JPH06118000A - Clinically inspecting method - Google Patents

Abstract

PURPOSE:To convert measured data obtained by measuring a color difference into a numeric value and to impart objectivity to an inspecting decision by measuring one of colorimetric data selected from a group consisting of X, Y, X colorimetric systems, CIELAB colorimetric system and CIFLUV colorimetric system by a colorimeter, and quantitatively analyzing substance to be inspected. CONSTITUTION:Substance to be inspected is quantitatively analyzed by measuring at least one colorimetric data selected from a group consisting of X, Y, Z, x and y of X, Y, X colorimetric systems, L*, a*, B* and aEQAb* and L*, u*, v* and AFuv* of CIELUV colorimetric system by a colorimeter in the case of coloring of the substance to be inspected. An inspecting method includes a carrier agglomeration method, a sedimentation method, and an oxygen immunity measuring method, etc., and colorimetric data is desirably set with an allowable limit value for removing influence of coloring due to specimen. A color difference between two objects obtained by a predetermined formula satisfactorily coincides with a magnitude of a color difference between the two sensitive objects, and if the difference is a predetermined value or less, the two objects can be decided to be the same color.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a clinical examination method. More specifically, the present invention relates to a clinical examination method,
Colors derived from the substance to be inspected are measured by a colorimeter to measure X, Y, Z, x, and y in the XYZ color system, L *, a *, b *, and ΔE _ab * in the CIELAB color system, and C.
L *, u *, v * and ΔE _uv in the IELUV color system
* Clinical characterized by measuring at least one value of colorimetric data selected from the group consisting of (hereinafter these values may be collectively referred to as colorimetric data), and quantifying the substance to be tested. Regarding inspection method.

Specimens to be subjected to the clinical examination method of the present invention are urine, feces, serum, plasma, blood, and other fluids of biological origin, and the percentages in this specification are by weight unless otherwise specified. It is a value, and quantification means imparting objectivity to normal quantification in clinical tests and visual judgment.

[0003]

2. Description of the Related Art Most of clinical examination methods are radioimmunoassay (RIA), enzyme immunoassay (EIA), latex agglutination method, immunoprecipitation method based on chemical method or immunological method.
The immunochromatography method, the immunofilter method and the like are known. Most chemical methods consist of ions in the sample,
To chemically quantify sugars, lipids, proteins, enzymes, etc.
An enzyme or a color-developing reagent is added to a sample and reacted to measure the degree of coloration.

The measurement of the amount of urea nitrogen in serum (diacetyl monooxime method) is as follows. DA
20 ml and 34 of M-TSC reagent (6.0 g of diacetyl monooxime and 0.3 g of thiosemicarbazide dissolved in 1 l of water)
20% to 3.0 ml of mixed solution containing 100 ml of phosphoric acid aqueous solution.
Add μl of sample, standard solution or blank (water), soak in boiling water bath for 20 minutes, and then cool with running water for 2-3 minutes. The absorbance at 540 nm is measured using the blank as a control, and the amount of urea nitrogen in the sample is determined from the calibration curve obtained from the standard solution.

RIA is a method of labeling an antibody or an antigen with an isotope and quantifying a target substance in a sample specifically and highly sensitively by an antigen-antibody reaction. For example, adding a sample to a solid-phased antibody, reacting with an antigen in the sample to form a solid phase-antibody-antigen complex, reacting with ¹²⁵ I-labeled antibody, and measuring radioactivity after washing The antigen is quantified by. However, this method has the disadvantages that it requires special facilities and measuring equipment, and is complicated and requires skill.

EIA is almost the same in principle as RIA, except that an enzyme-labeled antibody or antigen is used instead of isotope labeling. The analysis result is expressed by the intensity of the color of the reaction system derived from the enzyme reaction,
Quantify by qualitative comparison with a color sample with the naked eye, or by absorbance or reflection intensity measured using a photometer such as a spectrophotometer. For example, in the same manner as the above RIA, solid phase-antibody-
An antigen complex is formed and alkaline phosphatase
After reacting with a ze-labeled antibody and after washing, a substrate, phenol-phthalein-monophosphate-dichlorohexyl-ammonium salt, is added, and the color tone of the reaction system derived from the enzyme reaction product is quantified using the absorbance as an index. Recently, methods such as immunodot blot method in which a change in color tone as a measurement result appears on a solid phase have been developed.

