U.S. patent application number 10/763339 was filed with the patent office on 2004-08-05 for assay for detecting, measuring and monitoring the activities and concentrations of proteins and methods of use thereof.
Invention is credited to Doctor, Bhupendra P., Feaster, Shawn R., Gordon, Richard K..
Application Number | 20040152145 10/763339 |
Document ID | / |
Family ID | 22748877 |
Filed Date | 2004-08-05 |
United States Patent
Application |
20040152145 |
Kind Code |
A1 |
Feaster, Shawn R. ; et
al. |
August 5, 2004 |
Assay for detecting, measuring and monitoring the activities and
concentrations of proteins and methods of use thereof
Abstract
An assay for detecting, measuring, or monitoring the activity or
concentration of at least two proteins that have similar or
overlapping properties is disclosed. The assay comprises first
determining the sensitivity coefficients of the substrates for each
of the proteins in which the concentrations are to be determined.
This method may be used for detecting, measuring, or monitoring the
activity and concentration of AChE, BChE, or both in a test sample
which test sample may be whole and unprocessed blood or tissue.
Also disclosed are methods of using the assay to detect a subject's
exposure to an agent which affects cholinesterase, determine the
efficacy or progress of a treatment, determine the amount of
protection provided against exposure to an agent which affects
cholinesterase, or both, screen a subject for having a drug
sensitivity or a particular disease, detect a change in red blood
cell count of a subject, determine whether a candidate compound
affects cholinesterase. Also disclosed are devices and kits for
detecting, measuring, or monitoring the activities and
concentrations of AChE, BChE, or both.
Inventors: |
Feaster, Shawn R.; (Duluth,
GA) ; Gordon, Richard K.; (Potomac, MD) ;
Doctor, Bhupendra P.; (Potomac, MD) |
Correspondence
Address: |
ATTN: MCMR-JA (Ms. Elizabeth Arwine)
Office of the Staff Judge Advocate
U.S. Army Medical Research and Materiel Command
504 Scott Street
Fort Detrick
MD
21702-5012
US
|
Family ID: |
22748877 |
Appl. No.: |
10/763339 |
Filed: |
January 26, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10763339 |
Jan 26, 2004 |
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09848370 |
May 4, 2001 |
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6746850 |
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60202201 |
May 5, 2000 |
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Current U.S.
Class: |
435/7.92 ;
435/23 |
Current CPC
Class: |
G01N 33/557 20130101;
C12Q 1/005 20130101; G01N 33/567 20130101; C12Q 1/46 20130101 |
Class at
Publication: |
435/007.92 ;
435/023 |
International
Class: |
G01N 033/53; G01N
033/537; G01N 033/543; C12Q 001/37 |
Goverment Interests
[0002] This invention was made by employees and contractors of the
United States Army. The government has rights in the invention.
Claims
What is claimed is:
1. An assay for detecting, measuring or monitoring the activity or
concentration of a protein in a test sample, wherein the protein
belongs to a plurality of proteins and the plurality of proteins
have similar or overlapping properties towards a plurality of
substrates, comprising determining the activity or the
concentration of the protein in the test sample with a sensitivity
coefficient of each of substrate for the protein.
2. The assay of claim 1, further comprising adding each substrate
to test sample aliquots; and measuring reaction rates between the
protein and each substrate.
3. The assay of claim 1, wherein each sensitivity coefficient is
determined from a sensitivity coefficient sample by obtaining a
plurality of inhibited dilutions of the sensitivity coefficient
sample, wherein the plurality of inhibited dilutions comprise a
plurality of concentrations of the protein which are partially to
completely inhibited; exposing each inhibited dilution of the
plurality of inhibited dilutions to each substrate; measuring the
reaction rates between each uninhibited protein in each inhibited
dilution and each substrate; calculating the relationships between
the reaction rates of each uninhibited protein and each
concentration of the sensitivity coefficient sample at infinite
inhibitor concentration; and extracting each sensitivity
coefficient of each substrate for each protein from the calculated
relationships.
4. The assay of claim 3, wherein the plurality of inhibited
dilutions is obtained by obtaining a plurality of dilutions of at
least one inhibitor which selectively inhibits a protein belonging
to the plurality of proteins; obtaining a plurality of dilutions of
the sensitivity coefficient sample; and adding each dilution of the
inhibitor to each dilution of the sensitivity coefficient
sample.
5. The assay of claim 1, wherein the concentration or activity of
more than one protein in a test sample is detected, measured or
monitored.
6. The assay of claim 1, wherein the plurality of proteins comprise
acetylcholinesterase and butyrylcholinesterase.
7. The assay of claim 1, wherein the plurality of substrates is
selected from the group consisting of acetylcholine,
acetylthiocholine, butyrylcholine, butyrylthiocholine,
propionylcholine, and propionylthiocholine.
8. The assay of claim 1, wherein the plurality of substrates
comprise acetylthiocholine, butyrylthiolcholine, and
propionylthiocholine.
9. The assay of claim 4, wherein the inhibitor is huperzine-A,
tetraisopropyl pyrophosphoramide, or a combination thereof.
10. An assay for detecting, measuring or monitoring the activity or
concentration of acetylcholinesterase, butyrylcholinesterase, or
both in a test sample comprising determining the activity or the
concentration of acetylcholinesterase, butyrylcholinesterase, or
both in the test sample with sensitivity coefficients of each
substrate for acetylcholinesterase and butyrylcholinesterase.
11. The assay of claim 10, wherein the plurality of substrates is
selected from the group consisting of acetylcholine,
acetylthiocholine, butyrylcholine, butyrylthiocholine,
propionylcholine and propionylthiocholine.
12. The assay of claim 10, wherein the plurality of substrates
comprise acetylthiocholine, butyrylthiocholine, and
propionylthiocholine.
13. The assay of claim 10, wherein the test sample is a synthetic
sample or a natural sample.
14. The assay of claim 10, wherein the natural sample is a tissue,
fluid, or a membrane.
15. The assay of claim 10, wherein the sample is blood, serum,
lymph, cerebrospinal fluid, breast milk, interstitial or urine.
16. The assay of claim 10, wherein the sample is diaphragm, bone
marrow, brain, liver, muscle, adrenal and kidney.
17. The assay of claim 10, further comprising adding each substrate
to test sample aliquots; measuring the reaction rates between
acetylcholinesterase and each substrate; and measuring the reaction
rates between butyrylcholinesterase and each substrate.
18. The assay of claim 10, wherein the sensitivity coefficients are
determined from a sensitivity coefficient sample by obtaining a
plurality of dilutions of at least one inhibitor which selectively
inhibits either acetylcholinesterase or butyrylcholinesterase;
obtaining a plurality of dilutions of the sensitivity coefficient
sample; adding each dilution of the inhibitor to each dilution of
the sensitivity coefficient sample to obtain a plurality of
inhibited sensitivity coefficient samples; exposing each inhibited
sensitivity coefficient sample to each substrate; measuring the
reaction rates between acetylcholinesterase and each substrate;
measuring the reaction rates between butyrylcholinesterase and each
substrate; calculating the relationship between the reaction rates
of acetylcholinesterase and each concentration of the sensitivity
coefficient sample at infinite inhibitor concentration; calculating
the relationships between the reaction rates of
butyrylcholinesterase and each concentration of the sensitivity
coefficient sample at infinite inhibitor concentration; and
extracting each sensitivity coefficient of each substrate for
acetylcholinesterase and butyrylcholinesterase from the calculated
relationships.
19. The assay of claim 18, wherein the inhibitor is huperzine-A,
tetraisopropyl pyrophosphoramide, or a combination thereof.
20. The assay of claim 17, wherein measuring the reaction rates
comprises utilizing a chromogenic substrate and measuring the
absorbance of the reactions.
21. The assay of claim 10, wherein the test sample further
comprises an agent which affects the concentration or activity of
acetylcholinesterase, butyrylcholinesterase, or both.
22. The assay of claim 21, wherein the agent is removed from the
test sample prior to measuring the reaction rates.
23. A method of detecting or confirming whether a subject was
exposed to an agent which affects the concentration or activity of
acetylcholinesterase, butyrylcholinesterase, or both comprising
obtaining a test sample from the subject; measuring the reaction
rates between acetylcholinesterase and a plurality of substrates;
measuring the reaction rates between butyrylcholinesterase and the
plurality of substrates; and calculating the activity or the
concentration of acetylcholinesterase, butyrylcholinesterase, or
both with sensitivity coefficients of each substrate for
acetylcholinesterase and butyrylcholinesterase.
24. A method of determining the identity of an agent which affects
the concentration or activity of acetylcholinesterase,
butyrylcholinesterase, or both to which a subject was exposed
comprising obtaining a test sample from the subject; measuring the
reaction rates between acetylcholinesterase and a plurality of
substrates; measuring the reaction rates between
butyrylcholinesterase and the plurality of substrates; and
calculating the activity or the concentration of
acetylcholinesterase, butyrylcholinesterase, or both with
sensitivity coefficients of each substrate for acetylcholinesterase
and butyrylcholinesterase; and comparing the activities or the
concentrations with a database of activity and concentration
acetylcholinesterase and butyrylcholinesterase profiles for agents
which affect the concentration or activity of acetylcholinesterase,
butyrylcholinesterase, or both.
25. A method of determining the efficacy or monitoring the progress
of a treatment regime, wherein a subject is administered a compound
which affects the concentration or activity of
acetylcholinesterase, butyrylcholinesterase, or both comprising
obtaining a test sample from the subject; measuring the reaction
rates between acetylcholinesterase and a plurality of substrates;
measuring the reaction rates between butyrylcholinesterase and the
plurality of substrates; calculating the activity or the
concentration of acetylcholinesterase, butyrylcholinesterase, or
both with sensitivity coefficients of each substrate for
acetylcholinesterase and butyrylcholinesterase; and monitoring the
activities or the concentrations of acetylcholinesterase,
butyrylcholinesterase, or both as a function of time of the
treatment regime.
26. A method of determining whether a subject suffers from a drug
sensitivity or a disease which affects the activities or the
concentrations of acetylcholinesterase, butyrylcholinesterase, or
both comprising obtaining a test sample from the subject; measuring
the reaction rates between acetylcholinesterase and a plurality of
substrates; measuring the reaction rates between
butyrylcholinesterase and the plurality of substrates; calculating
the activity or the concentration of acetylcholinesterase,
butyrylcholinesterase, or both with sensitivity coefficients of
each substrate for acetylcholinesterase and butyrylcholinesterase;
and comparing the activities or the concentrations with a database
of activity and concentration acetylcholinesterase and
butyrylcholinesterase profiles which are typical of individuals
suffering from given drug sensitivities and individuals suffering
from given diseases which affect the activities or the
concentrations of acetylcholinesterase, butyrylcholinesterase, or
both.
27. A method of measuring the concentration of red blood cells in a
subject comprising obtaining a test sample from the subject;
measuring the reaction rates between acetylcholinesterase and a
plurality of substrates; measuring the reaction rates between
butyrylcholinesterase and the plurality of substrates; calculating
the activity or the concentration of acetylcholinesterase,
butyrylcholinesterase, or both with sensitivity coefficients of
each substrate for acetylcholinesterase and butyrylcholinesterase;
determining a relationship between standard concentrations of red
blood cells and the activities or the concentrations of
acetylcholinesterase, butyrylcholinesterase, or both; and using the
relationship to calculate the concentration of red blood cells of
the sample.
28. A method of screening for a candidate compound which affects
the concentration or activity of acetylcholinesterase,
butyrylcholinesterase, or both comprising obtaining a test sample;
measuring the reaction rates between acetylcholinesterase and a
plurality of substrates; measuring the reaction rates between
butyrylcholinesterase and the plurality of substrates; calculating
the activity or the concentration of acetylcholinesterase,
butyrylcholinesterase, or both with sensitivity coefficients of
each substrate for acetylcholinesterase and butyrylcholinesterase;
and determining whether the concentration or activity of
acetylcholinesterase, butyrylcholinesterase, or both changes.
29. A device for detecting, measuring or monitoring the activities
or concentrations of acetylcholinesterase, butyrylcholinesterase,
or both in a test sample wherein the device measures the reaction
rates between acetylcholinesterase and butyrylcholinesterase and at
least two substrates; and calculates the activities or the
concentrations of acetylcholinesterase, butyrylcholinesterase, or
both with sensitivity coefficients of each substrate for
acetylcholinesterase and butyrylcholinesterase.
30. The device of claim 26, further comprises a cartridge
comprising the reagents, buffers, substrates and standards for
measuring the reaction rates.
31. A kit for detecting, measuring or monitoring the activities or
concentrations of acetylcholinesterase, butyrylcholinesterase, or
both in a test sample comprising substrates for
acetylcholinesterase and butyrylcholinesterase.
32. The kit of claim 31, further comprising a device for measuring
the reaction rates between acetylcholinesterase and
butyrylcholinesterase and the substrates, and calculating the
activities or concentrations acetylcholinesterase and
butyrylcholinesterase.
33. The kit of claim 31, wherein the substrates for
acetylcholinesterase and butyrylcholinesterase include
acetylthiocholine, butyrylthiocholine, and
propionylthiocholine.
34. The kit of claim 31, further comprising a chromogenic
substrate.
35. A biosensor capable of detecting an agent which affects the
concentration or activity of acetylcholinesterase,
butyrylcholinesterase, or both which comprises a known mixture of
acetylcholinesterase and butyrylcholinesterase immobilized on a
support and a sealed chamber containing the known mixture of
acetylcholinesterase and butyrylcholinesterase.
36. A database of sensitivity coefficients for calculating the
activities or the concentrations of acetylcholinesterase,
butyrylcholinesterase, or both made by a method comprising
obtaining a plurality of inhibited dilutions of a sensitivity
coefficient sample, wherein the plurality of inhibited dilutions
comprise a plurality of concentrations of either
acetylcholinesterase or butyrylcholinesterase which is partially to
completely inhibited; exposing each inhibited dilution of the
plurality of inhibited dilutions to each substrate in a plurality
of substrates for acetylcholinesterase and butyrylcholinesterase;
measuring the reaction rates between acetylcholinesterase and each
substrate; measuring the reaction rates between
butyrylcholinesterase and each substrate; calculating the
relationship between the reaction rates of acetylcholinesterase and
each concentration of the sensitivity coefficient sample at
infinite inhibitor concentration; calculating the relationships
between the reaction rates of butyrylcholinesterase and each
concentration of the sensitivity coefficient sample at infinite
inhibitor concentration; and extracting each sensitivity
coefficient of each substrate for acetylcholinesterase and
butyrylcholinesterase from the calculated relationships.
