Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
2 ~
SALIVA TESTING AND FINGERPRINT
IDENTIFICATION METHO~ AND DEVICE
RELATED APPLICATIONS
This is a continuation-in-part application of
United States patent application Serial Number 07/467,~32
filed January 191 1990.
BACKGROUND OF INVENTION
~ he present invention is directed to an individual
testing method and device and more specifically to a method
and device for detecting the presence of specific antigens
or specific antibodies produced by drugs in a biological
fluid such as saliva and using the device to also positively
identify the individual tested by reproducing the
fingerprint of the person being tested. Previously drug
testing has been accomplished by testing individual fluid
samples such as urine or blood to determine the presence of
drugs in the body. Such testing procedures are very common
in the athletic world, prisons, courts of law, and in the
general workplace and are many times proscribed by contracts
between the individual and his/her employer or labor union
which represents the individual or group. A problem which
has occurred during such testing is that test fluids are
obtained from persons other than the person to be tested or
that test fluids become mixed, lost, or cannot be
specifically identified with that person after the test
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comes back with positive results. Another problem is that
the time for testing i5 generally too long to obtain results
which are timely.
Saliva i5 a transcellular fluid produced by
several paired salivary glands, particularly the parotid,
the submaxillary, the sublingual glands and some other small
glands.
The present invention attempts to overcome the
problems which are inherit in the prior art through the use
of a specifically designed fingerprint pad device which
tests for the presence of drugs or other specified agents in
the saliva as well as providing a fingerprint of the person
giving the test so that positive identification of the fluid
donor is irrefutably obtained.
It is known that there is a correlation between
cocaine levels in blood and levels of cocaine in saliva
tVol. 11, Journal of Analytical Toxicology,_ p. 36,
January/February 1987) and that cocaine can be found in
saliva after cocaine administration in concentrations equal
to or greater than those in plasma allowing for the
possibility of a relatively noninvasive means of chain
detection and monitoring (Vol. 34, Clinical Chemistry, p.
150B, No._7, 1988). It is also known that detection of
cocaine in saliva and urine is successful through the first
24 hours of collection for saliva and 4-5 days for urine
(Vol. 13, Journal_ of Analytical Toxicology, p. 65,
March/April 1989.
Furthermore, it has been proposed that morphine
and codeine usage can be found in testing human hair long
after drug levels in urine~ plasma and saliva are not
detectable and a comparison of same with drug levels in
biofluids is found in Vol. 14, Journal of Analytical
Toxicology, p. 1, January/February 1990.
Medium dosages of heroin in the range of 5-10 mg
per 70 kg has been found detectable in saliva 1 to 2 hours
after use as was dextromethorphan, while morphine has been
found detectable for 3 to 4 hours after the last morphine
dose. (Vol. lS, Clinical Pharmacology and Therapeutics, p.
579, No. 6, 1974).
A saliva collection device for the quantitative
determination of endogenous substances and therapeutic drugs
in disclosed in Vol. 37, Clinical Chemistry, pp.__ll4-115,
No. 1, 1991. The saliva i9 collected in the buccal cavity
by a specially designed device comprising an osmotically
active substance enclosed in a pouch consisting of a
semipermeable membrane to form a disc of about 35 mm
diameter.
The family of immunoassays works upon the single
principle that is the specific recognition of an antigen by
an antibody. The specific antigen detection and
quantification requires an antibody which recognizes the
uniqueness of an antigen. One unique binding site serves as
an identifying marker for that protein.
Thus detection can be direct where the antigen-
specific antibody is purified, labelled and used to bind
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directly to the antigen or indirect where the antigen-
specific antibody is unlabelled and need not be purified.
In indirect detection the binding to the antigen is detected
by a secondary reagent such as labelled anti-immunoglobulin
antibodies or labelled protein A. ~ variation that uses
aspects of both the direct and indirect methods modifies the
primary antibody by coupling to it a small chemical group
such as biotin or dinitrophenol (DNP) so that the modified
primary antibody can then be detected by labelled reagents
such as a biotin binding protein or haptene-specific
antibodies such as anti-DNP antibodies.
Antibodies which are immobilized (irreversibly
bound) on a membrane are well known in the art and such
antibodies are designed to have binding sites which have
high affinity for the epitopes of the antigens carried in
the saliva and vice versa. Covalent binding of protein to
the membrane surface offers a permanent bond which is
irreversible, so that once a protein like an antibody is
bound, it will not be desorbed during an assay. The
principle of affinity chromatography requires that a
successful separation of a biospecific ligand is available
and that it can be chemically immobilized to a
chromatographic bed material, the matrix. Numbers of
methods well known in the art have been used to couple or
immobilize the ligand to a variety of activated resins.
