Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1999-12-09
2002-05-14
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S963000, C435S004000
Reexamination Certificate
active
06387646
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to reagent compositions for measuring electrolytes such as calcium ions and chloride ions in body fluids, in particular blood and urine. More specifically, the present invention relates to reagent compositions for measuring the electrolytes by utilizing &agr;-amylases.
BACKGROUND OF THE INVENTION
Since the electrolyte level of the living body in the normal state is closely regulated by the metabolism, measurement of electrolyte level in body fluids is the most common biochemical clinical test for checking the function of the living body and diagnosing various diseases. For example, measurement of calcium ion level in serum is utilized for diagnosis of diseases involving hypocalcemia, such as hypoprotemia, hypophosphataemia, nephritis, nephrosis, vitamin D deficiency, hypoparathyroidism and rickets, and diseases involving hypercalcemia, such as bone tumors, Addison's disease, chronic pulmonary emphysema, hyperparathyroidism and renal failure. Further, measurement of chlorine ion level in serum is utilized for diagnosis of diseases involving hypochloremia, such as hypotonic dehydration, hyperglucocorticoidinemia and respiratory acidosis, and diseases involving hyperchloremia, such as hypertonic dehydration, renal tubular acidosis and respiratory alkalosis.
Known methods for measuring calcium ions in body fluids include (1) titration, (2) colorimetry, (3) atomic absorptiometry, (4) flame photometry, (5) electrode method, (6) enzyme method, etc.
The titration (1) is a chemical titration using oxalates or chelates. This method is disadvantageous in that it involves troublesome manipulations, causes operator-to-operator variation in measured value, and is incapable of treating a large amount of a sample in a short period of time. The colorimetry (2) utilizing o-CPC (orthocresolphthalein complexone) as the color developer can be performed with a general-purpose automatic analyzer, but this method is disadvantageous in that it involves temperature- or time-dependent variation in absorbance and is subject to the influence of magnesium ions.
The atomic absorptiometry (3) necessitates dilution of the sample and skilled manipulation to perform. The flame photometry (4) has problems with specificity and reproducibility. The electrode method (5) is disadvantageous in that it is subject to the influence of pH and necessitates apparatus that is difficult to maintain in constant conditions.
The enzyme method (6) includes (i) a method using phospholipase D (Japanese Unexamined Patent Publication No. 195297/1987), (ii) a method using calmodulin (Japanese Unexamined Patent Publication No. 36199/1987), (iii) a method using an &agr;-amylase (Japanese Examined Patent Publication No. 87798/1994), etc.
The method (i) encounters difficulties in preparing a uniform substrate and requires much time for the reaction. The method (ii) necessitate 100- to 1000-fold dilution of the sample. On the other hand, the method (iii) is free from the problems of the above methods (1) to (5) and more precise and easier than the enzyme methods (i) and (ii), and thus has been put into practice in the field of clinical testing. The method (iii) is a method for measuring calcium ions in body fluids wherein an inactivated &agr;-amylase is activated by calcium ions to decompose a sugar substrate, and the decomposition product is measured.
Conventional methods for measuring chlorine ions in body fluids include (A) coulometric titration, (B) ion electrode method, (C) colorimetry and (D) enzyme method. The coulometric titration (A) and ion electrode method (B) require a special apparatus, necessitate careful maintenance and control of the equipment, and have poor analysis efficiency. The colorimetry (C) includes a method using mercury thiocyanate and iron nitrate, which, however, produces waste liquid containing cyan or mercury and thus necessitates special waste liquid treatment, hence disadvantageous.
The enzyme method (D) includes (a) a method using an &agr;-amylase (Japanese Unexamined Patent Publications Nos. 176000/1991, 94698/1992, etc.), (b) a method using sarcosine oxydase (Japanese Unexamined Patent Publication No. 187296/1987) and other methods. The method (a) is a method for measuring chlorine ions in body fluids, wherein an inactivated &agr;-amylase is activated by chlorine ions to decompose a sugar substrate, and the decomposition product is measured. This method is superior in precision, handiness and analysis efficiency to the coulometric titration, ion electrode method and colorimetry, and thus has been put into practice in clinical testing.
In recent clinical testing, reagents are chiefly used in a solution form, for meeting the demand for improvement of precision and stability, cost reduction and labor saving. Conventionally, components unstable in a solution form, such as enzymes, are distributed as lyophilized, and are dissolved in the solvent packaged with the components, at the time of use. Recently, however, enzyme components have higher heat resistance, and can be improved in stability by optimizing pH and buffer conditions, and thus reagent compositions capable of being distributed in a solution form are available. The solution-form reagents are remarkably laborsaving in clinical testing, since it saves the manufacturer lyophilizing the reagents and the test operator dissolving the reagents.
As described above, the electrolyte measuring method utilizing an &agr;-amylase is superior to various other conventional methods in handiness and precision. In this method, the &agr;-amylase is usually inactivated in advance, by removing the target ions to be measured (such as calcium or chlorine ions) which are necessary for expression of the &agr;-amylase activity. The reagents used in this method contain a chelating agent which inhibits a blank reaction, serves as an antagonist to control the quantitativity, and masks interfering ions analogous to the target ions. Since such an inactivated &agr;-amylase is unstable in the presence of a chelating agent, the reagents have the problem that they cannot be stored in a solution form for a prolong period of time.
It is known that oligosaccharides such as maltose and &agr;-cyclodextrin, and their mixtures are useful for stabilizing inactivated &agr;-amylases in the presence of a chelating agent (Japanese Unexamined Patent Publication No. 113894/1994). However, the oligosaccharides are insufficient for stabilizing inactivated &agr;-amylases for a long period of time or at room temperature (18 to 37° C.) at which the measurement is to be conducted, although they are useful for stabilization for a short period of time (1 to 2 months) at a low temperature (2 to 8° C.).
Further, the solution-form reagents may have the problem of admixture of &agr;-amylases derived from outside the sample. For example, if human saliva is accidentally admixed into a solution-form reagent, &agr;-amylases contained in the saliva decompose the substrate, so that the reagent blank increases and the measurement sensitivity reduces, resulting in lowered quantitativity and impaired precision. Foreign matters such as saliva are likely to be admixed at the time of production or use of reagents, and even a trace amount of sweat- or saliva-derived &agr;-amylases, when admixed, affect the performance characteristics of reagents which may be stored in the form of a solution for a long period of time. Accordingly, &agr;-amylases used in the solution-form reagents need to be stabilized.
In order to cope with the problem of admixed &agr;-amylases derived from outside the sample, Japanese Unexamined Patent Publication No. 277096/1994 discloses a method wherein p-nitrophenyl-&bgr;-galactosyl-&agr;-maltopentaoside for use as an &agr;-amylase substrate is made into an aqueous solution at a pH of 2.0 to 5.5 for stabilization. This method can stabilize the &agr;-amylase substrate, but is not sufficiently useful since it imposes limitations on the pH values of the reagent solutions.
As discussed above, although solution-form reagents are mainly used in clin
Kawamura Yoshihisa
Kimata Shinsuke
Mizuguchi Katsuhiko
Leary Louise N.
Leydig , Voit & Mayer, Ltd.
Toyo Boseki Kabushiki Kaisha
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