The carrier agglutination method is a method in which the agglutination of carrier particles sensitized with an antigen or antibody and carrier particles caused by an antigen-antibody reaction with a target substance is qualitatively determined by the naked eye, or turbidity, absorbance,
Alternatively, the scattered light intensity is measured or quantified. For example, by adding an antigen to the antibody-sensitized latex dispersion and rocking it on a slide plate to generate agglomeration of latex particles, the agglomerate gradually grows to be visually recognizable. When no antigen is present, no agglutination occurs and the reaction system remains a white dispersion. Either the presence or absence of generated agglutination is visually determined, or the degree or speed of agglutination is quantified by measuring turbidity, absorbance, and scattered light intensity. This method is a highly sensitive, specific, convenient, and rapid method because the reaction is amplified using carrier particles.

The immunochromatographic method is a qualitative method in which an antigen-antibody reaction and a chromatographic method utilizing a diffusion phenomenon are combined, and the analysis result is visually judged. For example, an antibody is immobilized on a part of an insoluble matrix such as a glass filter, while a monoclonal antibody-sensitized gold colloid is mixed with a sample, and the antigen in the sample is bound to the antibody to form a gold colloid-monoclonal antibody. -Form an antigen complex.

When this complex is dropped onto the antibody-free portion on the matrix, it migrates in the matrix due to the diffusion phenomenon and reaches the immobilized antibody portion, colloidal gold-monochrome-
A null antibody-antigen-antibody-matrix complex is formed and the color of gold colloid appears. If the sample did not contain an antigen, the complex was not formed and the monoclonal antibody-sensitized gold colloid passed through the immobilized antibody portion,
The color of gold colloid does not appear. Therefore, the determination is made by observing with the naked eye whether or not there is coloring by the gold colloid.

The immunofilter method is a qualitative method in which an antigen-antibody reaction and a filtration method such as a membrane filter are combined, and the analysis result is visually judged. For example, an antibody is immobilized on a part of a membrane filter such as a nitrocellulose membrane, a monoclonal antibody-sensitized gold colloid is mixed with a sample, the antibody captures the antigen in the sample, and the formed gold colloid- The monoclonal antibody-antigen complex is dropped on the antibody-immobilized filter, and the immobilized antibody is allowed to react with the complex to form a gold colloid-monoclonal antibody-antigen-antibody-filter-complex. Colloidal color appears. When the antigen does not exist in the sample, the complex is not formed and the monoclonal antibody-sensitized gold colloid passes through the filter, and the color tone of the gold colloid does not appear. Therefore, the determination is made by observing with the naked eye whether or not there is coloring by the gold colloid.

In the modified EIA methods such as the immunochromatography method, the immunofilter method and the immunodot blot method,
Since the reaction proceeds almost only at the solid-liquid interface and the reagents are stored in a dry state, it has the advantages that the operation is very simple, the reaction time is short, and the analysis results can be stored.

As described above, in the conventional clinical examination methods, the above-mentioned macroscopic judgment, absorbance measurement, scattered light measurement, turbidity measurement and the like are adopted, and recently, on an insoluble matrix such as a glass filter. Alternatively, a dedicated reagent for producing a colored analysis result on a test paper such as a filter paper (for example, a multilayer analysis material disclosed in JP-A-57-67860)
A system for quantifying and quantitating the degree of coloring by measuring the reflection intensity using a discontinuous single wavelength light matched to the color tone with a dedicated device [a so-called dry chemistry system. For example, the Fuji Dry Chem System (manufactured by Fuji Photo Film Co., Ltd.) and the like have been developed and are commercially available.

[0013]

Most of the above methods for qualitative judgment with the naked eye have a more or less quantitative property with respect to the degree of reaction. For example, in the immunofilter method using the gold colloid, when the amount of the antigen reacting with the antibody on the filter is small, the amount of the gold colloid-monoclonal antibody bound thereto is small and the coloring becomes thin, On the contrary, when the amount of antigen is large, the coloring becomes dark. As described above, since the tint of the color continuously changes depending on the amount of the antigen, it is extremely difficult to make a clear negative or positive judgment visually with the cutoff value as a boundary. That is, in the determination using the presence or absence of coloration and the presence or absence of aggregation as an index, if the test substance is present in an amount near the cutoff value in the sample, it is difficult to determine negative or positive. It is carried out by subjectivity based on the experience of the person.

However, there is a possibility that a true determination may not be made due to factors such as the skill of the inspector, individual differences, physical condition, and illumination conditions at the time of determination. The same thing occurs in a method in which the test substance is semi-quantified, although it is determined by the naked eye. The purpose of laboratory tests is to diagnose diseases,
It is the judgment of the degree of the disease and the judgment of the therapeutic effect, and in order to accurately achieve this purpose, objective test data are necessary above all.

