponectin (ADN). , ATIII (antithrombin III), β2M (β 2 microglobulin), CEA (carcinoembryonic antigen), factor XIII (factor 13, F13), FDP (fibrin/fibrinogen degradation product), FRTN (ferritin), HCG ( Human chorionic gonadotropin), HPL (human placental lactogen), tPAI (tissue plasminogen activator/plasminogen activator inhibitor 1 complex), D dimer, SF (soluble fibrin), PPI (PIC, plasmin/plasmin inhibitor complex), TAT (Thrombin/antithrombin complex), Cys C (cystatin C), PSA (prostate specific antigen), PG (pepsinogen I/II), hemoglobin, Tf (urinary transferrin, fecal transferrin), urinary BTA (bladder tumor) Antigen), IRI (insulin), E2 (estradiol, estrogen), E3 (estriol), MMP-3 (matrix metalloproteinase-3), Mb (myoglobin), H-FABP (human heart-derived fatty acid binding protein), L -FABP (urinary L-type fatty acid binding protein), KL-6 (sialylated sugar chain antigen), RBP (retinol binding protein), type IV collagen, HCV antibody, HBs antigen, HBe antigen/antibody, HBc antibody, HIV antibody , HTLV-1 antibody, and the like, but are not limited thereto.
（２）抗体または抗原
本発明において使用することができる抗体または抗原は、前記測定項目において測定対象とする物質に対して特異的に反応する抗体であり、測定対象と結合する抗体である限り、特に限定されるものではない。また、ＲＦ等抗体を不溶性担体に固定化して自己抗体を検出する免疫学的反応を行うが、抗原として抗体を利用する場合も含むものとする。
特に、本発明において使用されるＤダイマーに対する抗体としては、少なくともＤダイマーと結合する抗体である限り、特に限定されるものではなく、モノクローナル抗体又はポリクローナル抗体を用いることができる。また、抗体の種類としては、免疫グロブリン分子自体のほか、抗体フラグメント、例えば、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、又はＦｖ等も使用可能である。これらのフラグメントは、公知の方法に従って前記抗体から得ることができる。
(2) Antibody or Antigen The antibody or antigen that can be used in the present invention is an antibody that specifically reacts with the substance to be measured in the above measurement item, and as long as it is an antibody that binds to the measurement target, It is not particularly limited. In addition, an antibody such as RF is immobilized on an insoluble carrier to perform an immunological reaction of detecting an autoantibody, and the case where an antibody is used as an antigen is also included.
In particular, the antibody against D dimer used in the present invention is not particularly limited as long as it is an antibody that binds to at least D dimer, and a monoclonal antibody or a polyclonal antibody can be used. In addition to the immunoglobulin molecule itself, antibody fragments such as Fab, Fab′, F(ab′) 2 or Fv can also be used as the type of antibody. These fragments can be obtained from the antibody according to known methods.
（３）不溶性担体
本発明において使用される不溶性担体としては、粒子、例えば、有機高分子粒子、無機物質粒子、若しくは赤血球、又はプレート、例えば、ＥＬＩＳＡ用プレートなどが挙げられる。有機高分子粒子としては例えば、不溶性アガロース、不溶性デキストラン、セルロース、ラテックス粒子が挙げられる。好適にはラテックス粒子を挙げることができ、ポリスチレン、スチレン−メタクリル酸共重合体、スチレン−グリシジル（メタ）アクリレート共重合体、スチレン−スチレンスルホン酸塩共重合体、メタクリル酸重合体、アクリル酸重合体、アクリロニトリルブタジエンスチレン共重合体、塩化ビニル−アクリル酸エステル共重合体、ポリ酢酸ビニルアクリレートなどの粒子を挙げることができる。また、無機物質粒子としてはシリカ、アルミナなどが挙げられる。また粒子の平均粒径は、測定機器などによって適宜選択されるが、０．０５〜０．５０μｍのものが挙げられる。
(3) Insoluble carrier Examples of the insoluble carrier used in the present invention include particles such as organic polymer particles, inorganic substance particles, or red blood cells, or plates such as ELISA plates. Examples of the organic polymer particles include insoluble agarose, insoluble dextran, cellulose and latex particles. Preferable examples of the latex particles include polystyrene, styrene-methacrylic acid copolymer, styrene-glycidyl (meth)acrylate copolymer, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer. Examples thereof include particles of coalesce, acrylonitrile butadiene styrene copolymer, vinyl chloride-acrylic acid ester copolymer, polyvinyl acetate acrylate and the like. Examples of the inorganic material particles include silica and alumina. The average particle size of the particles is appropriately selected depending on the measuring instrument and the like, and examples thereof include those of 0.05 to 0.50 μm.
特に、粒径の大きなラテックス粒子を、抗体又は抗原を固定化する担体として使用している測定試薬の場合、その凝集のしやすさに影響されて標準品の検量線が高濃度域では寝る傾向が見られ、測定レンジが狭まる傾向が見られる。そのため、本発明は特に比較的粒径の大きなラテックス粒子を使用した測定試薬において、特に有用である。ここで粒径が大きいとは、例えば、粒径０．１μｍ以上のラテックスを担体として使用した測定試薬の場合が好ましく、０．２μｍ以上がより好ましい。また、粒径の上限は、０．５０μｍまでであれば適宜選択して使用することができる。 In particular, in the case of measurement reagents that use latex particles with large particle sizes as carriers for immobilizing antibodies or antigens, the calibration curve of the standard product tends to lie in the high concentration range due to the easiness of aggregation. Can be seen, and the measurement range tends to narrow. Therefore, the present invention is particularly useful for a measuring reagent using latex particles having a relatively large particle size. Here, the large particle size is, for example, preferable in the case of a measurement reagent using a latex having a particle size of 0.1 μm or more as a carrier, and more preferably 0.2 μm or more. Further, the upper limit of the particle size can be appropriately selected and used as long as it is up to 0.50 μm.
