Method of assay of enzymatic activity

Method of assay of enzymatic activity, including projecting excitation light to a sample containing an enzyme, a substrate which forms a product by action of the enzyme, and a reference substance which is insensitive to the action of the enzyme but emits fluorescence; obtaining a first measured value of fluorescence intensity of the sample at a first wavelength region which includes fluorescence emitted by the substrate or the product at least, obtaining a second measured value of fluorescence intensity at a second wavelength region which is different from the first wavelength region for the first measured value and includes fluorescence emitted by the reference substance; and assaying the enzymatic activity from the ratio of the first measured value to the second measured value and apparatus for performing the method. The method assures high accuracy and high sensitivity of measurement in enzyme labeled immunoassay and enzyme labeled DNA hybridization.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a method of assay of activity of an enzyme 
employed as a label for immunoassay, etc. based on the detection of 
fluorescence intensity of a substance having been affected by the enzyme. 
The present invention also relates to an apparatus used for the assay of 
the activity of the enzyme. 
2. Description of the Related Art 
Conventionally known methods for assaying a minute quantity of a biological 
substance include labeled immunoassay and enzyme-labeled DNA 
hybridization. 
These methods are based on a principle that the biological substance is 
identified or determined by assaying the activity of an enzyme linked 
directly or indirectly to an immune complex formed through an immune 
reaction, or the like, according to detection of the change of the 
substrate. The known methods include measurement of the change of a light 
absorption coefficient, measurement of the change of fluorescence 
intensity, measurement of the change of light emission intensity, of a 
sample having been affected by enzyme activity, and so forth. From among 
these methods, the last two methods including measurement of enzyme 
activity by fluorescence or light emission are advantageous in that a 
higher sensitivity is achievable in shorter time than in the method which 
utilizes the light absorption for enzymatic activity assay. The 
fluorescence method is known to be highly sensitive for assaying enzymatic 
activity. 
Several methods have been reported for quantitative determination of 
biological substances on the basis of change of fluorescence intensity of 
a sample. The examples are a method in which the measurement is conducted 
at a specific point of time after the initiation of an enzymatic reaction 
and the enzymatic activity is derived from the measured data (a one-point 
method); a method in which the measurement is conducted at two points of 
time during the progress of a series of enzymatic reactions and the 
enzymatic activity is derived from the difference of the two measured data 
(a two-point method); and a method in which the measurement is conducted 
nearly continuously and the enzymatic activity was derived from the change 
rate of the continuously measured data (a rate method). 
In recent years, the significance and the importance of quantitative 
determination of a minute quantity of a substance have been given more 
attention, in particular, in the fields such as clinical diagnosis. 
Consequently, the determination of a minute quantity of a biological 
sample with high precision and high sensitivity has been increasingly 
required. To meet such requirement, proposals have been made regarding a 
system which is free from contamination of a sample by another sample, a 
detecting device for successive measurement of a plurality of samples with 
high sensitivity, and so forth. 
The methods and apparatus of prior arts, which have been proposed or been 
put into commercial practice for quantitative determination of biological 
substances with high sensitivity and high precision, have been confronted 
with limitation in improvement of precision of measurement more than a 
certain degree because of significant influence of air bubbles in the 
sample and meniscus of the sample liquid, and so forth. One reason for the 
above limitation in the improvement is that samples of biological 
substances to be measured are available only in a minute amount in most 
cases. 
For example, the presence of bubbles in the sample liquid gives a larger 
value of fluorescence measurement than a value measured in the absence of 
the bubbles, so that disappearance of the bubbles during the measurement 
will give, for example, a lower value in a rate method than the real 
value. On the other hand, the injection of the substrate, which is to be 
subjected to the reaction with a label enzyme, into a reaction vessel is 
required to be conducted as quickly as possible in order to treat quickly 
a number of samples. Further, any adhesion of a liquid drop at the tip of 
the nozzle needs to be avoided at the completion of dispensing of the 
sample liquid in order to obtain high accuracy of the volume to be 
dispensed. However, if the liquid ejection speed is made higher to satisfy 
the above requirements, the dispensed liquid tends to be foamed more. In 
the enzyme-labeled immunoassay, an immune reaction complex and an 
unreacted matter are generally separated from each other (B/F separation) 
before the substrate injection. If the concentration of surface active 
agent, which is usually contained in the washing solution, is made higher, 
more bubbles tend to be formed. 