On the other hand, in the quantitative measurement, the analysis result is digitized by measuring the transmitted light intensity or the reflected light intensity using a discontinuous single wavelength. The subject used for measurement generally has its own color tone. That is, it is yellow if the sample is urine, and red if it is serum or plasma.
When photometry is performed using such a specimen, it is necessary to select a wavelength that does not affect the measurement and that can measure only the coloration due to the presence of the target substance. However, there are individual differences in the color tone of these samples, and the sample sample of a patient may differ from the sample sample of a healthy subject. Therefore, the photometric amount derived from the color tone of the patient's sample may affect the true quantitative value, leading to an incorrect quantitative value.

In the prior art, in order to eliminate such an influence, a control system which has no coloration due to the presence of the target substance and can measure only the color tone of the sample is measured at the same time, and a blank value is mathematically determined. Correct by processing
Either the photometry with two wavelengths is performed to make the correction.

As another solution, an automatic analyzer is developed and put on the market, which is capable of performing both corrections by providing both a blank portion and a measurement portion on the test piece and automatically performing from the operation to the display of the quantitative result. ing. However, these devices are unique systems that must be used in combination with a dedicated reagent, and therefore cannot be measured unless a reagent corresponding to the required test item is commercially available. Further, since the system is in a black box, it is often impossible to trace whether the cause is the reaction system or the photometric system, even if the measurement result is questionable. Furthermore, the equipment is very expensive, so it is difficult to be general purpose.

As described above, various measures have been conventionally made to remove the influence of the color tone of the sample, but at the present time, the measured value is still measured in samples such as hemolyzed urine, strongly colored urine and abnormally colored urine. In fact, it is said that the effect of hemolysis in the vicinity of 547 nm cannot be avoided (test and technology, Vol. 18, p. 1451, 1990, etc.).

In view of the situation in the field of clinical examinations, the present inventors can add objectivity to the determination of clinical examinations and reliably remove the influence of the color tone caused by the sample. As a result of intensive research aimed at developing a new clinical test method, the principle of color difference measurement, which can quantify the difference in color between two samples, can be applied as a clinical test method. The inventors have found that the object can be achieved and have completed the present invention.

That is, the present invention has been made in view of the above situation, and imparts objectivity to the determination of the clinical test based on the subjectivity of the tester, and the object of the analysis result is determined. It is an object of the present invention to provide a clinical test method that removes the influence of the color tone of (3) and can be easily performed by a general-purpose colorimeter without using a dedicated device and reagent.

[0021]

The method of the present invention for achieving the above-mentioned object is a clinical test method, wherein the color development derived from the substance to be tested is measured by X in a XYZ color system by a colorimeter.
Y, Z, x, and y, L in the CIELAB color system
*, A *, b * and ΔE _ab * and at least one colorimetric data selected from the group consisting of L *, u *, v * and ΔE _uv * in the CIE LUV color system, and a substance to be inspected The present invention relates to a clinical test method, which is characterized by quantifying

Further, in the present invention, the clinical examination method is a carrier agglutination method, a sedimentation method, an enzyme immunoassay method, an immunochromatography method, or an immunofilter method, and the colorimetric data originates from the sample. It is a desirable mode that an allowable limit value is set in order to remove the influence of coloring.

Before describing the method of the present invention in detail, colorimetric data will be described. The color of an object that humans perceive with the naked eye can be quantified by the spectral characteristics of a light source used for illumination, the reflection characteristics of the object with respect to light, and the sensitivity characteristics of the eyes with respect to light. The International Commission on Illumination (CIE) has standardized the method of displaying colors in this way. According to this method, the color is calculated for the wavelength (λ) range 380 nm to 780 nm (industrial 400 nm to 700 nm) by the following equations (1) to (3). Displayed by value.

[0024]

[Equation 1]

[0025]

[Equation 2]

[0026]

[Equation 3]

Generally, in the conventional method of clinical examination, a target substance is made to develop a color and its absorbance, transmitted light,
The method of measuring reflected light etc. is mainly adopted, mainly using a colorimeter used in the fields of paint, printing, etc., an example of applying the principle of color difference measurement to the method of clinical examination is,
At least we haven't seen it in the field of clinical testing so far,
It was first proposed by the present inventors.

Here, S (λ) is the spectral distribution of standard light used for illumination, and R (λ) is the spectral reflection of the object.

[0029]

[Outer 1]

It represents a constant given by λ. Formulas (1)-(3)
Is a representation of the color of an object under standard light, when the observer sees it by replacing it with three numerical values.
Therefore, in the two colors of the object to be compared, if the tristimulus values of the two objects are equal under the same illumination, equal colors are established. However, if there is a difference in the tristimulus values of both objects, the same color difference is not perceived even if the distance in the tristimulus value space is constant and the regions in which the colors exist differ.
Therefore, in CIE, based on the idea that the color coordinate system is coordinate-converted into another color system so that the color difference is perceived to be almost constant in any region where the distance between two points is C,
The IELAB and CIELUV color systems are adopted and recommended.

