JPS6232344A - Method for measuring enzyme activity using reflection optical system - Google Patents

Abstract

PURPOSE:To measure an enzyme activity value with high accuracy in a method for measuring enzyme activity using a dry type analyzing element by determining the relation between a reflection optical density value and dye concn. value and making the correlation equation between the fluctuation of the dye concn. with time and the enzyme activity value. CONSTITUTION:The reflection optical density (ODR) of the dry type analyzing element is measured with standard solns. having different concns. [P] of dyes or chemical species which can give the dyes, then the correlation equation [P]=f(ODR) is determined. The reflection optical density is then measured at spaced time intervals at the prescribed temp. in an incubator and the concn. fluctuation DELTA[P] of the dye per unit time is determined. The reflection optical density is measured at spaced time intervals by using the standard soln. of which the enzyme activity is measured by a reference method. The correlation equation [E]=f(DELTA[P]) between the enzyme activity value [E] and the concn. fluctuation DELTA[P] of the dye is made. The reflection optical density of the sample of the enzyme in a vital body is measured by the dry type analyzing element and is substituted into the above-mentioned correlation equation; therefore, the enzyme activity is measured with the high accuracy.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of the Invention] The present invention relates to a method for measuring enzyme activity using a reflective optical system. More particularly, the present invention relates to a method for measuring enzyme activity in a liquid sample using a dry analytical element using a reflective optical system.

[Background of the Invention] In clinical testing, it is extremely important to measure enzyme activity in liquid samples such as human serum. In other words, enzyme activity values in body fluids are important knowledge when estimating the diseased area and the degree of damage to the affected area. Therefore, there is a strong demand for quick measurement with simple operation.

To answer this question, a dry analysis method based on a reflective optical system has been developed in place of the conventional solution method based on a transmission optical system, and its practical application has been progressing in recent years. . The above dry analysis method uses a dry analysis element in which the reagents necessary for the detection reaction are stored in a dry state inside the element of a sheet ball, and the liquid sample spotted on this dry analysis element and the above reagents are It is used to detect and measure reactions with other substances. Particularly, among the above-mentioned dry analytical elements, various types of integrated multilayer analytical elements ', L, which are characterized by multilayer coating that further improves ease of handling and analytical accuracy, are already commercially available.

When using a dry analytical element such as an integrated multilayer analytical element to measure the concentration of an analyte in a liquid sample, reflected optical density is measured directly using a collimated light source using the conventional end-point colorimetric method. If a calibration curve was created that correlated the dye concentration (hereinafter referred to as ODR) and the dye concentration, no problems would occur in normal measurements.

However, in order to measure enzyme activity using a dry analytical element, a rate method analysis method using multi-point anti-Q4 photometry is essential. For this purpose, first of all, it is necessary to convert the ODR to a transmitted optical density (hereinafter abbreviated as OUT) that achieves linearity, and research on this has been carried out for a long time. In theory, Williams (
It has become clear that ODR can be converted to On+ using the conversion formula proposed by William William S) and Clapper (Journal Fist of Force Society of America [J, 0ptical 5ocie
ty of As+erial Vol 43No.
7 595-599 (1953)), but in a practical system, the above conversion formula has various parameters that are complicated, so the value obtained by empirically converting ODR to OD+ (ODD')
is used. One of the conversion formulas is related to integrated multilayer analytical elements, H, G, Curse 'f- is clinical chemistry (C1inical Ghe+5istry).
) Vat 24 No. 81335-42 (1978)
The test paper type dry analysis element usually uses an integrating sphere that uses scattered light as a light source, and uses the Kubelker-Munk
The measured value of the reflected light is processed based on the equation of Zeitung Technologie and Physik [2, TI!ch, Physikl 125'93].
(1931)), bipedal photometric systems are generally used within a narrow optical density range that satisfies Beer's law.
, 7 (Masaya Suehiro; J J CL A Vo18
No. 4 (1983) 358-363).

To measure fermentation activity using a dry analytical element, a rate analysis method using multi-point reflection photometry is essential, as described above.

