Analytical test devices for competition assay for drugs of non-protein antigens using immunochromatographic techniques

An analytical test device for competition assay for particular non-protein antigens, such as antigens representing drugs of abuse, is disclosed. The analytical test device is a test kit housing having an opening for introduction of a body fluid sample and a flow path for the body fluid sample. A supply of microscopic colored latex particles is adjacent to the opening along the flow path. A chromatographic membrane support is within the test kit housing for exposing the colored latex particles to the body fluid sample. When non-protein antigens are not present in the body fluid specimen, the colored latex particles accumulate at a predetermined site on the chromatographic membrane by complexing of antibodies on the colored latex particles to a drug conjugate probe on the membrane support to leave a visually perceptible colored mark of the same color as the colored latex particles. When non-protein antigens are present in the body fluid specimen, complexing of the non-protein antigens to the supply of antibodies on the colored latex particles exhausts the antibody supply on the colored latex particles such that the colored latex particles cannot complex to the immobilized drug conjugate probe, leaving no visually perceptible colored mark at the predetermined site. Also disclosed is a process for analytical testing of a body fluid for the presence of non-protein antigens, and an analytical test assembly for simultaneous multiple competition assays.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The invention relates to analytical test devices which use 
immunochromatographic assays formulated on a competitive immunochemical 
protocol, and which determine small hapten, non-protein molecules such as 
those representing the presence of drugs of abuse. More particularly, the 
invention relates to self-contained analytical devices which require only 
the addition of a few drops of body fluid such as urine or other liquid to 
initiate a complex, multi-step immunoassay that produces a visually 
perceptible precipitin via antigen/antibody reactions, and which do not 
require instrumentation or sophisticated training to assess the results. 
The invention further relates to housing articles useful for packaging 
kits for these immunoassays, to novel reagents, and to methods for 
utilizing the novel reagents in the test devices which simultaneously can 
assay in a single device for up to five (5) of the National Institute of 
Drug Abuse (NIDA) designated drugs of abuse recommended in a drug screen. 
2. Prior Art 
The measurement of physiologically important substances in urine, serum and 
tissue with immunological principles is an important recent development in 
immunology (V. P. Butler, Pharmocol. Reviews (1978), 29, 103-184). In 
particular, drug-specific antibodies and antigens have been used in the 
development of a variety of immunological assay procedures for the 
detection and/or quantification of antibodies or antigens in the bodily 
fluids of humans and animals. Immunoassay methods have distinct advantages 
when compared, for example, with techniques using thin layer 
chromatography, gas chromatography, gas chromatography/mass spectroscopy 
or high performance liquid chromatography because of the high specific 
accuracy associated with immunoassays and their ease of use. 
Most immunological testing procedures are focused upon the antigen-antibody 
reaction or competition which provides an end point with an insoluble or 
agglutinated precipitate (E. A. Kabat, Methods in Enzymology, Vol. 70 
(1980), Academic Press, New York, pp. 3-49). Existing non-isotopic 
immunoassay test kits are based on enzyme-linked immunosorbent assay 
(ELISA), homogeneous enzyme multipled immunoassay techniques (EMIT), latex 
agglutination assays and fluorescent polarization immunoassay. More 
recently, membrane-based particle immunoassays have been reported for 
human pregnancy tests which measure the level of human chorionic 
gonadotropin (HCG) (UK Patent Application, GB 2,204,398A, Apr. 26, 1988). 
These assay procedures generally have the disadvantage of requiring 
multi-step protocols, multiple reagents, reagent storage problems, and the 
need of special designed instrumentation or specially trained personnel to 
assess and record the test results. Specific immunoassays using 
reagent-impregnated test strips and latex particles have been reported in 
U.S. Pat. Nos. 3,857,931; 4,094,647; 4,690,907; and 4,740,468; G.B. Patent 
1,589,234; European patents 0,225,054; 0,138,442; and 0,136,799; and in 
G.B. Patent Application 2,204,398A (Unilever). 
There is a need for a test and/or device which is simple to use and which 
can be used anywhere rather than only in a laboratory setting, which via 
competition of the analyte, or drug, and the analyte conjugate for limited 
antibody binding sites, can identify presence or absence of drugs of abuse 
in humans, and which does not require instrumentation to read the end 
results. The test devices of this invention will detect drugs of abuse, 
for example, in a stable immunoassay configuration using certain novel 
protein conjugates of these drugs of abuse, and the accompanying 
antibodies. There is also a need for a self-contained analytical testing 
device which can simultaneously perform assays for multiple drugs of abuse 
in a single device. 
SUMMARY OF THE INVENTION 
It is an object of the invention to provide a test device to identify the 
presence of small hapten, non-protein antigens such as drugs of abuse. 
It is another object of the invention to provide a self-contained 
analytical test device which requires only the addition of a few drops of 
test liquid to initiate an immunoassay which proceeds through a plurality 
of steps by inherent characteristics of the test device. 
It is a further object of the invention to provide an analytical test 
device which does not require instrumentation or sophisticated personnel 
to obtain and read the results. 
It is still another object of the invention to provide an easily 
ascertainable visual identification of the drugs and/or metabolites of 
drugs of abuse in urine processed using a simple test kit. 
It is still another object of the invention to provide an analytical test 
device which can simultaneously perform assays for multiple drugs of 
abuse. 
These and other objects are accomplished by a test device which uses 
various immunochemical based configurations embodied in an 
immunochromatographic system. The test devices use small antigen 
conjugates, have precipitin end points with hapten detection capabilities, 
contain chromatographic supports made of impregnated membranes, and offer 
accuracy as well as ease of use in non-laboratory settings. The test 
devices require only the addition of a few drops of urine, biological 
fluids or aqueous solutions. At least five drugs of abuse can be tested 
simultaneously in a single device. The five drugs of abuse recommended by 
the NIDA for a drug screen, namely amphetamines/methamphetamines, cocaine, 
opiates, phencyclidine, and cannabinoids, are thus readily detectable 
simultaneously using a simple test kit.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention improves and adopts certain known immunological 
techniques involving laboratory procedures (A. J. Weiss and L. A. 