抗体若しくはそのフラグメント又は抗原を不溶性担体に固定化させる方法としては、任意の公知の方法で実施することができ、不溶性担体に抗体又は抗原を物理的又は化学的に結合させることにより固定化することができる。 The antibody or fragment thereof or the antigen can be immobilized on the insoluble carrier by any known method, and the antibody or the antigen is immobilized on the insoluble carrier by physically or chemically binding to the insoluble carrier. You can
（４）緩衝液
本発明における緩衝液の濃度は、抗原抗体反応時に０．１９ｍｏｌ／Ｌ以上の濃度であればよく、好ましくは０．２ｍｏｌ／Ｌ以上、より好ましくは０．３ｍｏｌ／Ｌ以上、更に好ましくは０．４ｍｏｌ／Ｌ以上、最も好ましくは０．５ｍｏｌ／Ｌ以上あるいは０．７ｍｏｌ／Ｌ以上であることができ、好ましくは０．３〜１．０ｍｏｌ／Ｌである。
(4) Buffer Solution The concentration of the buffer solution in the present invention may be 0.19 mol/L or more during the antigen-antibody reaction, preferably 0.2 mol/L or more, more preferably 0.3 mol/L or more, It is more preferably 0.4 mol/L or more, most preferably 0.5 mol/L or more, or 0.7 mol/L or more, and preferably 0.3 to 1.0 mol/L.
本発明において使用される緩衝液としては、分析対象物質を失活させることがなく、かつ免疫反応を阻害しないようなイオン濃度やｐＨを有するものであればよい。具体的には、ｐＨ５〜１０、特にｐＨ６〜９に緩衝作用をもつものが好ましく、例えば、弱酸であるホウ酸、炭酸、リン酸、カルボン酸、等で調製される緩衝液やグッド緩衝液が挙げられ、カルボン酸をもつ化合物の例としてギ酸、酢酸、クエン酸、フタル酸、乳酸、リンゴ酸、酒石酸、マレイン酸などがあり、グッド緩衝液ではＭＥＳ、ＰＩＰＥＳ、ＡＣＥＳ、ＢＥＳ，ＴＥＳ、ＨＥＰＥＳ、などが挙げられる。一方、弱塩基では１級または２級または３級アミン、等で調製される緩衝液やグッド緩衝液が挙げられ、アミンをもつ化合物としてはエタノールアミン、Ｔｒｉｓ、Ｂｉｓ−Ｔｒｉｓ、Ｂｉｓ−Ｔｒｉｓ−Ｐｒｏｐａｎｅ、バルビタール、イミダゾール、などがあり、グッド緩衝液ではＡＤＡ、アセトアミドグリシン、トリシン、グリシンアミド、ビシン、コラミン塩酸、などが挙げられる。また、両性の化合物も緩衝作用があり、アミノ酸から調製されるグリシン緩衝液や、アセトアミドグリシン緩衝液、ＢＳＡのようなタンパク質もまたカルボキシル基やアミノ基をもつため、使用する濃度を調整すれば本発明に利用することができる。さらに、カルボン酸のポリマー体やアミンのポリマー体も本発明に使用でき、具体的にはポリグルタミン酸やポリリジンで調製した緩衝液などが挙げられる。好ましくは、抗原抗体反応に好適な中性付近に緩衝能を持つＡＣＥＳ、ＰＩＰＥＳ、ＢＥＳ、ＴＥＳ、ＨＥＰＥＳ、ＭＯＰＳ、Ｔｒｉｓ、Ｂｉｓ-Ｔｒｉｓ緩衝液であり、特に好ましくは、ＨＥＰＥＳ、ＭＯＰＳ、Ｔｒｉｓ又はＢｉｓ−Ｔｒｉｓである。
当業者であれば、被検試料の希釈直線性を確認することで、適宜、最適な緩衝液の濃度及び種類を選択することができる。
As the buffer solution used in the present invention, any buffer solution may be used as long as it does not inactivate the substance to be analyzed and has an ion concentration or pH that does not inhibit the immune reaction. Specifically, those having a buffer action at pH 5 to 10, particularly pH 6 to 9, are preferable. For example, a buffer solution or a Good buffer solution prepared with a weak acid such as boric acid, carbonic acid, phosphoric acid or carboxylic acid may be used. Examples of compounds having a carboxylic acid include formic acid, acetic acid, citric acid, phthalic acid, lactic acid, malic acid, tartaric acid, maleic acid, etc., and Good buffer solutions include MES, PIPES, ACES, BES, TES, HEPES, And so on. On the other hand, weak bases include buffer solutions and Good buffer solutions prepared with primary, secondary, or tertiary amines, and the amine-containing compounds include ethanolamine, Tris, Bis-Tris, and Bis-Tris-Propane. , Barbital, imidazole, and the like, and Good buffers include ADA, acetamidoglycine, tricine, glycinamide, bicine, colamin hydrochloride, and the like. In addition, amphoteric compounds also have a buffering action, and glycine buffer prepared from amino acids, acetamidoglycine buffer, and proteins such as BSA also have carboxyl groups and amino groups, so if the concentration used is adjusted, It can be used for inventions. Further, a polymer of carboxylic acid and a polymer of amine can be used in the present invention, and specific examples thereof include buffer solutions prepared with polyglutamic acid and polylysine. ACES, PIPES, BES, TES, HEPES, MOPS, Tris, and Bis-Tris buffer having a buffering capacity near neutrality suitable for antigen-antibody reaction are preferable, and HEPES, MOPS, Tris, and Bis are particularly preferable. -Tris.
Those skilled in the art can appropriately select the optimum concentration and type of buffer solution by confirming the dilution linearity of the test sample.
本発明では、抗原抗体反応時に、０．１９ｍｏｌ／Ｌ以上の濃度の緩衝液を存在させることができる限り、反応実施前の試薬の緩衝液濃度は特に限定されない。また、本発明では、必要に応じて、緩衝液に公知の成分を組み合わせることができ、増感剤、非特異反応抑制剤、安定化剤、界面活性剤、無機塩などを適宜添加することができる。 In the present invention, the buffer solution concentration of the reagent before the reaction is not particularly limited as long as the buffer solution having a concentration of 0.19 mol/L or more can be present during the antigen-antibody reaction. Further, in the present invention, if necessary, known components can be combined with the buffer solution, and a sensitizer, a non-specific reaction inhibitor, a stabilizer, a surfactant, an inorganic salt and the like may be appropriately added. it can.
