Chemiluminescent Detection, Quantification, and Activity Measurement Method of Peroxidase

Abstract

To provide a peroxidase detection, quantitation, and activity measurement method that can perform detection with higher sensitivity without preparing a special enhancer.SOLUTION: The present invention is a peroxidase activity measurement method using luminols and hydrogen peroxide as substrates. The peroxidase detection, quantitation, and activity measurement method includes dissolving a substance generating hydrophobic characteristics, such as high-concentration ammonium sulfate.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for detecting, quantifying, and measuring the activity of peroxidase using luminol as a substrate.

Background Art

[0002] In order to measure an antigen-antibody reaction, it is necessary to distinguish between an antigen-antibody conjugate and unreacted antigen and antibody. A method used for this is to bind a labeling substance to an antigen or antibody and measure the amount of the antigen or antibody by detecting the labeling substance after the reaction. Known labeling substances include radioisotopes, enzymes, phosphors, luminophores, chromogens, metal complexes, electrochemically active substances, and the like. In the method using an enzyme as a labeling agent, peroxidase or alkaline phosphatase is labeled, and quantification can be performed by color development (change in absorbance) using a chromogenic substrate. Also, even with the same labeled enzyme, chemiluminescence enzyme immunoassay (CLEIA) and fluorescent enzyme immunoassay (FEIA) have been developed, which measure using luminescence or fluorescence as an index by changing to a luminescent substrate or a fluorescent substrate. On the other hand, there is chemiluminescent immunoassay (CLIA) that uses a labeling agent that induces chemiluminescence without using an enzyme that is easily affected by temperature changes or the like. For example, luminol and Ru complexes are used as luminescent molecules. There is also electrochemiluminescent immunoassay (ECLIA) that applies a predetermined voltage using an electrode to induce chemiluminescence. All of these have already been put into practical use as highly sensitive measurement methods.

[0003] Even in the above measurement method, chemiluminescence of the luminol type catalyzed by peroxidase is particularly often used. Luminol emits light through an intermediate in the presence of hydrogen peroxide to produce a luminescent species. It has been discovered (Non-Patent Documents 1 to 4) that this chemiluminescence is enhanced by adding a phenol having a substituent at the p-position, such as p-iodophenol, which is an enhancer. Since then, various improved methods have been developed and it has come to be called Enhanced Chemiluminescence (ECL). The characteristics of ECL are as follows. In a weakly basic aqueous solution, p-iodophenoxyl radicals are efficiently generated by the reaction of the peroxidase intermediate in the 2-electron oxidation state generated by the reaction of peroxidase and hydrogen peroxide with a p-substituted phenol compound. The generated phenoxyl radicals efficiently react with luminol present as the luminol monoanion to be converted into a luminol diazaquinone intermediate. The luminol diazaquinone intermediate then reacts with an excess of hydrogen peroxide, and after passing through several intermediates, the final product, excited 3-aminophthalic acid, is generated and emits light during its relaxation process. In this way, the enhanced luminescence of luminol is induced by the action of the p-substituted phenol derivative. That is, in the chemiluminescence detection of peroxidase, the reason why the p-substituted phenol compound is useful as a luminescence enhancer (enhancer) is that the phenoxyl radicals efficiently produced by peroxidase accelerate the formation of the key intermediate in the luminescence reaction, the luminol diazaquinone intermediate.

[0004] In addition, excellent sensitizers to replace p-iodophenol have also been studied (Patent Documents 1, 2).

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

[0006] [Patent Document 1] Japanese Patent Application Laid - Open No. 2011 - 43447 [Patent Document 2] Japanese Patent Application Laid - Open No. 2 - 291299 [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] However, although the chemiluminescence method using these enhancers can obtain continuous luminescence and achieve a certain degree of high - sensitivity, it requires the preparation of special enhancers and also requires an organic solvent to dissolve the slightly water - soluble enhancer. Moreover, further sensitivity is required for the measurement of trace components. [Means for Solving the Problems]

