JPH0568655B2 - - Google Patents
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- Publication number
- JPH0568655B2 JPH0568655B2 JP59010493A JP1049384A JPH0568655B2 JP H0568655 B2 JPH0568655 B2 JP H0568655B2 JP 59010493 A JP59010493 A JP 59010493A JP 1049384 A JP1049384 A JP 1049384A JP H0568655 B2 JPH0568655 B2 JP H0568655B2
- Authority
- JP
- Japan
- Prior art keywords
- value
- concentration
- calibration curve
- standard solution
- log
- Prior art date
- 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.)
- Expired - Lifetime
Links
- 239000012086 standard solution Substances 0.000 claims description 55
- 238000011088 calibration curve Methods 0.000 claims description 47
- 238000005259 measurement Methods 0.000 claims description 31
- 238000000034 method Methods 0.000 claims description 25
- 238000002835 absorbance Methods 0.000 claims description 19
- 230000008105 immune reaction Effects 0.000 claims description 8
- 230000036046 immunoreaction Effects 0.000 claims 1
- 238000000149 argon plasma sintering Methods 0.000 description 10
- 239000000243 solution Substances 0.000 description 6
- 239000006185 dispersion Substances 0.000 description 5
- 230000001678 irradiating effect Effects 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Automatic Analysis And Handling Materials Therefor (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
Description
(イ) 産業上の利用分野
本発明は、免疫反応測定装置を用いて免疫反応
測定の際に使用する検量線の作成方法に関するも
のである。
(ロ) 従来技術
免疫反応の測定においては、抗原抗体反応液に
光を照射し、光の吸光度又は散乱光強度を測定
し、濃度測定を行なうのであるが、濃度未知の測
定対象を検出するものであるから、測定開始前に
反応容器に所定量の抗原の標準品（血清）と所定
量の抗体（抗血清）とを加え、所定時間反応させ
た後に光を照射し、光の吸光度又は散乱光強度を
分光光度計又はネフエロメータにより測定し、標
準品の濃度と吸光度又は散乱光強度との関係をグ
ラフにプロツトし、検量線を作成していた。
この種の検量線を作成するにあたつて、後述す
る所であるが免疫反応の場合、測定範囲が高濃度
域から低濃度域までの広い範囲にわたるもので、
かつ測定項目により濃度範囲が異なるため検量線
が曲線となり、測定項目によつて曲線の形状が変
るため、いろいろな曲線の近似式（例えば、２次
式、３次式、log，logit−log1次式、logit−log2
次式など）をもつており、測定項目毎に使い分け
をするため、操作が面倒になるという欠点があつ
た。
(ハ) 目的
本発明は、前記した従来技術の有する欠点を解
消するもので、標準液の濃度値の低い方から２番
目の標準液を基準にしてlogの多項式を用いて検
量線を作成することにより、標準液の濃度値に対
するlogをとつたものの分散度合が均等となり、
測定項目に拘わらず高濃度領域から低濃度領域に
わたる測定範囲に適用しうる近似式を用いての検
量線の作成方法を提供することを目的とする。
(ニ) 構成
本発明は、複数の標準液の濃度値xiをXi＝log
（１＋xi／ｋ）（ｋは測定項目により変更する）か
ら求め、前記した標準液に光を照射し、吸光度値
又は散乱光強度値yiにより決定される点（Xi，yi）
を用いて、２次式ｙ＝aX2＋bX＋ｃ、又は３次
式ｙ＝aX3＋bX2＋cX＋ｄによる最小自乗法近似
により係数ａ，ｂ，ｃ，ｄ又はａ，ｂ，ｃを求
め、Xiの分散がほぼ均等な検量線を作成するもの
である。
(ホ) 実施例
以下において、説明の便宜上、従来技術と本発
明の実施例とを比較しながら説明する。
第１図は免疫反応の濃度xiと測定値yiとの関係
を示し、第２図と第３図では実線により従来の検
量線作成方法により作成された検量線と、点線は
本発明により作成し、修正された検量線とを示
し、第４図はXi＝log（１＋xi）により得られたも
のでXiの分散が不均等なためうまく適合しない場
合の検量線を示し、第５図は本発明の検量線作成
方法により作成されたもので、最適のｋを用いて
Xiの分散が均等になつた検量線を示し、第６図は
ｋの値を自動的に標準液の濃度値の最低値std1に
決めた場合のXiの分散例を、第７図はｋの値を自
動的に標準液の濃度値の２番目に低い値std2に定
めた場合のXiの分散例を示す。
第１図に、３項目の免疫反応の測定において、
横軸に濃度xiを、縦軸に反応容器に光を照射した
ときの光の吸光度値又は光散乱強度yiをとり、濃
度xiと光の吸光度値又は光散乱強度値yiの関係を
近似式によらずに、単にその傾向を概念的に示し
たものである。
ところで、免疫反応の測定においては、前述し
たように測定項目の相違により高濃度域から低濃
