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Claims  |
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We claim:
1. Apparatus for the chemical analysis of an analyte comprising:
A. a first bibulous element containing a chemical reaction system that
includes all the reagents necessary to provide a liquid-transferable
chemical species within the first element upon the addition of an
analyte-containing liquid sample to the first element, the presence or
amount of which species is related to the presence or amount,
respectively, of analyte in the liquid sample;
B. a second bibulous element containing a detection system responsive to
the liquid-transferable chemical species to produce a perceptible signal;
and
C. support means carrying said bibulous elements in a normally spaced
relationship but enabling one or both of the bibulous elements to move in
a pre-determined path to bring the elements into liquid transferring
contact, thereby enabling any transferable chemical species to be
transferred to the second bibulous element and reacted with the detection
system to produce a perceptible signal.
2. The apparatus of claim 1 in which the support means supporting the
second bibulous element is sufficiently transparent as to enable the
perceptible signal to be perceived therethrough.
3. The apparatus of claim 1 wherein the support means supporting the first
bibulous element includes conduit means enabling a liquid sample to pass
therethrough into the first bibulous element.
4. The apparatus of claim 3 including a generally water-proof enclosure
having ports therein adjacent the support means and positioned to permit
the direct addition of a liquid sample to the first bibulous strip through
said conduit means.
5. The apparatus of claim 1 wherein said chemical reaction system comprises
a member of a ligand-receptor pair, of which the analyte is a pair member,
uniformly bound to the first bibulous element and a labeled
ligand-receptor pair member chosen to bind to the bound member of the
ligand-receptor pair or to analyte bound thereto in relation to the
quantity of analyte in said liquid sample and wherein the second bibulous
element includes a label detection system responsive to said label to
produce a detectable signal.
6. The apparatus of claim 1 wherein said chemical reaction system comprises
a member of the ligand-receptor pair, of which the analyte is a member,
uniformly bound to the first bibulous element and the liquid-transferable
chemical species is a labeled ligand-receptor pair member chosen to bind
to the bound member of the ligand-receptor pair or to analyte bound
thereto in relation to the quantity of analyte in said sample and the
second bibulous element includes a label detection system responsive to
said label to produce a detectable signal.
7. Apparatus for detecting an analyte in a liquid sample, comprising first
and second separate reaction zones, the first zone including a first
bibulous element and containing a chemical reaction system that includes
all the reagents necessary to provide a liquid-transferable chemical
species within the first bibulous element upon the addition of an
analyte-containing liquid sample to the first element, the presence or
amount of which species is related to the presence or amount,
respectively, of analyte in the liquid sample, and the second reaction
zone including a second bibulous element carrying a chemical reaction
system responsive to the transferable chemical species to produce a
visually perceptible signal; and support means carrying the reaction zones
and spacing the bibulous elements in adjacent, opposed, facing, aligned
orientation and enabling the elements to move into liquid-transferring
contact with one another.
8. The apparatus of claim 7 wherein the support means supporting the second
bibulous element is sufficiently transparent as to enable the visually
perceptible signal to be visually perceived therethrough.
9. The apparatus of claim 8 in which the support means includes conduit
means extending through its thickness and enabling a liquid sample to pass
therethrough into the first bibulous element.
10. The apparatus of claim 9 wherein the first zone includes a third
bibulous element carried by the support means in liquid-transferring
contact with the first bibulous element and positioned to be contacted by
the liquid sample passing through said conduit.
11. Apparatus for the chemical analysis of an analyte, comprising first and
second reaction zones, the first zone including a first bibulous element
and containing a chemical reaction system that includes all the reagents
necessary to provide a liquid-transferable chemical species within the
first bibulous element upon the addition of an analyte-containing liquid
sample to the first reaction zone, the presence or amount of which species
is related to the presence or amount, respectively, of analyte in the
liquid sample, and the second reaction zone including a second bibulous
element containing a chemical detection system responsive to the
transferable chemical species to produce a visually perceptible signal,
the apparatus including first and second support strips carrying the
respective first and second reaction zones, and connection means
connecting the first and second strips and orienting the same in spaced,
generally parallel planes with the bibulous elements carried in aligned,
facing, and spaced relationship to one another, the support strips and
connector means being so constructed and arranged as to enable the support
strips to be manually pinched towards one another to bring the respective
bibulous elements into liquid-transferring contact, the second support
strip being transparent to enable the visually perceptible signal to be
viewed therethrough.
