U.S. patent application number 10/652901 was filed with the patent office on 2004-06-24 for method for detection of 4-hydroxybutyric acid and its precursor(s) in fluids.
Invention is credited to Smith, Jack V..
Application Number | 20040121420 10/652901 |
Document ID | / |
Family ID | 27789314 |
Filed Date | 2004-06-24 |
United States Patent
Application |
20040121420 |
Kind Code |
A1 |
Smith, Jack V. |
June 24, 2004 |
Method for detection of 4-hydroxybutyric acid and its precursor(s)
in fluids
Abstract
A method for determining the presence or amount of
gamma-hydroxybutyrate or precursors in a sample, said method
comprising contacting said sample with an indicator which
specifically binds to gamma-hydroxybutyrate or precursors to form
an indicatorcomplex; and, measuring said indicatorcomplex to
determine the presence or amount of said gamma-hydroxybutyrate or
precursors in said sample.
Inventors: |
Smith, Jack V.; (Arden,
NC) |
Correspondence
Address: |
JACK V. SMITH
P.O. BOX 156
Arden
NC
28704
US
|
Family ID: |
27789314 |
Appl. No.: |
10/652901 |
Filed: |
August 28, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10652901 |
Aug 28, 2003 |
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09607026 |
Jun 29, 2000 |
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6617123 |
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Current U.S.
Class: |
435/18 ;
435/25 |
Current CPC
Class: |
C12Q 1/44 20130101 |
Class at
Publication: |
435/018 ;
435/025 |
International
Class: |
C12Q 001/34; C12Q
001/26 |
Claims
I claim:
1. A method for quantitatively determining gamma hydroxybutyrate
comprising: contacting a sample with the mixture of an enzyme
conjugated to gamma hydroxybutyrate, enzyme substrate, and a
buffer; determining a color change in the mixture; and correlating
a color change in the mixture with the amount of GHB in the
sample.
2. The method according to claim 1 wherein the enzyme is selected
from the group consisting of glycosidases, esterases, sulfatases,
phosphatases, hydroxylases and oxidoreductases.
3. The method according to claim 1 wherein the buffer is selected
from the group consisting of Hepes, citrate, borate, borax, sodium
tetraborate decahydrate, sodium perchlorate, sodium chlorate,
sodium carbonate, TRIS (Tris[hydroxymethyl]aminomethane, MES
(2-[N-Morpholino]ethanesulfonic acid), BIS-TRIS
(bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;
2-bis[2-hydroxyethyl]amino-2-[hydroxymethyl-1,3-propanediol), ADA
(N-[2-Acetamidol]-2-iminodiacetic acid;
N-[Carbaoylmethyl]iminodiacetc acid), ACES
(2-[(2-Amino-2-oxoethyl)amino]ethanesulfonic acid;
N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES
(PiperazineN-N'-bis[2-ethanesulfonic acid)];
1,4-Piperzinedethanesulfoic acid), MOPSO
(3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRIS PROPANE
(1,3-bis[tris(Hydroxymethyl)methylamino]propane), BES
(N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid;
2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS
(3-[N-Morpholino]propanesulfonic acid), TES
(N-tris[Hydroxymethyl]methyl-- 2-aminomethanesulfonic acid;
2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amin- o)ethanesulfonic
acid), DIPSO (3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxyp-
ropanesulfonic acid), TAPSO
(3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxy- propanesulfonic
acid), HEPPSO (N-[2-Hydroxythyl]piperazine-N'-[2Hydroxypro-
panesulfonic acid]), POPSO
(Piperazine-N,N'-bis[2-hydroxypropanesulfonic acid]), EPPS
(N-[2-Hydroxyethyl]piperazine-N'-[3-propanesulfonic acid), TEA
(triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine;
N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE
(N,N-bis[2-Hydroxyethyl]glycine), TAPS
(N-tris[Hydroxymethyl]methyl-3-ami- nopropanesulfonic acid;
([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-pr- opanesulfonic
acid), AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydro-
xypropanesulfonic acid), CHES (2-[N-Cyclohexylamino]ethanesulfonic
acid), CAPSO (3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic
acid), AMP 2-Amino-2-ethyl-1-propanol, CAPS
(3-[cyclohexylamino]-1-propanesulfonic acid), hydrochloric acid,
phosphoric acid, lactic acid, sulfuric acid, nitric acid, chromic
acid, boric acid, perchloric acid, potassium hydrogen tartrate,
potassium hydrogen phthalate, calcium hydroxide, phosphate,
bicarbonate, sodium hydroxide, potassium hydroxide, oxalate and
succinate.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a division of application Ser. No. 09/607,026, Filed
Jun. 29, 2000, now U.S. Pat. No. 6,617,123 granted Sep. 9,
2003.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] As the use of illicit drugs in this country has increased,
public concern over the problems associated with its effects has
grown into a major concern. This concern has led to workplace drug
testing in order to identify, treat, and remove active drug users
from the workforce. This trend started in the military, and spread
rapidly to law enforcement and any "safety-sensitive" private
sector jobs such as airline pilots, truck drivers, and active
crewmembers of public transportation. These initial strides into
drug testing in the workplace revealed the obtrusive incursion of
drug use and abuse in the daily lives of a significant portion of
Americans. Further research indicated the staggering costs to
public and private industry in terms of lost productivity,
increased health care costs, and human suffering and death due to
this scourge of drug abuse. As a result, drug testing has rapidly
spread to all areas of the public and private sector. The vast
majority of workplace drug testing has taken the form of urine
testing, because of ease of collection, low cost, and effective
indication of recent drug use. Other forms of testing include
analysis of blood, saliva, sweat, and hair.
[0004] Gamma-hydroxybutyrate (4-hydroxybutyrate, 4-hydroxybutyric
acid, Gamma-hydroxybutyric acid, 4-hydroxybutyric acid sodium salt,
GHB) was first used for anesthetic purposes in 1961, because it was
unpredictable and caused adverse effects, its use was discontinued.
Later, GHB was used by body builders for muscle building and weight
control. Presently, the U.S. DEA (Drug Enforcement Agency) is
investigating GHB to see if it should be a controlled substance.
The U.S. FDA (Food and Drug Administration) list GHB as an
unapproved drug except for investigational use in the treatment of
narcolepsy. Common names for GHB are Scoop, Georgia Home Boy,
Grievous Bodily Harm, Liquid Ecstasy, and Cherry Meth among others.
Its precursor GBL (gamma-butyrolactone) is used as a GHB substitute
and once ingested rapidly converts to GHB. The pharmacological
effects of both GHB and GBL are similar and the range of analgesic
effect (euphoria) are similar.
[0005] In the 1990's GHB has become a popular drug of abuse on
college campuses, bars, and dance clubs and is called the "date
rape" drug. The abuse of GHB has enormous sociological and economic
impact on our society. A typical "date rape" scenario is as
follows: The victim(s), usually women, are in a bar, they drink a
beverage that has been laced with GHB by a rapist, the victim then
becomes catatonic and is usually agreeable with anyone and everyone
around them. They can become unconscious and then of course are
susceptible to the rapist desires. Later, the victim(s) wakes up
completely disoriented, naked and robbed. This type of horrific
tragedy is occurring on a daily basis. The victims of "date rape"
are not only exposed to the physical assault of rape and robbery,
but to the contraction of diseases such as AIDS and STD's. The
damage caused and the consequences of such occurrences are
immeasurable.
[0006] Accordingly, a need exists for providing an easy and
convenient manner by which to make a determination of the presence
of GHB in urine, in a beverage, or other biological fluids or
liquids. A further need exists for a convenient manner by which
such determinations may be made by using rapid analysis manual
techniques (such as a dipstick or lateral flow devices) and
automated techniques that will advance the art significantly. And,
the most important need is for a device that would detect GHB using
just a single assay that does not require an extraction process or
lactone conversion. This would be a marked advancement in the art
and would result in the savings of millions of dollars to the drug
testing laboratories required to perform GC (gas chromatography) or
GCMS (gas chromatography mass spectrometry) testing for GHB and
obviously this savings would be passed on to the end user (the
businesses which initially request drug's of abuse assays on
perspective and current employees). To explain further, the
drug-testing laboratory would normally perform GC (gas
chromatography) or GCMS (gas chromatography mass spectrometry)
assay for GHB. The necessary time to perform these assays is
burdensome to the laboratory through cost for tech time, reagents,
and turnaround time to mention a few. The alternative to this would
be the significant advancement in the art that the present device
offers which is the capability to detect GHB without lengthy
extraction processes of the current art with a single assay.
[0007] 2. Description of the Related Art
[0008] This invention is in the field of toxicology. More
specifically, this invention provides test strips (i.e. dry
chemistry dipsticks, or on-site test modules utilizing thin layer
chromatography in a lateral flow format, or other similar
technology to the test strip) and liquid chemistry reagents for use
in the detection of GHB with a single assay in aqueous fluids to
include but not limited to urine, saliva, serum, blood, sweat
extracts, liquid homogenates of hair and liquids such as beverages,
soft drinks, mixed drinks to included alcohol, etc.
[0009] It is known that the polarity and small molecular size of
the GHB molecule and the lack of detectability of the GHB by UV
(ultra violet), chromatographic, and spectrophotometric means
complicates detection. GHB is relatively unstable and will form the
GHB lactone derivative when heated and under acidic conditions. GHB
can cause euphoria at less than 50 ug/mL to marked central
depression, sleep, coma and death. Currently, all of the methods to
detect GHB use and/or solid-phase extraction, liquid-liquid
extraction, silyl-derivatization, and then GCMS. These methods
mentioned are very time consuming, expensive (costing the
laboratories, companies ordering drug testing, and general public
millions of dollars per year), and labor intensive. The GC-MS,
assay is typically performed to verify the urines that screen
positive for drugs of abuse. The GC-MS analysis costs 100 times as
much as the initial screen ($100 vs $1). Every additional
unnecessary GC-MS performed drives up the overall cost of drug
testing.
[0010] The novel invention described herein describes a method to
determine the presence or absence of GHB and its precursor(s) in
urine or other fluids by liquid and dry chemistry test means which
has not been taught prior to the present art. It should be noted
that GHB is not normally found in urine.
[0011] There are no published, taught, or even mentioned methods of
the present arts technology to detect GHB or GBL in urine by the
present arts techniques.
[0012] Again, a thorough search of patents and research revealed no
relative art (i.e., prior art) with any correlation to this
technology. The art of testing for GHB or GBL in urine or other
fluids as previously delineated in the literature describe various
techniques including methods for solid-phase extraction,
liquid-liquid extraction, silyl-derivatization, then GCMS. No
reference, however, has described this new art as delineated here.
The previous art will be enumerated here to further illustrate the
unique advancement in the field of GHB and GBL detection. It has
been acknowledged in the art that random urinary sample matrices
are very complex, and consist of many urinary constituents, which
create strong buffering, and interference problems (e.g. cannibal
like enzymes such as protease). In addition, disease states will
significantly impact the nature of urinary contents. Urine is also
the repository of all of the body's waste products including excess
parent nutrients, vitamins, drugs, and their metabolites. These
waste chemicals vary from person to person and significantly
contribute to the individual uniqueness that makes assay design for
urinary constituents more difficult than any other body fluid. All
of these factors impact an assay's ability to obtain acceptable
precision and accuracy. The ability of an assay to analyze a
biological liquid such as saliva, therefore, rarely ever translates
to an effective assay for urine. Therefore the present invention's
ability to effectively cope with random urine samples and
biological fluids or other fluids such as a beverage(s) makes it
unique.
[0013] Patent, U.S. Pat. No. 3,603,957, discloses the use of assay
test strips, but fails to teach a method for the determination of
GHB or GBL of a test sample submitted for drugs of abuse testing.
It also doesn't teach a method to determine the presence or absence
of any substance such as GHB or GBL. The patent doesn't teach the
use of the present art's reaction formula to dry chemistry format
called a dipstick or lateral flow technology that not only is
completely novel, but prevents cross contamination between test
pads typically found on a test strip (dipstick). In addition, this
patent also failed to mention any methods for determination of GHB
or GBL by dipstick, lateral flow, colorimetric, liquid reagent
(automated) or other suitable means.
[0014] Another patent, U.S. Pat. No. 4,351,899, discloses the use
of assaying metabolic acids specifically beta-hydroxybutyric acid
and/or lactic acid, but fails to teach a method for the
determination of GHB or GBL in a sample of fluid. The patent
doesn't teach the use of a dry chemistry format utilizing lateral
flow and/or a lateral flow hybrid method or any use for automated
analyzers and has no bearing on the present invention. There is no
similarity between beta-hydroxybutyric acid and GHB and/or GBL. GHB
is an analgesic drug which can induce comas and death. GHB could
not be manufactured from beta-hydroxybutyrate. Beta-hydroxybutyrate
is a by-product of human metabolism, it is not a drug, and cannot
provide or cause an analgesic effect.
[0015] Another patent, U.S. Pat. No. 4,622,296, discloses a process
for measuring the activity of a dehydrogenase or the amount of
substrate reacted or enzyme such as dehydrogenase. This assay has
no bearing with regards to the present art. It makes no mention of
GHB or GBL and fails to teach a method for the determination of GHB
or GBL in a sample of fluid. The patent doesn't teach the use of a
dry chemistry format utilizing, dipsticks, lateral flow and/or a
lateral flow hybrid method or any use for automated analyzers and
has no bearing on the present invention. There is no similarity
between dehydrogenase and GHB and/or GBL. GHB is an analgesic drug
which can induce comas and death. GHB could not be manufactured
from a dehydrogenase. Dehydrogenase is an enzyme not GHB (a drug)
or GBL a by-product of GHB metabolism. Dehydrogenase is not a drug,
and cannot provide or cause an analgesic effect.
[0016] Another patent, U.S. Pat. No. 5,912,139, discloses a method
(test strip) for measuring the activity of a dehydrogenase or the
amount of substrate reacted. This assay has no bearing with regards
to the present art. It makes no mention of GHB or GBL and fails to
teach a method for the determination of GHB or GBL in a sample of
fluid. The patent doesn't teach the use of a dry chemistry format
utilizing lateral flow and/or a lateral flow hybrid method or any
use for automated analyzers and has no bearing on the present
invention. There is no similarity between a dehydrogenase and GHB
and/or GBL. GHB is an analgesic drug which can induce comas and
death. GHB could not be manufactured from dehydrogenase.
Dehydrogenase id an enzyme and is not a drug, and cannot provide or
cause an analgesic effect.
[0017] Another patent, U.S. Pat. No. 5,624,813, discloses a method
for measuring the activity of a NAD(P)-linked dehydrogenase
reactions. This method has no bearing with regards to the present
art. It makes no mention of GHB or GBL and fails to teach a method
for the determination of GHB or GBL in a sample of fluid. The
patent doesn't teach the use of a dry chemistry format utilizing
lateral flow and/or a lateral flow hybrid method or any use for
automated analyzers and has no bearing on the present invention.
There is no similarity between a NAD(P)-linked dehydrogenase and
GHB and/or GBL. GHB is an analgesic drug which can induce comas and
death. GHB could not be manufactured from dehydrogenase.
Dehydrogenase is an enzyme and is not a drug, and cannot provide or
cause an analgesic effect.
[0018] Another patent, EU #0 291 194, discloses a method for
pregnancy testing. This method has no bearing with regards to the
present art. It makes no mention of GHB or GBL and fails to teach a
method for the determination of GHB or GBL in a sample of fluid.
The patent doesn't teach the use of a dry chemistry format
utilizing lateral flow and/or a lateral flow hybrid method or any
use for automated analyzers and has no bearing on the present
invention. There is no similarity between pregnancy testing and GHB
and/or GBL. GHB is an analgesic drug which can induce comas and
death. GHB could not be manufactured from any pregnancy test or any
teaching of any pregnancy test. A pregnancy is a method to
determine if an individual is pregnant. This cannot provide a
method for the detection of GHB or GBL and is not a drug, and
cannot provide or cause an analgesic effect.
[0019] Another patent, U.S. Pat. No. 5,032,506, discloses a method
for measuring the activity of a NAD(P)H, HAD(P)-linked
dehydrogenase reactions. This method has no bearing with regards to
the present art. It makes no mention of GHB or GBL and fails to
teach a method for the determination of GHB or GBL in a sample of
fluid. The patent doesn't teach the use of a dry chemistry format
utilizing lateral flow and/or a lateral flow hybrid method or any
use for automated analyzers and has no bearing on the present
invention. There is no similarity between a NAD-linked
dehydrogenase and GHB and/or GBL. GHB is an analgesic drug which
can induce comas and death. GHB could not be manufactured from
dehydrogenase. Dehydrogenase is an enzyme and is not a drug, and
cannot provide or cause an analgesic effect.
[0020] Another patent, EU #0 226 427, discloses the method of
simultaneously inspecting quality of animal milk and health of the
animal secreting the milk and an indicator for beta-hydroxybutyric
acid, but fails to teach a method for the determination of GHB or
GBL in a sample of fluid. The patent doesn't teach the use of a dry
chemistry methods or techniques or format utilizing lateral flow
and/or a lateral flow hybrid method or any use for automated
analyzers and has no bearing on the present invention. There is no
similarity between beta-hydroxybutyric acid and GHB and/or GBL. GHB
is an analgesic drug which can induce comas and death. GHB could
not be manufactured from beta-hydroxybutyrate. Beta-hydroxybutyrate
is a by-product of human metabolism, it is not a drug, and cannot
provide or cause an analgesic effect
[0021] Another patent, U.S. Pat. No. 4,301,115, discloses the use
of assay test strips, and the ability of the assay strips to resist
cross contamination between reactant areas (chemically impregnated
test pads), but fails to teach a method for the determination of
GHB or GBL in a sample of fluid. The patent doesn't teach the use
of the dry chemistry format utilizing either a dipstick or lateral
flow device, liquid reagent (automated) method or mention any
methods for determination of GHB or GBL.
[0022] Another patent, U.S. Pat. No. 5,447,837, does mention the
use of assay test strips but again fails to disclose a method for
the determination of GHB or GBL. This is a method for detection of
an antigenic substance in human, biological samples. This patent
also fails to mention the use of a reaction formula that is
adaptable to the dry chemistry format utilizing either a dipstick
or lateral flow device. It also doesn't teach a method to determine
the presence or absence of any substance such as GHB or GBL. In
addition, this patent also failed to mention any methods for
determination of GHB or GBL by dry chemistry, liquid chemistry,
colorimetric, or other suitable means.
[0023] Published literature and the prior art describes techniques
such as ELISA that have been used to determine the presence of
drugs of abuse, but these technologies have no relevant bearing on
the present device. Previously taught technologies include
measurement of GHB or GBL using GC or GCMS techniques and the
required extraction procedures inherent (required) prior to
analysis by GC or GCMS.
[0024] Therefore, in a nutshell, the present device provides an
absolute novel approach to GHB or GBL testing and lateral flow
testing using dry chemistry test pads and automated liquid reagent
testing.
[0025] Not surprisingly, it is known and is illustrated here that a
great need exists in the field of drug testing for rapid,
economical, and effective method for the detection of GHB or GBL on
samples submitted for testing, whether liquid chemistry and/or dry
chemistry methodology using dipsticks or lateral flow test devices
(for single use and for on-site collections). The present invention
does detect GHB or GBL effectively with a single assay and
therefore and accordingly, the present device provides an easy and
convenient manner by which to make a determination of the presence
or absence of GHB or GBL in a fluid. The present art's use of
lateral flow also enables the removal of any interference of any
cross over of reagents or fluid from one test pad to another which
is one of the exclusive problems with dipsticks.
[0026] It is clear that a need exists for a convenient manner by
which a determination of GHB or GBL can be made utilizing a rapid
automated analysis utilizing a liquid reagent format of the present
device or manual analysis in the form of dry chemistry (dipstick)
and/or lateral flow test devices. These and other advances in the
current state of the art will become evident in view of the present
specification and claims.
SUMMARY OF THE INVENTION
[0027] Briefly stated, the present invention relates to the test
devices for detecting the presence of GHB and or its precursor GBL
(gamma-butyrolactone) in a liquid test sample and the methods for
making said devices. GBL is also used as a GHB substitute. Once
ingested, GHL is rapidly converted to GHB. GHB can also be
converted to GBL by acid catalysis. This invention is in the field
of drug testing. More specifically, this invention provides dry
chemistry test strips (i.e. dipsticks, or dry chemistry and lateral
flow [thin layer chromatography] test means) or automated or manual
liquid reagent means for use in the detection of GHB or GBL in
biological samples (e.g. urine, blood, serum, saliva, sweat
extracts, and hair homogenates) or other fluids such as beverages,
water, soft drinks, alcoholic drinks to name a few. This invention
achieves this goal by measuring the presence of GHB or GBL in a
test sample. And, this invention provides a unique method for
preventing cross contamination between test pads (reactant areas)
on dipsticks by the present inventions use of the dipstick test pad
and lateral flow device technology. This invention provides a
previously unavailable dry chemistry or liquid chemistry method for
determining GHB or GBL presence in a test sample by measuring the
presence of GHB or GBL.
[0028] The present invention encompasses a method that can utilize
several different techniques. The techniques would employ the
manual method using dry chemistry dipsticks and the method of
combining dry chemistry dipstick reactant areas (test pads) with
lateral flow thin layer chromatography or the method of using a
liquid reagent that is compatible with automated analyzers that
provide high speed quantitative analysis which would be much less
labor intensive than the manual methods providing a savings in time
and money. The widespread utility of the present art also provides
the drug testing laboratory, over-the-counter user, individual,
police agency, drug testing collection site (where the urine is
actually collected), or other users the choice of using the dry
chemistry (manual) or automated liquid means which ever method best
suits their situation or needs.