[0008] As a result of intensive research in pursuit of further high - sensitivity, the present inventor has found that in a method of using luminols and hydrogen peroxide as substrates for detecting and quantifying peroxidase by utilizing chemiluminescence, the sensitivity is improved by dissolving a substance that produces hydrophobic properties in the luminol reaction, which is an aqueous reaction, and thus completed the present invention. That is, the present invention has found that a substance that is water-soluble but produces hydrophobic properties when added promotes the coordination reaction of hydrogen peroxide to the enzyme peroxidase active site and the nucleophilic substitution reaction of hydrogen peroxide to the luminol azakino intermediate, thus leading to the present invention. As the substance that produces hydrophobic properties used in the present invention, when dissolved, it preferably forms salts with strong salting-out effects, such as ammonium ions, magnesium ions, sodium ions, etc. as cations, and sulfate ions, acetate ions, iodide ions, and chloride ions as anions, that is, anti-chaotropic ions. Among the cations, the effect of ammonium is particularly large, and examples of salts containing ammonium ions include ammonium sulfate and ammonium acetate. On the other hand, focusing on the anions, examples of salts containing sulfate ions include ammonium sulfate, sodium sulfate, and magnesium sulfate; examples of salts containing acetate ions include ammonium acetate and sodium acetate; and examples of salts containing chloride ions include ammonium chloride and sodium chloride. Among these substances, ammonium sulfate is particularly preferred. In addition to the above-mentioned inorganic salts, substances that form a micro-hydrophobic environment, such as glycerin and polyethylene glycol, may also be used.

[0009] When using ammonium sulfate, a high concentration of ammonium sulfate is preferred, and the concentration is 2.8 M or more, preferably 3.0 - 4.0 M, and more preferably 3 - 3.5 M. From the perspective of solubility, around 3.5 M is practically suitable. In the present invention, it is also preferable to use ethylenediaminetetraacetic acid (EDTA) together with ammonium sulfate. By dissolving EDTA, the background luminescence can be almost completely suppressed. The amount of EDTA added depends on the concentration of ammonium sulfate. For example, when the ammonium sulfate concentration is 3.0 - 3.5 M, it is 60.0 ppm or more and 5000 ppm or less. Preferably, it is 62.5 ppm - 500 ppm.

[0010] The chemiluminescence measurement method in the present invention is not particularly limited to the measurement object and measurement method as long as peroxidase is used as an enzyme and chemiluminescent substrates, oxidants, and salts that generate anti-cationotropic ions are used for the detection, quantification, and activity measurement of peroxidase. For example, as a specific binding reaction system using peroxidase as a labeled enzyme, it can be used in various analysis methods such as the primary antibody method, secondary antibody method, competitive analysis method, sandwich analysis method, homogeneous analysis method, heterogeneous analysis method, Western blot analysis method, and DNA probe method of enzyme immunoassay.

[0011] The peroxidase (POD) used in the present invention is not particularly limited, and examples include peroxidase extracted from horseradish, microorganisms, milk, white blood cells, etc. Among these, preferred are horseradish peroxidase and horseradish peroxidase (HRP). Also, peroxidase may be in a free state or in a complex state bound to a ligand (for example, antigen, antibody, hapten, protein A, avidin, biotin, etc.) used in immunoassay.

[0012] In the method of the present invention, a suitable amount of POD to be used is an amount necessary for the relationship between the amount of POD and the amount of chemiluminescence to be a linear relationship or a relationship close to linear. A particularly suitable amount of POD to be used is to be present in an amount in the range of pM to nM during the luminescence reaction. If the amount of POD is less than this range, the effect of the present invention will decrease. When the concentration of POD is high, the conventional luminol chemiluminescence is sufficient, and there is no need to use highly sensitive luminol chemiluminescence.

[0013] POD may be dissolved in a free state in a solution, or POD may be bound to an insoluble carrier. As the insoluble carrier, conventionally known substances can be used. That is, beads, tubes, fine particles, etc. formed of polymers such as polystyrene. Also, as a method for binding POD to an insoluble carrier, any of the conventionally known physical or chemical methods can be used, and there is no particular limitation.

[0014] Luminols, which are chemiluminescent substrates used in chemiluminescent reactions, preferably specifically include luminol, isoluminol, N-ethylisoluminol, N-(4-aminobutyl)-N-ethylisoluminol hemisuccinimide, N-(6-aminohexyl)-N-ethylisoluminol, 6-[N-(4-aminobutyl)-N-methylamino]-2,3-dihydro-1,4-phthalazinedione, and the like. Among them, luminol or isoluminol is preferred, and luminol is particularly preferred. Since commercially available reagent-grade luminol often contains hydrazine and sulfide ions, which are raw materials for production, it is preferable to use luminol that has been repeatedly recrystallized and purified.