12. A method for detecting an analyte that is a member of a ligand-receptor
pair contained in a liquid sample, the method employing;
(a) a first reaction zone comprising a bibulous element and having bound
thereto a member of the ligand-receptor pair;
(b) a labeled ligand-receptor pair member chosen to bind to the bound
member of the ligand-receptor pair or to analyte bound thereto in relation
to the quantity of analyte in the liquid sample, the label being part of a
signal-producing system;
(c) a second reaction zone comprising a bibulous element carrying a label
detection system responsive to said label to produce a detectable signal;
and
(d) support means supporting said reaction zones in a normally spaced
relationship but enabling one or both of the reaction zones to be moved in
a predetermined path to bring the bibulous elements thereof into
liquid-transferring contact;
said method comprising:
adding, to the first reaction zone, the liquid sample containing analyte
and the labeled ligand-receptor pair member, the amount of the latter
member remaining unbound and liquid-transferable in said first reaction
zone relating to the presence or quantity of analyte in said liquid
sample;
waiting a predetermined amount of time;
moving one or both of said reaction zones in said predetermined path to
bring said bibulous elements into liquid-transferring contact with one
another, permitting any unbound labeled ligand-receptor pair member to
transfer to the second reaction zone, the detection system in the latter
producing a detectable signal in response thereto; and
detecting said signal.
13. A method for detecting an analyte in a liquid sample comprising the
steps of:
(a) combining, in a first bibulous element a liquid sample and all the
reagents necessary to react with analyte in the sample to provide a
liquid-transferable chemical species within the first bibulous element,
the quantity of said liquid-transferable species provided being related to
the amount of analyte in the sample;
(b) after a predetermined amount of time moving the first bibulous element
along a predetermined path into liquid-transferring contact with a second
bibulous element containing a detection system responsive to the
liquid-transferable chemical species to produce a perceptible signal; and
(c) perceiving said signal.
14. The method of claim 13 further comprising moving a third bibulous
element containing a detection system responsive to the
liquid-transferable chemical species to produce a perceptible signal along
a predetermined path into liquid-transferring contact with the first
bibulous element of the first reaction zone to bring the first and third
bibulous elements into liquid-transferring contact independently of
bringing the first and second bibulous elements together and perceiving a
signal produced by the detection system of the third bibulous element
independently of perceiving the signal produced by the detection system of
the second bibulous element.
15. The method of claim 14 wherein said second elements are moved
sequentially into contact with the first bibulous element.
16. The method of claim 14 wherein said second bibulous elements are
simultaneously moved into contact with the first bibulous element.
17. The method of claim 14 in which said first bibulous element contains
adenosine triphosphate (ATP) and hexokinase which reacts with glucose to
provide a liquid-transferable species that is glucose or ATP, the
detection system of the second bibulous element comprises o-dianisidine,
glucose oxidase and horseradish peroxidase which together react with
glucose to produce a perceptible signal and the detection system of the
third bibulous element comprises Methylene Blue, glycerol kinase, glycerol
and glycerol phosphate dehydrogenase which together react with ATP to
produce a perceptible signal, the method further comprising the step of
adding to the first bibulous element a liquid sample suspected of
containing glucose. |
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Claims |
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Description |
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BACKGROUND OF THE INVENTION
A variety of accurate and reliable assays for measuring minute quantities
of analyte dissolved in a solution (e.g., hormones in biological fluids)
have been produced and described in the literature. Such assays have
commonly required, for their proper performance, a rather high degree of
technical and mechanical skill in the measurement of small amounts of
reagents, in following detailed procedures, and in using sophisticated
analytical equipment. There exists a need for a method of qualitatively
and desirably at least semiquantitatively detecting, in the field (that
is, outside the laboratory environment) the presence or absence of minute
quantities of materials on a rapid basis by persons who often may not be
technically skilled. For example, tests for various drug levels in human
biological fluids such as urine and blood serum desirably should be
available to and capable of use by law enforcement personnel or by
paramedics or other emergency medical personnel, inasmuch as it is often
of great diagnostic benefit to quickly determine the presence or absence
of particular drugs in the blood stream. A ready and effective assay is
needed for determining whether certain harmful substances are present in
food, such as penicillin in milk, marine toxins in seafood, etc. Needed
also are effective field tests for determining whether pollutants exceed
particular concentrations (e.g., salts of mercury in lake water).
