JP2739615B2 - Setting method of calibration curve in quantification method using dry immunoassay element - Google Patents
Setting method of calibration curve in quantification method using dry immunoassay elementInfo
- Publication number
- JP2739615B2 JP2739615B2 JP3175142A JP17514291A JP2739615B2 JP 2739615 B2 JP2739615 B2 JP 2739615B2 JP 3175142 A JP3175142 A JP 3175142A JP 17514291 A JP17514291 A JP 17514291A JP 2739615 B2 JP2739615 B2 JP 2739615B2
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- JP
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- Prior art keywords
- calibration curve
- concentration
- dry
- setting
- immunoassay
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 238000000034 method Methods 0.000 title claims description 34
- 238000003018 immunoassay Methods 0.000 title claims description 26
- 238000011002 quantification Methods 0.000 title claims description 5
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- 239000000427 antigen Substances 0.000 description 3
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Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明は、乾式免疫分析要素を用
いて生物体液中たとえば血液中とか尿中の特定物質の定
量を行う免疫分析方法における検量線の設定方法に関す
る。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for setting a calibration curve in an immunoassay method for quantifying a specific substance in a biological fluid such as blood or urine using a dry immunoassay element.
【０００２】[0002]
【従来の技術】生物体液たとえば血液や尿などに含まれ
る生体成分、薬物等の分析は、病態の診断や治療経過の
判定に非常に有用であり、臨床検査の分野で重要な役割
を果たしている。このような生物体液中の微量成分の分
析方法として、酵素免疫分析方法（ＥＩＡ）がある。酵
素免疫分析方法には、Ｂ／Ｆ分離が必要な非均一系とＢ
／Ｆ分離が不必要な均一系がある。均一反応系は抗体と
抗原が結合すると、標識酵素の酵素活性が何らかの干渉
を受けることに基づくもので、一般には抗原と抗体の結
合による阻害作用を利用する。2. Description of the Related Art The analysis of biological components and drugs contained in biological fluids such as blood and urine is very useful for diagnosing disease states and judging the course of treatment, and plays an important role in the field of clinical tests. . As a method for analyzing a trace component in such a biological fluid, there is an enzyme immunoassay (EIA). Enzyme immunoassays include heterogeneous systems that require B / F separation and B
There is a homogeneous system that does not require / F separation. The homogeneous reaction system is based on the fact that when an antibody and an antigen bind, the enzymatic activity of the labeling enzyme undergoes some interference, and generally utilizes the inhibitory action of the antigen and antibody binding.
【０００３】均一系酵素免疫分析方法の検量線は従来の
臨床化学検査のそれとは大きく異なり、一般に直線とな
らずほとんどの場合Ｓ字（シグモイダル）になる。この
ような検量線については、放射線免疫分析方法（ＲＩ
Ａ）についてこれまで十分な検討がなされている。酵素
免疫分析方法の場合も本質的に放射線免疫分析方法の検
量線の設定方法を適用することができ、その回帰方法
は、logistic曲線、logit−log変換式などとして知られ
ており、石川榮治ほか編、「酵素免疫測定法」、第２版
（医学書院、1982年発行(153〜164頁))などに紹介され
ている。[0003] The calibration curve of the homogeneous enzyme immunoassay is very different from that of the conventional clinical chemistry test. In general, it is not a straight line but an S-shaped (sigmoidal) in most cases. For such a calibration curve, a radioimmunoassay method (RI
A) has been sufficiently studied so far. In the case of the enzyme immunoassay method, the method of setting the calibration curve of the radioimmunoassay method can be essentially applied, and the regression method is known as a logistic curve, a logit-log conversion formula, and the like, and Eiji Ishikawa et al. And "Enzyme Immunoassay", 2nd Edition (Medical Publishing, 1982, pp.153-164).