増感剤としては特に限定されず、ポリビニルピロリドン、ポリアニオン、ポリエチレングリコール、多糖類等を挙げることができる。
非特異反応抑制剤としては、動物血清や抗体、抗体断片等を挙げることができる。
安定化剤としては、アルブミン、スキムミルク、ゼラチン等のタンパク質や、アジ化ナトリウム、チメロサーム、ケーソンＣＧ、プロクリン等の防腐剤を挙げることができる。
界面活性剤としては、非イオン性界面活性剤、陰イオン性界面活性剤、陽イオン性界面活性剤、両性界面活性剤が挙げることができる。
被検試料中の塩濃度の影響を抑えることなどを目的とし無機塩を添加することができる。無機塩としては塩化ナトリウム、塩化カルシウム等を挙げることができる。
The sensitizer is not particularly limited, and examples thereof include polyvinylpyrrolidone, polyanion, polyethylene glycol, and polysaccharides.
Examples of the non-specific reaction inhibitor include animal serum, antibody, antibody fragment and the like.
Examples of the stabilizer include proteins such as albumin, skim milk and gelatin, and preservatives such as sodium azide, thimerosam, caisson CG and proclin.
Examples of the surfactant include nonionic surfactants, anionic surfactants, cationic surfactants and amphoteric surfactants.
An inorganic salt can be added for the purpose of suppressing the influence of the salt concentration in the test sample. Examples of the inorganic salt include sodium chloride and calcium chloride.
（５）被検試料
本発明により分析可能な被検試料としては、例えば、液状生体試料、例えば、血液、血漿、血清、または尿等を挙げることができる。
(5) Test Sample As a test sample that can be analyzed by the present invention, for example, a liquid biological sample such as blood, plasma, serum, or urine can be mentioned.
（６）免疫学的に測定する方法
免疫学的に測定する方法としては、少なくとも０．１９ｍｏｌ／Ｌ以上の濃度の緩衝液の存在下で免疫反応を行うことを除けば、公知の免疫学的測定方法を使用することができる。具体的には凝集反応法、特にはラテックス凝集反応法、又はＥＬＩＳＡ法が挙げられる。
(6) Method of measuring immunologically As a method of measuring immunologically, a known immunological method is used except that the immunoreaction is performed in the presence of a buffer solution having a concentration of at least 0.19 mol/L or more. Measurement methods can be used. Specifically, an agglutination reaction method, particularly a latex agglutination reaction method, or an ELISA method can be mentioned.
ラテックス凝集反応法では、抗体（又は抗原）を感作したラテックス粒子（以下、単に「抗体感作ラテックス粒子」ともいう）を用いる。抗体感作ラテックス粒子は、被検試料中に存在する測定対象物質と抗原抗体反応を起こし、凝集を生ずる。従って、前記抗体感作ラテックスの一定量と被検試料の一定量とを混合し、一定時間後の吸光度の増大を適当な波長で光学的に測定することにより、被検試料中の測定対象物質の量を定量することができる。 In the latex agglutination reaction method, latex particles sensitized with an antibody (or an antigen) (hereinafter, also simply referred to as “antibody-sensitized latex particles”) are used. The antibody-sensitized latex particles cause an antigen-antibody reaction with the substance to be measured existing in the test sample to cause aggregation. Therefore, by mixing a fixed amount of the antibody-sensitized latex and a fixed amount of the test sample and optically measuring the increase in absorbance after a certain time at an appropriate wavelength, the substance to be measured in the test sample Can be quantified.
さらに、既知濃度の測定対象物質（例えば、Ｄダイマー）含有溶液と被検試料の各々について生じた凝集の度合を光学的に観察し比較することで、被検試料中の測定対象物質濃度が測定され得る。Ｄダイマーを例にとると、まず既知濃度のＤダイマー溶液を二濃度（Ｄダイマー濃度が０μｇ／ｍＬの溶液を含むのが好ましい）以上測定し、得られた光学密度変化量とＤダイマー濃度の関係から検量線を作成する。次に被検試料を測定しその光学密度変化量から検量線を利用して濃度を求め、その値をＤダイマー濃度とする。抗体感作ラテックス粒子の凝集の度合を光学的に検出する方法においては、測定は散乱光強度、吸光度または透過光強度を測定する光学機器で行う。測定波長は３００〜２４００ｎｍ、好ましくは３００〜１０００ｎｍ、より好ましくは３００〜８００ｎｍの範囲から適切な波長が選択される。測定方法については公知の方法に従い、用いる抗体感作ラテックス粒子の大きさあるいは濃度の選択、反応時間の設定により、散乱光強度、吸光度または透過光強度の増加もしくは減少を測定することにより行われる。また、これらの方法を併用することも可能である。 Furthermore, the concentration of the substance to be measured in the test sample can be measured by optically observing and comparing the degree of aggregation generated in each of the solution containing the substance to be measured (for example, D-dimer) having a known concentration and the test sample. Can be done. Taking D-dimer as an example, first, a D-dimer solution having a known concentration is measured at two concentrations (preferably containing a solution having a D-dimer concentration of 0 μg/mL), and the obtained optical density change amount and D-dimer concentration are measured. Create a calibration curve from the relationship. Next, the test sample is measured, the concentration is obtained from the amount of change in optical density using a calibration curve, and the value is defined as the D-dimer concentration. In the method of optically detecting the degree of aggregation of antibody-sensitized latex particles, the measurement is performed with an optical instrument that measures scattered light intensity, absorbance or transmitted light intensity. The measurement wavelength is 300 to 2400 nm, preferably 300 to 1000 nm, and more preferably 300 to 800 nm. The measuring method is carried out according to a known method by measuring the increase or decrease of scattered light intensity, absorbance or transmitted light intensity by selecting the size or concentration of the antibody-sensitized latex particles to be used and setting the reaction time. It is also possible to use these methods together.
免疫反応の条件は公知の条件が採用されるが、反応時の温度は１０〜５０℃、特に２０〜４０℃が好ましい。反応時間は適宜決定することができる。 Known conditions are adopted as the conditions for the immunoreaction, but the temperature during the reaction is preferably 10 to 50°C, particularly preferably 20 to 40°C. The reaction time can be appropriately determined.