According to Japanese Industrial Standard, Z8729-1
980 (display method of object color by L * a * b * color system and L * U * V * color system) and Z8730-1980 (color difference display method). When these two color systems are used to compare two colors, the difference between the two colors perceived by humans is the same if the distance between the two points is the same no matter where they are located in the color space. Are almost equal. The CIELAB color system is often used in normal color management.

Next, the CIELAB color system and the color difference will be described. From the following equations (4) to (6), L *, a * and b
Calculate *

[0033]

[Equation 4]

[0034]

[Equation 5]

[0035]

[Equation 6]

However, X / Xn, Y / Yn, Z / Zn
The above applies when both are greater than 0.008856.
Xn, Yn and Zn are tristimulus values of a perfect diffusion surface under the same illumination as the object color having the tristimulus values X, Y and Z,
It is normally standardized to Yn = 100. The meaning of each parameter is that the larger the value of a *, the stronger the degree of red, and the smaller the value, the stronger the degree of green. The larger b * is, the more yellow it is, and the smaller it is, the more blue it is. L * represents lightness, and the larger the value, the brighter. Next, tristimulus values X1 and Y1
, Z1 and the color difference between two objects having X2, Y2, Z2 is calculated by the following method. From formulas (4) to (6), L ₁
*, A ₁ *, b ₁ * and L ₂ *, a ₂ *, b ₂ * are calculated, and the color difference is obtained from the following equation (7).

[0037]

[Equation 7]

Here, L ₂ * -L ₁ * is ΔL *, a ₂ *
If -a ₁ * is Δa * and b ₂ * -b ₁ * is Δb *,
It is expressed as the following equation (8).

[0039]

[Equation 8]

The value of the color difference thus obtained agrees well with the magnitude of the perceptual color difference between the two objects. Therefore, if this color difference is less than a certain value, it can be determined that the two objects have the same color.

The colorimetric data in the method of the present invention are X, Y, Z, x, and y, CIE in the XYZ color system.
L *, a *, b * and ΔE _ab * in the LAB color system,
And at least one value of colorimetric data selected from the group consisting of L *, u *, v * and ΔE _uv * in the CIE LUV color system.

Next, the method of the present invention will be described in detail. Color development of the substance to be examined in the method of the present invention is carried out by a known chemical or immunological clinical examination method, preferably an immunological clinical examination method. The colorimetric data of color development derived from the substance to be inspected is measured by an ordinary method using a commercially available ordinary colorimeter and digitized to give objectivity to the visual judgment and quantitativeness.

Next, the method of the present invention will be specifically described by taking an immunofilter method using gold colloid as a carrier (hereinafter referred to as a gold colloid filter method) as an example.
The analysis result of this method is expressed as a reddish purple coloring on the filter according to the amount of the target substance in the sample, and the determination is made by observing the degree of this coloring with the naked eye.

In the method of the present invention, the intensity of the reddish purple coloring is quantified by colorimetric data using an ordinary colorimeter such as a colorimeter. In this case, it is convenient to use ΔE _ab * as the colorimetric data. Of course, since the intensity of reddish purple is measured, ΔL *, Δa *, Δb *, (Δa * ²
It is also possible to use + Δb * ² ) ^1/2 or the like as colorimetric data, and a parameter that best suits the conditions of the analysis system can be used as appropriate.

However, when the saturation or lightness of the color tone of the analysis result is low, or in the case of a reagent or measurement system in which the hue differs between the measured samples, it is preferable to use ΔE _ab * because there is less error. As a control, L *, a *, and b * of the filter operated by the sample that is known not to contain the target substance are measured and set as reference values, and ΔE _ab * between this and the sample is set. To measure. Since the shade of macroscopic red-purple coloration depends on the amount of the target substance in the sample, ΔE _ab * with the control also corresponds to this.

In the method of the present invention, when the naked eye judgment results vary depending on the examiner, the true judgment result obtained is statistically processed from the naked eye judgment results and quantitative values by a large number of examiners. , ΔE _ab *, a cutoff value can be set, and this can be matched with the macroscopic coloring density to add objectivity to the macroscopic judgment, and the judgment among the testers can be unified. Furthermore, a calibration curve is created in advance using ΔE _ab * for coloring using a sample in which the amount of the target substance is known,
Quantitative measurement is possible by measuring ΔE _ab * of a sample containing an unknown amount and reading the amount of the target substance from the calibration curve.