[0029] The present arts technique utilizes two dry chemistry
techniques, one is dipsticks, which is a carrier dependent, rapid
test that uses absorbent medium such as paper, which have been
impregnated with a chemical formulation to detect adulteration.
After dipping one (dipstick) into a liquid test sample, a reaction
takes place. Said resulting reaction will yield a color change
indicating a positive or negative result (i.e. presence or absence
of GHB or GBL). The other technique is the use of lateral flow in
combination with a dry chemical test pad. The lateral flow device
is a rapid test that uses absorbent medium such as paper, which has
been impregnated with the chemical formulation to detect GHB or
GBL. The paper, after impregnation, is then placed on a lateral
flow medium, such as nitrocellulose paper, glass fiber paper, or
other suitable wicking material that will deliver the test sample
to the impregnated paper. The lateral flow device works by dipping
one end of the lateral flow device (LFD) into a sample (urine for
example). The urine migrates up (along) the paper (or absorbent
material) to the reactive sites (test paper) containing reagents
(reactive ingredients). The urine constituents react with the assay
reagents during the migration process and yield visible results.
The urine can also be droppered onto the LFD and the sample will
then migrate along the paper.
[0030] The ease of use and rapid results obtained by the present
art's methodology illustrate the unique utility of this testing
technique. In addition, very little technical expertise is required
to perform this type of assay (no instrumentation required).
Furthermore, the early detection of GHB or GBL facilitates the
prevention of the "date rape" syndrome. This novel concept for GHB
or GBL monitoring provides an enormous savings of time and money.
The present arts method(s) of GHB or GBL testing utilizing these
techniques are currently not available and have never been
taught.
[0031] An important aspect of GHB or GBL testing in fluids is the
sensitivity of the test method. Both techniques taught here have an
effective sensitivity range comparable to the GC-MS target range.
The sensitivity can also be adjusted to indicate a large amount
present as would be the case if the sample tested were a soft drink
spiked with GHB or GBL. Obviously, the amount in a sample directly
spiked with GHB or GBL would be much higher in concentration than
the amounts found in urine. The dipstick test and LFD dipstick
hybrid (to be known as the LFD hybrid) have a quantitative to
qualitative assay range. The results are evaluated via one of
following categories: negative, positive, or quantitatively.
[0032] The present arts technique also utilizes a liquid chemistry
test means that allows for rapid analysis via an automated analyzer
that can yield high-speed quantitative results. This will result in
rapid test results, improved accuracy, lowered labor cost, and
better turn around on a high volume of test. This automated method
is only limited by the speed of the automated analyzer. Some
analyzers currently on the market are capable of over 10,000 test
per a hour. The ability of the present art to perform a single
assay on a high speed automated analyzer that is capable of
detecting GHB or GBL has never been present or taught in the prior
art.
[0033] It is currently known in the art that enzyme and
antigen/antibody reaction kinetics are related to the rate of
change in analytical, biological systems. The variables that affect
this rate of change include concentration of reactants and product,
temperature, pH, ionic strength, buffer strength, and other
parameters. The present art's innate and unique ability enables it
to determine the presence of GHB or GBL in fluids. As it is known
in the art urine is a very complex matrix and the measurement of
GHB or GBL in fluids such as urine has to take into account many
factors, which will affect the assay.
[0034] The composition of the formulation to be applied to the dry
chemistry dipstick, LFD hybrid and liquid chemistry method are
composed of indicator(s) (visible colorimetric), and buffer(s).
[0035] Briefly stated, the present invention relates to test
devices for measurement of GHB or GBL in urine but could also work
in other biological matrices such as blood, saliva, hair or other
fluids and the procedures for making said test means. This
invention is in the field of clinical diagnostics. More
specifically, this invention provides dry chemistry dipsticks
(DCD's or on-site test modules), thin layer lateral flow
chromatographic dry chemistry technology (LFD's), and the
combination of both in a unique hybrid that is not known prior to
the present art and liquid chemistry reagents for automated and
manual use. That is to say (in it's simplest terms) that this
unique hybrid (LFD) will encompass the use of a dry chemistry test
pad resting on the surface of a wicking material (such as
nitrocellulose) acting as a fluid delivery device. This new art can
utilize aqueous, biological specimens including urine, saliva,
sweat extracts, blood, serum and other fluids (such as water or
soft drinks, etc.). Thus, this invention provides a unique method
for GHB or GBL measurement utilizing rapid test devices including
the automated method as well as the DCD, and LFD methodology
thereby enabling in-home, workplace, and recreational testing
through over-the-counter (OTC) sales. This is an enormous
advancement in the art. These advances and improvements of the
present device over the prior art provides the public safety,
health care and drug testing industry with powerful new clinical
and diagnostic tools.
[0036] A thorough search of the literature reveals no relative art
resembling this technology; therefore, this invention is clearly a
novel creation, and is not obvious to anyone skilled in the art of
toxicology and clinical chemistry.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The instant invention is a single assay in the form of a
liquid chemistry reagent, dry chemistry dipstick or lateral flow
device in conjunction with using a dry chemistry test pad for the
detection of GHB or GBL in sample matrices consisting of urine and
other biological specimens (e.g. saliva, serum, blood, sweat
extracts, and hair homogenates) or fluids such as beverages such
water, soft drinks, beer, or mixed drinks. The GHB or GBL detection
assay that makes up the instant invention may take the form of dry
chemistry dipsticks or dry chemistry test pad lateral flow hybrid,
both of which are composed of some or all of the following
compounds: buffer(s) and color indicator(s), hereinafter referred
to as the adulteration reagent or the liquid automated reagent
designed for high speed automated analyzers also composed of some
or all of the following compounds: buffer(s) and color
indicator(s). It can be noted that the liquid reagent method could
also be used manually employing spectrophotometers or other types
of visual detection technology. Buffering of the reactants is
critical to the GHB or GBL reagent, because pH plays a vital role
in the reaction kinetics. In the case of the dipstick (or dry
chemistry dipstick, DCD) and the dipstick/lateral flow hybrid
(which can be known as the "DLFH" device), GHB or GBL reagent
components are impregnated on the test strip pad composed of solid,
absorbent carrier(s), usually known as test pads. In the case of
dipsticks, these test pads are typically affixed to a solid support
(usually plastic). This device is then submerged in the liquid test
sample, removed, and a measurable (i.e. visible) response is
observed. Or in the case of the DLFH, the dry chemistry test pad is
chemically impregnated identically to the dipstick. The test pad is
then placed in fluid (direct) contact with lateral flow paper (such
as nitrocellulose). This device is then exposed to a fluid (urine
for example). The urine then migrates to the location of the test
pad, saturates the test pad, and the reaction takes place.
[0038] The GHB or GBL reagents of the device constitute the heart
of the analytical response provided by it, and is comprised of one
or more reagent compositions responsive to any number of chemical
components made up of GHB or GBL or are analogs or precursors of
GHB. The reagents, in the broadest sense produces a detectable
manifestation of the presence of GHB or GBL; the detectable
manifestation can be a measurable response in the form of the
appearance or disappearance of a color, or the changing of one
color to another. Said measurable response may also be evidenced by
a change in the amount of light reflected or absorbed during the
reaction of interest. The analytical arts are replete with examples
of these types of detectable responses.
[0039] In the present invention, there is provided a dry chemistry
test strip in the form of a dipstick or DLFH for the detection of
GHB or GBL in urine (or other biological fluids including saliva,
sweat extracts, serum, blood, and hair homogenates) and fluids such
as beverages to included but not limited to water, soft drinks,
beer, or mixed drinks (possibly containing alcohol) comprising a
solid, carrier matrix in the form of a dry chemistry dipstick
containing an indicator compound and buffer.
[0040] The present technology does not predict or forecast the
obvious advancement in the art to encompass the present invention,
nor does it hint at the extraordinary improvement the present
invention provides in the field of GHB or GBL detection. While
urine is the sample matrix of choice for this instant invention
(and for the immunoassays currently in general use for drug abuse
screening) it is well within the scope of this novel invention to
utilize it in the analysis of other sample matrices including
saliva, sweat extracts, serum, hair homogenates, gastric contents,
cerebral spinal fluid, blood and fluids such as beverages to
included but not limited to water, soft drinks, beer, or mixed
drinks (possibly containing alcohol).
[0041] The remarkable discovery of the new art formula will require
the presence of an indicator(s) for GHB or GBL in urine (as well as
the other matrices mentioned) that was unknown prior to this art.
The newly discovered and suitable indicators and compounds that are
reactive to the presence of GHB or GBL are but, not limited to the
following; hydroxybutyrate dehydrogenase, esterase,
3-hydroxybutyrate dehydrogenase, 4-hydroxybutyrate dehydrogenase,
carboxyl esterase, carboxylic-ester hydrolase,
.beta.-hydroxybutyrate dehydrogenase, [R]-3-hydroxybutanoate, NAD
(nicotinamide adenine dinucleotide).sup.+ oxidoreductase, NAD
(.alpha.-nicotinamide adenine dinucleotide) or analogs of NAD such
as 3-acetylpyridine adenine dinucloetide, 3-acetylpyridine
hypoxanthine dinucleotide, .beta.-nicotinamide adenine
dinucleotide-agarose, nicotinamide 1,N.sup.6-ethenoadenine
dinucleotide, nicotinamide guanine dinucleotide, nicotinamide
hypoxanthine dinucleotide, nicotinic acid adenine dinucleotide,
3-pyridinealdehyde adenine dinucleotide, thionicotinamide adenine
dinucleotide and .alpha.-hydroxybutyrate dehydrogenase, NADP
(.alpha.-nicotinamide adenine dinucleotide phosphate) or analogs of
NADP such as .beta.-nicotinamide adenine dinucleotide phosphate,
3-acetylpyridine adenine phosphate, .beta.-nicotinamide adenine
dinucleotide 2',3'-Cyclic monophosphate, .beta.-nicotinamide
adenine dinucleotide 3'-phosphate, nicotinamide 1,N.sup.6-etheno
adenine dinucleotide phosphate, nicotinamide hypoxanthine
dinucleotide phosphate, thionicotinamide adenine dinucleotide
phosphate and anti-gamma-hydroxybutyrate, gamma-butyrolactone
dehydrogenase, anti-gamma-butyrolactone, alpha-nicotinamide adenine
dinucleotide phosphate, beta-nicotinamide adenine dinucleotide
phosphate and all analogs of the afore mentioned, and can be
selected from the following group consisting of NBT (nitro blue
tetrazolium), phenazine methosulfate, tetranitroblue tetrazolium,
napthol AS-TR phosphate, methylene blue, Fast red, napthol-AS-MX,
napthol AS-TR phosphate, thymol blue, bromcresol green, methyl red,
cresol red, metanil yellow, m-cresol purple, xylenol blue, thymol
blue, tropeolin OO, quinaldine red, .alpha.-dinitrophenol, methyl
yellow; dimethyl yellow, bromophenol blue, tetrabromophenol blue,
bromochlorophenol blue, Congo red, methyl orange,
p-ethoxychrysoidine hydrochloride, napthyl red, alizarin sodium
sulfonate, bromocresol green, .alpha.-dinitrophenol, methyl red,
lacmoid, chlorophenol red, benzoyl auramine G, bromocresol purple,
bromophenol red, p-nitrophenol, bromthymol blue, phenol red,
p-quinonemono(bis-4-oxyphenylmethide), neutral red, quinoline blue,
.alpha.-naphtholphthalein, tropeolin OOO; .alpha.-napthol orange,
ethyl bis(2,4-dinitrophenyl)acetate, di-o-cresolphthalide,
phenolphthalein, thymolphthalein, dimethylphenolphthalein, alizarin
yellow GG; salicyl yellow, alizarin yellow R, Nile blue,
2,4,6-trinitrophenylmethyl-nitramine, tropeolin O,
triphenylrosaniline sulfonic acid (sodium or potassium salt),
indigo carmine, nitrobenzene, bromcresol green, bromcresol purple,
bromchlorophenol blue, brilliant yellow, brilliant blue R,
brilliant cresyl blue ALD, brilliant blue G, brlliant black BN,
bromthymol blue, bromphenol red, bromphenol red, bromoxylenol blue,
coomasie blue, azolitmin, litmus, pyrogallosulfonphthalein,
pyrogallo red-molybdate, alcohol dehydrogenase, ABTS
(2,2'-azinobis(3-ethylbenzothiazoline-6-sulfo- nic acid)),
4-aminoantipyrine (4AAP), tetramethylbenzidine (TMB),
o-phenylenediamine (OPD), o-dianisidine, 5-aminosalicylic acid
(5AS), 3,3'-diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC),
4-chloro-1-napthol (4C1N), AEC (3-Amino-9-ethyl carbazole),
dimethyl-2,5-dihydroperoxyhexane,
Bis{4-[N-(3'-sulfo-n-propyl)-N-n-ethyl]-
amino-2,6-dimethylphenyl}methane (Bis-MAPS),
N-Ethyl-N-(2-hydroxy-3-sulfop- ropyl)-3-methoxyaniline (ADOS),
N-Ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (ALOS),
N-Ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline (MAPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS),
N-Ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS),
N-(3-sulfopropyl)anilin- e (HALPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-aniline (DAOS),
N-Ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline (DAPS),
N-Ethyl-N-(3-sulfopropyl)aniline (ALPS),
N-(2-hydroxy-3-sulfopropyl)-3,5-- dimethoxyaniline (HDAOS),
N-(3-sulfopropyl)-3,5-dimethoxyaniline (HDAPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAO),
N,N-Bis(4-sulfobutyl)-3,5-dimethylaniline (MADB), pyrogallol,
2,4-Dichlorophenol, N,N-Diethyl-m-toluidine, p-Hydroxybenzene
Sulfonate, N,N-Dimethylaniline,
3,5-Dichloro-2-Hydroxybenzenesulfonate,
2,4,6-tribromo-3-hydroxybenzoic acid, Sodium
N-Ethyl-N-(3-Sulfopropyl)-m-- Anisidine, hydroxybenzoic acid,
4-hydroxybenzoic acid,
N-Ethyl-N-(2-hydroxy-3-Sulfopropyl)-m-toluidine, AEC
(3-Amino-9-ethyl carbazole), 2-5, dimethyl-2,5-dihydroperoxyhexane,
Bis{4-[N-(3'-sulfo-n-propyl)-N-n-ethyl]amino-2,6-dimethylphenyl}methane
(Bis-MAPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline
(ADOS), N-Ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (ALOS),
N-Ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline (MAPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS),
N-Ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS), N-(3-sulfopropyl)
aniline (HALPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-anilin- e (DAOS),
N-Ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline (DAPS),
N-Ethyl-N-(3-sulfopropyl)aniline (ALPS),
N-(2-hydroxy-3-sulfopropyl)-3,5-- dimethoxyaniline (HDAOS),
N-(3-sulfopropyl)-3,5-dimethoxyaniline (HDAPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAO),
N,N-Bis(4-sulfobutyl)-3,5-dimethylaniline (MADB),
3-Methyl-2-benzothiazol- inonehydrazone, Dimethylaniline,
5-bromo-6-chloro-3-indoxyl-beta-D-galacat- opyranoside,
4-Aminophenyl-beta-D-galactopyranoside,
3-indoxyl-beta-D-galactopyranoside (blue),
5-Bromo-4-chloro-3-indoxyl-bet- a-D-galactopyranoside (blue),
5-Bromo-3-indoxyl-beta-D-galactopyranoside (blue),
6-chloro-3-indoxyl-beta-D-galactopyranoside (salmon),
6-Fluoro-3-indoxyl-beta-D-galactopyranoside,
8-Hydroxyquinoline-beta-D-ga- lactopyrano-side,
5-Iodo-3-indoxyl-beta-D-galactopyranoside (purple),
N-Methylindoxyl-beta-D-galactopyranoside,
2-Nitrophenyl-beta-D-galactopyr- anoside,
4-Nitrophenyl-beta-D-galactopyranoside, Naphthol
AS-BI-beta-D-galactopyranoside, 2-Naphthyl-beta-D-galactopyranoside
(yellow), 4-Methylumbelliferyl-beta-D-glucuronic acid,
beta-D-Galactosidase, Iodo-3-indoxyl-beta-D-galactopyranoside,
alpha-L-Galactosidase, Iodo-3-indoxyl-alpha-L-beta-Galactosidase,
glycosidase, beta-Cellobiosidase, cellobioside,
beta-D-Cellobiosidase,
5-Bromo-4-chloro-3-indoxyl-beta-D-cellobioside,
5-Bromo-6-chloro-3-indoxy- l-beta-D-cellobioside,
4-Nitrophenyl-beta-D-cellobioside, 1-Naphthyl-cellobioside,
4-Methylumbelliferyl-beta-D-cellobioside, Arabinosidase,
Fucosidase, Galactosaminidase, Glucosaminidase, Glucosidase,
Glucuronidase, Lactosidase, Maltosidase, Mannosidase, and
Xylosidase. Their corresponding substrates, Arabinopyranoside,
Fucopyranoside, Galactosaminide, Glucosaminide, Glucopyranoside,
Glucuronic acid, Lactopyranoside, Maltopyranoside, Mannopyranoside,
Xylopyranoside, 5-Bromo-4-chloro-3-indoxyl,
5-Bromo-6-chloro-3-indoxyl, 6-chloro-3-indoxyl, 5-Bromo-3-indoxyl,
5-Iodo-3-indoxyl, 3-indoxyl, 2-(6-Bromonaphthyl),
6-Fluoro-3-indoxyl 2-Nitrophenyl, 4-Nitrophenyl, 1-Naphthyl,
Naphthyl AS-BI, 2-Nitrophenyl-N-acetyl, 4-Nitrophenyl-N-acetyl,
4-Methylumbelliferyl, glycosidase enzyme, carboxyl esterase,
cholesterol esterase, sulfatases (e.g. Aryl sufatase), phosphatases
(e.g. Alkaline phosphatase), carboxyl esterase, 6-chloro-3-indoxyl
butyrate, aryl sulfatase, 5-bromo-4-chloro-3-indoxyl sulfate,
alkaline phosphatase, and 2-naphthyl phosphate. It is understood
that the present arts discovery of the use of indicators such as
the ones mentioned above or others that have not been mentioned
that are sensitive to GHB or GBL biological matrices such as urine
and other fluids are capable of producing a detectable response in
the presence of GHB or GBL are within the present art. Therefore
the use of GHB or GBL indicators that are not mentioned here would
fall within the spirit and scope of the present invention. These
indicators form indicatorcomplexes with GHB or GBL for determining
the presence or amount of gamma-hydroxybutyrate or
gamma-butyrolactone in a sample, said method(s) comprising
contacting said sample with an indicator which specifically binds
to gamma-hydroxybutyrate or gamma-butyrolactone to form an
indicatorcomplex; and, measuring said indicatorcomplex to determine
the presence or amount of said gamma-hydroxybutyrate or
gamma-butrylactone in said sample.
[0042] This new art formula will require appropriate buffering.
Suitable buffers may include any of the following (referred to here
by their commonly used acronyms): citrate, borate, borax, sodium
tetraborate decahydrate, sodium perchlorate, sodium chlorate,
sodium carbonate, TRIS (Tris[hydroxymethyl]aminomethane), MES
(2-[N-Morpholino]ethanesulfonic acid), BIS-TRIS
(bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;
2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl-1,3-propanediol), ADA
(N-[2-Acetamidol]-2-iminodiacetic acid;
N-[Carbaoylmethyl]iminodiacetc acid), ACES
(2-[(2-Amino-2-oxoethyl)amino]ethanesulfonic acid;
N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES
(PiperazineN-N'-bis[2-ethanesulfonic acid)];
1,4-Piperzinedethanesulfoic acid), MOPSO
(3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRIS PROPANE
(1,3-bis[tris(Hydroxymethyl)methylamino]propane), BES
(N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid;
2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS
(3-[N-Morpholino]propanesulfonic acid), TES
(N-tris[Hydroxymethyl]methyl-- 2-aminomethanesulfonic acid;
2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amin- o)ethanesulfonic
acid), HEPES (N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesu- lfonic
acid]), DIPSO
(3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesul- fonic acid),
TAPSO (3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxypropanesu-
lfonic acid), HEPPSO
(N-[2-Hydroxythyl]piperazine-N'-[2Hydroxypropanesulfo- nic acid]),
POPSO (Piperazine-N,N'-bis[2-hydroxypropanesulfonic acid]), EPPS
(N-[2-Hydroxyethyl]piperazine-N'-[3-propanesulfonic acid), TEA
(triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine;
N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE
(N,N-bis[2-Hydroxyethyl]glycine), TAPS
(N-tris[Hydroxymethyl]methyl-3-ami- nopropanesulfonic acid;
([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-pr- opanesulfonic
acid), AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydro-
xypropanesulfonic acid), CHES (2-[N-Cyclohexylamino]ethanesulfonic
acid), CAPSO (3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic
acid), AMP 2-Amino-2-ethyl-1-propanol, CAPS
(3-[cyclohexylamino]-1-propanesulfonic acid), hydrochloric acid,
phosphoric acid, lactic acid, sulfuric acid, nitric acid, chromic
acid, boric acid, perchloric acid, potassium hydrogen tartrate,
potassium hydrogen phthalate, calcium hydroxide, phosphate,
bicarbonate, sodium hydroxide, potassium hydroxide, oxalate or
succinate. Other buffers with an effective pK and pH range, and
capacity suitable for maintaining the sample-reagent mixture within
the required parameters of the assay's reaction mechanism may be
added to the above group.
[0043] Manufacture of the dry chemistry dipsticks may require the
addition of thickeners as taught in the art. Some compounds
commonly used for this purpose include: polyvinylpyrrolidone,
algin, carragenin, casein, albumin, methyl cellulose, and gelatin.
