1. Field of the Invention
The present invention relates to a method for quantitative measurement of a trace enzyme, and particularly to a method for photochemical measurement of the activity of a trace enzyme and a substrate used therein.
2. Prior Art Statement
Various methods have hitherto been developed for measuring the activity of a trace enzyme. For example, there are: a turbidimetric method wherein the decrease in turbidity caused by an enzyme reaction is traced using a suspension of high molecular weight substrate; an absorptiometric method wherein a high molecular weight substrate is decomposed or cleaved by an enzyme and, after precipitating and removing undecomposed substrate, soluble component is determined by an absorbance measurement; a method wherein a dye or fluorescent substance is previously bound to a high molecular weight substrate, an enzymatic reaction is effected to decrease the molecular weight of the dye or fluorescent substance, and the fractionated dye or fluorescent substance of lower molecular weight is measured; and a method of quantitative assay wherein, using a substrate which is designed to change in absorption spectrum, one forms a color or forms a fluorescent substance, based on a splitting-off or change in part of the substrate after an enzymatic reaction, and the resulting absorbancy or fluorescent intensity is measured, etc. (SEIKAGAKU JIKKEN KOZA (Lectures on Biochemical Experiments), vol. 5, subtitled "Study on Enzymes", edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin, 1975.)
Most of these methods, however, quantitatively determine an amount of enzyme on the order of, .mu.g/ml. Even utilizing a type of substrate releasing a fluorescent substance (e.g. derivatives of umbelliferone, etc.), which is recognized to be most sensitive among these conventional methods, it is only possible to measure an enzyme quantity on the order of ng/ml.
Therefore, in activity measurements of trace enzymes labelled in accordance with an enzyme immunoassay, the development of more stable and more highly sensitive enzyme activity measurement methods has been demanded.
Further, since the enzymes as biocomponents in blood, body fluids, urine and in tissues in the living body such as various organs, the brain, etc. mostly are present in a very small amount, except for certain enzymes (amylase, GOT, GTP, etc.) which exist in a large amount, such enzymes cannot be determined by conventional measurement methods. Therefore, a radioimmunoassay (hereinafter referred to as "RIA") which is an immunological measurement method using a radioactive isotope has recently been introduced. The principle of RIA is described in, for example, Kumahara and Shizume, RADIOIMMUNOASSAY, New Edition, pages 3 to 10 (1977), published by Asakura Publishing Co., Ltd., KISO SEIKAGAKU JIKKENHO (Basic Biochemical Experiments) (6), Biochemical Assay (1967), published by Maruzen Co., Ltd., Tokyo, METHODS IN ENZYMOLOGY, edited by Sidney P. Colowick et al., vols. I, II, III, V and VII, published by Academic Press, New York and The Enzyme, vols. 3, 4 and 5, Paul D. Boyer et al. (1071), published by Academic Press, New York.
However, RIA as a quantitative assay for enzymes has disadvantages such as: (1) since it is an immunoassay which detects a quantity of enzyme, there is a possibility that the activity of an enzyme -which is the functional characteristic of the enzyme- will not be actually reflected; (2) there is a possibility that analogous enzymes and precursors having a similar antigenic site might be included in analytical data; and (3) in the case where the enzyme to be measured, for example, such a label enzyme used for the enzyme immunoassay, is bound to an other component and not present in the free state, it is difficult to prepare an antibody against the aforesaid labelled antigen or antibody and the design for a method for measurement is practically difficult.
RIA has further disadvantages due to the use of radioisotopes. That is: (1) potential injury to the person dealing with radiation is a matter of concern; (2) special places and controls are required for storage and waste disposal of the radioactive substances used; (3) the amount of radiation from the radioisotope is reduced with the passage of time due to the half decay of the isotope; and (4) measurement of the amount of radioactivity requires expensive devices.
The information obtained by measuring enzyme activity using a specific substrate under given reaction conditions (e.g. concentration of substrate, total volume, reaction pH, reaction temperature, reaction time, ionic strength, salts co-existent, etc.) is generally classified as follows.
(1) The sum of enzyme activities having a catalytic action on the structure of the substrate in the system; or
(2) Under the condition of constant enzyme concentration, the degree of inactivation depending upon purity of the enzyme, the presence or absence of inhibitors, intensity of inhibition, denaturation, etc., i.e. measure of specific activity.
Finally, the purpose of measuring the activity of an enzyme as a component in the living body is frequently to obtain mainly information per (1) above and the purpose of activity measurements of enzyme-labelled materials exterior living body is to obtain mainly information per (1) and (2) above. As is well known, in any case, specific enzyme to the living body is to obtain mainly information per (1) and (2) above. As is well known, in any case, specific enzyme activity can only be measured by selecting a substrate corresponding to the enzyme specificity.