Therefore, in enzymatic activity measurement, in addition to the conversion to the above-mentioned OD+'', a conversion formula to an enzyme activity value based on the enzyme reaction rate method is required.

However, OD+' obtained using Cuba's formula has meaning as a physical quantity! ambiguous, and-・generally OD+'
The conversion to 2.0 is dependent on the parameters specific to the measurement system.

Therefore, even if a conversion formula to an enzyme activity value is created using OD-, the reliability of the conversion formula is low and it is difficult to make it applicable to all systems.

[Summary of the Invention] The name of the present invention is to introduce a completely separate concept to replace the value of ODD' in Cuba's equation as a conversion method to an enzyme activity value based on the enzyme reaction rate method, and to obtain an enzyme activity value. We found that it is effective and reached the J.

The purpose of the present invention is to convert ODR into a conventional ODD'
Instead of the value of , it should be made to correspond to a numerical value supported by concrete experimental values.

Furthermore, it is an object of the present invention to measure enzyme activity in a liquid sample using a dry analytical element using a reflective optical system, which includes the step of calculating a numerical value by adding one digit to a specific experimental value based on a new concept. The goal is to provide a method to do so.

The present invention provides a method for measuring enzyme activity in a liquid sample using a reflective optical system using a dry analytical element, in which: a step of experimentally determining the correlation equation (1) between the reflected optical density value of the dry analytical element on which the solution is spotted and the concentration value of the dye or the chemical species, using two or more standard solutions containing the solution; and (II) Measure the reflected optical density values of the two-sided dry analytical element on which the liquid sample has been spotted at two or more points separated by time, and calculate these measured values using the two-sided correlation formula (1). a step of converting the dye or chemical species into concentration values at the two or more points, and determining a concentration fluctuation value of the dye or chemical species per unit time from these dye concentration values; The present invention provides a method for measuring enzyme activity using the system.

[Effects of the Invention] The present invention replaces ODR with the conventionally used O -1- The dye concentration value or chemical species concentration value is a value supported by specific experimental values as shown in step (I) of the present invention.Therefore, the dye concentration value or chemical species concentration value in the present invention is Concentration values or chemical species concentration values are physical quantities with high specificity that can be applied to each measurement system, so they are highly reliable in processing measured values, and can be obtained by the enzyme activity measurement method of the present invention. The measured data is easy to process and interpret.

As a result of the above, the enzyme activity measuring method of the present invention has the effect of improving measurement accuracy by reducing the possibility of ii) causing errors in processing measured values compared to conventional methods.

[Detailed Description of the Invention] The dry analysis element of the reflective optical system used in the present invention is not particularly limited as long as it is used for measuring enzyme activity, and any known element can be used. Specific examples of dry analysis elements include test paper type test strips commercially available under the trade names of Ceralyzer from Ames Co., USA and Refrotron from Boehringer Mannheim Co., West Germany. I can list the ones that exist. In addition, as an integrated multilayer analysis element,
Examples include those commercially available under the trade names of Ektachem from Eastman Kodak Company in the United States and Drychem from Fuji Photo Film Co., Ltd., respectively. Especially when there are no other conditions that limit the type of dry analytical element, it is preferable to use an integrated multilayer analytical element from the viewpoint of ease of handling and analytical accuracy.

Moreover, instead of these commercially available products, dry analysis elements corresponding to conditions such as analysis purpose and test items can be created by referring to various literatures.

Enzyme activity test items for dry analysis elements include lipase,
Amylase, GGT (γ-glutamyl transferase), ALP (alkaline phosphatase), A
LT (alanine aminotransferase) and A
ST (aspartate-aminotransferase)
Amine transferases such as creatine kinase (
CK), lactate dehydrogenase (LDH), cholinesterase (ChE), etc., but the method of the present invention can generally be applied to the measurement of all enzyme activities.

The dry analysis element for enzyme activity measurement mentioned above contains the reagents necessary for producing dyes (pigments, colored products) based on the enzyme activity in the liquid sample, as well as the reagents necessary for other detection reactions. is preserved with.

