JP2681635B2 - Method to improve analysis sensitivity and accuracy - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for improving analytical sensitivity and / or accuracy, which is used in the technical fields of chemical analysis, enzyme analysis, immunoassay, etc. Is. Therefore, the present invention can be applied to analysis, measurement reagents, and clinical diagnostic agents. (Prior Art) Conventionally, when measuring enzyme activity, a method using a nitrophenol derivative as an indicator is known. However, in the method using this nitrophenol-based indicator, the enzyme is completely used at a pH at which the enzyme exhibits maximum activity. A decrease in sensitivity was unavoidable because it did not dissociate. These indicators also have a pH that measures their enzymatic activity.
In the vicinity, it also had a defect that the stability of absorbance against pH fluctuation was poor. In order to solve these drawbacks, a method has been developed in which the assay sensitivity is increased by using the inclusion compound cyclodextrin in the enzyme activity measurement (Patent Publication Sho 58-
501357), specifically, by using α-cyclodextrin, the maximum sensitivity pH was changed from 7.3 to 7.0 to increase the maximum sensitivity by about 50%, and as a result, the α-amylase activity in human serum was increased. It is disclosed that it is possible to measure. (Problems to be solved by the invention) However, in the method using cyclodextrin, in order to perform measurement and analysis while shifting the maximum sensitivity pH to a desired pH, a very large amount of cyclodextrin must be used. In that case, a large amount of cyclodextrin interferes with the measurement of a small amount of a component such as a component in the body fluid, and the disadvantage that an accurate measurement cannot be performed is inevitable. Further, the cyclodextrin-containing reagent used for that purpose cannot be used even if stored at room temperature because the phenomenon of crystallization and solidification occurs and the storage stability is extremely poor. Furthermore, such a biological reagent must be refrigerated because its components are altered at room temperature, but refrigeration further promotes crystallization and solidification development, which is very problematic in practice. (Means for Solving Problems) The present invention has been made to solve this drawback, and as a result of extensive studies from various fields, the advantage of cyclodextrin is not neglected particularly in the analysis of biological components. I reached the conclusion. As a result of repeated research to improve the cyclodextrin method, they found that the solubility was an important factor, and focused on cyclodextrin having high solubility. Therefore, when glucosyl-α-cyclodextrin was used as the easily soluble cyclodextrin and α-amylase was measured using p-nitrophenol as an indicator, a measurement result with excellent sensitivity and accuracy was obtained, and the reagent was stored. It was confirmed that there was no problem with sex. It was also confirmed that this excellent effect can be achieved with all inclusion compounds, and the present invention was completed based on this new finding. That is, the present invention is a method for improving the sensitivity and accuracy of the analysis, which uses the easily soluble inclusion compound having a high solubility as an important point. In the present invention, as the easily soluble inclusion compound, any inclusion compound that is excellent in solubility and easily soluble can be used, and examples thereof include glycosyl-α-cyclodextrin and maltosyl-α-cyclodextrin. Examples thereof include sugar derivatives of cyclodextrin. INDUSTRIAL APPLICABILITY The present invention can be widely used in a chemical analysis method, an enzymatic analysis method, and an immunoassay method using a nitrophenol derivative as an indicator. For example, in addition to analysis of hydroxycinnamic acid using nitrophenol, nitrophenyl (oligo ) It can be advantageously used for analysis of amylase and glycosidase using glycoside as a substrate, analysis of galactosidase using nilophenylgalactoside as a substrate, and analysis of mannosidase, fucosidase, and phosphatase also using mannoside, fucoside and phosphate as substrates. The principle of the enzymatic assay according to the present invention is that the p-nitrophenyl sugar substrate is contacted with, for example, α-amylase and then α-
The activity of α-amylase is measured by decomposing it into p-nitrophenol and glucose using glucosidase, glucoamylase, etc., and reading the yellow color of the p-nitrophenyl thus produced at the absorbance of 405 nm, for example. In the present invention, the easily soluble clathrate compound is added to this measurement system, but the addition timing is not particularly limited, a predetermined effect can be obtained at any time of the measurement system, and delicate operation for the addition time is Since it is not necessary at all, it is particularly advantageous in a busy bed leaving field, and the practical effect of the present invention is extremely high. The easily soluble clathrate compound does not need to be used in a large amount as in the conventional method, for example, maltosyl- or glucosyl-.
