JPH0518969A - Calibration curve setting method for immune analysis method - Google Patents

Abstract

PURPOSE:To obtain the calibration curve of a dry analytical element adopting a homogeneous-system enzyme immune analysis method as a simple regression formula with a less number of concentration levels of a calibrator liquid while the accuracy of measured results is maintained at a high level. CONSTITUTION:An immune analysis method measures the content of a specific substance content in the body fluid of an organism by obtaining a detectable signal by utilizing an immunoreaction. This calibration curve setting method sets one of the coordinate axes of the calibration curve as the reciprocal of detectable signal values and the other coordinate axis as the antilogarithm of the concentration value of the specific substance.

Description

Detailed Description of the Invention

[0001]

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 by utilizing an immune reaction.

[0002]

2. Description of the Related Art Analysis of biological components, drugs, etc. contained in biological fluids such as blood and urine is very useful for diagnosing pathological conditions and determining the course of treatment, and plays an important role in the field of clinical examination. .. Enzyme-linked immunosorbent assay (EIA) is available as a method for analyzing trace components in biological fluids. Enzyme-linked immunosorbent assay requires B / F separation
Some homogeneous systems do not require / F separation. The homogeneous reaction system is based on the fact that when an antibody and an antigen bind to each other, the enzyme activity of the labeling enzyme undergoes some interference, and in general, the inhibitory action by the binding of the antigen and the antibody is utilized.

The calibration curve of the homogeneous enzyme immunoassay method is significantly different from that of the conventional clinical chemistry test, and generally does not become a straight line but becomes an S-shape (sigmoidal) in most cases. Regarding such a calibration curve, a radioimmunoassay method (RI
Sufficient consideration has been made so far regarding A). In the case of the enzyme immunoassay method, the method of setting the calibration curve of the radioimmunoassay method can be applied essentially, and the regression method is known as a logistic curve, a logit-log conversion formula, etc., Eiji Ishikawa et al. Edited by "Enzyme Immunoassay", 2nd edition (Medical Shoin, published in 1982 (pages 153-164)).

On the other hand, in a clinical test in which a large number of specimen samples are handled and routineized, it is desired that simple and quick analysis can be performed. From such a viewpoint, a dry analytical element to which a homogeneous enzyme immunoreaction is applied has been proposed (JP-A-1-321360). This is a multi-layer dry analytical element (eg,
JP-A-49-53888, 59-77356, 59-102388, and U.S. Pat. No. 4,459,358), in the same layer or in different layers, a water-insoluble polymer substrate, an enzyme for the substrate and an antibody for a specific substance Is included.

[0005]

DISCLOSURE OF THE INVENTION The relationship between the reflection optical density and the concentration of a substance to be measured obtained by a dry analytical element to which a homogeneous enzyme immunoassay method is applied is a logistic curve, a logit-log conversion equation or a right-angled hyperbola which has been used so far. It is possible to regress using the method. However, when such a regression equation is used, the number of concentration levels of the calibrator liquid required for setting increases because there are many regression coefficients to be set. Moreover, since the regression is complicated, a lot of time is required for calculation. This is a drawback of halving the characteristics of the dry analytical element, such as simplicity and speed.

[0006] In setting the calibration curve in the dry analytical element to which the homogeneous enzyme immunoassay method is applied, it is preferable that the number of concentration levels of the calibrator solution required is as small as possible, and the regression equation of the calibration curve is as simple as possible. This makes it possible to simplify and speed up the operation for setting the calibration curve. For that purpose, it is ideal that the calibration curve is a straight line, but the calibration curve of the homogeneous enzyme immunoassay method is generally not a straight line, and almost always becomes an S-shape (sigmoidal). However, depending on the substance to be measured, the concentration range you want to measure is S-shaped (sigmoidal).
There is a case where it does not cover the entire area of and fit in a part of S-shape. It was also found that the shape of the calibration curve greatly changes depending on how the coordinate axes are set, and surprisingly the calibration curve in the measurement concentration range can be represented by a straight line depending on how the coordinate axes are set. The fact that the calibration curve can be represented by a straight line in the measurement concentration range has an advantage that not only when the calibration curve is linearly regressed, but also when it is regressed by a polynomial polynomial, the regression can be performed more accurately. In addition, even when regression is performed by the conventionally known linear interpolation method for each point, there is an advantage that the discontinuity between the straight lines, which is a drawback of this method, can be reduced.