The qualitative clinical test method applied here is not limited to the immunofilter method, and the colorimetric data is not limited to the CIELAB colorimetric parameters. Absent.

Generally, in a quantitative measurement system based on the use of a dedicated reagent and the measurement of the degree of coloration by a dedicated device to quantify it, an expensive device and a corresponding reagent are required. Therefore, it is difficult to introduce these systems in a laboratory where the number of measurement items is small. Further, even if only a reagent is obtained, and a reaction is performed using a sample to cause coloration, the reagent form is unique and often not compatible with the photometric portion of a general-purpose reflection intensity meter.

In the method of the present invention, when a quantification is carried out using such a reagent, the degree of coloration can be quantified by colorimetric data measured using a colorimeter.
As the colorimetric data, the most suitable parameters can be used according to the hue of coloration and other conditions. For example,
When measurement is performed using a commercially available reagent for dry chemistry system, a reaction is performed using a blank, a standard solution and a sample to cause coloration, and these are subjected to color measurement. If color difference is used as the colorimetric data, using the blank as a reference value, ΔE _ab * with the standard solution or the sample can be obtained, and the concentration of the sample can be read from the calibration curve prepared in advance from ΔE _qb * of the standard solution. it can.

In the conventional clinical test method based on photometry of the degree of coloration by the reaction, the color tone of the sample often influences the measurement result, as exemplified above. In this method, this effect can be reliably eliminated. For example, in general, in the measurement of urine by the immunofilter method, the yellow color tone of urine appears on the filter, even though the objective substance is not present in the sample and no reddish purple coloring is observed with the naked eye, and ΔE
_Ab * may show a high value due to the influence of yellow. Here, it is easily expected that the color tone that appears due to the reaction is reddish purple, Δa * is plus, and Δb * is minus. Further, when the color tone of the sample itself is yellow and no reddish purple coloration does not occur due to no antigen-antibody reaction, only the color tone of urine is colored on the filter, so Δa * is a low value near zero, It is expected that Δb * will show a positive value before the measurement.

Therefore, the allowable limit values are set in advance for Δa * and Δb *, and ΔE only for coloring caused by the target substance in urine is set.
_{If ab} * is measured and digitized, ΔE due to the influence of urine color tone
_{Higher ab} * can be excluded. That is, in this case, the permissible limit values are set to Δa * = 0 and Δb * = 0, and Δa *
ΔE _ab only for samples showing ≧ 0 or (and) Δb * ≦ 0
It would be good to measure *. A sample showing Δa * <0 and / or Δb *> 0 can be regarded as having no coloration due to the antigen-antibody reaction and only having coloration due to the color tone of urine. It is expressed as a low level below the sensitivity.

On the other hand, when the coloration of the reagent is limited to pure red, blue, yellow, green hues or achromatic colors such as black, white and gray, and the color tone of the sample is clearly different from the coloration of the reagent Can also be digitized using only the colorimetric parameters corresponding to each hue.

Conventionally, color difference measurement by a color difference meter has not been performed at all in the field of clinical examination, and no research results have been found that can be expected to be applicable to the field of clinical examination. Under the circumstances, the present inventors apply the principle of the color difference measurement, digitize the measurement data, and set an allowable limit value unique to the sample, thereby measuring only the color development caused by the sample. However, we succeeded in establishing a new clinical test method that made it possible to reliably remove the effect of the color tone of the sample on the measurement results, and such findings are those related to conventional normal clinical tests, and It cannot be expected from the general concept of colorimetric data.

[0054]

EXAMPLES Next, the method of the present invention will be described in more detail by way of examples, but the present invention is not limited to the following examples. The standard light used for the following colorimetry is D65 / 2. Example 1: Example of measurement of human luteinizing hormone in urine (hereinafter referred to as hLH) (1) Reagent A commercially available urinary hLH detection reagent (manufactured by Nippon Gene) by an immunofilter method using gold colloid as a carrier was used. .
According to the package insert of this reagent, the judgment is that if a circular reddish purple color with a diameter of 4 mm is visually observed on the filter after the reaction, 20 IU / l or more hLH is present in the sample. If not, it is supposed to be judged as less than 20 IU / l. In addition, the test method is the "conventional technology" described above.
It is the same as the description of the section.

As a control method, anti-microplate was used.
An ELISA method was used in which hLH monoclonal antibody was coated, the sample was reacted, and then enzyme-labeled anti-hLH antibody was reacted to quantify hLH in the sample from the enzyme activity. (2) Sample hLH (manufactured by UCB Bioproducts) was added to normal male pool urine at a ratio of 0, 20, 50, 75, or 100 IU / l to prepare a standard solution. As samples of unknown concentration, 11 urine samples from infertility outpatients were used.