Blankstein, Amer. Clin. Prod. Review (1987), 6, 8-19), including those 
reported in the above publications, to obtain analytical test devices 
applicable for the assay of non-protein antigens such as drugs of abuse. 
The test devices are quick and convenient to use by non-sophisticated 
personnel in non-laboratory settings, and perform assays for up to five 
drugs of abuse simultaneously. The multiple competition assays are housed 
in a single device and result in easily discernable visual end points that 
do not require instrumentation to interpret. 
The test devices contain all of the components for a specific 
immunoreaction, and provide an appropriate visual end point which detects 
or identifies the presence or absence of drugs of abuse in the test 
sample. 
The test devices of the invention comprise: 
a test kit housing having means for introduction of a body fluid sample and 
means defining a flow path for the body fluid sample; 
a supply of microscopic colored latex particles disposed adjacent to the 
means for introduction of the body fluid sample along the flow path, the 
colored latex particles becoming suspended in the body fluid sample and 
moving with flow of the body fluid sample along the flow path, the latex 
particles being sensitized with a supply of antibodies for the non-protein 
antigen at least on a surface thereof, said antibodies being responsive to 
said non-protein antigens and being operable to complex therewith; 
a chromatographic membrane support disposed within the test kit housing and 
being impregnated at a predetermined site along the flowpath downstream of 
the colored latex particles with an immobilized drug conjugate probe 
sensitive to said antibodies of the latex particles, and operable to 
complex therewith; and, 
means for exposing the colored latex particles to the body fluid sample for 
substantially complete reaction of the non-protein antigens in the body 
fluid sample with the antibodies for the non-protein antigen on the latex 
particles, prior to the body fluid sample reaching the predetermined site 
along the flow path; 
whereby when said non-protein antigens are not present in the body fluid 
sample the latex particles accumulate at the predetermined site by 
complexing of the antibodies on the latex particles with the drug 
conjugate probe on the membrane support to leave a visually perceptible 
colored mark, and when the non-protein antigens are present in the body 
fluid sample, complexing of the non-protein antigens to the supply of 
antibodies on the latex particles substantially exhausts the antibody 
supply on the latex particles such that the latex particles cannot complex 
to the immobilized drug conjugate probe, leaving no visually perceptible 
mark at the predetermined site. 
The elongated moisture impervious housing provides an opening for the 
introduction of body fluid samples to the test device. The opening in the 
moisture impervious housing permits the introduction of small quantities 
of human body fluids into the housing. An absorbent well containing an 
absorbent pad is in communicating relationship with the housing opening. 
The porous chromatographic membrane support located within the housing may 
be a nitrocellouse membrane such as that available from Schleicher & 
Schuell, Inc. or a nylon membrane such as that available from Amicon under 
the AUTOBLOC trademark. Such porous membranes have the natural ability to 
bind proteins, and immunoreagents can be applied directly to the membranes 
and immobilized thereon. The membranes are available in a broad range of 
pore sizes which provides a range of carrier materials which can be 
selected for test devices for particular drugs of abuse. Generally, the 
membranes have from about 1.0 to about 12.0 micron pore sizes, preferably 
from about 5 to 12 micron pore sizes. 
The porous chromatographic membrane support is impregnated with a specific 
antigen or probe such as a drug conjugate probe. For example, antigens of 
derivatives thereof for any of the five drugs of abuse recognized by the 
NIDA may be impregnated on the membrane. A benzoylecgonine derivative is 
the antigen which is impregnated to identify the presence or absence of 
cocaine in the tested body fluid sample. A carboxy methylmorphine 
derivative conjugate with bovine serum albumin is the antigen to detect 
the presence or absence of opiates such as morphine. A methamphetamine 
conjugate is used as the antigen to test for the presence or absence of 
amphetamines and methamphetamines, and a phencyclidine conjugate or a 
tetrahydrocannabinoid conjugate are respectively impregnated to determine 
the presence or absence of phencyclidine or cannabinoids in body fluids. 
The volume of drug conjugate probe immobilized on the membrane ranges from 
about 0.1 to about 10 microliters per centimeter. 
A second immobilized immuno reaction probe is applied to the membrane 
downstream of the drug conjugate probe. The second immuno reaction probe 
is a protein antigen and the second probe serves as a test or indicator 
that the analytical test device is operative. Any available protein 
antigen can be selected for the second immuno reaction probe. The protein 
is applied or impregnated on the membrane at a concentration of from about 
0.1 to about 10 milligrams per milliliter. 
The third component impregnated on the chromatographic membrane support is 
an area of colored microscopic latex spheres. The latex spheres are 
sensitized or coated with detecting antibodies for the non-protein antigen 
or drug of abuse or their metabolites, and an antibody for the second or 
protein probe. The latex spheres may be from about 0.1 to about 1 micron 
in diameter. The latex spheres may be colored in any desirable color such 
as red, blue or green, blue being the preferred color. The latex spheres 
are applied to the membrane in an area from about 1 to about 10 
millimeters in width. The latex spheres are applied to the membrane 
upstream of the immobilized drug conjugate probe, and in close proximity 
to the opening of the housing and the absorbent pad found therein. The 
latex spheres are immobile when applied to the membrane although, in 
contrast with the drug conjugate probe and the immuno reaction probe, the 
latex spheres become mobile when in contact with moisture. 
