U.S. patent application number 10/633572 was filed with the patent office on 2004-02-05 for method and device for obtaining and detecting immunologically active substances from the gas phase.
This patent application is currently assigned to Securetec Detektions-Systeme AG. Invention is credited to Binder, Florian, Ertl, Harald, Herrmann, Rupert, Hilpert, Reinhold, Josel, Hans-Peter, Klein, Christian, Maier, Josef, Oberpriller, Helmut, Ritter, Josef.
Application Number | 20040023289 10/633572 |
Document ID | / |
Family ID | 27669637 |
Filed Date | 2004-02-05 |
United States Patent
Application |
20040023289 |
Kind Code |
A1 |
Klein, Christian ; et
al. |
February 5, 2004 |
Method and device for obtaining and detecting immunologically
active substances from the gas phase
Abstract
A method is disclosed for obtaining and/or immunologically
detecting an analyte contained in a gas phase by immunologically
binding the analyte to a binding partner thereof contained in a
gas- and liquid-permeable first carrier matrix. Said method is
characterized in that a) the analyte-containing gas phase is
brought into contact with the first carrier matrix (immune
adsorber), b) the analyte is bound to the first binding partner
which is contained in the first matrix and not bound to the matrix,
and c) the complex of analyte and first binding partner and the
free first binding partner are eluted from the first matrix, d) the
eluted complex or the free first binding partner is determined as a
measure for the amount of analyte present.
Inventors: |
Klein, Christian; (Weilheim,
DE) ; Josel, Hans-Peter; (Weilheim, DE) ;
Herrmann, Rupert; (Weilheim, DE) ; Maier, Josef;
(Weilheim, DE) ; Ertl, Harald; (Gelting, DE)
; Oberpriller, Helmut; (Westerhamm, DE) ; Hilpert,
Reinhold; (Moorenweis, DE) ; Binder, Florian;
(Traunstein, DE) ; Ritter, Josef; (Muenchen,
DE) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
Securetec Detektions-Systeme
AG
Ottobrunn
DE
|
Family ID: |
27669637 |
Appl. No.: |
10/633572 |
Filed: |
August 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10633572 |
Aug 5, 2003 |
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|
08898085 |
Jul 22, 1997 |
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6605444 |
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08898085 |
Jul 22, 1997 |
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08325516 |
Oct 19, 1994 |
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Current U.S.
Class: |
435/6.14 ;
435/7.1 |
Current CPC
Class: |
G01N 33/558 20130101;
Y10S 435/97 20130101; Y10S 436/807 20130101; Y10S 435/971 20130101;
G01N 33/54386 20130101 |
Class at
Publication: |
435/6 ;
435/7.1 |
International
Class: |
C12Q 001/68; G01N
033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 20, 1993 |
DE |
P 43 35 780.6 |
Jul 22, 1994 |
DE |
P 44 25 963.8 |
Claims
1. A method for isolating an analyte contained in a gas phase,
comprising the steps of: a) providing a gas permeable first carrier
matrix, wherein a first binding partner specific for an analyte is
contained in said first carrier matrix but is not bound to said
first carrier matrix, b) contacting a gas phase suspected of
containing said analyte with said first carrier matrix such that
said analyte binds to said first binding partner, and c) eluting
any complex consisting of said analyte and said first binding
partner and any uncomplexed first binding partner from said first
carrier matrix.
2. The method according to claim 1, wherein after step c) said
analyte is released from said complex of said analyte and said
first binding partner.
3. A method for immunologically detecting an analyte contained in a
gas phase, comprising the steps of: a) providing a first carrier
matrix, wherein a first binding partner specific for an analyte is
contained in said first carrier matrix but is not bound to said
first carrier matrix, b) contacting a gas phase suspected of
containing said analyte with said first carrier matrix such that
said analyte binds to said first binding partner, c) eluting any
complex consisting of said analyte and said first binding partner
and any uncomplexed first binding partner from said first carrier
matrix, and d) determining any eluted complex or uncomplexed first
binding partner as a measure of the amount of analyte present.
4. The method according to claim 3, wherein said first carrier
matrix is gas- and liquid-permeable.
5. The method according to claim 3, further comprising labeling the
first binding partner, and determining any labeled eluted complex
or labeled uncomplexed first binding partner as a measure of the
amount of analyte present.
6. The method according to claim 5, wherein said first binding
partner is enzymatically labeled.
7. Method according to claim 3, further comprising, a) binding any
eluted first binding partner to a labeled second binding partner
specific for said first binding partner, and b) determining any
bound label as a measure of the amount of analyte present, wherein
said first binding partner is unlabeled.
8. The method according to claim 7, wherein said first binding
partner is bound to said labeled second binding partner in a second
carrier matrix and said determination of said label is in a third
carrier matrix.
9. The method according to claim 1, wherein said first carrier
matrix has a liquid content of 10 to 90%.
10. The method according to claim 3, wherein said first carrier
matrix has a liquid content of 10 to 90%.
11. The method according to claim 9, wherein said analyte is first
nonspecifically adsorbed to an essentially dry matrix and then a
liquid is added allowing an immune reaction between said analyte
and said first binding partner to occur.
12. The method according to claim 10, wherein said analyte is first
nonspecifically adsorbed to an essentially dry matrix and then a
liquid is added allowing an immune reaction between said analyte
and said first binding partner to occur.
13. The method according to claim 9, wherein said liquid is an
aqueous solution containing 0 to 30% polar organic solvent and 0 to
1% detergent.
14. The method according to claim 10, wherein said liquid is an
aqueous solution containing 0 to 30% polar organic solvent and 0 to
1% detergent.
15. The method according to claim 3, wherein said first binding
partner is an antibody or Fab' fragment thereof.
16. The method according to claim 3, wherein said first binding
partner is a receptor which binds said analyte.
17. The method according to claim 3, wherein said first carrier
matrix is a hydrophilic or hygroscopic material.
18. The method according to claim 17, wherein said hydrophilic or
hygroscopic material is the form of particles or fibers.