Examples of immobilization techniques which exhibit variable
linkage are those formed by the reaction of the reactive
groups on the support with amino, thiol, hydroxyl, and
carboxyl groups on the protein ligand. The selection of the
ligand is influenced by two factors. First, the ligand
should exhibit specific and reversible binding affinity for
the substance to be purified and secondly it should have
chemically modifiable groups which allow it to be attached
to the matrix without destroying its binding activity.
(Examples of such are Protein G manufactured by Pharmacia,
~ydrazide AvidGel Ax manufactured by BioProbe International,
and Actigel-ALD manufactured by Sterogene Bioseparation
Inc.)
When a definitive antibody for a given antigen is
available, it i8 used to identify the antigen in the sample
mixture. Once the antibody combines with the antigen, a
means is needed to recognize the complex. This has been
accomplished in the past by providing a labelled antibody,
such as an enzyme, enzyme link immunosorbent (ELISA) -type
assay so that the site is incubated with a chromogenic
substrate and a color is developed whose intensity is
proportional to the enzyme label present.
It is known in the prior art to use membranes in
immunoassay testing and also to use such membranes in
connection with an absorbent pad to permit the creation of a
self-contained packager which is easily disposable.~American
Clinical Products Review, June 1987) Such membrane
structures have been developed by Millipore Corp. and other
manufacturers. Various problems have occurred with the use
of such membranes which are primarily used in strip testing
due to different plastics which have been used and the
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degradation for absorption of the protein from the affinity
membrane.
It is therefore desirable to provide an easy to
handle disposable testing pad which holds a saliva sample
taken from under the tongue of the test patient with the
saliva then being finger pressed on a membrane substrate
having a specific immobilized antibody or antigen bed to
capture a concentrated amount of specified antigen or
labelled antibody from the saliva while simultaneously
providing the fingerprint indentation of the user to be
recorded for positive identification.
B IEF SUMMARY OF THE INVENTION
The invention is directed toward a saliva antigen
collection device for testing and identification. The
device is in the form of a support member with an absorbent
section having a permeable membrane test pad mounted thereon
which is coded with specific antibodies. The membrane bed
antibodies capture specific antigens carried by the saliva
to determine the presence in the body of specific drugs or
substances. It is, of course, apparent that antigens and
antibodies can be switched and either immobilized to capture
the other.
It is an object of the invention to collect
antigen and/or antibodies from saliva samples removed from
the body for testing and simultaneously use the fingerprint
pattern obtained from pressing the finger of the test
subject against the saliva coated pad to positively identify
the donor of the sample. PreviQusly such testing has been
accomplished by a series of tests which may involve shifting
of the fluid being tested to different containers and
removal of the fluid from the person being tested to a place
distant from the person which allows fluid misplacement and
substitution and questions as to the chain of title of the
tested fluid.
It is also an object of the invention to segregate
various areas of the pad and provide specific immobilized
antibodies which react to specific drugs or substances on
different predetermined separated areas so that a multiple
drug test can be given to the test subject with one
fingerprinting using the test subject's saliva.
In the accompanying drawings there is shown an
illustrated embodiment of the invention from which these and
other objectives, novel features and advantages will be
readily apparent.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic exploded view of the test
kit components of the invention;
Figure 2 i8 a schematic view of a swab being
placed under the tongue of the user;
Figure 3 is a schematic view of the saliva soaked
swab being placed on the support member of the invention;
Figure 4 is a schematic view of the saliva soaked
swab placed on the support member and solution carrying
labelled antibodies being added to the swab;
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Figure 5 is a schematic view of the finger of the
test subject engaging the swab so that the finger is covered
with a thin film of mixed saliva and solution complex before
touching the testing pad;
Figure 6 is a schematic view of the solution
complex covered finger of Figure 5 pressed against the test
pad;
Figure 7 i~ a top plan view of the finger pad
surface shown in Figure 1 having the capacity to test for
multiple drugs used with the present invention;
Figure 8 is a top plan view of the surface of the
pad of Figure 7 after a fingerprint has developed on the
surface showing a negative test;
Figure 9 is a top plan view of the surface of the
pad of Figure 7 after a fingerprint has developed on the
surface showing bad reagents;
Figure 10 is a top plan view of the surface of the
pad of Figure 7 after a fingerprint has developed on the
surface showing cocaine positive test results;
Figure 11 is a top plan view of the surface of the
pad of Figure 7 after a fingerprint has developed on the
surface showing opiates positive test results;
Figure 12 is a top plan view of the surface of the
pad of Figure 7 after a fingerprint has developed on the
surface showing PCP positive test results;
Figure 13 is a top plan view of the surface of the
pad of Figure 7 after a fingerprint has developed on the
surface showing THC positive test results;
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Figure 14 is a top plan view of the surface of the
pad of Figure 7 after a fingerprint has developed on the
surface showing methamphetamine positive test results; and
Figure 15 is a top plan view of the surface of the
pad of Figure 7 after a fingerprint has developed on the
surface showing alcohol positive test results.