度域にわたる広い範囲に及ぶ測定領域を測定する
必要があり、また測定項目によりその濃度範囲が
異なるものである。
そして、事前に検量線を作成する場合には、横
軸に濃度値xiを、縦軸に吸光度値又は光散乱強度
値yiをとり、標準液の入つている容器に光を照射
するが、標準液の濃度は既知であり、そしてその
ときに得られた吸光度値又は光散乱強度値との交
点をプロツトし、次に異なつた濃度を持つ標準液
の入つている容器に光を照射し、同様の手法によ
つてプロツトし、これらのプロツト点の値yiを適
当な近似式にあてはめて最小自乗近似法により検
量線を作成する。
従来は、標準液の数が３点以上に及ぶときは、
標準液の濃度値xiに対応する吸光度値又は光散乱
強度値yiを２次式ｙ＝ax2＋bx＋ｃにあてはめ、
最小自乗近似法により係数ａ，ｂ，ｃを求めて検
量線を作成し、標準液の数が４点以上に及ぶとき
は３次式ｙ＝ax3＋bx2＋cx＋ｄにあてはめ、最
小自乗近似法により検量線を作成する場合が多
い。
このようにして得られた検量線の例を第２図と
第３図に示す。
第２図において実線で示すものは標準液の数が
４点以上に及ぶ場合で、標準液の濃度値xiと、標
準液の入つている容器に光を照射し、得られた吸
光度値又は光散乱強度値yiを３次式ｙ＝ax3＋bx2
＋cx＋ｄにあてはめ、最小自乗近似法により係
数ａ，ｂ，ｃ，ｄを求めて作成された検量線であ
る。この検量線によると、プロツトされた測定点
の数が低濃度側に多く存在し、高濃度側に少しし
か存在せず、特にこの場合のように３次式近似の
場合は検量線に変曲点が生じ、高濃度域における
測定には適用できなくなつてしまうという欠点が
ある。
なお、点線で示すものは本発明の検量線作成方
法により修正された検量線を参考的に示すもの
で、その作成方法については後述するが、標準液
の濃度値xiをＸ＝log（１＋ｘ／ｋ）に代入して濃
度値xiを求め、前記標準液の入つている容器に光
を照射して吸光度値は光散乱強度値yiを求め、Xi
とyiを用いて３次式ｙ＝aX3＋bX2＋cX＋ｄによ
る最小自乗近似により係数ａ，ｂ，ｃ，ｄを求め
て検量線を作成したものである。そして、ｋの値
は測定項目により選定した適当な値である。
第３図において実線で示すものは標準液の数が
３点以上に及ぶ場合で、標準液の濃度値xiと、標
準液の入つている容器に光を照射し、得られた吸
光度値又は光散乱強度値yiを２次式ｙ＝ax2＋bx
＋ｃであてはめ、最小自乗近似法により係数ａ，
ｂ，ｃを求めて作成された検量線である。この検
量線は高濃度域における測定には適合している
が、低濃度域側においては最小自乗近似法により
求めた近似曲線上に標準液により求めたプロツト
点が位置せず、従つて低濃度域における実際の測
定に適用できない欠点がある。なお、点線で示す
ものは、本発明の検量線作成方法により修正され
た検量線を示す。この点線で示す検量線は、標準
液の濃度値xiをＸ＝log（１＋ｘ／ｋ）に代入して
濃度値xiを求め、標準液の入つている容器に光を
照射し、吸光度値又は光散乱強度値yiを求め、Xi
とyiを用いて２次式ｙ＝aX2＋bX＋ｃによる最小
自乗近似により係数ａ，ｂ，ｃを求めて作成され
たものである。また、ｋの値は測定項目により選
定した適当な値である。
そこで、前記した不都合な欠点を除くために、
最小自乗法による近似を行なう場合に、プロツト
点が高濃度域から低濃度奇異にわたつて見かけ上
均等に存在せしめるようにするため、第４図に示
すように標準液の濃度値xiをＸ＝log（１＋ｘ）の
式（ｋの値が１となつている場合である。）に代
入して濃度Ｘを求め、これをグラフにプロツトす
ることが提案された。なお、log（１＋ｘ）とした
理由は、標準液の濃度ｘが零、即ちｘ＝０のとき
に濃度Ｘが零、即ちＸ＝log1＝０となるように配
慮したからである。このように、標準液の濃度ｘ
をＸ＝log（１＋ｘ）をあてはめ、得られた濃度Ｘ
と、標準液に光を照射して得られた吸光度値又は
光散乱強度値ｙとを３次式ｙ＝aX3＋bX2＋cX＋
ｄに代入し、最小自乗法により係数ａ，ｂ，ｃ，
ｄを求め、検量線を作成する。
なお、点線で示したものは、本発明の検量線作
成方法により作成し、修正された検量線を示す。
しかしながら、このようにして検量線を作成し
ても測定項目により濃度範囲が、濃度ｘがｘ＝
10〜1000の範囲にわたる場合、濃度ｘがｘ＝
0.5〜10の範囲にわたる場合において、の場合
は最小濃度のstd値（標準値）と濃度ｘがｘ＝０
との間が離れ過ぎて均等にならないという欠点が
ある。
以下に、との場合を表１と表２に示す。
(a) Industrial Application Field The present invention relates to a method for preparing a calibration curve used in immune reaction measurement using an immune reaction measurement device. (b) Prior art In measuring immune reactions, the antigen-antibody reaction solution is irradiated with light and the absorbance or scattered light intensity is measured to measure the concentration, but there is no method that detects a target of unknown concentration. Therefore, before starting measurement, a predetermined amount of antigen standard (serum) and a predetermined amount of antibody (antiserum) are added to a reaction container, and after reacting for a predetermined time, light is irradiated and the absorbance or scattering of light is measured. The light intensity was measured using a spectrophotometer or nephelometer, and the relationship between the concentration of the standard product and the absorbance or scattered light intensity was plotted on a graph to create a calibration curve. When creating this type of calibration curve, as will be discussed later, in the case of immune reactions, the measurement range spans a wide range from high concentration to low concentration.