The typical range of concentration for these thickeners is about
0.5 to 5.0 g. per 100 ml. Wetting agents or surfactants are also
typically used in dry chemistry. For dry chemistry applications,
wetting agents aid in even distribution of the chemicals and
promote even color development. Acceptable wetting agents can be
hydrophilic polymers, or cationic, anionic, amphoteric, or nonionic
species. Some commonly used wetting agents include sodium
dodecyl-benzene sulphonate, sodium lauryl sulphate, benzalkonium
chloride, N-lauroylsarcosine sodium salt, Brij-35, Tween 20, Triton
X-100, dioctyl sodium sulphosuccinate, and polyethylene glycol
6000. Wetting agents can be added to dipstick impregnation
solutions in amounts of 0.5% to 5.0%, and 0.1% to 1.0% in liquid
reagents.
[0044] Color enhancers may be used such as sucrose, lactose,
glucose or other compounds. Color enhancement can be defined as
intensification and/or alteration in some manner the color that is
produced by the reaction to improve the measurement of the
detectable response.
[0045] The production of dry chemistry test strips for the present
invention can utilize any form of absorbent, solid phase carrier
including filter paper, cellulose or synthetic resin fleeces in
conjunction with liquid solutions of reagent compositions in
volatile solvents. This can be carried out in one or more
impregnation steps. Each impregnation may contain one or more of
the chemical compounds making up the assay reagent composition; the
exact procedure is dictated by the inter-reactivity of the assay
constituents and the order in which they may have to react with the
analyte of interest.
[0046] In the case of the DLFH, the lateral flow invention it can
utilize any form of absorbent, solid phase carrier that is capable
of transporting a fluid. These can include filter paper, cellulose
or synthetic resins. More specifically, the lateral flow material
can include cellulose, cellulose acetate, nitrocellulose, mixed
ester, teflon, polyvinylidene difluoride (PVDF),
polytetrafluoroethylene (PTFE), polysulfone, cotton linter,
non-woven rayon, glass fiber, nylon, ion exchange or other suitable
membranes or solid support.
[0047] After impregnation, the dipsticks are dried, cut into
strips, glued to a support structure (usually a flexible, flat,
plastic stick made up of polystyrene, vinyl polypropylene, and
polyester or other suitable support material) as part of a
"sandwich" composed of the handle, test pad, and a synthetic resin
film and/or a fine-mesh material in the manner described in German
Pat. No, 2,118,455. In addition, the instant invention may be
combined with the water-stable film as taught in U.S. Pat. No.
3,530,957 to produce a dipstick in which the excess sample fluid
can be wiped off in order to improve the accuracy and precision of
the results.
[0048] The sensitivity of the assay can be as low as 5 ug/mL, is
the cutoffs illustrated in the present art are merely illustrative.
The cutoff of 50.0 ug/mL is used because this is the range that an
analgesic (high) effect is felt by the user or victim.
[0049] The following examples are provided to further illustrate
the inventive aspects of the present discovery, and to further
exemplify preferred embodiments. As such, they are intended as
merely illustrative, and are not to be construed as limiting the
scope of the claims appended hereto.
EXAMPLE 1
[0050] This is a method for manufacturing a dry chemistry dipstick
(DCD, test strip) with a solid carrier for the detection of GHB in
urine samples.
[0051] Filter paper is successively impregnated with the following
solutions and dried at 25 degree C.:
[0052] Solution I
[0053] Tris-Base (Tris[hydroxymethyl]aminomethane) buffer 2.97
g
[0054] Glucose-6-phosphate 0.01M
[0055] NAD 0.3 M
[0056] distilled water added to 100 mL total volume of solution
[0057] pH the solution to a value between 1.0 and 12.5 preferably
8.0
[0058] lab notes: Buffer strength is preferably 10 mM or
greater
[0059] Solution 2
[0060] GHB (gamma hydroxybutyrate) conjugated to a dehydrogenase
(glucose-6-phosphate (G6P)) 5 mM
[0061] GHB antibody
[0062] Tris-HCl (Tris[hydroxymethyl]aminomethane hydrochloride)
buffer 0.01M
[0063] NBT (nitro blue tetrazolium) 0.01 mg/L
[0064] distilled water added to make 1000 mL total volume of
solution
[0065] lab notes: pH the solution to a value between 1.0 and 12.5
preferably 8.0
[0066] In this example a dipstick was prepared in accordance with
the instant invention. The device comprised a paper carrier or
solid matrix incorporated with the composition of solutions 1 and 2
above. Note that the concentrations of any of the following
examples can be varied to suit the dipstick device format
(dependent upon paper type, or use of semi-permeable membrane or
other suitable material). This example is carried out using the
following procedure. To produce the test means, a piece of Whatman
3 MM filter paper having approximate dimensions of 1 inch by 3
inches was impregnated with solution 1 by immersing the paper into
solution 1. The paper was then dried by using forced air not
exceeding 25.degree. C. A second piece of Whatman 1 MM filter paper
having approximate dimensions of 1 inch by 3 inches was impregnated
with solution 2 by immersing the paper into solution 2. The paper
was then dried by using forced air not exceeding 25.degree. C. The
dried papers are then laminated to each other by the use of a
non-reactive (neutral adhesive). The dried, laminated papers are
then applied to one side of a piece of double-sided adhesive
transfer tape commercially available from 3M Company, St. Paul,
Minn. 55144. The laminate is then slit into portions measuring 3
inches by 0.2 inches. One portion is then attached, via the unused
adhesive side to a polystyrene sheet measuring about 1.5 inches by
3 inches and the resulting laminate is slit parallel to its short
dimension to form test devices comprising a 1.5 inch oblong
polystyrene strip carrying a square of the impregnated papers at
one end, the other end serving as the handle. When the dipstick
thus obtained is dipped into a urine (note: other sample matrices
including saliva, sweat extracts, serum, hair homogenates, gastric
contents, cerebral spinal fluid, blood and fluids such as beverages
to included but not limited to water, soft drinks, beer, or mixed
drinks) will produce no uniform blue color development if no GHB is
present. Conversely, if any concentration of GHB is present in the
urine at a 0.1% v/v or greater a blue color will develop thus
confirming the presence of GHB.
[0067] In summary, Example 1 is as follows: the foregoing dry
chemistry test strip (dipstick) method for the GHB detection of in
a sample of urine comprises the steps of preparing a test means by
successively impregnating a solid, carrier matrix with reagent
solutions, drying the impregnated, solid test means, then dipping
said dried test means into urine, and finally observing any color
change in the presence or absence of GHB.
[0068] The following changes to the above reagent solutions will
remain within the scope and function of this invention and will
have similar results to the example above. The GHB indicator
reactive compound (reactive in this sense means that the GHB
conjugated to the dehydrogenase competes with free GHB if present
for the antibody) dehydrogenase (glucose-6-phosphate dehydrogenase
(G6PDH)) is sensitive the presence of glucose-6-phosphate in
solution. If there is no free GHB in the specimen then the
dehydrogenase will not react with the G6P because it will be bound
up by the GHB antibody that will bind to the GHB conjugated to the
G6PDH, note that during the G6PDH-G6P reaction NAD (cofactor) will
be converted to NADH and in the presence of NADH, NBT will change
from a colorless compound to a blue/purple color. If there is GHB
present in the sample it will be will bind with the GHB antibody
leaving free GHB-G6PDH conjugate free to react with G6P thus
causing the cascade reaction of the NAD conversion to NADH and the
subsequent conversion of the NBT indicator. In solution 1 the
dehydrogenase (G6PDH), which could be substituted with one or more
of the following compounds including hydroxybutyrate dehydrogenase,
esterase, 3-hydroxybutyrate dehydrogenase, 4-hydroxybutyrate
dehydrogenase, carboxyl esterase, carboxylic-ester hydrolase,
.beta.-hydroxybutyrate dehydrogenase, [R]-3-hydroxybutanoate, NAD
(nicotinamide adenine dinucleotide).sup.+ oxidoreductase, and
.alpha.-hydroxybutyrate dehydrogenase, anti-gamma-hydroxybutyrate,
alpha-nicotinamide adenine dinucleotide phosphate,
beta-nicotinamide adenine dinucleotide phosphate, dehydrogenase,
.alpha.-hydroxybutyrate dehydrogenase, anti-gamma-hydroxybutyrate,
alpha-nicotinamide adenine dinucleotide phosphate,
beta-nicotinamide adenine dinucleotide phosphate, oxidases,
reductases, oxidoreductases, transferases, hydrolases, lyases,
isomerases and ligases and all analogs of the afore mentioned can
be substituted with the following enzyme pairs to include but are
not limited to one or more of the following:
4-Aminophenyl-beta-D-galactopyranoside,
3-indoxyl-beta-D-galactopyranoside (blue),
5-Bromo-4-chloro-3-indoxyl-bet- a-D-galactopyranoside (blue),
5-Bromo-3-indoxyl-beta-D-galactopyranoside (blue),
6-chloro-3-indoxyl-beta-D-galactopyranoside (salmon),
6-Fluoro-3-indoxyl-beta-D-galactopyranoside,
8-Hydroxyquinoline-beta-D-ga- lactopyranoside,
5-Iodo-3-indoxyl-beta-D-galactopyranoside (purple),
N-Methylindoxyl-beta-D-galactopyranoside,
2-Nitrophenyl-beta-D-galactopyr- anoside,
4-Nitrophenyl-beta-D-galactopyranoside, Naphthol
AS-BI-beta-D-galactopyranoside, and
2-Naphthyl-beta-D-galactopyranoside (yellow). Fluorescent
substrates may also be utilized including
4-Methylumbelliferyl-beta-D-glucuronic acid. The colors noted in
the parentheses are those produced in the reaction described above.
The indicator substrate used in these examples must be matched to
the conformation of the galactosidase used (i.e. alpha or beta, and
dextrorotorary (D) or levorotorary (L)). For example,
beta-D-Galactosidase should be matched with the indicator/substrate
Iodo-3-indoxyl-beta-D-galactopyranoside; conversely,
alpha-L-Galactosidase would be matched with
Iodo-3-indoxyl-alpha-L-galact- opyranoside. Note that some
cross-reactivity does occur between stereo-isomers and, therefore,
it is possible to substitute these compounds where appropriate.
[0069] Substitution of the G6PDH with another enzyme would
necessitate a change of substrate indicator complex. If another
glycosidase was selected, it would have to be matched to the
appropriate substrate (e.g. beta-Cellobiosidase and a
cellobioside). Examples of substrates for beta-D-Cellobiosidase
include 5-Bromo-4-chloro-3-indoxyl-beta-D-cellobios- ide,
5-Bromo-6-chloro-3-indoxyl-beta-D-cellobioside,
4-Nitrophenyl-beta-D-cellobioside, 1-Naphthyl-cellobioside,
beta-Galactosidase and the fluorescent indicator,
4-Methylumbelliferyl-be- ta-D-cellobioside.
[0070] Other glycosidases which may be substituted for
Galactosidase and Cellobiosidase include the alpha and beta, and D
and L conformations of the following enzymes: Arabinosidase,
Fucosidase, Galactosaminidase, Glucosaminidase, Glucosidase,
Glucuronidase, Lactosidase, Maltosidase, Mannosidase, and
Xylosidase. Their corresponding substrates, Arabinopyranoside,
Fucopyranoside, Galactosaminide, Glucosaminide, Glucopyranoside,
Glucuronic acid, Lactopyranoside, Maltopyranoside, Mannopyranoside,
and Xylopyranoside may be bound to each of the following color
indicator groups: 5-Bromo-4-chloro-3-indoxyl,
5-Bromo-6-chloro-3-indoxyl, 6-chloro-3-indoxyl, 5-Bromo-3-indoxyl,
5-Iodo-3-indoxyl, 3-indoxyl, 2-(6-Bromonaphthyl),
6-Fluoro-3-indoxyl 2-Nitrophenyl, 4-Nitrophenyl, 1-Naphthyl,
Naphthyl AS-BI, 2-Nitrophenyl-N-acetyl, 4-Nitrophenyl-N-acetyl, and
4-Methylumbelliferyl moieties.
[0071] Other enzymes that can be used with the indicator groups
listed above. These include esterases (e.g. Carboxyl esterase, and
Cholesterol esterase), sulfatases (e.g. Aryl sufatase), and
phosphatases (e.g. Alkaline phosphatase). These enzymes can utilize
the indicator groups delineated above when conjugated to the
corresponding substrate. For example, Carboxyl esterase and
6-chloro-3-indoxyl butyrate, and Aryl sulfatase and
5-bromo-4-chloro-3-indoxyl sulfate, and Alkaline phosphatase and
2-naphthyl phosphate form enzyme-substrate pairs.
[0072] Other enzymes may be conjugated to GHB, and therefore
substituted for the species described above. This group now listed,
however, must utilize a substrate that is distinct and separate
from the indicator. This enzyme group may include any
dehydrogenase, oxidase, hydroxylase, or oxidoreductase. Each
grouping will utilize a specific indicator or group of indicators.
The dehydrogenases and hydroxylases will utilize a co-enzyme, a
color indicator and an electron carrier such as a-NAD
(a-Nicotinamide adenine dinucleotide), however this electron
carrier/acceptor can be replaced by the alpha or beta isomers of
any one of the following substitutes: nicotinamide adenine
dinucleotide, nicotinamide adenine dinucleotide 3'-phosphate,
nicotinamide adenine dinucleotide phosphate, triphosphopyridine,
nicotinamide 1-N1-ethenoadenine dinucleotide phosphate,
nicotinamide hypoxanthine dinucleotide, nicotinamide hypoxanthine
dinucleotide phosphate, nicotinamide mononucleotide, nicotinamide
N1-propylsulfonate, nicotinamide ribose monophosphate, or other
analogs of NAD.
[0073] Some dehydrogenases and hydroxylases and their substrate
pairs which can be used include Formaldehyde dehydrogenase and
Formaldehyde, Fructose dehydrogenase and Fructose,
Glucose-6-phosphate dehydrogenase and Glucose-6-phosphate, Glucose
dehydrogenase and Glucose, Glutamate dehydrogenase and Glutamate,
Glycerol dehydrogenase and Glycerol, Glycerol-3-phosphate
dehydrogenase and Glycerol-3-phosphate, Hydroxybutyrate
dehydrogenase and Hydroxybutyrate, Hydroxybenzoate hydroxylase and
4-Hydroxybenzoate, Lactate dehydrogenase and Lactate, Leucine
dehydrogenase and Leucine, Malate dehydrogenase and Malate,
Mannitol dehydrogenase and Mannitol, or any other dehydrogenase or
hydroxylase.
[0074] The use of oxidases to replace the gylcosidase also requires
a separate indicator, and peroxidase. Some oxidases and their
substrate pair which can be used include Acyl-CoA oxidase and
Acyl-CoA, Alcohol oxidase and Ethanol, Ascorbate oxidase and
Ascorbate, Cholesterol oxidase and Cholesterol, Choline oxidase and
Choline, Glucose oxidase and Glucose, Glycerophosphate oxidase and
Glycerophosphate, Xanthine oxidase and Xanthine, Uricase and Uric
acid, or any other oxidase or all analogs of the afore
mentioned.
[0075] The indicator NBT, which will develop color in the presence
of the reduction of NAD or NADPH can be replaced by one of the
following: phenazine methosulfate, tetranitroblue tetrazolium,
napthol AS-TR phosphate, methylene blue, Fast red, napthol-AS-MX,
napthol AS-TR phosphate or analogs thereof.
[0076] The Tris buffer in solution 1, may be substituted with one
or more of the following buffers: citrate, borate, borax, sodium
tetraborate decahydrate, sodium perchlorate, sodium chlorate,
sodium carbonate, MES (2-[N-Morpholino]ethanesulfonic acid),
BIS-TRIS (bis[2-Hydroxyethyl]imino- tris[hydroxymethyl]methane;
2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl-1,- 3-propanediol), ADA
(N-[2-Acetamidol]-2-iminodiacetic acid;
N-[Carbaoylmethyl]iminodiacetc acid), ACES
(2-[(2-Amino-2-oxoethyl)amino]- ethanesulfonic acid;
N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES
(PiperazineN-N'-bis[2-ethanesulfonic acid)];
1,4-Piperzinedethanesulfoic acid), MOPSO
(3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRIS PROPANE
(1,3-bis[tris(Hydroxymethyl)methylamino]propane), BES
(N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid;
2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS
(3-[N-Morpholino]propanesulfonic acid), TES
(N-tris[Hydroxymethyl]methyl-- 2-aminomethanesulfonic acid;
2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amin- o)ethanesulfonic
acid), HEPES (N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesu- lfonic
acid]), DIPSO
(3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesul- fonic acid),
TAPSO (3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxypropanesu-
lfonic acid), HEPPSO
(N-[2-Hydroxythyl]piperazine-N'-[2Hydroxypropanesulfo- nic acid]),
POPSO (Piperazine-N,N'-bis[2-hydroxypropanesulfonic acid]), EPPS
(N-[2-Hydroxyethyl]piperazine-N'-[3-propanesulfonic acid), TEA
(triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine;
N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE
(N,N-bis[2-Hydroxyethyl]glycine), TAPS
(N-tris[Hydroxymethyl]methyl-3-ami- nopropanesulfonic acid;
([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-pr- opanesulfonic
acid), AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydro-
xypropanesulfonic acid), CHES (2-[N-Cyclohexylamino]ethanesulfonic
acid), CAPSO (3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic
acid), AMP 2-Amino-2-ethyl-1-propanol, CAPS
(3-[cyclohexylamino]-1-propanesulfonic acid), hydrochloric acid,
phosphoric acid, lactic acid, sulfuric acid, nitric acid, chromic
acid, boric acid, perchloric acid, potassium hydrogen tartrate,
potassium hydrogen phthalate, calcium hydroxide, phosphate,
bicarbonate, sodium hydroxide, potassium hydroxide, oxalate or
succinate. Other buffers with an effective pK and pH range, and
capacity suitable for maintaining the sample-reagent mixture within
the required parameters of the assay's reaction mechanism may be
added to the above group.
EXAMPLE 2
[0077] This is a method for manufacturing a liquid, carrier-free
reagent for the adulteration detection of GHB in samples submitted
for drugs of abuse analysis.
[0078] Prepare a Solution Containing:
[0079] Solution I (R1)
[0080] Tris-Base (Tris[hydroxymethyl]aminomethane) buffer 2.97
g
[0081] Iodo-3-indoxyl-beta-D-galactopyranoside 0.01M
[0082] distilled water added to 1000 mL total volume of
solution
[0083] pH the solution to a value between 1.0 and 12.5 preferably
8.0
[0084] lab notes: Buffer strength is preferably 10 mM or
greater
[0085] Solution II (R2)
[0086] GHB (gamma hydroxybutyrate) conjugated to galactosidase 5
mM
[0087] GHB antibody
[0088] Tris-HCl (Tris[hydroxymethyl]aminomethane hydrochloride)
buffer 0.01M
[0089] distilled water added to make 1000 mL total volume of
solution
[0090] lab notes: pH the solution to a value between 1.0 and 12.5
preferably 8.0
[0091] In this example a dipstick was prepared in accordance with
the instant invention.
[0092] GHB Calibrator Formulations
[0093] Zero (0) Calibrator:
[0094] 1 liter of 0.2 micron filtered normal human urine with no
adulterants or drugs present, and 0.01% sodium azide.*
[0095] 50 ug/mL Calibrator:
[0096] 50 ug/mL GHB
[0097] 0.01 M Sodium Borate
[0098] 100 mL of 0.2 micron filtered normal human urine with no
adulterants or drugs present*
[0099] pH the solution to a value between 3.0 and 11.0 preferably
9.0.
[0100] lab notes:* Human urine can be substituted with distilled
water, synthetic urine or other suitable solvent. The bacterial
inhibitor sodium azide could be replaced with chloroamphenicol or
other suitable bacterial inhibitors that would inhibit the growth
of bacteria.
[0101] The reagent system of the instant invention (liquid reagent)
is intended for use on any automatic chemistry analyzers with open
channel capability including Olympus series, Hitachi 700 series,
Beckmans and many others. The reagent as outlined in Example 2 is
used in the following manner: the one component of the reagent
composition (R-1) is placed in the reagent compartment of the
analyzer; samples, calibrators, and controls are aliquoted into
sample cups, which are then placed on the analyzer. An aliquot of 5
uL of each specimen is then pipetted into a single, discrete
cuvette followed by the addition of 150 uL of the first reagent,
R-1, and mixed; A first spectrophotometer reading is then taken
followed by a second after a specified incubation period (i.e. one
minute for this example) at the specified wavelength (between 340
and 800 nm). The spectrophotometer readings are then recorded. In
this instance the assay is read at 415 nm. The absorbance of
samples, and controls are printed and then compared to the
calibrator's absorbance. The quantitative value for GHB
concentration is then calculated. Any concentration of GHB greater
than 50.0 ug/mL is considered positive for the presence of GHB.
[0102] Please note if the present art is not used as illustrated
that very significant increase in the cost of analysis, because a
GC-MS assay must then be performed to verify the presence of GHB.
The GC-MS analysis costs 100 times as much as the screen ($100 vs
$1). Every additional unnecessary GC-MS performed drives up the
overall cost of drug testing. Eliminating these additional,
unnecessary assays will save millions of dollars per year.
[0103] Specifications for running urine samples vary from
instrument to instrument. Listed below is an example of parameters
for the Hitachi 700 series analyzer. The settings are intended as
guidelines, and are set forth with the understanding that all those
skilled in the art would recognize that such parameters will vary
from instrument to instrument.