Accordingly, more and more demands have been made for comprehensive 
investigations to be made to find a novel measurement method which is free 
from the above problems in principle and enables improvement of the 
accuracy of the measurement. 
Apart from the above problems, a higher detection sensitivity is required 
in fluorescence measurement. For example, in immunoassay of TSH 
(thyroid-stimulating hormone), the lower detection limit has 
conventionally been at a level of about 0.1 .mu.IU/ml, but the limit is 
now required to be at a level of 0.01 .mu.IU/ml for diagnosis of disease 
state of an abnormal low value of TSH. Therefore, a higher detection 
sensitivity is required as well as the suppression of nonspecific reaction 
of the labeled enzyme. For the improvement of the detection sensitivity, 
the change of the real measured value has to be distinguished from the 
variation caused by external disturbance. 
SUMMARY OF THE INVENTION 
An object of the present invention is to provide a novel method of 
measurement in enzyme labeled immunoassay and enzyme labeled DNA 
hybridization, which is less affected by obstructions in improvement of 
measurement accuracy: such as the adverse effect by the presence of 
bubbles in the sample, influence of the meniscus, influence of agitation 
with magnetic particles with a vibrating magnetic field in the reaction 
vessel for purposes of stirring the sample, influence of variation of 
light source intensity with time, and so forth, and which enables the 
measurement with high accuracy, and high sensitivity; and to provide also 
the apparatus for the method. 
Another object of the present invention is to provide a method for the 
above measurement which is not affected by any variation in the quantity 
of light projected to the reaction vessel owing to variation in the 
relative position of the reaction vessel to the light source, and which 
can achieve high sensitivity of measurement consistently, and to provide 
also the apparatus for the method. 
A further object of the present invention is to provide a novel method for 
the measurement in enzyme labeled immunoassay and enzyme labeled DNA 
hybridization, which enables simplification and automation of the 
substrate dispenser and the sample agitator, which could not be simplified 
nor automated due to requirements for reducing measurement errors, without 
lowering the accuracy, and to provide also an apparatus for the method. 
The present invention provides a method of assay of enzymatic activity, 
comprising projecting excitation light to a sample containing an enzyme, a 
substrate which forms a product by action of the enzyme, and a reference 
substance which is insensitive to the action of the enzyme but emits 
fluorescence; obtaining a first measured value of the fluorescence 
intensity of the sample at a first wavelength region which includes 
fluorescence emitted by the substrate or the product at least, obtaining a 
second measured value of the fluorescence intensity at a second wavelength 
region which is different from the first wavelength region for the first 
measured value and includes at least the fluorescence emitted by the 
reference substance; and assaying the enzymatic activity from the ratio of 
the first measured value to the second measured value. 
The apparatus according to the present invention for performing the above 
method comprises light-projecting means for projecting excitation light to 
a sample containing a fluorescent substance which increases or decreases 
by action of an enzyme, and a reference substance which emits fluorescence 
and is insensitive to the action of the enzyme; first photosensing means 
for measuring fluorescence intensity of wavelength for a first measured 
value of fluorescence originating from the fluorescent substance out of 
the entire fluorescence emitted by the sample; second photosensing means 
for measuring fluorescence intensity at wavelength for a second measured 
value of fluorescence originating from the reference substance out of the 
entire fluorescence emitted by the sample; and computing means for 
computing the enzyme activity from input of the first measured value and 
the second measured value.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
The enzymatic activity can be assayed according to the change rate relative 
to time of the ratio derived by dividing a first measured value at a first 
wavelength region by a second measured value at a second wavelength region 
in comparison with a calibration curve prepared preliminarily by use of 
known samples. The change rate relative to time of the fluorescence 
intensity is preferably derived by the aforementioned two-point method, or 
derived nearly continuously by the aforementioned rate method in which the 
measurement is conducted nearly continuously to follow the progress of the 
enzymatic reaction, and therefrom the enzymatic activity is assayed. 
However, the method is not limited thereto. 
The feature of the method of the present invention is explained as below. 
It is assumed here that the substrate, the product formed by action of the 
enzymatic action, and the reference substance are all fluorescent. The 
case is considered where excitation light is projected to the system 
containing the substances. In this case, the intensity of the fluorescence 
emitted from each of the substances is proportional to the concentration 
of each fluorescent substance, and external disturbance such as bubbles 
affects all of the measured data. Therefore, the first measured value (FI 
1) obtained at the above specified wavelength and the second measured 
value (FI 2) obtained at another wavelength are respectively shown by the 
equations below. 