(3) Color difference measurement Using a color difference meter (manufactured by Nippon Denshoku Industries Co., Ltd., Σ-80 type), 0 IU /
Using a filter operated with the standard solution (1) as a reference value, the color difference between this and a portion where reddish purple coloration was observed was measured. Since the coloring of this reagent is reddish purple, and it is clear that Δa * becomes positive and Δb * becomes negative in the CIELAB color system, the following allowable limit values were set for Δa * and Δb *. . That is, Δa * ≧ 0 or Δb *
ΔE _ab * was calculated only in the case of ≦ 0, and in other cases (that is, in the case of Δa * <0 and Δb *> 0), no numerical value was calculated and the result was negative.

(4) Results The colorimetric data are shown in Table 1. With the naked eye, "+" indicates that reddish purple coloring was observed, "++" indicates strong coloring, and "-" did not. Sample numbers 10 and 11 are below the tolerance limit,
It was negative. In particular, Sample No. 10 exhibited a clear yellow color with the naked eye, and no reddish purple coloration was observed. As a result, if ΔE _ab * is set to a cutoff value between 1.40 and 1.49, the influence of the color tone of the sample can be reliably removed, which is consistent with the naked eye judgment, and the objectivity is given to the naked eye judgment. Turned out to get.

FIG. 1 shows ΔE measured using the hLH standard solution.
The relationship between the _ab * and Δa * values and the hLH concentration is shown. In the figure, the vertical axis and the horizontal axis represent ΔE _ab *, Δa *, and hLH concentration, respectively. ○ and ● in the figure are the values of ΔE _ab and hLH, respectively.
The relationship with the concentration and the relationship between the Δa * value and the hLH concentration are shown. As is clear from Fig. 1, these correlations are very good and have excellent linearity (regression line y = 0.0, respectively).
661x + 0.508, r = 0.990 and regression line y = 0.0336x + 0.0573, r =
A calibration curve was obtained, which was in agreement with the naked eye.

Table 2 shows the results of measuring the hLH concentration in the sample using this calibration curve. As is clear from Table 2, the concentration of hLH obtained from ΔE _ab * and Δa * and the concentration obtained from the control ELISA method were in very good agreement (regression line y = 1.05x-0.481, r = 0.853 and Regression line y
= 1.07x + 5.23, r = 0.797), but it was difficult to judge by the naked eye at a concentration near 20 IU / l.

As described above, according to the method of the present invention,
By measuring the coloration obtained as a result of the analytical reaction with the hLH qualitative reagent, setting the allowable limit value, quantifying it, and quantifying it, objectivity is given to the judgment with the naked eye, and it can also be used for quantitative applications. It turned out to be. Further, the influence of the color tone of the sample on the quantitative value could be reliably removed by setting the allowable limit value.

[0061]

[Table 1]

[0062]

[Table 2]

Example 2: Example of measurement of human chorionic gonadotropin in urine (hereinafter referred to as hCG) (1) Reagent Commercially available urinary hCG detection reagent based on the immunofilter method using gold colloid as a carrier (Manufactured by Nippon Gene Co., Ltd.) was used. With this reagent, if the urinary hCG concentration is 25 IU / l or more,
It is positive as judged by the naked eye.

(2) Specimens hCG (manufactured by UCB Bioproducts Co., Ltd.) was applied to two types of normal male pool urine at 0, 25, 50, 100, 250.
, 500, 1000, or 2000 IU / l, respectively.

(3) Color difference measurement Using a color difference meter (CR-321 manufactured by Minolta), 0 IU / l
The values of ΔE _ab * and Δa * were measured using the sample of No. 2 as a reference value.

(4) Results The results are shown in Table 3, and the sample 1 showed stronger coloring than the sample 2 as a whole. By setting the cutoff value near ΔE _ab * = 1, it was in good agreement with the naked eye determination, objectivity could be imparted to the naked eye determination, and the quantification was also good.

[0067]

[Table 3]

Example 3: Measurement example of human chorionic gonadotropin in urine (hereinafter referred to as hCG) (1) Reagent A commercially available urinary hCG detection reagent based on the immunochromatographic method using gold colloid as a carrier ( Boehringer Mannheim) was used. With this reagent, it is determined that the urinary hCG concentration is 150 IU / l or more when the red-purple line is visually confirmed on the chromatographic matrix, and is less than that when it is not confirmed. .

(2) Specimens hCG (manufactured by UCB Bioproducts, Inc.) was added to the normal male pool urine at 0, 500, 750, 1000, 300000 or
It was added at a rate of 500,000 IU / l.