（７）免疫学的測定試薬
免疫学的測定試薬としては、１試薬からなる試薬形態の他、第一試薬と第二試薬からなる２試薬系等の複数の試薬からなる試薬形態（すなわち、キット）であってもよい。１試薬系の試薬の場合は、被検試料と混合した際に０．１９ｍｏｌ／Ｌ以上となる濃度の緩衝液に、抗体又は抗原を固定化した不溶性担体を含む試薬であればよく、複数の試薬からなる試薬形態の場合は、免疫反応が開始される溶液中での緩衝液濃度が０．１９ｍｏｌ／Ｌ濃度以上となるように、各試薬の緩衝液濃度を決定すればよい。好ましくは測定精度の観点などから第一試薬と第二試薬からなる２試薬系がよく、以下に例示する。
(7) Immunological measurement reagent As the immunological measurement reagent, in addition to a reagent form consisting of one reagent, a reagent form consisting of a plurality of reagents such as a two-reagent system consisting of a first reagent and a second reagent (that is, a kit) ). In the case of a one-reagent type reagent, a reagent containing an insoluble carrier on which an antibody or an antigen is immobilized in a buffer solution having a concentration of 0.19 mol/L or more when mixed with a test sample may be used. In the case of a reagent form consisting of reagents, the buffer concentration of each reagent may be determined so that the buffer concentration in the solution in which the immune reaction is initiated is 0.19 mol/L or more. A two-reagent system composed of a first reagent and a second reagent is preferable from the viewpoint of measurement accuracy, and is exemplified below.
第一試薬は抗原抗体反応時の条件を整えるための緩衝液からなり、第二試薬は測定対象物質に対する抗体又は抗原を固定化した不溶性担体からなる。好ましい例としては、第一試薬は緩衝液、増感剤、非特異反応抑制剤及び無機塩からなり、第二試薬は抗体（又は抗原）感作ラテックス粒子からなる試薬が挙げられる。
各試薬に用いる緩衝液は、第一試薬と第二試薬とで同一の緩衝液を用いてもよいし、第一試薬と第二試薬とで異なる種類の緩衝液を用いてもよい。また、第二試薬には緩衝液を用いず、第一試薬にのみ緩衝液を用いてもよい。なお２種類以上の緩衝液を併用する場合には、個々の緩衝液の濃度の総和が０．１９ｍｏｌ／Ｌ以上となればよい。
各試薬の緩衝液濃度は、被検試料と第一試薬および第二試薬とを混合した際に、緩衝液の濃度が０．１９ｍｏｌ／Ｌ以上の濃度となるように決定すればよい。各試薬の液量は、測定に用いる自動分析装置によっても変動するが、当業者であれば、被検試料および第一試薬、第二試薬との液量比から、反応液中の緩衝液濃度が０．１９ｍｏｌ／Ｌ以上となるように、適宜、各試薬の緩衝液濃度を設定することができる。例えば、被検試料と第一試薬、第二試薬の液量比が、１／２０〜１／１０：１〜２：１の場合、第一試薬の緩衝液濃度は０．１〜１ｍｏｌ／Ｌであり、第二試薬の緩衝液濃度は０〜０．４ｍｏｌ／Ｌであることが好ましい。
The first reagent is composed of a buffer solution for adjusting the conditions at the time of antigen-antibody reaction, and the second reagent is composed of an insoluble carrier on which an antibody against a substance to be measured or an antigen is immobilized. As a preferred example, the first reagent comprises a buffer solution, a sensitizer, a non-specific reaction inhibitor and an inorganic salt, and the second reagent comprises a reagent comprising antibody (or antigen)-sensitized latex particles.
As the buffer solution used for each reagent, the same buffer solution may be used for the first reagent and the second reagent, or different kinds of buffer solutions may be used for the first reagent and the second reagent. Further, the buffer solution may not be used for the second reagent, and the buffer solution may be used only for the first reagent. When two or more kinds of buffer solutions are used in combination, the total concentration of the individual buffer solutions should be 0.19 mol/L or more.
The buffer solution concentration of each reagent may be determined so that the concentration of the buffer solution becomes 0.19 mol/L or more when the test sample is mixed with the first reagent and the second reagent. Although the volume of each reagent varies depending on the automatic analyzer used for measurement, those skilled in the art can determine the buffer concentration in the reaction solution from the volume ratio of the test sample and the first and second reagents. The concentration of the buffer solution of each reagent can be appropriately set so that is 0.19 mol/L or more. For example, when the liquid volume ratio of the test sample to the first reagent and the second reagent is 1/20 to 1/10:1 to 2:1, the buffer concentration of the first reagent is 0.1 to 1 mol/L. And the buffer concentration of the second reagent is preferably 0 to 0.4 mol/L.
上記測定試薬を用いて測定する場合は、第一試薬と被検試料を反応セル中で混合した後、第二試薬を添加して抗体感作ラテックス粒子の凝集度合いを光学的に測定する方法や、第一試薬と第二試薬を反応セル中で混合した後、被検試料を添加して抗体感作ラテックス粒子の凝集度合いを光学的に測定する方法等が採用される。 When measuring using the above measurement reagent, after mixing the first reagent and the test sample in the reaction cell, a method of optically measuring the degree of aggregation of antibody-sensitized latex particles by adding a second reagent or After mixing the first reagent and the second reagent in the reaction cell, a test sample is added to optically measure the degree of aggregation of the antibody-sensitized latex particles.
（８）測定範囲の拡大方法
本発明の測定における測定範囲の拡大方法は、測定対象物質に対する抗体若しくはそのフラグメント又は抗原を固定化した不溶性担体を用いて、被検試料中の測定対象物質の免疫学的測定において、０．１９ｍｏｌ／Ｌ以上の濃度の緩衝液の存在下で免疫反応を行うことによるものである。０．１９ｍｏｌ／Ｌ以上の濃度の緩衝液の存在下で免疫反応を行うことにより、測定対象物質の測定範囲が高濃度側に拡大し、低濃度の感度を維持したまま、より高濃度域の測定対象物質を正確に測定することができる。
なお、測定範囲の決定は、公知の方法により行うことができ、例えば、高濃度の被検試料の希釈系列を作製・測定し、希釈直線性が得られる範囲を測定範囲と設定する方法などが挙げられる。
当業者であれば、測定しようとする濃度範囲において、被検試料の希釈直線性を確認することで、適宜、最適な緩衝液の濃度や種類を選択することができる。
(8) Method for expanding the measurement range The method for expanding the measurement range in the measurement of the present invention is an immunization of a measurement target substance in a test sample using an insoluble carrier on which an antibody against the measurement target substance or a fragment thereof or an antigen is immobilized. This is because the immunological reaction was carried out in the presence of a buffer solution having a concentration of 0.19 mol/L or more in the biological measurement. By performing the immune reaction in the presence of a buffer solution having a concentration of 0.19 mol/L or more, the measurement range of the substance to be measured is expanded to the high concentration side, and the sensitivity of the low concentration is maintained, The substance to be measured can be measured accurately.