In addition, since the concentration range differs depending on the measurement item, the calibration curve becomes a curve, and the shape of the curve changes depending on the measurement item, so various curve approximations (for example, quadratic equation, cubic equation, log, log it - log1 The following equation, log it −log2
(e.g., the following formula), which has to be used for each measurement item, which has the disadvantage of making operation cumbersome. (c) Purpose The present invention solves the drawbacks of the prior art described above, and involves creating a calibration curve using a log polynomial based on the second standard solution from the lowest concentration value of the standard solution. By doing this, the degree of dispersion of the logarithm of the concentration value of the standard solution becomes equal,
It is an object of the present invention to provide a method for creating a calibration curve using an approximation formula that can be applied to a measurement range from a high concentration region to a low concentration region regardless of the measurement item. (d) Configuration The present invention provides concentration values x i of a plurality of standard solutions as X i = log
(1+x i /k) (k changes depending on the measurement item), irradiates the standard solution described above with light, and determines the point (X i , y i ) from the absorbance value or scattered light intensity value y i
, find the coefficients a, b, c, d or a, b, c by least squares approximation using the quadratic equation y=aX 2 +bX+c or the cubic equation y=aX 3 +bX 2 +cX+d, and calculate the coefficients a, b, c, d or a, b, c of X i. This creates a calibration curve with approximately equal variance. (e) Examples In the following, for convenience of explanation, a comparison will be made between the prior art and an example of the present invention. Figure 1 shows the relationship between the immune reaction concentration x i and the measured value y i , and in Figures 2 and 3, the solid line is the calibration curve created by the conventional calibration curve creation method, and the dotted line is the calibration curve created by the present invention. Figure 4 shows the calibration curve obtained by X i = log (1 + x i ) when the distribution of X i is uneven and does not fit well. Figure 5 was created using the calibration curve creation method of the present invention, using the optimal k.