In the case of α-cyclodextrin, a desired amount can be sufficiently achieved with a use amount of about 0.05 to 100 mmol / ml, preferably about 1 to 20 mmol / ml. Therefore, even a very small amount of biological components can be quantified accurately and quickly. When actually conducting an analysis using the method of the present invention,
It is convenient to preliminarily prepare an amylase analysis reagent prepared by dissolving a p-nitrophenol-bonded amylase substrate, a readily soluble inclusion compound, α-glucosidase, glucoamylase and the like in a buffer solution. This reagent is extremely superior to conventional products because it does not crystallize and solidify even when stored in a refrigerator for a long time. For example, in order to analyze amylase in human serum or urine using this reagent, the following operation may be performed. First, the reagent was placed in a cell and heated to 37 ° C; a sample was added to this and mixed; the color development of p-nitrophenol was read by the absorbance at around 400 nm, and the amylase activity can be measured according to a conventional method. is there. The present invention is characterized in that the sensitivity and accuracy of analysis can be improved by using an inclusion compound that is easily soluble in water. The details of the mechanism must be investigated later, but once the inclusion complex is formed by the inclusion compound and the nitrophenol derivative contained in the diagnostic reagent, the absorbance is increased, and pH
It is presumed to stabilize the absorbance against changes. Hereinafter, examples of the present invention will be described in detail. Example 1 Increase in Absorbance by Cyclodextrin Derivative 100 mmol / PIPES buffer (pH 7.0), 34 μmol / para-nitrophenol, and glucosyl-α-cyclodextrin or maltosyl-α-cyclodextrin 0-10 mmol / Were added at the respective ratios, and the absorbance at 405 nm was measured to obtain the results shown in FIG. Example 2 Stabilization of absorbance by cyclodextrin derivative 100 mmol / PIPES buffer, 34 μmol / para-nitrophenol, glucosyl-α-cyclodextrin,
Or 10 mm each of maltosyl-α-cyclodextrin
ol / added and measured for changes in pH and absorbance,
The results shown in FIG. 2 were obtained. Example 3 Measurement of amylase activity 1 mmol / benzylidene-paranitrophenyl maltoheptaoside, 20 U / ml glucoamylase, 10 U / ml α.
Glucosyl-α-cyclodextrin or maltosyl-α-cyclodextrin was added at 10 mmol / into 100 mmol / PIPES buffer (pH 7.0) containing glucosidase, 20 mmol / NaCl, 2 mmol / calcium acetate, and at 37 ° C., When the amylase activity was measured, the results shown in FIG. 3 were obtained. As is clear from this result, it is understood that amylase activity can be measured accurately and with high sensitivity by using the easily soluble inclusion compound. (Effects of the Invention) As described above, the method for improving the analytical sensitivity and accuracy of the present invention uses the cyclodextrin derivative that is easily soluble in water to stabilize the color development of the indicator nitrophenol derivative and the pH fluctuation. It has a remarkable effect that the sex is extremely increased. That is, it is possible to detect a trace amount of a target substance in a sample with high sensitivity. Therefore, the present invention is particularly effective for analysis of various trace components, and is particularly effective in the field of clinical diagnosis.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the relationship between the amount of cyclodextrin derivative added in Example 1 and the increase in its absorbance, and FIG. 2 shows the relationship between the amount of cyclodextrin derivative added in Example 2. It is the figure which showed the light absorbency change with respect to pH change. Further, FIG. 3 is a standard curve when measuring amylase activity in Example 3.

   ────────────────────────────────────────────────── ─── Continuation of front page    (56) Reference JP-A-57-102198 (JP, A)                 JP 59-219270 (JP, A)                 JP-A-61-200101 (JP, A)                 Special Table Sho 58-501357 (JP, A)                 Monthly food chemicals, 3 (4)               (1987) p. 64-69                 Science and Industry, 60 (11) (1986) P.               438-444

Claims (1)

(57) [Claims] Use of glucosyl-α-cyclodextrin and / or maltosyl-α-cyclodextrin as an easily soluble inclusion compound in a measurement system for detecting liberated nitrophenol, which improves the sensitivity and / or accuracy of the analysis. Method. 2. A measurement system for detecting liberated nitrophenol is a nitrophenol-based indicator having a convertible chemical or physical state and a clinical diagnostic agent containing components necessary for performing a desired analysis. A method as claimed in claim 1. 3. The concentration of glycosyl-α-cyclodextrin and / or maltosyl-α-cyclodextrin is 1 to 20 mmol.
The method according to claim 1 or 2, which is / ml. 4. The method of claim 2 wherein the nitrophenol indicator is para-nitrophenol. 5. The method according to claim 2, wherein the pH of the diagnostic agent is 6 to 8.