37. The database of claim 36, wherein the plurality of inhibited
dilutions is obtained by obtaining a plurality of dilutions of at
least one inhibitor which selectively inhibits either
acetylcholinesterase or butyrylcholinesterase; obtaining a
plurality of dilutions of the sensitivity coefficient sample; and
adding each dilution of the inhibitor to each dilution of the
sensitivity coefficient sample.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/202,201, filed 5 May 2000, naming Shawn
R. Feaster, Richard K. Gordon, and Bhupendra P. Doctor as
inventors, which is herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] 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.
[0005] 2. Description of the Related Art
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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, N.Y., all of which are herein incorporated by
reference.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] A pH-indicator colorimetric method, unlike the pH meter
method, comprises using acetylcholine as a substrate, and measuring
a pH change due to acetic acid produced by ChE in terms of the
molecular absorbance of the indicator. Indicators utilized include
phenol red, bromothymol blue, and m-nitrophenol. Although the
pH-indicator colorimetric method may be used to analyze many
samples, the reaction time is long, the pH is not kept constant,
and the obtained values are not sufficiently reproducible at low
and high values.
[0018] Assays based on thiocholine color formation utilize
acetylthiocholine, butylthiocholine or the like as a substrate. The
substrate yields thiocholine by the enzymatic reaction of ChE,
which then reacts with 5,5'-ithiobis-2-nitrobenzoic acid (DTNB) to
produce a yellow color which is measured by a colorimeter. Although
the thiocholine method has a high sensitivity, comprises simple
operations, and many samples may be analyzed, it is detrimentally
affected by the yellow coloration of bilimbin and hemoglobin in
whole blood and is unavoidably affected by compounds having a thiol
group such as glutathione. Additionally, the substrate itself is
somewhat unstable.
[0019] Coupled enzymatic methods utilize benzoylcholine,
orthotoluoylcholine or the like as a substrate. These substrate
yield betaine by choline oxidase. Then 4-aminoantipyrine is
subjected to oxidative condensation with phenol or the like which
produces hydrogen peroxide in the presence peroxidase to cause
color production. The enzymatic method is problematic since phenol
or 4-aminoantipyrine, which is used as the reagent for the
color-producing system, competitively inhibits ChE, and the amount
of these reagents is limited and sufficient color production is
difficult. Additionally, the use of hydrogen peroxide is affected
by the presence of bilirubin, reducing substances such as ascorbic
acid, and choline. Furthermore, benzoylcholine undergoes
non-enzymatic hydrolysis.
[0020] One UV method utilizes benzoylcholine as a substrate wherein
the decrease in amount of the substrate caused by hydrolysis due to
the enzymatic action of ChE at 240 nm is monitored. This UV method
is problematic as interference by serum components generally occurs
at 240 nm and benzoylcholine undergoes non-enzymatic hydrolysis and
the reaction can not be carried out in the optimum pH range of ChE.
Additionally, there is a large deviation of absorption coefficient
with respect to wavelength.
[0021] Another UV method utilizes p-hydroxybenzoylcholine as the
substrate wherein p-hydroxybenzoate hydroxylase is reacted with
p-hydroxybenzoic acid and the decrease in absorbance caused by the
oxidation of NADPH into NADP is monitored at 340 nm. This UV method
is problematic as it utilizes NADPH, which is expensive, unstable,
must be made frequently, and needs to be kept frozen.
[0022] As described above, these conventional methods for
determining the ChE activities and concentrations are cumbersome
employ reagents and techniques with inherent problems that
detrimentally affect precision and accuracy, and are ill suited for
high-throughput screening.
[0023] There exists a need for an assay and a device for the rapid,
accurate and precise detection and measurement of the activity and
concentration of at least two proteins, such as AChE and BChE,
having similar or overlapping properties towards a plurality of
substrates.
SUMMARY OF THE INVENTION
[0024] In some embodiments, the present invention relates to an
assay for detecting, measuring or monitoring the activity or
concentration of a protein in a test sample, wherein the protein
belongs to a plurality of proteins and the plurality of proteins
have similar or overlapping properties towards a plurality of
substrates, comprising determining the activity or the
concentration of the protein in the test sample with each
sensitivity coefficient of each substrate for the protein.
[0025] In the embodiments of the invention, the test sample may be
a synthetic sample or a natural sample. Natural samples include
tissues, fluids, or membranes. Fluids may include blood, serum,
lymph, cerebrospinal fluid, breast milk, interstitial or urine.
Tissues may include diaphragm, brain, liver, muscle, and
kidney.
[0026] The sensitivity coefficients are determined from a
sensitivity coefficient sample by obtaining a plurality of
inhibited dilutions of the sensitivity coefficient sample, wherein
the plurality of inhibited dilutions comprise a plurality of
concentrations of the protein which are partially to completely
inhibited; exposing each inhibited dilution of the plurality of
inhibited dilutions to each substrate; measuring the reaction rates
between each uninhibited protein in each inhibited dilution and
each substrate; calculating the relationships between the reaction
rates of each uninhibited protein and each concentration of the
sensitivity coefficient sample at infinite inhibitor concentration;
and extracting each sensitivity coefficient for each protein from
the calculated relationships.
[0027] In some embodiments, the plurality of proteins comprise
acetylcholinesterase and butyrylcholinesterase. In some
embodiments, the plurality of substrates comprise acetylcholine,
acetylthiocholine, butyrylcholine, butyrylthiocholine,
propionylcholine, and propionylthiocholine. In some embodiments,
the inhibitor is huperzine-A, tetraisopropyl pyrophosphoramide, or
a combination thereof.
[0028] In some embodiments, the invention relates to an assay for
detecting, measuring or monitoring the activity or concentration of
acetylcholinesterase, butyrylcholinesterase, or both in a test
sample comprising determining the activity or the concentration of
acetylcholinesterase, butyrylcholinesterase, or both in the test
sample with the sensitivity coefficients of each substrate for
acetylcholinesterase, butyrylcholinesterase, or both. The plurality
of substrates may comprise acetylcholine, acetylthiocholine,
butyrylcholine, butyrylthiocholine, propionylcholine, and
propionylthiocholine. Preferably, the substrates are
acetylthiocholine, butyrylthiocholine, and propionylthiocholine. In
these embodiments, the sensitivity coefficients are determined from
a sensitivity coefficient sample by obtaining a plurality of
dilutions of at least one inhibitor which selectively inhibits
either acetylcholinesterase or butyrylcholinesterase; obtaining a
plurality of dilutions of the sensitivity coefficient sample;
adding each dilution of the inhibitor to each dilution of the
sensitivity coefficient sample to obtain a plurality of inhibited
sensitivity coefficient samples; exposing each inhibited
sensitivity coefficient sample to each substrate; measuring the
reaction rates between acetylcholinesterase and each substrate;
measuring the reaction rates between butyrylcholinesterase and each
substrate; calculating the relationship between the reaction rates
of acetylcholinesterase and each concentration of the sensitivity
coefficient sample at infinite inhibitor concentration; calculating
the relationships between the reaction rates of
butyrylcholinesterase and each concentration of the sensitivity
coefficient sample at infinite inhibitor concentration; and
extracting each sensitivity coefficient of each substrate for
acetylcholinesterase and butyrylcholinesterase from the calculated
relationships. The inhibitor may be huperzine-A, tetraisopropyl
pyrophosphoramide, or a combination thereof. The reaction rates may
be measured by utilizing a chromogenic substrate and measuring the
absorbance of the reactions.
[0029] In some embodiments, the test samples may include an agent
which affects the concentration or activity of
acetylcholinesterase, butyrylcholinesterase, or both. The agent may
be removed from the test sample prior to measuring the reaction
rates.
[0030] In some embodiments, the present invention relates to a
method of detecting or confirming whether a subject was exposed to
an agent which -affects the concentration or activity of
acetylcholinesterase, butyrylcholinesterase, or both comprising
obtaining a test sample from the subject; measuring the reaction
rates between acetylcholinesterase and a plurality of substrates;
measuring the reaction rates between butyrylcholinesterase and the
plurality of substrates; and calculating the activity or the
concentration of acetylcholinesterase, butyrylcholinesterase, or
both with sensitivity coefficients of each substrate for
acetylcholinesterase and butyrylcholinesterase.
[0031] In some embodiments, the present invention relates to a
method of determining the identity of an agent which affects the
concentration or activity of acetylcholinesterase,
butyrylcholinesterase, or both to which a subject was exposed
comprising obtaining a test sample from the subject; measuring the
reaction rates between acetylcholinesterase and a plurality of
substrates; measuring the reaction rates between
butyrylcholinesterase and the plurality of substrates; and
calculating the activity or the concentration of
acetylcholinesterase, butyrylcholinesterase, or both with
sensitivity coefficients of each substrate for acetylcholinesterase
and butyrylcholinesterase; and comparing the activities or the
concentrations with a database of activity and concentration
acetylcholinesterase and butyrylcholinesterase profiles for agents
which affect the concentration or activity of acetylcholinesterase,
butyrylcholinesterase, or both.
[0032] In some embodiments, the present invention relates to a
method of determining the efficacy or monitoring the progress of a
treatment regime, wherein a subject is administered a compound
which affects the concentration or activity of
acetylcholinesterase, butyrylcholinesterase, or both comprising
obtaining a test sample from the subject; measuring the reaction
rates between acetylcholinesterase and a plurality of substrates;
measuring the reaction rates between butyrylcholinesterase and the
plurality of substrates; and calculating the activity or the
concentration of acetylcholinesterase, butyrylcholinesterase, or
both with sensitivity coefficients of each substrate for
acetylcholinesterase and butyrylcholinesterase; and monitoring the
activities or the concentrations of acetylcholinesterase,
butyrylcholinesterase, or both as a function of time of the
treatment regime.
[0033] In some embodiments, the present invention relates to a
method of determining whether a subject suffers from a drug
sensitivity or a disease which affects the activities or the
concentrations of acetylcholinesterase, butyrylcholinesterase, or
both comprising obtaining a test sample from the subject; measuring
the reaction rates between acetylcholinesterase and a plurality of
substrates; measuring the reaction rates between
butyrylcholinesterase and the plurality of substrates; and
calculating the activity or the concentration of
acetylcholinesterase, butyrylcholinesterase, or both with
sensitivity coefficients of each substrate for acetylcholinesterase
and butyrylcholinesterase; and comparing the activities or the
concentrations with a database of activity and concentration
acetylcholinesterase and butyrylcholinesterase profiles which are
typical of individuals suffering from given drug sensitivities and
individuals suffering from given diseases which affect the
activities or the concentrations of acetylcholinesterase,
butyrylcholinesterase, or both.
[0034] In some embodiments, the present invention relates to a
method of measuring the concentration of red blood cells in a
subject comprising obtaining a test sample from the subject;
measuring the reaction rates between acetylcholinesterase and a
plurality of substrates; measuring the reaction rates between
butyrylcholinesterase and the plurality of substrates; and
calculating the activity or the concentration of
acetylcholinesterase, butyrylcholinesterase, or both with
sensitivity coefficients of each substrate for acetylcholinesterase
and butyrylcholinesterase; determining a relationship between
standard concentrations of red blood cells and the activities or
the concentrations of acetylcholinesterase, butyrylcholinesterase,
or both; and using the relationship to calculate the concentration
of red blood cells of the sample.
[0035] In some embodiments, the present invention relates to a
method of screening for a candidate compound which affects the
concentration or activity of acetylcholinesterase,
butyrylcholinesterase, or both comprising obtaining a test sample;
measuring the reaction rates between acetylcholinesterase and a
plurality of substrates; measuring the reaction rates between
butyrylcholinesterase and the plurality of substrates; and
calculating the activity or the concentration of
acetylcholinesterase, butyrylcholinesterase, or both with
sensitivity coefficients of each substrate for acetylcholinesterase
and butyrylcholinesterase; and determining whether the
concentration or activity of acetylcholinesterase,
butyrylcholinesterase, or both changes.
[0036] In some embodiments, the present invention relates to a
device for detecting, measuring or monitoring the activities or
concentrations of acetylcholinesterase, butyrylcholinesterase, or
both in a test sample wherein the device measures the reaction
rates between acetylcholinesterase and butyrylcholinesterase and at
least two substrates; and calculates the activities or the
concentrations of acetylcholinesterase, butyrylcholinesterase, or
both with sensitivity coefficients of each substrate for
acetylcholinesterase and butyrylcholinesterase. The device may
further comprise a cartridge comprising the reagents, buffers,
substrates and standards for measuring the reaction rates.
[0037] In some embodiments, the-present invention relates to a kit
for detecting, measuring or monitoring the activities or
concentrations of acetylcholinesterase, butyrylcholinesterase, or
both in a test sample comprising substrates for
acetylcholinesterase and butyrylcholinesterase. The kit may further
comprise a device for measuring the reaction rates between
acetylcholinesterase and butyrylcholinesterase and the substrates,
and calculating the activities or concentrations
acetylcholinesterase and butyrylcholinesterase. The substrates for
acetylcholinesterase and butyrylcholinesterase may include
acetylthiocholine, butyrylthiocholine, and propionylthiocholine.
The kit may also include a chromogenic substrate. The kit may also
include directions.
[0038] In some embodiments, the present invention relates to a
biosensor capable of detecting an agent which affects the
concentration or activity of acetylcholinesterase,
butyrylcholinesterase, or both wherein the comprises a known
mixture of acetylcholinesterase and butyrylcholinesterase
immobilized on a support and a sealed chamber containing the known
mixture of acetylcholinesterase and butyrylcholinesterase.
[0039] In some embodiments, the present invention relates to a
database of sensitivity coefficients for calculating the activities
or the concentrations of acetylcholinesterase,
butyrylcholinesterase, or both made by a method comprising
obtaining a plurality of inhibited dilutions of a sensitivity
coefficient sample, wherein the plurality of inhibited dilutions
comprise a plurality of concentrations of either
acetylcholinesterase or butyrylcholinesterase which is partially to
completely inhibited; exposing each inhibited dilution of the
plurality of inhibited dilutions to each substrate in a plurality
of substrates for acetylcholinesterase and butyrylcholinesterase;
measuring the reaction rates between acetylcholinesterase and each
substrate; measuring the reaction rates between
butyrylcholinesterase and each substrate; calculating the
relationship between the reaction rates of acetylcholinesterase and
each concentration of the sensitivity coefficient sample at
infinite inhibitor concentration; calculating the relationships
between the reaction rates of butyrylcholinesterase and each
concentration of the sensitivity coefficient sample at infinite
inhibitor concentration; and extracting each sensitivity
coefficient of each substrate for acetylcholinesterase and
butyrylcholinesterase from the calculated relationships.
DESCRIPTION OF THE DRAWINGS
[0040] This invention is further understood by reference to the
drawings wherein:
[0041] FIG. 1 is a graph which illustrates that AChE is inhibited
by small concentrations of 2-PAM, an oxime and part of the United
States Army's current treatment regime for organophosphate and
pesticide poisoning, while BChE is relatively unaffected.