Figure 6 shows an example of the distribution of X i when the value of k is automatically determined to the lowest concentration value std 1 of the standard solution, and Figure 7 shows an example of the distribution of X i . shows an example of the distribution of X i when the value of k is automatically set to the second lowest value std 2 of the concentration value of the standard solution. Figure 1 shows that in the measurement of three items of immune response,
The horizontal axis is the concentration x i , and the vertical axis is the light absorbance value or light scattering intensity y i when the reaction container is irradiated with light, and the relationship between the concentration x i and the light absorbance value or light scattering intensity value y i It simply shows the tendency conceptually, without using an approximation formula. By the way, in measuring immune reactions, as mentioned above, it is necessary to measure a wide range of measurement from high concentration to low concentration due to the difference in measurement items, and the concentration range differs depending on the measurement item. be. When creating a calibration curve in advance, take the concentration value x i on the horizontal axis and the absorbance value or light scattering intensity value y i on the vertical axis, and irradiate the container containing the standard solution with light. , the concentration of the standard solution is known, and the intersection with the absorbance value or light scattering intensity value obtained at that time is plotted, and then the containers containing standard solutions with different concentrations are irradiated with light. , using the same method, and applying the values y i of these plot points to an appropriate approximation formula to create a calibration curve using the least squares approximation method. Conventionally, when the number of standard solutions is three or more,
Applying the absorbance value or light scattering intensity value y i corresponding to the concentration value x i of the standard solution to the quadratic equation y = ax 2 + bx + c,
Create a calibration curve by determining the coefficients a, b, and c using the least squares approximation method, and when the number of standard solutions is 4 or more, apply the cubic equation y = ax 3 + bx 2 + cx + d and use the least squares approximation method. A calibration curve is often created. Examples of calibration curves obtained in this manner are shown in FIGS. 2 and 3. In Figure 2, the solid line indicates the case where there are four or more standard solutions, and the concentration value x i of the standard solution and the absorbance value obtained by irradiating the container containing the standard solution with light or The light scattering intensity value y i is calculated using the cubic formula y = ax 3 + bx 2
+cx+d, and the coefficients a, b, c, and d are calculated using the least squares approximation method. According to this calibration curve, there are many plotted measurement points on the low concentration side and only a few on the high concentration side, and especially in the case of cubic approximation as in this case, there is an inflection in the calibration curve. This method has the disadvantage that it causes dots and cannot be applied to measurements in high concentration ranges. Note that the dotted line indicates for reference the calibration curve corrected by the calibration curve creation method of the present invention, and the creation method will be described later. /k) to obtain the concentration value x i , irradiate the container containing the standard solution with light, obtain the absorbance value, and obtain the light scattering intensity value y i .
A calibration curve was created by calculating the coefficients a, b, c, and d by the least squares approximation using the cubic equation y=aX 3 +bX 2 +cX+d using and y i . The value of k is an appropriate value selected depending on the measurement item. In Figure 3, the solid line indicates the case where there are three or more standard solutions, and the concentration value x i of the standard solution and the absorbance value obtained by irradiating the container containing the standard solution with light or The light scattering intensity value y i is calculated using the quadratic formula y = ax 2 + bx
+c, coefficients a, by least squares approximation method
This is a calibration curve created by determining b and c. This calibration curve is suitable for measurements in the high concentration range, but in the low concentration range, the plot point determined using the standard solution is not located on the approximate curve determined by the least squares approximation method, and therefore It has the disadvantage that it cannot be applied to actual measurements in the area. Note that the dotted line indicates a calibration curve corrected by the calibration curve creation method of the present invention. The calibration curve shown by this dotted line is obtained by substituting the concentration value x i of the standard solution into X = log (1 + x / k) to obtain the concentration value x i , irradiating the container containing the standard solution with light, and calculating the absorbance value. Alternatively, find the light scattering intensity value y i and calculate X i
It was created by calculating the coefficients a, b, and c by the least squares approximation using the quadratic equation y=aX 2 +bX+c using and y i . Further, the value of k is an appropriate value selected depending on the measurement item. Therefore, in order to eliminate the above-mentioned disadvantages,
When performing approximation using the least squares method, in order to make the plot points appear evenly from the high concentration region to the low concentration region, the concentration value x i of the standard solution is changed to It has been proposed that the concentration Note that the reason for using log(1+x) is that consideration was given so that when the concentration x of the standard solution is zero, that is, x=0, the concentration X becomes zero, that is, X=log1=0. In this way, the concentration of the standard solution x
Applying X=log(1+x) to the obtained concentration
and the absorbance value or light scattering intensity value y obtained by irradiating the standard solution with light, using the cubic formula y=aX 3 +bX 2 +cX+
d and use the least squares method to find the coefficients a, b, c,
d and create a calibration curve. Note that the dotted line indicates a calibration curve created and corrected by the calibration curve creation method of the present invention. However, even if a calibration curve is created in this way, the concentration range may vary depending on the measurement item, and the concentration x may vary depending on the measurement item.
If the concentration x ranges from 10 to 1000, then x=
In cases ranging from 0.5 to 10, if the minimum concentration std value (standard value) and concentration x are x = 0
The disadvantage is that the distance between the two is too far and it is not even. The cases of and are shown below in Tables 1 and 2.