EQU (FI 
1)=p.times.(k1.times.[Substrate]+k2.times.[Product]+k3.times.[Reference]) 
EQU (FI 
2)=p.times.(k4.times.[Substrate]+k5.times.[Product]+k6.times.[Reference]) 
wherein p is a coefficient of the fluorescence variation affecting the 
entire sample resulting from bubbles and other causes; [Substrate], 
[Product], and [Reference] are respectively the concentration of the 
substrate, the product, and the reference substance; and k1 to k6 are 
proportion coefficients corresponding to the respective wavelengths. 
Therefore, the ratio of (FI 1) to (FI 2) in the present invention is shown 
by Formula (1) below. This formula is transformed to Formula (2) to 
represent the substrate concentration as a function of the product 
concentration. Thus the concentration of the product is derived from the 
concentration of the reference substance and the proportion coefficients 
k1 to k6. 
##EQU1## 
where [Substrate].sub.0 is the initial concentration of the substrate 
added to the reaction vessel, and is a known value. Thus, it is understood 
that the influence of the variation of the fluorescence intensity is 
canceled by taking the ratio of two measured values according to Formula 
(1) or Formula (2). 
The above formulas are shown to describe generally the case where the 
substrate, the product, and the reference material emit respectively 
fluorescence by irradiation of excitation light. However, other cases 
where k1=0, or k1=k4=k5=0 may be selected by selecting the wavelength for 
the first measured value or for the second measured value, including the 
case where the coefficient or coefficients are not completely zero but 
practically can be considered as being zero. The purpose of the present 
invention is achieved essentially by measuring the fluorescence intensity 
of the enzymatic reaction product as the one measured value (e.g., the 
first measured value) and the fluorescence intensity of the reference 
substance as the other measured value (e.g., the second measured value). 
The wavelength of the excitation light in the present invention is selected 
so as to excite at least one of the substrate and the product, as well as 
the reference substance. 
The fluorescence wavelength for obtaining the first measured value 
(hereinafter referred to as the "first fluorescence wavelength") is 
selected from the wavelength range of the fluorescence emitted from the 
substrate or the product, and the fluorescence wavelength for obtaining 
the second measured value (hereinafter referred to as the "second 
fluorescence wavelength") is selected from the wavelength range of the 
fluorescence emitted from the reference substance. The wavelength herein 
covers a required wavelength region. 
The wavelength of the excitation light and the first fluorescence 
wavelength are selected such that the change of the fluorescence 
characteristics caused by the change of the substrate into the product is 
reflected in the measured fluorescence intensity. 
It is preferable that the reference substance contained in the sample has a 
fluorescence peak wavelength departed at least 50 nm from a fluorescence 
peak wavelength of the substrate and/or the product formed by the 
substrate. 
Thus when the respective fluorescences of the product and the reference 
substance are measured, it is preferable to use a reference substance 
having a fluorescence peak wavelength departed at least 50 nm from the 
fluorescence peak wavelength of the product. 
Also when the fluorescences of the substrate and the reference substance 
are measured, it is preferable to use a reference substrate having a 
fluorescence peak wavelength departed at least 50 nm from the fluorescence 
peak wavelength of the substrate. 
As an example for the combination, 4 MU (fluorescence wavelength 450 nm) 
produced from 4 MUP by alkaline phosphatase is used in combination with 
dansylhydrazine (fluorescence wavelength 500 nm). 
With the combinations mentioned above it is possible to minimize the 
measurement error caused by the fluorescence peaks and to obtain a high 
degree of accuracy of the measurement. However it is more preferable to 
use a reference substance having a fluorescence peak wavelength departed 
at least 50 nm from the fluorescence peak wavelengths of the substrate and 
the product. This combination enables a high degree of accuracy of 
measurement in all cases where the fluorescence of the substrate is 
measured and where fluorescence of the product is measured. 
The enzyme includes a free enzyme of natural origin, an enzyme linked to a 
free antigen, antibody, or DNA, an enzyme bonded indirectly to an inner 
wall of a small container or a surface of a sheet as at obtained by enzyme 
labeled immunoassay or enzyme labeled DNA hybridization, and the like. 
