JPS6146116B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel highly sensitive method for measuring peroxidase activity by fluorescence analysis. Peroxidase is an enzyme that catalyzes the oxidation reaction of various hydrogen donors in the presence of peroxides such as hydrogen peroxide (H ₂ O ₂ ), and various peroxidases derived from plant and animal tissues and peroxidases produced by microorganisms are already known. It is being The peroxidases used in the present invention include these various types of peroxidases, but among them, peroxidases derived from plants are the main target, and in particular, peroxidase derived from horseradish (hereinafter abbreviated as HRP) is commonly used. The most representative. Among plant peroxidases, HRP has been extracted, separated, and purified for the longest time, and has been used in biochemical analysis methods for the direct determination of substrates such as H ₂ O ₂ and hydrogen donors, as well as sugars, amino acids, organic bases, and cholesterol. , polyamines, etc.
It is widely used in the field of clinical chemistry, aiming at measuring trace amounts of the target substance by combining the oxidase reaction that generates H ₂ O ₂ and the quantification of H ₂ O ₂ generated by peroxidase. Therefore, by being able to measure peroxidase activity with high sensitivity, it is possible not only to detect peroxidase itself with high sensitivity, but also to measure trace amounts of the substrate (H ₂ O ₂ or hydrogen donor) involved in this reaction, and even more importantly, to detect the peroxidase itself with high _sensitivity . It has become possible to measure ultra-trace amounts of the above-mentioned target analyte based on the micro-measurement described in ₂ , which is of great significance in terms of clinical diagnosis.
On the other hand, in the enzyme immunoassay method that has developed rapidly in recent years as a means for measuring ultra-trace amounts of various trace components in living organisms, peroxidase such as HRP is often used as an enzyme for labeling antigens or antibodies. At this time, in the enzyme immunoassay method, as is clear from the principle of the measurement system, if the activity of the labeled enzyme can be measured with high sensitivity, it is possible to measure the amount of the labeled enzyme in trace amounts, and as a result, it is possible to measure the amount of the labeled enzyme. Ultra-trace quantification of various antigens and antibodies is achieved. Establishing a highly sensitive method for measuring peroxidase activity in this way is important in the field of enzyme immunochemical measurement, in combination with antigen-antibody reactions, as well as in the field of simple lifetime chemical analysis in combination with oxidases, as mentioned above. This enables a wide range of applications such as Conventionally, visible absorption spectrometry has been exclusively used as a method for measuring the activity of various peroxidases including HRP, but a common characteristic of visible absorption spectrometry is that its measurement sensitivity is quite low. Meanwhile, Guilbault et al. reported a new fluorescence analysis method using homovanillic acid as a substrate [Guilbault et al.
-bault, GGetal: Anal, Chem39 (No. 2), 271
(1967), ibid, 40 (No. 1), 190-196 (1968),
ibid, 40 (No. 8), 1256-1263 (1968)]. This fluorescence analysis method converts homovanillic acid, a stable non-fluorescent substrate, into a highly fluorescent substance by the coexistence of H ₂ O ₂ in a neutral to weakly alkaline solution, and then converts the substance into a highly fluorescent substance. Peroxidase activity is determined by measuring intensity, and although the measurement sensitivity is generally higher than when using conventional visible absorption spectroscopy, it is still insufficient in many of the aforementioned cases using peroxidase. It was hot. Under these circumstances, the present inventor has conducted extensive research in order to improve the shortcomings of the known methods, and as a result, the present inventor has developed a method that improves the conventional method by conducting the enzyme reaction under acidic conditions when measuring peroxidase activity by fluorescence analysis. We have previously completed a new method for measuring peroxidase activity that is relatively sensitive and accurate (Japanese Patent Application No. 52-66518). In addition to these prior inventions, by improving the quantitative ratio of the two types of substrates, we have created a reaction solution containing a substrate in which the molar ratio of hydrogen donor and peroxide is 1:0.02 to 1. We have completed the present invention by discovering that peroxidase activity can be measured with extremely high sensitivity, accuracy, and reproducibility by carrying out an enzyme reaction in a sterile medium, and that extremely stable results can be obtained. The enzyme reaction in the present invention is carried out under acidic conditions, but the pH in this case only needs to be 7 or less, and in order to expect the best measurement results, it is preferable to select a specific pH range under acidic conditions. Usually, a pH of 3.5 to 5.0 is adopted due to the relationship with the type of acid used to prepare the buffer solution for setting the specific pH range. In this case, the present invention can be applied regardless of the types of acids and alkalis used as materials for buffer preparation, or their mutual combination. The molar ratio of hydrogen donor to peroxide used in the present invention is 1:0.02 to 1, and within this range, it is particularly preferably about 0.04 to 0.5. In addition, the enzyme reaction solution contains the hydrogen donor at or near saturation, while the peroxide
It is preferable to contain 0.5 μg since peroxidase activity can be measured with higher sensitivity. Hydrogen donors used in the present invention include homovanillic acid, P-oxyphenylacetic acid, P-
Cresol, tyramine, etc., while peroxides
H ₂ O ₂ , alcohol peroxide, etc. can be selected as appropriate from those used in the field of this fluorescence analysis method.
Usually homovanillic acid and H ₂ O ₂ are used. Hereinafter, the present invention will be explained in detail with reference to Examples. Example 1 Correlation between HRP amount and optimal amount of H ₂ O ₂ 0.1M-citric acid- _{Ma 2} B ₄ O ₇ buffer (PH4.0) 50
μl, HRP (Type Vi, RZ: approx. 2.75, activity: approx.
260 Purpurogallin Units/mg, Sigma Chemical
Co., Ltd. (purchased from the United States) enzyme solution of 1×10 ⁰ to 1×10 ^-3 units/ml 10μ, bovine serum albumin 12.5
mg/ml aqueous solution 20μ, 0.25 homovanillic acid aqueous solution
After incubation for 40 minutes at 27.7°C with gentle shaking, a reaction mixture consisting of 50μ and 50μ of an aqueous solution containing the following amount of H ₂ O ₂ was 1.25%-
The reaction was stopped by adding 50μ of KCN solution, and 0.1N-NaOH2.5 was further added, and the fluorescence intensity was measured using a Hitachi spectrofluorescence temperature model 204S (excitation wavelength 320nm, fluorescence wavelength 425 nm), the results shown in Table 1 were obtained. At this time, for each amount of HRP used, the fluorescence intensity shown in the H ₂ O ₂ 25 μ area was set as 100, and that of the other areas was expressed as a relative value.