【表】【table】
【表】
表１、表２において、第２段に示すstd系列
とstd系列は高濃度域を測定すべき項目の標準
液と低濃度域を測定すべき項目の標準液であり、
それぞれの標準液の濃度ｘをlog（１＋ｘ）にあて
はめて求めた値を第３段に示し、第４段にはlog
（１＋ｘ）の差を示す。
さて、表１、表２を参照すると、std系列に
おいては標準液濃度31.3と０とのlog（１＋ｘ）の
差1.62と、std系列の標準液濃度0.63と０との
log（１＋ｘ）の差0.21と比較すると、std系列
のそれは!?かに大となり、第４図に点線で示す本
発明による検量線からはずれて実線で示すstd系
列の０から31.3の範囲が低下した近似曲線とな
つてしまい、低濃度域では実線に使用できないと
いう欠点がある。
第５図は、本発明の検量線作用方法により作成
されたもので、最適のｋを用いXiの分散が均等に
なつた場合の検量線を示す。標準液の濃度値xiを
Ｘ＝log（１＋ｘ／ｋ）の式に代入してXiを求め、
そして標準液の入つている容器に光を照射して吸
光度値又は散乱吸光度値yiを求め、得られたXiと
yiを用いて３次式ｙ＝aX3＋bX2＋cX＋ｄによる
最小自乗近似法により係数ａ，ｂ，ｃ，ｄを求め
て検量線を作成したものである。なお、ｋの値は
測定項目により最適のものを選定して使用する
が、表３に測定項目によるｋの値を例示的に示
す。[Table] In Tables 1 and 2, the std series and std series shown in the second row are the standard solutions for the items that should be measured in the high concentration range and the standard solutions for the items that should be measured in the low concentration range,
The third row shows the values obtained by applying the concentration x of each standard solution to log(1+x), and the fourth row shows the log
It shows the difference of (1+x). Now, referring to Tables 1 and 2, in the STD series, the difference in log (1 +
Compared to the difference of log(1+x) of 0.21, that of the standard series is much larger!, and the range from 0 to 31.3 of the standard series, shown as a solid line, deviates from the calibration curve according to the present invention, shown as a dotted line in Figure 4, and decreases. This has the disadvantage that it cannot be used as a solid line in the low concentration range. FIG. 5 shows a calibration curve created by the calibration curve operation method of the present invention when the optimal k is used and the variance of X i is made equal. Substitute the concentration value x i of the standard solution into the formula X = log (1 + x / k) to find X i ,
Then, the absorbance value or scattered absorbance value y i is determined by irradiating the container containing the standard solution with light, and the obtained X i and
A calibration curve was created by determining coefficients a, b, c, and d using yi using the cubic equation y=aX 3 +bX 2 +cX+d using the least squares approximation method. Note that the optimum value of k is selected and used depending on the measurement item, and Table 3 exemplarily shows the value of k depending on the measurement item.
【表】
第４図に示された従来技術により作成された検
量線の持つ不都合な点を除去するために、本発明
の検量線作成方法により検量線を作成した場合の
濃度値Xiの分散度合について第６図と第７図を参
照して説明する。
本発明においては、Ｘ＝log（１＋ｘ／ｋ）の式
を用いる。ここにおいて、ｋの値は表３に示すよ
うに測定項目によつて任意に選定し、変えること
ができるが、ｋの設定を自動的に行なえるように
する場合に、ｋの値として標準液の濃度値の最小
値から２番目の値をstd濃度値（標準濃度値）と
して用いることもできる。
一般に、市販されている試薬に添付されている
標準液は測定上限域と測定下限域との濃度値の標
準液を含んでいる場合があり、最小あるいは最大
のstd濃度値を用いることが不適当な場合がある。
即ち、Ｘ＝log（１＋ｘ／ｋ）の式において、最
小のstd濃度値を基準値とし、これを２倍にして
ｋの値とした場合に第４図に示す従来技術による
よりもより良好であるが、各標準液の濃度値stdi
に対し、Xi＝log（１＋stdi／2std1）の濃度値は表
４〜表６と第６図に示すように均等に分散しなく
なる。
以下に、表４〜表６において具体例により得ら
れた数値を示す。[Table] Variance of concentration values X The degree will be explained with reference to FIGS. 6 and 7. In the present invention, the formula X=log(1+x/k) is used. Here, the value of k can be arbitrarily selected and changed depending on the measurement item as shown in Table 3, but when setting k automatically, The second value from the minimum density value can also be used as the standard density value (standard density value). Generally, the standard solution attached to a commercially available reagent may contain a standard solution with a concentration value between the upper measurement limit and the lower measurement limit, so it is inappropriate to use the minimum or maximum standard concentration value. There are cases where That is, in the equation of X=log(1+x/k), if the minimum standard density value is taken as the reference value and this is doubled to take the value of k, the result is better than that of the conventional technique shown in FIG. However, the concentration value of each standard solution std i
On the other hand, the density values of X i =log(1+std i /2std 1 ) are no longer evenly distributed as shown in Tables 4 to 6 and FIG. Below, numerical values obtained in specific examples in Tables 4 to 6 are shown.