The latex spheres are coated with a detecting antibody for the non-protein 
antigen. The detecting antibody will vary depending upon the drug of abuse 
which is detected in the housing. The latex spheres will also be coated 
with an antibody for the immuno reactive antigen and probe downstream of 
the drug conjugate probe. The latex spheres preferably are bathed with a 
protein agent to coat the entire circumference of the sphere to facilitate 
movement of the spheres as they traverse the chromatographic membrane. 
To use the analytical test device to detect the presence of or absence of 
non-protein antigens such as drugs of abuse in body fluids, a body fluid 
sample, such as a sample of urine is introduced to the opening within the 
moisture impervious housing. The body fluid sample is absorbed on the pad 
beneath the opening and capillary action results in contact between the 
body fluid sample and the colored latex spheres. The urine or body fluid 
combines with the spheres after a short time of incubation, perhaps three 
to five minutes, for example. In the event that the body fluid sample 
being tested contains a drug of abuse or its metabolite, it will 
immediately bind with or complex with the antibody present on the latex 
sphere. Should this occur, there will be no free detecting antibody 
present on the latex spheres as they migrate or traverse the membrane and 
reach the immobilized drug conjugate probe. This will prevent the 
attachment of the colored latex spheres to the site of the drug conjugate 
probe on the membrane, a fact which will be indicated by the absence of a 
colored line in the area of the drug conjugate probe. Therefore, a 
positive test urine sample will be demonstrated or indicated by the 
absence of the colored line or indication at the area of the drug 
conjugate probe. 
The latex spheres will continue their migration and traversal of the 
membrane where the protein antibody on the latex spheres will complex with 
the protein antigen immobilized on the second probe. The result of this 
complex will be the appearance of a colored line on the probe, a fact 
which demonstrates the viability of the test device. This means that a 
negative test urine sample will produce two colored lines on the membrane, 
whereas, as stated above, a positive test urine sample will produce only 
one line. 
No special reagents or conditions are required for completion of the 
analytical test. Moreover, there is no need for sophisticated personnel to 
complete the test. 
The invention comprises several designs for the devices and analytical 
procedures. FIGS. 1a and 1b show an analytical test device 100 which has 
two pieces of plastic that are welded to fabricate a plastic housing of a 
test device. The plastic used for these devices may be clear or opaque, 
for example of a polymer or copolymer material as known to those skilled 
in the art of mold or cavity casting. Within this device is a porous 
chromatographic membrane 102 which is impregnated with immunochemical 
agents and has an absorbent pad placed at slot 103. A reception cavity 107 
communicates with the membrane through a passage channel 108. An aperture 
for an air equalization outlet is located at 104. When an outside sleeve 
105 is displaced by gripping along a serrated side 106, a view window area 
is revealed, and this action can be arranged to create an air displacement 
or vacuum by expanding a compartment downstream from a sample well, which 
thereby draws or pulls the aqueous mixture containing the analyte away 
from the reception cavity and into a path leading toward the predetermined 
site of the antigen at which a visual mark may be produced. This action 
initiates movement of the liquid through the passage channel 108 and onto 
the porous membrane 102. The passage channel 108 preferably has a coarse 
surface to effectively prevent the free flow of the sample and reagent 
mixture toward the self-contained membrane until such vacuum pulling 
action is initiated to commence flow. 
When the aqueous mixture is drawn into the passage channel 108, the passage 
channel assists in at least one of mixing and incubation of the analyte 
solution and immunochemical components of the test kit, contained in the 
well 107 or in the channel, in the kit as provided. This device allows for 
the deposition of the reagents onto the membrane. The reagents may include 
for example, antibody-coated colored particles, which can be freeze dried 
in a carrier buffer comprising protein stabilizers. 
Sample urine solutions, for example, are added dropwise to the reception 
cavity 107 to suspend the particles and solubilize the other reagents in 
the well. After sufficient time for mixing and incubation (3 to 5 minutes, 
for example), the aqueous mixture is allowed to enter the passage channel 
and come in contact, via capillary action, with the carrier membrane after 
the movement of the sleeve 105 is completed. The stop catch 109 allows for 
the sleeve to move the appropriate length, whereupon the sleeve is 
stopped. The colored particles pass with flow assisted by capillary action 
along the membrane. When the colored particles complex, they are 
immobilized on the membrane through specific antibody/antigen reactions 
with antibodies or antigens provided on the membrane at predetermined 
location. This produces a precipitin color end point defining for example 
a line, specific design or symbol which is visible at said location. 
In the competition assay technique, when the test sample is positive for a 
particular analyte or hapten, such as, for example, a drug of abuse or a 
metabolite of a drug of abuse, the antibody which might be absorbed on a 
latex particle on the membrane, for example, this specific test analyte or 
hapten, is blocked due to previous absorption of the analyte in the test 
sample. This prevents the formation of a precipitin end point. 
A reference immuno reaction probe or communicant can be added to the 
chromatographic support, flowing to a point downstream of the 
predetermined location. The appearance of a color end point then informs 
the user of the completion of an analytical test run as well as indicating 
the stability or viability of the test components. This reference probe or 
communicant line can be constructed, for example, with a second 
antibody/antigen reaction or with other configurations such as, for 
example, an avidin/biotin interaction. 