Therefore, the r-dye J in the present invention is specifically determined according to the type of dry analytical element used in the practice of the present invention. When using the integrated multilayer analytical element for GGT'fFJ property measurement contained in the developing layer, the "dye" in the present invention corresponds to P-nitroaniline liberated from γ-glutamyl-P-nitroanilide due to GGT activity. do.

In addition, in the present invention, the chemical species J capable of providing a dye through an r-conjugation reaction is a product of the enzyme reaction whose activity is to be measured or a chemical reaction coupled thereto, or a coenzyme produced, and the chemical species J is The use of the above chemical species in place of the dye in each step of the present invention is because the resulting dye is poorly soluble in water and it is difficult to prepare a standard solution of the dye. This is particularly effective in cases where

For example, in LDH measurement, etc., in a reaction known as a P-type reaction, cotinamide dinucleotide (reduced form) is generated in the system, so the electron transport chain is involved in this reaction. CLINICA CH
IMICA AC A 12 (19E15)
(See pages 210 to 215), the formazan dye produced in such a system is poorly soluble, making it difficult to prepare a dye standard solution. In such cases, the stability is relatively high,
NADH, which is easily available as a high-purity reagent, was selected as a chemical species capable of producing a dye through a conjugation reaction.

Examples of other "chemical species" include:
Even in the ALT or AST dry enzyme analysis element using POP (pyruvate oxidase) enzyme, which is already known in the US Pat. In such systems, hydrogen peroxide or pyruvic acid may be selected as the dye-providing species, although it is preferred to form poorly soluble dyes. In the above case,
Particularly preferred is pyruvic acid, which is stable in a crystalline state and into which a sample can be input.

The above dye water used in the step (I) of the present invention
It is preferable to prepare the liquid sample so that the viscosity condition is similar to that of a liquid sample. Therefore, when the liquid sample is a biological fluid, generally Prepared using a solution of protein such as fulbumin.

At least two standard solutions containing dyes or chemical species at different concentrations are used as the standard solution, but the reliability of the correlation formula (1) between the reflected optical density value of the dry analysis element and the concentration value of the dye or chemical species is In order to improve the results, it is preferable to use as many standard solutions as possible.

When spotting the above-mentioned standard solution on a dry analytical element (including other sample application modes such as +1 sample application), the spotted amount is preferably within the range of the amount of liquid sample required by the dry analytical element used. The method is carried out using the same amount of liquid sample applied. If the dry analysis element does not have a liquid sample metering effect (metering effect), the amount of the standard solution spotted must be the same as the amount of the liquid sample spotted.

The reflection optical density value corresponding to the dye concentration value or the chemical species concentration value is measured by applying a standard solution to a dry analytical element and measuring the reflection density after a certain period of time using a reflection densitometer. Furthermore, L
The above measurement is preferably carried out under the same conditions as the measurement conditions (measuring equipment, incubation, etc.) of a dry analytical element on which a liquid sample is deposited, which will be described below.

Correlation equation (1) is created from the reflective optical density value of the standard solution measured as described above and the dye concentration value or chemical species concentration value. There is no particular restriction on the form of correlation equation (1), but 1
The dye concentration value or chemical species concentration value ([P]) is a function of the above reflective optical density value (ODR) ([P] = f (ODR
)) is preferable for data processing. Particularly when the data is processed by a computer or the like, it is preferable that L is an integer of 0 or more, and An is a parameter.

In step (II) of the present invention, the operation of measuring the reflected optical density value of the dry analytical element on which a liquid sample has been spotted at two or more points separated by time is generally performed by keeping the dry analytical element at a constant temperature in an incubator. The method involves uniformly heating the sample and measuring the reflected optical density on the dry analytical element during that time. The above photometry can be carried out using a reflection densitometer attached to the incubator, but photometry is possible even if the incubator and reflection densitometer are not integrated.

The temperature during incubation is generally 25°C,
The reaction is carried out at 30° C., 37° C., etc., but there is no need to be particularly limited, however, it is preferable to control the temperature fluctuation within ±0.2° C. within the reaction time.

The reflected optical density measurements described above are converted into dye concentration values or chemical species concentration values using the correlation equation (1) described above.