19. A device for obtaining and/or immunologically detecting an
analyte contained in a gas phase, comprising a) a gas- and
liquid-permeable carrier matrix containing a first binding partner
of an analyte in elutable form, and b) a capture system for an
eluate in which a complex consisting of said analyte and said first
binding partner or a noncomplexed first binding partner can be
determined or isolated.
20. The device according to claim 19, further comprising a vacuum
pump to draw a sample gas containing an analyte across said gas-
and liquid-permeable carrier matrix, and an eluting liquid.
21. The device according to claim 19, wherein said gas- and
liquid-permeable carrier matrix has a gas permeability between 10
ml/min to 10 l/min.
22. The device according to claim 19, wherein said gas- and
liquid-permeable carrier matrix is in a planar form or in the form
of laminar layers.
23. A method for detecting the presence of a substance in a gas
phase in a gas permeable, enclosed package, comprising the steps of
a) drawing a sample of a gas surrounding a gas permeable, enclosed
package suspected of containing a substance of interest, b)
contacting a first carrier matrix with said sample of gas, wherein
a first binding partner, specific for an analyte of said substance,
is contained in said first carrier matrix but not bound to said
first carrier matrix, c) binding any analyte of said substance
present in said sample of gas to said first binding partner, d)
eluting any complex consisting of said analyte and said first
binding partner and any uncomplexed first binding partner from said
first carrier matrix, and e) determining any eluted complex or
uncomplexed first binding partner as a measure of the presense of
said substance.
24. The method according to claim 23, wherein said substance is
selected from the group consisting of nitroglycol, nitroglycerin,
nitropenta, hexogen, octogen, tetranitromethane, trinitrotolulene,
trinitrobenzene, trinitroanisol, triaminotrinitrotolulene,
hexanitrostilben, polycyclic aromatic hydrocarbons, polychlorated
biphenyls, herbicides and pesticides.
25. The method according to claim 23, wherein said substance is an
illegal drug of abuse.
26. The method according to claim 25, wherein said substance is
selected from the group consisting of cocaine, heroin, cannabinol,
cannabidiol and tetrahydrocannabinol.
27. The method according to claim 23, wherein said enclosed package
is luggage.
Description
[0001] Subject matter of the invention is a method and device for
obtaining and detecting immunologically active substances (e.g.
drugs of abuse) from the gas phase by means of an immunological
procedure.
[0002] The detection of immunologically active substances from the
gas phase has gained particular importance for the detection of
drugs of abuse, such as cocaine and cannabinoids.
[0003] To date, various techniques and technologies (X-ray
instruments, GC-MS coupling, GC with chemiluminescence detectors)
are used to carry out stationary tests for the presence of illegal
drugs of abuse, e.g. in baggage. However, size and weight of these
systems and the high costs involved contradict the common use of
these techniques. Principally, all portable instruments whose
function is based on ion mobility spectroscopy (IMS) are suitable
for use anywhere. They involve, however, problems with respect to
selectivity and sensitivity. Moreover, these instruments are only
suitable to detect contamination which is present in the form of
particles. The only method for the selective detection of narcotics
in the gas phase is currently the use of drug-sniffing dogs. The
disadvantage involved with the use of such animals is, however, the
short time for which the animals are available, the fact that they
can be distracted, the presence of irritating substances, and the
high costs. A portable instrument for the selective detection of
narcotics which can be used under various conditions would, hence,
constitute an enormous progress in the fight against illegal drug
trafficking.
[0004] Biosensors making use of the principle of an immunological
reaction between the analyte and binding partners contained in the
biosensor would be potentially suitable for such tasks due to the
high selectivity and specificity of the immunological reactions. A
biosensor of this kind has been described by Ngen-Ngwainbi, J., et
al. in J. Am. Chem. Soc. 108 (1986), 5444-5447. In this literature
reference, an antibody to a cocaine metabolite (benzoylecgonine) as
a reactive component of the sensor is used as a Piezo transducer
with a resonance frequency of 9 MHz. The antibody is immobilized
through physical adsorption on the surface of the sensor. The lower
detection limit is at 0.5 ppb corresponding to 2.times.10.sup.-1
mol/l in gas phase (for cocaine and cocaine-HCl). As the mass
sensitivity of the transducer is very low, the performance of such
an instrument is not suitable for use in the practice.
[0005] Another method is described in JP-A-0374460. In this
literature reference, drug molecules from the gas phase are
adsorbed to a polyethersulfone membrane, then eluated and detected
in the eluate in an immunological reaction. Again, the sensitivity
is very low.
[0006] Another method is described in DE-A 41 21 493. According to
this reference, immobilized antibodies and labeled tracers (which
correspond to the analyte) are bound to one another and present in
a permeable, non-transparent partial area of the carrier. The
analyte is added and replaces labeled tracers prior to binding to
the antibody. The free labeled tracer diffuses then into a
transparent part of the carrier. The progress of the tracer
diffusion can be monitored as it causes a discoloration, since the
label used in the tracer is a dye. Again, this is a method which
exhibits a very low sensitivity.
[0007] The detection limits necessary for a successful detection of
drugs of abuse from the gas phase cannot be reached with these
methods.
[0008] Cocaine, for example, has a saturation concentration of
6.times.10.sup.-12 mol/l gas phase at 20.degree. C. In the
practice, however, concentrations are considerably lower so that
the lower detection limit of a method necessary for successful
detection ranges between 10.sup.-13 to 10.sup.-15 mol/l gas
phase.
[0009] As it is not possible to measure such low concentrations
directly from the gas phase, a device for determining
immunologically active substances from the gas phase advantageously
comprises an adsorption unit and a detection unit.
[0010] In the fight against drugs, it is also necessary to detect
particle-like drugs or drugs that are bound to particles in
addition to drug molecules which occur freely in the gas phase
(edited by BKA. "Internationales Symposium Detektion von
Rauschgift", Wiesbaden 1991).
[0011] It is an object of the present invention to provide a method
of obtaining immunologically active substances from the gas phase
which can be used to convert the substance to be detected from the
gas phase into a form suitable for subsequent detection
reactions.