The labeled enzyme for enzyme labeled immunoassay and enzyme labeled DNA 
hybridization is exemplified typically by alkaline phosphatase, 
.beta.-galactosidase, and the like. The substrate which changes its 
fluorescence intensity by the action of the alkaline phosphatase is 
exemplified by 4-methylumbelliferyl phosphate. This substrate is converted 
to 4-methylumbelliferone by the action of the above enzyme. The substrate 
which changes its fluorescence intensity by the action of the 
.beta.-galactosidase is exemplified by 4-methylumbelliferyl galactoside. 
This substrate is converted to 4-methylumbelliferone by the action of the 
above enzyme. For example, when excitation light of wavelength of 365 nm 
(see FIG. 4(a)) is projected to the above product (4-methylumbelliferone) 
in alkaline medium (pH 10), the 4-methylumbelliferone emits fluorescence 
having maximum intensity at about 450 nm (see FIG. 4(b)), while the 
fluorescence emitted by 4-methylumbelliferyl phosphate or 
4-methylumbelliferyl galactoside is negligible. Accordingly, the 
fluorescence intensity emitted by the product can be measured by 
projecting the excitation light of 365 nm to a sample, and measuring 
fluorescence at the wavelength around 450 nm from the entire fluorescence 
of the sample. 
The reference substance, which is characteristic to the present invention, 
to be added to a sample containing the substrate and the product as 
mentioned above is selected from the substances which emit fluorescence in 
a spectrum pattern different from that of the above fluorescence spectrum 
which is changed by the enzymatic action. 
In the case of the above typically exemplified 4-methylumbelliferone, the 
reference substance is preferably selected from the substances which emit 
fluorescence, for example, of wavelength of 500 nm or more on receiving 
excitation light of 365 nm. The examples of such reference substances are 
shown below. 
______________________________________ 
(Ex.sup.max) 
(Em.sup.max) 
(Structural formula) nm nm 
______________________________________ 
1) Dansyl compounds: 
(Example) Dansylsulfonic acid 
325 550 
##STR1## 
Dansyl chloride 325 550 
##STR2## 
Dansylamino acids 
(Example) Dansyl-L-alanine 
325 550 
##STR3## 
Dansylhydrazine 340 525 
##STR4## 
2) 4-Dimethylamino-1-naphthoylnitrile 
350 530 
##STR5## 
3) N-(1-anilinonaphthyl)-4-maleimide 
350 530 
##STR6## 
4) 7-Benzylamino-4-nitrobenz-2-oxa-1,3- 
340 530 
diazole (R = H) 7-(p-Methoxy- 
or 
benzylamino)-4-nitrobenz-2-oxa-1,3- 
458 
diazole (R = OCH.sub.3) 
##STR7## 
______________________________________ 
The above substances have the maxima of the excitation spectra not at 365 
nm. However, the dansyl-L-alanine shown above has a breadth in its 
excitation spectrum as shown in FIG. 5(a). Therefore, projection of 
excitation light of 365 nm causes the emission of fluorescence having the 
maximum at about 550 nm (FIG. 5(b)). 
In the case where the enzymatic reaction product is 4-methylumbelliferone 
and the reference substance used is dansyl-L-alanine, the fluorescence 
spectra of the both substances partly overlap with each other (FIG. 4(b) 
and FIG. 5(b)). If the light of about 450 nm is selected as the first 
fluorescence wavelength, the light includes a slight portion of the 
fluorescence of the reference substance. However, the rate of change with 
time of the ratio of the first measured value obtained at the first 
fluorescence wavelength to the second measured value obtained at the 
wavelength of 500 nm or more is not affected thereby, which is the 
characteristic of the present invention. 
Further in the aforementioned case, the second measured value obtained at 
the second fluorescence wavelength include a portion of the fluorescence 
of 4-methlumbelliferone. However, the effect of the present invention is 
appropriately exhibited by selecting the second fluorescence wavelength 
and the concentration of the reference substance such that the influence 
of the 4-methylumbelliferone is not large. Specifically, for example, in 
practicing the above case, the concentration of the reference substance to 
be incorporated in the sample is preferably selected within the range to 
give sufficient fluorescence in intensity at the second fluorescence 
wavelength and not to cause excessively large absorption of the excitation 
light by the reference substance. Generally the absorption ratio should 
not be more than 50%. In the case where the fluorescence spectrum of the 
substrate or the product overlaps with the excitation spectrum of the 
reference substance, the fluorescence emitted by the substrate or the 
product is re-absorbed by the reference substance. In this case, the 
concentration of the reference substance is preferably selected within the 
range that the ratio of the re-absorption is not large, generally not more 
than 50%. 