DETAILED DESCRIPTION OF THE DRAWINGS
The preferred embodiment and best mode of the
invention is shown in Figures 1-15. In the invention, a
swab 20 is removed from a sterile swab package 22 which is
packaged with or attached to support member 30. The swab is
is placed under the tongue 101 of the test subject 100 to
obtain a saliva specimen as is shown in Figure 2. The swab
after it is soaked with saliva is then placed in an
incubation channel 31 and basin 32 cut in support member 30.
The support member 30 has an ab~orbent section 34 which
includes an absorbent pad 36 upon which is mounted a
membrane 38 impregnated with specified antibody or antigen
which will capture specific antigen or antibodies produced
in the human body by a drug, biological agent or substance
which has been taken by the individual. The swab 20 and
subsequently membrane 38 is then incubated with a
chromogenic substrate solution 40 as shown carried in bottle
42 and a color fingerprint 44 is developed whose intensity
is proportional to the enzyme present. The addition of the
chromogenic substrate to the saliva soaked swab eliminates
the need to add another liquid to develop the color on the
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membrane surface. Thus when the membrane 38 is incubated
with a chromogenic substrate pressed into it by the coated
finger a reverse fingerprint will be developed by the
deposit of colloidal gold particles.
Many of commonly used substrates for binding
antibodies or enzymes are large and/or have a large number
of charged groups.
A secondary antibody is premixed in the solution
added to the saliva and complexes with the antigen in the
saliva prior to applying the fingertip to the membrane to
produce the fingerprint. This secondary antibody is
provided with microsome labelling which colors the
fingerprint in negative or reverse order of a normal print
in that the valleys contain a majority of colloidal
particles. The enzyme in the conjugate serves as an
indicator that upon reaction with substrate demonstrates the
presence of unknown in the sample. The chromogenic
substrate provides a detection for the conjugate enzyme and
the color produced is proportional to the amount of the
unknown in the sample.
The preferred substrate used in the present
invention is that of colloidal gold. Gold is biologically
inert and has very good charge distribution. It is now
becoming widely available in many useful forms. Its
detection can be enhanced using several silver deposition
methods available commercially. Colloidal gold also can be
detected easily in electron microscopy applications and can
be prepared in discrete and uniform size ranges, permitting
double-labelling experiments. Several commercial companies
have introduced silver enhancement kits that do not require
using a darkroom, permitting development to be monitored
under the microscope or by the naked eye. Colloidal gold
particles bind tightly but not covalently to proteins at pH
values around the protein's pI . Colloidal gold particles
conjugated with a wide range of anti-immunoglobulin
antibodies, protein A or streptavidin are available
commercially. Because some of the bound protein may slowly
dissociate from the gold particles, the colloid can be
washed if desired before use to remove free protein.
Gold labels give higher resolution than enzyme-
based methods and avoid the problems of substrate
preparation and endogenous enzyme activity. Until recently
the gold labels lacked sensitivity at the level of light
microscopy, but the recent development of the photochemical
silver method of amplification has overcome this problem.
With the silver enhancement method, the gold
particleR become coated in metallic silver and yield a
black-brown label, best visualized by bright-field optics.
Gold labelling methods are compatible with many
histochemical strains. Gold labelling reactions are very
readily controlled, as the appearance of staining can be
monitored directly and continuously under the microscope or
with the naked eye.
~ he membrane 38 which is preferably of latex ha a
surface 50 which as shown in Figure 7 is divided up into
separate segment test areas which are provided immobilized
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ligands such as antibodies or antigens which attach to
predetermined labelled ligands carrying specific substances.
The test se~ment areas are shown reacting or holding cocaine
molecules 51, opiates 52, PCP 53, THC 54, methamphetamine 55
and ~lcohol 56 althou~h other substances can be substituted.
Presence of one or more of the aforenoted drugs or
predetermined substance in the saliva provides the
corresponding segment with a negative no color or inhibition
assay. A control center 57 and outer ring 58 are provided
to provide backup accuracy. The central control area is
porous to prevent fluid entrapment on the surface of the
membrane.