[0042] FIG. 2 is a graph which illustrates that AChE is inhibited
while BChE is stimulated by small concentrations of HI-6, an oxime
and part of the treatment regimes of Non-United States militaries
for organophosphate and pesticide poisoning.
[0043] FIG. 3A1 is a graph demonstrating an ex vivo titration of
Hartley guinea pig blood AChE as a function of racemic Huperzine-A
concentration.
[0044] FIG. 3B1 is a graph which shows the concentration of BChE in
Hartley guinea pig blood as a function of titration with
tetraisopropylphosphoramide (Iso-OPMA).
[0045] FIG. 3A2 is a graph demonstrating an ex vivo titration of
human blood AChE as a function of racemic Huperzine-A (rac Hup-A)
concentration.
[0046] FIG. 3B2 is a graph which shows the concentration of BChE in
human blood as a function of titration with
tetraisopropylphosphoramide (Iso-OPMA).
[0047] FIG. 3C2 is a graph demonstrating the ex vivo titration of
human blood AChE with a mixture of rac Hup-A and Iso-OMPA, wherein
the results have been plotted as a function of the rac Hup-A
concentration.
[0048] FIG. 3D2 is a graph demonstrating the ex vivo titration of
human blood BChE with a mixture of rac Hup-A and Iso-OMPA, wherein
the results have been plotted as a function of the Iso-OMPA
concentration.
[0049] FIG. 4 is a graph demonstrating the simultaneous ex vivo
inhibition of Rhesus monkey whole blood AChE and BChE with 2.5
.mu.M pyridostigmine bromide, PB, as a function of time.
[0050] FIG. 5 is a graph demonstrating the simultaneous ex vivo
titration of human blood AChE and BChE with the chemical threat
agent soman, GD.
[0051] FIG. 6A shows a graph that illustrates that a small AChE
antagonist such as soman (GD) can be effectively removed by using
spin column purification.
[0052] FIG. 6B shows a graph that illustrates that a small BChE
antagonist such as GD can be effectively removed by using spin
column purification.
[0053] FIG. 6C illustrates that spin columns do not retain the AChE
and BChE contained in thoroughly hemolysed whole blood samples.
[0054] FIG. 6D demonstrates that spin column chromatography
effectively removed free unbound pyridostigmine bromide from a
complex matrix of human blood.
[0055] FIG. 7A shows a graph, which illustrates that the
cholinesterase assay of the present invention, the COBAS/FARA, and
the TestMate OP methods produce colinear titrations for an average
population for human AChE.
[0056] FIG. 7B shows a graph, which illustrates that the
cholinesterase assay of the present invention, the COBAS/FARA, and
the TestMate OP methods produce colinear titrations for an average
population for human BChE.
[0057] FIG. 7C shows a graph, which illustrates that for any given
individual the cholinesterase assay of the current invention
produces results for human AChE that are more colinear than the
COBAS/FARA or TestMate OP methods.
[0058] FIG. 7D is a graph showing that the concentrations of human
AChE obtained with the COBAS/FARA and TestMate OP methods can be
converted to those of the current invention by the use of a simple
linear function.
[0059] FIG. 7E is a graph showing that the concentrations of human
BChE obtained with the COBAS/FARA and TestMate OP methods can be
converted to those of the current invention by the use of a simple
linear function.
[0060] FIG. 8A shows a graph illustrating the pharmacokinetics as
reflected in the AChE and BChE concentrations for Harley guinea
pigs injected intramuscularly with 20 .mu.g/kg body weight of
pyridostigmine bromide.
[0061] FIG. 8B shows a graph illustrating the dose dependent
in-vivo peak inhibition of pyridostigmine bromide for Hartley
guinea pigs.
[0062] FIG. 9A depicts the response of the AChE and BChE
concentration/activity of Hartley guinea pig blood to prolonged
exposure at -80.degree. C.
[0063] FIG. 9B depicts the effect of repetitive freeze-thawing on
the concentrations of AChE and BChE contained in Hartley guinea pig
whole blood.
[0064] FIG. 10A is a graph showing the ex vivo titration of Hartley
guinea pig blood with rac Hup-A at four dilutions of blood.
[0065] FIG. 10B is a replot of the parameters, Vc and Vr, obtained
from the fits in FIG. 10A.
[0066] FIG. 11 is a plot which shows the absorbancy of Hartley
guinea pig blood as a function of blood dilution at 415 and 445
nm.
[0067] FIG. 12 demonstrates that automation of the assay of the
present invention yielded substantially similar results to that of
the manual method detailed in Example 2 below.
[0068] FIG. 13 shows that the assay of the present invention can be
ported to other laboratories without introducing a bias in the
sample results.
[0069] FIG. 14 is a plot of packed red blood cell (RBC) AChE
activity as a function of their parent whole blood (WB) values as
determined by the assay of the present invention.
[0070] FIG. 15 depicts that substantially the same AChE (Panel A)
and BChE (Panel B) activities are derived for human whole blood
samples obtained from either an intravenous draw or a finger prick
sampling.
[0071] FIG. 16 demonstrates that the assay of the present invention
provides substantially the same results when performed six times
during one day or once over three successive days. Panel A depicts
a single runs data and demonstrates that the assay is linear over
nearly two orders of magnitude. Panel B displays the processed
inter and intra day variability.
[0072] FIG. 17 demonstrates that the assay of the current invention
is highly sensitive as changes in activity of about 1.5% are
readily apparent.
[0073] FIG. 18 depicts the peak resolution of 4-thiopyridine and
that of the major hemoglobin band in Hartley guinea pig blood and
further demonstrates that a mixture of 4,4'-dithiopyridine (the
chromogenic substrate used in the assay of the present invention),
does not significantly alter the blood sample.
DETAILED DESCRIPTION OF THE INVENTION
[0074] The present invention generally relates to an assay for
detecting, measuring, or monitoring the activity or concentration
of at least two proteins in a sample, which have similar or
overlapping properties towards a plurality of substrates. As used
herein, "similar or overlapping properties" means that the proteins
react with the same plurality of substrates. For example, the
proteins hydrolyze the same plurality of substrates. At a minimum,
there should be one substrate for each protein. In preferred
embodiments, the number of substrates used equals one substrate for
each protein plus one. For example, if the concentrations or
activities of two proteins, which have similar or overlapping
properties, are to be determined, then at least three substrates
are preferred. However, it is possible to conduct the assay with
exactly the same number of substrates as proteins.
[0075] Generally, the present assay comprises first determining the
sensitivity coefficients of the substrates for each of the proteins
in which the concentrations are to be determined. The sample from
which the sensitivity coefficients are to be determined will be
hereinafter referred to as the "sensitivity coefficient sample" and
the sample from which the activities or concentrations of the
proteins are to be determined by using the sensitivity coefficients
will be hereinafter referred to as the "test sample". Use of the
terms "sample" or "samples" alone may refer to either the test or
sensitivity coefficient sample or samples.
[0076] The sensitivity coefficients of the substrates for each of
the proteins in which the concentrations are to be determined are
specific for a given population, species or sample group.
Therefore, if a test sample is obtained from a human subject, the
sensitivity coefficients must be determined for humans. Likewise,
if a test sample is obtained from a Sprague Dawley rat, the
sensitivity coefficients must be determined for Sprague Dawley
rats. Additionally, if a test sample is processed in a particular
manner, the sensitivity coefficients should be determined with at
least one sensitivity coefficient sample processed in the same or a
substantially similar manner such that the test sample and the
sensitivity coefficient sample do not have characteristics from
each other which characteristics would affect how the proteins
react with the substrates.
[0077] The sensitivity coefficient sample is preferably a pooled
sample comprising a plurality of samples obtained from a plurality
of representatives of the given population, species, or sample
group. It is important to note that this assay may be applied to
any test sample belonging to a given population, species or sample
group so long as the sensitivity coefficients are determined from a
sensitivity coefficient sample obtained from at least one
representative of the given population, species or sample
group.
[0078] It is also important to note that a first person may
determine the sensitivity coefficients with a first pooled sample
to measure the activities or concentrations of proteins in a test
sample. A second person may determine the sensitivity coefficients
with a second pooled sample and obtain sensitivity coefficients
that are different from the first pooled sample. If the second
person uses the sensitivity coefficients determined by the second
pooled sample to measure the activities or concentrations of the
proteins in the same test sample as the first person, the second
person should obtain concentrations and activities that are the
same as the first person's concentrations and activities.
[0079] Once the sensitivity coefficients are determined, the
sensitivity coefficients need not be determined again for the given
population, species or sample group. However, if the
characteristics of the test sample differ significantly from the
characteristics of the sensitivity coefficient sample, the
sensitivity coefficients should be determined again from a
sensitivity coefficient sample that has the same or substantially
similar characteristics of the test sample to compensate for
unforeseen complications due to non-routine sample processing.
[0080] Both the test sample and the sensitivity coefficient sample
may be synthetic such as a mixture of chemical reagents and
proteins or biological which includes tissues, biological fluids
and membranes. However, if the test sample is a particular
biological fluid, the sensitivity coefficient must be determined
from a sensitivity coefficient sample of the particular biological
fluid unless it is known that properties of the proteins remain
unchanged irrespective of source (ie., tissue, biological fluid,
and membranes). The assay of the present invention may be applied
to samples obtained from eukaryotes or prokaryotes. The assay may
be applied to samples obtained from any organism.
[0081] The sensitivity coefficients are determined by optimizing
the concentration range of the sensitivity coefficient sample, and
adding various concentrations of an inhibitor selective for the
proteins in which the concentrations are to be determined. At a
minimum, there may be one less selective inhibitor per protein. For
example, if the sensitivity coefficients are to be determined for
two proteins, which have similar or overlapping properties, then at
least one selective inhibitor is used.
[0082] The inhibitors are then added to several dilutions of the
sensitivity coefficient sample. The inhibited diluted sensitivity
coefficient samples are exposed to the substrates for the given
proteins. Preferably, there should be one substrate for each
protein plus one additional substrate, although having the same
number of substrates as proteins is acceptable. For example, if the
sensitivity coefficients are to be determined for two proteins,
which have similar or overlapping properties, then three substrates
for the proteins are preferably used. The substrates may or may not
be specific for a given protein.
[0083] After the substrates are added, the rates or progression of
the reactions between each protein and each substrate are
simultaneously measured. The contribution of each protein to the
reaction rate of each substrate is calculated as a function of the
concentration of the sensitivity coefficient sample at an infinite
inhibitor concentration. This calculation results in a linear
relationship in which the sensitivity coefficients may be extracted
from the calculated slopes. These sensitivity coefficients are then
used to calculate the concentrations of the proteins in a test
sample obtained from a subject belonging to the same population or
species from which the sensitivity coefficients were
determined.
[0084] For example, FIG. 10A is a graph which shows the ex vivo
titration of Hartley guinea pig blood with rac Hup-A at four
dilutions of blood. The substrate used to monitor the extent of
inhibition was acetylthiocholine (ATC). The data has been fit to
the equation explained in Example 3 below. The fit parameters are
used to generate the sensitivity coefficient data for AChE and BChE
with respect to ATC. From this plot, the control activities (Vc)
and residual activities (Vr) at infinite inhibitor concentration
are obtained. FIG. 10B is a replot of the parameters, Vc and Vr,
obtained from the fits in FIG. 10A. The slope of the Vr replot is
the sensitivity coefficient for Hartley guinea pig blood BChE, and
the difference in the slopes, i.e., slope Vc-slope Vr, is the
sensitivity coefficient for AChE.
[0085] As an example, the present invention may be used to detect,
measure, or monitor the activity and concentration of AChE, BChE,
or both in a test sample. Generally, the assay for detecting,
measuring, or monitoring the activity and concentration of AChE,
BChE, or both in a test sample comprises first determining the
sensitivity coefficient of an AChE substrate and the sensitivity
coefficient of a BChE substrate. The assay for detecting,
measuring, or monitoring the activity and concentration of AChE,
BChE, or both in a blood sample provides greater than about 99%
accuracy and less than about 1% precision in less than about five
minutes. Again these sensitivity coefficients are specific. for the
test sample to be analyzed.
[0086] The sensitivity coefficients are determined by optimizing
the concentration range of the sensitivity coefficient sample, and
adding a selective AChE or a selective BChE inhibitor to several
sensitivity coefficient sample dilutions. Suitable inhibitors
include tetraisopropylphosphoramide (Iso-OMPA) (Sigma Chemical Co.
Mo.), racemic huperzine-A (rac Hup-A) (CalBiochem-NovaBiochem
Corporation, San Diego, Calif.), echothiophate (phospholine iodide)
(Wyeth-Ayerst Laboratories, St. Davids, Pa.), ethopropazine,
tacrine (Cognex) (Sigma, St. Louis, Mo.), E2020 (Aricept) (Eisai
Inc. Teaneck, N.J.), edrophonium (Sigma, St. Louis, Mo.), or any
other selective inhibitor for AChE or BChE known in the art. The
inhibitors only need be selective over the concentration range used
for the titration. Thus, suitable inhibitors may be selective
inhibitors and need not be specific inhibitors. For example, if
extremely high concentrations rather than nanomolar concentrations
of Hup-A are used, BChE would also be titrated.
[0087] The inhibited sensitivity coefficient samples are then
exposed to at least one AChE substrate, at least one BChE substrate
and an additional substrate. None of these substrates needs to be
specific for either protein. After the substrates are added, the
catalytic rate of hydrolysis is measured either serially or
simultaneously for all of the substrates. In preferred embodiments,
the rates are measured simultaneously since the turnaround time is
minimized, and temporal sample artifacts are minimized as in the
case of transient or reversible inhibitors.
[0088] Next the contribution of AChE and BChE to the control sample
is calculated as a function of the concentration of the sensitivity
coefficient sample at an infinite inhibitor concentration. This is
accomplished by plotting the residual activities at infinite
inhibitor concentration as a function of sensitivity coefficient
sample concentration for each substrate. The slopes from the
resulting lines for each substrate are the sensitivity coefficients
for the protein that was unaffected by the addition of the
inhibitor. Furthermore, the sensitivity coefficients for the other
protein are calculated by subtracting the aforementioned slopes
from the corresponding control reactions for each substrate. See
FIG. 10B. The sensitivity coefficients are then used to calculate
the concentration of AChE and BChE in a test sample obtained from a
subject belonging to the same population or species from which the
sensitivity coefficient sample was obtained.
[0089] To confirm the sensitivity coefficients obtained by using a
particular selective inhibitor of AChE or BChE, the method
described above may be repeated by using a second selective
inhibitor such as tetraisopropylphosphoramide (so-OMPA). Preferably
the second inhibitor completely inhibits the other protein. Similar
analysis of these rates as a function of serial dilution produces
identical results.