Any sample may be subjected to the assay of the present invention, provided 
that the enzyme labeled immunoassay or the enzyme labeled DNA 
hybridization is applicable to the sample. In most cases, the sample in a 
small container having a cup shape is brought into an assay apparatus. 
The apparatus for the assay of enzymatic activity of the present invention 
includes a light-projecting means for projecting excitation light to a 
sample containing a fluorescent substance which increases or decreases by 
action of an enzyme, and a reference substance which emits fluorescence 
and is insensitive to the action of the enzyme; a first photosensor means 
for measuring fluorescence intensity of wavelength for a first measured 
value of fluorescence originating from the fluorescent substance out of 
the entire fluorescence emitted by the sample; a second photosensor means 
for measuring fluorescence intensity at wavelength for a second measured 
value of fluorescence originating from the reference substance out of the 
entire fluorescence emitted by the sample; and a computing means for 
computing the enzyme activity from input of the first measured value and 
the second measured value. 
The aforementioned light-projecting means is generally an optical system 
constituted by suitably combining a light source, a lens, a filter, a 
mirror, a condenser lens, etc. The light source is preferably of a flasher 
type, since it enables elimination of the influence of external disturbing 
light, etc. from the signal obtained from the photosensor by synchronous 
wave detection at the light-receiving side to take out exclusively the 
fluorescence component emitted by the sample, thus enabling more reliable 
detection of fluorescence intensity. As the flashing light source, a 
discharge lamp or a fluorescence lamp which flashes at a frequency ranging 
from tens of Hz to several hundred Hz is advantageous rather than a lamp 
flashed by a mechanical chopper in view of its durability in long time of 
continuous operation. The power source may be an AC source or a pulse 
source operated at the aforementioned frequency, or a high frequency AC 
source of several kHz to several ten kHz which is switched on and off at 
the above frequency. 
The photosensor means of the apparatus for enzymatic activity assay 
includes a first photosensor means and a second photosensor means. Each of 
the photosensor means is constituted by combining a light-receiving 
optical system including a mirror, a condenser lens, a filter, a 
photosensor such as a photodiode and a photomultiplier, and an electric 
circuit such as an amplifier. As described above, if a light 
source-flashing means is employed, a wave detector is also used which 
detects synchronously electric signals from a photosensor by utilizing 
clock signals for flashing the light source such as a fluorescence lamp. 
The apparatus for assay of enzymatic activity of the present invention may 
be of a system in which the fluorescence emitted from the sample is 
detected in a direction opposite to the projection of the excitation light 
to the sample, or otherwise may be of a system in which the direction of 
the fluorescence detection is perpendicular to the projection of the 
excitation light. For efficient continuous assay of enzymatic activity of 
a number of samples, an embodiment of a top-top type is preferred in which 
the optical path of the light-receiving system is superposed on the 
optical path of the light-projection system. In an example, a dichroic 
mirror or the like is placed in an optical path of a light-projecting 
system to reflect the light from the light source downward, and the 
fluorescence emitted upward by the sample is divided into the light 
components of the first fluorescence wavelength and the second 
fluorescence wavelength with a filter, a half-mirror, or a dichroic mirror 
placed in the optical path of the light receiving system. Thus, the 
respective light components can be detected by separate photosensors. In 
particular, the use of a dichroic mirror is advantageous in that 
excitation light and fluorescence are effectively utilized with reduced 
loss by selecting the property thereof in accordance with the wavelength 
of the excitation light and the fluorescence. 
The light source, the photosensor, the filter, etc. are placed in suitable 
positions in accordance with the properties of the employed elements such 
as a dichroic mirror. 
FIG. 6(a) to FIG. 6(c) illustrate roughly examples of embodiments of the 
optical system of the top-top type as mentioned above. The light from a 
light source 501 is projected through a half-mirror or a dichroic mirror 
507, 508 to a sample 1 in a sample container 2. Fluorescence emitted by 
the sample is introduced through filters 505, 506 to photosensors 503, 
504. In FIG. 6(b) and FIG. 6(c), the symbol numbers correspond 
respectively to the ones in FIG. 6(a) provided that the numeral 10 or 20 
is added thereto, respectively. 