[0015] In the present invention, the luminescence reaction is preferably carried out in a weakly basic solution, and particularly preferably at pH 7-9. Any type of buffer solution can be used as long as it satisfies the above pH. Preferred examples include phosphate buffer, glycine / NaOH buffer, Tris / HCl buffer, Tris / acetic acid buffer, carbonate buffer, barbital buffer, borate buffer, and the like.

[0016] The principle of the present invention is considered as follows. The outline of the luminol-HRP-H2O2 reaction is shown in Figure 5. First, in the present invention, it is considered that the protoheme iron of POD forms a low-spin complex in a system where high-concentration ammonium sulfate exists, which easily forms a coordination bond with the lone pair of electrons on the oxygen atom of hydrogen peroxide. From the spectroscopic data obtained so far, it is known that the protoheme iron of POD is usually in a high-spin state with five coordination in the resting state. In the absence of ammonium sulfate, it remains in the high-spin state regardless of pH, but in the presence of ammonium sulfate, it shifts to the low-spin form at weakly basic pH. That is, usually a high-spin complex has a lower coordination bond efficiency compared to a low-spin complex, but in the present invention, by shifting to the low-spin form, an effect of enhancing the reaction efficiency, that is, promoting the coordination bond to the protoheme iron at the active center, occurs. This is considered to be effective for the reaction of H2O2 and HRP in the first stage (First step of the reaction of HRP with H2O2). Next, in the presence of high-concentration ammonium sulfate, many water molecules form hydrogen bonds with high-concentration sulfate ions and ammonium ions, stabilizing the structure of water. Despite being an aqueous solution, a so-called hydrophobic microenvironment is formed, and as a result, it is considered that the efficiency of the nucleophilic reaction (Addition of HOOH(H2O2),followed by the formation of luminol dioxetane product) of the lone pair of electrons on the oxygen atom of H2O2 to the luminol diazaquinone intermediate is increased. The micro-hydrophobic environment, of course, also has an effect on the reaction of First step of the reaction of HRP with H2O2, removing the water molecules at the active site, and this effect also promotes the coordination bond.

[0017] The present invention also provides a POD detection / quantification / activity measurement solution comprising luminol, hydrogen peroxide, POD, and ammonium sulfate. The concentration of ammonium sulfate is 2.8 M or more as described above, and preferably 3.0 M to 3.5 M. The concentrations of luminol and hydrogen peroxide need to be set to optimal values for the quantification of POD. For example, when POD is in the range of pM to nM, hydrogen peroxide at 10 mM to 100 mM and luminol at 2 mM to 10 mM are preferred. Also, the pH is set to an optimal value of 8 to 9 for the detection / quantification of POD.

Effects of the Invention

[0018] According to the present invention, highly sensitive analysis of POD can be performed without using an enhancer.

Brief Description of the Drawings

[0019]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0020] Hereinafter, the present invention will be described in detail. The general conditions in the experiments of the present invention are as follows. For the reaction, first, 1 μL of 1×10 -6 M to 1×10 -9 M of HRP was placed in a 1 mL cuvette, and then 1000 μL of a solution obtained by mixing equal amounts of a luminol solution and a hydrogen peroxide solution was added to start the reaction. In particular, the concentration of HRP in the reaction solution used for the evaluation of the present invention was 1×10 -7 M and 5×10 -9 M. In this case, the HRP concentrations in the reaction mixtures were 1×10 -10 M or 5×10 -12It is M. The above luminol solution is a mixed solution of 1 vol of 0.75 M sodium hydroxide containing 30 mM luminol and 5 vol of a pH 8.5 tris(hydroxymethyl)aminomethane (Tris) (0.1 M) buffer solution containing various concentrations of ammonium sulfate. The feature of this luminol solution is that it is designed so that the pH of the finally obtained reaction mixture becomes optimal. On the other hand, the hydrogen peroxide solution is an aqueous ammonium sulfate solution containing various concentrations of 100 mM hydrogen peroxide (the ammonium sulfate concentration is the same as the concentration used in the preparation of the luminol solution). The pH of the reaction mixed solution is 8.3 - 8.8. However, the pH of the system without ammonium sulfate is about 12 because the pH buffering action by high-concentration ammonium sulfate does not work. In the evaluation of the effect of ammonium sulfate (AS), in the above reaction system, the chemiluminescence of luminol was examined by changing the ammonium sulfate (AS) concentration (M). Specifically, the luminescence intensity was recorded and evaluated as a luminescence spectrum with the reaction time as a variable. The measurement of the luminescence spectrum was recorded 5 times at 1-minute intervals starting from 10 s (0.17 min) after the start of the reaction, including the immediately following one. That is, 0.17 min, a00; 1.17 min, a01; 2.17 min, a02; 3.17 min, a03; 4.17 min; the third-digit number was denoted as a. For example, in Figure 1, 0420!900 → 0.17 min, 901 → 1.17 min, 902 → 2.17 min, 903 → 3.17 min, 904 → 4.17 min are represented. Figure 1(a) shows the emission spectra when AS = 0.0 M, (b) when AS = 0.9 M, (c) when AS = 1.8 M, (d) when AS = 2.3 M, (e) when AS = 2.5 M, (f) when AS = 2.8 M, (g) when AS = 3.0 M, and (h) when AS = 3.2 M is added. As shown in Figure 1, it can be seen that as the concentration of AS increases, the intensity of the emission spectrum increases. In the system containing 3.2 M ammonium sulfate, the intensity increases by about 100 times compared to the system without it. In the figure, in the system without adding ammonium sulfate, weak luminescence is observed, but under this condition, as described above, the pH is high and iron(III) ions are released from HRP, and it is considered that this is due to the manifestation of its catalytic action, and it is not the luminescence based on the catalytic action of HRP targeted here. By adjusting the concentrations of the above luminol solution and hydrogen peroxide solution, an increase in intensity of about 200 times is considered possible.