[0090] It is noted, however, that use of only one inhibitor is
sufficient. For example, if only HupA is used, the slope of rac
Hup-A equals the sensitivity coefficient of BChE, and the slope of
the control minus the slope of Hup-A equals the sensitivity
coefficient for AChE. If only Iso-OMPA is used, the slope of the
Iso-OMPA equals the sensitivity coefficient of AChE, and the slope
of the control minus the slope of Iso-OMPA equals the sensitivity
coefficient of BChE.
[0091] It is preferred that at least one of the substrates be of
those normally used in clinical screening assays, since a wealth of
information is available for these substrates. Suitable AChE
substrates include acetylcholine and acetylthiocholine (ATC).
Preferably, the AChE substrate is acetylthiolcholine. It is
preferred that the BChE substrate be one normally used in clinical
screening assays. Suitable BChE substrates include butyrylcholine,
and butyrylthiocholine (BTC). Preferably, the BChE substrate is
butyrythiolcholine. It is noted, however, that other suitable
substrates include propionylthiolcholine (PTC),
acetyl-.sup.14C-choline, benzoylcholine, orthotoluoylcholine,
p-hydroxybenzoylcholine, indophenyl acetate, indoxyl acetate,
2,6-dichloroindophenyl acetate, resorufm acetate or butyrate, other
cholinesterase ester analogs, and other cholinesterase thioesters
analogs may be used as a substrate by AChE, BChE, or both. A
suitable substrate should possess specific AChE and BChE
affinities, similar or overlapping AChE and BChE affinities, or
both.
[0092] Suitable chromogenic substrates include
5,5'-dithiol-bis(2-nitroben- zoic acid) (DTNB), 4,4'-dithiopyridine
(DTP), disulfide analogs thereof, and
7-diethylamino-3-(4'-malemidylphenyl)-4-methyl courmarin (CPM).
Preferably, the chromogenic substrate is DTP or a compound that
does not have a maximum absorption at wavelengths that overlap with
the absorbancies native to the sample. For example, DTP has a
maximum absorbance at 324 nm and does not overlap with the
absorbance range of about 375 nn to about 480 nm of hemoglobin in a
whole blood sample. See e.g., FIG. 18. Thus, the absorbance of the
whole blood sample at 415 nm, 445 nm, or any other wavelength
between 375 nm and 480 nm may be used as a normalization marker for
hemoglobin content in the sample. Thus, one may account for
individual variations in red blood cell concentrations.
Additionally, higher concentrations of blood may be used since
blood does not absorb significantly in the region of 324 nm. Thus,
the assay is no longer limited by instrumentation.
[0093] Both the test sample and the sensitivity coefficient sample
may be synthetic such as a mixture of chemical reagents and
proteins or natural which includes tissues, fluids and membranes.
The samples may be processed or, more important unlike other
conventional assays, unprocessed. The samples may be obtained from
any subject or source in which AChE, BChE, or both are expected to
be present. The fluids may be biological fluids which include
blood, serum, lymph, interstitial, cerebrospinal fluid, breast
milk, urine or any other fluid containing AChE, BChE, or both.
Preferably, if the samples are blood, the samples are treated with
any suitable anticoagulant known in the art. Preferred
anticoagulants do not affect the concentrations and activities of
AChE and BChE. Finger prick blood samples and intravenous blood
samples produce the same or substantially similar results, thereby
allowing relatively non-invasive blood sampling. The tissues
include diaphragm, brain, liver, muscle, kidney, heart, lung,
intestine, adrenal, or any other tissues possessing AChE, BChE, or
both.
[0094] This procedure may be applied to tissues, and has been done
successfully for Hartley guinea pig diaphragm. To prepare a tissue
sample, the tissue is thoroughly homogenized using standard
techniques known in the art. There is no need to separate the
tissue from the supernatant, since excellent results were obtained
by using the whole homogenate. In fact, the whole homogenate is a
better representation of the sample than just the extract.
[0095] The sensitivity coefficient sample range is optimized using
a wavelength and concentration that provides a linear relationship
between the sensitivity coefficient sample concentration and
absorbance. For example, FIG. 11 is a plot which shows the
absorbancy of Hartley guinea pig blood as a function of blood
dilution at 415 and 445 nm. FIG. 11 demonstrates that a linear
response of absorbance as a function of blood concentration may be
obtained by using a hyperchromic shift from the peak maximum of
hemoglobin.
[0096] For a blood or tissue sensitivity coefficient sample, the
preferred range of absorbance is highly dependent on the analytical
instrument. For example, using a the Molecular Devices SpectraMax
Plus microplate reader (Molecular Devices Corporation, Sunnyvale,
Calif.) a linear range of measurements can be achieved from
approximately 0.01 to 4.0 absorbance units from about 200 nm to
about 1000 nm. This allows blood dilutions from about 8 to 5000
fold to be used. On other instruments, however, the range may be
only 0.01 to 1.0, necessitating a significantly smaller working
range. One of ordinary skill in the art may determine by standard
techniques the preferred range for the particular analytical
instrument used.
[0097] Next, sensitivity coefficients are determined for several
dilutions of the sensitivity coefficient sample optimized by sample
and population normalization. See e.g., FIGS. 10A, 10B, and 11. The
practical dilution range for human whole blood samples is from
about 600 to 4000. Then the sensitivity coefficients are used to
calculate the concentration or activity of AChE, BChE, or both in a
test sample.
[0098] The assay of the present invention may be used to determine
or confirm exposure to an agent that affects the concentration or
activity of AChE, BChE, or both. For example, the assay may be used
to analyze a test sample obtained from a subject to determine if or
confirm that the subject was assaulted with a nerve agent. The
assay may be used to confirm suspected cholinesterase poisoning due
to organophosphates, organophosphites, carbamates, or the like. The
assay may also be used to determine whether a subject was exposed
to a particular agent as a particular agent may affect
cholinesterase concentrations in a manner that may be
distinguishable from the cholinesterase concentrations caused by
other agents. Once exposure is determined or confirmed, an
appropriate containment, decontamination, treatment or a
combination thereof may be initiated.
[0099] The assay of the present invention may also be used to
determine the efficacy or progress of a treatment wherein a
compound which affects the AChE, BChE, or both is administered to a
subject suffering from an abnormal concentration or activity of
AChE, BChE, or both. By monitoring the ChE content as a function of
time of the treatment, one may determine the effect the treatment
has on the concentration or activity of AChE, BChE, or both and, if
desired, modify the treatment to have the desired affect.
[0100] The assay may be used to monitor the concentration or
activity of AChE, BChE, or both in a subject exposed to a compound
which affects the concentration or activity of AChE, BChE, or both.
In particular, the simultaneously monitoring the AChE and BChE
concentrations or activities of a test sample can provide early
detection of compounds which affect the concentration or activity
of AChE, BChE, or both such as nerve agents, chemical warfare
agents, organophosphates (OPs), pesticides, and insecticides. Since
AChE and BChE have different affinities for particular compounds,
it is possible to determine which compound or type of compound is
present.
[0101] To accomplish this, an activity and concentration profile
for each possible compound would be established. The profile would
indicate how a given compound affects the activities and
concentrations of AChE and BChE as a function of time and compound
concentration. Then first responders would be able to confirm
exposure to a nerve agent, a chemical warfare agent, an
organophosphate, a pesticide, or insecticide and initiate
appropriate containment and decontamination measures. In a similar
manner, a sensor could be used at a given location to monitor
pesticides and insecticides or to detect a biochemical or chemical
warfare attack.
[0102] The assay of the present invention may be used to determine
the amount of protection provided against exposure to a compound
which affects the concentration or activity of AChE, BChE, or both
such as a nerve agent in a subject by the administration of a
protective inhibitor such as pyridostigmine bromide (e.g. FIG. 8A)
or physostigmine.
[0103] The assay may be used to screen individuals for sensitivity
to a drug. For example, an individual may be screened for
succinylcholine sensitivity before general anesthesiology. This
could be accomplished by ex vivo dosing of a patient's blood sample
with the therapeutic level of succinylcholine used in surgery. The
ratio of inhibition of this sample to that of the normal population
would indicate whether the patient possesses the phenotypic BChE
sensitivity.
[0104] Likewise, a subject may be screened for a disease such as
cirrhosis of the liver or chronic drug abuse as these disease
states selectively alter the concentration of AChE or BChE
circulating in the blood. In particular, since AChE is
biosynthesized in the liver, any disease state affecting liver
function may exhibit a change in concentration of AChE. Also,
chronic cocaine use has been demonstrated to decreases the plasma
concentration of BChE. Therefore, one of ordinary skill in the art
could monitor the treatment of chronic cocaine abusers by
monitoring the blood levels of BChE as a function of time.
Furthermore, any other disease state that selectively alters the
levels or activities of AChE, BChE, or both, could likewise be
screened for and monitored.
[0105] The change in red blood cell count of a subject may also be
determined as the assay of the present invention may be used to
detect a change in AChE concentration of about 2%, preferably about
1.5%. See e.g., FIG. 17. Since about 10% to about 12% of a
subject's total blood volume is removed during blood donation and
the levels of AChE and red blood cells are decreased after blood
donation. The assay of the present invention can be used to screen
individuals to determine if they are able to donate or if they
donated blood recently. Likewise, the present invention may be used
to determine if a subject suffers from anemia, thalassemias,
spherocytosis, hemoglobin SS, hemolytic anemia, paroxysmal
nocturnal hemoglobinuria, or megaloblastic aneami since these
diseases either cause an increase or decrease in red blood cells
count.
[0106] The assay may be used to determine whether a candidate
compound affects the concentration or activity of AChE, BChE, or
both. Any one interested in screening for a therapeutic agent could
implement the assay of the invention in a much more relevant media
such as blood. This would allow the determination of the effect
that the candidate compound has, if any, on AChE, BChE, or both.
Primary neuron cultures may also be used to screen for a
therapeutic agent that may be neuroprotective. Candidate compounds
to be screened may include those capable of providing nerve agent
prophylaxis and those that transiently inhibit AChE, BChE, or both.
For candidate compound screening, a stopped timed assay is
preferred since the effect that the candidate demonstrates as a
function of time is crucial and may be missed if a single arbitrary
endpoint type assay is performed.
[0107] In addition to the stopped time assay, the effect of
dilution on an inhibited sample must also be measured, since
reversible non-covalently modifying compounds may be missed. This
would occur in vitro since in the stopped time assay these
compounds would display no catalytic turn-over and hence no
activity return. In vivo due to elimination or clearance by the
body, these reversible compounds would dissociate from AChE, BChE,
or both and the activity of these proteins would increase. Body
clearance can be mocked by dilution.
[0108] The assay of the invention may be adapted for use in a
biosensor capable of detecting a agent such as a nerve agent, a
chemical warfare agent, an organophosphate, an organophosphite, a
pesticide, an insecticide, a carbamate, and the like. For example,
a biosensor may contain known mixtures of AChE and BChE immobilized
on a support which may then be placed in a given location or
environment. Simultaneous monitoring and comparison of the rates of
given substrates for AChE and BChE to that of a sealed chamber
containing the same mixture of AChE and BChE, would provide real
time information on the appearance or presence of the agent. The
agent may be identified by comparing the rates of inhibition of
AChE and BChE to those of a predefined database of rates for a
variety of agents which affect the concentration or activity of
AChE, BChE, or both. This biosensor may be used remotely from a
given location to provide a buffer zone of early warning and
detection. Alternately, first responders to a suspected chemical
attack could use this biosensor to confirm and initiate appropriate
containment and decontamination measures.
[0109] One may desire to remove any contaminants or compounds that
may interfere with determining the activities and concentrations of
AChE, BChE, or both in the test sample. Removal of a compound or a
contaminant is desired when the presence of said compound changes
activities and concentrations of AChE, BChE, or both in the test
sample. A compound may potentially interfere with the assay of the
present invention in that the compound may selectively alter the
activity of AChE, BChE, or both (see e.g., FIGS. 1 and 3), or the
compound may alter the molar extinction coefficient of the
chromogenic substrate. For example, if a blood test sample is
analyzed to determine whether or not the subject from which the
test sample was obtained was exposed to a nerve agent, a moderate
decrease in the concentration of AChE, BChE, or both would be
expected. However, as illustrated in FIG. 1, if the subject is
administered an oxime, such as 2-PAM, the concentration of AChE is
inhibited but BChE is unaffected. These alterations may cause the
concentrations of AChE to appear to fall below the normal
concentration range for individuals not exposed to a nerve agent.
Even though the subject was not exposed to a low concentration or a
small amount of a nerve agent a false positive would result.
Alternatively, as shown in FIG. 2, treatment with an oxime, such as
HI-6, which selectively inhibits AChE and stimulates BChE may cause
the concentration of AChE, BChE, or both appear to be within a
concentration range typical of individuals exposed to low levels or
concentrations of a nerve agent, even though the subject was
actually exposed to a much higher concentration. Thus, it would be
desirable to remove the oxime from the test sample before
analysis.
[0110] The removal of a compound or a contaminant may be done where
one desires to monitor the concentration of AChE, BChE, or both in
subject being treated with a compound, such as an oxime. For
example, before one monitors the concentration of AChE, BChE, or
both in a subject being treated with an oxime, one should remove
the oxime from the test sample before analysis.
[0111] The removal of a compound or a contaminant may also remove
the agonists or antagonists of AChE, BChE, or both. The removal of
an agonist or antagonist does not affect the assay of the present
invention where the agonist or antagonist (1) is irreversibly bound
to AChE, BChE, or both and the binding results in an increase or a
decrease in the cholinesterase activity, or (2) exhibits a slow
disassociation or turnover rate with respect to the time scale of
the assay and sample preparation. The removal of an agonist or
antagonist will affect the assay of the present invention where the
agonist or the antagonist exhibits a fast disassociation or
turn-over rate. If the removal of an agonist or antagonist will
affect the assay, one may analyze the test sample before removing
the potentially interfering compound or contaminant and then
analyze the test sample after removing the compound or contaminant.
In any event, none of the currently accepted clinical methods
remove said compounds prior to analysis.
[0112] The potentially interfering compound or contaminant may be
removed by any suitable methods known in the art. For example, a
spin column may be used to rapidly remove any free ligand from the
complexed form by size exclusion. See e.g., FIG. 6B2.
[0113] Relative to the prior art assays, the assay of the present
invention is rapid, accurate, and precise. Since the assay of the
present invention is fast relative to prior art assays, the present
invention may be adapted for use with high-throughput screening
platforms such as the Biomeck 2000 (Beckman Coulter, Inc,
Fullerton, Calif.) or any other such system known in the art. The
present assay does not rely on the addition of selective AChE or
BChE inhibitors, employs minimally invasive sampling techniques
such as pricking the subject's finger, and provides results in less
than about six minutes.