The present invention provides apparatuses and methods which are unique in
that they can in large measure be readily and easily used or performed in
the field by minimally trained personnel.
SUMMARY OF THE INVENTION
The present invention provides an apparatus and method for the detection of
analytes, the invention making use of first and second normally separated
and spaced reaction zones, each comprising a bibulous element. Support
means are provided to carry the bibulous elements in a normally spaced
relationship, the support means providing means for moving one or both of
the bibulous elements in a predetermined path or paths so as to bring them
into liquid-transferring contact with one another. Prior to performing an
assay with the invention, the bibulous strips are desirably maintained dry
and are spaced from one another, as mentioned, by the support means.
In one embodiment, a liquid sample suspected of containing an analyte is
added to the apparatus and specifically to the first bibulous reaction
zone. Broadly speaking, the first reaction zone may contain a chemical
system that responds to the analyte, if any, in the liquid sample to
provide a liquid-transferrable chemical species, the presence or amount of
which is related to the presence or amount of the analyte. The apparatus
may have two, three or more zones. For example, a liquid analyte sample
added to a zone may entrain and carry with it another chemical species for
ultimate liquid transfer to the first bibulous element. As will now be
understood, once the bibulous elements are brought together into
liquid-transferring contact, the transferrable detectable chemical
species, if any, will be transferred to the second bibulous element,
resulting in the production of a detectable signal.
Although the apparatus and method of the invention are applicable, in a
broad sense, to a wide variety of analytes and chemical detection systems,
in its preferred embodiment, (and particularly when small amounts of an
analyte are to be detected), the invention makes use of ligand-receptor
pairs. The first reaction zone, in this embodiment, may have bound to it
one member ("pair member") of a ligand-receptor group comprising,
commonly, a pair or set of pairs. Added to the first reaction zone, in
addition to the analyte contained in a liquid sample, is a labeled pair
member chosen to bind to the first reaction zone in relation to the
quantity of analyte in the liquid sample which binds thereto, the label
being part of a signal-producing system. The bibulous element of the
second reaction zone may carry a label-detection system that is responsive
to the label to produce a detectable signal. The invention, in this
embodiment, is used by adding to the first reaction zone the liquid sample
and the labeled pair member. The presence or amount of the pair member
that remains unbound and hence remains liquid-transferrable in the first
reaction zone relates to the presence or quantity of analyte in the liquid
sample. One or both of the reaction zones are moved in said predetermined
path to bring the bibulous elements into liquid-transferring contact with
one another to permit any unbound labeled pair member to transfer to the
second reaction zone, the detection system in the latter responding by
producing a detectable signal.
DESCRIPTION OF THE DRAWING
FIG. 1 is a plan view of an apparatus of the invention;
FIG. 2 is a cross-sectional view taken along line 2--2 of FIG. 1;
FIG. 3 is a view of the apparatus shown in FIG. 1, folded upon itself;
FIG. 4 is a view of a modified apparatus of the invention;
FIG. 4A is a broken-away cross-sectional view taken along line 4A--4A of
FIG. 4;
FIGS. 5A, B, C and D schematically represent an apparatus of the invention
in sequential stages of its use;
FIGS. 6A, B and C schematically represent another apparatus of the
invention in various stages of its use;
FIG. 7 is a broken-away, perspective view of a portion of another
embodiment of an apparatus of the invention;
FIG. 8 is a perspective view of the completed apparatus of FIG. 7;
FIG. 9 is a cross-sectional view taken along line 9--9 of FIG. 8;
FIG. 10 is a perspective view similar to that of FIG. 8 but taken from the
opposite side;
FIG. 11 is a broken-away, cross-sectional view similar to that of FIG. 9
but showing a step in the use of the device shown therein;
FIG. 12A is a top diagramatic view of another embodiment of an apparatus of
the invention, suitable for dipping into a liquid sample;
FIG. 12B is a side view of the embodiment shown in FIG. 12A;
FIG. 13 is a perspective view of the device shown in FIG. 12 and depicting
one step in the analysis process;
FIG. 14 is a view similar to that of FIG. 13 but showing another step in
said process; and
FIG. 15 is a view similar to FIGS. 13 and 14 but showing said steps being
carried out simultaneously.