In the division processing of the computing device according to the present 
invention, a first electrical signal obtained by a first light-receiving 
system and a second electrical signal obtained by a second light-receiving 
system may be processed in an analog manner, or otherwise the above 
electrical signals may be once converted by A/D converters to digital 
signals and then be processed by a digital computing circuit such as a 
digital computer. 
FIG. 7 illustrates schematically a signal processing system of a measuring 
apparatus having the above-described constitution. FIG. 7(a) illustrates 
an example employing a light source 70 continuously lighted by a DC power 
source 80. FIG. 7(b) illustrates another example employing a light source 
70 flashed by a pulse source 81. The light introduced to photosensors 63, 
64 is amplified as electrical signals 71, 72 by amplifiers 73, 74, 
converted to digital signals by A/D converters 75, 76, and then processed 
by a digital computing circuit 77 such as a computer to calculate, for 
example, the change rate as a function of time and to obtain division 
quotient. 
In FIG. 7(b), a clock-generating circuit 82 synchronizes the pulse source 
81 with a synchronous wave detecting circuits 83, 84. 
Naturally, the electrical signals 71, 72 may be inputted to an analog 
division circuit. The outputs therefrom being converted to digital signals 
by A/D converters, and being processed by a digital computing circuit such 
as a computer to calculate the above change rate as a function of time. 
The present invention is described in more detail by reference to examples 
shown in the drawings. 
EXAMPLE 1 
FIG. 3 illustrates an example of a measuring apparatus for the assay of the 
present invention (an embodiment having a top-top type of optical system). 
In the drawing, a cup-shaped sample container 2 (top end diameter: 10 mm, 
volume: 1 ml) holds a small amount--several tens to hundreds .mu.l--of a 
sample liquid 1. The sample in the container passes through processes of a 
substrate dispense and an enzyme reaction, which are not shown in the 
drawing, and transferred to the place for fluorescence detection. FIG. 3 
illustrates a state that the sample container 1 is set in a measurement 
position. The product formed from the substrate was a product from 
4-methylumbelliferyl phosphate by action of alkaline phosphatase, the 
product being 4-methylumbelliferone having fluorescence characteristics 
shown in FIG. 4(a) and FIG. 4(b), and the reference substance was 
dansyl-L-alanine having fluorescence characteristics shown in FIG. 5(a) 
and FIG. 5(b). 
A light source 61 emits excitation light of the light-projecting optical 
system. The light passed through a filter 62 which allows the light in the 
wavelength range of from 320 nm to 380 nm to transmit, and then the light 
is reflected by a half-mirror 67 to be projected downward as the 
excitation light to the sample 1 in the sample container 2. 
Fluorescence caused by the irradiation of the exciting light and emitted by 
the sample liquid 1 passes through the half-mirror 67 and is separated by 
the dichroic mirror 68 into the light including the first fluorescence 
wavelength and the light including the second fluorescence wavelength. The 
dichroic mirror 68 is designed to have a property that it transmits light 
having wavelength of 530 nm or longer but reflects light having wavelength 
of from 420 nm to 530 nm. 
The light of the first fluorescence wavelength separated by the dichroic 
mirror 68 is introduced to a filter 65 which transmits light of from 450 
nm to 510 nm, and selected wavelength of light is introduced to a 
photosensor 63. On the other hand, the light of the second fluorescence 
wavelength proceeds upward and is introduced to a filter 66 which 
transmits light of 600 nm or longer, and the selected wavelength of light 
is introduced to a photosensor 64. 
With the measuring apparatus having the constitution described above, the 
fluorescence of a sample liquid was measured which contained a biological 
substance labeled with alkaline phosphatase, 4-methylumbelliferyl 
phosphate as the substrate, and dansyl-L-alanine as the reference 
substance mixedly. The alkaline phosphatase was used in an appropriate 
quantity, 4-methylumbelliferyl phosphate was at a concentration of 1 mM, 
and dansyl-L-alanine was at a concentration of 100 .mu.g/ml. 