The present invention is a method for setting a calibration curve which provides a simple and highly accurate measurement result in an immunoassay method using a dry analytical element to which a homogeneous enzyme immunoreaction is applied (a new calibration curve is prepared). Method).

[0008]

The object of this kind is to set the coordinate axes of a calibration curve in an immunoassay method in which a detectable signal is obtained by utilizing an immune reaction to quantify a specific substance in a biological fluid. This can be achieved by a method of setting a calibration curve in the immunoassay method, characterized in that one is performed by the reciprocal of the detectable signal value and the other is performed by the true number of the concentration value of the specific substance.

The immunoassay method is a method of analyzing an antigen or an antibody by utilizing an antigen-antibody reaction, and includes an enzyme immunoassay method, a radioimmunoassay, a fluorescent immunoassay, a laser immunoassay, etc., depending on the detection means. In addition, it is divided into a competitive method and a non-competitive method depending on the reaction mode.
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 can be applied regardless of the type of immunoassay, but can be preferably applied to enzyme immunoassay,
Particularly, it is useful for an analytical method using a dry analytical element to which a homogeneous enzyme immunoassay method is applied.

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 analytical element to which the homogeneous enzyme immunoassay method is applied, It is necessary to obtain the relationship between the detectable signal obtained by the measurement and the concentration of the substance to be measured 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 the substance to be measured at several concentration levels is spotted on the dry analytical element. The spotted liquid undergoes an immunoreaction and a color reaction in the dry analysis element to give a coloring concentration according to the concentration of the substance to be measured in the liquid. In practice, after the spotting, the dry analytical element is kept at a constant temperature for a certain period of time (incubation) to allow the color development reaction to proceed, and then the dry analytical element is irradiated with irradiation light to measure the amount of reflected light in a specific wavelength range. Then, the reflection optical density corresponding to the concentration of the substance to be measured in the calibrator liquid is obtained. The relationship between the obtained reflection optical density and the concentration of the substance to be measured is obtained as a regression equation and used as a calibration curve for quantifying the substance to be measured.

Conventionally, the logarithm of the detected signal value and the concentration value of the analyte was used on the coordinate axis of the calibration curve, but in the method of the present invention, let's quantify it as the reciprocal of the detected signal value. The feature is that the true number of the concentration value of the specific substance is used as the coordinate axis. The detected signal value is determined according to each analysis system and the type of measuring equipment,
For example, when a color-developing system is used in the enzyme immunoassay method, it is an optical density, a reflection optical density, etc. (generally, a difference in density for eliminating the influence of background). The radioimmunoassay corresponds to radiation intensity, and the fluorescence immunoassay corresponds to fluorescence intensity.

The method of setting the calibration curve of the present invention will be described with reference to the model diagram (FIG. 1). The vertical axis is the logarithm of the concentration value of the specific substance (analyte, ie, analyte),
When the true value of the signal value (or the value obtained by subtracting the background value from the signal value) detected on the horizontal axis is taken as the immunoassay method,
In particular, in EIA, the calibration curve is generally represented by a sigmoidal curve like the model diagram of FIG. 1 and the solid line of FIG. 1B or 1C.

In the case of the sigmoidal calibration curve, it is as follows. 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 set on the calibration curve (two points marked with a circle), and a straight line passing through these two points. (Fig. 1
B, the two-dot chain line in FIG. 1C). Within the analyte quantitation range, this line only coincides with the sigmoid calibration curve at two or three points, and in most quantification ranges this line deviates significantly from the sigmoidal calibration curve. Therefore,
Obviously, this straight line cannot be used as a calibration curve.