(3) Color difference measurement: 0 IU / l using a color difference meter (Σ-80 type manufactured by Nippon Denshoku Industries Co., Ltd.)
The color difference between the matrix operated with the sample liquid of Example 1 and the portion where linear coloring was observed was measured by the same method as in Example 1.

(4) Results The results are shown in Table 4, and the measurement range of this reagent was 15
0 IU / l or more, but 500 I when measured by the method of the present invention
The color density was already saturated at U / l. Also 3000
When it was more than 00IU / l, the phenomenon of prozone occurred and the coloring became thin. The colorimetric data was also in good agreement with the naked eye judgment, and it was possible to add objectivity to the naked eye judgment.

[0072]

[Table 4]

Example 4 Measurement Example of Urea Nitrogen in Serum (hereinafter referred to as BUN) (1) Reagent A commercially available reagent (manufactured by Konica) exclusively for the dry chemistry system was used. The principle of measurement with this reagent is that urea in serum is reacted with orthophthalaldehyde and the carbonium ion of the reaction product is reacted with N-1-naphthyl-N'-diethylethylenediamine under strong acidity to give a deep green color. Colored. All the reagents involved in these reactions were present on the solid phase in a dry state, and the reaction was completed by dropping 10 μl of the sample and incubating at 37 ° C. for 7 minutes. The degree of coloration was quantified and quantified by measuring the reflection intensity at a wavelength of 650 nm using a dedicated device.

(2) Specimens 9 healthy human sera were used, and purified water was used as a blank.

(3) Color difference measurement The reaction was carried out according to the operation method of this reagent, and the color reaction part was subjected to the same reaction using a blank as a sample using a color difference meter (CR-321 type manufactured by Minolta Co., Ltd.). ΔE as a reference value
_Ab * was measured. As a control method, the measurement was performed using a dry chemistry system (manufactured by Konica) exclusively for this reagent.

(4) Results The results are shown in Table 5. The quantitative value of BUN measured using a dedicated device and Δ measured by the method of the present invention.
Very good agreement with E _ab * (y = 1.18x + 20.5, r = 0.98
Four). Therefore, by using the color difference meter, BUN could be easily measured without using an expensive dedicated device.

[0077]

[Table 5]

Example 5: Example of measurement of antibody titer of rabbit anti-bovine serum albumin (hereinafter referred to as rabbit anti-BSA) (1) Immunodot blotting method Bovine serum albumin (BSA) was added to a phosphate buffer (PBS) of pH8. )
Was dissolved at a concentration of 5 μg / ml, and 50 μl of this solution was placed on a nitrocellulose membrane in 12 places in a circular shape with a diameter of 4 mm, and
Incubated at ℃ for 2 hours. After washing with PBS, add 100 μl of 3% gelatin and incubate again at 37 ° C for 2 hours.
I got it. After washing with PBS, add rabbit anti-BSA antiserum to PB.
Diluted 10 × 2 ² times S, then further diluted to 10 × 2 ^13, was added to each 50μl to each processing part. Incubate at 37 ℃ for 30 minutes, wash with PBS containing 0.05% Tween20 (Tween-PBS), add 50 μl of horseradish peroxidase-labeled goat anti-rabbit IgG antibody diluted in PBS, and incubate at 37 ℃. so
Incubated for 30 minutes. After washing with Tween-PBS, the substrate solution (4-chloro-1-naphthol was dissolved in methanol,
Then, the whole nitrocellulose membrane was dipped in (added hydrogen peroxide) and left for about 30 minutes. A brown spot, which was visually dependent on the concentration of rabbit anti-BSA, appeared.

(2) Color difference measurement 10 × by using a color difference meter (manufactured by Nippon Denshoku Industries Co., Ltd., Σ-80 type)
The color difference between the portion operated with the 2 ^13- fold diluted solution and the portion where circular coloring was observed was measured by the same method as in Example 1.

(3) Results The results are shown in Table 6, and all the colorimetric data were in good agreement with the naked eye judgment. Further, the numerical difference between the positive judgment and the negative judgment was large, and the cutoff could be easily performed, and the naked eye judgment could be provided with objectivity. Further, any of the colorimetric data had good correlation with the antiserum dilution ratio, and it was possible to impart quantification.