FIGS. 2a-2m illustrate another embodiment of a test device according to the 
invention. A top piece of the housing 201, reception cavity 203, reception 
cavity opening 204, air outlet 202, bottom half of the device 205, frame 
for receiving the chromatographic support membrane, and absorbent pad 
location 207 are included. Test sample liquid is delivered into the 
reception cavity opening 204 of the device 200. The sample well may or may 
not have an absorbent pad therein. Additionally or alternatively the 
device can define dish-like sample well 208, as shown in FIG. 2e, which 
facilitates sample capillary migration. As shown in FIG. 2f, the sample 
well is inclined such that it slopes into the membrane chamber. When the 
device 200 is fully assembled, the top and bottom pieces are welded along 
a junction line, and a removable tape covers the air outlet 202. This 
configuration produces a partially closed system that prevents premature 
migration of the aqueous solution of the sample and the mobile colored 
particles, down the membrane to the predetermined site of the fixed 
antigens/antibodies before proper mixing, incubation and equilibration of 
the liquid carried components of the test is achieved. After a sample or 
sample reagent are introduced into the device, sufficient time and/or 
mixing is permitted to ensure that tests are accurate. The migration can 
then be initiated by lifting the tape at the air outlet to create a 
solvent flow down the membrane. As the liquid sample migrates through the 
porous support, the colored particles form an indicator visual end point 
from the specific antigen/antibody reaction. The presence of free analyte 
in the sample solution competes with the immobilized binding sites of the 
antibody or antigen specific for the analyte, and the formation of 
precipitin end point is inhibited. 
FIG. 3a depicts the holder design 300 for use with two plastic housings, 
such as, for example, devices 100, 200, 408, 500 and 608. The two external 
wells 301 are located in the holder 300 adjacent to each test device. The 
two slots 302 hold two plastic housing test devices. The external well 301 
is used to deposit particles on which antibody or antigen are absorbed. 
When a liquid test sample is added to the well charged with the particles, 
the previously freeze dried particle mass is then suspended in the aqueous 
test sample. After a period of incubation, for example, 3 to 5 minutes, 
the test mixture is then transferred to the corresponding test device. 
Another version of the holder is illustrated in FIG. 3d. The holder 304 
has four slots in which four different test device units are contained, 
such as, for example, devices 100, 200, 408, 500 and 608. FIG. 3g shows a 
complete test device assembly having four test devices fitted in a holder. 
FIGS. 4a-4k show device 408 which is another version of a test device. The 
chromatographic membrane 409 is essentially suspended in a chamber 405 
between bottom piece 404 and top piece 400. Teeth 410 extended from the 
top and bottom pieces and hold the membrane between their opposing ends. 
When a liquid sample is deposited in the opening 401, the aqueous solution 
percolates through the absorbing or filter pad 402 onto and down the 
membrane pretreated with probes or communicant materials. An absorbing pad 
406 is in contact with the membrane or porous material to receive the test 
sample fluid after the liquid has traveled through the membrane. 
FIG. 5 represents a different configuration of a test device 500 in which 
an aqueous sample is first allowed to mix with the latex particles in the 
sample well 501 to facilitate equilibration, incubation and mixing of the 
imaunochemicals with the sample. The mixture is then introduced or 
channeled into the capillary passage 502 for movement via capillary action 
toward a membrane located at 503. During the migration of the test sample 
mixture down the capillary passage, the analytes in the sample or the 
particles containing antigen and/or antibody have time to mix, react and 
complex as a prelude to migrating through the porous material that 
contains the probes or communicants which give the precipitin end point as 
described for device 100. The absorbent pad is found at 504. The test 
device represented in FIGS. 6a-6c is a further extension of the device 
described in FIG. 5 except that the capillary passage 602 is extended to 
go completely around the membrane housed at 603 to allow for additional 
incubation of the reactants. The test device 608 contains an absorbing pad 
604 at the end of the chromatographic path. The top piece of the device 
605, sample well opening 606, bottom piece 600 and reception cavity 601 
are as marked. 
A further extension of the test devices and configurations allows more than 
one assay to be accomplished in a single housing unit such as 100, 200, 
408, 500 and 608. In this configuration, two or more analytes can be 
assayed on the same membrane by the addition of the antibodies/antigens 
specific for each test such that appropriate probe or communicant end 
points can be ascertained which are distinct for each analyte. This 
membrane has antigen conjugate probe lines corresponding to the specific 
antibodies/antigens that are absorbed on the latex particles. The 
housings, holders, test protocols and end points are as described 
previously for the assays. 
Another embodiment of the multiple test configurations is illustrated in 
FIGS. 7a-7c. Three test devices 705 are placed in a single holder 700 
which has bottom piece 701 and top piece 702. The test devices share a 
common reception cavity 703. A liquid test sample is introduced into the 
reception cavity through a central opening 704. Each test strip contains a 
specific analyte-conjugate probe and the corresponding antibody is coated 
on latex particles. These strips are placed in the plastic holder in a 
symmetrical pattern and are individually marked for each specific test. In 
this design, only a single test sample is applied to the common reception 
cavity for the multiple analyte testing. The aqueous sample permeates down 
to the different test strips which allows rehydration of deposited 
particles. The colored latex particles migrate only through the capillary 
channel defined by the porous membrane strip. Reference or communicant 
areas are separated from other analytes with an external reference added. 
Each chromatographic strip is cut in size to take from 40 microliters to 
100 microliters of aqueous sample. The test is usually completed within 
five to ten minutes. A minus sign or some other symbol placed in each view 
window area indicates qualitatively the results of the test. Absence of 
the minus sign, or reference symbol, is an indication that the sample is 
positive on the particular test. 
In another multiple test assembly represented by FIGS. 8a-8c, six test 
devices 805 are assembled in a single holder 800 for the simultaneous 
testing of, for example, benzoylecgonine, morphine, marijuana, 
amphetamine, and phencyclidine. The holder has bottom piece 801 and top 
piece 802. An opening 804 leads to a common reception cavity 803. The test 
devices and methods are as previously described. A negative result for a 
single drug or analyte in the multiple test is indicated by the presence 
of multiple precipitin lines or symbols for the negatives. A test that is 
positive for an analyte or drug will not show a precipitin end point, and 
can easily be distinguished from the negative results. 