【表】【table】
【表】【table】
【表】
ここで、表４〜表６に示す数値Xiを第６図にお
ける線ａ〜ｃで示すと、線ａにおいては倍倍希釈
した標準液を８点、即ち表４に示す6.25〜800の
濃度液を用い、標準液の濃度値において最低の濃
度値6.25を基準値とし、Xi＝log（１＋stdi／２
（6.25））の式から求めたXiを順次プロツトしたも
のであり、線ｂにおいては標準液を５点、即ち表
５に示す50〜500の濃度液を用い、標準液の最低
の濃度値50を基準値とし、Xi＝log（１＋stdi／２
（50））の式からXiを順次プロツトしたものであ
り、線ｃにおいては標準液を５点、即ち表６に示
す５〜100の濃度液を用い、標準液の最低の濃度
値５を基準値とし、Xi＝log（１＋stdi／２（５））
の式からXiの値を求めてプロツトしたものを示
す。なお、点線ｐは、表４から表６に示す最低の
濃度値を基準としたときの基準線を示す。
そこで、標準液の濃度の低い方から２番目の標
準液の濃度値を基準に選んだ場合には、各標準液
の濃度値stdiに対するXi＝log（１＋stdi／std2）の
濃度値の分散は以下に示す表７〜９と第７図に示
するように均等に近くなる。[Table] Here, when the numerical values X i shown in Tables 4 to 6 are shown by lines a to c in Fig. 6, in line a, the standard solutions diluted twice are measured at 8 points, that is, from 6.25 to 6.25 shown in Table 4. Using a concentration solution of 800, the lowest concentration value of 6.25 among the concentration values of the standard solution is used as the reference value, and X i = log (1 + std i /2
(6.25)) is plotted in sequence, and in line b, the standard solution is used at five points, that is, the concentration solutions of 50 to 500 shown in Table 5 are used, and the lowest concentration value of the standard solution is With 50 as the standard value, X i = log (1 + std i /2
The X As the standard value, X i = log (1 + std i /2 (5))
The value of X i calculated from the equation is shown and plotted. Note that the dotted line p indicates a reference line based on the lowest density values shown in Tables 4 to 6. Therefore, if the concentration value of the second standard solution from the one with the lowest concentration is selected as the standard, the concentration value of X i = log (1 + std i / std 2 ) for the concentration value std i of each standard solution The dispersion of is nearly equal as shown in Tables 7 to 9 below and FIG.
【表】【table】
【表】【table】
【表】
表７から表９に示す数値Xiを第７図の線ｄ〜ｆ
で示すと、線ｄにおいて倍倍希釈した標準液を８
点、即ち表７に示す6.25〜800の濃度液を用い、
標準液の最低から２番目の濃度値12.5を基準値と
し、Xi＝log（１＋stdi／12.5）の式から求めたXi
を順次プロツトしたものであり、線ｅにおいては
標準液を５点、即ち表８に示す50〜500の濃度液
を用い、標準液の最低から２番目の濃度値100を
基準値とし、Xi＝log（１＋stdi／100）の式から求
めたXiをプロツトしたものであり、線ｆにおいて
は標準液を５点、即ち表９に示す５〜100の濃度
液を用い、標準液の最低から２番目の濃度値25を
基準値とし、Xi＝log（１＋stdi／25）の式から求
めたXiを順次プロツトしたものである。なお、点
線ｑは標準液の濃度の最低から２番目のものを基
準値としたときの基準線を示す。
第６図および表４〜表６に示される差の値と、
第７図および表７〜表９に示される差の値とを比
較してみると、表５と表８に示すもの同志には差
異がないが、表４と表７、表６と表９においては
表７と表９の示すものの方が表４と表７に示すも
のよりも、ほぼ均等に分布していることがわか
る。
(ヘ) 効果
以上説明したように本発明によると、複数の標
準液の濃度値xi（ｉ＝１，２，３，…）に対しXi
＝log（１＋xi／ｋ）を求め、それらの標準液の入
つている容器に光を照射し、得られた吸光度値又
は散乱光強度値yiにより決定されるXi，yiを用い
て３次式ｙ＝aX3＋bX2＋cX＋ｄ又はｙ＝aX2＋
bX＋ｃによる最小近似法により係数ａ，ｂ，ｃ，
ｄ又はａ，ｂ，ｃを決定し、均等な分散度合を有
する実際の測定に適合した検量線を作成すること
ができる。[Table] The values X i shown in Tables 7 to 9 are
In line d, the diluted standard solution is 8 times
Using a solution with a concentration of 6.25 to 800 shown in Table 7,
Using the second lowest concentration value of the standard solution, 12.5, as the reference value, X i was calculated from the formula X i = log (1 + std i /12.5)