[0081]

[Table 6]

Example 6: Estorol measurement example (1) Reagent A commercially available urinary estriol measuring reagent (manufactured by Morinaga Milk Industry Co., Ltd.) based on the latex aggregation inhibition method was used. The sample and the antibody test solution were mixed on a slide plate, and the latex test solution was added dropwise thereto, and the sample was shaken for 2 minutes and visually judged. 50 times, 100 times,
If a 200-fold diluted sample is used and aggregation inhibition is observed in the 200-fold diluted sample, the urinary estriol is 20 μg /
If agglutination inhibition is observed in a 100-fold or more diluted sample of 100 ml or more,
10 to 20 μg / ml, 5 to 10 μg / ml for 50-fold diluted sample, 5 μg / ml if inhibition was not observed in any diluted sample
It is a semi-quantitative reagent that is determined to be the following. Therefore, the cut-off value is 0.10 μg / ml in urinary estriol concentration.
The obtained analytical results were visually judged by two judges.

(2) Specimens Estoril was added to the normal male pool urine to adjust the concentration to 0.
It was adjusted to 08, 0.09 and 0.12 μg / ml.

(3) Color difference measurement The reaction with this reagent was carried out on a transparent glass plate, and using a color difference meter (Nippon Denshoku Industries Co., Ltd., Σ-80 type), 0.12 μg / ml sample and 0.08 and 0.09 were used. The ΔE _ab * of μg / ml was measured.

(4) Results The results are shown in Table 7, and ΔE _ab * was quantified. The visual determination results of 0.09 μg / ml were different between the judge 1 and the judge 2. Due to the performance of this reagent, 0.
Since the sample of 09 μg / ml is negative, if the cutoff value is set between ΔE _ab * of 0 and 1.70 in the judgment by the colorimetric data, objectivity is given to the judgment by the naked eye, and the We were able to give unity to the judgment. In Table 7, when aggregation inhibition was observed, it was indicated as +, and when it was not observed, it was indicated as-.

[0086]

[Table 7]

Example 7: Example of measurement of rabbit anti-trypsin inhibitor antibody titer (1) Hemagglutination reaction Rabbit anti-human trypsin inhibitor-antiserum was added to PBS (pH 7.
100 μl of the solution diluted 1000 times in 5) was added to a U-bottomed microtitration plate and diluted every two times with PBS to prepare a dilution series up to 1000 × 2 ¹¹ times. A suspension containing human trypsin inhibitor-sensitized erythrocytes at a concentration of 1% in veronal buffer (pH 7.5) containing 0.1% gelatin 50
μl was added to each well containing each diluted solution, mixed by pipetting and allowed to stand for 1 hour. Red blood cells agglutinated in the well where the antigen-antibody reaction occurred, and the red blood cells could be visually confirmed as a spread of a red color on the entire bottom surface of the well. It settled in the form of dots.

(2) Color difference measurement Δa * using a color difference meter (CR-321 manufactured by Minolta Co., Ltd.)
Was measured. A * of sedimentation image of antiserum 1000 × 2 ¹¹ times dilution
Was used as a reference value.

(3) Results The results are shown in Table 8, which is in good agreement with the visual judgment of the red color depth at the bottom of the wells. In Table 8, + means that a light red diffusion was observed on the entire bottom surface of the well, and-means that a deep red dot-like sedimentation was observed at the bottom of the well.

[0090]

[Table 8]

[0091]

INDUSTRIAL APPLICABILITY The present invention has applied a principle of color difference measurement to a clinical test method for the first time, and established a new clinical test method characterized by digitizing and quantifying measurement data by color difference measurement, The effects of the present invention are as follows.

(1) Objectivity can be added to a qualitative clinical test based on the naked eye. (2) Quantitativeness can be added to a qualitative clinical test based on the naked eye.

(3) In the quantitative clinical test method, the influence of the color tone of the sample, which is often seen in the measurement results, can be completely eliminated. (4) Without being limited to dedicated equipment and dedicated reagents,
It can be easily measured using a general-purpose reagent by using a general-purpose reagent.

[Brief description of drawings]

FIG. 1 shows ΔE _ab * measured using hLH standard solution.
And the relationship between Δa * value and hLH concentration.

Claims (3)

[Claims] 1. In a clinical examination method, the color development derived from a substance to be examined is measured by a colorimeter such that X, Y, Z, x, and y in the XYZ color system, L *, a * in the CIELAB color system, b * and ΔE _ab *, and at least one value of colorimetric data selected from the group consisting of L *, u *, v * and ΔE _uv * in the CIE LUV color system are measured to quantify the substance to be inspected. A clinical examination method characterized by the following. 2. The clinical examination method is a carrier agglutination method, a sedimentation method,
The clinical test method according to claim 1, which is an enzyme immunoassay method, an immunochromatographic method, or an immunofilter method. 3. The clinical test method according to claim 1, wherein the colorimetric data has an allowable limit value set in order to remove the influence of coloring caused by the sample.