DESCRIPTION OF PREFERRED EMBODIMENTS
As used herein, "ligand-receptor binding pair" or "ligand-receptor pair"
refers to a pair of compounds of which one, a "receptor" is capable of
recognizing a particular spacial and polar organization of the other
("ligand") or portion thereof, and is capable of binding to that compound.
For various ligands, illustrative receptors forming the other half of a
ligand-receptor pair include antibodies, enzymes, lectins, Fab fragments,
and the like. Commonly, the receptor will be an antibody and the analyte
or analyte derivative will act as an antigen or hapten. As used herein,
"analyte derivative" means a chemical derivative of an analyte that
retains the capacity to bind to the other member of a ligand receptor pair
in competition with the analyte.
By "labeled pair member" or "labeled ligand-receptor pair member" is meant
a conjugate of one ligand-receptor pair member with a chemical label such
as an enzyme or other detectable chemical species, the conjugate retaining
the capacity to bind to the other member of the ligand-receptor pair and
the enzyme or other detectable label continuing to have the capacity of
being detected by a detector system (which may be a separate chemical
reaction system) to provide a perceptible signal. "Detector", "label
detector" and the like, refers to a chemical system that provides
perceptible signals, commonly electromagnetic radiation or absorption of
the same leading to perceptible fluorescence, color changes and the like,
when contacted with a specific enzyme or other label.
Speaking broadly, the apparatus and method of the invention may be used
with a large variety of known chemical analysis techniques that involve at
least two distinct reactions of which one, in a liquid medium, provides a
liquid-transferrable chemical species, the presence or amount of which is
related to the presence or amount of analyte in a liquid sample that is
added to the apparatus. A separate second reaction involves the detection
of the transferrable chemical species to produce a detectable signal.
The invention thus is useful for detecting a broad range of analytes that
can be suspended or dissolved in a liquid carrier. Such analytes include
inorganic elements and their compounds (usually salts), organic monomers
and polymers including macromolecules and assemblages thereof such as
subcellular organelles (chromosomes, nuclei, chloroplasts, cell
membranes), viruses, bacteria, fungi and other microorganisms. Excellent
lists of analytes which are part of specific immunological binding pairs
are set out in U.S. Pat. Nos. 4,374,925 and 3,817,837, the teachings of
which are incorporated herein by reference. Analytes of particular
interest include common drugs such as barbiturates and opiates, and
various toxins found in food, water and air including natural toxins
(microbial, plant, insect, reptillian, etc.) and synthetic (man-made)
toxins or poisons. Natural toxins include the marine toxins such as
saxitoxin and other paralytic shellfish toxins, ciguatoxin, brevetoxin,
palytoxin and the like. Other toxins include mycotoxins (for example,
trichothecenes, aflatoxins, patulin, ochratoxins and zearalonone).
Synthetic toxins include nerve agents such as Soman
(methylphosphonofluoridic acid, 1,2,2-tri-methylpropyl ester) and Sarin
(methylphosphonofluoridic acid, 1-methyl-ethyl ester) and pesticides
(e.g., Paraoxon (phosphoric acid diethyl-4-nitrophenyl ester), Furadan,
2,3-dihydro-2,2-dimethyl-7-benzo-furanol methylcarbamate, C.sub.12
H.sub.15 NO.sub.3, a trademarked product of FMC Corporation, and
Malathion, [(Dimethoxyphosphinothioyl)thio]butanedioic acid diethyl ester,
C.sub.10 H.sub.19 O.sub.6 PS.sub.2 (a product of American Cyanamid).