[0114] The present invention also relates to devices for detecting,
measuring, or monitoring the activities and concentrations of AChE,
BChE, or both, as the present assay may be adapted for use with
diagnostic devices and computer software. Examples of suitable
devices include hand held devices such as the commercially
available i-STAT.RTM. system available from I-STAT Corporation,
(Princeton, N.J.) or the Test-Mate OPT. unit available from EQM
Research (Cincinnati, Ohio) as well as any other such device.
[0115] The present assay may be readily adapted to work with a
device whose detection platforms are amperometric, UV/Visable,
fluorescent or other. For example, an amperometric-based device,
such as the i-STAT.RTM. system, may be adapted by replacing the
chromogenic substrate with a substrate that produces a given
equivalent of hydrogen peroxide per catalytic cycle which can be
monitored amperometrically using standard methods known in the art.
Further a micro-fluidic cartridge such as those available for use
with the i-STAT.RTM. system may be developed or modified to
comprise all the reagents, such as buffer, standards and
substrates, for performing the assay of the present invention. A
sterile lancet for blood sampling may also be included with or in
the device. The device may be programmed or designed to
automatically perform all the necessary test sample dilutions when
the cartridge is inserted.
[0116] The present assay may be used for high throughput screening
and adapted for use with benchtop equipment such as the Biomeck
2000 or other such systems known in the art. For example, A Biomeck
2000 possessing circulating reagent reservoirs, single and
multi-channel pipettes, a gripper tool for labware movement and
placement, a plate and tip stacker carousel, and an integrated
mictrotiter plate reader would allow all necessary sample
dilutions, equipment placement, reagent addition, and velocity
measurements comprised in the present invention to be carried out
automatically.
[0117] The assay of the invention may also be used for normalizing
sample data for direct comparison to that of a given population by
measuring an internal standard property of the sample and
referencing that value to that of the given population, this
normalization constant is then used to directly modify the measured
concentrations of activity of acetylcholinesterase,
butyrylcholinesterase, or both.
[0118] In the following examples, acetylthicholine iodide (ATC),
propionylthiocholine iodide (PTC), butyrylthiocholine iodide (BTC),
4,4'-dithiopyridine (DTP), and tetraisopropylphosphoramide
(Iso-OMPA) were purchased from Sigma Chemical Co. Racemic
huperzine-A (rac Hup-A) was purchased from
CalBiochemical-NovaBiochem Corporation (San Diego, Calif.). Water
was polished to 18.2 M.OMEGA. by passage through a Millipore water
purification system (Millipore, Bedford, Mass.). Intra-venous blood
was obtained from ten human volunteers and stored in heparin
vacutainers.RTM. (BD Vacutainer Systems, Annapolis, Md.).
Intra-venous blood was obtained from ten Rhesus monkeys (Walter
Reed Army Insititute of Research, Division of Veterinary Medicine,
Silver Spring, Md.) and stored in heparin vacutainers.RTM.. Whole
blood samples from 10 Sprague Dawley rats (Charles River
Laboratories, Wilmington, Mass.) were obtained and stored in
heparin vacutainers.RTM.. Trunk blood obtained from 10 Hartley
guinea pigs (Charles River Laboratories, Wilmington, Mass.) was
stored in the presence of EDTA. All blood samples were refrigerated
at 4.degree. C. until used.
[0119] While the detailed description and following examples are
directed to an assay for acetylcholinesterase,
butyrylcholinesterase, or both, the present invention is not
limited to acetylcholinesterase and butyrylcholinesterase, but
includes any assay for any protein which belongs to a plurality of
proteins which have similar or overlapping properties towards a
plurality of substrate.
EXAMPLE 1
Sample Preparation for Cholinesterase Assay
[0120] A. Blood Sample
[0121] A sample of blood is obtained from a subject and
appropriately treated with a suitable anticoagulant known in the
art. If the blood sample is to be stored and screened later and the
sample is time sensitive, it may be flash frozen in a liquid
nitrogen bath and stored at -80.degree. C. A sample that is not
time sensitive may be stored at 4.degree. C.
[0122] When ready for screening, 20 .mu.L of the blood sample is
transferred to a 200 .mu.L PCR tube containing 140 .mu.L of 18.2
M.OMEGA. water with a positive displacement pipette. Then the
sample is mixed thoroughly by any suitable method such as pipetting
or vortexing. When mixed thoroughly, the sample may be analyzed as
set forth in Example 2. It is important to note that the actual
dilutions to be used are specific for a given population, species
or sample group. The dilutions used here were for 20 .mu.L of
Hartley guinea pig blood diluted with 140 .mu.L of water or 10
.mu.L of human blood diluted with 190 .mu.L of water.
[0123] B. Tissue Sample
[0124] It is preferred that the tissue sample is obtained from a
CO.sub.2 anesthetized subject since some anesthetics inhibit AChE,
BChE, or both. The tissue sample is flash frozen on powdered dry
ice. The tissue sample or a fraction thereof is weighed and minced.
The minced sample is quantitatively transferred to a plastic tube
and 4 volumes (w/v) of 50 mM sodium phosphate buffer at pH 8.00 is
added. The sample is homogenized 5 times for about 3 seconds each
with an electric homogenizer at full RPM. The crude homogenate is
transferred to a glass ground hand homogenizer and thoroughly
pulverized as per the manufacture's directions. A 160 .mu.L aliquot
of the sample is transferred to a 200 .mu.L PCR tube and may be
analyzed as set forth in Example 2. The remaining homogenate may be
stored for later use. Again, it is important to note that the
actual dilutions to be used are specific for a given population,
species or sample group. The dilutions used here were for Hartley
guinea pig diaphragm.
EXAMPLE 2
Cholinesterase Assay
[0125] The following stock reagents ATC, PTC, BTC, DTP and buffer
were prepared and stored at -20.degree. C. until needed, or stored
at 4.degree. C. when in use: ATC=30 mM acetylthiocholine prepared
in 18.2 M.OMEGA. water, PTC=30 mM propionylthiocholine prepared in
18.2 M.OMEGA. water, BTC=30 mM butyrylthiocholine prepared in 18.2
M.OMEGA. water, DTP=6 mM 4,4'-dithiopyridine prepared in 10% HPLC
grade methanol/50 mM sodium phosphate buffer, pH 8.00, buffer=50 mM
sodium phosphate buffer, pH 8.00.
[0126] The following working reagents A, B, D and P were prepared
and stored at 25.degree. C. or room temperature: A=1.0 mM
acetylthiocholine and 200 .mu.M 4,4'-dithiopyridine (8.40 mL of
buffer, 300 .mu.L of ATC, 300 .mu.L of DTP), P=1.0 mM
propionylthiocholine and 200 .mu.M 4,4'-dithiopyridine (8.40 mL of
buffer, 300 .mu.L of ATC, 300 .mu.L of DTP), B=1.0 mM
butyrylthiocholine and 200 .mu.M 4,4'-dithiopyridine (8.40 mL of
buffer, 300 .mu.L of ATC, 300 .mu.L of DTP), D=200 .mu.M
4,4'-dithiopyridine (8.40 mL of buffer, 300 .mu.L of 18.2 M.OMEGA.
water, 300 .mu.L of DTP).
[0127] A microtiter plate spectophotometer such as Molecular
Devices Spectramax Plus microtiter plate spectrophotometer
available from was used. Two experiments were performed on the same
plate. For the first experiment, it was indicated that it was a
kinetic assay and the parameters set were: 1) 324 nm wavelength, 2)
60 second pre-read shaking, 3) 3 second shaking between reads, 4) 4
minute collection time, and 5) linear least squares data analysis.
For the second experiment, it was indicated that it was an endpoint
assay and the parameters set were 1) two wavelengths, 415 nm and
445 nm and 2) 5 second pre-read shaking.
[0128] Test samples obtained from either Hartley guinea pigs or
humans were mixed five times by pipetting. 10 .mu.L of each test
sample was dispensed into each column of a 96 well microtiter plate
(i.e., 8 test samples were dispensed into 12 columns=96 wells). 290
.mu.L aliquots of working reagent D (control) were added to columns
1-3, 290 .mu.L aliquots of working reagent A (acetylthiocholine)
were added to columns 4-6, 299 .mu.L aliquots of working reagent P
(propionylthiocholine) were added to columns 7-9, and 290 .mu.L
aliquots of working reagent B (butyrylthiocholine) were added to
columns 10-12 with a multichannel electronic pipette.
[0129] The absorbencies and the kinetic rates of the test samples
were obtained. To account for well-to-well variation due to
pipetting error within a sample (i.e., the twelve wells that
constitute one row of a standard 96 well microtiter plate), each
well rate was multiplied by a correction factor. This correction
factor was the ratio of the average absorbency of the test sample,
ie., the average of the twelve wells (A.sub.415 for human or
A.sub.445 for guinea pig) to the observed absorbance for the well
being treated. The ensuing values were used to calculate the
concentrations of AChE and BChE by solving the following three sets
of equations:
ATC rate=x.sub.1[AChE]+y.sub.1[BChE]
BTC rate=x.sub.3[AChE]+y.sub.3[BChE] Equation Set 1
ATC rate=x.sub.1[AChE]+y.sub.1[BChE]
PTC rate=x.sub.2[AChE]+y.sub.2[BChE] Equation Set 2
PTC rate=x.sub.2[AChE]+y.sub.2[BChE]
BTC rate=x.sub.3[AChE]+y.sub.3[BChE] Equation Set 3
[0130] These equations may be solved by any method known in the art
such as linear combination. The sensitivity coefficients,
x.sub.1-x.sub.3 and y.sub.1-y.sub.3, were determined as described
in Example 3. In the above equations, the sensitivity coefficients
for AChE are x.sub.1, X.sub.2, and X.sub.3 and correspond to ATC,
PTC, and BTC, respectively. Similarly, y.sub.1, y.sub.2, and
y.sub.3 denote the BChE sensitivity coefficients. All rates were
corrected for spontaneous hydrolysis of DTP by blood and are
expressed in terms of change in absorbance with respect to time
(e.g., mAbs/min). The units of the sensitivity coefficients are
mAbs/min/sample dilution, and the concentrations of AChE and BChE
obtained via equation sets 1 through 3 are unitless pure
numbers.
[0131] Final numerical processing began with evaluating the mean
and standard deviation for AChE and BChE from the three
independently determined concentrations of AChE and BChE. These
values were transformed from pure numbers into mAbs/min/sample
dilution by multiplying the mean and standard deviation by the
appropriate sensitivity coefficient. For example, to convert the
calculated concentration of AChE into mAbs/min/sample dilution, the
mean and standard deviation were multiplied by x.sub.1, the ATC
AChE sensitivity coefficient for AChE. In a similar manner, the
calculated concentration of BChE was transformed into
mAbs/min/sample dilution, by multiplying each value by y.sub.3, the
BTC sensitivity coefficient for BChE. It is important to realize
that any of the protein's sensitivity coefficients could be used
for this process (ie., x.sub.1-x.sub.3 for AChE/y.sub.1-y.sub.3 for
BChE), however, the final results will represent the turnover of
that sensitivity coefficient's corresponding substrate (e.g.,
X.sub.2 turnover of PTC by AChE).
[0132] The data was then corrected to a 1-cm pathlength by taking
into account the pathlength to volume ratio of a well in the
microtiter plate being used (ie., the 300 .mu.L total well volume
corresponded to 0.89 cm). In addition all test sample dilutions
were accounted which included the dilutions of the sample due to
sample processing, see Example 1, and reagent addition, see above.
In the case of guinea pig blood, 20 .mu.L of blood was mixed with
140 .mu.L of water producing an 8-fold dilution. In addition to
this dilution, 290 .mu.L of working reagent was mixed with 10 .mu.L
of sample for a 30-fold dilution. Therefore, the sample was diluted
a total of 240 fold. In the case of human blood, 10 .mu.L of blood
was mixed with 190 .mu.L of water producing a 20-fold dilution. In
addition to this dilution, 290 .mu.L of working reagent was mixed
with 10 .mu.L of sample for a 30-fold dilution. Therefore, the
sample was diluted a total of 600 fold. Thus, the concentrations of
AChE and BChE determined above were divided by the pathlength and
multiplied by the total dilution. Finally, the data was converted
from mAbs/min to U/mL, wherein 1 U/mL corresponds to the turnover
of 1 .mu.mol of substrate/min at 1 mM substrate concentration using
standard methods known in the art.
[0133] Moreover, the results can be normalized to the average
population by multiplying the AChE and BChE concentrations by the
ratio of the calculated or predetermined average population
A.sub.415 (A.sub.445) to that of the sample's A.sub.415 (A.sub.445)
(i.e., the average absorbency for all twelve wells in one row
corresponding to the sample in question). This method accounts for
volumetric errors introduced by the technician, since both AChE and
BChE are being modified by the same ratio. Alternatively, one could
selectively multiply the AChE results by the aforementioned ratio
to account for hematocrit variations. Likewise, since plasma has
absorption around 240 nm, a similar correction could be applied
selectively to the BChE values.
[0134] Table 1A shows that the precision of the assay for Hartley
guinea pig is constant at 0.003 U/mL for both AChE and BChE
corresponding to a precision of less than about 0.8% and about 0.3%
for uninhibited AChE and BChE, respectively. Due to the constant
nature of the error, increasing the extent of inhibition increases
the uncertainty associated with knowing the true value, however, a
working range of inhibition from about 0% to about 99% is still
clearly demonstrated for both AChE and BChE.
1 TABLE 1A AChE (U/mL) BChE (U/mL) Average STD Average STD [rac
Hup-A] nM 56 0.006 0.004 0.907 0.004 28 0.019 0.002 0.929 0.002 14
0.055 0.003 0.912 0.001 7.03 0.136 0.002 0.892 0.002 3.52 0.214
0.002 0.907 0.002 1.76 0.334 0.002 0.930 0.003 0.88 0.462 0.001
0.913 0.002 0.00 0.610 0.007 0.925 0.004 [Iso-OMPA] nM 320 0.605
0.007 0.048 0.003 160 0.630 0.003 0.071 0.001 80 0.616 0.001 0.080
0.001 40 0.635 0.004 0.143 0.001 20 0.657 0.004 0.279 0.005 10
0.585 0.004 0.565 0.003 5 0.597 0.005 0.699 0.001 0 0.582 0.008
0.945 0.004
[0135] FIG. 3A1 is a graph that shows the concentration of AChE and
BChE in Hartley guinea pig blood as a function of titration with
rac Hup-A. FIG. 3B1 is a graph that shows the concentration of AChE
and BChE in Hartley guinea pig blood as a function of titration
with Iso-OPMA. Note that FIG. 3A1 illustrates the selective nature
of rac Hup-A, and FIG. 3B1 likewise illustrates the selective
nature of Iso-OMPA.