[0021] Figure 2 is a plot of the chemiluminescence intensity of luminol vs. the time after the start of the reaction under various ammonium sulfate (AS) concentration conditions. The area of each spectrum is used as the integrated intensity, and the time after the start of the reaction is used as the variable. It can be seen that when the AS concentration is 3.0 M or more, the chemiluminescence intensity particularly increases. It can be seen from Figure 2 that when the ammonium sulfate concentration is 2.8 M or more, this effect becomes significant. That is, it can be said that there is a threshold for the manifestation of the effect. This result indicates that the effect of ammonium sulfate is significantly involved in the reaction mechanism. That is, it can be concluded that high-concentration ammonium sulfate contributes to the increase in the overall reaction rate by promoting the two types of reaction paths as described above, resulting in an increase in the luminescence intensity. Figure 3 shows the results of detecting and quantifying HRP under the optimal conditions (concentrations in the reaction mixture; AS = 3.2 M, luminol = 2.5 mM, H2O2 = 50 mM). HRP at the pM level can be clearly detected and quantified with a high S / N ratio. It can be clearly detected and quantified with a [S / N] ratio.

[0022] Next, the effect of the present invention was examined by further dissolving EDTA in ammonium sulfate. The concentrations (common to all) in the reaction mixture were ammonium sulfate = 3.2 M, luminol = 2.5 mM, and H2O2 = 50 mM. The EDTA concentration in the reaction mixture is shown in Fig. 4. The graphs in the left column ((a) column) of Fig. 4 contain the enzyme HRP (= 1 × 10 -10 M), while the graphs in the right column ((b) column) do not contain HRP. It can be seen that in the system where EDTA is dissolved, the background luminescence is almost completely suppressed. Therefore, the luminescence of the left graph can be regarded as the luminescence due to the catalysis of HRP. The lowermost graph in the right column is obtained from the system without adding EDTA. Luminescence is observed even though there is no HRP. This is presumably due to the influence of impurity metal ions contained in ammonium sulfate. This background is almost completely eliminated by the masking effect of impurity metal ions by EDTA originally added as a stabilizer for hydrogen peroxide. Furthermore, it is clear that EDTA does not have a negative effect on the effect of ammonium sulfate on the luminol-H2O2-HRP chemiluminescence.

[0023]

Industrial Applicability

[0024] According to the present invention, it can be used for the detection and quantification of peroxidase or the chemiluminescence assay of its activity, which is useful for the detection of all types of analytes, such as biological macromolecules and organic molecules. In addition, the present invention can be applied to the detection of proteins and nucleic acids using assays on a membrane substrate, such as dot blotting, Western blotting, and Southern blotting.

Claims

【Claim 1】 A method for detecting, quantifying, and measuring the activity of peroxidase using luminols and hydrogen peroxide as substrates, characterized in that ammonium sulfate with a concentration of 3.0 M to 3.5 M is dissolved.

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