[0012] Another object of the invention is the provisision of a
method of determining immunologically active substances from the
gas phase which exhibits a high sensitivity and allows quick
adsorption or adsorption and determination.
[0013] Immunologically active substances from the gas phase are
substances which occur freely in the gas phase, particles of these
substances or substances bound to particles which can be obtained
from the gas phase.
[0014] Subject matter of the invention is a method of obtaining an
analyte contained in a gas phase by immunologically binding this
analyte to a binding partner of said analyte contained in a gas-
and liquid-permeable first matrix. The method is characterized in
that
[0015] a) the analyte-containing gas phase is brought into contact
with the first carrier matrix (immune adsorber),
[0016] b) the analyte is bound to the first binding partner which
is contained in the first matrix and not bound to the matrix,
and
[0017] c) the complex consisting of analyte and first binding
partner and uncomplexed first binding partner are eluted from the
first matrix, and
[0018] d) optionally the analyte is released from the complex.
[0019] Another subject matter of the invention is a method of
immunologically detecting an analyte contained in the gas phase by
immunologically binding the analyte to a binding partner of said
analyte contained in a gas- and liquid-permeable first carrier
matrix. This method is characterized, in that
[0020] a) the analyte-containing gas phases brought into contact
with the first carrier matrix (immune adsorber),
[0021] b) the analyte is bound to the first binding partner which
is contained in the first matrix and not bound to the matrix,
and
[0022] c) the mixture consisting of complex of analyte and first
binding partner and free (uncomplexed) first binding partner is
eluted from the first matrix and
[0023] d) the eluted complex or the free first binding partner are
determined as a measure for the amount of analyte present.
[0024] Suitable binding partner of the analyte are antibodies which
may be monoclonal or polyclonal antibodies, or fragments (e.g. Fab,
Fab') thereof. Also suitable are receptors which are present in the
body, as these bind drugs, e.g. a cannabinoid receptor (Matsuda et
al., Nature 346 (1990) 561-564).
[0025] Preferred carrier matrices consist of hydrophilic or
hygroscopic materials, e.g. based on cellulose, modified cellulose
such as cellulose nitrate or cellulose acetate, hydroxyalkylated
cellulose, or modified and unmodified cellulose crosslinked with
substances such as epichlorhydrin. Also suitable are glass fiber
matrices and matrices consisting of polyester. These materials can
either be used solely or in combination with other compound
materials with a hydrophilic portion in the carrier matrix
prevailing.
[0026] In a preferred manner, the first carrier materials and,
optionally, other carrier materials are structured so as to form
particles (e.g. pearl-like, see DD-A 296 005) or fibers, such as
filter papers on cellulose basis (EP-A 374 684, EP-A 0 470 565).
Other materials used for the construction of the test carriers are
described in EP-A 0 374 684, EP-A 0 353 570 and EP-A 353 501.
[0027] The first carrier matrix must be gas-permeable to allow
enrichment of the immunologically active substance from the gas
phase. For pressure gradients above the adsorber (200-500 mbar)
which are technically easy to implement, the gas permeabilities
advantageously range between 1 m/min and 100 l/min, preferably
between 100 ml/min and 20 l/min and particularly preferred between
500 ml/min and 10 l/min.
[0028] In addition to keeping back the analyte, another essential
requirement of first matrix (immune adsorber) is to allow the
immune reaction to occur between the binding partner and the
immunologically active substance, e.g. via diffusion. To accomplish
this, the first matrix can contain the necessary liquids either
before the beginning of the adsorption or the first matrix can be
essentially dry and the immune reaction can be allowed to occur
after adding liquid. An essentially dry matrix as used in the
presently claimed invention, is a matrix in which there is no
measurable amount of liquid.
[0029] The preferred liquid in the first matrix is water. If the
first matrix contains liquid already before the adsorption, the
contents thereof ranges between 10 and 90% of the total weight of
the first matrix. The water used can have a percentage of up to 30
wt.-% of organic substances as a dissolving agent (e.g.
dimethylsulfoxide, glycerol).
[0030] In order to increase the solubility of the binding partners,
it is preferred to add 0.01 to 1% of detergents (e.g. Tween 20 M,
Tween 80.TM., octylglucoside, polydocanol and/or Synperonic.TM.,
e.g. F 68).
[0031] If the adsorption is followed by the detection of the
analyte which is based on the detection of the labeled binding
partner, it is possible to use in accordance with the invention all
those labels that have been described for immunoassays. They
include, for example, enzyme labels, fluorescence dye labels,
radioactive labels, labels with gold, molecules capable of
generating luminescence or selenium dioxide or labels with dyed
polymers, such as latex particles.
[0032] In accordance with the invention, however, it is preferred
to use an enzyme as a label which is then detected in an enzymatic
color reaction. Particularly preferred enzymes include
.beta.-D-galactosidase, alkaline phosphatase, or peroxidase. They
can be detected by using such substrates as
chlorophenolred-.beta.-D-galactoside, p-nitrophenyl-phosphate,
AMPPD (disodium-3-(methoxyspirof
1,2-dioxetane-3,2'-{3.3.1.1.3,7]decane} phenylphosphate) or
ABTS.TM.. In another preferred embodiment of the invention, a
fluorescence dye is used as a label where the fluorescence quantum
yield or the depolarization capacity changes due to the binding to
the antibody in dependency upon the concentration of the analyze
contained in the sample liquid. When enzyme labels are used, it is
particularly preferred to increase the sensitivity during the
detection of the label by using an amplification system with an
amplification reaction as described, for example, in DD222896,
DD222897, and DD280790.
[0033] Particularly preferred labels are those which allow
detection of luminescence, e.g. enzyme labels of alkaline
phosphatase combined with, for example, ANMPPD
(disodium-3-(methoxyspiro{1,2-dioxetane-3,2'-tricyclo-
[3.3.1.1.3,7] decane}phenylphosphate), or CSBD (disodium
3-(methoxyspiro{1,2-dioxetane-3-2'-(5-chloro)-tricyclo[3.3.1.1.3,7]decane-
}phenylphosphate or peroxidase with luminol; when molecules capable
of luminescence are used, aequorin (Biochemistry 1992, Vol. 31,
page 1433-1442) where luminescence is triggered by calcium ions; or
acridinium ester, where luminescence is triggered by peroxide.