A chromogenic substrate provides a sensitive
detection method for the enzyme. The following tables I and
II ~et forth chromogenic substrates yielding water-insoluble
products and water-soluble products that can be used in the
invention in place of the colloidal gold substrate
previously noted.
TABLE I
Chromogenic Sub~trates Yield~ng Water-In~oluble Product~
Abbrev- Starting Final
Enzyme Substrate iationColor Color
_
Horseradish
Peroxidase Diaminobenzidene DABClear Brown
Diaminobenzidene DAB/Clear Grey/
with nickel nickelBlack
enhancement
3-Amino-9- AEC Clear Red
ethylcarbazole
4-Chloro-l- --- Clear Blue
naphthol
Alkaline Naphthol-AS-BI NABP/FR Clear Red
13
Phosphatase phosphate/fast
red TR
Naphthol-AS-MX- NAMP/FR Clear Red
phosphate/fast
red TR
Naphthol-AS-BI- NABP/NF Clear Red
phosphate/new
fuchsin
Bromochloroindolyl BCIP/NBT Clear Purple
phosphate/nitro-
blue tetrazolium
5-Bromo-4-chloro- BCIG Clear Blue
3-indolyl-B-d-
galactopyranoside
B-Galactosidase Naphthol AS-BI-B- NABG Clear Red
d-galactopyrano-
side
14
TA~LE II
Chromogenic ~bstrates Yielding ~atar-~oluhle Product~
Abbrev- Starti~g Final
Enzyme _ Substrate _iation Color _o or
Horseradish 2,2'-Azinodi[eth- AsTs Clear Green
Peroxidase ylbenzthiazoline]
sulfonate
o~Phenylene OPD Clear Brown
diamine
3,3',5,5'-Tetra- TMB Clear Yellow
methylbenzidine
Alkaline p-Nitrophenol PNPP Clear Yellow
Phosphatase phosphate
B-Galactosidase 0-Nitrophenyl-B- ONPG Clear Yellow
d-galactophyran-
oside
Generally, the color produced is proportional to the
amount of unknown in the sample, providing the unknown is
the limiting component of the system. The BCIP,NBT
Phosphates Substrate System generates a dark purple stain on
membrane sites bearing phosphatose. Alkaline phosphatase
catalyze the dephosphorylation of 5-bromo-4-chloro-3 indolyl
phosphate which initiates a reaction cascade resulting in
intense color formation.
Binding of an antibody can be detected by a
variety of reagent systems as is the case for antigen bound
to the antibodies of the membrane. For instance, I-labelled
antimouse immunoglobulin or I-labelled protein A may be
used. Antimouse immunoglobulin conjugated directly to
alkaline phosphatase or to peroxidase may be used, together
with appropriate chromogenic substrates. The biotin-
avidin peroxidase system together with appropriate
~s;~ ~
chromogenic substrates. The biotin--avidin peroxidase system
(for example, the Vectastain AB~ system supplied by Vector
Laboratories) is particularly sensitive.
The solid phase membrane~ eliminate handling,
allow the product configuration to be cut in the desired
shape or format for placement on a base, and provides
faster kinetics and increased protein binding. Protein
binding to solid plastic substrates has been found to be a
non-stoichiometric process and varies greatly by the type of
plastic used. Binding is not specific and generally occurs
through electrostatic and hydrophobic interreactions between
plastic and proteins. Membrane substrates overcome many of
the problems inherent in solid phase immunoassays as they
combine the qualities of a solid substrate with a range of
expanded capabilities and, due to their porosity and
consequential large surface area, have a high protein
binding capacity. A protein binding capacity is increased
by using smaller pore sized membranes whose total binding
surface increases for an equivalent frontal surface.
Membranes which can be used in the present
invention in addition to the noted latex can be constructed
of nitrocellulose, nylon, cellulose or IAM produced by
Millipore Inc~ The choice of adsorbing matrix depends on
the physical properties such as sensitivity, binding
capacity, stability or bound molecules and compatibility
with the assay system.