The measurement range can be determined by a known method. For example, a method of preparing and measuring a dilution series of a high-concentration test sample and setting a range in which dilution linearity is obtained as a measurement range can be used. Can be mentioned.
Those skilled in the art can appropriately select the optimum concentration and type of buffer solution by confirming the dilution linearity of the test sample in the concentration range to be measured.
以下、実施例によって本発明を具体的に説明するが、これらは本発明の範囲を限定するものではない。 Hereinafter, the present invention will be described in detail with reference to Examples, but these do not limit the scope of the present invention.
《実施例１：ラテックス凝集法によるＤダイマー測定試薬の作製》
（１）第一試薬の調製
濃度を、０．１ｍｏｌ／Ｌ、０．２ｍｏｌ／Ｌ、０．４ｍｏｌ／Ｌ、０．６ｍｏｌ／Ｌ、１ｍｏｌ／Ｌに調製したＨＥＰＥＳ緩衝液（ｐＨ７．２）にウシ血清アルブミン（ＢＳＡ）を添加したものを、第一試薬とした。
<<Example 1: Preparation of D-dimer measuring reagent by latex aggregation method>>
(1) Preparation of First Reagent In HEPES buffer solution (pH 7.2) adjusted to a concentration of 0.1 mol/L, 0.2 mol/L, 0.4 mol/L, 0.6 mol/L, 1 mol/L. What added bovine serum albumin (BSA) was made into the 1st reagent.
（２）第二試薬の調製
抗Ｄダイマー抗体をＭＥＳ緩衝液（ｐＨ６．０）に懸濁し、この抗体液にポリスチレンラテックス（ＪＳＲ社）を添加して４℃にて一晩撹拌した。上記混合物に、ＢＳＡを含むトリス塩酸緩衝液（ｐＨ８．０）を添加し、４℃にて３０分間撹拌した後、遠心分離を実施した。得られた沈殿物を、防腐剤を含む水に懸濁し、抗Ｄダイマー抗体感作ポリスチレンラテックス液（第二試薬）を得た。
(2) Preparation of second reagent The anti-D dimer antibody was suspended in MES buffer (pH 6.0), polystyrene latex (JSR) was added to this antibody solution, and the mixture was stirred at 4°C overnight. Tris-hydrochloric acid buffer solution (pH 8.0) containing BSA was added to the above mixture, stirred at 4° C. for 30 minutes, and then centrifuged. The obtained precipitate was suspended in water containing a preservative to obtain an anti-D dimer antibody-sensitized polystyrene latex solution (second reagent).
（３）Ｄダイマー標準液の調製
Ｄダイマーの調製は、主にStephanie A.OlexaとAndrei Z.Budzynskiの方法（1978）、Circulation,Suppl.58,119,Olexa et al.の方法（1979）、及びBiochim.Biophys.Acta 576,39〜50に準じて行った。ヒトフィブリノーゲン（Enzyme Research Laboratories社）に、ウシトロンビン（持田製薬社）および塩化カルシウムを加え、３７℃２時間反応させフィブリノーゲンをフィブリンに変換させた。これを遠心分離し、フィブリンを非凝固性物質から分離した。フィブリンは塩化カルシウムを含むトリス塩酸緩衝液ｐＨ７．８に浮遊させた。３７℃環境下で浮遊液にヒトプラスミン（Chromogenix社）を添加した。アプロチニン（Pentapharm.社）を加えて分解反応を停止させた後、リジンセファロースカラムを通過させプラスミンを除去した。この通過液は分子量の異なるＤダイマーの混合物である。
(3) Preparation of D-dimer standard solution D-dimer was mainly prepared by the method of Stephanie A. Olexa and Andrei Z. Budzynski (1978), the method of Circulation, Suppl. 58, 119, Olexa et al. (1979), and Biochim. Biophys. Acta 576, 39-50. Bovine thrombin (Mochida Pharmaceutical Co., Ltd.) and calcium chloride were added to human fibrinogen (Enzyme Research Laboratories) and reacted at 37° C. for 2 hours to convert fibrinogen into fibrin. This was centrifuged to separate fibrin from non-coagulable material. Fibrin was suspended in a Tris-HCl buffer solution pH 7.8 containing calcium chloride. Human plasmin (Chromogenix) was added to the suspension in a 37° C. environment. Aprotinin (Pentapharm.) was added to stop the decomposition reaction and then passed through a lysine sepharose column to remove plasmin. This passing liquid is a mixture of D dimers having different molecular weights.
次に塩化カルシウムを含むトリス塩酸緩衝液ｐＨ７．５（溶液Ａ）で平衡化したセファクリルＳ−３００（S-300）カラムに先に得られた通過液を、溶液Ａで展開する分子ふるいクロマトグラフィーによって分画した。この分画をＳＤＳ−ＰＡＧＥによる電気泳動法、ウェスタンブロット法によってＤダイマー分画を同定、分離して、精製Ｄダイマーを得た。
得られた精製Ｄダイマーを、０、２、８、３２、４８、６０μｇ／ｍＬとなるように、ＢＳＡを含むトリス塩酸緩衝液（ｐＨ８．０）にて調製し、Ｄダイマー標準液とした。
Next, a Sephacryl S-300 (S-300) column equilibrated with Tris-hydrochloric acid buffer solution pH 7.5 (solution A) containing calcium chloride was used to develop the passing solution previously obtained using solution A, and molecular sieve chromatography was performed. Fractionated by. This fraction was identified and separated by a DDS-PAGE electrophoresis and Western blotting method to obtain a purified D-dimer.
The purified D-dimer thus obtained was adjusted to 0, 2, 8, 32, 48, and 60 μg/mL with a Tris-hydrochloric acid buffer solution (pH 8.0) containing BSA to obtain a D-dimer standard solution.