[0034] The antibodies used in accordance with the invention are
manufactured according to known methods of immunizing suitable
animals with the corresponding immunogen. The antibodies are
obtained after immunization and those antibodies are selected which
exhibit the highest possible specificity and affinity for the
analyte. The expert is also familiar with the manufacture of
monoclonal antibodies and antibody fragments.
[0035] The determination is carried out such that the complex of
analyte and first binding partner as well as free first binding
partner from the first matrix are eluted in a first step. In this
eluate, the analyte can be determined with all known
immunoassays.
[0036] In a preferred embodiment, the carrier essentially comprises
four zones (FIG. 1):
[0037] 1. a first carrier matrix (immune adsorber) containing first
binding partner (conjugate of antibody and labels) which is not
bound to the carrier,
[0038] 2. a second carrier matrix (capture matrix), in which the
non-analyte-bound conjugate is bound to immobilized analyte
analogs, and is optionally determined.
[0039] 3. a third carrier matrix (detection field), in which the
label is preferably determined.
[0040] 4. optionally, the storage container holding liquid suitable
for the elution can be disposed before the first carrier matrix. In
a preferred manner, this is a carrier fleece.
[0041] To implement the determination, a pressure difference is
created to draw or pump the gas to be analyzed into the first
carrier matrix. After sufficient enrichment of the analyte on the
immune adsorber, and a possible formation of a complex between
analyte and binding partner, the elution liquid is transferred from
the storage container into the first carrier matrix, for example by
applying pressure: In this first step, it is also possible to have
a complex formation between analyte and binding partner. The
soluble components of the first carrier matrix elute in the second
carrier matrix (capture matrix), where the amount of receptor which
has not entered an immunological binding with the analyte is bound
to excess, carrier-bound analyte analog. The conjugate of analyte
and specific receptor continue to travel to the third carrier
matrix in which the label is detected.
[0042] If, for example, the conjugate of antibody and acridinium
ester is used on the first carrier matrix (immune adsorber),
detection in the detection field is achieved by measuring
luminescence triggered by alkaline peroxide. If the conjugate of an
antibody and alkaline phosphatase is used on the first carrier
matrix, luminescence is measured after diffusion into the second
carrier matrix (capture matrix) using a luminogenic AP substrate
such as Lumiphos (disodium
3-(methoxyspiro{1,2-dioxetane-3-2'-tricyclo[3.3.1.1.3,7]decane}
phenylphosphate or a chromogenic AP substrate (X-Gal, NBT) in the
detection field. If a conjugate of antibody and gold sol is used as
conjugate, detection is carried out visually in the third carrier
matrix following a chromatography via a capture matrix. Such
capture matrices are described, for example, in EP-A 0052769, EP-A
0167171 and EP-A 0470565.
[0043] If the conjugate used is one of an antibody and aequorin,
luminescence is measured in the third carrier matrix by means of
calcium ions used to impregnate third carrier matrix or by adding
calcium ions.
[0044] In another embodiment, the first binding partner (antibody)
is not labelled and the mixture of analyte-antibody-complex and
free antibodies w hich was eluted from the first matrix is
incubated with a second solid phase coated with analyte analogs.
Subsequently second labelled antibodies to the first analyte
antibodies (preferably to the constant part thereof) are used to
incubate and wash the whole mixture and to detect the amount of
bound or free first antibodies eluted from the first matrix in a
substrate reaction, e.g. with ABTS.TM.. If a conjugate of analyte
antibodies and label (e.g. peroxidase) is used instead of the
unlabelled first analvte antibody, the incubation and washing step
with the secondary detection conjugate of peroxidase and antibody
to the analyte antibody can be omitted. Further, it is also
possible to use an analyte antibody-acridiniumester-conjugate
instead of an analyte antibody enzyme conjugate and to carry out a
luminometric detection by reacting it with alkaline peroxide
solution.
[0045] The conjugates of antibody and labels can be applied on the
carrier materials in a simple wetting procedure. It is also
possible to apply conjugate solution on the dried carrier, to allow
the solution to be adsorbed and to separate excess liquid by
letting it drop, by centrifuging it or rapidly aspirating air. In a
preferred manner, the conjugate is applied on the carrier in a
buffered solution, such as PBS, or other conventional buffers with
a pH of 5-9. To improve the permeability of the carrier and to
improve its stability, the solution may also contain non-denaturing
detergents, such as Tween 20, Tween 80, octylglucoside,
polydocanol, Synperonic F 68, or organic solvents, such as glycerol
DMSO, and/or polyethyleneglycol and/or protein additives, such as
bovine serum albumin or nonspecific IgG.
[0046] For the elution, it is advantageous to use buffered solution
such as PBS or other conventional buffers in the pH range between 5
and 9.
[0047] Another subject matter of the invention is a device for
obtaining and/or detecting an immunologically active substance in a
gas phase, said device comprising
[0048] a) a gas- and liquid-permeable carrier matrix containing a
first binding partner of the analyte in an elutable form,
[0049] b) a capture system for such an eluate in which the complex
of analyte and first binding partner, or nonbound first binding
partner is isolated, and, if necessary, determined in a subsequent
reaction.
[0050] The matrix of the immune adsorber in accordance with the
invention can be planar, such as a fleece or a membrane, or have
the form of laminar layers (e.g. in the form of columns). In case
of planar matrices, it is possible to use round or otherwise shaped
surfaces with an area between 2 mm.sup.2 and 100 cm.sup.2 and a
depth between 0.1 mm and 10 mm when volume currents of 1 ml to 100
l/min, preferably 100 ml to 20 1/min, particularly preferred 500
ml/min to 10 l/min are used for the adsorption. It is particularly
preferred to use matrices with a surface area between 0.2 and 5
cm.sup.2 and 0.5 to 2 mm in depth. If deeper layers are used, it is
preferred to use column-like geometries having an area between 3
mm.sup.2 and 25 cm.sup.2 and layer thicknesses between 3 mm and 20
cm. A preferred area is one between 10 mm.sup.2 and 100 mm.sup.2
and 1 to 10 cm layer depth. The form of the matrix material can
also be a sphere or a fiber; it can have irregular kernels or be a
gel.