【０００４】一方、多数の検体試料を取扱いルーティン
化している臨床検査では、簡便、迅速に分析できること
が望まれている。このような観点から、均一系酵素免疫
反応を適用した乾式分析要素が提案されている（特開平
１−321360）。これは、多層乾式分析要素（たとえば、
特開昭49−53888、同59−77356、同59−102388、米国特
許4,459,358)の同一層或いは別々の層に水不溶性の高分
子基質と、その基質に対する酵素と特定物質に対する抗
体との結合物を含有させたものである。[0004] On the other hand, in a clinical test in which a large number of sample samples are handled and routinely performed, it is desired that the analysis can be performed easily and quickly. From such a viewpoint, a dry analysis element to which a homogeneous enzyme immunoreaction is applied has been proposed (JP-A-1-321360). This is a multi-layer dry analytical element (for example,
JP-A-49-53888, JP-A-59-77356, JP-A-59-102388, U.S. Pat.No.4,459,358) .A conjugate of a water-insoluble polymer substrate in the same layer or in a separate layer, and an enzyme against the substrate and an antibody against a specific substance. Is contained.
【０００５】[0005]
【発明が解決しようとする課題】均一系酵素免疫分析方
法を適用した乾式分析要素で得られる反射光学濃度と測
定物質濃度の関係をこれまで用いられているｌｏｇｉｓ
ｔｉｃ曲線、ｌｏｇｉｔ−ｌｏｇ変換式や直角双曲線法
を用いて回帰することは可能である。しかしながら、こ
のような回帰式を用いる場合は設定すべき回帰係数が多
いために、設定に必要なキャリブレータ液の濃度レベル
数が多くなってしまう。また、回帰が複雑なために計算
に多くの時間を必要としてしまう。このようなことは、
乾式分析要素の簡便性、迅速性といった特徴を半減させ
てしまう欠点となっている。The relationship between the reflection optical density and the concentration of a substance obtained by a dry analytical element to which a homogeneous enzyme immunoassay method is applied is described by using logis which has been used so far.
It is possible to perform regression using a tic curve, a logit-log conversion formula or a rectangular hyperbolic method. However, when such a regression equation is used, there are many regression coefficients to be set, so that the number of concentration levels of the calibrator solution required for the setting increases. In addition, the calculation requires a lot of time due to the complicated regression. Such a thing,
This is a disadvantage that the characteristics of the dry analysis element, such as simplicity and quickness, are halved.
【０００６】均一系酵素免疫分析方法を適用した乾式分
析要素における検量線の設定では、必要なキャリブレー
タ液の濃度レベル数はできるだけ少なく、そして検量線
の回帰式はできるだけ単純なものであるのがよい。それ
によって検量線を設定するための操作の簡便化と迅速化
を達成することができる。そのためには、検量線が直線
であるのが理想的であるが、均一系酵素免疫分析方法の
検量線は一般に直線とならず、ほとんどの場合Ｓ字（シ
グモイダル）になってしまう。しかしながら、測定物質
によっては、測定したい濃度範囲がＳ字（シグモイダ
ル）の全領域に渡らず、Ｓ字の一部分で納まってしまう
場合がある。また、検量線の形は座標軸の設定の仕方に
よっても大きく変わり、意外にも座標軸の設定の仕方に
よって測定濃度範囲の検量線を直線で表現できることが
わかった。測定濃度範囲で検量線を直線に表現できるこ
とは、検量線を直線回帰するときはもとより、整次多項
式で回帰するときも、より正確に回帰できるというメリ
ットがある。また、これまで知られている各点間直線補
間法で回帰する場合も、この方法の欠点である各直線間
の不連続性を軽減できるというメリットもある。In setting a calibration curve in a dry analytical element to which a homogeneous enzyme immunoassay method is applied, it is preferable that the number of necessary concentration levels of the calibrator solution be as small as possible and the regression equation of the calibration curve be as simple as possible. . Thereby, the operation for setting the calibration curve can be simplified and speeded up. For that purpose, it is ideal that the calibration curve is a straight line. However, the calibration curve of the homogeneous enzyme immunoassay method is generally not a straight line, and in most cases, becomes an S-shape (sigmoidal). However, depending on the substance to be measured, the concentration range to be measured may not cover the entire area of the S-shape (sigmoidal) and may fall within a part of the S-shape. In addition, it was found that the shape of the calibration curve varies greatly depending on the way of setting the coordinate axes, and surprisingly, the calibration curve of the measured concentration range can be expressed as a straight line by the way of setting the coordinate axes. The fact that the calibration curve can be expressed as a straight line in the measured concentration range has the advantage that the regression can be performed more accurately not only when the calibration curve is linearly regressed but also when the regression is performed using an order polynomial. Also, in the case of performing regression by the known inter-point linear interpolation method, there is an advantage that the discontinuity between the respective straight lines, which is a drawback of this method, can be reduced.