The invention is described primarily with reference to a competition assay 
wherein any antigens in the sample (representing presence of a drug of 
abuse or the like) bind to the colored latex particles which have been 
treated with antibodies responsive to the antigen. The competition upon 
which this embodiment of the test is based is thus a competition for a 
limited number of antibody-bearing bonding sites, between latex bodies 
whose antibodies have been exhausted by presence of the antigen in the 
sample, and latex bodies whose antibodies have not been blocked by 
presence of the antigen and remain able to bind to antigen conjugates. It 
is also possible, and within the scope of the invention, to base the test 
on a competition between the antigens in the sample and functionally 
identical antigens or antigen conjugates fixed on the latex particles, for 
the limited number of binding sites of antibodies fixed on the membrane 
support. Inasmuch as the mobility of antigens in the body fluid sample is 
substantially greater than the mobility of antigen conjugates on the latex 
particles suspended in the body fluid sample, the antigens in the body 
fluid sample (representing the presence of drugs of abuse or the like) 
tend to find the binding sites on the membrane support before the latex 
particles reach the binding sites. As a result, a more direct competition 
between the relatively more mobile liquid-suspended antigens and the 
relatively less mobile latex-fixed antigens also provide distinct visually 
identifiable results depending on the presence or absence of the antigens 
in the body fluid sample. In the event the antigens are not present, the 
colored latex particles bearing the antigens eventually flow to and 
complex with the fixed antibodies at the binding sites, leaving a distinct 
color mark. In the event antigens are present in the body fluid sample, 
these antigens are free and relatively mobile, and bind to the fixed 
antibodies before the less-mobile latex particles arrive at the binding 
sites. When a latex particle eventually arrives at a given binding site, 
the binding site has already been exhausted by free antigens in the body 
fluid sample (which of course are not colored), and no colored mark 
results. 
The following examples are meant to illustrate the invention, and are only 
representative embodiments of the scope of the invention. 
The antigens which can be used in the method of this invention are related 
to the antigen/antibody conplexation reaction for each of the individual 
immunological assays. Antigens for the cocaine assay are exemplified by 
Formula I: 
##STR1## 
Where A=single bond, --CH.dbd.CH--, or CH.sub.2 ; 
B=NHCO, OCO, or --CH.dbd.CH; 
R.sup.1 =H, CH.sub.3, --CH.sub.2 CH.sub.3, NHCH.sub.2 COOH; 
R.sup.2 =--CO-- R.sup.3, --CO(CH.sub.2).sub.2 COR.sup.3 ; 
R.sup.3 =serum albumin (bovine, human); hemocyanin, ovalbumin, synthetic 
polypeptides, serum globulins. 
The antigens for the amphetamine/methamphetamine assay are exemplified by 
Formula II: 
##STR2## 
A=single bond, --COCH.sub.2 O--, --CO(CH.sub.2).sub.n --(CH.dbd.CH).sub.m 
-- 
R.sup.3 =same as defined in formula (I); 
n=0 to 3; 
m=0 or 1. 
The antigens for the morphine/opiate assay for conjugates of derivatives 
reported in Science (1972), 176, 1143-1144, for example. 
The antigens for the tetrahydrocannabinoids are conjugates of derivatives 
reported in FEBS Letters (1975), 55, 257-260, for example. 
The antigens for the phencyclidine assay are conjugates of derivatives 
reported in Res. Comm. Chem. Pathol. Pharmacol. (1979), 25, 547-557, for 
example. 
EXAMPLE 1 
Blue Colored latex particles (Bangs Laboratories, Inc., with diameter of 
from 0.075 microns, stock code P000750PR, up to a diameter of 0.899 
microns, #P0008990 CB) are washed with a mixed bed of ion exchange resins 
(Bio-Rad AG 501-X8, 20-50 mesh). A benzoylecgonine derivative was then 
covalently bound to the carboxylated latex by the method described (R. S. 
Molday, et al., J. Cell. Biol. (1975), 64 75-88). The antigen-coated latex 
beads were suspended in a carrier buffer containing glycerol (0.5%) and 
polyethylene glycol 6000 (2% wt/vol). This latex suspension was then 
applied to a porous membrane support (Schleicher & Schuell, Inc. #30270 
and #65790; for 8 and 12 micron pore size nitrocellulose membrane, 
respectively) that was already coated with an antibody specific for 
benzoylecgonine, at a concentration of from 0.5 or 4 milligrams per 
milliliter. On another site or section of the membrane, a second, 
different antibody was absorbed, that is an IgG molecule used to mark the 
latex particles, and which was applied at a concentration of from 0.2 to 8 
milligrams per milliliter. 
The volumes of each of the antibody solutions absorbed onto the membrane 
range from 0.1 to 10 microliters per centimeter. The membranes were then 
sliced into strips that are from 0.5 to centimeter wide to about 5 
centimeters long which are then assembled in housing device 200, for 
example. To run an assay with this device and configuration, four or five 
drops of urine are added to the sample well. When the pull tab is removed, 
the urine will travel down the membrane, to, first, mobilize and suspend 
the latex particles, which then flow over the two protein probe or 
communicant lines. If benzoylecgonine is not present in the urine sample, 
the probe containing the benzoylecgonine specific antibody will complex 
with the benzoylecgonine conjugate on the colored latex beads to produce a 
visual precipitin end point. As the latex continues to move down the 
membrane, the second marker protein will give a precipitin complex to 
demonstrate the viability of the entire test system. If benzoylecgonine is 
present in the urine sample, the free benzoylecgonine, a metabolite of 
cocaine, will compete for the antibody binding sites to prevent the 
benzoylecgonine bound to the latex from complexation at the antibody probe 
site on the membrane. Thus, for a sample that contain benzoylecgonine, the 
latex beads will pass over the antibody probe line and no colored 
precipitin or complex end point is observed. A negative test result shows 
up with two color precipitin end point lines. A positive test result shows 
only one line, for example, corresponding to the reference or control 
probe or communicant interaction. 