The measured value A of the first fluorescence wavelength and the measured 
value B of the second fluorescence wavelength detected thereby are shown 
in FIG. 1. The ratio of the first measured value at the first fluorescence 
wavelength to the second measured value at the second fluorescence 
wavelength (relative fluorescence intensity) is shown as a function of 
time in FIG. 2. In FIG. 1 and FIG. 2, the denotations (1) to (4) shows the 
time when air is blown artificially into the sample liquid and the bubbles 
formed thereby disappeared or moved away, causing changes of the measured 
fluorescence intensity. 
From the comparison of FIG. 1 with FIG. 2, it is understood that the 
fluorescence intensity changes greatly by blow of air but the ratio of the 
first measured fluorescence to the second measured fluorescence is not 
affected by the presence of the air bubbles. Thus it is confirmed that the 
present invention enables precise detection of the change of fluorescence 
intensity emitted by a fluorescent substance without the adverse effect of 
the presence of air bubbles in the sample. 
EXAMPLE 2 
The experiment below was conducted in order to compare the result of the 
measurement obtained by the method of the present invention with a 
conventional method. 
An apparatus AIA-1200 (made by Tosoh Corporation) was employed which has 
the same constitution as the one employed in Example 1. 
A solution was preliminarily prepared by dissolving 26 mg of 
4-methylumbelliferyl phosphate in 100 ml of an aqueous solution containing 
4.5 g of 2-amino-2-methyl-1-propanol and further dissolving therein 0.1 
mg/ml of dansyl-L-alanine (10 mg to 100 ml of the substrate solution). 
Into a sample container having several magnetic particles (about 1.5 mm in 
diameter) for stirring, 50 .mu.l of alkaline phosphatase was dispensed 
manually by a pipetter, and further thereto 200 .mu.l of the above, 
preliminarily prepared solution was dispensed manually by a pipetter. 
The container was immediately placed below the detector. The fluorescence 
intensity was measured every 2 seconds, beginning several seconds after 
the addition of the solution for 100 seconds at the first and the second 
wavelengths. The rate was derived according to the calculation formula (3) 
by a least square method (Test Nos. 1 to 10). The results are shown in 
Table 1. Throughout the measurement, the magnetic particles were moved in 
reciprocation by magnetic vibration. In the experiments of Test Nos. 1 and 
9, air was introduced into the liquid in the container just before the 
detection operation. 
EQU [(Sig(4MU)-Sig(4MU.sub.0)]/[(ref(DLA)-ref(DLA.sub.0)] (3) 
where Sig(4MU) is a measured value of fluorescence intensity at the first 
wavelength (substantially the fluorescence intensity of 
4-methylumbelliferone); Sig(4MU.sub.0) is a measured value for the empty 
container at the first wavelength; ref(DLA.sub.0) is a measured value of 
fluorescence intensity at the second wavelength (substantially the 
fluorescence intensity of the reference substance); and ref(DLA.sub.0) is 
a measured value for the empty container at the second wavelength. 
The same tests were conducted in the same manner as above except that the 
alkaline phosphatase was not added, and the fluorescence intensity was 
measured and the rate was calculated (Test Nos. 11 to 19). The results are 
shown in Table 2. In the experiments of Test Nos. 13 and 15, air was 
introduced into the liquid in the container just before the detection 
operation. In the experiments, the agitation magnetic beads used had been 
obtained from a commercial immunodiagnostic reagent (E Test "TOSOH" made 
by Tosoh Corporation). Although the beads were washed sufficiently before 
the use, a slight amount of alkaline phosphatase was believed to remain 
adhered to the surface thereof. 
For comparison, for the respective cases where the alkaline phosphatase is 
added and not added, the rates were calculated from the measured 
fluorescence intensity only at the first wavelength according to a 
conventional method by use of the formula (4) by a least square method 
(No. 1 to No. 10, and No. 11 to No. 19). The result are show in Table 1 
and Table 2. 
EQU [{Sig(4MU)-Sig(4MU.sub.0)}/ref(UV)].times.50 (4) 
where Sig(4MU) and Sig(4MU.sub.0) mean the same as in the formula (3), and 
the term "ref(UV)" is for canceling the variation of the source light with 
time by dividing the measured value by the light intensity and the term 
(.times.50) is a factor for convenience to facilitate the comparison of 
the data with the measured value of the present invention. 