[0104] The suggested specifications for the Hitachi 700 series are
as follows:
[0105] Parameter Settings for the Hitachi 700 Series
1 Test: [GHB] Assay code: [1 POINT] [50]-[0] Sample volume: [5] [5]
R1 volume [125] [100] [NO] R2 volume [125] [100] [NO] Wavelength
[0] [415] Calib. Method: [Linear] [0] [0] Std. (1) Conc.-POS:
[0.0]*-[1]* assigned calibrator value Std. (2) Conc.-POS: [50.0]-[2
] assigned calibrator value Std. (3) Conc.-POS: [ ]-[ ] Std. (4)
Conc.-POS: [ ]-[ ] Std. (5) Conc.-POS: [ ]-[ ] Std. (6) Conc.-POS:
[ ]-[ ] SD Limit: [999] Duplicate Limit: [32000] Sensitivity Limit:
[0] ABS. Limit (INC/DEC): [32000] [INCREASE] Prozone Limit: [0]
[lower] Expected Value: [0.0]-[1.0] Tech. Limit: [0]-[1000]
Instrument Factor [1.0] Note: this assay is to be performed at the
same temperature used for the DAU testing, usually 37 degrees
Centigrade. However, this can vary without affecting the assay. The
temperature could be between refrigerated to 45 degrees
Centigrade.
[0106] Thus as described above, an unknown urine submitted for
drugs of anaylsis for GHB will produce a value of less than the 0.0
ug/mL if no GHB is present. Conversely, if the sample has a
concentration of greater than 50.0 ug/mL than the sample is
positive for GHB.
[0107] To summarize more specifically Example 2, the automated
method for the detection of adulteration of an unknown sample of
urine submitted for drugs of abuse testing comprising the steps of
placing aliquots of an unknown urine (or other biological sample
i.e. serum, whole blood, cerebral spinal fluid, gastric fluid, hair
homogenates, sweat extracts, saliva or other biological fluid and
other fluids such as beverages, water, etc.) and calibrator to be
tested in automated analyzer sampling cups, placing the cups in a
sampling tray within an automated analyzer, transferring the
aliquots of sample and calibrator to cuvettes mounted within the
automated analyzer, injecting a first reagent composition (R-1)
comprising an indicator and buffer in an aqueous medium into the
cuvettes, and mixing sample and reagents, and reading absorbance
values of reaction mixture composed of reagents and test samples
(said test samples include urine specimens, controls, and
calibrator) at specified intervals, in accordance with a
preprogrammed code introduced into the automated analyzer, at a
preprogrammed monochromatically specified wavelength, and comparing
absorbance of the first reagent composition plus the unknown
samples with that of the first reagent composition plus the
calibrator containing a zero reference point (normal urinary
matrix), and thereby determining quantitatively the presence or
absence of GHB.
[0108] The following changes to the above reagent solutions will
remain within the scope and function of this invention and will
have similar results to the example above. The GHB indicator
reactive compound (reactive in this sense means that the GHB
conjugated to the galactosidase competes with free GHB if present
for the antibody) galactosidase is sensitive the presence of
iodo-3-indoxyl-beta-D-galactop- yranoside in solution. If there is
no free GHB in the specimen then the galactosidase will not react
with the iodo-3-indoxyl-beta-D-galactopyrano- side because it will
be bound up by the GHB antibody that will bind to the GHB
conjugated to the galactosidase. If there is GHB present in the
sample it will be will bind with the GHB antibody leaving free
GHB-galactosidase conjugate free to react with
iodo-3-indoxyl-beta-D-gala- ctopyranoside thus causing the
development of color which can be monitored by the
spectrophotometer at the specified wavelength. In solution 1 the
galactosidase, which could be substituted with one or more of the
following compounds including hydroxybutyrate dehydrogenase,
esterase, 3-hydroxybutyrate dehydrogenase, 4-hydroxybutyrate
dehydrogenase, carboxyl esterase, carboxylic-ester hydrolase,
.beta.-hydroxybutyrate dehydrogenase, [R]-3-hydroxybutanoate, NAD
(nicotinamide adenine dinucleotide).sup.+ oxidoreductase, and
.alpha.-hydroxybutyrate dehydrogenase, anti-gamma-hydroxybutyrate,
alpha-nicotinamide adenine dinucleotide phosphate,
beta-nicotinamide adenine dinucleotide phosphate, dehydrogenase,
oxidases, reductases, oxidoreductases, transferases, hydrolases,
lyases, isomerases, glycosidases, phosphatases, sulfatases, ligases
and all analogs of the afore mentioned can be substituted with the
following can be substituted with the following enzyme pairs to
include but are not limited to one or more of the following:
4-Aminophenyl-beta-D-galactopyranoside,
3-indoxyl-beta-D-galactopyranosid- e (blue),
5-Bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside (blue),
5-Bromo-3-indoxyl-beta-D-galactopyranoside (blue),
6-chloro-3-indoxyl-beta-D-galactopyranoside (salmon),
6-Fluoro-3-indoxyl-beta-D-galactopyranoside,
8-Hydroxyquinoline-beta-D-ga- lactopyrano-side,
5-Iodo-3-indoxyl-beta-D-galactopyranoside (purple),
N-Methylindoxyl-beta-D-galactopyranoside,
2-Nitrophenyl-beta-D-galactopyr- anoside,
4-Nitrophenyl-beta-D-galactopyranoside, Naphthol
AS-BI-beta-D-galactopyranoside, and
2-Naphthyl-beta-D-galactopyranoside (yellow). Fluorescent
substrates may also be utilized including
4-Methylumbelliferyl-beta-D-glucuronic acid. The colors noted in
the parentheses are those produced in the reaction described above.
The indicator substrate used in these examples must be matched to
the conformation of the galactosidase used (i.e. alpha or beta, and
dextrorotorary (D) or levorotorary (L)). For example,
beta-D-Galactosidase should be matched with the indicator/substrate
Iodo-3-indoxyl-beta-D-galactopyranoside; conversely,
alpha-L-Galactosidase would be matched with
Iodo-3-indoxyl-alpha-L-galact- opyranoside. Note that some
cross-reactivity does occur between stereo-isomers and, therefore,
it is possible to substitute these compounds where appropriate.
[0109] Substitution of the glactosidase with another enzyme would
necessitate a change of substrate indicator complex. If another
glycosidase was selected, it would have to be matched to the
appropriate substrate (e.g. beta-Cellobiosidase and a
cellobioside). Examples of substrates for beta-D-Cellobiosidase
include 5-Bromo-4-chloro-3-indoxyl-b- eta-D-cellobioside,
5-Bromo-6-chloro-3-indoxyl-beta-D-cellobioside,
4-Nitrophenyl-beta-D-cellobioside, 1-Naphthyl-cellobioside,
beta-Galactosidase and the fluorescent indicator,
4-Methylumbelliferyl-be- ta-D-cellobioside.
[0110] Other glycosidases which may be substituted for
Galactosidase and Cellobiosidase include the alpha and beta, and D
and L conformations of the following enzymes: Arabinosidase,
Fucosidase, Galactosaminidase, Glucosaminidase, Glucosidase,
Glucuronidase, Lactosidase, Maltosidase, Mannosidase, and
Xylosidase. Their corresponding substrates, Arabinopyranoside,
Fucopyranoside, Galactosaminide, Glucosaminide, Glucopyranoside,
Glucuronic acid, Lactopyranoside, Maltopyranoside, Mannopyranoside,
and Xylopyranoside may be bound to each of the following color
indicator groups: 5-Bromo-4-chloro-3-indoxyl,
5-Bromo-6-chloro-3-indoxyl, 6-chloro-3-indoxyl, 5-Bromo-3-indoxyl,
5-Iodo-3-indoxyl, 3-indoxyl, 2-(6-Bromonaphthyl),
6-Fluoro-3-indoxyl 2-Nitrophenyl, 4-Nitrophenyl, 1-Naphthyl,
Naphthyl AS-BI, 2-Nitrophenyl-N-acetyl, 4-Nitrophenyl-N-acetyl, and
4-Methylumbelliferyl moieties.
[0111] Other enzymes that can be used with the indicator groups
listed above. These include esterases (e.g. Carboxyl esterase, and
Cholesterol esterase), sulfatases (e.g. Aryl sufatase), and
phosphatases (e.g. Alkaline phosphatase). These enzymes can utilize
the indicator groups delineated above when conjugated to the
corresponding substrate. For example, Carboxyl esterase and
6-chloro-3-indoxyl butyrate, and Aryl sulfatase and
5-bromo-4-chloro-3-indoxyl sulfate, and Alkaline phosphatase and
2-naphthyl phosphate form enzyme-substrate pairs.
[0112] Other enzymes may be conjugated to GHB, and therefore
substituted for the species described above. This group now listed,
however, must utilize a substrate that is distinct and separate
from the indicator. This enzyme group may include any
dehydrogenase, oxidase, hydroxylase, or oxidoreductase. Each
grouping will utilize a specific indicator or group of indicators.
The dehydrogenases and hydroxylases will utilize a co-enzyme, a
color indicator and an electron carrier such as a-NAD
(a-Nicotinamide adenine dinucleotide), however this electron
carrier/acceptor can be replaced by the alpha or beta isomers of
any one of the following substitutes: nicotinamide adenine
dinucleotide, nicotinamide adenine dinucleotide 3'-phosphate,
nicotinamide adenine dinucleotide phosphate, triphosphopyridine,
nicotinamide 1-N1-ethenoadenine dinucleotide phosphate,
nicotinamide hypoxanthine dinucleotide, nicotinamide hypoxanthine
dinucleotide phosphate, nicotinamide mononucleotide, nicotinamide
N1-propylsulfonate, nicotinamide ribose monophosphate, or other
analogs of NAD.
[0113] Some dehydrogenases and hydroxylases and their substrate
pairs which can be used include Formaldehyde dehydrogenase and
Formaldehyde, Fructose dehydrogenase and Fructose,
Glucose-6-phosphate dehydrogenase and Glucose-6-phosphate, Glucose
dehydrogenase and Glucose, Glutamate dehydrogenase and Glutamate,
Glycerol dehydrogenase and Glycerol, Glycerol-3-phosphate
dehydrogenase and Glycerol-3-phosphate, Hydroxybutyrate
dehydrogenase and Hydroxybutyrate, Hydroxybenzoate hydroxylase and
4-Hydroxybenzoate, Lactate dehydrogenase and Lactate, Leucine
dehydrogenase and Leucine, Malate dehydrogenase and Malate,
Mannitol dehydrogenase and Mannitol, or any other dehydrogenase or
hydroxylase.
[0114] The use of oxidases to replace the glycosidase also requires
a separate indicator, and peroxidase. Some oxidases and their
substrate pair which can be used include Acyl-CoA oxidase and
Acyl-CoA, Alcohol oxidase and Ethanol, Ascorbate oxidase and
Ascorbate, Cholesterol oxidase and Cholesterol, Choline oxidase and
Choline, Glucose oxidase and Glucose, Glycerophosphate oxidase and
Glycerophosphate, Xanthine oxidase and Xanthine, Uricase and Uric
acid, or any other oxidase or all analogs of the afore
mentioned.
[0115] The Tris buffer in solution 1, may be substituted with one
or more of the following buffers: citrate, borate, borax, sodium
tetraborate decahydrate, sodium perchlorate, sodium chlorate,
sodium carbonate, MES (2-[N-Morpholino]ethanesulfonic acid),
BIS-TRIS (bis[2-Hydroxyethyl]imino- tris[hydroxymethyl]methane;
2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl-1,- 3-propanediol), ADA
(N-[2-Acetamidol]-2-iminodiacetic acid;
N-[Carbaoylmethyl]iminodiacetc acid), ACES
(2-[(2-Amino-2-oxoethyl)amino]- ethanesulfonic acid;
N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES
(PiperazineN-N'-bis[2-ethanesulfonic acid)];
1,4-Piperzinedethanesulfoic acid), MOPSO
(3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRIS PROPANE
(1,3-bis[tris(Hydroxymethyl)methylamino]propane), BES
(N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid;
2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS
(3-[N-Morpholino]propanesulfonic acid), TES
(N-tris[Hydroxymethyl]methyl-- 2-aminomethanesulfonic acid;
2[2-Hysroxy-1, 1-bis(hydroxymethyl)-ethyl]ami- no)ethanesulfonic
acid), HEPES (N-[2-Hydroxyethyl]piperazine-N'-[2-ethanes- ulfonic
acid]), DIPSO (3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesu-
lfonic acid), TAPSO
(3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxypropanes- ulfonic
acid), HEPPSO (N-[2-Hydroxythyl]piperazine-N'-[2Hydroxypropanesulf-
onic acid]), POPSO (Piperazine-N,N'-bis[2-hydroxypropanesulfonic
acid]), EPPS (N-[2-Hydroxyethyl]piperazine-N'-[3-propanesulfonic
acid), TEA (triethanolamine), TRICINE
(N-tris[Hydroxymethyl]methyllycine;
N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE
(N,N-bis[2-Hydroxyethyl]glycine), TAPS
(N-tris[Hydroxymethyl]methyl-3-ami- nopropanesulfonic acid;
([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-pr- opanesulfonic
acid), AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydro-
xypropanesulfonic acid), CHES (2-[N-Cyclohexylamino]ethanesulfonic
acid), CAPSO (3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic
acid), AMP 2-Amino-2-ethyl-1-propanol, CAPS
(3-[cyclohexylamino]-1-propanesulfonic acid), hydrochloric acid,
phosphoric acid, lactic acid, sulfuric acid, nitric acid, chromic
acid, boric acid, perchloric acid, potassium hydrogen tartrate,
potassium hydrogen phthalate, calcium hydroxide, phosphate,
bicarbonate, sodium hydroxide, potassium hydroxide, oxalate or
succinate. Other buffers with an effective pK and pH range, and
capacity suitable for maintaining the sample-reagent mixture within
the required parameters of the assay's reaction mechanism may be
added to the above group.
EXAMPLE 3
[0116] This example will illustrate in detail the exact method for
manufacturing the lateral flow GHB method. Keep in mind this method
could be utilized for any general chemistry "test pad" or pads that
are currently used or will be used in the art. In the case of DLFH
technology, the manufacturing process includes impregnating onto an
absorbent, solid carrier (e.g. paper) called in this example, the
"test pad", in exactly the same manner as Example 1 with similar
constituents. The test pad, once impregnated, is dried, then
mounted onto a solid support (nitrocellulose membrane) that is
capable of transporting (through lateral flow) liquid to the test
pad from the point of application of a test sample. In simpler
terms, the device is dipped into a liquid or the liquid sample is
placed on the device at the bottom or starting point for the assay.
The liquid migrates from the starting application point to the
opposite end of the nitrocellulose lateral flow paper, during which
the test pad becomes saturated with the sample. The reaction takes
place on the test pad and color develops. The developed color is
then compared to a color chart with known concentrations of GHB
that has the appropriate colors relative to each specific
concentration of GHB(s). For example a specific color for 0.0 ug/mL
GHB, 50.0 ug/mL, 100.0 ug/mL, etc., for comparison. The results are
then recorded. Note, the test pad must be an absorbent (wicking)
material that permits migration of sample up the solid absorbent
test pad and allows analytes and reactants to interact.
[0117] Absorbent material is successively impregnated with the
following solutions and dried at 25 degree C.:
[0118] Solution 1
[0119] NAD 0.3 M
[0120] hydroxybutyrate dehydrogenase 5 mM
[0121] Tris-HCl (Tris[hydroxymethyl]aminomethane hydrochloride)
buffer 0.01M
[0122] NBT (nitro blue tetrazolium) 0.01 mg/L
[0123] distilled water added to 100 mL total volume of solution
[0124] pH the solution to a value between 1.0 and 12.5 preferably
8.0
[0125] lab notes: Buffer strength is preferably 10 mM or
greater
[0126] In this example, the lateral flow device is prepared in
accordance with the instant invention. The lateral flow device is
comprised of a paper carrier matrix (S&S, 593 grade filter
paper) impregnated with the compositions of solution 1. The paper
is then cut into test pads 5 mm by 5 mm. Note that said
concentrations of any of the above constituents can be varied to
suit the DLFH lateral flow/dipstick device format (e.g. dependent
upon paper type, and inclusion of semi-permeable membranes or other
innovations utilized in dry chemistry technology). The paper is
then dried using forced air. The dried impregnated test pad is then
placed at approximately 35 mm (in the middle) of a 5 mm wide by 70
mm long nitrocellulose membrane (S&S FastTrack.TM. NC) and
makes fluid contact with nitrocellulose lateral flow paper. The
nitrocellulose membrane is capable of transporting a liquid by
capillary action or wicking from one end of the lateral flow device
to the other in approximately 60 seconds. In this example, the DLFH
has the dimensions of 5 mm wide by 70 mm long and can be backed by
or in contact with strips of glass fiber filter material (e.g.
S&S 30 grade) to aid in controlling the wicking action, or
other solid support material can be used.
[0127] Again, to completely illustrate the present device the
starting point or origin at which the sample is placed on the test
device is 5 mm from one end of the strip, and 35 mm from the site
of where the test pad is placed in fluid contact with the strip.
For simplicity, this example will have the 5 mm by 5 mm impregnated
test pad placed on top of the lateral flow paper and thus be in
fluid contact with the said paper.
[0128] The mechanics of how the present art's LFD and dipstick test
pad hybrid may be explained is as follows. The starting point or
origin at which the sample is placed on the test device is 5 mm
from one end of the strip, and 35 mm from site where the chemically
impregnated test pad is in fluid contact with the lateral flow
paper. The test pad can be placed on top of the lateral flow paper
making fluid contact with the lateral flow paper from the bottom
side of the test pad, or the lateral flow paper can touch the paper
from the side of the test pad and remain in fluid contact with the
test pad. Or the lateral flow paper can rest on top of the edge of
test pad or be attached and in fluid contact with the test pad in
some other manner. One of the novel advantages in using a hybrid
device made of lateral flow material and a dry chemistry test pad
is the lack of cross contamination from one pad to the next from
excessive fluid, as is inherent in the prior art. For illustration,
currently there are available many different types of dry chemistry
test strips available, such as the Miles Laboratories, Inc.
MULTISTIXref. This device and many other like it has multiple
reagents test pads with different chemistries impregnated onto each
pad on a single support membrane backing (usually plastic). Because
of the relative proximity of these pads to each other on the same
device it is easy for cross contamination to occur, causing
unreliable results. This is called "runover" (i.e. when a reagent
from one pad runs over another adjacent test pad). The present arts
eliminates runover. The applicant's novel approach to the solution
of runover has not been taught prior to the present art and is the
result of extensive research and development.
[0129] Result interpretation can be explained as follows. If the
sample is positive, with a concentration of 50.0 ug/mL GHB or more,
the following occurs. A drop of urine (approximately 50 uL) is
applied at the starting point or origin of the strip. The urine
then migrates to the opposite or terminal end of the strip. As the
urine migrates across the lateral flow material (nitrocellulose)
and comes into contact with the test pad (filter paper), the urine
will saturate the pad and cause a chemical reaction between the
impregnated chemicals and GHB in the urine. A blue color will
develop on the test pad indicating a positive (greater than 50.0
ug/mL GHB) for the presence of high levels of GHB. This color can
then be compared to a color chart showing the different colors from
colorless (white background)) to a dark blue depending upon the
concentration of the GHB(s), if greater than 50.0 ug/mL. The
reaction on the test pad is immediate thus the test results can be
observed immediately.
[0130] If the sample is negative, with a concentration of less than
50.0 ug/mL of GHB present the following occurs. A drop of urine
(approximately 50 uL) is applied at the starting point or origin of
the strip. The urine then migrates to the opposite or terminal end
of the strip. As the urine migrates across the lateral flow
material and comes into contact with the test pad, the urine will
saturate the pad and cause a chemical reaction between the
impregnated chemicals and GHB. However, this example is for a
negative result, thus, no reaction occurs and no color develops,
indicating a negative result. This negative result color can then
be compared to a color chart showing the different colors from no
color developed (negative) to dark blue depending upon the
concentration of the GHB, if greater than 50 ug/mL GHB. The
reaction on the test pad is immediate thus the test results can be
observed immediately.
[0131] Changes to the above reagent solution of example 3 can be
made and still remain within the scope and function of this
invention and will have similar results to examples 1 and 2 above.
The indicator(s) and buffer(s) of example 3 can be replaced by all
the examples and possible substitutions as illustrated in example
1.
[0132] This brief description of the present art illustrates a
completely enabled device that would allow an individual,
physician, patient, and/or technician to quickly and easily
determine the presence of the GHB in urine, providing a much needed
advancement the art of GHB testing.
[0133] To briefly explain the present device as taught. The present
art includes a device for the detection of GHB in a sample of urine
submitted for drugs of abuse testing the steps comprise of
preparing a dry chemistry test means by successively impregnating a
solid, carrier matrix with reagent solutions containing an
indicator and a buffer, and drying the impregnated, solid carrier
matrix. Finally, by dipping said dry chemistry test means into
urine, one can observe the detectable response in the form of a
color developed in the presence or absence of GHB. This present art
also illustrates a unique device that will prevent cross
contamination (runover) of test pads on the same dipstick, as well
as a unique dry chemistry test pad lateral flow device hybrid.
These methods can incorporate detectable responses in the visible
color range to the human eye or in the visible light spectrum.
These methods have a wide sample choice other than urine, and can
be replaced by any biological sample including serum, whole blood,
cerebral spinal fluid, gastric fluid, hair homogenates, sweat
extracts, saliva or other biological fluid and other fluids such as
water, beverages (beer, soft drinks, etc.), to include alcohol
drinks.
EXAMPLE 4
[0134] This is a method for manufacturing a liquid, carrier-free
reagent for the adulteration detection of GBL in samples submitted
for drugs of abuse analysis.