In addition, the reflected optical density value of the dry analysis element on which the liquid sample is spotted is measured at two or more points separated by time, but generally the amount of dye produced per time is a constant value (corresponding to the initial rate of the enzyme reaction). Measure at two points within the range that shows the value. When dye concentration values are measured at two points, the difference in dye concentration values per unit time is generally taken as the dye concentration fluctuation value per unit time.

In measuring enzyme activity, generally, a calibration curve or correlation equation (2) is created by correlating the enzyme activity value with the time variation of dye concentration or the time variation of chemical species concentration. In particular, in the present invention, the calibration curve or correlation equation (2) is created using two or more standard solutions whose enzyme activity values have been measured by the standard method.
spot on the dry analytical element, measure the reflection density at two or more points separated by time, calculate the temporal fluctuation of the dye concentration or the chemical species concentration using the correlation formula (1), and then Step (III) of creating a calibration curve or correlation formula (2) by correlating the enzyme activity value determined by the method with the time variation of the dye concentration or the time variation of the chemical species concentration due to the standard solution.
It is preferable to carry out using.

The enzyme standard solution used in the above step (III) also includes:
The enzyme standard solution described in F., which is preferably prepared so that the conditions such as viscosity are similar to the liquid sample, can be easily prepared using a commercially available standard enzyme sample.

At least two enzyme standard solutions having different values of enzyme activity are used as the enzyme standard solution, but the reliability of the calibration curve or correlation formula (2) between the time fluctuation of the dye concentration or the time fluctuation of the chemical species concentration and the enzyme activity value is In order to improve the results, it is preferable to conduct the test using as many enzyme standard solutions as possible.

In step (III), the reflection optical density value is measured.

It is preferable to carry out the measurement under the same conditions as the measurement conditions (measuring equipment, incubation': J) of the dry analytical element on which the liquid sample is spotted as described in step (II).

In step 1-, step (III), the reflected optical density value of the -1-2 dry analytical element on which the L enzyme standard solution is spotted is measured at two or more points separated by time, but generally, the reflected optical density value is measured per hour. Measurement is performed at two points within a range in which the amount of dye produced or the amount of chemical species produced is a constant value (a value corresponding to the initial rate of enzyme reaction). When the dye concentration value or the chemical species concentration value is measured at two points, the difference between the dye concentration value or the chemical species concentration value per unit time is generally taken as the dye $-1e degree fluctuation value per unit time.

A calibration curve or correlation equation (2) is created from the variation value per unit time of the dye concentration value or chemical species concentration value of the enzyme standard solution and the enzyme activity value measured as described in 1 below. A calibration curve allows you to visually grasp the relationship between the two, so it is advantageous in understanding and examining the relationship, but it is not suitable for microcomputers.
In the case of automatic processing using, etc., it is generally preferable to express the relationship between the two names as a correlation expression.

There is no particular restriction on the format of the L-letter correlation equation (2), but the above enzyme activity value ([E]) is the median time variation value (Δ[P]) of the dye concentration value or chemical species concentration value. Function ([E]
=f(Δ[P])) is preferable in terms of data processing.

Especially when data is processed by computer etc. (Δ[
P])n; where n is an integer of 0 or more, and B is a parameter).

The enzyme activity value in the liquid sample is calculated using the measured values and relational expressions obtained as described above. Specifically, for example, the analyzer may be equipped with the correlation equation (1) obtained in step (I) above or the calibration curve or correlation equation (2) obtained in step (III) above, and the liquid sample may be Enzyme activity can be calculated by placing a spot on a dry analytical element and measuring the reflected optical density at two or more points separated by time.

The method for measuring enzyme activity of the present invention will be explained in more detail below according to Examples, but the present invention is not limited thereto.

The dry analytical element (integrated multilayer analytical element) used in the examples was manufactured as follows.

[Integrated multilayer analytical element for measuring GGT activity] The surface of a transparent polyethylene terephthalate support (thickness: 180 gm) is treated to make it hydrophilic, and a coating solution of the following component is applied on the treated surface, and dried to form a dry film. A water absorption layer with a thickness of 15 JLm was formed.

