Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1999-11-29
2002-07-16
Wilson, James O. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S201000, C514S002600, C514S058000, C536S103000
Reexamination Certificate
active
06420129
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a reagent composition for the determination of electrolytes in body fluid, particularly blood or urine, for example, electrolytes such as calcium or chloride ions. More particularly, it relates to a reagent composition for the determination of electrolytes by making use of &agr;-amylase activity.
BACKGROUND ART
In general, the concentrations of electrolytes in a living body, such as calcium and chloride ions, are strictly controlled by metabolism. Therefore, the determination of electrolytes in body fluid is the most standard analysis in the clinical examination on biochemistry, as the barometer of living body functions, and the diagnosis of various diseases is carried out by their determination. For example, the determination of the amount of calcium ions in sera is used for the diagnosis of hypocalcemic diseases such as hypoproteinemia, hypophosphatemia, nephritis, nephrosis, vitamin D deficiency, hypoparathyroidism, and rachitis; or for the diagnosis of hypercalcemic diseases such as bone tumors, Addison's disease, pulmonary emphysema, hyperparathyroidism, and renal insufficiency. The determination of the amount of chloride ions in sera is used for the diagnosis of hypochloremic diseases such as hypotonic dehydration, hyperglucocorticoidosis, and respiratory acidosis; or for the diagnosis of hyperchloremic diseases such as hypertonic dehydration, tubular acidosis, and respiratory alkalosis.
The determination of electrolytes by making use of &agr;-amylase activity is based on the following principles. In the case of calcium ions, inactive &agr;-amylase is activated by calcium ions to decompose a saccharide substrate; and the determination of calcium ions in body fluid is achieved by the determination of a decomposition product (e.g., JP-B 6-87798). In the case of chloride ions, inactive &agr;-amylase is activated by chloride ions as well to decompose a saccharide substrate; and the determination of chloride ions in body fluid is achieved by the determination of a decomposition product (JP-A 3-176000 and JP-A 4-94698).
In these methods, &agr;-amylase is usually used in inactive form by the previous elimination of calcium or chloride ions necessary for the expression of its activity. In these methods, reagents for determination further contain a chelating agent for the purpose of avoiding blank reactions, controlling quantitative reliability by its use as a competitive inhibitor, or masking similar contaminated ions besides the target. The. inactive &agr;-amylase is, however, unstable in the presence of a chelating agent, which causes serious problems that the reagents cannot be stored as solutions for a long period of time and there occurs a fluctuation in the values determined.
As the means of making &agr;-amylase stable, there have been known so far, for example, those involving the addition of: calcium ions, chloride ions, or albumin (“Rinsho-byouri (clinical pathology)”, vol. 37, no. 10, p. 1155 (1990)); amino acids (JP-A 51-26284); alkali metal salts of acids (JP-A 57-29286); methionine (JP-A 63-17690); aluminum salts (JP-A 1-104173); urea (“The Enzyme”, 3rd ed., vol. 5, pp. 235-271 (1971)); guanidine hydrochloride (J. Biol. Chem., vol. 244, pp. 48-54 (1969)); and dithiothreitol or mercaptoethanol (Biochem. Soc. Trans., vol. 18, pp. 310-311 (1990)). These means are, however, insufficient for making inactive &agr;-amylase stable, and particularly in the determination of electrolytes in the presence of a chelating agent, &agr;-amylase begins to be remarkably deactivated just after the preparation of a reagent, which causes a decrease in sensitivity with the lapse of time, resulting in an unfavorable influence upon the values determined; therefore, these means are not suitable for practical use.
In the meantime it is well known that &agr;-cyclodextrin, &bgr;-cyclodextrin, or &ggr;-cyclodextrin is useful as a stabilizing agent for proteinase or glycosidase (JP-A 59-104556 and JP-A 1-117786). It is also well known that oligosaccharides such as maltose and &agr;-cyclodextrin, or mixtures thereof, are used as stabilizing agents for inactive &agr;-amylase (JP-A 6-113894). By this means, reagents for determination can stand up to low temperature (2-8° C.) storage for a short period of time (1-2 months); however, it still cannot be said that they have satisfactory stability during long-term storage or at room temperature (18-37° C.) in practical use.
Furthermore, oligosaccharides consisting of 1 to 3 monosaccharides connected in sequence, such as maltose, are products formed from substrates by &agr;-amylase; therefore, from the viewpoint of the principle of determination, they have an unfavorable influence upon the sensitivity and quantitative reliability, which places restrictions on their amounts for use and leads to the drawback that, when they are used as stabilizing agents, other kinds of performance will become worse instead of better. On the other hand, oligosaccharides such as &agr;-cyclodextrin, &bgr;--cyclodextrin, and &ggr;-cyclodextrin have the drawbacks that their solubility at low temperatures is poor; they cause the deposition of crystals or the occurrence of white turbidity during storage; and they are not suitable for long-term storage.
For these reasons, the present state of methods for the determination of electrolytes by making use of inactive &agr;-amylase is that high solution stability enabling reagents for determination to stand up to their distribution in the market as liquid reagents presently becoming the main type of reagents for clinical examination has not yet been obtained.
Under these circumstances, an objective of the present invention is to provide a composition for enzymatic determination of electrolytes, which has excellent stability, quantitative reliability, and accuracy.
DISCLOSURE OF THE INVENTION
The present inventors have intensively studied to attain the above objective. They have found that the use of a cyclodextrin derivative as a stabilizing agent for inactive &agr;-amylase results in excellent solution stability during long-term storage from low temperatures to room temperature that is at the level for practical use without any unfavorable influence upon the reaction of &agr;-amylase, and they have further found that the stability as the above objective can be obtained even in low concentrations of a cyclodextrin derivative by the combined use of an SH group containing compound, thereby completing the present invention.
A system for the determination with &agr;-amylase has been well known, in which glucosyl-&agr;-cyclodextrin and/or maltosyl-&agr;-cyclodextrin are used as easily soluble clathrate compounds for p-nitrophenyl succharide substrates and the detection is carried out for nitrophenol liberated by the action of &agr;-amylase activity (JP-B 2681635). In this system, the &agr;-amylase is in active form and no chelating compound is used. Therefore, it does not suggest the stability of inactive &agr;-amylase in the reagent composition of the present invention as a liquid reagent in the presence of a chelating compound.
Thus, the present invention provides a reagent composition for the determination of electrolytes, characterized in that it comprises: (a) inactive &agr;-amylase; (b) a chelating agent; (c) a substrate for &agr;-amylase; and (d) a cyclodextrin derivative.
The present invention further provides a reagent composition for the determination of electrolytes, which comprises: (a) inactive &agr;-amylase; (b) a chelating agent; (c) a substrate for &agr;-amylase; and (d) a cyclodextrin derivative, and which may optionally further comprise (e) an SH group containing compound or a salt thereof.
The reagent composition for the determination of electrolytes according to the present invention achieves the determination of electrolytes in a sample by utilizing the fact that inactive &agr;-amylase is activated by ions such as calcium or chloride ions, which are activating agents for &agr;-amylase, and by the determination of a decomposition product formed by the action of the a
Asano Shigeki
Kawamura Yoshihisa
Kimata Shinsuke
Kenyon & Kenyon
Maier Leigh C.
Toyo Boseki Kabushiki Kaisha
Wilson James O.
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