[0135] Prepare a Solution Containing:
[0136] Solution I (R1)
[0137] Hepes buffer 2.97 g
[0138] NAD 0.3 M
[0139] gamma-butyrolactone dehydrogenase 5 mM distilled water added
to make 1000 mL total volume of solution
[0140] lab notes:
[0141] a) pH the solution to a value between 1.0 and 12.5
preferably 6.5
[0142] b) Buffer strength is preferably 0.01 Molar or greater
[0143] c) NBT needs to be in solution at a concentration 0.001 mg/L
or greater
[0144] GBL Calibrator Formulations
[0145] Zero (0) Calibrator:
[0146] 1 liter of 0.2 micron filtered normal human urine with no
adulterants or drugs present, and 0.01% sodium azide.*
[0147] 50 .mu.g/mL Calibrator:
[0148] 50 ug/mL GBL
[0149] 0.01 M Tris HCl
[0150] 100 mL of 0.2 micron filtered normal human urine with no
adulterants or drugs present*
[0151] pH the solution to a value between 3.0 and 11.0 preferably
5.0.
[0152] lab notes:* Human urine can be substituted with distilled
water, synthetic urine or other suitable solvent. The bacterial
inhibitor sodium azide could be replaced with chloroamphenicol or
other suitable bacterial inhibitors that would inhibit the growth
of bacteria.
[0153] The reagent system of the instant invention (liquid reagent)
is intended for use on any automatic chemistry analyzers with open
channel capability including Olympus series, Hitachi 700 series,
Beckmans and many others. The reagent as outlined in Example 5 is
used in the following manner: the one component of the reagent
composition (R-1) is placed in the reagent compartment of the
analyzer; samples, calibrators, and controls are aliquoted into
sample cups which are then placed on the analyzer. An aliquot of 5
uL of each specimen is then pipetted into a single, discrete
cuvette followed by the addition of 150 uL of the first reagent,
R-1, and mixed; A first spectrophotometer reading is then taken
followed by a second after a specified incubation period (i.e. one
minute for this example) at the specified wavelength (between 340
and 800 nm). The spectrophotometer readings are then recorded. In
this instance the assay is read at 340 nm. It is noted that at this
wavelength that the conversion (reduction) of NAD to NADH is
observed. The absorbance of samples, and controls are printed and
then compared to the calibrator's absorbance. The quantitative
value for GBL concentration is then calculated. Any concentration
of GBL greater than 50.0 ug/mL is considered positive for the
presence of GBL.
[0154] Please note if the present art is not used as illustrated
that very significant increase in the cost of analysis, because a
GC-MS assay must then be performed to verify the presence of GBL.
The GC-MS analysis costs 100 times as much as the screen ($100 vs
$1). Every additional unnecessary GC-MS performed drives up the
overall cost of drug testing. Eliminating these additional,
unnecessary assays will save millions of dollars per year.
[0155] Specifications for running urine samples vary from
instrument to instrument. Listed below is an example of parameters
for the Hitachi 700 series analyzer. The settings are intended as
guidelines, and are set forth with the understanding that all those
skilled in the art would recognize that such parameters will vary
from instrument to instrument.
[0156] The suggested specifications for the Hitachi 700 series are
as follows:
[0157] Parameter Settings for the Hitachi 700 Series
2 Test: [GBL] Assay code: [1 POINT] [50]-[0] Sample volume: [5] [5]
R1 volume [150] [100] [NO] R2 volume [ 0] [100] [NO] Wavelength [0]
[340] Calib. Method: [Linear] [0] [0] Std. (1) Conc.-POS:
[0.0]*-[1]* assigned calibrator value Std. (2) Conc.-POS: [50.0]-[2
] assigned calibrator value Std. (3) Conc.-POS: [ ]-[ ] Std. (4)
Conc.-POS: [ ]-[ ] Std. (5) Conc.-POS: [ ]-[ ] Std. (6) Conc.-POS:
[ ]-[ ] SD Limit: [999] Duplicate Limit: [32000] Sensitivity Limit:
[0] ABS. Limit (INC/DEC): [32000] [INCREASE] Prozone Limit: [0]
[lower] Expected Value: [0.0]-[1.0] Tech. Limit: [0]-[1000]
Instrument Factor [1.0] Note: this assay is to be performed at the
same temperature used for the DAU testing, usually 37 degrees
Centigrade. However, this can vary without affecting the assay. The
temperature could be between refrigerated to 45 degrees
Centigrade.
[0158] Thus as described above, an unknown urine submitted for
drugs of anaylsis for GBL will produce a value of less than the 0.0
ug/mL if no GBL is present. Conversely, if the sample has a
concentration of greater than 50.0 ug/mL than the sample is
positive for GBL.
[0159] To summarize more specifically Example 5, the automated
method for the detection of adulteration of an unknown sample of
urine submitted for drugs of abuse testing comprising the steps of
placing aliquots of an unknown urine (or other biological sample
i.e. serum, whole blood, cerebral spinal fluid, gastric fluid, hair
homogenates, sweat extracts, saliva or other biological fluid and
other fluids such as beverages, water, etc.) and calibrator to be
tested in automated analyzer sampling cups, placing the cups in a
sampling tray within an automated analyzer, transferring the
aliquots of sample and calibrator to cuvettes mounted within the
automated analyzer, injecting a first reagent composition (R-1)
comprising an indicator and buffer in an aqueous medium into the
cuvettes, and mixing sample and reagents, and reading absorbance
values of reaction mixture composed of reagents and test samples
(said test samples include urine specimens, controls, and
calibrator) at specified intervals, in accordance with a
preprogrammed code introduced into the automated analyzer, at a
preprogrammed monochromatically specified wavelength, and comparing
absorbance of the first reagent composition plus the unknown
samples with that of the first reagent composition plus the
calibrator containing a zero reference point (normal urinary
matrix), and thereby determining quantitatively the presence or
absence of GBL.
[0160] The following changes to the above reagent solutions will
remain within the scope and function of this invention and will
have similar results to the example above. The indicator in the
solution 1, GBL, which is the indicator reactive compound (reactive
in this sense means that gamma-butyrolactone dehydrogenase is
sensitive the presence of GBL and will react with GBL during which
NAD is reduced to NADH) in solution 1 is gamma-butyrolactone
dehydrogenase, which could be substituted with one or more of the
following compounds including NAD (nicotinamide adenine
dinucleotide).sup.+, oxidoreductase, anti-GBL, alpha-nicotinamide
adenine dinucleotide phosphate, beta-nicotinamide adenine
dinucleotide phosphate, ketone group sensitive indicators and all
analogs of the afore mentioned.
[0161] The Hepes buffer in solution 1, may be substituted with one
or more of the following buffers: citrate, borate, borax, sodium
tetraborate decahydrate, sodium perchlorate, sodium chlorate,
sodium carbonate, MES (2-[N-Morpholino]ethanesulfonic acid),
BIS-TRIS (bis[2-Hydroxyethyl]imino- tris[hydroxymethyl]methane;
2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl-1,- 3-propanediol), ADA
(N-[2-Acetamidol]-2-iminodiacetic acid;
N-[Carbaoylmethyl]iminodiacetc acid), ACES
(2-[(2-Amino-2-oxoethyl)amino]- ethanesulfonic acid;
N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES
(PiperazineN-N'-bis[2-ethanesulfonic acid)];
1,4-Piperzinedethanesulfoic acid), MOPSO
(3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRIS PROPANE
(1,3-bis[tris(Hydroxymethyl)methylamino]propane), BES
(N,N-bis[2-Hydroxyethyl]-2-aminoethanesulfonic acid;
2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS
(3-[N-Morpholino]propanesulfonic acid), TES
(N-tris[Hydroxymethyl]methyl-- 2-aminomethanesulfonic acid;
2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amin- o)ethanesulfonic
acid), TRIS (Tris[hydroxymethyl]aminomethane, DIPSO
(3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic acid),
TAPSO (3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxypropanesulfonic
acid), HEPPSO
(N-[2-Hydroxythyl]piperazine-N'-[2Hydroxypropanesulfonic acid]),
POPSO (Piperazine-N,N'-bis[2-hydroxypropanesulfonic acid]), EPPS
(N-[2-Hydroxyethyl]piperazine-N'-[3-propanesulfonic acid), TEA
(triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine;
N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE
(N,N-bis[2-Hydroxyethyl]glycine), TAPS
(N-tris[Hydroxymethyl]methyl-3-ami- nopropanesulfonic acid;
([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-pr- opanesulfonic
acid), AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydro-
xypropanesulfonic acid), CHES (2-[N-Cyclohexylamino]ethanesulfonic
acid), CAPSO (3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic
acid), AMP 2-Amino-2-ethyl-1-propanol, CAPS
(3-[cyclohexylamino]-1-propanesulfonic acid), hydrochloric acid,
phosphoric acid, lactic acid, sulfuric acid, nitric acid, chromic
acid, boric acid, perchloric acid, potassium hydrogen tartrate,
potassium hydrogen phthalate, calcium hydroxide, phosphate,
bicarbonate, sodium hydroxide, potassium hydroxide, oxalate or
succinate. Other buffers with an effective pK and pH range, and
capacity suitable for maintaining the sample-reagent mixture within
the required parameters of the assay's reaction mechanism may be
added to the above group, however acidic buffers are preferred.
EXAMPLE 5
[0162] This example will illustrate in detail the exact method for
manufacturing the lateral flow GBL method. Keep in mind this method
could be utilized for any general chemistry "test pad" or pads that
are currently used or will be used in the art. In the case of DLFH
technology, the manufacturing process includes impregnating onto an
absorbent, solid carrier (e.g. paper) called in this example, the
"test pad", in exactly the same manner as Example 1 with similar
constituents. The test pad, once impregnated, is dried, then
mounted onto a solid support (nitrocellulose membrane) that is
capable of transporting (through lateral flow) liquid to the test
pad from the point of application of a test sample. In simpler
terms, the device is dipped into a liquid or the liquid sample is
placed on the device at the bottom or starting point for the assay.
The liquid migrates from the starting application point to the
opposite end of the nitrocellulose lateral flow paper, during which
the test pad becomes saturated with the sample. The reaction takes
place on the test pad and color develops. The developed color is
then compared to a color chart with known concentrations of GBL
that has the appropriate colors relative to each specific
concentration of GBL(s). For example a specific color for 0.0 ug/mL
GBL, 50.0 ug/mL, 100.0 ug/mL, etc., for comparison. The results are
then recorded. Note, the test pad must be an absorbent (wicking)
material that permits migration of sample up the solid absorbent
test pad and allows analytes and reactants to interact.
[0163] Absorbent material is successively impregnated with the
following solutions and dried at 25 degree C.:
[0164] Solution 1
[0165] NAD 0.3 M
[0166] gamma-butyrolactone dehydrogenase 5 mM
[0167] Phosphate buffer 0.01M
[0168] NBT (nitro blue tetrazolium) 0.01 mg/L
[0169] distilled water added to 100 mL total volume of solution
[0170] pH the solution to a value between 1.0 and 12.5 preferably
8.0
[0171] lab notes: Buffer strength is preferably 10 mM or
greater
[0172] In this example, the lateral flow device is prepared in
accordance with the instant invention. The lateral flow device is
comprised of a paper carrier matrix (S&S, 593 grade filter
paper) impregnated with the compositions of solution 1. The paper
is then cut into test pads 5 mm by 5 mm. Note that said
concentrations of any of the above constituents can be varied to
suit the DLFH lateral flow/dipstick device format (e.g. dependent
upon paper type, and inclusion of semi-permeable membranes or other
innovations utilized in dry chemistry technology). The paper is
then dried using forced air. The dried impregnated test pad is then
placed at approximately 35 mm (in the middle) of a 5 mm wide by 70
mm long nitrocellulose membrane (S&S FastTrack.TM. NC) and
makes fluid contact with nitrocellulose lateral flow paper. The
nitrocellulose membrane is capable of transporting a liquid by
capillary action or wicking from one end of the lateral flow device
to the other in approximately 60 seconds. In this example, the DLFH
has the dimensions of 5 mm wide by 70 mm long and can be backed by
or in contact with strips of glass fiber filter material (e.g.
S&S 30 grade) to aid in controlling the wicking action, or
other solid support material can be used.
[0173] Again, to completely illustrate the present device the
starting point or origin at which the sample is placed on the test
device is 5 mm from one end of the strip, and 35 mm from the site
of where the test pad is placed in fluid contact with the strip.
For simplicity, this example will have the 5 mm by 5 mm impregnated
test pad placed on top of the lateral flow paper and thus be in
fluid contact with the said paper.
[0174] The mechanics of how the present art's LFD and dipstick test
pad hybrid may be explained is as follows. The starting point or
origin at which the sample is placed on the test device is 5 mm
from one end of the strip, and 35 mm from site where the chemically
impregnated test pad is in fluid contact with the lateral flow
paper. The test pad can be placed on top of the lateral flow paper
making fluid contact with the lateral flow paper from the bottom
side of the test pad, or the lateral flow paper can touch the paper
from the side of the test pad and remain in fluid contact with the
test pad. Or the lateral flow paper can rest on top of the edge of
test pad or be attached and in fluid contact with the test pad in
some other manner. One of the novel advantages in using a hybrid
device made of lateral flow material and a dry chemistry test pad
is the lack of cross contamination from one pad to the next from
excessive fluid, as is inherent in the prior art. For illustration,
currently there are available many different types of dry chemistry
test strips available, such as the Miles Laboratories, Inc.
MULTISTIX.RTM.. This device and many other like it has multiple
reagents test pads with different chemistries impregnated onto each
pad on a single support membrane backing (usually plastic). Because
of the relative proximity of these pads to each other on the same
device it is easy for cross contamination to occur, causing
unreliable results. This is called "runover" (i.e. when a reagent
from one pad runs over another adjacent test pad). The present arts
eliminates runover. The applicants novel approach to the solution
of runover has not been taught prior to the present art and is the
result of extensive research and development.
[0175] Result interpretation can be explained as follows. If the
sample is positive, with a concentration of 50.0 ug/mL GBL or more,
the following occurs. A drop of urine (approximately 50 uL) is
applied at the starting point or origin of the strip. The urine
then migrates to the opposite or terminal end of the strip. As the
urine migrates across the lateral flow material (nitrocellulose)
and comes into contact with the test pad (filter paper), the urine
will saturate the pad and cause a chemical reaction between the
impregnated chemicals and GBL in the urine. A blue color will
develop on the test pad indicating a positive (greater than 50.0
ug/mL GBL) for the presence of high levels of GBL. This color can
then be compared to a color chart showing the different colors from
colorless (white background)) to a dark blue depending upon the
concentration of the GBL(s), if greater than 50.0 ug/mL. The
reaction on the test pad is immediate thus the test results can be
observed immediately.
[0176] If the sample is negative, with a concentration of less than
50.0 ug/mL of GBL present the following occurs. A drop of urine
(approximately 50 uL) is applied at the starting point or origin of
the strip. The urine then migrates to the opposite or terminal end
of the strip. As the urine migrates across the lateral flow
material and comes into contact with the test pad, the urine will
saturate the pad and cause a chemical reaction between the
impregnated chemicals and GBL. However, this example is for a
negative result, thus, no reaction occurs and no color develops,
indicating a negative result. This negative result color can then
be compared to a color chart showing the different colors from no
color developed (negative) to dark blue depending upon the
concentration of the GBL, if greater than 50 ug/mL GBL. The
reaction on the test pad is immediate thus the test results can be
observed immediately.
[0177] Changes to the above reagent solution of example 6 can be
made and still remain within the scope and function of this
invention and will have similar results to examples 3 and 4 above.
The indicator(s) and buffer(s) of example 5 can be replaced by all
the examples and possible substitutions as illustrated in example 3
and 4.
[0178] This brief description of the present art illustrates a
completely enabled device that would allow an individual,
physician, patient, and/or technician to quickly and easily
determine the presence of the GBL in urine, providing a much needed
advancement the art of GBL testing.
[0179] To briefly explain the present device as taught. The present
art includes a device for the detection of GBL in a sample of urine
submitted for drugs of abuse testing the steps comprise of
preparing a dry chemistry test means by successively impregnating a
solid, carrier matrix with reagent solutions containing an
indicator and a buffer, and drying the impregnated, solid carrier
matrix. Finally, by dipping said dry chemistry test means into
urine, one can observe the detectable response in the form of a
color developed in the presence or absence of GBL. This present art
also illustrates a unique device that will prevent cross
contamination (runover) of test pads on the same dipstick, as well
as a unique dry chemistry test pad lateral flow device hybrid.
These methods can incorporate detectable responses in the visible
color range to the human eye or in the visible light spectrum.
These methods have a wide sample choice other than urine, and can
be replaced by any biological sample including serum, whole blood,
cerebral spinal fluid, gastric fluid, hair homogenates, sweat
extracts, saliva or other biological fluid and other fluids such as
water, beverages (beer, soft drinks, etc.), to include alcohol
drinks.
EXAMPLE 6
[0180] This is a method for manufacturing a dry chemistry dipstick
(test strip) with a solid carrier for the GHB detection of in urine
samples submitted for drugs of abuse analysis. Filter paper is
successively impregnated with the following solutions and dried at
25 degree C.:
[0181] Solution I
[0182] EPPS (N-[2-Hydroxyethyl]piperazine-N'-[3-propanesulfonic
acid), buffer 0.97 g
[0183] distilled water added to 100 mL total volume of solution
[0184] pH the solution to a value between 1.0 and 12.5 preferably
8.0
[0185] lab notes: Buffer strength is preferably 0.01 Molar or
greater
[0186] Solution 2
[0187] esterase 0.01 g/L
[0188] thymol blue 0.01 g/L
[0189] distilled water added to make 1000 mL total volume of
solution
[0190] lab notes: esterase needs to be in solution at a
concentration 0.001 g/L or greater
[0191] In this example a dipstick was prepared in accordance with
the instant invention. The device comprised a paper carrier or
solid matrix incorporated with the composition of solutions 1 and 2
above. Note that the concentrations of any of the following
examples can be varied to suit the dipstick device format
(dependent upon paper type, or use of semi-permeable membrane or
other suitable material). This example is carried out using the
following procedure. To produce the test means, a piece of Whatman
3 MM filter paper having approximate dimensions of 1 inch by 3
inches was impregnated with solution 1 by immersing the paper into
solution 1. The paper was then dried by using forced air not
exceeding 25.degree. C. A second piece of Whatman 1 MM filter paper
having approximate dimensions of 1 inch by 3 inches was impregnated
with solution 2 by immersing the paper into solution 2. The paper
was then dried by using forced air not exceeding 25.degree. C. The
dried papers are then laminated to each other by the use of a
non-reactive (neutral adhesive). The dried, laminated papers are
then applied to one side of a piece of double-sided adhesive
transfer tape commercially available from 3M Company, St. Paul,
Minn. 55144. The laminate is then slit into portions measuring 3
inches by 0.2 inches. One portion is then attached, via the unused
adhesive side to a polystyrene sheet measuring about 1.5 inches by
3 inches and the resulting laminate is slit parallel to its short
dimension to form test devices comprising a 1.5 inch oblong
polystyrene strip carrying a square of the impregnated papers at
one end, the other end serving as the handle. When the dipstick
thus obtained is dipped into a urine submitted for drugs of abuse
testing, and no green color develops then no GHB is present.
Conversely, if any concentration of GHB is present in the urine at
a 50 ug/mL or greater a green-blue color will develop thus
confirming the presence of GHB.
[0192] In summary, Example 6 is as follows: the foregoing dry
chemistry test strip (dipstick) method for the GHB detection in a
sample of urine submitted for drugs of abuse testing comprises the
steps of preparing a test means by successively impregnating a
solid, carrier matrix with reagent solutions, drying the
impregnated, solid test means, then dipping said dried test means
into urine, and finally observing any color change in the presence
or absence of GHB.
[0193] The reaction as illustrated by example 6 can best be
understood as follows. When esterase reacts with GHB an acid and
alcohol are produced as by-products of the reaction. As more GHB is
present, more acid and alcohol is generated by the reaction between
GHB and esterase. The detection method for this pathway can take
two different directions. The change in pH of the solution can be
monitored by the use of a pH indicator or the production of alcohol
can be monitored. Example 7 solutions 1 and 2 above illustrate the
pH monitoring pathway. Example of the alcohol monitoring pathway
will follow.
[0194] The following changes to the above reagent solutions will
remain within the scope and function of this invention and will
have similar results to the example above. The GHB reactive
indicator in the solution 1, esterase, could be substituted with
one or more of the hydroxybutyrate dehydrogenase, 3-hydroxybutyrate
dehydrogenase, 4-hydroxybutyrate dehydrogenase, carboxyl esterase,
carboxylic-ester hydrolase, .beta.-hydroxybutyrate dehydrogenase,
[R]-3-hydroxybutanoate, NAD (nicotinamide adenine
dinucleotide).sup.+ oxidoreductase, and a-hydroxybutyrate
dehydrogenase, anti-gamma-hydroxybutyrate, alpha-nicotinamide
adenine dinucleotide phosphate, beta-nicotinamide 7adenine
dinucleotide phosphate and all analogs of the afore mentioned.