Coating liquid for water absorption layer formation: Alkali-treated deionized gelatin Log octylphenoxy-polyethoxyethanol 0.5g Water 100+
nJ11.2-bis(vinylsulfonylacetamido)ethane 0.15g Next, the following coating solution was applied on the water absorption layer, and the dry film thickness was 3p.
An adhesive layer of m was formed.

Coating liquid for adhesive layer formation: Gelatin 12g water
290g nonylphenoxyφ polyglycitol 1.3g A 0.4% aqueous solution of nonylphenoxyboliglycidol was applied onto the above adhesive layer, and then a polyethylene terephthalate spun yarn (36 gauge, 50 denier) was attached to form a spreading layer.

A coating amount of 120+n is applied to the above development layer with the substrate coating liquid described in r.
It was coated and dried so that it became J1/m'.

Preparation of substrate coating solution: Add γ-glutamyl-P-nitroanilide 2 to the F2 solution.
．． 28g to 1m9 of 2N hydrochloric acid. and ethanol 1+nJ
1 and then add about 1.3 liters of 6N hydrochloric acid.
The pH was adjusted to 3 using +n statement, and used as a substrate coating solution.

[Solution] Tris(hydroxymethyl) aminomethane 3.03g Glycylglycine 0.651g Cetyltrimethylammonium bromide 0.500g Water 20.
0 g 10% polyacrylamide 25 g Furthermore, the following titanium dioxide coating solution was applied to the above development layer in a coating amount of 112 m/g, and then dried.

Titanium dioxide coating 4j liquid: Anatase type titanium dioxide 2.5g 0.5% methylcellulose 50m An integrated multilayer analytical element for measuring GGT activity was prepared by the method described above.

[A I, P integrated multilayer analysis f element J transparent polyethylene terephthalate support (thickness 2 180μ,
The surface of (m) was subjected to a wood-carrying treatment, and a coating solution having the following composition was applied to the treated surface -1- and dried to form a loose # layer with a dry film thickness of 15 μm.

Coating solution for forming buffer layer: Alkali-treated deionized gelatin 24g Water 240
g Nonylphenoxy O Polyglycitol 1.6g Poly(N-[(dimethylamine)propyl]acrylamide) 18% aqueous solution (monotropic molecular weight: 100,000) 30g N-(2-hydroxyethyl) Ethylenediamine- N, N', N'-triacetic acid 2 mmol Zn (CH3Coo)2 2H201 mmol Mg5o4 7H Add 5N NaOH aqueous solution to a solution of 202 mmol or more to adjust the pH to 11
．． Adjusted to 0.

On top of the above buffer layer, apply the following coating solution to a dry film thickness of 3.6 g.
The film was coated in an amount of m and dried to form a dura mater layer.

Coating liquid for forming dura layer: Gelatin 12g water
268 g Nonylphenoxy polyglycitol 1.3 g 1.2-bis(vinylsulfonylacetamido)ethane 0.72 g Water/acetone On the above hardening layer, apply the following coating solution to a dry film thickness of 3 m. It was applied as an adhesive layer.

Discard for forming adhesive layer: Gelatin 12g water
286. 7g nonylphenoxy polyglycitol 1.3g 0.4% nonylphenoxy polyglycitol aqueous solution was applied on the above adhesive layer, and then a tricot knitted fabric made of 36 gauge 50D polyethylene terephthalate spun yarn was crimped and developed. layered.

Apply a coating solution with the following composition to the above development layer at a rate of 120m/ml.
I applied it so that it looked like this and dried it.

Coating solution containing self-developing substrate: p-nitrophenylphosphate bis[tris(hydroxymethyl) aminomethane] salt 50 mmol polyvinylpyrrolidone (average molecular weight 10,000) 6 g ethanol
22g acetone
An integrated multilayer analytical element for measuring ALP activity was prepared using a method using 22 g or more.

[Example 1] A 7% human albumin aqueous solution containing p-nitroaniline at various concentrations shown in Table 1 below was applied to the above-mentioned integrated multilayer analytical element for measuring GGT activity, and the reflection density was measured after a certain period of time. was measured through a 400 nm interference filter.