In the calibration curve setting method of the present invention, for example, the vertical axis represents the logarithm of the analyte concentration and the horizontal axis represents the reciprocal of the detected signal value. When the two axes are taken in this way, the calibration curve becomes a curved calibration curve (solid line) with a gentle curvature close to a straight line, as shown in FIG. 1A. Two points corresponding to the upper limit value (H) and the lower limit value (I) of the analyte quantification range are set on the calibration curve (two points marked with a circle), and a straight line passing through these two points (two-dot chain line in FIG. 1A). )pull. Within the quantitation range of the analyte, this straight line lies near the curvilinear calibration curve of gentle curvature. Therefore,
When this straight line is used as a calibration curve, it is clear that the obtained concentration value of the analyte has a small error from the value estimated to be the true value.

As described above, since the calibration curve can be represented by a straight line in the measured concentration range of the analyte, there is a merit that the calibration curve can be more accurately regressed both when the calibration curve is linearly regressed and when it is regressed by a polynomial of order. .. Further, even when the regression is performed by the conventionally known linear interpolation method between the respective points, there is an advantage that the discontinuity between the respective straight lines, which is a drawback of this method, can be reduced or eliminated. In addition, calibration curve correction work to correct differences due to different lots of immunoassay reagents or due to different lots of dry analysis elements (difference between lots or batches) can be performed with a smaller number of calibrator solution concentrations. There is an advantage that it can be carried out (it can be carried out with the number of concentration levels being 1 or 2). Furthermore, there is also an advantage that the calibration curve itself for correcting the difference between lots can be implemented by extremely simple mathematical or numerical processing.

The method of calculating the concentration of the specific substance from the signal value obtained for each sample using this calibration curve may be the same as the conventional method.

The preferred embodiment of the present invention includes the following embodiments. The method for setting a calibration curve according to claim 1, wherein the immunoassay method is an enzyme immunoassay method. The method for setting a calibration curve according to claim 1, wherein the immunoassay method is an immunoassay method using a dry analysis element. The method for setting a calibration curve according to claim 1, wherein the enzyme immunoassay method is a homogeneous enzyme immunoassay method.

[0018]

Example Preparation of multi-layer dry slide for CRP analysis A colorless transparent polyethylene terephthalate (PET) sheet (support) having a thickness of 180 μm and provided with a gelatin subbing layer, and a cross-linking agent with the following coating amount. The containing reagent solution was applied and dried to form a reagent layer. Alkali-treated gelatin 14.5g / m ² Nonylphenoxypolyethoxyethanol 0.2g / m ² (oxyethylene unit average 9 to 10) Glucose oxidase 5000u / m ² Peroxidase 15000u / m ² Glucoa Mirrorza 5000u / 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 0.1g / m ²

An adhesive layer was applied on the reagent layer so as to have the following coating amount, and dried to provide. Alkali-treated gelatin 14.5g / m ² Bis [(vinylsulfonylmethylcarbonyl) amino] methane 0.1g / m ²

Then, the following reagent-containing aqueous solution was applied to the surface of the adhesive layer so that the coating amount was as follows, the gelatin layer was swollen, and a PET spun yarn equivalent to 50 denier was woven with 36 gauge of about 250 μm. The tricot knitted fabric was laminated by applying pressure uniformly and lightly to provide a porous spreading layer. Nonylphenoxypolyethoxyethanol 0.15 g / m ² (oxyethylene unit average 9 to 10) Bis [(vinylsulfonylmethylcarbonyl) amino] methane 0.4 g / m ²

Next, a substrate was applied so as to have the following coating amount and dried to form a substrate layer. Carboxymethylated starch 4g / m ² Nonylphenoxypolyethoxyethanol 0.2g / m ² (Oxyethylene unit average 9-10 included)

Furthermore, 3 mg / amylase-anti-CRP / IgG binding product was added to the tricot knitted fabric layer, which is also a substrate layer and a spreading layer.
ethanol solution was applied as a coating amount of m ^2,
After impregnation and drying, a multilayer dry analytical element for CRP analysis was obtained.