[0195] The pH indicator of solution 2, thymol blue could be
replaced with one of the following compounds such as bromcresol
green, methyl red, cresol red, metanil yellow, m-cresol purple,
xylenol blue, thymol blue, tropeolin OO, quinaldine red,
a-dinitrophenol, methyl yellow; dimethyl yellow, bromophenol blue,
tetrabromophenol blue, bromochlorophenol blue, Congo red, methyl
orange, p-ethoxychrysoidine hydrochloride, napthyl red, alizarin
sodium sulfonate, bromocresol green, ?-dinitrophenol, methyl red,
lacmoid, chlorophenol red, benzoyl auramine G, bromocresol purple,
bromophenol red, p-nitrophenol, bromthymol blue, phenol red,
p-quinonemono(bis-4-oxyphenylmethide), neutral red, quinoline blue,
a-naphtholphthalein, tropeolin OOO; a-napthol orange, ethyl
bis(2,4-dinitrophenyl)acetate, di-o-cresolphthalide,
phenolphthalein, thymolphthalein, dimethylphenolphthalein, alizarin
yellow GG; salicyl yellow, alizarin yellow R, Nile blue,
2,4,6-trinitrophenylmethylnitramine- , tropeolin O,
triphenylrosaniline sulfonic acid (sodium or potassium salt),
indigo carmine, nitrobenzene, bromcresol green, bromcresol purple,
bromchlorophenol blue, brilliant yellow, brilliant blue R,
brilliant cresyl blue ALD, brilliant blue G, brlliant black BN,
bromthymol blue, bromphenol red, bromphenol red, bromoxylenol blue,
coomasie blue, azolitmin, litmus, pyrogallosulfonphthalein, and
pyrogallo red-molybdate.
[0196] The EPPS buffer in solution 1, may be substituted with one
or more of the following buffers: citrate, borate, borax, sodium
tetraborate decahydrate, sodium perchlorate, sodium chlorate,
sodium carbonate, (Tris[hydroxymethyl]aminomethane), MES
(2-[N-Morpholino]ethanesulfonic acid), BIS-TRIS
(bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;
2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl-1,3-propanediol), ADA
(N-[2-Acetamidol]-2-iminodiacetic acid;
N-[Carbaoylmethyl]iminodiacetc acid), ACES
(2-[(2-Amino-2-oxoethyl)amino]ethanesulfonic acid;
N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES
(PiperazineN-N'-bis[2-ethanesulfonic acid)];
1,4-Piperzinedethanesulfoic acid), MOPSO
(3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRIS PROPANE
(1,3-bis[tris(Hydroxymethyl)methylamino]propane), BES
(N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid;
2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS
(3-[N-Morpholino]propanesulfonic acid), TES
(N-tris[Hydroxymethyl]methyl-- 2-aminomethanesulfonic acid;
2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amin- o)ethanesulfonic
acid), DIPSO (3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxyp-
ropanesulfonic acid), TAPSO
(3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxy- propanesulfonic
acid), HEPPSO (N-[2-Hydroxythyl]piperazine-N'-[2Hydroxypro-
panesulfonic acid]), POPSO
(Piperazine-N,N'-bis[2-hydroxypropanesulfonic acid]), TEA
(triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine- ;
N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE
(N,N-bis[2-Hydroxyethyl]glycine), TAPS
(N-tris[Hydroxymethyl]methyl-3-ami- nopropanesulfonic acid;
([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-pr- opanesulfonic
acid), AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydro-
xypropanesulfonic acid), CHES (2-[N-Cyclohexylamino]ethanesulfonic
acid), CAPSO (3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic
acid), AMP 2-Amino-2-ethyl-1-propanol, CAPS
(3-[cyclohexylamino]-1-propanesulfonic acid), Hepes, hydrochloric
acid, phosphoric acid, lactic acid, sulfuric acid, nitric acid,
chromic acid, boric acid, perchloric acid, potassium hydrogen
tartrate, potassium hydrogen phthalate, calcium hydroxide,
phosphate, bicarbonate, sodium hydroxide, potassium hydroxide,
oxalate or succinate. Other buffers with an effective pK and pH
range, and capacity suitable for maintaining the sample-reagent
mixture within the required parameters of the assay's reaction
mechanism may be added to the above group.
[0197] Now, if in example 6, solution 2 were formulated as follows,
then the by product of the reaction (GHB and esterase) alcohol is
acted upon by the alcohol oxidase. Solution 2 in this example is
then used in the same manner as solution 2 previously in example 6
and applied to the matrix as previously illustrated.
[0198] Solution 2
[0199] esterase 0.01 g/L
[0200] alcohol oxidase 0.01 mM
[0201] ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid))
0.01%
[0202] distilled water added to make 1000 mL total volume of
solution
[0203] lab notes: alcohol oxidase needs to be in solution at a
concentration 0.001 mM or greater
[0204] In this reaction the alochol oxidase is specific for
methanol and not ethanol (which is the alcohol found in beverages).
Methanol would be poisonous.
[0205] Therefore, in this example a dipstick was prepared in
accordance with the instant invention. The device comprised a paper
carrier or solid matrix incorporated with the composition of
solutions 1 of example 6 above and the second example of solution 2
above containing the alcohol oxidase. Note that the concentrations
of any of the following examples can be varied to suit the dipstick
device format (dependent upon paper type, or use of semi-permeable
membrane or other suitable material). This example is carried out
using the following procedure. To produce the test means, a piece
of Whatman 3 MM filter paper having approximate dimensions of 1
inch by 3 inches was impregnated with solution 1 by immersing the
paper into solution 1. The paper was then dried by using forced air
not exceeding 25.degree. C. A second piece of Whatman 1 MM filter
paper having approximate dimensions of 1 inch by 3 inches was
impregnated with solution 2 by immersing the paper into solution 2.
The paper was then dried by using forced air not exceeding
25.degree. C. The dried papers are then laminated to each other by
the use of a non-reactive (neutral adhesive). The dried, laminated
papers are then applied to one side of a piece of double-sided
adhesive transfer tape commercially available from 3M Company, St.
Paul, Minn. 55144. The laminate is then slit into portions
measuring 3 inches by 0.2 inches. One portion is then attached, via
the unused adhesive side to a polystyrene sheet measuring about 1.5
inches by 3 inches and the resulting laminate is slit parallel to
its short dimension to form test devices comprising a 1.5 inch
oblong polystyrene strip carrying a square of the impregnated
papers at one end, the other end serving as the handle. When the
dipstick thus obtained is dipped into a urine submitted for drugs
of abuse testing, and no uniform green-blue color develops then no
GHB is present.
[0206] Conversely, if any concentration of GHB is present in the
urine at a 50 ug/mL or greater a blue-green color will develop thus
confirming the presence of GHB.
[0207] In summary, Example 6 is as follows: the foregoing dry
chemistry test strip (dipstick) method for the GHB detection in a
sample of urine submitted for drugs of abuse testing comprises the
steps of preparing a test means by successively impregnating a
solid, carrier matrix with reagent solutions, drying the
impregnated, solid test means, then dipping said dried test means
into urine, and finally observing any color change in the presence
or absence of GHB.
[0208] The reaction as illustrated by example 6 can best be
understood as follows. When esterase reacts with GHB an acid and
alcohol are produced as by-products of the reaction. As more GHB is
present, more acid and alcohol is generated by the reaction between
GHB and esterase. The detection method for this pathway can take
two different directions. The change in pH of the solution can be
monitored by the use of a pH indicator or the production of alcohol
can be monitored. Example 7 solution 1 and solution 2 (containing
alcohol oxidase) illustrates using the alcohol monitoring pathway.
Alcohol oxidase in this example will produce as a by product of the
reaction, hydrogen peroxide.
[0209] The following changes to the above reagent solutions will
remain within the scope and function of this invention and will
have similar results to the example above. The GHB reactive
indicator in the solution 1, esterase, could be substituted with
one or more of the hydroxybutyrate dehydrogenase, 3-hydroxybutyrate
dehydrogenase, 4-hydroxybutyrate dehydrogenase, carboxyl esterase,
carboxylic-ester hydrolase, .beta.-hydroxybutyrate dehydrogenase,
[R]-3-hydroxybutanoate, NAD (nicotinamide adenine
dinucleotide).sup.+ oxidoreductase, and a-hydroxybutyrate
dehydrogenase, anti-gamma-hydroxybutyrate, alpha-nicotinamide
adenine dinucleotide phosphate, beta-nicotinamide adenine
dinucleotide phosphate and all analogs of the afore mentioned.
[0210] The alcohol reactive enzyme of solution 2, alcohol oxidase
could be replaced with one or more of the following compounds such
as alcohol dehydrogenase, NAD, NADP, or any other enzyme or
antibody reactive to the production of alcohol as a result of the
interaction of esterase and GHB.
[0211] The EPPS buffer in solution 1, may be substituted with one
or more of the following buffers: citrate, borate, borax, sodium
tetraborate decahydrate, sodium perchlorate, sodium chlorate,
sodium carbonate, (Tris[hydroxymethyl]aminomethane), MES
(2-[N-Morpholino]ethanesulfonic acid), BIS-TRIS
(bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;
2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl-1,3-propanediol), ADA
(N-[2-Acetamidol]-2-iminodiacetic acid;
N-[Carbaoylmethyl]iminodiacetc acid), ACES
(2-[(2-Amino-2-oxoethyl)amino]ethanesulfonic acid;
N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES
(PiperazineN-N'-bis[2-ethanesulfonic acid)];
1,4-Piperzinedethanesulfoic acid), MOPSO
(3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRIS PROPANE
(1,3-bis[tris(Hydroxymethyl)methylamino]propane), BES
(N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid;
2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS
(3-[N-Morpholino]propanesulfonic acid), TES
(N-tris[Hydroxymethyl]methyl-- 2-aminomethanesulfonic acid;
2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amin- o)ethanesulfonic
acid), DIPSO (3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxyp-
ropanesulfonic acid), TAPSO
(3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxy- propanesulfonic
acid), HEPPSO (N-[2-Hydroxythyl]piperazine-N'-[2Hydroxypro-
panesulfonic acid]), POPSO
(Piperazine-N,N'-bis[2-hydroxypropanesulfonic acid]), TEA
(triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine- ;
N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE
(N,N-bis[2-Hydroxyethyl]glycine), TAPS
(N-tris[Hydroxymethyl]methyl-3-ami- nopropanesulfonic acid;
([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-pr- opanesulfonic
acid), AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydro-
xypropanesulfonic acid), CHES (2-[N-Cyclohexylamino]ethanesulfonic
acid), CAPSO (3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic
acid), AMP 2-Amino-2-ethyl-1-propanol, CAPS
(3-[cyclohexylamino]-1-propanesulfonic acid), Hepes, hydrochloric
acid, phosphoric acid, lactic acid, sulfuric acid, nitric acid,
chromic acid, boric acid, perchloric acid, potassium hydrogen
tartrate, potassium hydrogen phthalate, calcium hydroxide,
phosphate, bicarbonate, sodium hydroxide, potassium hydroxide,
oxalate or succinate. Other buffers with an effective pK and pH
range, and capacity suitable for maintaining the sample-reagent
mixture within the required parameters of the assay's reaction
mechanism may be added to the above group.
[0212] The indicator used in solution 2, ABTS
(2,2'-azinobis(3-ethylbenzot- hiazoline-6-sulfonic acid)) is an
oxygen acceptor and is sensitive to the presence of hydrogen
peroxide and can be substituted with one or more of the following:
4-aminoantipyrine (4AAP), tetramethylbenzidine (TMB),
o-phenylenediamine (OPD), o-dianisidine, 5-aminosalicylic acid
(5AS), 3,3'-diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC),
4-chloro-1-napthol (4C1N), or other suitable compound that produces
an observable color for the peroxidase/peroxide reaction. Other
such compounds may include, AEC (3-Amino-9-ethyl carbazole), 2-5,
dimethyl-2,5-dihydroperoxyhexane,
Bis{4-[N-(3'-sulfo-n-propyl)-N-n-ethyl]-
amino-2,6-dimethylphenyl}methane (Bis-MAPS),
N-Ethyl-N-(2-hydroxy-3-sulfop- ropyl)-3-methoxyaniline (ADOS),
N-Ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (ALOS),
N-Ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline (MAPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS),
N-Ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS),
N-(3-sulfopropyl)anilin- e (HALPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-aniline (DAOS),
N-Ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline (DAPS),
N-Ethyl-N-(3-sulfopropyl)aniline (ALPS),
N-(2-hydroxy-3-sulfopropyl)-3,5-- dimethoxyaniline (HDAOS),
N-(3-sulfopropyl)-3,5-dimethoxyaniline (HDAPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAO),
N,N-Bis(4-sulfobutyl)-3,5-dimethylaniline (MADB), and pyrogallol.
Also, 4-aminoantipyrine can be paired with a number of compounds to
create a violet to violet-blue color complex in the presence of the
peroxide/peroxidase reaction. These compounds include
2,4-Dichlorophenol, N,N-Diethyl-m-toluidine, p-Hydroxybenzene
Sulfonate, N,N-Dimethylaniline,
3,5-Dichloro-2-Hydroxybenzenesulfonate,
2,4,6-tribromo-3-hydroxybenzoic acid, Sodium
N-Ethyl-N-(3-Sulfopropyl)-m-Anisidine, hydroxybenzoic acid,
4-hydroxybenzoic acid,
N-Ethyl-N-(2-hydroxy-3-Sulfopropyl)-m-toluidine, AEC
(3-Amino-9-ethyl carbazole), 2-5, dimethyl-2,5-dihydroperoxyhexane,
Bis{4-[N-(3'-sulfo-n-propyl)-N-n-ethyl]amino-2,6-dimethylphenyl}methane
(Bis-MAPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline
(ADOS), N-Ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (ALOS),
N-Ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline (MAPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS),
N-Ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS),
N-(3-sulfopropyl)anilin- e (HALPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS),
N-Ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline (DAPS),
N-Ethyl-N-(3-sulfopropyl)aniline (ALPS),
N-(2-hydroxy-3-sulfopropyl)-3,5-- dimethoxyaniline (HDAOS),
N-(3-sulfopropyl)-3,5-dimethoxyaniline (HDAPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAO),
N,N-Bis(4-sulfobutyl)-3,5-dimethylaniline (MADB), and pyrogallol.
Another indicator pair that may be utilized consists of
3-Methyl-2-benzothiazolin- onehydrazone and Dimethylaniline.
EXAMPLE 7
[0213] This is a method for manufacturing a liquid, carrier-free
reagent for the adulteration detection of GHB in samples submitted
for drugs of abuse analysis.
[0214] Prepare a solution containing:
[0215] Solution I (R1)
[0216] phosphate buffer 1.0 M
[0217] esterase
[0218] distilled water added to make 1000 mL total volume of
solution
[0219] lab notes:
[0220] a) pH the solution to a value between 1.0 and 12.5
preferably 8.0
[0221] b) Buffer strength is preferably 0.01 Molar or greater
[0222] c) NBT needs to be in solution at a concentration 0.001 mg/L
or greater
[0223] Solution II (R2)
[0224] phosphoric acid 1.0 M
[0225] sodium dichromate 10.0 mg/dL
[0226] lab notes: a) pH final solution to a value of 2.0 (range of
1.0 14.0)
[0227] GHB Calibrator Formulations
[0228] Zero (0) Calibrator:
[0229] 1 liter of 0.2 micron filtered normal human urine with no
adulterants or drugs present, and 0.01% sodium azide.*
[0230] 50 ug/mL Calibrator:
[0231] 50 ug/mL GHB
[0232] 0.01 M Sodium Borate
[0233] 100 mL of 0.2 micron filtered normal human urine with no
adulterants or drugs present*
[0234] pH the solution to a value between 3.0 and 11.0 preferably
9.0.
[0235] lab notes:* Human urine can be substituted with distilled
water, synthetic urine or other suitable solvent. The bacterial
inhibitor sodium azide could be replaced with chloroamphenicol or
other suitable bacterial inhibitors that would inhibit the growth
of bacteria.
[0236] The reagent system of the instant invention (liquid reagent)
is intended for use on any automatic chemistry analyzers with open
channel capability including Olympus series, Hitachi 700 series,
Beckmans and many others. The reagent as outlined in Example 2 is
used in the following manner: the one component of the reagent
composition (R-1) is placed in the reagent compartment of the
analyzer; samples, calibrators, and controls are aliquoted into
sample cups which are then placed on the analyzer. An aliquot of 5
uL of each specimen is then pipetted into a single, discrete
cuvette followed by the addition of 150 uL of the first reagent,
R-1, and mixed; Then the second reagent, R-2 is added to the
cuvettes for each sample (urine, unknown, calibrator, standard,
quality control, etc.,), and mixed; then A first spectrophotometer
reading is then taken followed by a second after a specified
incubation period (i.e. one minute for this example) at the
specified wavelength (between 340 and 800 nm). The
spectrophotometer readings are then recorded. In this instance the
assay is read at 600 nm. The absorbance of samples, and controls
are printed and then compared to the calibrator's absorbance. The
quantitative value for GHB concentration is then calculated. Any
concentration of GHB greater than 50.0 ug/mL is considered positive
for the presence of GHB.
[0237] Please note if the present art is not used as illustrated
that very significant increase in the cost of analysis, because a
GC-MS assay must then be performed to verify the presence of GHB.
The GC-MS analysis costs 100 times as much as the screen ($100 vs
$1). Every additional unnecessary GC-MS performed drives up the
overall cost of drug testing. Eliminating these additional,
unnecessary assays will save millions of dollars per year.
[0238] Specifications for running urine samples vary from
instrument to instrument. Listed below is an example of parameters
for the Hitachi 700 series analyzer. The settings are intended as
guidelines, and are set forth with the understanding that all those
skilled in the art would recognize that such parameters will vary
from instrument to instrument.
[0239] The suggested specifications for the Hitachi 700 series are
as follows:
[0240] Parameter Settings for the Hitachi 700 Series
3 Test: [GHB] Assay code: [1 POINT] [50]-[0] Sample volume: [5] [5]
R1 volume [150] [100] [NO] R2 volume [150] [100] [NO] Wavelength
[0] [600] Calib. Method: [Linear] [0] [0] Std. (1) Conc.-POS:
[0.0]*-[1]* assigned calibrator value Std. (2) Conc.-POS: [50.0]-[2
] assigned calibrator value Std. (3) Conc.-POS: [ ]-[ ] Std. (4)
Conc.-POS: [ ]-[ ] Std. (5) Conc.-POS: [ ]-[ ] Std. (6) Conc.-POS:
[ ]-[ ] SD Limit: [999] Duplicate Limit: [32000] Sensitivity Limit:
[0] ABS. Limit (INC/DEC): [32000] [INCREASE] Prozone Limit: [0]
[lower] Expected Value: [0.0]-[1.0] Tech. Limit: [0]-[1000]
Instrument Factor [1.0] Note: this assay is to be performed at the
same temperature used for the DAU testing, usually 37 degrees
Centigrade. However, this can vary without affecting the assay. The
temperature could be between refrigerated to 45 degrees
Centigrade.
[0241] Thus as described above, an unknown urine submitted for
drugs of anaylsis for GHB will produce a value of less than the 0.0
ug/mL if no GHB is present. Conversely, if the sample has a
concentration of greater than 50.0 ug/mL than the sample is
positive for GHB.
[0242] To summarize more specifically Example 7, the automated
method for the detection of adulteration of an unknown sample of
urine submitted for drugs of abuse testing comprising the steps of
placing aliquots of an unknown urine (or other biological sample
i.e. serum, whole blood, cerebral spinal fluid, gastric fluid, hair
homogenates, sweat extracts, saliva or other biological fluid and
other fluids such as beverages, water, etc.) and calibrator to be
tested in automated analyzer sampling cups, placing the cups in a
sampling tray within an automated analyzer, transferring the
aliquots of sample and calibrator to cuvettes mounted within the
automated analyzer, injecting a first reagent composition (R-1)
comprising an GHB reactive compound and buffer in an aqueous medium
into the cuvettes, mixing sample and reagent, then second reagent
composition (R-2) comprising the alcohol indicator is an aqueous
medium into the cuvettes and again the mixing of sample and
reagents occurs, and reading absorbance values of reaction mixture
composed of reagents and test samples (said test samples include
urine specimens, controls, and calibrator) at specified intervals,
in accordance with a preprogrammed code introduced into the
automated analyzer, at a preprogrammed monochromatically specified
wavelength, and comparing absorbance of the first reagent
composition plus the unknown samples with that of the first reagent
composition plus the calibrator containing a zero reference point
(normal urinary matrix), and thereby determining quantitatively the
presence or absence of GHB.
[0243] The following changes to the above reagent solutions will
remain within the scope and function of this invention and will
have similar results to the example above. The indicator in the
solution 1, GHB, which is the indicator reactive compound (reactive
in this sense means that esterase is sensitive (reactive) the
presence of GHB and will react with GHB during which alcohol and
acid is produced) in solution 1 is esterase, which could be
substituted with one or more of the following compounds including
hydroxybutyrate dehydrogenase 3-hydroxybutyrate dehydrogenase,
4-hydroxybutyrate dehydrogenase, carboxyl esterase,
carboxylic-ester hydrolase, 13-hydroxybutyrate dehydrogenase,
[R]-3-hydroxybutanoate, NAD (nicotinamide adenine
dinucleotide).sup.+ oxidoreductase, and a-hydroxybutyrate
dehydrogenase, anti-gamma-hydroxybutyrate, alpha-nicotinamide
adenine dinucleotide phosphate, beta-nicotinamide adenine
dinucleotide phosphate and all analogs of the afore mentioned.