[0051] The capture system can also be planar (carrier matrix),
column-like or configured as a container to hold liquid.
[0052] The carriers are preferably made of several essentially
adjacent capillary active test fields (carrier matrices) which are
in fluid contact with each other, so as to form a line of
transportation along which liquid is propelled by means of
capillary forces and through the immune adsorber toward a detection
field. The number of test fields per se is irrelevant and depends
whether the reagents necessary for the immunological reaction are
separately applied to the fields or together.
[0053] As described in EP-A 0 374 684, a glueing agent is used to
attach the test fields on a carrier foil. To allow gas to traverse,
the carrier foil is provided with holes such that the test field
(first carrier matrix) comes to lie above the openings. The glued
carrier foil is then cut into individual carriers where the gas
penetration openings are essentially in the center below the first
carrier matrix.
[0054] FIGS. 1a) and 1b), wherein
[0055] 1) is a fleece for receiving elution liquid,
[0056] 2) is an immune-adsorbing matrix above an adsorption opening
(3) of the carrier foil (6),
[0057] 3) is an adsorption opening
[0058] 4) is a capture matrix
[0059] 5) is a detection matrix
[0060] show an example of a test carrier.
[0061] In another embodiment, the device also contains at least one
buffer field, which is in contact with immune adsorber, capture
matrix, and/or detection field. It contains auxiliary substances to
adjust the conditions for an optimal reaction (e.g. ion strength,
pH value). The buffer field consists of a porous material,
preferably a fleece made of cellulose, polyester, or nylon.
Suitable carrier materials and methods for applying the conjugate
are described in EP-A 0353570. Particularly suitable materials are
porous materials on polyester, cellulose, or glass fiber basis.
[0062] The analyte analog can be immobilized in the capture matrix
according to known methods, for example, chemically or by immune
precipitation. In a preferred manner, however, a biological binding
partner such as (strept)avidine is bound to the material of the
capture field and the antibody, and/or the peptide in accordance
with the invention is added while bound to another biological
binding partner, for example biotin. Immobilization of the analyte
analog or the antibody is then achieved by binding the biological
binding partner (e.g. biotin/streptavidin binding). Such methods of
immobilization are described in EP-A 0374684.
[0063] If necessary, the detection zone can preferably be provided
with a capture matrix above or below it (cf. EP-A 0353500). The
detection zone preferably contains a reagent system with a
detection substrate suitable for the label which is subject to an
observable change upon contact with the labeled component or it
serves for direct detection of the conjugate, if a label is used
which can be directly determined (e.g. fluorescence label). If it
contains a substrate, this zone is preferably made of a dissolvable
film or a tissue or a fleece which is hydrophobically blocked. Such
carriers are described in EP-A 0353501. When it reaches the end of
the field of the substrate, a sample liquid is thus eluted from the
carrier and the detection reaction is triggered.
[0064] Examples for detectable substances include as drug molecules
cocaine, heroin, cannabinol, cannabidiol, tetrahydrocannabinol;
examples for explosives include nitroglycol, nitroglycerin,
nitropenta, hexogen, octogen, tetranitromethane, trinitrotoluene,
trinitrobenzene, trinitroanisol, triaminotrinitrotoluene,
hexanitrostilben, and polycyclic aromatic hydrocarbons,
polychlorated biphenyls, herbicides, and pesticides (such as
atracin, parathion, simacin) and odoriferous compounds (e.g.
terpineol, limes, caryophylls, camphor; farnesol).
[0065] A preferred embodiment (FIG. 2) of the device comprises the
following elements:
[0066] 1. immune adsorber in accordance with the invention
[0067] 2. vacuum pump
[0068] 3. valve
[0069] 4. three-way valve
[0070] 5. detection unit
[0071] 6. signal processing and display
[0072] 7. storage device with eluting agent
[0073] 8. pressure pump
[0074] 9. glider
[0075] 10. drive unit for glider
[0076] 11. recess for measuring solution
[0077] 12. power supply
[0078] 13. operating console
[0079] 14. gas outlet
[0080] 15. sample gas inlet
[0081] 16. housing
[0082] 17. gas-like sample molecules, sample molecules bound to
particles, or particle-like sample.
[0083] In a first working cycle, a vacuum pump 2 is used to draw in
sample gas containing molecules 17 to be adsorbed through sample
inlet 15 across immune adsorber 1.
[0084] The gas will be exhausted by the device through outlet 14.
If necessary, sample particles which adhere to the material to be
analyzed or particles to which sample molecules are adsorbed are
brought into the intake current by means of blowing or brushing. To
accomplish this, valve 3 is closed, valve 4 between immune adsorber
and vacuum pump is open, and glider 9 is opened prior to sample
intake. Once the adsorber 1 is charged with the amount of sample
gas to be analyzed, drive 10 is activated to close glider 9.
Subsequently, valve 3 is opened and at the same time valve 4 is
switched to allow free passage between adsorber and detection unit.
Pressure pump 8 is now used to transport eluting agent from the
storage container 7 through the immune adsorber. The eluting liquid
which, at the outlet from adsorber 1, now contains the conjugate of
analyte molecule 17 and the labeled antibody, and, depending on the
amount of analyte contained in the sample gas, also unbound labeled
antibody, is now made available to another unit. This is a
detection unit 5 in which the amount of analyte molecules 17
present in the sample is determined in a subsequent (immunological)
reaction. Then, pressure pump 8 is again activated to transport
eluting agent from storage container 7 through the system. The
system is thus washed and at the same time used measuring solution
is transported to a receptacle 11. The signals contained in the
detection unit are processed and displayed by means of a function
unit 6 which is operated via operating field 13. Power supply to
all components is effected via an exchangeable storage battery 12
and all components are disposed in a housing 16.