In the examples that follow, it will be noted that the analytical reactions
by and large fall into two broad groups of which one, applicable primarily
to inorganic analytes, involves stoichiometric determinations and the
other, applicable primarily to organic materials, involves the use of
ligand-receptor binding pairs. In the field of stoichiometric inorganic
determinations, the invention is particularly applicable to detecting the
presence of an analyte in a concentration above a predetermined
concentration. For example, it may be desirable to determine whether an
analyte that is a pollutant is present in a concentration above a specific
upper concentration that may have been set by a regulatory agency.
Inorganic stoichiometric determinations are particularly suitable for
analyses that involve volumetric precipitation methods, and
complexiometric methods including chelatometric techniques such as may be
used for metal ion determinations. In general, such analyses provide for a
reaction of the analyte in the sample with a stoichiometrically
predetermined amount of reagent to provide a liquid-transferrable species,
the presence or amount of which is related to the presence or amount of
analyte in the sample.
As to the preferred embodiment of the invention which employs
ligand-receptor pairs, reference is made to U.S. Pat. No. 4,391,904 which
sets forth a variety of binding pairs, the teaching of which patent is
hereby incorporated by reference. It will be understood that the invention
in its broader aspect is not dependent upon the selection of any
particular chemical reaction system, but, being in the nature of apparatus
and method, is applicable to a variety of such systems.
The "bibulous elements" that are employed herein may be made of filter
paper or other fibrous, particulate or porous material that has the
capacity to absorb and be wetted by the liquid of the analyte-containing
sample. The bibulous elements provide spatially defined and contained
reaction zones within which reactions may occur in a liquid environment.
The elements hence should not be soluble in the liquid (normally aqueous
solutions) containing the sample. Although the elements may swell
somewhat, they should be capable of generally holding their shape against
substantial deformation even when saturated with liquid. The bibulous
elements need not be flexible or compressible. It is desired, however,
that at least the first bibulous element be compressible so that, when it
is brought into contact with and pressed against the second element, it
will tend to decrease in volume and hence liberate liquid which can then
be more readily transferred into the second bibulous element. The first
bibulous element in the preferred embodiment desirably includes or is made
of a reactive material to which a ligand-receptor pair member can be
bound. To promote storageability of apparatuses of the invention, the
bibulous elements desirably are not reactive in the dry state with any of
the reactants that they contain. The second bibulous element desirably is
white or is at least light in color so that color changes or other
visually perceptible signals can be readily observed. The bibulous
elements may take the form of small discs of filter paper, although
fabric, glass wool, polyurethane foams and other materials that can absorb
at least small quantities of liquid may be used. The first and second
bibulous elements must have exposed or open faces through which liquid may
pass when these elements are brought into liquid-transferring contact.
Similarly, the support means which supports the bibulous elements in a
normally spaced relationship and which permits them to move in a
predetermined path into liquid-transferring contact with one another
desirably is of a material which does not react with either the bibulous
elements nor the reactants carried by these elements. Preferably, the
support means comprises one or more strips or other appropriate shapes of
a polymeric material such as polyethylene or polypropylene, the surfaces
of which are generally hydrophobic and are not easily wetted with aqueous
solutions. Thus, when a support of the type described carries several
bibulous elements, each of which may become saturated with a liquid
sample, the tendency of liquid to transfer across the surface of the
support from one bibulous element to another is reduced.
The size of apparatuses of the present invention may vary as desired.
Commonly, however, the bibulous elements may be discs having diameters of,
for example, about 6 mm, and the supports that carry bibulous elements
desirably are sized to be held in the hand. Although the bibulous elements
carried by the support means may be moved into contact with one another
through the use of a mechanical device such as a pair of opposed rollers,
in its simpler form the bibulous elements are supported by the support
means in such a fashion as to permit them to be moved, in the
predetermined path, by finger pressure, the elements being gently
"pinched" together to cause liquid transfer therebetween. As will be
evident from the description that follows, the predetermined path followed
by one or both bibulous elements desirably is arcuate or straight. In one
embodiment, the first and second bibulous strips may be so oriented as to
be brought into liquid-transferring contact when one or both of the
elements swell upon the addition of liquid thereto.