《実施例２：緩衝液濃度の違いによる被検試料の希釈直線性の確認》
（１）測定方法
実施例１（３）で調製したＤダイマー標準液７μＬに、ＨＥＰＥＳ緩衝液の濃度が異なる第一試薬を１２０μＬ添加混合して３７℃で約５分間保持した後、第二試薬を１２０μＬ添加して撹拌し、約１０分間経過するまでの間の吸光度変化を波長７００ｎｍにて測定し、検量線を作成した。上記吸光度測定は、全自動分析機日立７１７０を用いて行った。
次に、前記標準液の代わりに、ＢＳＡを含むトリス塩酸緩衝液（ｐＨ８．０）にて血漿検体を２倍、４倍、８倍希釈したものを測定し、上記で作成した検量線を用いて測定値（検量線より導き出した被検試料中のＤダイマーの濃度）を得た。結果を表１に示す。
<<Example 2: Confirmation of dilution linearity of test sample due to difference in buffer concentration>>
(1) Measurement method To 7 μL of the D-dimer standard solution prepared in Example 1(3), 120 μL of the first reagent having a different concentration of HEPES buffer was added and mixed, and the mixture was kept at 37° C. for about 5 minutes, and then the second reagent. Was added and stirred, and the change in absorbance until about 10 minutes elapsed was measured at a wavelength of 700 nm to prepare a calibration curve. The absorbance measurement was performed using a fully automatic analyzer Hitachi 7170.
Next, instead of the standard solution, a 2-fold, 4-fold, 8-fold dilution of the plasma sample with Tris-hydrochloric acid buffer solution (pH 8.0) containing BSA was measured, and the calibration curve prepared above was used. To obtain a measurement value (concentration of D-dimer in the test sample derived from the calibration curve). The results are shown in Table 1.
なお、表１において、抗原抗体反応時のＨＥＰＥＳ緩衝液の濃度を０．０５ｍｏｌ／Ｌ、０．１ｍｏｌ／Ｌ、０．２ｍｏｌ／Ｌ、０．３ｍｏｌ／Ｌ、０．５ｍｏｌ／Ｌと表示しているが、抗原抗体反応時の正確な緩衝液濃度は、それぞれ０．０４８ｍｏｌ／Ｌ（＝０．１ｍｏｌ／Ｌ×［１２０μＬ／（１２０μＬ＋７μＬ＋１２０μＬ）］）、０．０９７ｍｏｌ／Ｌ、０．１９ｍｏｌ／Ｌ、０．２９ｍｏｌ／Ｌ、０．４９ｍｏｌ／Ｌである。 In Table 1, the concentration of the HEPES buffer solution at the time of antigen-antibody reaction is shown as 0.05 mol/L, 0.1 mol/L, 0.2 mol/L, 0.3 mol/L, 0.5 mol/L. However, the exact buffer concentration at the time of antigen-antibody reaction is 0.048 mol/L (=0.1 mol/L×[120 μL/(120 μL+7 μL+120 μL)]), 0.097 mol/L, 0.19 mol/L, It is 0.29 mol/L and 0.49 mol/L.
測定値に希釈率を乗じて算出した計算値を、希釈前の測定値と比較したところ、ＨＥＰＥＳ緩衝液の濃度が高いほどその差は小さくなり、免疫反応時の緩衝液濃度を０．２ｍｏｌ／Ｌ（正確には０．１９ｍｏｌ／Ｌ）以上にすることによって、測定値と計算値の差が±１０％以内となった。ＨＥＰＥＳ緩衝液の濃度を０．５ｍｏｌ／Ｌとして測定した際に得られた４７．２４μｇ／ｍＬという数値は、希釈試験における測定値と計算値の差が、いずれの希釈率においても５％以内であることから、実験に供した血漿検体のＤダイマー濃度をほぼ正確に算出していると考えられる。一方、ＨＥＰＥＳ緩衝液の濃度を０．０５ｍｏｌ／Ｌとして測定した際に得られた３７．６３μｇ／ｍＬという数値は、希釈試験における測定値と計算値の差が、いずれの希釈率においても１０％以上あることから、実験に供した血漿検体のＤダイマー濃度を正確に算出していると考え難い。以上のことから、測定系で使用する緩衝液の濃度を高くすることで希釈直線性が改善し、得られる測定値自体も高く変化して正しく算出されることが観察された。 When the calculated value calculated by multiplying the measured value by the dilution rate was compared with the measured value before dilution, the higher the HEPES buffer concentration was, the smaller the difference was, and the buffer concentration during the immune reaction was 0.2 mol/mol. By setting L (more accurately, 0.19 mol/L) or more, the difference between the measured value and the calculated value was within ±10%. The value of 47.24 μg/mL obtained when the concentration of the HEPES buffer solution was measured as 0.5 mol/L was within 5% at any dilution rate because of the difference between the measured value and the calculated value in the dilution test. Therefore, it is considered that the D-dimer concentration of the plasma sample used in the experiment is calculated almost accurately. On the other hand, the numerical value of 37.63 μg/mL obtained when the concentration of the HEPES buffer solution was measured as 0.05 mol/L was 10% at any dilution rate because of the difference between the measured value and the calculated value in the dilution test. From the above, it is unlikely that the D-dimer concentration of the plasma sample used in the experiment is accurately calculated. From the above, it was observed that by increasing the concentration of the buffer solution used in the measurement system, the linearity of dilution was improved, and the obtained measurement value itself also changed highly and was calculated correctly.