[0085] The antibody antigen complex from the first carrier matrix
is transferred to the other carrier matrices and/or reaction
vessels preferably by mechanically squeezing the solvent which is
present in the first carrier matrix, by applying and pressing
through an additional eluting agent via pressure or aspiration.
Instead of using mechanical pressure, the transport of additional
eluting agent can also be continued by means of capillary
forces/concentration gradients/hydrostatic pressure.
[0086] It is preferred to remove the solvent by means of squeezing
or to pass through additional eluting agents by pressing or drawing
which requires pressure or the applicaiton of a vacuum.
EXAMPLE 1
[0087] a) Preparation of Benzoylecgonine Maleimidoethylamide
[0088] In 200 ml of dried acetonitrile, 1 g N-hydroxysuccinimide
and 1.8 g dicyclohexylcarbodiimide are added to 2.4 g
benzoylecgonine hydrochloride, which is then stirred for 3 hours.
The precipitate is removed by filtration, the filtrate is
evaporated, taken up in nitromethane and again filtered. After
evaporating the solvent, it is triturated with ether. The result is
1.13 g of benzoylecgonine succinimidylester. Together with 0.47 g
maleimidoethylamine hydrochloride (cf. WO 90/15798), this product
is taken up in 100 ml dried acetonitrile. 1.1 g of triethylamine
are added and stirred for 12 hours at room temperature. The
reaction mixture is evaporated, taken up in 50 ml ethylacetate and
extracted 3 times with sodium hydrogen carbonate solution. The
ethyl acetate phase is evaporated and the product is converted into
the hydrochloride by taking it up in 10 ml of dioxane saturated
with HCl. The result is again filtered, washed with ether and
produces 1 g of benzoylecgonine maleimidoethylamide
hydrochloride.
[0089] b) Preparing the Cocaine Immunogen
[0090] Contained in 25 ml of 0.1 mol potassium phosphate buffer at
pH 8.5, 300 mg of bovine serum albumin are reacted with 106.6 mg of
S-acetvithiopropionic acid succinimidylester, dissolved in 5 ml of
dioxane, over a period of 3 hours at room temperature. The modified
bovine serum albumin is separated from low-molecular reaction
products in a gel chromatography over ACA 202 using 0.1 mol
potassium phosphate buffer, pH 8.5. The result is a solution of
310.5 mg of product in 57.5 ml potassium phosphate buffer, pH
8.5.
[0091] An amount of solution which corresponds to 100 mg of the
modified bovine serum albumin is reacted with 4.7 ml of a 1 M
hydroxylamine solution. Subsequently, 49.4 mg benzoylecgonine
maleimidoethylamidehydroc- hloride are added and allowed to react
for 12 hours at 4.degree. C. The resulting cocaine immunogen is
separated from the low-molecular reaction products in a gel
chromatography over ACA 202 using 0.1 mol potassium phosphate
buffer, pH 8.5. The results are 95 mg of cocaine immunogen
dissolved in 0.1 molar potassium phosphate buffer, pH 8.5.
[0092] c) Obtaining Antibodies to Cocaine
[0093] 10 sheep were immunized with the cocaine immunogen in a
complete Freund's adjuvans. The dose administered to each animal
was 200 .mu.g for the first and for each following immunization.
Immunization were carried out in monthly intervals. The resulting
sera were assayed in a microtiter plate assay for the presence of
antibodies to cocaine. To do this, streptavidin-coated microtiter
plates were incubated with
benzoylecgonine-[N'-biotinylaminocaproyl-(3,6-dioxa-8-aminooctyl)amide],
prepared from benzoylecgonine succinimidylester and
N-(biotinylaminocaproyl)-1,8-diamino-3,6-dioxaoctane, washed, then
again incubatred with a sera to be analyzed, washed and, for the
purpose of detection, incubated with a conjugate of peroxidase and
a rabbit-anti-sheep-immunoglobulin, washed and a substrate was
added. The relative affinities to cocaine were determined
corresponding to example 12 of EP-A 0547 029. Sera from S 4987 with
a good affinity to cocaine were selected for additional tests.
[0094] d) Preparing Conjugates of Antibodies to Cocaine and
Alkaline Phosphatase
[0095] Polyclonal sheep antibodies to cocaine, DE-purified
(PAB<cocaine>S-IgG(DE)) are isolated from delipidized raw
serum (sheep) according to methods which are known to the expert
using ammonia sulfate precipitation and DEAE-sepharose
chromatography.
[0096] Immuneresorptive Purification of PAB<BZE>S-IgG(DE)
[0097] Preparaing of a Cocaine Immune Adsorber
[0098] The cocaine polyhapten of example 1b (without biotinylation
step) is bound to a glutardialdehyde-activated affinity adsorbens
(activated Spherosil, Boehringer Mannheim, Cat.-No. 665 525) in
accordance with the specifications of the manufacturer.
[0099] Immunesorption
[0100] The (PAB<cocaine>S-IgG(DE)) is dialyzed against
PBS/azide (50 mmol/l potassium phosphate, pH 7.5, 150 mmol/l sodium
chloride, 0.1% sodium azide) and then added over a suitably
dimensioned adsorption column (depending on the binding capacity of
the adsorption and the titer of the IgG(DE)) over a period. of 2 h
at room temperature. After washing nonbound protein with PBS/azide,
the bound antibody is eluted with 1 M propionic acid at room
temperature.
[0101] The eluate is dialyzed against 30 mM sodium phosphate
buffer, pH 7.1.
[0102] Preparation of PAB<BZE>S-IgG(IS)-AP Conjugates
[0103] Activating the IgG
[0104] The immunosorptively purified IgG is incubated at a
concentration of 10 mg of protein/ml in 30 mM sodium phosphate
buffer, pH 7.1, with a 5-fold molar excess of
maleimido-hexanoyl-N-hydroxy-succinimide ester (MHS) at 25.degree.
C. over a period of 1 hour. The mixture is stopped by adding
100-fold molar excess of L-lysine/HCl as compared to MHS and
dialyzed against 10 mM potassium phosphate, 50 mM sodium chloride,
10 mM MgCl.sub.2, pH 6.1.