【０００７】本発明は、均一系酵素免疫反応を適用した
乾式分析要素を用いた免疫分析方法において、簡便でし
かも正確度の高い測定結果を与える検量線の設定方法
（新たに検量線を作成する方法）を提供することを目的
とする。According to the present invention, there is provided an immunoassay method using a dry analytical element to which a homogeneous enzyme immunoreaction is applied, wherein a method for setting a calibration curve which gives a simple and highly accurate measurement result (a new calibration curve is prepared). Method).
【０００８】[0008]
【課題を解決するための手段】このような目的は、均一
系酵素免疫反応を適用した乾式分析要素を用いて生物体
液中の特定物質の定量を行う免疫分析方法において、検
量線の座標軸の設定を、一方を該方法で測定される前記
分析要素に現れた検知可能な信号値の逆数で、そしても
う一方を特定物質の濃度値の真数で行うことを特徴とす
る、乾式免疫分析要素を用いた定量方法における検量線
の設定方法によって達成することができた。SUMMARY OF THE INVENTION An object of the present invention is to provide an immunoassay method for quantifying a specific substance in a biological fluid using a dry analytical element to which a homogeneous enzyme immunoreaction is applied. Dry immunoassay element, wherein one is performed with the reciprocal of the detectable signal value appearing on the analysis element measured by the method, and the other is performed with the exact number of the concentration value of the specific substance. This was achieved by the method of setting the calibration curve in the quantification method used.
【０００９】免疫分析方法は、抗原抗体反応を利用して
抗原または抗体を分析する方法であり、検出手段により
酵素免疫分析方法、ラジオイムノアッセイ、蛍光イムノ
アッセイ、レーザイムノアッセイ等がある。また、反応
形態により競合法と非競合法に分けられ、さらにＢ／Ｆ
分離が必要な非均一系とＢ／Ｆ分離が不要な均一系に分
けられる。本発明の方法は、特に均一系酵素免疫分析方
法を適用した乾式分析要素を用いた分析法に適用される
ものである。The immunoassay is a method for analyzing an antigen or an antibody by utilizing an antigen-antibody reaction, and includes an enzyme immunoassay, a radioimmunoassay, a fluorescence immunoassay and a laser immunoassay depending on the detection means. In addition, the reaction method is divided into a competitive method and a non-competitive method.
It is divided into a non-homogeneous system that requires separation and a homogeneous system that does not require B / F separation. The method of the present invention is particularly applied to an analytical method using a dry analytical element to which a homogeneous enzyme immunoassay method is applied.
【００１０】他の免疫分析方法においても一般にそうで
あるが、均一系酵素免疫分析方法を適用した乾式分析要
素を用いて生物体液たとえば血液中、尿中の特定物質の
定量をおこなうには、予め測定により得られる検知可能
な信号と測定物質の濃度との関係を検量線として得てお
く必要がある。この操作は、例えば以下のように行われ
る。いくつかの濃度レベルの測定物質の濃度既知溶液
（キャリブレータ液）をそれぞれ乾式分析要素上に一定
量点着する。点着された液は乾式分析要素中で免疫反
応、発色反応をし、液中の測定物質の濃度に応じた発色
濃度を与える。実際には、点着後乾式分析要素を一定時
間、一定温度に保って（インクベーション）発色反応を
進行させた後、光を乾式分析要素に照射し、特定波長域
で反射光量を測定してキャリブレータ液の測定物質濃度
に応じた反射光学濃度を求める。得られた反射光学濃度
と測定物質濃度の関係を回帰式として求め、測定物質定
量のための検量線とする。As is generally the case with other immunoassay methods, in order to quantify a specific substance in a biological fluid such as blood or urine using a dry analysis element to which a homogeneous enzyme immunoassay is applied, It is necessary to obtain the relationship between the detectable signal obtained by the measurement and the concentration of the measured substance as a calibration curve. This operation is performed as follows, for example. A fixed amount of a solution (calibrator solution) having a known concentration of a measurement substance at several concentration levels is spotted on the dry analytical element. The spotted solution undergoes an immunoreaction and a color development reaction in a dry analytical element, and gives a color development concentration corresponding to the concentration of the test substance in the solution. In practice, after the spotting, the dry analytical element is kept at a constant temperature for a certain period of time (incubation) to advance the color development reaction, and then the light is irradiated to the dry analytical element, and the amount of reflected light is measured in a specific wavelength range. The reflection optical density is determined according to the concentration of the substance to be measured in the calibrator solution. The relationship between the obtained reflection optical density and the measured substance concentration is obtained as a regression equation, and is used as a calibration curve for quantitatively determining the measured substance.