[Table] Example 2 Correlation between chemically modified HRP specimen and optimal amount of H ₂ O ₂ HRP (Basic Isoenzyme Grade 1-c,
RZ: 3.4, Activity: 360 Purpurogallin Units/mg.
(Purchased from Toyobo Seki KK) 2× ^-3 unit of 10μ
, this HRP preparation was subjected to the chemical modification method of PKNaKane et al. [PKNaKane, A.Kawaoi: J.Histochem,
Cytochem, 22.1084-1091 (1974), Yasuyuki Kawai
PKNakane; Protein Nucleic Acid Enzyme 20 (No.11) , 1007~
1013 (1975)], 10μ of an aldehyde-type preparation of DNP-modified HRP (approximately 1.5 HRP activity)
Examples include 10μ of HRP.TSH complex (containing approximately 2.1×10 ^-3 units as HRP activity) prepared by reacting this aldehyde form with TSH ^, a peptide hormone. The same operation as in 1 was performed to obtain the results shown in Table 2.

[Table] Example 3 Amount of hydrogen donor and HRP activity Homovanillic acid was selected as a representative hydrogen donor, and 50μ of its aqueous solution (including the amount shown below) was combined with the amount of H ₂ O ₂ , the other substrate, at two levels. The other operations were the same as in Example 1, and the results shown in Table 3 were obtained.