[0244] The phosphate buffer in solution 1 and phosphoric acid
buffer of solution, may be substituted with one or more of the
following buffers: citrate, borate, borax, sodium tetraborate
decahydrate, sodium perchlorate, sodium chlorate, sodium carbonate,
MES (2-[N-Morpholino]ethanesulfonic acid), BIS-TRIS
(bis[2-Hydroxyethyl]imino- tris[hydroxymethyl]methane;
2-bis[2-Hydroxyethyl]amino-2-[hydroxymethyl-1,- 3-propanediol), ADA
(N-[2-Acetamidol]-2-iminodiacetic acid;
N-[Carbaoylmethyl]iminodiacetc acid), ACES
(2-[(2-Amino-2-oxoethyl)amino]- ethanesulfonic acid;
N-[2-Acetamido]-2-aminoethanesulfonic acid), PIPES
(PiperazineN-N'-bis[2-ethanesulfonic acid)];
1,4-Piperzinedethanesulfoic acid), MOPSO
(3-[N-Morpholinol]-2-hydroxypropanesulfonic acid), BIS-TRIS PROPANE
(1,3-bis[tris(Hydroxymethyl)methylamino]propane), TRIS, BES
(N,N-bis[2-Hydroxyethyl]-2-aminoethaesulfonic acid;
2-bis(2-Hydroxyethyl)amino]ethanesulfonic acid), MOPS
(3-[N-Morpholino]propanesulfonic acid), TES
(N-tris[Hydroxymethyl]methyl-- 2-aminomethanesulfonic acid;
2[2-Hysroxy-1,1-bis(hydroxymethyl)-ethyl]amin- o)ethanesulfonic
acid), HEPES (N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesu- lfonic
acid]), DIPSO
(3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesul- fonic acid),
TAPSO (3-[N-tris(Hydroxyethyl)methylamino]-2-hydroxypropanesu-
lfonic acid), HEPPSO
(N-[2-Hydroxythyl]piperazine-N'-[2Hydroxypropanesulfo- nic acid]),
POPSO (Piperazine-N,N'-bis[2-hydroxypropanesulfonic acid]), EPPS
(N-[2-Hydroxyethyl]piperazine-N'-[3-propanesulfonic acid), TEA
(triethanolamine), TRICINE (N-tris[Hydroxymethyl]methyllycine;
N-[2-Hydroxy-1-1-bis(hydroxymethyl)etyyl]glycine), BICINE
(N,N-bis[2-Hydroxyethyl]glycine), TAPS
(N-tris[Hydroxymethyl]methyl-3-ami- nopropanesulfonic acid;
([2-Hdroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-pr- opanesulfonic
acid), AMPSO (3-[(1,1-Dimethyl-2-hydroxyethyl)amino]-2-hydro-
xypropanesulfonic acid), CHES (2-[N-Cyclohexylamino]ethanesulfonic
acid), CAPSO (3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic
acid), AMP 2-Amino-2-ethyl-1-propanol, CAPS
(3-[cyclohexylamino]-1-propanesulfonic acid), hydrochloric acid,
phosphoric acid, lactic acid, sulfuric acid, nitric acid, chromic
acid, boric acid, perchloric acid, potassium hydrogen tartrate,
potassium hydrogen phthalate, calcium hydroxide, phosphate,
bicarbonate, sodium hydroxide, potassium hydroxide, oxalate or
succinate. Other buffers with an effective pK and pH range, and
capacity suitable for maintaining the sample-reagent mixture within
the required parameters of the assay's reaction mechanism may be
added to the above group, however acidic buffers are preferred.
[0245] The indicator of the presence of alcohol in solution sodium
dichromate may be replaced with one or more of the following:
sulfuric acid, sodium salicylate, sodium hydroxide, iodine,
potassium permanganate or other alcohol reactive indicators or
analogs of the afore mentioned.
EXAMPLE 8
[0246] The following procedure is a method for manufacturing a dry
chemistry test strip (DCD), for the determination of GHB in a test
sample.
[0247] Filter paper is impregnated with the following solutions and
dried at 25 degree C.:
[0248] Solution 1
[0249] 30.2 G PIPES (1,4-Piperazinediethanesulfonic acid)
[0250] 0.05 Units/mL beta-Galactosidase/anti-GHB (enzyme conjugated
to the anti-GHB)
[0251] add to 900 mL D.I. water, mix, adjust pH to 6.8, Q.S. to
1000 mL
[0252] Solution 2
[0253] 0.01 M 5-bromo-6-chloro-3-indoxyl-beta-D-galactopyranoside
(Magenta-beta-D-Gal)
[0254] 1 mL (0.1%) DMSO
[0255] dissolve in 900.0 mL distilled water, mix, and Q.S. to 1000
mL.
[0256] In this example, a dipstick is prepared in accordance with
the instant invention as described in Example 1, however. Solution
1 and 2 are both incorporated into the test device by immersing the
test paper into solution 1; drying the paper; then immersing the
test paper into solution 2; the paper is then dried by using forced
air not exceeding 60 degrees C. If a two-part test pad "sandwich"
is used, the pad with solution #1 must be on top and the pad with
solution 2 is on the bottom. The dipstick thus obtained will
produce a magenta color when exposed to GHB at a concentration of
50 ug/mL or greater. In fact, the intensity of the magenta color is
proportional to the concentration of the GHB, present in the
sample. This test device, therefore, effectively identifies the
presence of GHB in urine by the measurement of the GHB in the urine
sample used for illustrative purposes in this example.
[0257] To summarize Example 8 more specifically, the foregoing dry
chemistry test strip (DCD) method to measure the GHB concentration
in a urine sample (or other matrices) for the determination of
presence of absence of GHB using said sample, the method comprising
the steps of preparing a test means by successively impregnating an
absorbent carrier matrix with reagent solutions, drying said test
means, dipping completed test means into test sample, and
determining the quantity of GHB present in said test sample by
comparing the relative intensity of the color (magenta) produced by
the reaction to a color chart with color blocks referenced to
specific concentrations of GHB.
[0258] Changes to the foregoing solutions could be made and still
have similar results. In addition, changes to the foregoing
solutions or use of the identical solutions as illustrated could be
used in an automated analyzer and produce the same results. The
foregoing solutions could be combined together, or reduced to
include only 1. The concentrations of said constituents may also be
changed and still remain within the scope of the invention. The
buffer may be replaced with any one or more of those constituents
enumerated in Example 1.
[0259] The indicator substrate complex in the solution
5-bromo-6-chloro-3-indoxyl-beta-D-galacatopyranoside, could be
substituted with one or more of the following:
4-Aminophenyl-beta-D-galac- topyranoside,
3-indoxyl-beta-D-galactopyranoside (blue),
5-Bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside (blue),
5-Bromo-3-indoxyl-beta-D-galactopyranoside (blue),
6-chloro-3-indoxyl-beta-D-galactopyranoside (salmon),
6-Fluoro-3-indoxyl-beta-D-galactopyranoside,
8-Hydroxyquinoline-beta-D-ga- lactopyranoside,
5-Iodo-3-indoxyl-beta-D-galactopyranoside (purple),
N-Methylindoxyl-beta-D-galactopyranoside,
2-Nitrophenyl-beta-D-galactopyr- anoside,
4-Nitrophenyl-beta-D-galactopyranoside, Naphthol
AS-BI-beta-D-galactopyranoside, and
2-Naphthyl-beta-D-galactopyranoside (yellow). Fluorescent
substrates may also be utilized including
4-Methylumbelliferyl-beta-D-glucuronic acid. The colors noted in
the parentheses are those produced in the reaction described above.
The indicator substrate used in these examples must be matched to
the conformation of the galactosidase used (i.e. alpha or beta, and
dextrorotorary (D) or levorotorary (L)). For example,
beta-D-Galactosidase should be matched with the indicator/substrate
Iodo-3-indoxyl-beta-D-galactopyranoside; conversely,
alpha-L-Galactosidase would be matched with
Iodo-3-indoxyl-alpha-L-galact- opyranoside. Note that some
cross-reactivity does occur between stereo-isomers and, therefore,
it is possible to substitute these compounds where appropriate.
[0260] Substitution of the beta-Galactosidase with another enzyme
would necessitate a change of substrate indicator complex. If
another glycosidase was selected, it would have to be matched to
the appropriate substrate (e.g. beta-Cellobiosidase and a
cellobioside). Examples of substrates for beta-D-Cellobiosidase
include 5-Bromo-4-chloro-3-indoxyl-b- eta-D-cellobioside,
5-Bromo-6-chloro-3-indoxyl-beta-D-cellobioside,
4-Nitrophenyl-beta-D-cellobioside, 1-Naphthyl-cellobioside, and the
fluorescent indicator,
4-Methylumbelliferyl-beta-D-cellobioside.
[0261] Other glycosidases which may be substituted for
Galactosidase and Cellobiosidase include the alpha and beta, and D
and L conformations of the following enzymes: Arabinosidase,
Fucosidase, Galactosaminidase, Glucosaminidase, Glucosidase,
Glucuronidase, Lactosidase, Maltosidase, Mannosidase, and
Xylosidase. Their corresponding substrates, Arabinopyranoside,
Fucopyranoside, Galactosaminide, Glucosaminide, Glucopyranoside,
Glucuronic acid, Lactopyranoside, Maltopyranoside, Mannopyranoside,
and Xylopyranoside may be bound to each of the following color
indicator groups: 5-Bromo-4-chloro-3-indoxyl,
5-Bromo-6-chloro-3-indoxyl, 6-chloro-3-indoxyl, 5-Bromo-3-indoxyl,
5-Iodo-3-indoxyl, 3-indoxyl, 2-(6-Bromonaphthyl),
6-Fluoro-3-indoxyl 2-Nitrophenyl, 4-Nitrophenyl, 1-Naphthyl,
Naphthyl AS-BI, 2-Nitrophenyl-N-acetyl, 4-Nitrophenyl-N-acetyl, and
4-Methylumbelliferyl moieties.
[0262] The glycosidase enzyme conjugated to the anti-GHB in the
example above can also be replaced by other types of enzymes whose
substrates are compatible with the indicator groups listed above.
These include esterases (e.g. Carboxyl esterase, and Cholesterol
esterase), sulfatases (e.g. Aryl sufatase), and phosphatases (e.g.
Alkaline phosphatase). These enzymes can utilize the indicator
groups delineated above when conjugated to the corresponding
substrate. For example, Carboxyl esterase and 6-chloro-3-indoxyl
butyrate, and Aryl sulfatase and 5-bromo-4-chloro-3-indoxyl
sulfate, and Alkaline phosphatase and 2-naphthyl phosphate form
enzyme-substrate pairs.
[0263] Other enzymes may be conjugated to the anti-GHB, and
therefore substituted for the species described above. This group
now listed, however, must utilize a substrate that is distinct and
separate from the indicator. This enzyme group may include any
dehydrogenase, oxidase, hydroxylase, or oxidoreductase. Each
grouping will utilize a specific indicator or group of indicators.
The dehydrogenases and hydroxylases will utilize a co-enzyme, a
color indicator and an electron carrier such as a-NAD
(a-Nicotinamide adenine dinucleotide) or NADP, however these
electron carrier/acceptors can be replaced by the alpha or beta
isomers of any one of the following substitutes: nicotinamide
adenine dinucleotide, nicotinamide adenine dinucleotide
3'-phosphate, nicotinamide adenine dinucleotide phosphate,
triphosphopyridine, nicotinamide 1-N1-ethenoadenine dinucleotide
phosphate, nicotinamide hypoxanthine dinucleotide, nicotinamide
hypoxanthine dinucleotide phosphate, nicotinamide mononucleotide,
nicotinamide N1-propylsulfonate, nicotinamide ribose monophosphate,
or other analogs of NAD or NADP.
[0264] Some dehydrogenases and hydroxylases and their substrate
pairs which can be used include Formaldehyde dehydrogenase and
Formaldehyde, Fructose dehydrogenase and Fructose,
Glucose-6-phosphate dehydrogenase and Glucose-6-phosphate, Glucose
dehydrogenase and Glucose, Glutamate dehydrogenase and Glutamate,
Glycerol dehydrogenase and Glycerol, Glycerol-3-phosphate
dehydrogenase and Glycerol-3-phosphate, Hydroxybutyrate
dehydrogenase and Hydroxybutyrate, Hydroxybenzoate hydroxylase and
4-Hydroxybenzoate, Lactate dehydrogenase and Lactate, Leucine
dehydrogenase and Leucine, Malate dehydrogenase and Malate,
Mannitol dehydrogenase and Mannitol, or any other dehydrogenase or
hydroxylase.
[0265] The use of oxidases to replace the glycosidase also requires
a separate indicator, and peroxidase. Some oxidases and their
substrate pair which can be used include Acyl-CoA oxidase and
Acyl-CoA, Alcohol oxidase and Ethanol, Ascorbate oxidase and
Ascorbate, Cholesterol oxidase and Cholesterol, Choline oxidase and
Choline, Glucose oxidase and Glucose, Glycerophosphate oxidase and
Glycerophosphate, Xanthine oxidase and Xanthine, Uricase and Uric
acid, or any other oxidase.
[0266] A few color indicators that can be utilized with peroxidase
include pyrogallol, ABTS (2,2'-Azinobis(3-ethylbenzthiazoline)
sulfonic acid), 3,3',5,5'-Tetramethylbenzidine, ortho-Dianisidine,
3,3'-Diaminibenzidine, AEC (3-Amino-9-ethyl carbazole), 2-5,
dimethyl-2,5-dihydroperoxyhexane,
Bis{4-[N-(3'-sulfo-n-propyl)-N-n-ethyl]amino-2,6-dimethylphenyl}methane
(Bis-MAPS), N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline
(ADOS), N-Ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (ALOS),
N-Ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline (MAPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS),
N-Ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS),
N-(3-sulfopropyl)anilin- e (HALPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS),
N-Ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline (DAPS),
N-Ethyl-N-(3-sulfopropyl)aniline (ALPS),
N-(2-hydroxy-3-sulfopropyl)-3,5-- dimethoxyaniline (HDAOS),
N-(3-sulfopropyl)-3,5-dimethoxyaniline (HDAPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAO), and
N,N-Bis(4-sulfobutyl)-3,5-dimethylaniline (MADB). An indicator pair
may also be used. One such pair is
3-Methyl-2-benzothiazolinonehydrazone and Dimethylaniline. Another
pair combines 4-aminoantipyrine with a number of compounds to
create a violet to violet-blue color complex in the presence of the
peroxide/peroxidase reaction. These compounds include phenol,
2,4-Dichlorophenol, N,N-Diethyl-m-toluidine, p-Hydroxybenzene
Sulfonate, N,N-Dimethylaniline,
3,5-Dichloro-2-Hydroxybenzenesulfonate, Sodium
N-Ethyl-N-(3-Sulfopropyl)-m-Anisidine, and
N-Ethyl-N-(2-hydroxy-3-Sulfopr- opyl)-m-toluidine. An example of
this assay procedure would substitute glucose oxidase for
galactosidase in the antibody-enzyme conjugate in R-1; the R-2
would then contain glucose as the substrate and ABTS (reduced) as
the indicator. The R-2 would also contain peroxidase, because the
product of the reaction between glucose oxidase and glucose yields
peroxide. The peroxidase oxidizes any peroxide thus produced,
thereby releasing an oxygen atom; this oxygen, in turn, reacts with
ABTS, and converts it from the colorless, reduced form to its blue,
oxidized form. The intensity of the blue color produced is
proportional to the GHB concentration present in the specimen.
Clearly, peroxidase may be conjugated to the antibody, and the
indicators noted above used with it and its substrate,
peroxide.
[0267] The use of oxireductases to replace glycosidase also
requires a separate indicator including NADPH oxidoreductase and
NADPH, or any oxidoreductase. The NADPH oxireductase reduces the
NADPH in the presence of Flavin mononucleotide (FMN). This reaction
may be observed visually by utilizing the same color indicators as
delineated for the dehydrogenases, or measured
spectrophotometrically at 340 nm.
[0268] The antigens used in this example and the prior examples may
be substituted with any one or more of the following anti-GHB (I or
II), or any monoclonal or polyclonal antibodies of the same. All of
these reactants can be used and will produce a detectable response
in the presence of GHB.
EXAMPLE 9
[0269] The following procedure is a method for manufacturing a dry
chemistry test strip, (DCD) for the determination of GHB in a test
sample by measurement of its GHB concentration. Filter paper is
impregnated with the following solutions and dried at 25 degree
C.:
[0270] Solution 1
[0271] 2-[N-Morpholino]ethanesulfonic Acid buffer (MES) 0.1 M
[0272] anti-GHB is conjugated to horseradish peroxidase
[0273] 900 mL D.I. water, mix, adjust pH to 6.0, and Q.S. to 1000
mL with D.I. water
[0274] Solution 2
[0275] 2-[N-Morpholino]ethanesulfonic Acid buffer 0.1 M
[0276] Tetramethylbenzidine, (TMB) 500 mg
[0277] Urea-Peroxide, 5.0 g
[0278] 900 mL D.I. water, mix, and adjust pH between 5.0 and 7.0,
preferably 6.0
[0279] Q.S. to 1000 mL with D.I. water
[0280] lab note: the techniques for producing these types of
conjugated antibodies is well known in the art.
[0281] This assay utilizes an antigen/antibody reaction with the
antibody conjugated to peroxidase. When antibody which is
conjugated to the peroxidase binds to its target antigen, it
releases the peroxidase which is then free to react with peroxide
and the chromogen, TMB, resulting in formation of a blue-green
colored complex. This color reaction yields a visible color change.
Therefore, the GHB concentration is proportional to the intensity
of the blue-green color produced.
[0282] The test device in this example is manufactured in the same
manner as that in Example 9. If this device is constructed using
two reaction pads, the reaction pad containing solution 2 must be
on the bottom half of the "sandwich". In addition, it may be
necessary to separate the two pads with a semipermeable
membrane.
[0283] Changes to the foregoing solutions could be made and still
have similar results. In addition, changes to the foregoing
solution or use of the identical solutions could be utilized in an
automated analyzer and produce the same results. The foregoing
solutions could be combined together, or reduced to only 1. The
concentrations of said constituents may also be changed and still
remain within the scope of the invention. Obviously, the same
substitution groups for anti-GHB are possible as already
demonstrated in examples 1-9 and this includes the buffers as noted
in the prior examples also apply to this example. The urea peroxide
was chosen, because it is more stable than simple peroxide. It is
obvious, however, that one may utilize any peroxide-containing
compound to act as a substrate to peroxidase.
[0284] The TMB may be replaced by any suitable compound that will
produce an observable color as part of the peroxidase/peroxide
reaction. Other such compounds include ABTS
(2,2'-Azino-di-(3-ethylbenzthiazolinesulfonic acid) diammonium
salt, AEC (3-Amino-9-ethyl carbazole), 2-5,
dimethyl-2,5-dihydroperoxyhexane,
Bis{4-[N-(3'-sulfo-n-propyl)-N-n-ethyl]-
amino-2,6-dimethylphenyl}methane (Bis-MAPS),
N-Ethyl-N-(2-hydroxy-3-sulfop- ropyl)-3-methoxyaniline (ADOS),
N-Ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (ALOS),
N-Ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline (MAPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS),
N-Ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS),
N-(3-sulfopropyl)anilin- e (HALPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS),
N-Ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline (DAPS),
N-Ethyl-N-(3-sulfopropyl)aniline (ALPS),
N-(2-hydroxy-3-sulfopropyl)-3,5-- dimethoxyaniline (HDAOS),
N-(3-sulfopropyl)-3,5-dimethoxyaniline (HDAPS),
N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAO),
N,N-Bis(4-sulfobutyl)-3,5-dimethylaniline (MADB), and pyrogallol.
Also, 4-aminoantipyrine can be paired with a number of compounds to
create a violet to violet-blue color complex in the presence of the
peroxide/peroxidase reaction. These compounds include
2,4-Dichlorophenol, N,N-Diethyl-m-toluidine, p-Hydroxybenzene
Sulfonate, N,N-Dimethylaniline,
3,5-Dichloro-2-Hydroxybenzenesulfonate, Sodium
N-Ethyl-N-(3-Sulfopropyl)-- m-Anisidine, and
N-Ethyl-N-(2-hydroxy-3-Sulfopropyl)-m-toluidine. Another indicator
pair that may be utilized consists of 3-Methyl-2-benzothiazolin-
onehydrazone and Dimethylaniline.
[0285] In addition, it is possible to conjugate other enzymes to
antibodies or antigens. Consequently, these conjugated pairs can
also be substituted into the test reaction together with an
appropriate indicator compound. Therefore, this assay may include
any enzyme capable of being conjugated to an antibody or
antigen.
[0286] To further describe the preferred test method for
determining the presence of GHB by the measurement in an unknown
test sample, the assay system can take the form of a dipstick
(DCD), lateral flow device (LFD), or an aqueous liquid reagent that
is composed of a buffer and an indicator that produces a color or
change in the intensity of color or absorbance in the UV or visible
spectrum in the presence of GHB. The antibodies (such as anti-GHB,
anti-anti-GHB, anti-IgG or others). The anti-GHB antibodies can
also include IgA, IgD, IgE, and IgM. The buffers used may be any
one or more compounds selected from the following group and
enumerated by their common names: citrate, hepes, tris (trizma),
taps, popso, tes, pipes, mopso, tricine, mops, mes, bicine, bes,
caps, epps, dipso, ches, capso, ampso, aces, ada, bis-tris-propane,
tapso, heppso, tea, amp, phosphate, phthalate, succinate,
hydrochloric acid, sulfuric acid, nitric acid, acetic acid, sodium
hydroxide, and potassium hydroxide. In addition, as taught the test
sample can be any biological fluid from the following group: urine,
serum, whole blood, saliva, cerebral spinal fluid, gastric
contents, and extracts of hair or sweat. This art as taught herein
can employ an aqueous-based liquid reagent for measuring the
concentration of GHB, said test method comprising the steps of
placing the reagent in the reagent compartment of the chemistry
autoanalyzer, aliquoting samples, calibrators, and controls into
sample cups and placing them on the chemistry autoanalyzer,
transferring an aliquot of each sample, calibrator, and control
into single, discrete cuvettes mounted within the chemistry
autoanalyzer, aliquoting a specified volume of the reagent
composition into each cuvette and mixing, incubating the reaction
mixture for a specified time interval, measuring and recording
absorbance values of the reaction mixtures with the chemistry
autoanalyzer's spectrophotometer at the specified wavelength (from
340 to 800 nm) at preprogrammed time intervals, and comparing
absorbance values of samples and controls to those of the
calibrators in the form of a standard curve thereby quantitating
the GHB if present.