【００１１】従来、この検量線の座標軸に検知された信
号値の真数とアナライトの濃度値の対数が用いられてい
たが、本発明の方法においては検知された信号値の逆数
と定量しようとする特定物質の濃度値の真数を座標軸と
して用いるところに特徴がある。検知される信号値は各
分析系と測定機器の種類等に応じて定まるものであり、
例えば、酵素免疫分析方法で発色系を利用した場合には
光学濃度、反射光学濃度等（一般にはバックグラウンド
の影響を排除するため濃度差）である。Conventionally, the true number of the detected signal value and the logarithm of the analyte concentration value have been used on the coordinate axis of the calibration curve. However, in the method of the present invention, the reciprocal of the detected signal value will be determined. It is characterized in that the true number of the concentration value of the specific substance is used as a coordinate axis. The detected signal value is determined according to the type of each analysis system and measurement device, etc.
For example, when a color system is used in the enzyme immunoassay, the optical density, the reflection optical density, and the like (generally, a density difference to eliminate the influence of the background) are used.
【００１２】本発明の検量線の設定方法をモデル図（図
１）にもとづいて説明する。縦軸に特定物質（分析対象
物質、すなわちアナライト（ａｎａｌｙｔｅ））濃度値
の対数、横軸に検知される信号値（又は信号値からバッ
クグラウンド値を差引いた値）の真数をとったとき免疫
分析法、ことにＥＩＡでは一般にモデル図である、図１
Ｂ又は図１Ｃの実線のように検量線はシグモイダル曲線
で表される。A method for setting a calibration curve according to the present invention will be described with reference to a model diagram (FIG. 1). The vertical axis represents the logarithm of the specific substance (analyte), and the horizontal axis represents the logarithm of the detected signal value (or the value obtained by subtracting the background value from the signal value). FIG. 1 is a model diagram generally used for immunoassays, especially for EIA.
The calibration curve is represented by a sigmoidal curve like B or the solid line in FIG. 1C.
【００１３】シグモイダル検量線の場合には、次のよう
になる。例えば図１の縦軸のアナライトの定量範囲のほ
ぼ上限値（Ｈ）と下限値（Ｉ）に対応する２点を検量線
の上にとり（○印の２点）、この２点を通る直線（図１
Ｂ、図１Ｃの２点鎖線）を引く。アナライトの定量範囲
内では、この直線はシグモイド検量線と２点又は３点で
一致するだけであって、大部分の定量範囲ではこの直線
はシグモイド検量線から大きくはなれている。従って、
この直線を検量線として用いることができないことは明
らかである。In the case of a sigmoidal calibration curve, the following is obtained. For example, two points corresponding to the upper limit value (H) and the lower limit value (I) of the quantitative range of the analyte on the vertical axis of FIG. 1 are plotted on a calibration curve (two points indicated by ○), and a straight line passing through these two points (Figure 1
B, a two-dot chain line in FIG. 1C) is drawn. In the quantification range of the analyte, this straight line only coincides with the sigmoid calibration curve at two or three points, and in most quantification ranges, the straight line deviates greatly from the sigmoid calibration curve. Therefore,
Obviously, this straight line cannot be used as a calibration curve.