[0105] Activating Alkaline Phosphatase
[0106] The alkaline phosphatase (EIA quality, Boehringer Mannheim,
Cat. No. 567 744) is, at a concentration of 10 mg of protein/ml in
30 mM triethanolamine, 3 M NaCl, 0.1 mM ZnCl.sub.2, 1 mM
MgCl.sub.2, pH 7.0, incubated for 1 hour at 25.degree. C. with a
30-fold molar excess of succinimidyl acetylthiopropionate (SATP).
The reaction mixture is stopped at 10 mM by adding L-lysine/HCl and
dialyzed against 10 mM potassium phosphate, 50 mM NaCl, pH 7.5. 1 M
hydroxylamine solution, pH 7.5, at 20 mM and 0.1 M EDTA solution up
to 0.5 mM are added to deacetylate the protected SH group and the
mixture is incubated for 15 min at 25.degree. C. The activated AP
solution is immediately used for the coupling.
[0107] Coupling
[0108] The solutions of the activated AP and the activated IgG are
mixed in equimolar amounts, and the pH is adjusted to 6.8-7.0, and
the AP concentration is adjusted to 5 mg/ml using redistilled
water. After 3 hours of reaction at 25.degree. C., the mixture is
stopped by sequentially adding N-ethylmaleimide (up to 5 mM, 30
min, 25.degree. C.) and 1 M hydroxylamine solution, pH 7.5 (at 20
mM, 1 h, 25.degree. C.). The conjugate solution is dialyzed against
50 mM triethanolamine/HCl, 150 mM NaCl, 1 mM MgCl.sub.2, 0.1 mM
ZnCl.sub.2, pH 7.6, and spiked up to 10 mg/ml bovine serum albumin
and 3 M NaCl.
[0109] e) Preparing a Biotinylated Cocaine Polyhapten
[0110] At a concentration of 25 mg/ml in phosphate buffer, pH 8,
rabbit IgG is reacted with the 6-fold molar amount of
S-acetylthiopropionic acid succinimidyl ester, dissolved in
dimethylsulfoxide. After 1 h at 25.degree. C., the reaction is
stopped by adding a solution of 1 mol/l lysine. Then a dialysis is
carried out against 0.1 mol/l potassium phosphate buffer, pH 6
using 1 mmol/l EDTA. Subsequently, the pH is adjusted to 7.8 and
incubated with 1 mol/l hydroxylamine solution, pH 7.5 at 20 mmol/l
for 1 h at 25.degree. C. To accomplish the coupling, a 5-fold molar
excess of benzoylecgonine maleimidoethylamidehydrochloride,
dissolved in dimethylsulfoxide, and is under stirring added to the
solution of the rabbit IgG which is modified with sulfhydryl
groups. After incubating for 2 hours at 25.degree. C., the reaction
is stopped by successively adding 0.1 mol/l cystein solution up to
1 mmol/l and 0.5 mol/l iodine acetamide solution up to 5 mmol/l.
The mixture is dialyzed overnight against 0.1 mol/l potassium
phosphate buffer, pH 8.5, and via membrane filtration concentrated
to a protein concentration of 10 mg/ml. Then, the resulting cocaine
polyhapten is biotinylated with an 8-fold molar excess of
biotinylcaproic acid succinimidyl ester, dissolved in
dimethylsulfoxide. The mixture is dialyzed against 20 mmol/l sodium
acetate, pH 4.3, and purified via FPLC.
EXAMPLE 2
[0111] a) Preparing a First Carrier Matrix (Immune Adsorber) with a
Conjugate Consisting of Anti-Cocaine Antibody and Alkaline
Phosphatase
[0112] A fleece was prepared which consists of 80 parts polyester
fibers with a linear density of 3.3 dtex and a length of 4 mm, 20
parts viscose staple fiber with a linear density of 1.7 dtex and a
length of 3 mm, and 20 parts polyvinyl alcohol fibers with a length
of 4 mm. The fiber materials polyester, viscose staple fiber and
polyvinyl alcohol were broken open/separated in mixing tubs at a
density of 0.3% using fully deionized water. The fiber material was
then pumped to a circulating sieve. While water was removed from
the fiber mixture, i.e. drawn off by producing a vacuum, fibers
oriented themselves on the sieve side and were contact-dried as a
fleece with a drying contents of approximately 20% over drying
cylinder. The result is a fleece with an area weight of 80
g/cm.sup.2 and a thickness of 0.32 mm.
[0113] On the fleece, circular disks with a diameter of 18 mm were
punched out and each was impregnated with 20 .mu.l of a solution of
the concentration 500 ng/ml of the conjugate of anti-cocaine
antibody and alkaline phosphatase (free first binding partner)
which was prepared according to example 1 d. The result is an
embodiment of the immune adsorber in accordance with the
invention.
[0114] b) Measuring Cocaine from the Gas Phase
[0115] The immune adsorbers in accordance with the invention are
placed into a support according to FIG. 3 The support with the
immune adsorber mounted thereto is connected to a flow-through
controlled vacuum pump (type GSA 50-01). In the drawing, the
reference numerals mean the following:
[0116] 1) plastic tube
[0117] 2) guide for fleece-like adsorber material
[0118] 3) fleece-like adsorber material
[0119] 4) suction direction toward the vacuum pump
[0120] The cocaine gas phase is provided by means of a test stand
where nitrogen gas is passed through an also heated up base body of
solid cocaine (free base) at 20.degree. C. The entire gas conduct
of the test stand is carried out under isothermal conditions to
ensure uniform gas quality. The presence of a constant gas quality
at the output of the test stand is determined with a reference
analysis instrument (introducing the test gas in several
consecutively disposed condensation trap, analysis with GC/MS). At
a given temperature of 20.degree. C., a cocaine concentration of
approximately 2 ng/l is maintained which corresponds to the
saturation of concentration of cocaine to be theoretically expected
for this temperature (Lawrence et al., Can. J. Chem. 62, 1984). In
an additional dilution segment, the cocaine gas is diluted with
nitrogen to produce 1% of the saturation concentration, i.e. 20
pg/l or 6.6.times.10.sup.-14 mol/l of cocaine.