[0136] Table 1B and FIG. 3A2 panels A-D show representative data
for human whole blood titrated rac Hup-A, Iso-OMPA, and combination
mixtures of rac Hup-A and Iso-OMPA. The table demonstrates several
other key details of the assay. First, the precision of the assay
for human blood is constant at about 0.01 U/mL regardless of
inhibitor for both AChE and BChE. This corresponds to a precision
of less than about 0.83% and about 0.34% for uninhibited AChE and
BChE, respectively. Second, due to the constant nature of the
error, increasing the extent of inhibition increases the
uncertainty associated with knowing the true value, however, a
working range of inhibition from about 0% to about 99% is still
clearly demonstrated for both AChE and BChE. Third, the inter run
variability was about 1.9% and about 1.0% for AChE and BChE,
respectively. The AChE value was obtained by evaluating the % CV
for all AChE samples in the presence and absence of Iso-OMPA (ie.,
Iso-OMPA does not affect AChE concentration). Likewise, the BChE
value refers to the % CV for all BChE values obtained in the
presence and absence rac Hup-A. Finally, mixtures of selective AChE
and BChE inhibitors produce identical results to those obtained
with the isolated pure inhibitor. See FIG. 3A2.
2 TABLE 1B Inhibitor/Inhibitor Mixture Iso-OMOA & Hup-A Hup-A
Iso-OMPA [Hup-A], nM [Iso-OMPA], nM Average Stdev Average Stdev
Average Stdev AChE, 56 1280 0.292 0.027 0.315 0.003 4.181 0.015
U/mL 28 640 0.354 0.005 0.397 0.010 4.085 0.020 14 320 0.640 0.007
0.642 0.008 4.079 0.023 7 160 1.158 0.010 1.108 0.014 3.974 0.020 4
80 1.809 0.001 1.761 0.010 3.992 0.024 2 40 2.459 0.005 2.378 0.021
4.010 0.017 1 20 3.172 0.016 3.067 0.015 3.980 0.015 0 0 3.947
0.025 4.029 0.020 3.973 0.006 BChE, 56 1280 2.676 0.011 0.847 0.003
0.875 0.007 U/mL 28 640 2.609 0.005 1.567 0.005 1.491 0.008 14 320
2.638 0.006 2.068 0.004 2.054 0.010 7 160 2.633 0.009 2.347 0.012
2.318 0.018 4 80 2.617 0.016 2.495 0.009 2.440 0.009 2 40 2.612
0.013 2.563 0.009 2.509 0.009 1 20 2.600 0.005 2.586 0.007 2.572
0.005 0 0 2.653 0.007 2.633 0.002 2.636 0.006
[0137] In Table 1B, inhibitor concentrations correspond to those in
undiluted whole blood. The samples were incubated at room
temperature for three hours.
[0138] FIG. 3A2 is a graph that shows the concentration. of AChE
and BChE in human blood as a function of titration with rac Hup-A.
FIG. 3B2 is a graph that shows the concentration of AChE and BChE
in human blood as a function of titration with Iso-OPMA. FIG. 3C2
is a graph that depicts the concentration of AChE as a function of
rac Hup-A contained in the combined inhibitor mixtures. FIG. 3D2
illustrates the response of BChE as a function of Iso-OMPA
concentration present in the combined mixtures. Note that FIGS.
3A1, 3A2 and 3C2 illustrate the selective nature of rac Hup-A,
while FIGS. 3B1, 3B2 and 3D2 similarly illustrate the selective
nature of Iso-OMPA
EXAMPLE 3
Sensitivity Coefficient Determiination: Method 1
[0139] The sensitivity of AChE and BChE towards ACT, PTC, and BTC
was established as detailed below for Hartley guinea pig.
[0140] A stock solution of 900 nM rac Hup-A was prepared in 18.2
M.OMEGA. water. A stock solution of 5.12 .mu.M Iso-OMPA was
prepared in 18.2 M.OMEGA. water. A stock solution of 900 nM Hup-A
and 5.12 .mu.M Iso-OMPA (Hup-A/Iso-OMPA) was prepared in 18.2
M.OMEGA. water.
[0141] Serial dilutions of the Hup-A stock solution were prepared
and resulted in concentrations of 900, 450, 225, 113, 56, 28, 14,
and 0 nM of Hup-A. Serial dilutions of the Iso-OMPA stock solution
were prepared and resulted in concentrations of 5120, 2560, 1280,
640, 320, 160, 80, and 0 nM of Iso-OMPA. Serial dilutions of the
Hup-A/Iso-OMPA stock solution were prepared and resulted in
concentrations of 900, 450, 225, 113, 56, 28, 14, and 0 nM of Hup-A
and 5120, 2560, 1280, 640, 320, 160, 80, and 0 nM of Iso-OMPA,
respectively.
[0142] For each species, ten different whole blood samples were
obtained and then pooled together. Each whole blood sensitivity
coefficient sample was the pooled whole blood sample and
represented an average sample for each given species. Serial
dilutions of each sensitivity coefficient sample were prepared in
18.2 M.OMEGA. water and resulted in concentrations of 0.5, 0.25,
0.125, 0.063, 0.031, 0.016, 0.008, and 0.004 (volume:volume).
[0143] Generally, each sensitivity coefficient sample, the serial
dilutions described above, was titrated with the inhibitor, rac
Hup-A. Then the activity of an aliquot. of each sensitivity
coefficient sample after a three-hour incubation at room
temperature was measured in the presence of acetylthiocholine. This
was repeated for propionylthiocholine (PTC), butyrylthiocholine
(BTC), and finally 4,4'-dithiopyridine (DTP). At infinite inhibitor
concentration, the activity of the AChE component was selectively
eliminated, and the residual activity was solely from BChE.
Analysis of the measured substrate rates (ATC, PTC, BTC) in the
absence and presence of infinite inhibitor, corrected for
background hydrolysis (DTP), as a function of serial dilution
produced linear relationships corresponding to control (Vc) and
residual (Vr) rates, respectively. See FIGS. 10A-B for ATC/rac
Hup-A results. The slope of each Vr line represented the
sensitivity of BChE for each substrate. The sensitivity of AChE for
each substrate was obtained by subtracting the sensitivity of BChE
from the corresponding slopes of the control reactions, Vc.
[0144] The previously described titration was repeated using
Iso-OMPA. This time the slope of the line for the residual
activities (i.e., in the presence of infinite inhibitor, Vr)
represented the sensitivity of AChE for each substrate. The
sensitivity of BChE for each substrate was obtained by subtracting
the sensitivity of AChE from the corresponding slopes of the
control reactions, Vc.
[0145] Specifically, the stock and working reagents as set forth in
Exarnple 2 were used. A microtiter plate spectophotometer such as
Spectramax Plus microtiter plate spectrophotometer was used. Two
assays were performed on each sample. The first was a kinetic assay
possessing the following parameters: 1) 324 nm wavelength, 2) 60
second pre-read shaking, 3) 3 second shaking between reads, 4) a 4
minute collection time, and 5) linear least squares data analysis.
Upon completion of the first assay, the second, an endpoint assay,
was done using the following parameters: 1) two wavelengths, 415 nm
and 445 nm and 2) a 5 second pre-read shaking.
[0146] The activity of the control vs. the concentration of each
blood sensitivity coefficient sample was determined. The A.sub.415
and A.sub.445 vs. blood concentration were determined and the most
appropriate range of blood concentrations was used. It is desirable
to have the high end linear over 4 minutes and have enough signal
over the low end such that the titration with Hup-A/Iso-OMPA can
clearly be resolved from the baseline. See FIGS. 10A-B ATC/rac
Hup-A results. It is also desirable to consider a blood range in
which the relationship of A.sub.415 or A.sub.445 vs. the blood
concentration is linear in order to normalize the data. See FIG.
11.
[0147] After the appropriate concentration range was determined, 4
or 5 serial dilutions of the pooled whole blood spanning the
appropriate concentration were prepared. The volume of each
dilution was 4 mL.
[0148] 150 .mu.L aliquots of each blood dilution were mixed with 10
.mu.L aliquots of each dilution of Hup-A, Iso-OMPA, and
Hup-A/Iso-OMPA and incubated at room temperature for 3 hours on a
plate rocker. After incubation, the sensitivity coefficient samples
were mixed five times in the PCR tubes by pipetting. 10 .mu.L of
each sensitivity coefficient sample were dispensed into each column
of a 96 well microtiter plate (i.e., 8 sensitivity coefficient
samples were dispensed into 12 columns=96 wells, corresponding to
one blood dilution at eight inhibitor concentration). 290 .mu.L
aliquots of working reagent D (background) were added to columns
1-3, 290 .mu.L aliquots of working reagent A (acetylthiocholine)
were added to columns 4-6, 290 .mu.L aliquots of working reagent P
(propionylthiocholine) were added to columns 7-9, and 290 .mu.L
aliquots of working reagent B (butyrylthiocholine) were added to
columns 10-12 with a multichannel electronic pipette. The rates of
hydrolysis and the absorbancies of the sensitivity coefficient
samples were measured as described above. Each substrate rate was
corrected for spontaneous background hydrolysis of DTP. The
activity of the control vs. the concentration of each blood
sensitivity coefficient sample was determined and plotted on a
graph. This was repeated for each remaining blood dilution and
inhibitor/inhibitor mixtures. See FIG. 10A for ATC/rac Hup-A
results.
[0149] Each titration for each substrate and each inhibitor (Hup-A,
Iso-OMPA, Hup-A/Iso-OMPA) was fitted to the following equation: 1 V
obs = ( Vc - Vr ) K I K I + [ I ] + Vr
[0150] in which Vc and Vr refer to the velocities at 0 and infinite
inhibitor concentration, respectively, and K.sub.I refers to the
observed inhibition constant. Vc, Vr, and K.sub.I are obtained by
fitting the observed velocities vs. inhibitor concentration using
non-linear least squares fitting procedures known in the art. The
control activities, Vc, and the residual activities, Vr, were
tabulated and used in subsequent calculations.
[0151] For each substrate the control activities, Vc, vs. blood
dilution, and the residual activity, Vr, vs. blood dilution were
plotted on a graph for each type of inhibitor. See FIG. 10B for
ATC/rac Hup-A results. At an absorbance of 415 nm vs. the blood
concentration, the average slope and intercept for: human were
31.785 and 0.027; Rhesus monkey were 30.460 and 0.038. At an
absorbance of 445 nm vs. the blood concentration, the average slope
and intercept for: Hartley guinea pig were 4.500 and 0.027 (FIG.
11); Sprague Dawley rat were 5.269 and 0.017. These values permit
normalizing any given sample to that of the average population as
previously described in Example 2. All measured velocities
possessed units of mAbs/min, while all measured absorbances are in
standard absorbance units.
[0152] The sensitivity coefficient samples (ie., the pooled whole
blood serial dilutions previously described) titrated with the
inhibitor solutions comprising both Hup-A and Iso-OMPA previously
detailed established that about 100% of the ChE activity was due to
AChE and BChE.
[0153] The sensitivity coefficients of AChE and BChE for ATC, PTC,
and BTC for Hartley guinea pig as determined for each specific
cholinesterase inhibitor are set forth in Table 2. The average of
said sensitivity coefficients are also tabulated. It is important
to note that these values may not be identical to those determined
by another lab since the pooled blood sample will not have an
identical cholinesterase composition due to individual sample
population variation of AChE and BChE. However, this is not
critical to the assay of the present invention since these values
only reflect the content of AChE and BChE of the pooled sample. The
actual concentrations of each protein as determined by Example 2,
however, remain the same or substantially similar regardless of the
sensitivity coefficients used, provided that the sensitivity
coefficients were obtained from the same species being tested.
3 TABLE 2 AChE BChE Coefficient Coefficient (mAbs/min/ (mAbs/min/
[blood]) [blood]) Species Substrate Value Error Value Error Hartley
Guinea Pig: rac Hup-A Titration ATC 444 8 270 4 PTC 226 16 518 12
BTC 43 13 515 9 Hartley Guinea Pig: Iso-OMPA Titration ATC 418 14
297 16 PTC 194 36 550 37 BTC 0 1 557 10 Hartley Guinea Pig: Average
of rac Hup-A & Iso-OMPA Titrations ATC 431 19 284 19 PTC 210 23
534 23 BTC 21 30 536 30
EXAMPLE 4
Sensitivity Coefficient Determination: Method 2
[0154] The sensitivity of AChE and BChE towards ATC, PTC, and BTC
was established using a modification of the procedure outline in
Example 3 for Hartley guinea pig, human, Rhesus monkey, and Sprague
Dawley rat. The advantage of this method is faster processing time
and one-third less sensitivity coefficient sample needs to be
obtained.
[0155] Specifically the procedure is analogous to that in Example 3
except that only the Hup-A titration is performed. All remaining
steps including sample preparation, data collection, and data
analysis remain the same. The sensitivity coefficients of AChE and
BChE for ATC, PTC, and BTC for Hartley guinea pig, human, Rhesus
monkey, and Sprague Dawley rat blood determined using this method
are set forth in Table 3.
4 TABLE 3 AChE BChE Coefficient Coefficient (mAbs/min/ (mAbs/min/
[blood]) [blood]) Species Substrate Value Error Value Error Human
ATC 2162 68 841 17 PTC 1071 32 1417 16 BTC 82 3 1466 37 Rhesus
monkey ATC 1599 99 314 17 PTC 844 25 514 85 BTC 0 0 661 45 Hartley
Guinea Pig ATC 294 12 321 3 PTC 151 22 554 15 BTC 0 4 558 3 Sprague
Dawley Rat ATC 282 21 103 8 PTC 158 15 134 8 BTC 0 0 62 2
EXAMPLE 5
Stopped Time Assay
[0156] The assay of the present invention may be used to determine
the kinetics of inhibition for a non-selective inhibitor, such as a
non-selective cholinesterase inhibitor, or a selective inhibitor.
It is noted that extremely short time intervals of about 30 seconds
or the limiting speed of the human technician may be monitored by
the assay of the present invention.
[0157] Rhesus monkey blood was used in a modified stopped-time
assay wherein the activities and concentrations of AChE and BChE
were determined with 8 different concentrations of pyridostigmine
bromide (PB) were determined as a function of time. Serial PB
dilutions were prepared in whole blood and resulted in
concentrations of 10.0, 5.0, 2.5, 1.25, 0.63, 0.31, 0.16, and 0
.mu.M of pyridostigmine bromide.
[0158] 10 .mu.L aliquots of the eight resulting mixtures were
transferred into 200 .mu.L PCR tubes. The PCR tubes were flash
frozen on dry ice at 0, 1, 2, 3, 4, 5, 10, 15, 20, 30, 60, 90, and
at periods of time up to about 12500 minutes to prevent further
inhibition or activity return. Then each test sample was assayed
for ATC, PTC and BTC activity according to Example 2. Then the
concentrations of AChE and BChE were calculated for each time
interval and test sample. FIG. 4 is a graph that shows the
activities of AChE and BChE in Rhesus monkey blood affected by 2.5
.mu.M of PB as a function of time.