【００１４】本発明の検量線の設定方法においては、例
えば縦軸にアナライト濃度の真数、横軸に検知される信
号値の逆数をとる。このように２軸をとると図１Ａのよ
うに、検量線は直線に近い、ゆるい曲率の曲線状検量線
（実線）になる。アナライトの定量範囲のほぼ上限値
（Ｈ）と下限値（Ｉ）に対応する２点を検量線の上にと
り（○印の２点）、この２点を通る直線（図１Ａの２点
鎖線）を引く。アナライトの定量範囲内で、この直線は
ゆるい曲率の曲線状検量線の近傍に位置する。従って、
この直線を検量線として用いた場合に、得られるアナラ
イトの濃度値は真の値と推定される値からの誤差は小さ
いことは明らかである。In the method of setting a calibration curve according to the present invention, for example, the vertical axis represents the true number of the analyte concentration, and the horizontal axis represents the reciprocal of the detected signal value. When the two axes are taken in this manner, as shown in FIG. 1A, the calibration curve becomes a curved calibration curve (solid line) having a close curvature and a gentle curvature. Two points corresponding to the upper limit value (H) and the lower limit value (I) of the quantitative range of the analyte are plotted on the calibration curve (two points indicated by ○), and a straight line passing through these two points (the two-dot chain line in FIG. 1A) )pull. Within the quantitative range of the analyte, this straight line is located in the vicinity of a curved calibration curve having a low curvature. Therefore,
When this straight line is used as a calibration curve, it is clear that the obtained analyte concentration value has a small error from a value estimated as a true value.
【００１５】このように、アナライトの測定濃度範囲で
検量線を直線で表現できることにより、検量線を直線回
帰する場合にも整次多項式で回帰する場合にも、より正
確に回帰できるメリットがある。また、これまで知られ
ている各点間直線補間法で回帰する場合も、この方法の
欠点である各直線間の不連続性を軽減又は排除できるメ
リットもある。さらに、免疫分析試薬のロットのちがい
による差違又は乾式分析要素のロットのちがいによる差
違（ロット間差又はバッチ間差）を補正するための検量
線の補正作業が、より少ないキャリブレータ液濃度レベ
ル数で実施できる（濃度レベル数が１、２で実施でき
る）というメリットがある。さらに、またロット間差を
補正するための検量線の補正そのものをきわめて簡単な
数式処理又は数値処理で実施できるというメリットもあ
る。As described above, since the calibration curve can be represented by a straight line in the measured concentration range of the analyte, there is an advantage that the regression can be performed more accurately both when the calibration curve is linearly regressed and when the regression is performed using an order polynomial. . Also, in the case of performing regression by the known linear interpolation method between points, there is also an advantage that the discontinuity between the respective straight lines, which is a disadvantage of this method, can be reduced or eliminated. Furthermore, the calibration curve correction operation for correcting the difference due to the difference between the lots of the immunoassay reagent or the difference between the lots of the dry analysis element (difference between lots or batches) requires less calibrator solution concentration levels. There is an advantage that it can be implemented (it can be implemented with one or two density levels). In addition, there is an advantage that the calibration curve itself for correcting the difference between lots can be implemented by a very simple mathematical processing or numerical processing.
【００１６】この検量線を用いて各試料について得られ
た信号値から特定物質の濃度を算出する方法は従来と同
様でよい。The method of calculating the concentration of a specific substance from the signal value obtained for each sample using this calibration curve may be the same as the conventional method.