さらに、検量線の形状と被検試料の希釈系列のプロットの形状を確認した。まず、８倍希釈した血漿検体の測定値に希釈率を乗じた計算値を原液理論値とした。原液理論値を各希釈倍率で割り、各希釈倍率における理論値を算出した（表２）。その理論値を横軸に、吸光度を縦軸にプロットした（図１（０．０５ｍｏｌ／Ｌ）、図２（０．２ｍｏｌ／Ｌ）、図３（０．５ｍｏｌ／Ｌ））ところ、緩衝液濃度が０．０５ｍｏｌ／Ｌの場合、理論値が５．４７μｇ／ｍＬ及び１０．９４μｇ／ｍＬでは検量線上にプロットされるが、２１．８８μｇ／ｍＬでは検量線よりも高値となり、４３．７６μｇ／ｍＬでは検量線よりも低値となった。緩衝液濃度が高いほど検量線との距離が小さくなり、０．２ｍｏｌ／Ｌ（正確には０．１９ｍｏｌ／Ｌ）以上の緩衝液濃度では、理論値で５．５４から４４．３２μｇ／ｍＬのすべての点が検量線上にプロットされた。緩衝液の濃度を高くすることで、検量線の形状に血漿検体の希釈特性が近づき、希釈直線性が改善されていることが分かった。これにより、より高濃度域まで測定範囲を拡大することができた。 Furthermore, the shape of the calibration curve and the shape of the plot of the dilution series of the test sample were confirmed. First, the calculated value obtained by multiplying the measured value of the 8-fold diluted plasma sample by the dilution ratio was used as the stock solution theoretical value. The theoretical value of the stock solution was divided by each dilution ratio to calculate the theoretical value at each dilution ratio (Table 2). The theoretical value was plotted on the horizontal axis and the absorbance was plotted on the vertical axis (FIG. 1 (0.05 mol/L), FIG. 2 (0.2 mol/L), FIG. 3 (0.5 mol/L)). When the concentration is 0.05 mol/L, the theoretical value is plotted on the calibration curve at 5.47 μg/mL and 10.94 μg/mL, but at 21.88 μg/mL, the value is higher than the calibration curve and 43.76 μg/mL. In mL, the value was lower than the calibration curve. The higher the buffer concentration, the smaller the distance from the calibration curve, and at a buffer concentration of 0.2 mol/L (more accurately, 0.19 mol/L) or higher, the theoretical value is 5.54 to 44.32 μg/mL. All points were plotted on the calibration curve. It was found that by increasing the concentration of the buffer solution, the dilution characteristics of the plasma sample approached the shape of the calibration curve, and the dilution linearity was improved. As a result, the measurement range could be expanded to a higher concentration range.
《実施例３：緩衝液の違いによる被検試料の希釈直線性の確認》
ＨＥＰＥＳ緩衝液の代わりにトリス塩酸緩衝液（ｐＨ８．０）を用い、抗原抗体反応時のトリス塩酸緩衝液の濃度が正確に０．０５ｍｏｌ／Ｌ、０．１ｍｏｌ／Ｌ、０．２ｍｏｌ／Ｌ、０．３ｍｏｌ／Ｌ、０．５ｍｏｌ／Ｌとなるように、以下のように第一試薬および第二試薬を調製した。それ以外は、実施例２と同様に測定を行った。なお、評価に用いた被検試料は実施例２と異なる血漿検体を用いた。
<<Example 3: Confirmation of dilution linearity of test sample due to difference in buffer solution>>
Tris-hydrochloric acid buffer solution (pH 8.0) was used instead of the HEPES buffer solution, and the concentration of the Tris-hydrochloric acid buffer solution during the antigen-antibody reaction was exactly 0.05 mol/L, 0.1 mol/L, 0.2 mol/L, The first reagent and the second reagent were prepared as follows so that the amounts were 0.3 mol/L and 0.5 mol/L. Other than that, it measured like Example 2. The test sample used for evaluation was a plasma sample different from that in Example 2.
結果を表４に示す。免疫反応時の緩衝液濃度を０．２ｍｏｌ／Ｌ以上にすることによって、測定値と計算値の差が±１０％以内となり、血漿検体の希釈直線性が改善できることが確認できた。 The results are shown in Table 4. It was confirmed that by setting the buffer concentration during the immune reaction to 0.2 mol/L or more, the difference between the measured value and the calculated value was within ±10%, and the linearity of dilution of the plasma sample could be improved.
《実施例４：フェリチン測定試薬及びフェリチン標準液の調製》
（１）第一試薬の調製
濃度を、０．１ｍｏｌ／Ｌ、１ｍｏｌ／Ｌ、２ｍｏｌ／Ｌに調製したトリス塩酸緩衝液にウシ血清アルブミン（ＢＳＡ）を添加して、ｐＨ８．５に合わせたものを第一試薬とした。
<<Example 4: Preparation of ferritin measuring reagent and ferritin standard solution>>
(1) Preparation of First Reagent Concentrated to pH 8.5 by adding bovine serum albumin (BSA) to a Tris-hydrochloric acid buffer solution adjusted to a concentration of 0.1 mol/L, 1 mol/L, 2 mol/L Was the first reagent.
（２）第二試薬の調製
抗フェリチン抗体をＭＯＰＳ緩衝液（ｐＨ７．１）に懸濁し、この抗体液にポリスチレンラテックスを添加して４℃にて一晩撹拌した。上記混合物に、ＢＳＡを含むトリス塩酸緩衝液（ｐＨ８．０）を添加し、４℃にて３０分間撹拌した後、遠心分離を実施した。得られた沈殿物を、防腐剤を含む水に懸濁し、抗フェリチン抗体感作ポリスチレンラテックス液（第二試薬）を得た。
(2) Preparation of Second Reagent The anti-ferritin antibody was suspended in MOPS buffer (pH 7.1), polystyrene latex was added to this antibody solution, and the mixture was stirred overnight at 4°C. Tris-hydrochloric acid buffer solution (pH 8.0) containing BSA was added to the above mixture, stirred at 4° C. for 30 minutes, and then centrifuged. The obtained precipitate was suspended in water containing a preservative to obtain an anti-ferritin antibody-sensitized polystyrene latex solution (second reagent).
（３）フェリチン標準液の調製
精製フェリチン（ccbiotech社）を０、５０、２００、４００、７００、１０００ｎｇ／ｍＬとなるように、ＢＳＡを含むトリス塩酸緩衝液（ｐＨ８．０）にて調製し、フェリチン標準液とした。
(3) Preparation of ferritin standard solution Purified ferritin (ccbiotech) was prepared with Tris-hydrochloric acid buffer solution (pH 8.0) containing BSA at 0, 50, 200, 400, 700, 1000 ng/mL. Ferritin standard solution was used.
《実施例５：緩衝液濃度の違いによる被検試料の希釈直線性の確認》
（１）測定方法
フェリチン標準液１１μＬに、トリス塩酸緩衝液の濃度が異なる第一試薬を９０μＬ添加混合して３７℃で約５分間保持した後、第二試薬を９０μＬ添加して撹拌し、約１０分間経過するまでの間の吸光度変化を主波長５７０ｎｍ、副波長８００ｎｍにて測定し、検量線を作成した。上記吸光度測定は、全自動分析機日立７１８０を用いて行った。
次に、前記標準液の代わりに、生理食塩水にて血漿検体を４／５倍、２／５倍、１／５倍に希釈したものを測定し、上記で作成した検量線を用いて測定値（検量線より導き出した被検試料中のフェリチン濃度）を得た。結果を表５に示す。
<Example 5: Confirmation of dilution linearity of test sample by difference in buffer concentration>
(1) Measurement method To 11 μL of the ferritin standard solution, 90 μL of the first reagent having a different Tris-HCl buffer concentration was added and mixed, and the mixture was kept at 37° C. for about 5 minutes, and then 90 μL of the second reagent was added and stirred, The change in absorbance until the lapse of 10 minutes was measured at a main wavelength of 570 nm and a sub wavelength of 800 nm to prepare a calibration curve. The absorbance measurement was performed using a fully automatic analyzer Hitachi 7180.
