Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2001-05-04
2004-06-08
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
Reexamination Certificate
active
06746850
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention.
The invention relates to an assay and a device for detecting and measuring the activities and concentrations of at least two proteins having similar properties or overlapping properties. In particular, the invention relates to an assay and a device for detecting and measuring the activities and concentrations of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), or both in a sample.
2. Description of the Related Art.
Cholinesterases (ChEs) are highly polymorphic carboxylesterases of broad substrate specificity, involved in the termination of neurotransmission in cholinergic synapses and neuromuscular junctions. Some ChEs terminate the electrophysiological response to the neurotransmitter acetylcholine by rapidly degrading it, while the precise function of others is unknown. ChEs are classified into acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) according to their substrate specificity and sensitivity to selective inhibitors. See Massoulie, J., et al., (1982) Ann. Rev. Neurosci. 5:57-106, which is incorporated herein by reference.
AChE is one of nature's most elegantly engineered proteins. AChE accelerates the hydrolysis of acetylcholine, a neurotransmitter, at nerve—nerve and neuromuscular junctions. BChE is found in mammalian blood, plasma, liver, pancreas, intestinal mucosa and the white matter of the central nervous system. BChE is also known as pseudocholinesterase and is sometimes referred to as serum cholinesterase as opposed to red blood cell cholinesterase, true cholinesterase, or AChE. BChE catalyzes the hydrolysis of a number of choline esters.
BChE also degrades cocaine ingested by a subject. Generally, cocaine is well tolerated by the majority of the population. However, acute cocaine abuse is related to a small incidence of sudden death. See Clouet, D. et al., Mechanisms of Cocaine Abuse and Toxicity, NIDA Research Monograph 88; and Johanson, C. and Fischman, M. W., (1989) Pharmacol. Rev. 41:3, which are both incorporated herein by reference. Although the physiological basis for sudden death due to acute cocaine abuse is not known, it is possible that abnormal BChE activity and amounts may contribute to a subject's sensitivity to cocaine. See Stewart, D. J. et al., (1979) Clin. Pharmacol. Ther. 25:464; Jatlow, P., (1979) Anesth. Anag., 58:235; Anton, A. H., (1988) Drug Intell. Clin. Pharm. 22:914; and Devenyl, P., (1989) Ann. Int. Med. 110:167, all of which are incorporated herein by reference.
BChE hydrolyzes and inactivates muscle relaxants such as succinylcholine and related anesthetics. About 5% of the population have an abnormal genotype for BChE, which results in a severe deficiency in BChE activity and amounts. When a subject having an abnormal genotype for BChE is administered succinylcholine for inducing general anesthesia prior to surgery, the subject may experience a prolonged apnea as compared to a subject having a normal genotype for BChE during which the subject is unable to breathe and must be artificially ventilated until the succinylcholine is degraded by secondary mechanisms. As this condition is a potentially life-threatening situation, a subject may be screened for abnormal BChE activity and amounts and then administered BChE before, during, or after general anesthesia. Clearly, it would be desirable to periodically measure the subject's amounts, activities, and sensitivities of BChE, AChE, or both.
Succinylcholine sensitivity may also result from an abnormal BChE concentration or activity caused by pregnancy, diseases such as liver disease and hepatitis, or medications. See Wildsmith, J. A. W., (1972) Anesthesia 27:90; Weissman, D. B., et al., (1983) J., Anesth. Analg. 62:444; Singh, D.C., et al., (1976) J. Ind. Med. Assoc. 66:49; and Foldes, F. F., Enzymes in Anesthesiology, (1978) Springer-Verlag, NY, all of which are herein incorporated by reference.
As succinylcholine and cocaine sensitivity and other diseases such as Alzheimer's disease, glaucoma, and myasthenia gravis or any other such disease may be treated by regulating the concentrations or activities of AChE, BChE, or both, it would be desirable to detect, measure and monitor the concentrations and activities of AChE and BChE.
Nerve agents, chemical warfare agents, organophosphates (OPs), pesticides, insecticides, and other such noxious chemicals exert their toxic effects by inhibiting AChE, BChE, or both. Plasma BChE and erythrocyte AChE provide some protection to synaptic AChE from these neurotoxins by scavenging free circulating AChE toxins, BChE toxins, or both prior to absorption into the central and peripheral nervous systems. Only the non-scavenged neurotoxins are capable of attacking synaptic AChE. Therefore, a subject's susceptibility to these neurotoxins may be determined by measuring the concentrations and activities of AChE and BChE in the subject. Additionally, exposure to these neurotoxins may be determined by measuring the concentration and activity of AChE, BChE, or both in a subject suspected of being exposed.
As the concentrations and activities of AChE and BChE are affected by certain disease states and exposure to nerve agents, chemical warfare agents, organophosphates (OPs), pesticides, insecticides, anesthetics, and cocaine, it would be desirable to use the concentrations or activities of AChE, BChE, or both, as indicators of a subject's (1) sensitivity to a drug or chemical, (2) exposure to a nerve agent, a chemical warfare agent, an organophosphate, a pesticide, or insecticide, or (3) disease state.
Unfortunately, the prior art methods for detecting and measuring the concentrations and activities of AChE and BChE are often problematic and inaccurate. Prior art methods have significant drawbacks which include wide statistical error, long clinical turn around times, lack of standardization, the inability to reliably compare results between laboratories, use invasive sampling techniques, are not approved by the United States Food and Drug Administration, use somewhat large blood volumes, and necessitate processing the samples prior to testing, or both. Prior art methods include assays commonly known as gasometric (manometric), Michel, micro-Michel, pH stat, Ellman, and micro-Ellman. These techniques analyze carbon dioxide formation, change in pH, chromophore formation, peroxidase activity, and ultraviolet (UV) absorption. These prior art methods normally determine either the amount of AChE or BChE, but not both simultaneously as red blood cells, plasma, or selective inhibitors are used to measure one or the other. Methods utilizing selective inhibition will not accurately account for samples exposed to certain chemical agents or oximes. Additionally, methods utilizing selective inhibition prevent the simultaneous analysis of AChE and BChE within the same sample, thereby doubling the analysis time and introducing potential errors.
Generally, methods based on gas analysis comprise using acetylcholine as a substrate, bringing acetic acid produced by the enzymatic action of ChE into contact with sodium bicarbonate, and quantitatively determining the carbon dioxide gas produced. This method is problematic as it is cumbersome and difficult to employ high-throughput screening of many samples. Additionally, use of acetylcholine as a substrate is disadvantageous because acetylcholine tends to undergo non-enzymatic hydrolysis and has no high substrate specificity. Furthermore, to achieve greater sensitivity, radioactive sodium bicarbonate has been used which generates regulated waste. This is environmentally unfriendly and increases the cost of the assay.
A pH meter method, like the gas analysis method, comprises using acetylcholine as a substrate, and measuring a pH change due to acetic acid produced by the enzymatic action of ChE by means of a pH meter. The pH meter method suffers from problems similar to the gas method, as well as requiring frequent standardization.
A pH-indicator calorimetric method, unlike the pH meter method, comprises using acetylcholine as a
Doctor Bhupendra P.
Feaster Shawn R.
Gordon Richard K.
Arwine Elizabeth
Saucier Sandra E.
The United States of America as represented by the Secretary of
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