【００１７】[0017]
【００１８】[0018]
【実施例】ＣＲＰ分析用多層乾式スライドの作製 ゼラチン下塗層が設けられている厚さ180μｍの無色透
明ポリエチレンテレフタレート（ＰＥＴ）シート（支持
体）上に、下記の被覆量になるように架橋剤含有試薬溶
液を塗布し、乾燥して試薬層を設けた。 アルカリ処理ゼラチン 14.5g/m2 ノニルフェノキシポリエトキシエタノール 0.2g/m2 （オキシエチレン単位平均９〜10含有） グルコースオキシダーゼ 5000u/m2 ペルオキシダーゼ 15000u/m2 グルコアミラーザ 5000u/m2 2-（4-ヒドロキシ-3,5-ジメトキシフェニル）-4-〔-4-(ジメチルアミノ）フェ ニル〕-5-フェネチルイミダゾール(ロイコ色素）酢酸塩 0.38g/m2 ビス〔（ビニルスルホニルメチルカルボニル）アミノ〕メタン 0.1g/m2 EXAMPLE Preparation of a multilayer dry slide for CRP analysis On a 180 μm thick colorless and transparent polyethylene terephthalate (PET) sheet (support) provided with a gelatin undercoat layer, a cross-linking agent was prepared in the following coating amount. The reagent solution was applied and dried to form a reagent layer. Alkali-treated gelatin 14.5 g / m 2 Nonylphenoxy polyethoxyethanol 0.2 g / m 2 (containing an average of 9 to 10 oxyethylene units) Glucose oxidase 5000 u / m 2 Peroxidase 15000 u / m 2 Glucoamiraza 5000 u / m 2 2- (4 -Hydroxy-3,5-dimethoxyphenyl) -4-[-4- (dimethylamino) phenyl] -5-phenethylimidazole (leuco dye) acetate 0.38 g / m 2 bis [(vinylsulfonylmethylcarbonyl) amino] Methane 0.1 g / m 2
【００１９】この試薬層の上に、下記の被覆量になるよ
うに接着層を塗布し、乾燥して設けた。 アルカリ処理ゼラチン 14.5g/m2 ビス〔（ビニルスルホニルメチルカルボニル）アミノ〕メタン 0.1g/m2 On this reagent layer, an adhesive layer was applied so as to have the following coating amount, and was provided by drying. Alkali-treated gelatin 14.5 g / m 2 bis [(vinylsulfonylmethylcarbonyl) amino] methane 0.1 g / m 2
【００２０】ついで接着層の表面に下記の被覆量になる
ように下記試薬含有水溶液を塗布し、ゼラチン層を膨潤
させ、その上に50デニール相当のＰＥＴ紡績糸36ゲージ
編みした厚さ約250μｍのトリコット編物布地をほぼ一
様に軽く圧力をかけてラミネートして多孔性展開層を設
けた。 ノニルフェノキシポリエトキシエタノール 0.15g/m2 （オキシエチレン単位平均９〜10含有） ビス〔（ビニルスルホニルメチルカルボニル）アミノ〕メタン 0.4g/m2 Next, the following reagent-containing aqueous solution was applied to the surface of the adhesive layer so as to have the following coating amount, the gelatin layer was swollen, and a PET spun yarn equivalent to 50 denier, 36 gauge knitted, was about 250 μm thick. The tricot knitted fabric was laminated almost uniformly under light pressure to provide a porous spreading layer. Nonylphenoxypolyethoxyethanol 0.15 g / m 2 (containing an average of 9 to 10 oxyethylene units) bis [(vinylsulfonylmethylcarbonyl) amino] methane 0.4 g / m 2
【００２１】次に、下記の被覆量になるように基質を塗
布、乾燥して基質層を設けた。 カルボキシメチル化澱粉 4g/m2 ノニルフェノキシポリエトキシエタノール 0.2g/m2 （オキシエチレン単位平均９〜10含有）Next, the substrate was applied to the following coating amount and dried to form a substrate layer. Carboxymethylated starch 4 g / m 2 Nonylphenoxypolyethoxyethanol 0.2 g / m 2 (containing 9 to 10 average oxyethylene units)
【００２２】さらに、基質層兼展開層であるトリコット
編物布地層にアミラーゼ−抗ＣＲＰ・IgＧ結合物を3mg/
m2の被覆量となるようにしてエタノール溶液を塗布し、
含浸させ乾燥させてＣＲＰ分析用多層乾式分析要素を得
た。Further, 3 mg / amylase-anti-CRP.IgG conjugate was added to the tricot knitted fabric layer serving as the substrate layer and the spreading layer.
ethanol solution was applied as a coating amount of m 2,
Impregnation and drying gave a multilayer dry analytical element for CRP analysis.
【００２３】得られた分析要素を15mm四方のチップに裁
断し、特開昭57−63452に記載のスライドの枠に収め
て、ＣＲＰ分析用多層乾式分析スライドとした。The obtained analytical element was cut into a 15 mm square chip and placed in a slide frame described in JP-A-57-63452 to obtain a multilayer dry analytical slide for CRP analysis.