EXAMPLE 6
Chemical Warfare Agent Titration
[0159] 16 Biorad P6 spin columns (Bio-Rad Laboratories Hercules,
Calif.) were prepared according to the manufacture's directions.
Serial dilutions of soman (GD) were prepared in saline and resulted
in concentrations of 1.00.times.10.sup.-6, 8.00.times.10.sup.-7,
6.40.times.10.sup.-7, 5.12.times.10.sup.-7, 4.10.times.10.sup.-7,
3.28.times.10.sup.-7, 2.62.times.10.sup.-7, 2.10.times.10.sup.-7,
1.68.times.10.sup.-7, 1.34.times.10.sup.-7, 1.07.times.10.sup.-7,
8.59.times.10.sup.-8, and 0 M of GD.
[0160] 200 .mu.L aliquots of the GD serial solutions were placed
into fourteen 1.5 mL microfuge tubes. Then 100 .mu.L aliquots of
human whole blood were added to each of the 14 tubes. The resulting
blood solutions were mixed by vortexing and then incubated at room
temperature for 2 hours. The cholinesterase assay as described in
Example 2 was performed.
[0161] The concentrations of AChE and BChE were calculated for each
test sample as described in Example 2. FIG. 5 is a graph that shows
the concentrations of AChE and BChE in human blood as a function of
titration with GD. FIG. 5 is intended to illustrate that the
specific effects exerted on AChE and BChE by a relatively
non-specific antagonist can be monitored using the procedure
detailed in Example 2. Note that only the linear portion of each
titration are depicted for clarity.
EXAMPLE 7
Oxime Titration
[0162] A. 2-PAM
[0163] A stock solution of 2-PAM prepared in 18.2 M.OMEGA. water
was prepared. Serial dilutions of the stock solution were prepared
having the following concentrations: 0.5, 0.25, 0.125, 0.063,
0.016, 0.008, and 0.000 M. Next, 10 .mu.L aliquots of each dilution
were added to the wells of a microtiter plate followed by the
addition of 150 .mu.L aliquots of 8.times. diluted Hartley guinea
pig blood. After thorough mixing, the test samples were assayed as
described in Example 2. The background hydrolysis of DTP, ATC, PTC,
and BTC was measured without blood present. These values were
subtracted from the blood values prior to calculating the
concentrations of AChE and BChE as per Example 2. The results were
calculated and graphed as illustrated in FIG. 1.
[0164] B. HI-6
[0165] A stock solution of HI-6 prepared in 18.2 M.OMEGA. water was
prepared. Serial dilutions of the stock solution were prepared
having the following concentrions: 0.450, 0.225, 0.112, 0.056,
0.028, 0.014, 0.007, and 0.000 M. Next, 10 .mu.L aliquots of each
dilution were added to the wells of a microtiter plate followed by
the addition of 150 .mu.L aliquots of 20.times. diluted Rhesus
monkey blood. After thorough mixing, the test samples were assayed
as described in Example 2. The background hydrolysis of DTP, ATC,
PTC, and BTC was measured without blood present. These values were
subtracted from the blood values prior to calculating the
concentrations of AChE and BChE as per Example 2. The results were
calculated and graphed as illustrated in FIG. 2.
EXAMPLE 8
Removal of Impurities in Samples
[0166] To demonstrate the efficacy of removing small organic
molecules from a blood matrix using spin columns based on size
exclusion chromatography, three separate experiments were
preformed. In the first experiment, Biorad P6 spin columns removed
high concentrations of small organic molecules. In the second
experiment, variously diluted thoroughly lysed whole blood samples
were applied to individual Biorad P6 spin columns. The effluents
from these columns as well as their parent blood dilutions were
assayed for cholinesterase content as described in Example 2. In
the third experiment, A Biorad P6 spin column was used to separate
PB inhibited ChE in whole human blood from free excess PB. A
stopped time assay was performed on the column effluent as well as
an untreated matched control. The assay described in Example 2 was
used to monitor the increase in AChE concentration as a function of
time.
[0167] The results clearly demonstrate the feasibility of applying
thoroughly lysed whole blood samples to spin columns with little
cholinesterase retention and full uncomplexed ligand removal. The
following examples demonstrate one potential method for removing
interfering compounds from a sample. It is noted, however, that
other methods known in the art may be used to remove interfering
compounds from a sample.
[0168] A. Removal of GD by Biorad P6 Spin Columns
[0169] To determine whether spin columns can effectively remove an
impurity such as GD from a test sample, Biorad P6 spin columns were
used as per the manufacturer's directions.
[0170] Sixteen GD dilutions possessing the following concentrations
were prepared in saline: 1.0.times.10.sup.-3, 5.0.times.10.sup.-4,
2.5.times.10.sup.-4, 1.3.times.10.sup.-4, 6.3.times.10.sup.-5,
3.1.times.10.sup.-5, 1.6.times.10.sup.-5, 7.8.times.10.sup.-6.
3.9.times.10.sup.-6, 2.0.times.10.sup.-6, 9.8.times.10.sup.-7,
4.9.times.10.sup.-7, 2.4.times.10.sup.-7, 1.2.times.10.sup.-7,
6.1.times.10.sup.-8, and 0 M. A total of 150 .mu.L of each GD
dilution was prepared. 100 .mu.L aliquots of each dilution were
applied to each of sixteen prepared spin columns as per the
manufacturer's directions. The columns were centrifuged at
1000.times.g for 2 minutes and the effluent was collected. Next, 20
.mu.L of each GD solution's effluent as well as 20 .mu.L of each GD
dilution was applied to a microtiter plate. 100 .mu.L aliquots of
human whole blood were applied to each of the 32 microtiter wells
followed by thorough mixing. After incubation at room temperature
for 2 hours, the concentration of AChE and BChE contained within
each sample was determined as described in Example 2. FIGS. 6A and
6B show graphs which demonstrate that the spin columns are capable
of removing up to 100 nmol (ie., 100 .mu.L of 1.0.times.10.sup.-3
M) of a small organic molecule such as GD.
[0171] B. Thoroughly Lysed Whole Blood Applied to Biorad P6 Spin
Columns
[0172] Four month old human blood test samples, thoroughly
hemolyzed, were diluted with water and resulted in blood
concentrations of 1.0, 0.545, 0.298, 0.162, 0.089, and 0.048
(volume:volume). A 100 .mu.L aliquot of each test sample was added
to a prepared spin column as per the manufacture's directions. The
columns were centrifuged for 2 minutes at 1000.times.g. The
cholinesterase assay as described in Example 2 was performed on
each column effluent and a fraction of the blood remaining in each
original matched test sample. The cholinesterase levels contained
in each sample were plotted and are depicted in FIG. 6C. Thus,
cholinesterase from thoroughly hemolyzed human whole blood samples
was not retained by the Biorad P6 spin columns.
[0173] FIGS. 6A, 6B, and 6C demonstrate that the spin column method
effectively removes GD and that AChE and BChE contained in
thoroughly hemolyzed whole blood samples are not retained on Biorad
P6 spin columns. Thus, small interfering compounds such as oximes
can easily be removed from test samples, such as blood samples, by
this method.
[0174] C. Removal of Free Uncomplexed PB Via Biorad P6 Spin
Columns
[0175] One hundred fifty microliters of human blood was treated
with enough PB to achieve about 75% inhibition of AChE at the end
of a thirty minute incubation at room temperature. A control sample
was treated with an equal volume of water. These samples were
prepared as is common in the art. After incubation, a 100
microliter fraction of each test blood sample were applied to
separate Biorad P6 spin columns prepared as per the manufacture's
directions. Both columns, one containing the PB inhibited blood the
other containing the matched control, were centrifuged at
1000.times.g for two minutes. After centrifugation, the
cholinesterase activity of each effluent was monitored at 15, 30,
60, 120, 180, 240, 300, 360, 420, and 1680 minutes post column. The
results are depicted in FIG. 6D as the ratio of the inhibited to
control activity as a function of time. FIG. 6D illustrates that
spin column separation can be used to separate uncomplexed ligands
from a complex sample matrix such as blood. Without spin column
chromatography, the return to normal activity would not have
occurred during the monitored time frame. See FIG. 4 for a similar
example without size exclusion chromatography.
[0176] Thus, spin column chromatography can be used to quickly and
efficiently remove small interfering compounds from test samples
such as blood without retaining cholinesterase within the column's
matrix.
EXAMPLE 9
Comparison with COBAS/FARA and Test-Mate OP.TM. Methods
[0177] The cholinesterase assay of the present invention as
described in Example 2 was compared with a standard clinical assay,
COBAS/FARA (Roche Diagnostics Corporation, Indianapolis, Ind.), and
the accepted field assay of the United States Army, the TestMate OP
method, technical bulletin 296.
[0178] A vial of dilute GD, 10 mM in saline, was stored frozen
until further dilutions were prepared. 200 .mu.L of saline was
added to a microtube and set aside. The GD was thawed and two
dilutions were prepared to achieve a target dilution of 1 .mu.M.
200 .mu.L of the 1 .mu.M GD in saline was pipetted into a microtube
and set aside. Then, serial dilutions were prepared and resulted in
concentrations of 1000, 800, 640, 512, 410, 328, 262, 210, 168,
134, 107, 86, 69, 0 nM of GD. Fresh human whole blood was collected
from ten subjects by a phlebotomist in heparin Vacutainer.RTM..
[0179] Next, 200 .mu.L aliquots of each GD concentration were
transferred to 1.5 mL microfuge tubes. This was repeated 10 times,
one for each human subject, at each GD concentration (i.e., a total
of 140 microfuge tubes or 10 sets of 14 GD concentrations). To each
of the 14 tubes within the GD sample set, one milliliter of a
particular subject's blood was added with a positive displacement
pipettor. The tubes were capped and mixed by inversion. This
process was repeated for the remaining nine human subject blood
test samples. All 140 test samples were incubated overnight at room
temperature, and then the cholinesterase assay as described in
Example 2 was performed.
[0180] A fraction of the remaining test samples were analyzed for
AChE and BChE content using the procedure of the Test-Mate OPTM
system as per the manufacturer's directions. The remainder of each
test sample was centrifuged for 5 minutes at 14,000 RPM. Plasma
from each tube was carefully removed and placed into appropriately
labeled microtubes for cholinesterase analysis. The remaining red
blood cells (RBCs) in each tube were mixed and diluted 50 fold by
placing 20 .mu.L of RBCs into microtubes containing 980 .mu.L of 1%
Triton X-100 in saline. The plasma was diluted 15 fold by placing
68 .mu.L plasma into microtubes containing 932 .mu.L of 1% Triton
X-100 in saline. Then the COBAS/FARA assay was performed. Each
blood test sample was analyzed in triplicate with each specific
cholinesterase assay. In other words, the test samples were
analyzed 3.times. for erythrocyte (AChE) and plasma (BChE)
cholinesterase activity.
[0181] The average concentrations of AChE as determined by the
cholinesterase assay of Example 2 were 3.88, 3.25, 3.15, 2.98,
2.75, 2.55, 2.23, 1.87, 1.58, 1.11, 0.64, 0.36, 0.13, and 0.04 U/mL
for each serial dilution, respectively. The average concentrations
of AChE as determined by the COBAS/FARA assay were 12.97, 11.73,
10.99, 10.61, 10.07, 9.14, 7.80, 6.69, 5.47, 4.01, 2.46, 1.58,
0.61, and 0.34 U/mL for each serial dilution, respectively. The
concentrations of AChE as determined by the Test-Mate OP.TM. method
were 2.74, 2.43, 2.23, 2.05, 1.85, 1.61, 1.41, 1.12, 0.75, 0.41,
0.15, 0.04, 0.00, and 0.00 U/mL for each serial dilution,
respectively. These concentrations were plotted as shown in FIG.
7A. In FIG. 7A, it is important to note that for clarity only the
linear titration range is depicted.
[0182] The average concentrations of BChE as determined by the
cholinesterase assay of Example 2 were 2.38,2.17, 2.12, 2.07, 1.99,
1.90, 1.77, 1.65, 1.47, 1.28, 0.95, 0.71, 0.35, and 0.14 U/mL each
serial dilution, respectively. The average concentrations of BChE
as determined by the COBAS/FARA assay were 6.12, 6.13, 6.02, 5.41,
5.21, 4.98, 4.63, 4.19, 3.67, 3.06, 2.29, 1.61, 0.75, and 0.20 U/mL
for each serial dilution, respectively. The concentrations of BChE
as determined by the Test-Mate OP.TM. method were 1.02, 1.00, 0.90,
0.75, 0.76, 0.68, 0.60, 0.52, 0.41, 0.31, 0.14, 0.06, 0.00, and
0.00 U/mL for each serial dilution, respectively. These
concentrations were plotted as shown in FIG. 7B. In FIG. 7B, it is
important to note that for clarity only the linear titration range
is depicted.
[0183] FIGS. 7A and 7B clearly demonstrate that all three methods
produce co-linear trends for both AChE and BChE, however, the
depicted figures are a reflection of the average population.
Therefore, large sample numbers can mask significant individual
deviations. This is illustrated in FIG. 7C for the Test-Mate OP.TM.
and the COBAS/FARA methods. In fact, for any given individual
sample, the results determined by the methodology of this invention
are more co-linear than those of the other two techniques and
therefore more reliable and accurate. FIGS. 7A and 7B also
illustrate that the cholinesterase assay of the present invention
produces titrations that are more tightly distributed around the
mean than the COBAS/FARA or Test-Mate OP.TM. assays. The average
population distributions for the COBAS/FARA, Test-Mate OP.TM., and
the current invention are 13%, 12%, and 9%, respectively for AChE
and 24%, 30%, and 19%, respectively for BChE.
[0184] Since each absolute value for AChE and BChE are different
for each assay conducted, the average results obtained from the
COBAS/FARA and Test-Mate OP.TM. methods were plotted as a function
of the average AChE/BChE concentration determined from Example 2.
FIGS. 7D and 7E clearly demonstrate the linear relationship between
the two established assay methods and that of the current invention
for both AChE and BChE. FIGS. 7D and 7E also demonstrate that
results from established methods can be converted to those of the
current invention by applying a simple linear transformation. This
allows the conversion of cholinesterase databases constructed using
prior methods to be converted to the values of the current
invention once validation between the methods has been
established.
EXAMPLE 10
In-Vivo Monitoring
[0185] The cholinesterase assay of the present invention as
described in Example 2 was used to assess the extent of AChE and
BChE inhibition in Hartley guinea pigs induced by intramuscular
(IM) injection of pyridostigmine bromide (PB) (Sigma, St. Louis,
Mo.) as a function of time. The experiment was repeated several
times at various PB doses to determine the peak inhibition time and
the extent of AChE inhibition as a function of IM PB dose.