The correlation between the concentration [P] of p-nitroaniline and the reflected optical density (ODA) was approximated by the following correlation formula (11).

[P] −Ao +AI(ODR)+A2(ODR)2
+A3(ODR)3 (11) Correlation formula (11)
Each parameter AO1AI, A2 and A3 in
had the following values.

Ao = -3,3646013 AI = 10.26816957A2 = -
11, 290208 A, + = 7.45991396L The dye concentration calculated from the ODR using the correlation equation is listed in Table 1 below, together with the ODA and the dye concentration actually spotted.

Table 1 ODR dye concentration Dye concentration (calculated value) (actual value) 0.525 -0.006mM 0mM0, 7
33 1 , 033 10.872 1
．． 950 20.995 3.023
31.083 3.989 41.161
5.012 51, 284 6,
997 6 Then, using 6 levels of samples (shown in Table 2 below) in which bovine kidney-derived GGT was added to Persatol ENPlug, each reflection density value was measured 2 minutes and 5 minutes after spot incubation, and after partial incubation. And the amount of p-nitroaniline dye produced after 5 minutes was calculated using the above conversion formula (it).

The correlation between the GGT activity of each standard solution and the amount of P-nitroaniline dye produced during the incubation time of 2 to 5 minutes (Δ[P]) is expressed by the following multidimensional equation (21).
It was approximated as

GGT activity (U/L) = iB 2 (Δ[P])” (21) Each parameter Bo, B+ in the above correlation equation (21)
, B2, B3, Bs and B5 had the following values.

Bo = -23,610628 B+ = 381.1602245 B2 = -36,60469387B3 = 32
1.2894.98 RA = -9n 6-7 9 R9678Bs
= 42.7391708 The above correlation equation (21
), and the amount of p-nitroaniline dye produced between 2 and 5 minutes (Δ[P]).
And the GGT activity value in each control along with the value actually measured by the solution method (measured with RAlooO) as shown in the following 2nd table.
Record in the table.

Table 2 Controls a-h G GT Concentration Δ[P] GG
T concentration No. (actual value) (calculated value) 1 11 0.091 11,
02 141 0.405 141 , 0
3 293 0.700 292 , 94
602 1.183 602 ,05
1148 1.915 1-147 , 96
1556 2.319 1556 , 0 above G
GGT activity was measured for 85 sera from 7+ε subjects using an analytical element for GT activity measurement, and each reflected optical density was processed using correlation equations (11) and (21) in Section 1. GGT activity was quantified. Also RA-1
GGT activity was measured by a solution method using 000 (Technicon), and the correlation with the above measured values was examined.

Both measured values were determined by the method of the present invention for Y and RA1000 for Z.
The following good correlation was obtained when using the measured values according to the method.

Y=0.968X+4.9 (II=85)γ=0.9
99 Syx=7.5 Therefore, although the method of the present invention uses a reflective optical system, it was found that analytical accuracy comparable to the solution method using a transmitting optical system can be obtained.

[Example 2] The standard solution in Example 1 was prepared using p-nitrophenol instead of p-nitroaniline, and the above-mentioned A
Using an integrated multilayer analytical element for measuring LP activity, a correlation formula rF12) between the reflected optical density value (ODR) and the above dye (P-nitrophenol) concentration value was created.

[P] =CO+CI(ODR)+C2(ODR)2+
C3(ODA)3+Ca(ODR)'+C3(ODR
)5+C6(ODR)6+CI(ODR)'+C5(O
DR) Each parameter C in sE correlation equation (12)
oc8 had the following value.

Co = -23,72762631 C+ = 88.67719551 C2 = -87,66953215 C3 = -51,50979621 C4 = 119.3958341 C5 = 2.993599581 Cb = -89,33292527 C7 = 50. 35986471 Ca = -7,765508396 Then, using 6 levels of samples in which human placenta-derived ALP was added to Persatol EN Plus.

Each reflection density value was measured 2 minutes and 5 minutes after spotting incubation, and the amount of p-nitrophenol dye produced after spotting and 5 minutes was calculated using the above conversion formula (12).