【００２４】検量線の作製 ＣＲＰ濃度が既知の２種のＣＲＰ標準血清のそれぞれ10
μｌを緩衝液(0.2ＭＭＥＳバッファー、pH6.0) 200μｌ
で希釈したＣＲＰ緩衝溶液を上記分析スライドのトリコ
ット編物布地層上にそれぞれ10μｌ点着し、各分析スラ
イドを密閉容器中で37℃に保って、中心波長650nmの可
視光でＰＥＴ支持体側から反射光学濃度を測定した。点
着から４分後および６分後の反射光学濃度の差（ΔＯＤ
r6-4）をＣＲＰ標準血清の既知濃度とともに表１に示
す。Preparation of calibration curve Each of two types of CRP standard sera with known CRP concentrations
200 μl of buffer (0.2MMES buffer, pH 6.0)
10 μl of each of the CRP buffer solutions diluted in the above was applied onto the tricot knitted fabric layer of the analytical slide, and each analytical slide was kept at 37 ° C. in a closed container, and reflected optically from the PET support side with visible light having a center wavelength of 650 nm. The concentration was measured. Difference in reflection optical density 4 minutes and 6 minutes after spotting (ΔOD
r 6-4 ) are shown in Table 1 together with known concentrations of CRP standard serum.
【００２５】[0025]
【表１】 [Table 1]
【００２６】表１のＣＲＰ濃度（Ｃ）とΔＯＤr6-4につ
いて、表２に示す設定の座標軸でそれぞれをプロット
し、２つのプロットを通る直線をもとめることで検量線
を得た。The CRP concentration (C) and ΔODr 6-4 in Table 1 were plotted on the coordinate axes set in Table 2, and a calibration curve was obtained by obtaining a straight line passing through the two plots.
【００２７】[0027]
【表２】 [Table 2]
【００２８】常用法と相関 ＣＲＰ測定の常用法である免疫比濁法によりＣＲＰ濃度
が測定されている50の検体（ＣＲＰ濃度は、０〜10.2mg
/dL)のそれぞれ10μｌを緩衝液(0.2Ｍ ＭＥＳバッファ
ー、pH6.0) 200μｌで希釈した溶液を上記分析スライド
のトリコット編物布地層上にそれぞれ10μｌ点着し、各
分析スライドを密閉容器中で37℃に保って、中心波長65
0nmの可視光でＰＥＴ支持体側から反射光学濃度を測定
し、点着から４分後および６分後の反射光学濃度の差
（ΔＯＤr6-4）を求めた。Correlation with conventional method 50 samples whose CRP concentration was measured by immunoturbidimetry, which is a common method of CRP measurement (CRP concentration was 0 to 10.2 mg
/ dL) was applied onto a tricot knitted fabric layer of the above-mentioned analysis slides by applying 10 μl of a solution obtained by diluting 10 μl of each with 200 μl of a buffer solution (0.2 M MES buffer, pH 6.0). ℃, center wavelength 65
The reflection optical density was measured from the PET support side with visible light of 0 nm, and the difference (ΔODr 6-4 ) between the reflection optical densities 4 minutes and 6 minutes after spotting was determined.
【００２９】それぞれの検体について得られたΔＯＤr
6-4を、上記の本発明の検量線と比較例１、２の検量線
を用いてＣＲＰ濃度に換算した。ΔODr obtained for each sample
6-4 was converted to a CRP concentration using the above-mentioned calibration curve of the present invention and the calibration curves of Comparative Examples 1 and 2.
【００３０】常用法の免疫比濁法で測定されたＣＲＰ濃
度と表２のそれぞれの検量線を用いて得られたＣＲＰ濃
度の相関を求めた。相関係数(r)と傾き(a)を表３に示
す。The correlation between the CRP concentration measured by a conventional immunoturbidimetric method and the CRP concentration obtained using the respective calibration curves in Table 2 was determined. Table 3 shows the correlation coefficient (r) and the slope (a).
【００３１】本発明の検量線で得られた相関図と比較例
１の検量線で得られた相関図をそれぞれ図２、図３に示
す。FIGS. 2 and 3 show a correlation diagram obtained by the calibration curve of the present invention and a correlation diagram obtained by the calibration curve of Comparative Example 1, respectively.
【００３２】[0032]
【表３】 [Table 3]
【００３３】表３、図２に示されているように、本発明
の座標軸にすることで、単純な直線の検量線でも正確な
ＣＲＰ測定が可能であることは明らかである。As shown in Table 3 and FIG. 2, it is clear that accurate CRP measurement is possible even with a simple linear calibration curve by using the coordinate axes of the present invention.