EXAMPLE 2 
A nylon membrane (Autobloc , Amicon) with a pore size of 5 to 10 microns 
was used as the solid phase support. A carboxymethyl morphine derivative 
(B. H. Wainer, et al., Science (1972), 176, 1143-1144), was conjugated to 
bovine serum albumin, and this protein-hapten conjugate at a molar ratio 
of 1:10 to 1:20 was absorbed onto the membrane at a concentration of 
between 0.2 and 10 milligrams per milliliter. The probe line was formed 
with about 0.1 to 10 microliters per centimeter of this conjugate solution 
being applied in a 1 millimeter width line. Anti bovine serum albumin 
antibody at an IgG concentration of 0.1 to 8 milligrams per milliliter was 
used to form a reference communicant line, for example, and was added at a 
rate of from 0.1 to 10 microliters per centimeter on the 0.5 to 1 
centimeter membrane. Colored latex particles are treated with an aqueous 
solution of antibody specific for morphine and were then applied to a 
sucrose foundation on the membrane at a site 2 to 3 centimeters away from 
the probe lines. After the loaded nylon membranes were completely dried, 
they were placed into device 408 with two absorbent pads on each end of 
the membrane, and the complete test device was sealed in a moisture 
barrier bag with a desiccant such as, for example, silica gel or molecular 
sieves. To assay for opiates, 4 or 5 drops of test urine sample are 
introduced into the reception cavity. After about 10 minutes, the latex 
spheres have completely traversed the membrane. The sample is read as 
positive or negative for opiates as described in Example 1. 
EXAMPLE 3 
Polyclonal antibody to amphetamines/methamphetamines were absorbed onto 
colored latex particles in the concentrations described in Examples 1 and 
2, and then deposited and dried in the sample well in device holder (300). 
The membrane was prepared by the addition of methamphetamine probe line 
made with a methamphetamine conjugate (L. T. Cheng, et al., FEBS Letters 
(1973), 36, 339-342) and the reference antibody as described in Example 2. 
The test for amphetamines/methamphetamine is performed by adding 5 drops 
of the urine test sample to the reception cavity containing the latex 
carriers. The suspended latex in the test urine is allowed to stand at 
ambient temperature for 5 minutes with or without occasional mixing or 
stirring. The suspension is then transferred to the membrane in device 200 
which is contained in holder 300. The end point is read as illustrated in 
Example 1. A thorough preincubation with urine that is positive for 
amphetamine/methamphetamine will preclude or prevent the antibody absorbed 
on the latex from binding to the amphetamines/methamphetamine probe line. 
The reference line or control is not subject to analyte/antibody 
complexation because the reference antibody and antigen are not present in 
the specimen to offer a competition. 
EXAMPLE 4 
The procedure of Example 3 is followed except that the colored latex 
particles are dried in the reception cavities of devices 408 or 608 and 
are kept dry in sealed bags. As in Example 3, the assembly in these 
devices includes a porous membrane of nitrocellulose, or nylon and an 
absorbent pad. Latex particles to which antibody to phencyclidine are 
absorbed and dried in the device 408 and 608 which also contained the 
membrane charged with phencyclidine conjugate (L. S. Rosenberg and H. V. 
Vunakis, Res. Comm. in Chem. Phathology and Pharmacology (1979), 25, 
547-557). The devices are equilibrated to ambient temperature before a 
test run if the devices had been refrigerated. About 5 drops of test urine 
are added to the reception cavity, the latex is brought into suspension by 
stirring and the incubation of latex with urine and/or analyte-containing 
urine is allowed to incubate for 5 to 10 minutes. Then the urine sample 
suspensions are introduced to the capillary channel with a dropper for 
migration to the membrane enclosed in devices 408 or 608 which are 
prepared by procedures described in Examples 1 and 2. If necessary, 
additional drops of urine are added to maintain a flow through the 
membrane to the pad. The precipitin end points for samples negative or 
positive for phencyclidine are observed as described in Example 1. 
EXAMPLE 5 
The procedure for the determination of marijuana metabolites in the urine 
is similar as described in Example 3 except that a tetrahydrocannabinoid 
conjugate (M. Cais., et al., FEBS Letters (1975) 55 257-260) is attached 
to the 10 micron pore size nylon membrane in the presence of a 
bifunctional linking agent such as, for example, glutataldehyde 0.01% to 
0.1%. The appropriate antibodies selected for this test, have an ELISA 
titre of over 1/4000 with an affinity constant greater than 
l.times.10.sup.9 M.sup.-1. Polyclonal antibody was first purified to the 
gamma globuline fraction with a DEAE ion exchange column, then dialysed 
against 50 mM phosphate buffer saline PH 7.4. The dialysate was then 
diluted in the same buffer to from 0.1 to 10 milligrams per milliliter. 
The antibody solution at these concentration ranges is employed to coat 
latex particles through a passive absorption procedure (C. F. Nathan and 
Z. A. Cohn, J. Exp. Med. (1981) 154, 1539-53). The latex is post blocked 
with two volumes of normal rabbit serum. Monoclonal antibody against 
11-nor-delta-9-tetrahydrocannabinoid-9-carboxylic acid is produced by 
immunizing animals with an immunogen conjugate tetrahydrocannabinoid 
keyhole limpet hemocyanin molecule. This antibody is purified through a 
protein-A affinity column, (Pierce Chemical Co. #21008). The 
immunoglobulin fraction is added at a concentration of from 0.1 to 10 
milligrams per milliliter. The optimum concentration for latex coating is 
determined by the actual test sensitivity and visibility of the precipitin 
end point. Devices 408 and 608 are used to determine cannabinoid and 
metabolites in the urine as described in Example 4. 