[0186] Specifically each experiment consisted of the following.
Stock solutions of PB were prepared in saline such that an
injection of a 100 .mu.L aliquot of said solution IM into an adult
male Hartley guinea pig of a known weight produced doses of 5, 10,
20, and 40 .mu.g/kg body weight PB. At time 0, 100 .mu.L of a
particular PB stock solution was injected IM into an adult male
Hartley guinea pig of a predetermined weight achieving the desired
PB dose. At times 0, 15, 30, 60, 90, 120, 150, 180, 240 and 300
minutes the guinea pig was bled through an implanted carotid artery
catheter. At the specified time, the catheter was opened and two
drops of blood were discarded. This blood represented the void
volume of the catheter. 20 .mu.L of the next drop of blood was
collected and transferred to a 200 .mu.L PCR tube containing 8 U of
heparin (8 .mu.L of 1000 U/mL heparin, VWR Scientific, Bridgeport,
N.J.). Following thorough mixing, the blood samples were flash
frozen on powdered dry ice and stored at -80.degree. C. until the
completion of the experiment.
[0187] At the end of each experiment, all of the samples were batch
analyzed for AChE and BChE concentration and activity as described
in Example 2. However, a slight modification of Example 2 was used
in that 132 .mu.L instead of 140 .mu.L of 18.2 M.OMEGA. water was
added to each frozen blood sample. The overall dilution due to
sample preparation, however, was still 8 fold (ie., 20 .mu.L blood
in a total volume of 160 .mu.L).
[0188] All data was normalized to percent control activity by
dividing the AChE and BChE concentrations by those determined at
time 0. The percent activity data sets for a given PB dose were
averaged together for each time point and the standard deviations
for each time point was also calculated. The average AChE and BChE
activities as a function of time were plotted and fit to standard
equations known in the art. FIG. 8A represents the average data set
for the 20 .mu.g/kg dose. Using standard equations known in the
art, the peak inhibition time for the PB dose range investigated
was determined to be about 30 minutes. Therefore, the extent of
inhibition at 30 minutes was calculated from the theoretical fits
to determine peak inhibition. Peak inhibition was plotted as a
function of PB dose and is depicted in FIG. 8B.
[0189] This experiment demonstrates that the assay of the current
invention is capable of monitoring the pharmacokinetics and
pharmocodynamics of in-vivo administered compounds that affect the
concentration or activities of AChE, BChE, or both. Therefore, due
to the unique characteristics of the method of this invention, it
can easily be extended to any other in-vivo experiment designed to
monitor the concentrations or activities of AChE, BChE, or both in
whole blood or any other biological tissue, fluid, or sample
containing AChE, BChE, or both. In addition, the assay set forth in
this invention can be used to monitor the progress of a treatment
regime, since periodic monitoring of the concentrations of AChE,
BChE, or both as a function of time would be required. This
parallels the time course nature of this in-vivo experiment.
EXAMPLE 11
Monitoring the Stability of AChE and BChE in a Whole Blood
[0190] The cholinesterase assay of the present invention as
described in Example 2 was used to monitor the stability of whole
blood AChE and BChE to extreme freezing. The goal of this
experiment was to determine if Hartley guinea pig whole blood could
be stored at -80.degree. C. for prolonged periods of time without
altering the cholinesterase activity. In addition, the effect of
repetitive freeze thawing on dry ice was also investigated. These
two issues are of great concern for batch sample processing of time
sensitive samples.
[0191] A. Prolonged Exposure to -80.degree. C.
[0192] Eight 20 .mu.L aliquots of heparin treated Guinea pig whole
blood were added to eight 200 .mu.L PCR tubes. Seven of these were
flash frozen on powdered dry ice then stored at -80.degree. C. The
eighth sample was marked as time zero, and analyzed for
cholinesterase content as per example 2. At times 0.5, 1, 2, 4, 24,
48, 72, and 144 hours, one of the PCR tubes was removed from the
deep freezer and assayed for cholinesterase content again as
described in Example 2. The AChE and BChE levels were plotted as a
function of time frozen, and the results are depicted in FIG.
9A.
[0193] This experiment clearly illustrates that prolonged exposure
to harsh temperatures does not alter the activities or
concentrations or AChE or BChE contained in guinea pig whole blood.
The average AChE and BChE concentrations for this experiment were
0.71.+-.0.04 U/mL and 0.96.+-.0.04 U/mL, respectively, while the
respective control values were 0.72.+-.0.01 U/mL and 0.97.+-.0.01
U/mL.
[0194] B. Repetitive Freeze Thawing
[0195] A 450 .mu.L aliquot of heparin treated Hartley guinea pig
whole blood was frozen over dry ice and repeatedly frozen then
thawed. During each thawing, a 20 .mu.L aliquot was diluted to a
fmal volume of 160 .mu.L using 18.2 M.OMEGA. water. Each sample was
then assayed as described in Example 2 for the concentrations and
activities of AChE and BChE. The results were then plotted as a
function of the number of times frozen then thawed. The results are
depicted in FIG. 9B. As is clearly demonstrated by FIG. 9B,
repetitive freezing does not alter guinea pig blood cholinesterase
content. The average AChE and BChE concentrations for this
experiment were 0.68.+-.0.04 U/mL and 0.94.+-.0.04 U/mL,
respectively, while the respective control values were
0.630.+-.0.008 U/mL and 0.994.+-.0.006 U/mL. This fact when
compared to A above allows even greater flexibility in experimental
design and sample storage for subsequent batch analysis.
[0196] As demonstrated by the previous two examples, the method as
detailed in the invention is capable of monitoring the stability of
a biological sample. In addition, this method could be extended to
any other sample containing AChE, BChE, or both in order to assess
sample stability or the effect a particular processing step causes
on the stability of the proteins in a sample.
EXAMPLE 12
Validation of Automated Cholinesterase Assay
[0197] The cholinesterase assay of the current invention as set
forth in Example 2 was ported to an automated platform. This was
accomplished by interfacing a Molecular Devices SpectraMax Plus to
a Beckman-Coulter Biomek 2000 liquid handling workstation. The
Biomek 2000 was then programmed to perform all of the necessary
plate handling, sample preparation, and reagent additions as
described in Example 2. To demonstrate that the manual and
automated methods produced comparable results, the cholinesterase
levels of a series of serial dilutions of human whole blood were
measured via Example 2 and the Biomek 2000 ported method described
above. The serial dilutions were prepared in 18.2 M.OMEGA. water
and included relative blood concentrations of 1.0 (undiluted whole
blood), 0.75, 0.56, 0.42, 0.32, 0.24, 0.18, and 0.13. The AChE and
BChE activities were plotted as a function of relative blood
concentration for both methods and are depicted in FIG. 12. The
AChE slopes determed via linear least squares analysis were
3.91.+-.0.05 and 3.90.+-.0.02 U/mL/blood dilution for the Biomek
2000 and manual methods, respectively. The similarly determined
slopes for BChE were 1.80.+-.0.02 and 1.79.+-.0.01 U/mL/blood
dilution for the Biomek 2000 and manual methods, respectively. As
shown by the slopes and FIG. 12, essentially no bias was introduced
by porting the method to the Biomek 2000 platform as comparable
results were obtained.
EXAMPLE 13
Inter-Lab Validation
[0198] The automated cholinesterase assay of the present invention
as described in Example 12 was implemented at the United States
Army Medical Research Institute of Chemical Defense (USAMRICD) as
well as the Walter Reed Army Institute of Research (WRAIR). To
validate the assay, each lab independently prepared all stock
reagents as detailed in Example 2. Next, blood samples from eight
human volunteers were titrated ex vivo with six different doses of
GD to produce cholinesterase inhibition ranging from about 0% to
about 75% inhibition by standard methods in the art. After an
overnight incubation, both labs independently measured the AChE
activity for each sample. The results were graphed as "ICD AChE"
versus "WRAIR AChE". See FIG. 13. Linear least squares analysis of
the data produced a slope of 0.983.+-.0.006. The slope indicates
that less than about a 1.7% bias exists between the two institutes
which is most likely due to inter run variability since it is less
than the inter run variability of about 1.9% for human blood
reported in Example 2. Finally, this example illustrates that the
assay of the current invention is easily ported to other
facilities.
EXAMPLE 14
Comparable Results for Whole Blood and Packed Red Blood Cells
[0199] To demonstrate the assay of the current invention produces
comparable results for both whole blood and packed red blood cells,
fresh blood from eight human volunteers was titrated ex vivo with
six different doses of GD to produce cholinesterase inhibition
ranging from about 0% to about 75% inhibition by methods standard
in the art. Roughly half of the sample was centrifuged in order to
separate the RBCs from the plasma by methods standard in the art.
After an overnight incubation, all samples were independently
assayed for AChE activity. Data processing included population
normalization as explained in Example 2.
[0200] The results were graphed as "RBC AChE" versus "WB AChE" as
shown in FIG. 14 which is a plot of packed red blood cell (RBC)
AChE activity as a function of their parent whole blood (WB) values
as determined by the assay of the present invention. FIG. 14
illustrates that that the assay produces the substantially the same
results for both RBC and WB samples. This is unlike other assays
known in the art. The range of cholinesterase levels was achieved
by titrating human whole blood with the nerve agent GD as is common
in the art. Linear least squares analysis of the data produced a
slope of 1.03.+-.0.02. The slope indicates that less than about a
3% bias exists between the two institutes which is most likely due
to inter run variability since it is about the same magnitude as
that for the inter run variability of about 1.9% for human blood
reported in Example 2.
EXAMPLE 15
Equality of Intravenous and Finger Prick Methods of Sample
Collection
[0201] To determine if sample collection produced different
results, blood was collected intravenously, IV blood, by a
phlebotomist using heparin-coated vacutainers by methods standard
in the art. At the same time, ten microliters of blood was
collected with a positive displacement pipette from a lancet finger
prick, FP blood, of the same five individuals. The FP blood was
immediately diluted 20 fold with 18.2 M.OMEGA. water. The AChE and
BChE activities were measured as per Example 2, except that the FP
blood sample was already considered processed. The results were
plotted as individual bar charts for AChE and BChE. See FIG. 15.
The average AChE activity for the five volunteers was about
4.3.+-.0.6 and about 4.3.+-.0.5 for the IV blood and FP blood
samples, respectively. The average BChE activity for the five
volunteers was about 2.3.+-.0.5 and about 2.6.+-.0.6 for the IV
blood and FP blood samples, respectively. Thus, different blood
sources, IV blood or IP blood, provides comparable results for the
cholinesterase activity determined by Example 2.
EXAMPLE 16
Inter and Intra Day Assay Variability
[0202] To determine the inter and intra day variability of the
present invention of Example 2 as implemented on the Biomek 2000
system detailed previously, fresh human whole blood was diluted
serially with 18.2 M.OMEGA. water. Next, the cholinesterase levels
of each blood dilution was determined as explained in Example 2 as
implemented on the Biomek 2000 workstation. This was repeated six
times on one day, and then once a day for three consecutive days.
The AChE and BChE values were plotted as a finction of blood volume
followed by linear least squares analysis of only the linear
portion of each data set. See FIG. 16A for a representative plot.
Next, the slopes from each trial were plotted as a bar graph for
both the inter and intra day data. See FIG. 16B. The % CV for the
six intraday runs was about 0.5% and about 1.1% for AChE and BChE,
respectively. While the % CV for the three interday determinations
was about 1.7% and about 1.5% for AChE and BChE, respectively.
These results show that the present invention provides the
determination of highly precise cholinesterase values.
Additionally, FIG. 16A demonstrates that the assay of the present
invention is linear over about two orders of magnitude that
translates into a linear range of detection of about 0% to about
99% which is consistent with that previously reported in Example
2.
EXAMPLE 17
Robustness of the Assay with Respect to Substrate Concentration
[0203] To determine the effect of substrate variation on the assay
of the current invention as implemented on a Biomek 2000 liquid
handling workstation, human whole blood sample serial dilutions
were assayed in the presence of various concentrations of each
substrate (i.e., ATC, PTC, BTC). The concentration of each
substrate was independently varied from about 20% below normal to
about 20% above normal in 10% increments. Normal is defined as the
concentration of the stock solutions as in Example 2. This resulted
in a matrix comprising 125 different combinations of ATC, PTC, and
BTC (i.e., 5 ATC levels (-20%, -10%, normal, 10%, 20%).times.5 PTC
levels.times.5 BTC levels=125).
[0204] Each of the 125 elements of the substrate matrix was used to
determine the AChE and BChE levels in each of the eight serial
blood dilutions. The AChE and BChE results were plotted as a
function of blood dilution, and the slopes of the resulting linear
relationships were determined via linear least squares analysis.
The slopes for AChE and BChE were normalized to that of the normal
method (ie., substantially the same conditions of Example 2). The
125 normalized values produced an average of about 100.+-.5% and
about 103.+-.7% for AChE and BChE, respectively. These results show
that the assay of Example 2 is extremely robust with respect to
substrate variation since about a 40% swing in any individual
substrate produced no statistically observable deviation in the
calculate cholinesterase activities.
EXAMPLE 18
Percent AChE Lost During Blood Donation
[0205] The assay of the present invention was used to screen an
individual for loss in AChE activity as a function of time due to
blood donation. In this experiment, a volunteer donated one unit of
whole blood. Blood AChE levels and the blood sample's absorbance at
415 nm, A.sub.415, were measured as per Example 2 at 30 minutes
prior to donation and at 30, 60, 90, 120, 180, and 300 minutes post
donation. The percent loss in AChE activity and A.sub.415 were
calculated based on the 30 minute pre-donation levels. The results
were graphed and are depicted in FIG. 17. FIG. 17 shows the percent
loss in human AChE activity following donation of one standard unit
of blood as well as the loss in hemoglobin as reflected by the
decrease in the absorbance at 415 nm (A.sub.415). FIG. 17
illustrates two important points. First, the loss of AChE activity
tracks identically to the loss in A.sub.415 which is a crude
measure of hematocrit since hemoglobin, a normal component of RBCs,
absorbs maximally at 415 nm. Second, the assay of Example 2 is
capable of measuring minute changes in AChE activity, about 1.5%.
Thus, this assay may be used to monitor subtle changes, about 1.5%,
in AChE levels such as subtle changes resulting from pesticide
poisoning, blood loss during surgery, or the like.
[0206] To the extent necessary to understand or complete the
disclosure of the present invention, all publications, patents, and
patent applications mentioned herein are expressly incorporated by
reference therein to the same extent as though each were
individually so incorporated.
[0207] Having thus described exemplary embodiments of the present
invention, it should be noted by those skilled in the art that the
within disclosures are exemplary only and that various other
alternatives, adaptations, and modifications may be made within the
scope of the present invention. Accordingly, the present invention
is not limited to the specific embodiments as illustrated herein,
but is only limited by the following claims.
* * * * *