With reference to the drawing, a simple apparatus of the invention is shown
in FIGS. 1-3 and is designated (10). A plastic strip (12), typifying
support means, is provided with a groove intermediate its ends forming a
crease line (14) upon which the strip can be folded upon itself as shown
in FIG. 3. To the upper surface of the strip at one side of the crease
line (14) are adhered (by means of adhesive tape having adhesive on both
surfaces) first bibulous elements (16), typified as filter paper discs.
Similarly adhered to the upper surface of the plastic strip at the other
side of the crease line (14) are second bibulous elements (18) typified by
filter paper discs. The bibulous elements (16) and (18) are so spaced from
the crease line (14) and are so arranged that when the plastic strip is
folded upon itself as shown in FIG. 3, the bibulous elements traverse
predetermined paths (arcuate, in this example) and become aligned with one
another as shown in FIG. 3. Further movement (pinching together) of the
elements will bring them into contact with one another, whereupon liquid
in the first bibulous elements may be transferred to the second bibulous
elements. It will be understood that the apparatus shown in FIGS. 1-3 can
be stored in its unfolded condition within a suitable, desirably
moisture-proof, envelope of protective material.
In the simple embodiment shown in FIGS. 1-3, a typical test may be made for
the presence of NaCl in an aqueous solution such as perspiration.
The detection of NaCl present in body fluids such as perspiration in excess
of normal levels is important in the diagnosis of cystic fibrosis. The
following example illustrates how the embodiment of FIGS. 1-3 may be
employed to determine the amount of NaCl in a given volume of human
perspiration. This example also typifies use of the method and device of
the invention with inorganic materials using stoichiometrically controlled
amounts of reactants, and makes use of standard chemical techniques (the
Volhard determination) for determining the presence of chloride ion.
To bibulous elements A, B and C in FIG. 1 are added small quantities of
Iron III ammonium sulfate and a predetermined concentration of silver
nitrate, except that to bibulous element B is added silver nitrate in
substantial excess. Elements D, E and F are identical in that each
contains a predetermined concentration of KSCN plus buffer. In addition,
bibulous element C is provided with a quantity of sodium chloride in
excess of that contained in a given sample volume of normal perspiration.
The test is conducted as follows. Small but predetermined sample volumes of
human perspiration containing NaCl are added to each of bibulous elements
A, B and C. Chloride ion in the perspiration reacts stoichiometrically
with the silver ion, yielding AgCl as an insoluble precipitate; all of the
chloride ion in element B, of course, is precipitated due to the excess of
silver nitrate in that element. The volume of perspiration added to each
of the bibulous elements is enough to thoroughly wet the element and
preferably to substantially saturate it.
The apparatus is then folded upon itself as shown in FIG. 3, and the
opposing bibulous elements are pinched together to, permit fluid flow
between them. As a result (with reference to bibulous elements A and F),
fluid transfer causes the ferric ammonium sulfate indicator and any
unreacted silver ion to transfer to bibulous element F wherein the silver
ion reacts stoichiometrically with the KSCN to yield AgSCN. Any remaining
SCN ion reacts with the ferric ion, forming the red Fe(SCN).sup.2+. By
adjusting the silver nitrate concentrations such that the normal
physiological concentration of chloride ion in perspiration precipitates a
given amount of silver nitrate yielding a known concentration of free
silver ion, and adjusting the KSCN concentration to consume this excess
silver ion, the test is adjusted so that the sample containing a normal
level of sodium chloride will provide no colored response, whereas the
sample with an elevated concentration of sodium chloride will provide a
dark red response. Concurrently, the previous addition of NaCl to the
bibulous element C provides a red signal in the bibulous element D
regardless of the amount of sodium chloride in the patient's perspiration,
indicating that the test is operable for large quantities of sodium
chloride. Because of the large excess of silver nitrate in element B, no
color can form in corresponding element E. Thus, the formation of red
signal in element D and of no color in element E provide an indication
that the apparatus is operable.