EXAMPLE 6 
A test for benzoylecgonine, the metabolite of cocaine, was configured in 
device 408 using from 5 to 12 micron pore size nitrocellulose membrane, 
and following the procedure described in Example 2. The colored latex 
particles were coated with antibody to benzoylecgonine through a double 
coating procedure. That is, anti-mouse IgG is coated first on the 
particles, then the mouse anti-benzoylecgonine is absorbed. A 
benzoylecgonine conjugate was absorbed on the membrane as the communicant 
or probe for the end point as the preceeding examples, a reference probe 
was added to the porous support. The assay was run exactly as described in 
Example 2. 
Other assays for opiates, amphetamines/methamphetamines, cannabinoids and 
phencylidine, for example, can be configured using the same formulations 
but using appropriate antibodies, antigens and antigen conjugates for each 
desired analyte test. 
EXAMPLE 7 
A dual test for benzoylecgonine and morphine on one porous membrane was 
accomplished in device 408 by using the procedure described in Example 2 
except that nitrocellulose (8 microns) was used in place of the nylon 
porous support. Colored latex particles were individually coated with 
antibody for each analyte, and the coated latex particles were then mixed. 
The latex particles were of the same color, but different colored 
particles could be used for each analyte, or, alternatively, the colored 
latex particles were treated sequentially with each antibody according to 
specific antibody titres and absorptivity. Appropriate and distinct probe 
lines, prepared from a specific analyte-containing molecule conjugated to 
either bovine serum albumin, keyhole limpet hemocyanin or a polypeptide 
for each analyte, were applied to the membrane at different sites as 
described in Examples 1 and 2. The test was performed exactly as described 
in Example 1, and each probe or communicant area was read for a specific 
analyte test. With this configuration, benzoylecgonine was detected at the 
300 to 400 nanogram per milliliter level, and morphine was detected below 
300 nanograms per milliliter in urine. 
EXAMPLE 8 
Preparation of Benzoylecgonine--BSA Conjugate 
A mixture of 4.0 g (15.1 mmole) of 6-(Carbobenzyloxyamino) caproic acid, 
4.07 g (31 mmole) of 1-hydroxybenzotriazole hydrate, and 35 mL of 
1,2-dimethoxyethane was treated all at once with 3.11 g (15.1 mmole) of 
1,3-dicyclohexylcarbodiimide. The mixture was stirred for one hour as the 
urea precipitated. Then, N-ethylmorpholine was added to adjust the pH to 
7.1-7.2. 4-Aminobenzoic acid (2.18 g, 15.1 mmole) was added, and the 
suspension was stirred at ambient temperature for 18 hours. The reaction 
was poured into iced dilute hydrochloric acid solution (300 mL), the 
precipitate was filtered and the aqueous layer was extracted with ethyl 
acetate (5.times.50 mL). The combined organic extracts were washed with 
brine, dried (Na.sub.2 SO.sub.4) and the solution was concentrated to 
about 50 mL and chilled. The isolated solid was recrystallized from 
methanol giving 1.93 g (33%) of a 4-(6-carbobenzyloxyaminocaproyl) 
aminobenzoic acid, m.p. 179.degree.-181.degree. C. 
A solution of 1.93 g (4.5 mmole) of the above compound in 20 mL of dry 
tetrahydrofuran and 5 mL of 1,2-dimethoxylethane was treated with 0.52 g 
(2.5 mmole) of 1,3-dicyclohexylcarbodiimide. This mixture was stirred and 
heated at 45.degree. C. for 72 hours. Then, triethylamine (0.6 mL), 
4-dimethylaminopyridine (250 mg) and ecgonine hydrochloride (0.55 g, 2.4 
mmole) were added, and the reaction was concentrated in vacuo to a dark, 
semi-solid residue which was taken up in a minimum of dichloromethane and 
applied atop a column packed with 60.0 g of Merck "60" silica gel. The 
column was eluted with a gradient of 20 to 40% of methanol in ethyl 
acetate. The homogeneous fractions showed an Rf on silica gel plates of 
0.16 in 3:1 methanol/acetate containing 1% of NH.sub.4 OH solution. There 
was obtained 230 mg of [4-(6-carbobenzyloxy-aminocaproyl) aminobenzoyl] 
ecgonine as an amorphous powder. 
This ecgonine derivative (70 mg) was dissolved in 2.5 mL of 32% hydrobromic 
acid dissolved in acetic acid. The resulting solution was stirred at 
5.degree. C. for one hour, and then allowed to warm to room temperature. 
After being stirred an additional 3 hours, ether (50 mL) was added and the 
supernatant was decanated from the white precipitate. This process of 
washing with ether was repeated three times. The white solid was dried in 
vacuo, and the hygroscopic hydrobromide salt was dissolved in 6 mL of 5% 
of dimethyformamide in water, and the pH was adjusted to 7.0-7.1 with 5% 
aqueous tetramethylammonium hydroxide. Then, 50 mg of N-hydroxysuccinimide 
was dissolved in 4 ml of a 25% solution of 1,2-dimethoxylethane in water 
and this was added to the reaction mixture. The mixture was then cooled to 
5.degree. C. and 70 mg of bovine serum albumin was added followed by 70 mg 
of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. After one 
hour, an additional 15 mg of the diimide was added and stirring was 
continued at from 5.degree. to 15.degree. C. for 18 hours. This mixture 
was then dialyzed against 0.1M NaCl (2.times.4 liters) for 48 hours to 
give the conjugate solution which was used as is for the probe line for 
the benzoylecgonine/cocaine assay. 