TABLE 1 
______________________________________ 
No. R.sub.old (/sec) 
R.sub.new (/sec) 
______________________________________ 
1 0.232 0.256 
2 0.267 0.257 
3 0.276 0.259 
4 0.277 0.256 
5 0.280 0.256 
6 0.270 0.248 
7 0.236 0.224 
8 0.291 0.250 
9 0.267 0.253 
10 0.285 0.252 
Average 0.268 0.251 
CV 6.9% 3.8% 
______________________________________ 
Note: 
R.sub.old : Measured values by a conventional method 
R.sub.new : Measured values by the method of the invention 
CV: Coefficient of variation 
TABLE 2 
______________________________________ 
No. R.sub.old (/sec) 
R.sub.new (/sec) 
______________________________________ 
11 0.120 .times. 10.sup.-3 
0.157 .times. 10.sup.-3 
12 0.098 .times. 10.sup.-3 
0.082 .times. 10.sup.-3 
13 0.733 .times. 10.sup.-3 
0.315 .times. 10.sup.-3 
14 0.099 .times. 10.sup.-3 
0.129 .times. 10.sup.-3 
15 -1.05 .times. 10.sup.-3 
0.023 .times. 10.sup.-3 
16 -0.112 .times. 10.sup.-3 
0.119 .times. 10.sup.-3 
17 0.131 .times. 10.sup.-3 
0.134 .times. 10.sup.-3 
18 0.175 .times. 10.sup.-3 
0.026 .times. 10.sup.-3 
19 -0.289 .times. 10.sup.-3 
0.038 .times. 10.sup.-3 
Average -0.0105 .times. 10.sup.-3 
0.144 .times. 10.sup.-3 
SD 0.0449 .times. 10.sup.-3 
0.0998 .times. 10.sup.-3 
______________________________________ 
Note: 
R.sub.old : Measured values by a conventional method 
R.sub.new : Measured values by the method of the invention 
SD: Standard deviation 
From the results in Table 1, the method of the present invention is 
confirmed to give measurement results with a smaller coefficient of 
variation (CV value) and higher accuracy than the conventional method. 
From the results in Table 2, the method of the present invention is 
confirmed to give much smaller standard deviations (SD value) than the 
conventional method. 
To show clearly the variation of the measured values caused by air-blowing 
just before the measurement, for the case where the alkaline phosphatase 
is added, the calculation results of the present invention are shown in 
(A) and (B) in FIG. 8, and the calculation results of the conventional 
method, as (a) and (b) in FIG. 8; and for the case where the alkaline 
phosphatase is not added the calculation results of the present invention 
are shown in (C) and (D) in FIG. 8, and the calculation results of the 
conventional method, as (c) and (d) in FIG. 9. From these Figs., the 
calculation results are greatly disturbed in the conventional method (a to 
d) at the point of time of air-blowing, while, the calculation results of 
the present invention (A to D) are not affected such disturbance. 
As described above, the method of assay of enzymatic activity and the 
apparatus therefor of the present invention are advantageous in that 
highly precise measurement is made possible with extremely little error 
caused by bubbles in the sample and meniscus of the sample in the cases 
where highly precise assay of enzymatic activity is required such as in 
enzyme labeled immunoassay and enzyme labeled DNA hybridization. 
The method of assay of enzymatic activity and the apparatus therefor are 
also advantageous in that the substrate dispensing means, the agitation 
means for enzymatic reaction, and other means which had to be designed and 
handled carefully to decrease the measurement error in automated 
apparatuses for enzyme labeled immunoassay and enzyme labeled DNA 
hybridization can be simply constructed. 
In an example of a conventional type of measuring apparatus, the measured 
fluorescence values are corrected by a sensor which detects the 
excitation-light intensity in consideration of deterioration with time of 
the lamp to emit the exciting light: for example, the correction being 
such that the detected fluorescence value is divided by emitted light 
intensity. In such a system, variation of light flux introduced to the 
sample container caused by variation of the relative position of the 
sample container to the lamp cannot be corrected, even though the 
deterioration with time of the intensity of the light emitted by the lamp 
can be corrected. On the contrary in the present invention, the 
measurement value is derived as the ratio of two fluorescence intensities 
at one point of time, so that the intensity of the light source does not 
directly affect the measurement results, and the measurement precision is 
greatly improved advantageously. 
As described above, in the present invention, influence of external 
disturbance is excluded, and high sensitivity of the measurement can be 
realized.