It would be desirable under some circumstances, e.g., involving drug
overdoses, to determine which of several common drugs or drug types have
been used. For example, one may wish to detect the presence, in urine or
blood serum, of barbiturates (e.g., phenobarbital), opiates (e.g.,
heroin), or tricyclic antidepressants (e.g., nortriptyline). A suitable
apparatus for this purpose is depicted in FIGS. 4 and 4A, the apparatus
utilizing, as in the previously described apparatus, support means in the
form of a plastic strip (12) having a crease line (14) intermediate to its
ends to permit the strip to be folded upon itself.
As shown, bibulous elements are carried by the plastic strip, the first
bibulous elements being designated generally (16) and the second bibulous
elements (18). For ease of description, each bibulous element can further
be identified by a letter (designating its vertical column) and a number
(designating its horizontal row). The bibulous elements (18) are adhered,
in the manner described above, to the plastic strip. The bibulous elements
(16), however, are carried in apertures (20) formed in the plastic strip
(FIG. 4A). Additional discs (22) of bibulous material are carried by the
apertures (20) adjacent the outer surface of the plastic strip and in
liquid-transferring contact with the elements 16. In this manner, urine or
other liquid suspected of containing a particular drug may be added to the
bibulous elements (16) from the outer surface (24) (FIG. 4A) of the
plastic strip, the liquid passing through the bibulous element (22) and
carrying reactants stored in that element into the bibulous element (16).
The bibulous elements in each of rows 1, 2 and 3 are to be used for
detecting the drugs phenobarbital, heroin or a heroin derivative, and
nortriptyline, respectively. The elements (16) (18), and (22) are prepared
by separately impregnating them with aqueous solutions of reactants and
then freeze-drying the elements. One surface of each of the elements (18)
is then adhered to the strip (12) as described above, and the elements
(16) and (22) may be adhered to the plastic strip by means of small pieces
of polyester tape bearing adhesive on both sides. The elements (16) and
(22) are carried in liquid-transferring contact with one another, as shown
in FIG. 4A.
The bibulous elements (18) each were prepared as follows: Approximately 6.3
mm discs of Whatman brand #17 chromatography paper were dried at
105.degree. C. for an hour, cooled in a desiccator, and then transferred
to a large tube. To the tube was added one gram of 1,1-carbonyldiimidazole
("CDI") and 35 ml. of dry dioxane, and the discs were rocked for 45
minutes at room temperature. The discs thereafter were washed several
times with cold deionized water and again several times with cold 0.1M
borate solution, pH 9.0. A solution of 35 ml of the borate solution
containing 10 mg of Horseradish Peroxidase was added to the reaction tube
and rocked in contact with the discs for 20 hours at 4.degree. C. The
discs then were washed several times with cold phosphate-buffered saline
solution ("PBS"), and were then stored in PBS at 4.degree. C. A solution
of 10% B-D-Glucose, 0.5% polyethylene glycol ("PEG") 4000, and one mg/ml
2,2-azinodi-(3-ethylbenzthiazoline sulphonic acid) was prepared, and 20
microliters of the solution was pipetted onto each of the discs prepared
above. The discs were then frozen at -70.degree. C. and lyophilized.
With respect to row 1 (testing for phenobarbital) of the bibulous elements
(16), element 1A was prepared as follows: Approximately 6.3 millimeter
diameter discs of Whatman #17 chromatography paper were activated by
reacting them with CDI as described above, followed by washing with cold
borate solution as also described. 1.0 ml. of the anti-phenobarbital IgG
fraction of rabbit serum was added to 100 of the thus prepared discs in a
35 ml. 0.1M borate (pH 9.0) solution and reacted for 20 hours at 4.degree.
C. The discs were then washed several times with cold PBS and were stored
in PBS at 4.degree. C. The 100 discs were blotted lightly and transferred
to a lyophilization flask to which was added 2 ml of a 4 mg/ml albumin in
0.5% PEG 4000/PBS solution. The discs were frozen at -70.degree. C. and
lyophilized.
Bibulous element 1B was identically prepared, except that 5 micrograms of
sodium phenobarbital was added directly to element 1B just prior to the
lyophilization step.
The elements (22) used in connection | | |