In line e, standard solutions are used at 5 points, that is, solutions with concentrations of 50 to 500 shown in Table 8 are used, and the second concentration value from the lowest of the standard solutions, 100, is used as the reference value, and X i =log(1+std i /100).In line f , standard solutions were used at 5 points, that is, solutions with concentrations ranging from 5 to 100 shown in Table 9, and the lowest concentration of the standard solutions was plotted. Using the second density value 25 as the reference value, X i obtained from the formula X i =log(1+std i /25) is successively plotted. Note that the dotted line q indicates a reference line when the second lowest concentration of the standard solution is taken as the reference value. The difference values shown in FIG. 6 and Tables 4 to 6,
Comparing the difference values shown in Figure 7 and Tables 7 to 9, there is no difference between those shown in Tables 5 and 8, but Tables 4 and 7, and Tables 6 and 9. It can be seen that those shown in Tables 7 and 9 are more evenly distributed than those shown in Tables 4 and 7. (F) Effect As explained above, according to the present invention, for the concentration values x i (i=1, 2, 3,...) of a plurality of standard solutions,
=log(1+x i /k), irradiate the container containing these standard solutions with light, and use X i , y i determined from the obtained absorbance value or scattered light intensity value y i Cubic formula y=aX 3 +bX 2 +cX+d or y=aX 2 +
Coefficients a, b, c, by the minimum approximation method using bX + c,
By determining d or a, b, and c, it is possible to create a calibration curve that has an even degree of dispersion and is suitable for actual measurements.
第１図は免疫反応の濃度xiと測定値yiとの関係
を示すグラフ、第２図は従来技術による３次式近
似により作成れたもので、高濃度域がうまく適合
しない場合の検量線図、第３図は従来技術による
２次式近似により作成されたもので、低濃度域が
うまく適合しない場合の検量線図、第４図はXi＝
log（１＋xi）により得られたもので、Xiの分散が
不均等なためうまく適合しない場合の検量線図、
第５図は本発明の検量線作成方法により作成され
たもので、最適のｋを用いてXiの分散が均等にな
つた場合の検量線図、第６図は本発明の検量線作
成方法の実施例により検量線を作成する場合に、
標準液の最小濃度値を基準値とし、これを２倍に
した値をｋとする場合のXiの分散度合いを示す
図、第７図は本発明の検量線作成方法の実施例に
より検量線を作成する場合に、標準液の最小濃度
値から２番目に低い値をｋとした場合のXiの分散
度合いを示す図である。
図中、横軸のｘは濃度値、縦軸のｙは吸光度値
又は散乱光強度値を示す。
Figure 1 is a graph showing the relationship between the immune reaction concentration x i and the measured value y i , and Figure 2 is a graph created by cubic approximation using conventional technology. The diagram, Figure 3, was created by quadratic approximation using conventional technology, and the calibration curve in the case where the low concentration region does not fit well, and Figure 4 is the calibration curve when X i =
log(1+x i ), which is a calibration curve when the distribution of X i is uneven and does not fit well,
Figure 5 is a calibration curve created using the calibration curve creation method of the present invention, and is a calibration curve when the variance of X i is made equal using the optimal k.Figure 6 is a calibration curve created using the calibration curve creation method of the present invention. When creating a calibration curve according to the example of
Figure 7 is a diagram showing the degree of dispersion of X i when the minimum concentration value of the standard solution is the reference value and the value doubled is k. FIG. 3 is a diagram showing the degree of dispersion of X i when k is the second lowest value from the minimum concentration value of the standard solution when creating the standard solution. In the figure, x on the horizontal axis represents the concentration value, and y on the vertical axis represents the absorbance value or the scattered light intensity value.