[0287] This art as taught in previous examples can also employ a
dry chemistry test strip (DCD) method for measuring the GHB
concentration in a test sample, the method comprising the steps of
preparing a test means by successively impregnating a carrier
matrix with reagent solutions, drying said test means, dipping
completed test means into test sample, and determining the quantity
of GHB present in said test sample by comparing the relative
intensity of the color produced by the reaction to a color chart
with color blocks referenced to specific concentrations of GHB.
[0288] This art as taught in previous examples can also employ a
dry chemistry, lateral flow device (LFD) for measuring the GHB
concentration in a test sample, the method comprising the steps of
preparing a test means by successively impregnating a solid,
absorbent carrier matrix with liquid, reagent solutions at specific
locations on the test means, drying said test means, dipping
completed test means into test sample or pipetting test sample onto
the test means, and determining the quantity of GHB in the test
sample by comparing the relative intensity (completeness) of color
developed to a standard chart.
EXAMPLE 10
[0289] The following procedure is a method for manufacturing a dry
chemistry, lateral flow test strip for the determination of GHB.
This example will also illustrate the utility of incorporating the
use of creatinine concentration (as determined by colorimetric
assay, DCD, LFD, antibody/antigen, etc. . . . ) on the same sample
measured for GHB and the enhanced clinical significance of the GHB
value. Absorbent material is successively impregnated with the
following solutions and dried at 25 degree C.:
[0290] Solution 1
[0291] 0.05 M Phosphate buffer pH 7.2
[0292] 30 fmol/L anti-GHB
[0293] Solution 2
[0294] 0.05 M Phosphate pH 7.2
[0295] 30 fmol/L 50 ug/mL of GHB bound to red microparticles
[0296] In this example, the lateral flow device is prepared in
accordance with the instant invention. The lateral flow device is
comprised of a paper carrier matrix impregnated with the
compositions of solutions 1 and 2 as follows. Note that said
concentrations of any of the above constituents can be varied to
suit the lateral flow/dipstick device format (e.g. dependent upon
paper type, and inclusion of semi-permeable membranes or other
innovations utilized in dry chemistry technology). Production of
this test device is carried out using the following procedure. The
test device made up of a solid support which includes an absorbent
material capable of transporting a liquid by capillary action or
wicking (e.g. nitrocellulose 5.0u, S&S brand) in this example
having dimensions of 5 mm by 70 mm and can be backed by or in
contact with strips of glass fiber (e.g. Whatman GF/A) to aid in
controlling the wicking action. In this example, the device uses an
GHB cutoff of 50 ug/mL.
[0297] The starting point or origin at which the sample is placed
on the test device is 5 mm from one end of the strip, and 30 mm
from this origin is the buffered solution 1 containing anti-GHB
bound to the test strip 35 mm from the bottom edge of said test pad
in a line approximately 1 mm wide by 5 mm long thereby extending
from one side of the device to the other side forming the A, "assay
line". A second buffered solution consisting of red colored
particles bound (i.e. irreversibly coupled, conjugated, or
covalently linked) to 50.0 ug/mL GHB is applied to the strip
approximately 5 mm from the starting point (or 10 mm from the lower
edge of the test strip) in a concentration as to make certain that
assay line forms a solid visual line to achieve effective
results.
[0298] A solid case made of plastic or other suitable material may
be used to conceal and protect the device except for a assay line
"window" (hole in the device for viewing the results of the
analysis); and a window for sample application at the origin. This
case may be composed of plastic, wood, cardboard, or other suitable
material.
[0299] If the sample is positive, with a concentration of 50 ug/mL
GHB or more the following occurs. A drop of urine (approximately 50
uL) is applied at the starting point or origin of the strip. The
urine then migrates to the opposite or terminal end of the strip.
The free GHB present (in a concentration of 50 ug/mL or greater of
GHB) in the urine starts to migrate to the assay line and binds all
of the bound anti-GHB at the assay line. The red particles bound
with GHB will migrate with the urine toward the terminal end of the
strip away from the starting point. These colored complexes will
not bind to the line of anti-GHB bound at the 10 mm "A" line or
assay window because all of the free GHB has already bound up all
of the active sites of the bound (immobilized) anti-GHB at the
assay line. The migrating red colored particle complexes,
therefore, continue migrating up the device and disappear from
view. Thus, no solid red line is formed indicating that a
concentration of 50 ug/mL or more of GHB is present.
[0300] If the sample is negative, with a concentration of less than
50 ug/mL GHB present, the following occurs. The free (unbound) red
GHB microparticles complexes migrate up to the "A" assay line and
bind to the anti-GHB conjugated (immobilized) to the test strip at
that location thereby forming a solid (complete) red line assay
line. The formation of a solid red line indicates a negative for
the presence of GHB in concentration of greater than 50 ug/mL.
[0301] The test strip can be placed on top of, or backed, with
glass fiber (e.g. Whatman GF/A) in order to control (i.e. speed up,
or slow down the "wicking" speed) and held in place by an adhesive
or other means. This brief description of the present art
illustrates a completely enabled device that would allow a
physician, patient, and/or technician to determine rapidly the
presence or absence of GHB in a urine or other suitable fluid.
[0302] If analysis is performed on a 24 hour urine collection, no
further analysis is required. Proper 24 hour urine collections are
difficult and inconvenient for the patient, however, the above test
can also be performed using a random specimen. Consequently, a
novel addition to further improve the ease of use and the accuracy
of the present device requires an additional assay on the same
random or spot urine used for the GHB assay. This additional assay
is for creatinine, cystatin C or any other steady state marker
consistently excreted in human urine. This analyte value can be
used to "normalize" or correct the GHB test result for the amount
of water present in the sample. Water content of a random urine
sample is affected by the diurnal variations, diet, diuretics (e.g.
caffeine, sugar, etc. . . . ) and short term fluid consumption
(water consumed over the previous 2 to 3 hours). The amount of
creatinine excreted by a normal, healthy individual is relatively
consistent from day to day, and hour to hour; any GHB would also be
excreted at a consistent rate from hour to hour. Creatinine or
Cystatin C is, therefore, ideal for adjusting or normalizing the
amount of GHB found in a random urine. Specifically, if for example
the creatinine concentration is high the subject has consumed very
little water over the previous few hours, and the GHB value will be
elevated; if the subject has consumed a large volume of water just
prior to testing, the creatinine value will be low and the GHB
marker will also be depressed.
[0303] This present art incorporates the unique invention of the
GHB analysis and determining the concentration of a steady state
marker such as creatinine or Cystatin C ratio (GHB/creatinine). The
following formula may be used to adjust the GHB value according to
the creatinine concentration, and thereby produce the
GHB/creatinine ratio (i.e. G/C ratio). This method requires
division of the GHB value by the creatinine concentration of the
sample. This yields a normalized GHB value for a random sample. The
method of measuring creatinine in urine by LFD is hitherto unknown
in the art until the present device and examples of this
methodology will follow. If analysis is being performed via
automated chemistry, a number of well known methods are currently
available. This ratio provides the most convenient way to normalize
the GHB value and allow the user, even an untrained one, to obtain
a corrected GHB value.
[0304] The following is a detailed description of how the
GHB/creatinine ratio is used. Obviously, in the case of testing the
sample with aqueous, liquid reagents on an automated chemistry
analyzer system quantitative results would be obtained for both
analytes. The GHB value is then divided by the creatinine
concentration. If this ratio is equal to, or greater than 0.27,
then GHB presence is confirmed. Values lower than 0.27 are
considered negative for GHB for this example.
[0305] In the example above, the device detects 50 ug/mL of GHB or
more in the urine, so positives are considered 50, and negatives
are zero. Typical creatinine values range from 45 to 180 mg/dl.
Therefore, if the GHB result is positive and the creatinine value
is less than 185 mg/dl, then the corrected result is still positive
(50/185=0.27); the ratio is inversely proportional to the
creatinine value (i.e. as the creatinine drops, the ratio
increases). Obviously the higher the ratio, the more GHB present.
Therefore, a semi-quantitative GHB/creatinine ratio can be obtained
by assuming any positive is 50 ug/mL of GHB and dividing it by the
creatinine quantitation (e.g. 50/60=0.833 ratio). On the other
hand, if the creatinine concentration is higher than 185, then the
true GHB value may be falsely elevated, and a new sample should be
tested because this could be interpreted as a false positive.
[0306] Conversely, if the GHB value is negative, and the creatinine
value is 157 mg/dl or higher, then the sample is clearly negative
(25 ug/mL/157 mg/dl=0.159). On the other hand if the creatinine
value is lower than 20 mg/dl creatinine the assay should be
repeated. It is well known in the art that a creatinine of less
than 20 mg/dl is a dilute specimen and a false negative could occur
with this specimen (25/20=1.25, a positive).
[0307] Another factor that can and should be taken into account is
kidney function as determined by the protein/creatinine ratio. If
the protein/creatinine ratio is normal (less than 3.0, as known in
the art), then the assay is not affected by the ability of the
kidneys to clear creatinine or other steady state marker such as
cystatin C and allow for an accurate assessment of the urine
concentration. If the protein/creatinine ratio is greater than 3.0,
then the assay can be affected by the kidney function. The
GHB/creatinine ratio may be corrected for kidney dysfunction by
dividing it by the protein/creatinine-ratio (i.e. GHB/K ratio), and
determining appropriate ranges. Preliminary data suggests that an
GHB/K ratio of 0.05 or higher is negative, and an GHB/K ratio of
less than 0.05 indicates a positive for GHB.
[0308] To summarize Example 10 more specifically, the foregoing
lateral flow/dry chemistry test strip (LFD) method for measuring
the GHB concentration in a random urine sample, the method
comprising the steps of preparing a test means by successively
impregnating a solid, absorbent, carrier matrix with liquid reagent
solutions at specific locations on said test means, drying said
test means, dipping completed test means into test sample or
pipetting sample onto the test means, and determining the quantity
of GHB in said test sample by comparing the relative intensity
(completeness) of the assay line produced by the reaction. Also,
the assay can include the determination of creatinine to determine
the GHB/creatinine ratio (G/C ratio) to improve the validity of the
test result. It is understood that the above example was purely
illustrative, and that the relative positions of the control and
assay lines could be relocated without changing the spirit, scope,
or intent of the instant invention.
[0309] Changes to the foregoing solutions could be made and still
have similar results. The foregoing solutions could be combined
together, or reduced to include only 1 solution for impregnation.
The concentrations of said constituents may also be changed and
still remain within the scope of the invention. The antibody to GHB
can be to can be replaced with antigens in appropriate positions to
make for a different format than explained in the example.
Anti-Anti-GHB could be used which is the antibody to the GHB
antibody. The foregoing was merely illustrative of the
possibilities of this novel and unique invention. In addition,
anti-GBL could be used instead of anti-GHB and GBL could be
conjugated to red-microparticles the assay would work for the
detection of GBL in exactly the same manner as the GHB lateral flow
method.
[0310] The buffer(s) used in example 11, may be substituted with
any one or more of the buffers as illustrated in example 1.
[0311] The colored particles used in example 10 could be replaced
with particles of any color, and made from many types of materials
including rubber, latex, plastics, synthetic solids, metals, or
other suitable material that will form a solid platform or
substrate for the covalent attachment (binding) of a reactive
compound, antibody, and/or antigen to it.
[0312] This Example's formulation could also include any one or
more of the surfactants, thickeners, or interference-removing
compounds disclosed above in this embodiment. Optional compounds
for removal of interfering substances include mono, di, tri, and
tetra sodium salts of EDTA or EGTA. Optional thickeners include
polyvinylpyrrolidone, algin, carrageenin, casein, albumin, methyl
cellulose, and gelatin in concentrations ranging from 0.5 to 5 g.
per 100 ml. Optional surfactants may include long chain organic
sulphates or sulphonates (e.g. Brij-35, Tween 20, Triton X-100,
dioctyl sodium sulphosuccinate, and sodium lauryl sulphate).
[0313] The subject invention provides an extraordinary and novel
method for quantitating the presence of GHB in biological
specimen(s) (i.e. urine, blood, serum, saliva, hair and sweat
extracts, and cerebrospinal fluid) and other fluids such as water,
beverages (to include but not be limited to soft drinks, beer,
mixed drinks, etc.) in order to determine the presence of GHB.
[0314] In addition, the absolute novelty of creatinine, cystatin C,
or other renal clearance marker measurement by the use of a DCD or
LFD is of enormous value to medical diagnostics, enforcement
agencies, and public safety and health organizations; its utility
when applied to aqueous, liquid form and modified for use on
automated clinical chemistry analyzers is also of great value for
the same reasons. All in all, the ability of the present art to
analyze urine for GHB measurement via dry chemistry dipsticks,
lateral flow devices, and aqueous, liquid reagents while
simultaneously enabling the user to normalize the results with the
sample's creatinine, cystatin C or other renal clearance marker
concentration as described herein is a substantial and significant
improvement over the prior art.
[0315] To further elaborate the present art so that it is clearly
understood the present art is a method for determining the presence
of GHB on an unknown test sample, said test method being composed
of a buffer and indicator that produces a detectable response or a
change in the absorbance or intensity of a color or line in the UV
or visible spectrum in the presence or absence of GHB. These
methods can use all the buffers, indicators, microparticles
(metallic or other matrix), and components as taught in examples 1
through 10. The methods in examples 1 through 10 as taught can be
employed as aqueous liquid reagents for measuring the concentration
of GHB on a test specimen, said test methods comprise the steps of
placing the reagent in the reagent compartment of the chemistry
autoanalyzer and aliquoting samples, calibrators, and controls into
sample cups and placing them on the chemistry autoanalyzer, then
transferring an aliquot of each sample, calibrator, and control
into single, discrete cuvettes mounted within the chemistry
autoanalyzer, aliquoting a specified volume of the reagent
composition into each cuvette and mixing, incubating the reaction
mixture for a specified time interval, and measuring and recording
absorbance values of the reaction mixtures with the chemistry
autoanalyzer's spectrophotometer at the specified wavelength (from
340 to 800 nm) at preprogrammed time intervals, and comparing
absorbance values of samples and controls to those of the
calibrators in the form of a standard curve thereby quantitating
the amount of GHB present. The methods as taught can also employ a
dry chemistry test strip (DCD) method to measure the GHB
concentration in a test sample, the method comprising the steps of
preparing a test means by successively impregnating an absorbent
carrier matrix with reagent solutions, drying said test means,
dipping completed test means into test sample, and determining the
quantity of GHB present in said test sample by comparing the
relative intensity of the color produced by the reaction to a color
chart with color blocks referenced to specific concentrations of
GHB. The methods as taught can also be used for any general
chemistry "test pad" or pads that are currently used or will be
used in the art like in the DCD device a further integration in a
device such as the DLFH, in this advance technology, the
manufacturing process includes impregnating onto an absorbent,
solid carrier (e.g. paper) called in this example, the "test pad",
in exactly the same manner as with the DCD's with similar
constituents. The test pad, once impregnated, is dried, then
mounted onto a solid support (nitrocellulose membrane) that is
capable of transporting (through lateral flow) liquid to the test
pad from the point of application of a test sample. In simpler
terms, the device is dipped into a liquid or the liquid sample is
placed on the device at the bottom or starting point for the assay.
The liquid migrates from the starting application point to the
opposite end of the nitrocellulose lateral flow paper, during which
the test pad becomes saturated with the sample. The reaction takes
place on the test pad and color develops. The developed color is
then compared to a color chart with known concentrations of GHB
that has the appropriate colors relative to each specific
concentration of GHB. The results are then recorded. Note, the test
pad must be an absorbent (wicking) material that permits migration
of sample up the solid absorbent test pad and allows analytes and
reactants to interact. In this example the inventor illustrates the
ability of the present device to use any GHB reaction indicator
that will produce a detectable response in the presence of GHB.
[0316] The methods can also employ a dry chemistry lateral flow
device (LFD) for measuring the GHB concentration in a test sample,
the method comprising the steps of preparing a test means by
successively impregnating a solid, absorbent carrier matrix with
liquid reagent solutions at specific locations on said test means,
drying said test means, dipping completed test means into test
sample or pipetting test sample onto the test means, and
determining the quantity of GHB present in said test sample by
comparing the relative intensity of the assay line produced by the
reaction to a standard chart, or by comparing the relative
intensity of the assay line produced by the reaction to the control
line. The method examples as taught utilizing a spectrophotometer
can employ wavelengths from 340 to 800 nm.
[0317] The methods as the present art teaches can also improve
analytical value of the GHB concentration of a test sample by
employing creatinine, cystatin C, or specific gravity
concentrations which can be used to normalize the sample for
accurate determination of GHB. This normalization of the GHB
concentration requires that it be divided by the creatinine,
cystatin C, or specific gravity concentration of the same test
sample thereby yielding the GHB to creatinine, cystatin C, or
specific gravity ratio. Thus, all the methods of the present art as
taught are for analyzing a sample using a dry chemistry dipstick,
dipstick/lateral flow hybrid, lateral flow device, or aqueous
liquid reagent to determine the concentration of GHB in an
individual's random urine sample in order to determine if the
individual's has ingested GHB, and normalizing or correcting this
assay value with the sample's creatinine, cystatin C, or specific
gravity concentration.
[0318] Changes to the above reagent solution of example 10 can be
made and still remain within the scope and function of this
invention and will have similar results to examples 1 through 10
above. The indicator(s) and buffer(s) of example 10 can be replaced
by all the examples and possible substitutions as illustrated in
examples 1 through 10. It can be noted that in any of the previous
examples that the reagent solutions 1 and 2 can be combined to form
one reagent solution and still be functional.
[0319] Changes to the above compound(s) used in the example(s) can
be exchanged with analogs or functional derivatives of the
compound(s) and still within the scope of the appended claims.
[0320] This brief description of the present art illustrates a
completely enabled device that would allow a physician, patient,
individual, enforcement agency, bar tender, parent, and/or
technician to quickly and easily determine the presence of GHB or
GBL in urine, providing a much needed advancement in the art of
drug testing.
[0321] To briefly explain the present device as taught. The present
art includes a device for the detection of GHB in a sample of urine
submitted for drugs testing the steps comprise of preparing a dry
chemistry test means by successively impregnating a solid, carrier
matrix with reagent solutions containing an indicator and a buffer,
and drying the impregnated, solid carrier matrix. Finally, by
dipping said dry chemistry test means into urine, one can observe
the detectable response in the form of a color developed in the
presence or absence of GHB. This present art also illustrates a
unique device that will prevent cross contamination (runover) of
test pads on the same dipstick, as well as a unique dry chemistry
test pad lateral flow device hybrid. These methods can incorporate
detectable responses in the visible color range to the human eye or
in the visible light spectrum. These methods have a wide sample
choice other than urine, and can be replaced by any biological
sample including serum, whole blood, cerebral spinal fluid, gastric
fluid, hair homogenates, sweat extracts, saliva or other biological
fluid and other fluid to include water, soft drinks, beverages,
beer, mixed drinks, drinks with alcohol, etc.
[0322] As completely described and enabled the methods for
determining the presence or amount of gamma-hydroxybutyrate (GHB)
or gamma-butyrolactone in a sample, said methods comprising
contacting said sample with an indicator which specifically binds
to GHB or GBL to form an indicatorcomplex; and, measuring said
indicatorcomplex to determine the presence or amount of said GHB or
GBL in said sample.
[0323] The type of sample for the methods of examples 1 through 10
can be selected from the following group consisting of urine,
serum, whole blood, cerebral spinal fluid, gastric fluid, hair
homogenates, sweat extracts, saliva, water, beverages, beer,
alcoholic drinks, or soft drinks.
[0324] To further delineate the foregoing teachings the methods for
the detection of GHB or GBL in a sample can also comprise the steps
of preparing a dry chemistry test means by successively
impregnating a solid, carrier matrix with reagent solutions
containing an indicator and a buffer, and drying the impregnated,
solid carrier matrix, and finally placing sample onto said dry
chemistry test means, and observing the detectable response in the
form of a color developed in the presence or absence of GHB or
GBL.
[0325] And the methods as completely taught for the detection of
GHB in a sample can comprise the steps of placing aliquots of an
unknown urine and calibrator to be tested in automated analyzer
sampling cups, placing the cups in a sampling tray within an
automated analyzer, transferring the aliquots of sample and
calibrator to cuvettes mounted within the automated analyzer,
injecting a first reagent composition (R-1) comprising an indicator
and buffer in an aqueous medium into the cuvettes, mixing sample
and reagent, reading the absorbance values of reaction mixture
composed of reagents and test samples (said test samples include
unknown specimens, controls, and calibrator) at specified
intervals, in accordance with a preprogrammed code introduced into
the automated analyzer, at a preprogrammed monochromatically
specified wavelength, and comparing absorbance of the first reagent
composition plus the unknown samples with that of the first reagent
composition plus the calibrator containing a zero reference point,
and thereby determining the presence or absence of GHB.
[0326] It is understood that variations or modifications in the
following embodiments may be made by someone skilled in the art
without departing from the spirit and scope of the invention. All
such modifications and variations are to be included within the
scope of the invention as defined in the appended claims:
[0327] Referenced U.S. Patents:
[0328] U.S. Pat. No. 4,351,899
[0329] U.S. Pat. No. 4,622,296
[0330] U.S. Pat. No. 5,032,506
[0331] U.S. Pat. No. 5,624,813
[0332] U.S. Pat. No. 5,912,139
[0333] U.S. Pat. No. 5,447,837
[0334] U.S. Pat. No. 4,301,115
[0335] U.S. Pat. No. 3,603,957
[0336] Referenced EU Patents:
[0337] 0 291194
[0338] 226 427
* * * * *