EXAMPLE 9 
Preparation of Cinnamoylecgonine--BSA Conjugate 
The procedure of Example 8 was followed except that 4-aminocinnamic acid 
was substituted for 4-aminobenzoic acid to afford 
4-(6-carbobenzyloxyaminocaproyl) aminocinnamic acid, mp 
142.degree.-144.degree. C. The preparation of the cinnamoylecgonine-BSA 
conjugate was exactly as described in Example 8 for the 
benzoylecgonine-BSA conjugate by substituting the above cinnamoyl 
derivative for the benzoyl derivative. 
EXAMPLE 10 
Preparation of a Phenylacetylecgonine-BSA Conjugate 
The procedure for Example 8 was followed except that 4-aminophenylacetic 
acid was substituted for 4-aminobenzoic acid to afford 
4-(6-carbobenzyloxyaminocaproyl) aminophenylacetic acid, mp 
136.degree.-138.degree. C. The preparation of the phenylacetyl-BSA 
conjugate was exactly as described in Example 8 for the 
benzoylecgonine-BSA conjugate by substituting the above phenylacetyl 
derivative for the benzoyl derivative. 
EXAMPLE 11 
Preparation of an Amphetamine/Methamphetamine-BSA Conjugate 
In a dry round bottom flask was placed 1.0 g (6.05 mmole) of 
4-hydroxymethamphetamine, 1.62 g (1.05 mL, 7.26 mmole) of N-methylbis 
(trifluoroacetamide) and 2 mL of sieve-dried dimethylformamide. This 
mixture was stirred and heated at 65.degree. C. for 2.5 hours, and then 
the mixture was stirred at room temperature for 18 hours. The reaction was 
diluted with iced brine, the product was extracted repeatedly with ethyl 
acetate and the water-washed organic extracts were dried over Na.sub.2 
SO.sub.4, concentrated and azeotroped with toluene to afford about 1.5 g 
of yellow, oily N-trifluoroacetamido-4-hydroxymethamphetamine. 
The above trifluoroacetamide (1.5 g) was dissolved in 5 mL of anhydrous 
1,2-dimethoxyethane, the solution was cooled to 5.degree. C. and sodium 
hydride (60% in mineral oil) (0.344 g, 8.6 mmole) was added. After 
stirring the cool suspension for one-half hour at room temperature, 
t-butyl bromoacetate (1.68 g, 8.6 mmole) in 2 mL of DME was added 
portionwise, and the reaction mixture was stirred at room temperature for 
18 hours. The reaction was poured into ice water, extracted with ethyl 
acetate, and the washed, dried and concentrated solution gave 2.3 g of 
4-(t-butylcarboxy) methoxy-N-trifluoracetamidomethamphetamine as a liquid 
which showed an Rf of 0.83 on silica gel with 3:1 hexane/ethyl acetate. 
The above t-butyl ester (2.32 g, 4.94 mmole) was dissolved in 3 mL of 
dichloromethane, and 0.5 mL of 1,3-dimethoxybenzene was added. The 
solution was cooled in ice and trifluoroacetic acid (5 mL), which was 
previously cooled, was added. The reaction was then stirred at room 
temperature for 2 days. The solvents were evaporated at room temperature 
at 10 mm of pressure, and the dark residue was taken up in ether and the 
product was then extracted into 10% solution sodium carbonate (4.times.15 
mL). The aqueous extracts were acidified with dilute HCl to pH 2, and the 
product was extracted into ethyl acetate. The water-washed, dried and 
concentrated solution gave 1.92 g of crude, oily 
4-carboxymethoxy-N-trifluoroacetamidomethamphetamine. 
The above free acid (1.92 g, 4.81 mmole) was dissolved in 4 mL of 
1,2-dimethoxyethane and 2 mL of tetrahydrofuran and 0.61 g (5.29 mmole) of 
N-hydroxysuccimimide and 1.09 g (5.29 mmole) of 
1,3-dicyclohexylcarbodiimide were added. This mixture was stirred at room 
temperature overnight, N-ethylmorpholine (2.5 mL) and methyl 
6-aminocaproate (1.05 g, 7.23 mmole) were added. Stirring was continued 
for an additional 24 hours, and the reaction was diluted with brine and 
the product extracted into dichloromethane. The organic extracts were 
washed with cold IN HCI, brine and water and the dried concentrated 
product was chromatographed over 45 g of silica gel in a gradient of 50% 
to 80% of ethyl acetate in hexanes to provide 300 mg of the light pinkish, 
oily 4-[(carbomethoxypentyl) carboxamido] 
methoxy-N-trifluoroacetamidomethamphetamine, with an Rf on silica thin 
layer plates of 0.58 with 2:1 ethyl acetate/hexanes. 
This methyl ester (300 mg, 0.58 mmole) was dissolved in 8 mL of methanol 
and a solution of 3.63 g of potassium hydroxide in 4 mL of water was 
added. After being stirred at room temperature for 2 days, the methanol 
was evaporated, 5 mL of water was added and the pH was adjusted to 6.8-7.0 
with dilute HCI to give a volume of about 30 mL. Then, 
1,2-dimethoxylethane (20 mL) was added followed by 134 mg (1.164 mmole) of 
N-hydroxsuccinimide. The mixture was cooled to 10.degree. C. and 43 mg of 
BSA were added followed by 126 mg (0.918 mmole) of 
1-ethyl-3-(3-dimethylaminopropyl) carbodimide hydrochloride which was 
added in portions over 30 minutes. The reaction was stirred at 10.degree. 
C. for 4 hours and an additional 50 mg of EDCI was added. After 7 hours of 
stirring at 10.degree. C., the reaction was then stirred at room 
temperature for 2 hours, and glycine (250 mg) was added to quench the 
reaction. This mixture was then dialyzed against 0.1M NaCI at room 
temperature for 48 hours (2.times.4 liters) to give the 
amphetamine/methamphetamine conjugate solution used in the assay.