【００３４】[0034]
【発明の効果】本発明の検量線の座標軸の設定によっ
て、均一系酵素免疫反応を適用した乾式分析要素の検量
線を、測定結果の正確度を十分保ちながら、少ないキャ
リブレータ液の濃度レベル数で簡単な回帰式として得る
ことができる。By setting the coordinate axes of the calibration curve of the present invention, the calibration curve of the dry analytical element to which the homogeneous enzyme immunoreaction is applied can be performed with a small number of calibrator solution concentration levels while maintaining the accuracy of the measurement result sufficiently. It can be obtained as a simple regression equation.
【図１】図１Ａは、本発明の検量線の設定方法を説明す
るためのモデル図である。 図１Ｂ、図１Ｃは免疫分析方法における従来の検量線を
説明するためのモデル図である。 実 線：系の持つ検量線 ２点鎖線：２つの濃度既知のアナライト溶液（アナライ
ト濃度Ｌ及びＨ）で設定した直線の検量線FIG. 1A is a model diagram for explaining a method of setting a calibration curve according to the present invention. 1B and 1C are model diagrams for explaining a conventional calibration curve in an immunoassay method. Solid line: calibration curve of the system 2-dot chain line: linear calibration curve set with two analyte solutions of known concentrations (analyte concentrations L and H)
【図２】本発明の座標軸の検量線を用いた場合の免疫比
濁法との相関を示すグラフである。FIG. 2 is a graph showing a correlation with an immunoturbidimetric method when a calibration curve of a coordinate axis according to the present invention is used.
【図３】比較例１の座標軸の検量線を用いた場合の免疫
比濁法との相関を示すグラフである。FIG. 3 is a graph showing a correlation with an immunoturbidimetry when a calibration curve of a coordinate axis of Comparative Example 1 is used.
Claims (1)
要素を用いて生物体液中の特定物質の定量を行う免疫分
析方法において、検量線の座標軸の設定を、一方を該方
法で測定される前記分析要素に現れた検知可能な信号値
の逆数で、そしてもう一方を特定物質の濃度値の真数で
行うことを特徴とする、乾式免疫分析要素を用いた定量
方法における検量線の設定方法1. An immunoassay method for quantifying a specific substance in a biological fluid using a dry analytical element to which a homogeneous enzyme immunoreaction is applied, wherein a coordinate axis of a calibration curve is set and one of the axes is measured by the method. A method for setting a calibration curve in a quantification method using a dry immunoanalytical element, characterized in that the reciprocal of a detectable signal value appearing in the analytical element and the other is performed by an exact number of a concentration value of a specific substance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3175142A JP2739615B2 (en) | 1991-07-16 | 1991-07-16 | Setting method of calibration curve in quantification method using dry immunoassay element |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3175142A JP2739615B2 (en) | 1991-07-16 | 1991-07-16 | Setting method of calibration curve in quantification method using dry immunoassay element |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0518969A JPH0518969A (en) | 1993-01-26 |
| JP2739615B2 true JP2739615B2 (en) | 1998-04-15 |
Family
ID=15991024
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3175142A Expired - Fee Related JP2739615B2 (en) | 1991-07-16 | 1991-07-16 | Setting method of calibration curve in quantification method using dry immunoassay element |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2739615B2 (en) |
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|---|---|---|---|---|
| JP5037439B2 (en) * | 2008-06-23 | 2012-09-26 | 株式会社堀場製作所 | Analysis equipment |
| JP5841403B2 (en) * | 2011-10-28 | 2016-01-13 | Kddi株式会社 | Color density measuring apparatus, color density measuring method, and color density measuring program |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SE7610939L (en) * | 1976-10-01 | 1978-04-02 | Bonnierfoeretagen Ab | PROCEDURE FOR QUANTITATIVE DETERMINATION OF ANTIGEN OR ANTIBODY |
| JPS60172596A (en) * | 1984-02-17 | 1985-09-06 | 呉羽化学工業株式会社 | Sheet for analyzing data |
| JPH0283448A (en) * | 1988-09-20 | 1990-03-23 | Fuji Photo Film Co Ltd | Immunoassay |
-
1991
- 1991-07-16 JP JP3175142A patent/JP2739615B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0518969A (en) | 1993-01-26 |
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