The correlation equation (22) between the ALP activity [E] for each standard solution and the amount of p-ditrophenol dye produced during the spot incubation time of 2 to 5 minutes (Δ[P]) is shown below. show.

[E] =Do +D+ (Δ[P]) (22
) Each parameter Do and Dl in the above correlation equation (22) had the following values.

Do=-8 D+=667 Using the above analytical element for measuring ALP activity, patient serum 101
The ALP activity of each example was measured, and the ALP activity of each individual was quantified by processing the measured value of each reflective optical density using the above correlation equations (12) and (22). Also R.A.
-1000ALP method (Technicon) was used for measurement using a solution method, and the correlation with the above measured values was investigated.
Both measured values were determined by using the method of the present invention for Y and RC100O for Z.
The following good correlation was obtained when using the measured values according to the method.

Y=1.148X+19.1 (II=85)γ=0.
994 Syx=10.7 Therefore, it was found that the method of the present invention in the above-mentioned example also provided analysis accuracy comparable to the solution method using a transmission optical system.

Claims (1)

[Claims] 1. A method of measuring enzyme activity in a liquid sample using a reflective optical system using a dry analytical element that has a function of producing a dye based on the enzyme activity in the spotted liquid sample. (I) Using two or more standard solutions containing the dye or a chemical species capable of producing the dye through a conjugate reaction at different concentrations, the reflection optical density value of the dry analytical element on which this solution is spotted and the above A step of experimentally determining the correlation equation (1) with the concentration value of the dye or the chemical species; and (II) measuring the reflected optical density value of the dry analytical element on which the liquid sample is deposited at two or more time intervals. These measured values are converted into concentration values of the dye or chemical species at the two or more points using the above correlation formula (1), and from these concentration values, the dye or chemical species per unit time is calculated. A method for measuring enzyme activity using a reflective optical system, the method comprising: determining a concentration variation value of; 2. In addition to the steps (I) and (II) above, (III)
Two or more standard solutions whose enzyme activity values have been measured using the standard method are spotted on the above dry analysis element, and the reflection density is measured photometrically at two or more points separated by time, using the above correlation formula (1). Calculate the time variation of the dye concentration or the chemical species concentration, and correlate the enzyme activity value determined by the standard method with the time variation of the dye concentration or chemical species concentration using the standard solution to create a calibration curve. 2. The measuring method according to claim 1, further comprising the step of creating a correlation equation (2). 3. Using an analyzer that incorporates the correlation formula (1) obtained in advance in step (I) above, a liquid sample is spotted on a dry analysis element, and the reflected optical density is measured at two or more points separated by time. 3. The measuring method according to claim 1 or 2, comprising the step of calculating the enzyme activity by photometrically measuring the enzyme activity. 4. Using an analyzer that incorporates the calibration curve or correlation formula (2) obtained in advance in step (III) above, spot the liquid sample on the dry analytical element, and measure it at two or more points separated by time. 3. The measuring method according to claim 2, comprising the step of calculating the enzyme activity by photometrically measuring reflected optical density. 5. The enzyme activity measuring method according to claim 1 or 2, wherein the dry analytical element is an integrated multilayer analytical element. 6. In the step (II) above, the reflected optical density value of the dry analytical element on which the liquid sample has been spotted is measured at two points separated by a certain period of time, and the measured value is calculated by the above correlation formula (1). The method of the present invention is characterized in that it is a step of converting the concentration value of each dye or chemical species at the above two points using The method for measuring enzyme activity according to scope 1. 7. The enzyme activity according to claim 1 or 2, wherein in the correlation formula (1), the concentration value of the dye or chemical species is expressed as a function of the reflective optical density value. Measuring method. 8. The method for measuring enzyme activity according to claim 7, wherein the function is an algebraic function. 9. The above calibration curve or correlation equation (2) is a correlation equation (2), and in the above correlation equation (2), the enzyme activity value is expressed as a function of the time variation of the dye concentration or the time variation of the chemical species concentration. The method for measuring enzyme activity according to claim 2, characterized in that: 10. The enzyme activity measuring method according to claim 9, wherein the function is an algebraic function.