The preferred membrane used is polystyrene latex
because the fingerprint color is retained. In adsorption of
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protein to polystyrene latex, the reaction tube is prepared
with 25 mg of washed latex, 1.0 mg/ml antibody and lOmM Tris
Buffer, pH 7.8 Q.S. to 2.0 ml total volume. The reaction
tube is incubated for 2 hours at 37C with rotation. The
tube is then (a) spun at 2,000-4,000 XG (depending on size
of latex) to remove unbound antibody and (b) the beads are
resuspended in 3.0 ml of deionized water and the latex is
washed three times repeating steps ta) and (b) above and the
washed latex is resuspended in 500 ul of PBS (pH 7.4~ plus
1.0% BSA plus 4% sucrose and .05% azide, and the sonicate is
bathed if necessary to disperse the latex. Membranes, such
aæ nylon and cellulose, can be modified to create surface
sites for covalent binding of proteins. Nitrocellulose is
one of the most commonly used membranes due to it's high
affinity for proteins and cellular macromolecules. In IAM,
polyvinylidenedifluoride (PVDF), the base polymer of IAM is
hydrophobic and binds proteins. IAM permits a high degree
of control over the extent of protein binding and the user
can reproducibly immobilize nanogram to microgram quantities
of protein on the surface to suit various assay
requirements. Binding the protein to IAM surfaces occurs
primarily though the epsilon amino group of lysine, which
contrasts the binding proteins to nitrocellulose, nylon or
plastic where the bonding is ionic or hydrophobic.
Another type of membrane which can be used in the
invention which has previously been noted is nitrocellulose
which provides an excellent matrix for blotting proteins and
nucleic acids. The nitrocellulose may be cut into whatever
17 ~ r3 r
shape is required and has the useful characteristic that the
amount of protein in a fingerprint will be clearly visible.
Pure nitrocellulose adsorbs proteins, nucleic acids and
other cellular antigens. These adsorbed substances often
retain antigen-antibody binding activity and can be
visualized using ultrasensitive, enzyme amplified
immuno~taining methods so that a chromogenic stain marks the
location of the adsorbed materials. This approach uses a
technique called Dot ELISA, (which also can be utilized with
the Nylon, IAM, plastic membranes) whereby nanogram amounts
of protein are directly applied to nitrocellulose. One
important advantage of Dot ELISA is the ability to perform
multiple enzyme immunoassays in a single test procedure
using as little as one microliter of antigen or capture
antibody solution. Nanogram amounts of capture antibodies
dotted onto a single membrane can be used to screen
simultaneously for a variety of antigens. In a Dot ELISA
procedure the reactant is diluted in coating solution and
dotted onto the damp membrane. While the optimal
concentration will vary from reactant to reactant, for
complex antigens 0.1 - 1.0 mg/ml is suitable. Following
membrane blotting excess binding sites are blocked by
thoroughly soaking both sides of the membrane in
Diluent/Blocking Solution. Any of a variety of reservoirs
can be used. The Diluent/Blocking Solution contains 1%
bovine serum albumin (BSA~ in phosphate buffered saline
which protects adsorbed protein from surface denaturation.
Following the blocking step, membranes can be stored dry at
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refrigeration temperatures for several months without loss
of activity. The adsorption of an antigen or capture
antibody onto the nitrocellulose membrane can be
accomplished by Antigen Detection ELISA, which is the
simplest method for detection of antigen, Indirect Antibody
ELISA which is capable o~ detecting either antibody or
antigen, depending on which is defined as the unknown or
Antibody Sandwich ELISA which is accomplished by adsorption
of an antigen or capture antibody, washing each reagent of
any free or unattached reactant and adding another reagent
to build step by step a molecular sandwich on the membrane
surface which is completed by the addition of an enzyme-
antibody conjugate. The construction of such membrane
surfaces is clearly shown by a bulletin of Kirkegaard &
Perry Laboratories, Inc. 1985 entitled ELISAmate (TM)
Enzymne Immunoassay Test System for Detection of Antigens or
Antibodies on Membranes which is incorporated in this
application by reference.
In operation of the test device, a swab 20 is
removed from its sterile pack 22 which may be attached to or
included with the test kit. The swab 20 is placed under the
tongue lOl of the test subject 100 for one minute. The swab
20 is placed in the incubation channel 31 and basin 32 which
forms the swab holder of the support member 30. Three drops
of solution 40 provided with a microsome gold label or
silver treated gold label are added to the saliva swab and
one minute is allowed for absorption and mixing. The index
finger llO or thumb of the test subject is pressed against
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the swab in its swab holder as shown in Figure 5 for 10
seconds and the finger or thumb is lifted off the swab and
immediately pressed and held on the test pad 36 for 30
seconds. The finger or thumb is lifted off the test pad and
after two minutes the result is read on membrane surface 50.
In the foregoing description, the invention has
been described with reference to a particular preferred
embodiment, although it is to be understood that specific
details shown are merely illu~trative and the invention may
be carried out in other ways without departing from the true
spirit and scope of the following claims:
