JPH0573397B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for quantifying components in biological samples such as blood, urine and other body fluids, and tissue sections in clinical tests or biochemical research. (Prior Art) As methods for analyzing components in biological samples, absorption photometry, fluorescence photometry, electrode methods, and the like have been effectively and widely used. However, each analytical method has a limit in detection sensitivity, and detection is difficult for situations in clinical tests or biochemical research where detection and quantification of components with lower concentrations than in the past are required. There are cases where the sensitivity requirement cannot be met as a law. Detection and quantification methods using the principles of chemiluminescence and bioluminescence have attracted attention because of their high sensitivity. While the sensitivity limit of spectrophotometry is μM, such luminescence methods have a limit of 1/1000 or less. Therefore, this can meet the demand for reducing the amount of sample to be analyzed when the content of the target substance in the sample is low and when the sample is valuable. Furthermore, in the case of the conventional absorption method or fluorescence method, a light source and a device for converting it into monochromatic light are required, and their handling must be strict, but in the case of the luminescence method, the handling is complicated due to the use of such devices. It doesn't require much time. Methods for quantifying hydrogen peroxide using chemiluminescent methods such as the luminol method and the lucigenin method are well known, but these reactions have the disadvantage that they are not sensitive enough to quantify hydrogen peroxide unless they are carried out under alkaline conditions. There is. When this chemiluminescence method is used for biochemical tests in the field of clinical testing, it is strongly influenced by components in biological samples such as serum and urine, which not only weakens the luminescent activity but also causes variations in measured values, making it unreliable. Not placed. For this reason, efforts have been made to dilute the biological sample, but this results in a contradiction in that the sample amount is reduced, which in turn lowers the measurement sensitivity. (Problems to be Solved by the Invention) The present invention provides a method for quantifying trace components, ie, substrates or enzyme activities, in a biological sample using a chemiluminescent method without being interfered with by substances present in the biological sample. . (Means for Solving the Problems) The present invention allows an oxidizing enzyme to act on a substrate in a biological sample or a substance generated by an enzymatic reaction with an enzyme in the biological sample, and then oxidizes the generated hydrogen peroxide under an oxidation catalyst. An oxidizable non-fluorescent substance is reacted to convert the non-fluorescent substance into a fluorescent substance, and then the fluorescent substance is reacted with oxalic acid diester and hydrogen peroxide under conditions of inhibition of the oxidation catalyst. This is a method for quantifying biological components using a chemiluminescent method, which is characterized by quantifying substrate or enzyme activity in a produced sample by measuring the amount of luminescence produced. The present invention consists of the following process. Process: Allowing an enzyme to act on a substrate, which is a biological component, or allowing a substrate to act on an enzyme, which is a biological component, through a single or multiple enzymatic reaction process, and finally causing an oxidizing enzyme to act. A process that produces hydrogen peroxide. Process: A process in which hydrogen peroxide obtained in the process is treated with a non-fluorescent substance and an oxidation catalyst to produce a fluorescent substance. Process: A process in which oxalic acid diester and hydrogen peroxide are applied to the fluorescent substance obtained in the process to generate light and measure the intensity of this light. Biological components include substrates or enzymes contained in blood, urine, saliva, lacrimal fluid, and other body fluids and tissues. Specific examples of substrates in biological samples include uric acid, cholesterol esters, free cholesterol, triglycerides, pretocin, polyamines such as cataverine, spermine, and spermidine, lactate, pyruvate, free fatty acids, creatine, and creatinine. Enzymes in the produced sample include polyamine oxidase (eg pretosine oxidase), cholinesterase, α-amylase, and GPT. Examples of oxidases include the following. Uricase (uric acid), cholesterol oxidase (cholesterol ster, free cholesterol),
glycerophosphate oxidase (triglyceride),
Polyamine oxidase (polyamine), lactate oxidase (lactate), pyruvate oxidase (pyruvate, GPT), acyl-CoA oxidase (free fatty acids), sarcosine oxidase (creatine, creatinine), choline oxidase (cholinesterase), glucose oxidase (α-amylase) ), NAD〓H oxidase (substrates or enzymes known to be measured using a dehydrogenase reaction), etc. In the present invention, various known hydrogen peroxide production reactions can be applied. An example will be shown next, but the present invention is not limited thereto. Production of hydrogen peroxide from uric acid Uric acid + O ₂ + 2H ₂ O uricase ----→ Allantoin + H ₂ O Production of hydrogen peroxide from ₂ cholesterol ester Cholesterol ester + H ₂ O cholesterol esterase -------------------- ―――→ Cholesterol + fatty acid Cholesterol + O ₂ cholesterol oxidase ――――――――――――→ △ ⁴ -Cholestenone + H ₂ O ₂ Production of hydrogen peroxide from putrescine Putrescine + O ₂ + H ₂ O putrescine oxidase ――――――――――――→ 4-aminobutyraldehyde + NH ⁺ ₄ + H ₂ O ₂ Generates peroxide from triglyceride Triglyceride + 3H ₂ O lipase ――――→ Glycerol ⁺ 3 fatty acids Glycerol + ATP glycerol kinase - ――――――――――→ Mg ² + L-α-glycerophosphate + ADP L-α-glycerophosphate + O ₂ glycerophosphate oxidase ――――――――――――→ H ₂ O ₂ + dihydroxy Generation of hydrogen peroxide by the action of acetone phosphate cholinesterase Benzoyl coin cholinesterase――――――――――→ Choline + Benzoic acid Choline + 2O ₂ +H ₂ Ocholine oxidase――――――――――→ Betaine + 2H ₂ Generation of peroxide by O ₂ α-amylase action Maltopentaose + H ₂ Oα-amylase――――――――→ Maltose + Maltotriose Maltose + Maltotriose + 3H ₂ Oα-glucosidase―――――――― ――→ Glucose + O ₂ + H ₂ O glucose oxidase ――――――――――→ H ₂ O ₂ + Gluconic acid The oxidation catalyst used in the process is:
Examples include peroxidase, microperoxidase, hemin, hematin, and the like. Non-fluorescent substances refer to those that are oxidized and converted into fluorescent substances by the hydrogen peroxide produced in the above process and the oxidation catalyst, but include the leuco form of fluorescein, such as leuco-2', 7'- Dichlorofluorescein diacetate or the like or the leuco form of rhodamine B can be suitably used. In addition to these compounds, the reduced forms of leucolhodamine, leucoeosin, etc. are oxidized to produce fluorescent groups, homovanillic acid, p-cresol, 3-(p
-Hydroxyphenyl)propionic acid, tyramine, etc., which undergo oxidative condensation and emit fluorescence in the presence of hydrogen peroxide. Among the leuco substances, leuco-2',7'-dichlorofluorescein diacetate (LDADCF) has particularly excellent stability and can be easily converted into leuco-2, the non-fluorescent substance referred to in the present invention, by hydrolysis with alkali or esterase. It is a suitable compound that can be easily handled as it can be converted into ',7'-dichlorofluorescein (LDCF). Processes and processes generally involve reactions in aqueous systems and can be carried out sequentially or simultaneously. The induction of hydrogen peroxide from a substrate in a sample by an enzymatic reaction, and the induction of hydrogen peroxide by the action of an enzyme in a sample, are known reaction systems as exemplified above, and these can be carried out in one step or in multiple steps.
Although it is carried out by step operations, it is convenient and convenient to carry out at least the final hydrogen peroxide production reaction in a simultaneous operation in conjunction with the process. The oxalic acid diester used in the process is bis(2,4,6-trichlorophenyl)
These include oxalate, bis(2,4-dinitrophenyl) oxalate, and the like. The process of the invention is carried out under conditions of inhibition of the oxidation catalyst used in the process. This is because an excessive amount of hydrogen peroxide is added from outside the reaction system during the process, so if the reaction is carried out without inhibiting the oxidation catalyst, an excessive amount of fluorescent material will be generated in the process.
This will increase the luminescence background. A process is therefore necessarily operationally separate from previous processes and performed sequentially. To inhibit the oxidation catalyst, sodium cyanide,
Known inhibitors such as potassium cyanide, sodium azide, etc. can be used, but the purpose (inhibition) can preferably be achieved by using an organic solvent. This is because it is necessary to carry out the process reaction in an organic solvent containing water in order to increase the amount of light emission, that is, increase the sensitivity. Therefore, organic solvents are selected from the viewpoints of inhibiting the oxidation catalyst and increasing the amount of light emitted. Examples of the organic solvent include ethyl acetate, acetone, and acetonitrile. The organic solvent can be used alone or in combination
It may be a mixture of more than one species. The reaction takes place at a temperature of 20-40℃ and a neutral or alkaline pH.
Do it below. The amount of hydrogen peroxide added in the process is 0.1~
It is 50mM. The amount of luminescence can be measured using a commercially available measuring device equipped with a detector such as a conventional photomultiplier or photocounter. For example, the peak luminescence amount is counted using a Lumiphotometer TD4000 (manufactured by Lab Science). According to the present invention, it is possible to measure a wide variety of substrates or enzymes in biological samples. Examples of substrates include cholesterol esters, cholesterol, glucose, triglycerides, putrescine, cataverine, spermine, polyamines such as spermidine, uric acid, lactate, pyruvate, free fatty acids, creatine, and creatinine, which are known to be measured using an oxidase reaction. There is a substrate that has been treated. Examples of enzymes include enzymes known to be measured by combining oxidase reactions, such as amylase, cholinesterase, and glutamatepyruvate transaminase. In addition, substrates or enzymes known to be measured using dehydrogenase reactions are also NAD(D)H.
It can be measured by using oxidase or by generating hydrogen peroxide under light irradiation using methylene blue from NADH as a sensitizer. Examples of such substrates include triglycerides, bile acids, glucose, and cholesterol, and examples of enzymes include amylase, transaminase, and lactate dehydrogenase. In addition, peroxides present in biological samples, such as lipid peroxides, are led to fluorescent substances by the process without going through any process, and react with oxalate diester and hydrogen peroxide in the process to generate light. Then, measure the luminescence intensity. (Effects of the Invention) According to the present invention, trace components in a biological sample can be quantified with high sensitivity without interference from coexisting substances in the biological sample. That is, in the spectrophotometric method,
10 ^-6 M, 10 -7 M for fluorophotometry, and 10 ^-7 M for electrode method.
While trace components up to 10 ^-6 M can be measured, the present invention can also measure trace components up to 10 ^-9 M. Further, in the present invention, it is possible to measure the absolute amount of trace components without depending on the shape of the measurement cell. Furthermore, the present invention is not affected by turbidity in the sample. (Example) Next, the present invention will be explained with reference to an example. Example 1 Quantification of uric acid Preparation of fluorescent reagent: 5 mg of leuco-2',7'-dichlorofluorescein diacetate was dissolved in 10 ml of ethanol, and 40 ml of 0.01 N NaOH was added to hydrolyze the acetyl group. Add to this 75 ml of 2% β-
25mM phosphate buffer (PH7) containing cyclodextrin was added and stirred, and further 75ml of 25mM phosphate buffer (PH7) was added to bring the total volume to 200ml. This fluorescent substance (non-fluorescent substance) is stable for two days at room temperature. Preparation of enzyme reagents: Horseradish radish peroxidase and uricase were added to 25 mM phosphate buffer PH7 at a concentration of 5 U/ml and 1.5 U/ml in purpurogalin units, respectively. Preparation of luminescence reagent: 0.5m dissolved in ethyl acetate
M bis(2,4,6-trichlorophenyl)oxalate (TCPO) and 2mM dissolved in water
The total amount of H ₂ O ₂ was prepared using acetonitrile. Furthermore, before measurement, it was diluted to 1/5 with acetonitrile and used as a luminescent reagent. Preparation of uric acid: Uric acid was dissolved in 25 mM phosphate buffer in the range of 5 x 10 ^-6 mg/dl to 5 x 10 ^-2 mg/dl, including a control without uric acid. For the measurement procedure, take 3 ml of fluorescent reagent,
Add 1 ml of enzyme solution, add 1 ml of uric acid sample, and add 30
Samples that fluoresced after incubating for 1 hour at ℃
0.5 ml of the sample was taken, 0.5 ml of luminescent reagent was added to it using a syringe, and the peak of luminescence was read using a measuring device.
The results are shown in FIG. The measurement sensitivity is 10 ^-6 mg/dl (5.95×
10 ^-9 M) and ranges from 10 ^-6 mg/dl to ^{10 -3} mg/dl
It was a straight line. Note that by containing acetonitrile in the luminescent reagent, peroxidase in the enzyme reagent is inhibited. Next, we will show the correlation with the absorption method when serum is used as a biological reagent. The spectrophotometric measurement was performed using a commercially available uric acid measuring reagent, Uricolor Ace (manufactured by Toyobo Co., Ltd.). In the measurement according to the present invention, the serum is diluted 100 times in advance, and then 1 ml of the diluted serum solution is added to the reaction system in which the enzyme solution is added to the fluorescence reagent described above (the diluted serum solution is added instead of the uric acid sample), and the following steps are repeated in the same manner. It was measured with As shown in Figure 2, the result is y=0.987x+
A good correlation of 0.098r=0.9991 was obtained. Example 2 Cholinesterase Activity Measurement A fluorescent reagent and a luminescent reagent were prepared in the same manner as in Example 1. Preparation of substrate reagent: benzoylcholine chloride 2.4m
M was dissolved in a N/50 aqueous hydrochloric acid solution. Preparation of enzyme reagent: choline oxidase 5U/ml,
5U/ml of peroxidase was dissolved in 0.1M Tris buffer (PH7.55). Preparation of stop solution: Neostigmine methyl sulfate 12m
M was dissolved in water. Preparation of standard solution: A 5 mM aqueous solution of choline chloride was prepared. It was further diluted in the same manner as the serum below. Sample dilution: Serum samples were diluted 500 times with 0.1M Tris buffer (PH7.55). Measurement procedure: Take 20μ of each diluted sample, diluted standard solution or water (blank) and add 1ml of enzyme reagent.
After prewarming at 37℃ for 5 minutes, add substrate reagent 1.
After heating at 37°C for exactly 5 minutes, 2 ml of stop solution was added, and fluorescent reagents (leuco-2',
Add 3 ml of a reagent containing phosphate buffer containing β-cyclodextrin to an ethanol solution containing 7'-dichlorofluorescein diacetate.
Warmed for 20 minutes. 0.5ml of this fluorescent sample
Add 0.5 ml of luminescent reagent (a reagent prepared by mixing bis(2,4,6-trichlorophenyl)oxalate (TCPO) dissolved in ethyl acetate and hydrogen peroxide dissolved in water with acetonitrile) into it using a syringe. The peak of luminescence was read using a measuring device. Note that by containing acetonitrile in the luminescent reagent, peroxidase in the enzyme reagent is inhibited. Choline oxidase activity in the serum sample was calculated using the following formula. ChE activity value (IU/) = Number of luminescence counts in serum sample - Blank count / Number of luminescence counts in standard solution - Blank count x 1000 On the other hand, the same serum sample was measured using the reagent kit Diacolor using absorptiometric method. , CHE (Toyobo Co., Ltd.)
The results were measured and compared using the one-point assay method of The results are shown in Table 1, and correlated data was obtained.

【table】

【table】 [Brief explanation of drawings]

FIG. 1 shows the uric acid concentration and luminescence intensity (relative values) obtained by the method of the present invention. FIG. 2 shows the correlation between the measurement of uric acid in serum by the method of the present invention and the conventional absorption method.

Claims (1)

[Claims] 1. An oxidizing enzyme is applied to a substrate in a biological sample or a substance generated by an enzymatic reaction by an enzyme in a biological sample, and the generated hydrogen peroxide is reacted with an oxidizable non-fluorescent substance in the presence of an oxidation catalyst. The non-fluorescent substance is converted into a fluorescent substance, and then oxalic acid diester and hydrogen peroxide are added to the fluorescent substance to cause a reaction under conditions of inhibiting an oxidation catalyst, and the amount of luminescence produced is measured. Or a method for quantifying biological components using chemiluminescence, which is characterized by quantifying enzyme activity.