Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2001-06-22
2004-11-16
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
Reexamination Certificate
active
06818415
ABSTRACT:
BACKGROUND OF THE INVENTION
1. The Field of the Invention
The present invention relates to methods, compositions, and assays for the determination of chemical components in biological samples. More particularly, the present invention relates to calorimetric assays based on the sodium activation of amylase.
2. The Relevant Technology
Chloride ion is the major extracellular negative ion in the human body. Its main function is to maintain electrical neutrality by acting as a counter-ion to sodium. Accordingly, chloride ion levels often accompany sodium losses and excesses. Chloride ion also helps regulate acid-base balances by entering cells in response to rising carbon dioxide levels. As carbon dioxide increases, bicarbonate moves from the intracellular space to the extracellular space. In response, chloride tends to enter the cells.
Therefore, there are various circumstances where it is important to analyze serum and other bodily fluids to determine the amount of chloride ion. For example, hyperchloremia (high serum chloride) may indicate chronic hyperventilation, Cushing's syndrome, dehydration, eclampsia, excess infusion of normal saline, kidney dysfunction, metabolic acidosis, or renal tubular acidosis. Hypochloremia (low serum chloride) may indicate Addison's disease, burns, chronic respiratory acidosis (chronic hypoventilation), congestive heart failure, excessive sweating, gastric suction, over hydration, salt-losing nephritis, syndrome of inappropriate ADH secretion, or vomiting.
In response to this need, there have been developed various methods and devices to analyze bodily fluids and determine the amount of chloride ion in a sample. One early method was a calorimetric test of free chlorine. It detected the presence of the chloride ion by using a soluble silver salt and tolidine. However, this method lacked the precision required by most applications.
The need for more precise chloride ion measurements has led to the development of more accurate tests through a variety of methods. Most current methods for the determination of chloride ion are based on either electric or chemical methods. Electric methods include coulometric titration and the ion selective electrode method. The coulometric titration method is considered the most reliable method, if not the quickest. It can be performed with either manual or semi-automatic methods, but is difficult to perform in an automatic analytical system. The ion selective electrode method can have specificity problems and is prone to interference from proteins and surfactants.
The principal chemical method is colorimetry, wherein the concentration of the chloride ion is measured according to changes in color density. The colorimetry method is considered the most effective test for simple applications because it is less complicated than coulometric or ion selective electrode methods. The most common chloride determination colorimetry methods react mercuric thiocyanate and chloride ion to produce thiocyanate ion, which then forms a complex with ferric ion. The result is a characteristic red orange color, which deepens as the concentration of the chloride ion becomes higher, thus enabling colorimetry. Nevertheless, this method has drawbacks because both mercuric ions and the thiocyanate ions are harmful to the environment. As a result, each use of the reagents creates additional waste that requires costly care and treatment. Therefore, there is a need for calorimetric methods to determine chloride ion concentration that avoid the mercuric and thiocyanate ions.
U.S. Pat. No. 5,229,270 to Ono et al. (hereinafter “Ono”) discloses an environmentally safe quantitative assay and reagent for the determination of chloride ion in bodily fluids. Ono uses a reagent which contains deactivated &agr;-amylase, a compound capable of chelating calcium ion, a calcium chelate ion, a calcium chelate compound, and an &agr;-amylase measuring substance. The method comprises the steps of: (a) contacting a bodily fluid sample suspected of containing chloride ions with a reagent which comprises a compound capable of forming a chelate with a calcium ion, deactivated &agr;-amylase, a calcium chelate compound, and an &agr;-amylase activity-measuring substance; (b) determining the quantity of &agr;-amylase activity formed due to the presence of chloride ions in the bodily fluid sample, which is directly proportional to the amount of chloride ions present in the bodily fluid sample; and (c) determining the quantity of the chloride ions from the quantity of the &agr;-amylase activity by referring to a calibration curve. This method is capable of automation and has high ion specificity.
It has previously been shown that &agr;-amylase contains one chloride ion binding site per molecule. One early approach at describing the chloride ion effects on &agr;-amylase is described in Lifshitz, Ruth, Levitski, Alexander,
Identity and Properties of the Chloride Effector Binding Site in Hog Pancreatic &agr;
-
Amylase
, Biochemistry, Vol. 15, No. 9, 1976. (hereinafter, “Lifshitz”). Lifshitz is specifically directed to determine the chloride ion binding site in &agr;-amylase. In reaching their conclusions, Lifshitz discusses various compounds and their effects upon the chloride ion binding site. For example, Lifshitz teaches that calcium-free &agr;-amylase is unable to bind chloride ion. Similarly, Lifshitz tested sodium fluoride (fluoride ion) and sodium acetate (acetate ion) to determine their effect upon the chloride ion affinity of deactivated &agr;-amylase. Lifshitz determined that neither fluoride ion nor sodium acetate had any appreciable effect upon the chloride ion affinity of deactivated &agr;-amylase. Lifshitz at 1990.
Notwithstanding the prior methods for assaying chloride ions using calcium-activated &agr;-amylase, there remains a continuing need for alternative systems for assaying chloride.
BRIEF SUMMARY OF THE INVENTION
It is an object of the present invention to provide alternative methods, compositions, and assays to determine the quantity of chloride ion in biological samples.
It is another object of the present invention to provide alternative methods, compositions, and assays to determine the quantity of sodium ion in biological samples.
It is a further object of the invention to provide a chloride assay that is environmentally safe.
It is a yet a further object of the invention to provide a chloride assay that is simple to perform yet accurate.
In accordance with the present invention, there is provided a method for the determination of chloride ion in biological fluids. To perform clinical assays incorporating the discoveries of the present invention, one method for determining the concentration of chloride ions in samples comprises first preparing an enzyme reagent which includes &agr;-amylase that is substantially calcium-free and an &agr;-amylase activity detecting substrate. Next, the enzyme reagent, sodium ion, and a sample containing chloride ion to be assayed are combined, wherein the sodium ion is present in a higher concentration than the chloride ion. The quantity of &agr;-amylase activated due to the presence of sodium ions and chloride ions in the sample is then assayed. Finally, the quantity of the chloride ions is determined by reference to the assay of &agr;-amylase.
A composition for use in determining the concentration of chloride ions in bodily fluid samples comprises &agr;-amylase that is substantially calcium-free, an &agr;-amylase activity detecting substrate, and sodium ion, wherein the concentration of sodium ion is higher than that of the chloride ion to be assayed.
Yet another aspect of the invention comprises a method for determining the concentration of sodium ions in bodily fluid samples. The method comprises first preparing an enzyme reagent which includes &agr;-amylase that is substantially calcium-free and an &agr;-amylase activity detecting substrate. Next, the enzyme reagent, chloride ion, and a sample containing sodium ion to be assayed are combined, wherein the chloride ion is present in a higher concentration t
Abaxis, Inc.
Gray Cary Ware & Freidenrich LLP
Saucier Sandra E.
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