The term "specificity" used throughout the specification is art-recognized and defines selective reactivity between substances, e.g. of an enzyme with its corresponding substrate.
Japanese Patent Publication Nos. 1118/1986 (U.S. Pat. No. 4,414,325) and 1119/1986 (U.S. Pat. No. 4,414,323) disclose a method for quantitative measurement of a trace enzyme, wherein an enzyme substrate having therein a photographically active material (a spectroscopically sensitizing dye or a fogging agent) as a labelling material is used so that the activity of a trace enzyme is photochemically determined.
The method disclosed in U.S. Pat. No. 4,414,323 is a method for measurement of an emzyme activity and/or a quantity of an enzyme comprising the steps of providing a synthetic substrate comprising at least one photographically active labelling structure (B) serving as a labelling material by contacting it with silver halide grains to form nuclei capable of being developed (a spectroscopically sensitizing dye or a nucleating agent (fogging agent)) and at least one structure (A) to be specifically contacted with the enzyme to be measured; bringing either the reaction product containing the photographically active labelling structure (B) formed by the enzyme reaction or the unreacted synthetic substrate into contact with silver halide followed by development; and measuring the amount of developed silver and/or the amount of colored dye obtained as an optical density.
The method referred to in the preceding paragraph provides not only a very high sensitivity but also is effectively applied to the situation where an enzyme is in the form of a conjugate or complex thereof with other organic materials (e.g. polymers, latexes, microcapsules, membranes including membranes in living bodies and ion exchange membranes, bacteria, microorganisms, components in living bodies such as hormones, peptides, proteins, lipoproteins, glycoproteins, glucosides and lipids, toxic substances, drugs, antibiotics, etc.).
In a method for quantitative determination of an enzyme activity, the sensitivity of the method depends mainly on the following two factors. The first factor is the absolute sensitivity of the substance used for generating the detected signal, and the second factor is how to detect the signal from the product of the enzymatic reaction.
As to the absolute sensitivity limited by the used substance, the limit for detection is an the order of 10.sup.-6 mole/l when a dye is used as the product of the enzymatic reaction, and the limit for detection is an the order of 10.sup.-9 mole/l when a fluorescent material is used as detectable signal. On the other hand, when a photographically active material is used as above-described, the limit for detection is improved to the order of 10.sup.-11 mole/l.
In the actual determination of an enzyme activity while using a photographically active material, the limit for detection depends on the density of the produced signal material which is detectable as having a significant difference from the background. In the colorimetric method wherein a dye is used, the density of the background does not affect the result of determination and the sensitivity of the method is affected only by the molecular absorption coefficient of the used signal material. In the method wherein a fluorescent material is used, the density of the background is often increased by the purity of energized light, dusts contained in the medium, entangled particles, dispersed materials or contamination of other fluorescent materials to lower the sensitivity to the order of 10.sup.-8 mole/l. The sensitivity of the method wherein a photographically active material is used depends on the purity of the product of the enzymatic reaction separated from the unreacted synthetic substrate.
In the method wherein a photographically active material is used, contamination with materials exhibiting similar functions as that of the used photographically active material need not be taken into account in view of the structure of the photographically active material. This is because the background is induced by the contaminating unreacted synthetic substrate in the step of detecting the photographically active material in the product of the enzymatic reaction. In general, a substrate is used in a concentration of from 10.sup.-2 to 10.sup.-6 mole/l in ordinary enzymatic reaction, and the substrate is used in a relatively high concentration when a trace enzyme is to be detected. Although 10.sup.-7 to 10.sup.-9 mole/l of a product of the enzymatic reaction should be detected in the low sensitivity range (10.sup.-7 to 10.sup.-9 mole/l), separation can be effected relatively easily by the conventional methods wherein the differences in chemical and physical properties between the reaction product of the enzymatic reaction and the unreacted synthetic substrate are detected. For example, both components may be separated from each other, utilizing their difference in absorptive property to silver halide or using another proper separation method (for example, ion exchange chromatography, affinity chromatography, high speed liquid chromatography, TLC, salting out, centrifugal separation, co-precipitation with a polymer, decantation, ultrafiltration, use of an adsorbent such as activated charcoal, etc.) The details thereof are described in DATABOOK OF BIOCHEMISTRY, Chapter 10, second separate volume, edited by Japanese Biochemical Society, published by tokyo Kagaku Dojin, 1980.
In the maximum sensitivity range, it is necessary to detect 10.sup.-9 to 10.sup.-11 mole/l of a reaction product of enzymatic reaction. Namely, it becomes necessary to separate 10.sup.-9 to 10.sup.-11 mole/l of a pure reaction product from a solution or suspension containing 10.sup.-2 to 10.sup.-6 mole/l of a synthetic substrate.