The obtained analytical element was cut into 15 mm square chips and placed in a slide frame described in JP-A-57-63452 to obtain a multilayer dry analytical slide for CRP analysis.

Preparation of calibration curve 10 kinds of CRP standard sera each of which has a known CRP concentration
200 μl of buffer solution (0.2MMES buffer, pH 6.0)
10 μl each of the CRP buffer solution diluted with the above was spotted on the tricot knitted fabric layer of the above analysis slide, each analysis slide was kept at 37 ° C. in a closed container, and reflected from the PET support side with visible light having a central wavelength of 650 nm. The concentration was measured. Difference in reflection optical density 4 minutes and 6 minutes after spotting (ΔOD
r _6-4 ) is shown in Table 1 together with known concentrations of CRP standard serum.

[0025]

[Table 1]

The CRP concentrations (C) and ΔODr _{6-4 in} Table 1 were plotted on the coordinate axes set in Table 2 respectively, and a straight line passing through the two plots was obtained to obtain a calibration curve.

[0027]

[Table 2]

Correlation with conventional method 50 samples whose CRP concentration is measured by the immunoturbidimetric method which is a conventional method for CRP measurement (CRP concentration is 0 to 10.2 mg).
10 μl each of 10 μl / dL) was diluted with 200 μl of buffer (0.2 M MES buffer, pH 6.0), and 10 μl of each solution was spotted on the tricot knitted fabric layer of the above analysis slide, and each analysis slide was placed in a closed container. Keep at ℃, center wavelength 65
The reflective optical density was measured from the PET support side with 0 nm visible light, and the difference (ΔODr _6-4 ) in the reflective optical density 4 minutes after the spotting and 6 minutes after the spotting was determined.

ΔODr obtained for each sample
_6-4 was converted into a CRP concentration using the calibration curve of the present invention and the calibration curves of Comparative Examples 1 and 2.

The correlation between the CRP concentration measured by the conventional immunoturbidimetric method and the CRP concentration obtained using each calibration curve in Table 2 was determined. Table 3 shows the correlation coefficient (r) and the slope (a).

2 and 3 show the correlation chart obtained by the calibration curve of the present invention and the correlation chart obtained by the calibration curve of Comparative Example 1, respectively.

[0032]

[Table 3]

As shown in Table 3 and FIG. 2, by using the coordinate axes of the present invention, it is clear that accurate CRP measurement can be performed even with a simple linear calibration curve.

[0034]

EFFECTS OF THE INVENTION By setting the coordinate axes of the calibration curve of the present invention, the calibration curve of the dry analytical element adapted to the homogeneous enzyme immunoassay method can be used to obtain a small number of concentration levels of the calibrator solution while sufficiently maintaining the accuracy of the measurement result. Can be obtained as a simple regression equation.

[Brief description of drawings]

FIG. 1A is a model diagram for explaining a method for setting a calibration curve of the present invention. 1B and 1C are model diagrams for explaining a conventional calibration curve in the immunoassay method. Solid line: calibration curve of the system Two-dot chain line: linear calibration curve set with two analyte solutions of known concentrations (analyte concentrations L and H)

FIG. 2 is a graph showing the correlation with the immunoturbidimetric method when using the calibration curve of the coordinate axes of the present invention.

FIG. 3 is a graph showing the correlation with the immunoturbidimetric method when the calibration curve of the coordinate axes of Comparative Example 1 is used.

Claims (1)

Claim: What is claimed is: 1. An immunoassay method for quantifying a specific substance in a biological fluid by obtaining a detectable signal by utilizing an immune reaction. A method for setting a calibration curve in an immunoassay method characterized by performing the reciprocal of the possible signal value and the other with the true value of the concentration value of the specific substance.