Claims (1)
…）についてXi＝log（１＋xi／ｋ）（ｋは測定項
目により変更する）から濃度値xiを求め、前記標
準液xiに光を照射して吸光度値又は散乱光強度値
yiから決定される（Xi，yi）を用いて２次式ｙ＝
aX2＋bX＋ｃ又は３次式ｙ＝aX3＋bX2＋cX＋ｄ
による最小自乗法近似により係数ａ，ｂ，ｃ，ｄ
又はａ，ｂ，ｃを求めて検量線を作成する免疫反
応測定に使用する検量線の作成方法。 ２ 前記したXi＝log（１＋xi／ｋ）におけるｋの
値を標準液の濃度値の低い方から２番目の濃度値
とする特許請求の範囲第１項記載の免疫反応測定
に使用する検量線の作成方法。[Claims] 1. Concentration values x i (i=1, 2, 3,
...), calculate the concentration value x i from X i = log (1 + x i /k) (k changes depending on the measurement item), irradiate the standard solution x i with light, and calculate the absorbance value or scattered light intensity value.
Using (X i , y i ) determined from y i , the quadratic formula y=
aX 2 +bX+c or cubic formula y=aX 3 +bX 2 +cX+d
The coefficients a, b, c, d are calculated by least squares approximation by
Or, a method for creating a calibration curve for use in immunoreaction measurement, in which a, b, and c are determined and a calibration curve is created. 2. Calibration used for immune reaction measurement according to claim 1, in which the value of k in X i =log(1+x i /k) is the second concentration value from the lowest concentration value of the standard solution. How to create lines.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59010493A JPS60154161A (en) | 1984-01-24 | 1984-01-24 | Formation of calibration curve used for immunoreaction measurement |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59010493A JPS60154161A (en) | 1984-01-24 | 1984-01-24 | Formation of calibration curve used for immunoreaction measurement |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS60154161A JPS60154161A (en) | 1985-08-13 |
| JPH0568655B2 true JPH0568655B2 (en) | 1993-09-29 |
Family
ID=11751706
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP59010493A Granted JPS60154161A (en) | 1984-01-24 | 1984-01-24 | Formation of calibration curve used for immunoreaction measurement |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS60154161A (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0692969B2 (en) * | 1986-07-30 | 1994-11-16 | 株式会社シノテスト | Immunological measurement method |
| JP2795403B2 (en) * | 1986-07-30 | 1998-09-10 | 株式会社 シノテスト | Immunoassay method and device |
| DE3842580A1 (en) * | 1988-12-17 | 1990-06-21 | Behringwerke Ag | METHOD FOR IMPROVING THE ACCURACY AND REPRODUCIBILITY OF THE MEASURED DATA OF IMMUNOMETRIC TESTS |
| JP4648739B2 (en) * | 2005-03-29 | 2011-03-09 | 株式会社堀場製作所 | Analysis apparatus and calibration method |
| JP6104746B2 (en) * | 2013-07-23 | 2017-03-29 | 株式会社日立ハイテクノロジーズ | Automatic analyzer and analysis method |
| JP6309062B2 (en) * | 2016-09-09 | 2018-04-11 | 新コスモス電機株式会社 | Gas detector |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS518984A (en) * | 1974-06-10 | 1976-01-24 | Dokutooru Pee Kiraa Ag | |
| JPS55154438A (en) * | 1979-05-21 | 1980-12-02 | Hitachi Ltd | Method for absorptiometric analysis analyzing |
| JPS5638859B2 (en) * | 1974-03-23 | 1981-09-09 | ||
| JPS56151342A (en) * | 1980-04-25 | 1981-11-24 | Hitachi Ltd | Analyzer utilizing light |
| JPS58109837A (en) * | 1981-12-24 | 1983-06-30 | Olympus Optical Co Ltd | Compensating method of calibration curve |
| JPS58211663A (en) * | 1982-06-02 | 1983-12-09 | Hitachi Ltd | Automatic analyzer |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5920665Y2 (en) * | 1979-08-31 | 1984-06-15 | 株式会社島津製作所 | Atomic absorption spectrometer |
-
1984
- 1984-01-24 JP JP59010493A patent/JPS60154161A/en active Granted
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5638859B2 (en) * | 1974-03-23 | 1981-09-09 | ||
| JPS518984A (en) * | 1974-06-10 | 1976-01-24 | Dokutooru Pee Kiraa Ag | |
| JPS55154438A (en) * | 1979-05-21 | 1980-12-02 | Hitachi Ltd | Method for absorptiometric analysis analyzing |
| JPS56151342A (en) * | 1980-04-25 | 1981-11-24 | Hitachi Ltd | Analyzer utilizing light |
| JPS58109837A (en) * | 1981-12-24 | 1983-06-30 | Olympus Optical Co Ltd | Compensating method of calibration curve |
| JPS58211663A (en) * | 1982-06-02 | 1983-12-09 | Hitachi Ltd | Automatic analyzer |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS60154161A (en) | 1985-08-13 |
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