Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Patent
1996-05-03
1998-06-16
Gitomer, Ralph
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
435962, C12Q 134, G01N 3353
Patent
active
057668701
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a method of quantitative determination of sodium ions using a chelating agent such as ethylenediamine-tetraacetic acid or the like, which is applicable to clinical examinations.
BACKGROUND ART
There is known a method of quantitative determination of sodium ions existing in a sample collected from a living organism by using the .beta.-galactosidase reaction, the method causes the increase in the activity of the enzyme in proportion to the amount of the sodium ions existing in the sample in which from 0.2 to 5 mM of Cryptofix 221 (trade name) is added to the reaction system in order to prevent the saturation Chemistry, Vol. 34, p. 2295, 1988!. The use of lithium ions or a small amount, from 0 to 20 mM of a lithium salt of ethylene glycol bis(.beta.-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) in the quantitative determination method using the .beta.-galactosidase reaction Laid-Open No. 1-503596 (WO88/08127)!.
Conventional binding reagents, cryptand, crown ether, etc., which have heretofore been used in the method of quantitative determination of sodium ions using .beta.-galactosidase, are expensive, and it is said that such binding reagents detract from the stability of .beta.-galactosidase. In addition, since the reaction speed of cryptand with sodium ions is low, the reaction system using cryptand takes a long period of time until it reaches a stationary state and therefore rapid measurement is difficult. For these reasons, it is said that the accuracy in the quantitative determination of sodium ions by the use of cryptand is low.
Moreover, regarding the method of using such known binding reagents and lithium ions, the range of the concentration of sodium ions that can be quantitatively determined by the method is narrow and the linearity of the calibration curves in the method is extremely bad. Therefore, the method needs a particular calculable assay device.
DISCLOSURE OF THE INVENTION
In general, it is known that if a large amount of a chelating agent such as ethylenediamine-tetraacetic acid or the like exists in enzymatic reaction, the chelating agent interferes with the enzymatic reaction and it lowers the stability of the enzyme used and lowers the reproducibility of determined values, and it has heretofore been said that the addition of a large amount of a chelating agent to the quantitative determination method using enzymatic reaction lowers the determination accuracy. Contrary to the prior art knowledge, we, the present inventors have found that even when a large amount of a chelating agent is present in the enzymatic reaction using .beta.-galactosidase, the chelating agent does neither interfere with the enzymatic reaction nor lower the stability of the enzyme used but the linearity of the calibration curves in the method is improved and the accuracy in the quantitative determination by the method is improved. On the basis of these findings, we have completed the present invention.
Specifically, the present invention provides a method of quantitative determination of sodium ions in a sample using .beta.-galactosidase, in which the .beta.-galactosidase reaction is conducted in the presence of a particular chelating agent.
The chelating agent for use in the present invention indicates at least one chelating agent selected from 1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid (CyDTA), ethylenediamine-tetraacetic acid (EDTA) , triethylenetetramine-hexaacetic acid (TTHA), diethylenetriamine-N,N,N',N",N"-penteacetic acid (DTPA), 1,3-diaminopropan-2-ol-N,N,N',N'-tetraacetic acid (DPTA-OH), ethylenediamine-N,N'-dipropionic acid dihydrochloride (EDDP), acid (IDA), hydroxyimino-diacetic acid (HIDA), nitrilo-triacetic acid (NTP) and combinations of these. In general, the chelating agent is employed at a concentration of from 1 to 500 mM. More preferably, CyDTA is used at from 2 to 400 mM, EDTA is at from 25 to 400 mM, TTHA is at from 25 to 400 mM, and DTPA is at from 10 to 400 mM.
The sample containing sodium ions may be any sample that is mis
REFERENCES:
patent: 5384247 (1995-01-01), Berry et al.
Hill, J. Effects of Various Concentrations of Na+ and Mg+2 on the Activity of Beta-Galactosidase, Biochimica et Biophysica Acta 250 530-537, 1971.
Berry et al., "Enzymatic Determination of Sodium in Serum," Clin. Chem. 1988, 34, 2295-2298, 1988.
Clinical Chemistry, vol. 34, No. 11 (1988) 2295-2298.
Aoyama Norihito
Shigenobu Kayoko
Gitomer Ralph
Kyowa Medex Co., Ltd.
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