Next, instead of the standard solution, a plasma sample diluted with physiological saline to 4/5 times, 2/5 times, or 1/5 times was measured and measured using the calibration curve prepared above. The value (ferritin concentration in the test sample derived from the calibration curve) was obtained. The results are shown in Table 5.
測定値に希釈率を乗じて算出した計算値を、希釈前の測定値と比較したところ、トリス塩酸緩衝液の濃度が高いほどその差は小さくなり、免疫反応時の緩衝液濃度を高くすることによって、いずれの希釈率においても測定値と計算値の差が小さくなった。トリス塩酸緩衝液の濃度が低い程、測定値と計算値の差が大きくなり、実験に供した血漿検体のフェリチン濃度を正確に算出していると考え難い。以上のことから、測定系で使用する緩衝液の濃度を高くすることで希釈直線性が改善し、得られる測定値自体も高く変化して正しく算出されることが観察された。 The calculated value calculated by multiplying the measured value by the dilution rate was compared with the measured value before dilution.The higher the concentration of Tris-HCl buffer, the smaller the difference, and the higher the buffer concentration during immune reaction should be. As a result, the difference between the measured value and the calculated value was small at any dilution rate. The lower the concentration of Tris-HCl buffer, the larger the difference between the measured value and the calculated value, and it is unlikely that the ferritin concentration of the plasma sample used in the experiment is accurately calculated. From the above, it was observed that by increasing the concentration of the buffer solution used in the measurement system, the linearity of dilution was improved, and the obtained measurement value itself also changed highly and was calculated correctly.
さらに、検量線の形状と被検試料の希釈系列のプロットの形状を確認した。まず、５倍希釈した血漿検体の測定値に希釈率を乗じた計算値を原液理論値とした。原液理論値を各希釈倍率で割り、各希釈倍率における理論値を算出した。その理論値を横軸に、吸光度を縦軸にプロットした（図４（０．０５ｍｏｌ／Ｌ）、図５（０．５ｍｏｌ／Ｌ））ところ、緩衝液濃度が０．０５ｍｏｌ／Ｌの場合、理論値が１０５ｎｇ／ｍＬ及び１９５ｎｇ／ｍＬでは検量線上にプロットされるが、３６１ｎｇ／ｍＬ以上では検量線から外れる結果となった。一方で、緩衝液濃度が高いほど検量線との距離が小さい結果となった。例えば、緩衝液濃度が０．５ｍｏｌ／Ｌ（正確には０．４７ｍｏｌ／Ｌ）の場合には、理論値で１０５から４５４ｎｇ／ｍＬのすべての点が検量線上にプロットされた。
また、上記検体とは別検体を使用して１．０ｍｏｌ／Ｌ（０．９４ｍｏｌ／Ｌ）の緩衝液を使用して測定した場合においても、検量線の形状に検体の希釈特性が近付き、希釈直線性が改善される傾向が観察された。
Furthermore, the shape of the calibration curve and the shape of the plot of the dilution series of the test sample were confirmed. First, a calculated value obtained by multiplying the measured value of a 5-fold diluted plasma sample by the dilution rate was used as a stock solution theoretical value. The theoretical value of the stock solution was divided by each dilution ratio to calculate the theoretical value at each dilution ratio. The theoretical value was plotted on the horizontal axis and the absorbance was plotted on the vertical axis (FIG. 4 (0.05 mol/L), FIG. 5 (0.5 mol/L)). As a result, when the buffer concentration was 0.05 mol/L, When the theoretical value was 105 ng/mL and 195 ng/mL, it was plotted on the calibration curve, but when it was 361 ng/mL or more, the result deviated from the calibration curve. On the other hand, the higher the buffer concentration, the smaller the distance from the calibration curve. For example, when the buffer concentration was 0.5 mol/L (correctly 0.47 mol/L), all theoretical points of 105 to 454 ng/mL were plotted on the calibration curve.
In addition, when a sample other than the above sample is used and measured using a 1.0 mol/L (0.94 mol/L) buffer solution, the dilution characteristics of the sample approach the shape of the calibration curve and A trend towards improved linearity was observed.
以上より、緩衝液の濃度を高くすることで、検量線の形状に血漿検体の希釈特性が近づき、希釈直線性が改善されていることが分かった。これにより、より高濃度域まで測定範囲を拡大することができた。 From the above, it was found that, by increasing the concentration of the buffer solution, the dilution characteristic of the plasma sample approaches the shape of the calibration curve, and the linearity of dilution is improved. As a result, the measurement range could be expanded to a higher concentration range.
本発明の測定を用いることにより、広い測定範囲で正確に測定することが可能となる。特に、Ｄダイマーのような凝固線溶分野における検査試薬においては、深部静脈血栓症（ＤＶＴ）の除外診断や播種性血管内凝固症候群（ＤＩＣ）の診断に有用である。また、フェリチン測定試薬においては、貧血、リンパ腫、肝炎、リウマチ、腎疾患等の各種疾患の病態把握等において有用である。
以上、本発明を特定の態様に沿って説明したが、当業者に自明の変形や改良は本発明の範囲に含まれる。
By using the measurement of the present invention, it is possible to measure accurately in a wide measurement range. In particular, a test reagent in the field of coagulation/fibrinolysis such as D-dimer is useful for exclusion diagnosis of deep vein thrombosis (DVT) and diagnosis of disseminated intravascular coagulation (DIC). In addition, the ferritin measuring reagent is useful for understanding the pathological condition of various diseases such as anemia, lymphoma, hepatitis, rheumatism, and renal disease.
Although the present invention has been described above according to the specific mode, modifications and improvements obvious to those skilled in the art are included in the scope of the present invention.
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