[Table] Example 4 Combination of mutually optimal amounts of hydrogen donor and H ₂ O ₂ 10μ of the aldehyde type preparation of DNP-modified HRP used in Example 2 (containing 2 × 10 ^-4 units of HRP activity), and HRP.TSH complex 10μ (HRP
The same procedure as in Example 1 was carried out, except that the peroxidase enzyme source (containing 3 × 10 ^-4 units as active) was used as a peroxidase enzyme source, and the amount of homovanillic acid as a hydrogen donor and the amount of H ₂ O ₂ as a peroxide were appropriately combined. The results shown in Table 4 were obtained.

[Table] Example 5 Optimum _amount of _H2O2 for various hydrogen donors 0.1M-citric acid-NaoH buffer (PH4.0) 50μ
and HRP10μ (containing 5 × 10 ^-4 units), 50μ of a 13.72mM solution of one of the various hydrogen donors shown in the following table (concentration equivalent to 0.25% solution of homovanillic acid shown in Example 1), and 2 The same operation as in Example 1 was performed except that the H ₂ O ₂ amounts were combined to obtain the results shown in Table 5 (HRP activity ratio). At this time, since the fluorescent substance produced by the enzyme action differs depending on the type of hydrogen donor used, the excitation wavelength (λex) and fluorescence wavelength that give the maximum value for each type of hydrogen donor are determined. (λem) was determined in advance and then the fluorescence intensity was measured.

[Table] As is clear from the results in Tables 1 to 5, the improvement effect of the quantitative ratio of two types of substrates and the concentration used when measuring peroxidase activity is greater when the enzyme action is carried out under the acidic pH range. As long as it has a buffering capacity that can maintain an optimum pH during enzymatic action, it will be effective regardless of the type of acids and alkalis used to prepare the buffer. The two types of substrates used in this case include various hydrogen donors such as homovanillic acid and p-oxyphenylacetic acid, oxyaromatic compounds such as p-cresol, aromatic amines such as tyramine, and H ₂ O ₂ . A variety of peroxides are useful, including any substrate and any combination of hydrogen donor and peroxide that can be used in the field of fluorometry. As a practical example, in general, the hydrogen donor is kept at a concentration close to saturation, while the amount of the other H ₂ O ₂ is approximately 0.5 to 2.5 μg in each test tube. The best measurement results can be expected if the enzymatic reaction is carried out in a reaction solution in which the molar ratio of peroxide to peroxide is 1:0.04 to 0.05. The phenomenon of increasing the sensitivity of peroxidase activity measurement by improving the quantitative ratio and concentration of the two types of substrates that constitute the constituent factors of the present invention is due to the RZ value, which reflects the source of the enzyme and its degree of purification. Various peroxidases and their respective isozymes, regardless of molecular group heme proteins, as well as native peroxidase itself, artificially chemically modified preparations, peroxidase labeled with various antigens and antibodies, and immobilization. The same is true for peroxidase. In particular, even if native peroxidase is used, the effects of the present invention will be even greater when the amount used is extremely small, when it is chemically modified or otherwise labeled, or even when it is immobilized on an insoluble carrier. It's large. On the other hand, if the present invention is appropriately combined with other operational measures in conventional fluorescence analysis methods, such as micronization of cells used in measuring fluorescence intensity, peroxidase activity can be detected even in extremely small amounts of samples. Measurable. Therefore, by implementing the present invention in addition to the previous application, peroxidase activity and various biological trace components can be measured more easily by simply using an ordinary type of fluorescence analyzer installed in ordinary examination rooms. The clinical significance of the present invention is extremely great because it allows measurements to be made with better accuracy, sensitivity, reproducibility, and stability within a short period of time with simple operations.

Claims (1)

[Claims] 1. When measuring peroxidase activity by fluorescence analysis, an enzyme reaction is carried out under acidic conditions in a reaction solution containing a substrate with a molar ratio of hydrogen donor to peroxide of 1:0.02 to 1. A method for measuring peroxidase activity, characterized in that the method comprises: 2. The method according to claim 1, wherein the reaction solution is a solution containing the hydrogen donor at or near saturation. 3. The method according to claim 1, wherein the reaction solution is a solution containing 25 to 0.5 μg of peroxide.