[0121] At the support, a through-put of 5 l/min is set which is
checked with a flow meter (rotameter) disposed in the gas conduit.
Here, a pressure decrease of approximately 140 mbar is observed.
With the given flow rate, diluted cocaine gas is applied to the
adsorber material at the output of the test stand for 30 sec, 1
min, 2 min, 4 min, and 8 min. This corresponds to total amounts of
50, 100, 200, 400, and 1000 ng of cocaine with which the immune
adsorber comes into contact.
[0122] The immune adsorbers are then shaken with 400 .mu.l of
elution buffer (50 mmol/l HEPES, 0.9% NaCl, 0.05% Tween 20, pH 6.8)
for a period of 1 min.
[0123] The wells of a microtiter plate are then incubated with 100
.mu.l of a solution of 10 ng benzoylecgonine
-[N'-biotinylaminocaproyl-(3, 6-dioxa-8-aminooctyl)amide] in
elution buffer for 60 min and then washed 3 times with washing
buffer (0.9% NaCl+0.05% Tween 20).
[0124] Portions of 100 .mu.l of the immune adsorber eluate, which
contains free first binding partner (conjugate of anti-cocaine
antibody and alkaline phosphatase) and complex of free first
binding partner and cocaine, depending on the amount of adsorbed
cocaine, are added into the wells of the microtiter plate,
incubated for 60 min and then washed 3 times with washing
buffer.
[0125] To carry out the detection reaction, 100 .mu.l of substrate
solution (0.4 mmol/l CSBD disodium
3-(methoxyspiro{1,2-dioxetane-(5-chlor-
o)-3,2'-tricyclo[3.3.1.1..sup.3 7]decane}phenylphosphate, 10%
Sapphire enhancer in 0.1 M diethanolamine, 1 mmol/l MgCl.sub.2, pH
10.0) were added and luminescence was measured for 60 sec.
[0126] Measurement Results
1 Amount of cocaine applied Luminescence [p] [rel. units/60 sec] 0
6767.2 50 5602.3 100 4766.8 200 3901.7 400 3834.3 800 3871.0
[0127] The resulting calibration curve is shown in FIG. 4.
[0128] In a comparative test, using an exactly weighed amount of
cocaine in the immunoassay, the yield of cocaine measured after
adsorption and elution referred to cocaine applied amounted to
79%.
EXAMPLE 3
[0129] a) Structure of Carrier (FIG. 1)
[0130] Carrier Fleece (1)
[0131] The polyester fleece was manufactured by Binzer, Hatzfeld,
Federal Republic of Germany. It is a pure polyester fleece,
reinforced with 10% kuralon. The thickness is 1.0-1.2 mm, the
suction capacity amounts to 1800 ml/m.sup.2.
[0132] First Carrier Matrix (Immune Adsorber) (2)
[0133] A mixed fleece consisting of 80 parts polyester and 20 parts
viscose staple fiber, reinforced with 20 parts of kuralon, with a
thickness of 0.32 mm and an adsorption capacity of 500 ml/m.sup.2
is impregnated with the following solution and then dried:
3.8.times.10.sup.-8 mol/l of conjugate consisting of anti-cocaine
antibody and alkaline phosphatase (free first binding partner),
which was manufactured according to example 1d and 3 mmol/l NaCl, 1
mmol/l MgCl.sub.2, 0.1 mmol/l ZnCl.sub.2, 30 mmol/l triethylamine,
pH 7.6.
[0134] Second Carrier Matrix (Capture Matrix) (4)
[0135] A fleece consisting of 100% linters, reinforced with 20%
etadurin having a thickness of 0.35 mm and an adsorption capacity
of 372 ml/m.sup.2 is impregnated with the following solution and
then dried: 10 mmol/l sodium phosphate, pH 7.5, polymerized
streptavidin 200 mg/l (manufactured according to example 1c, EPS 0
331 127).
[0136] The preimpregnated fleece is then again impregnated and then
dried: 10 mmol/l sodium phosphate, pH 7.5, 200 mg/l biotinylated
cocaine polyhapten according to example 1c.
[0137] Third Carrier Matrix (Detection Field) (5)
[0138] A fleece made of 100% linters, reinforced with 2% etadurin
and a thickness of 0.35 mm and a suction capacity of 372 ml/m.sup.2
is used.
[0139] Carrier matrices were glued onto a carrier foil of 6 mm in
width as shown in FIG. 1. The carrier foil has an aspiration
opening (3) of3 mm in diameter, which is disposed in the center
below the first carrier matrix (immune adsorber).
[0140] b) Measuring Cocaine from the Gas Phase
[0141] The carriers were placed into a support featuring a guide
for a tube with an O-ring to seal it. As described in example 2b,
the tube is connected to a vacuum pump. While the gas sample is
collected, the tube with the O-ring below the first carrier matrix
is pressed against the carrier foil such that the sample gas flows
through the first carrier matrix and the opening in the carrier
foil.
[0142] Cocaine gas with a saturation of 0.1%, i.e. 2 pg/l or
6.6.times.10.sup.-15 mol/l of cocaine (manufactured according to
example 2b) is aspirated across the first carrier matrix (immune
adsorber) of the carrier at a flow rate of 2 l/min for a period of
15 sec (total amount: 1 pg) and for 2.5 min (total amount: 10
pg).
[0143] Cocaine gas with a saturation of 10%, i.e. 200 pg/1 or
6.6.times.10.sup.-13 mol/l of cocaine (manufactured according to
example 2b) is aspirated across the first carrier matrix (immune
adsorber) of the carrier at a flow rate of 1 l/min for a period of
30 sec (total amount: 100 pg) and at a flow rate of 10 l/min for 30
sec (total amount: 1000 pg).
[0144] Then, the carrier is removed from the support and the
carrier fleece is immersed in the buffer solution (150 mmol/l NaCl,
50 mmol/l potassium phosphate buffer, pH 7.2) for a period of 10
sec. The liquid taken up by the carrier fleece chromatographs to
the third carrier matrix (detection field). Using a DIN 821
puncher, a circular part is punched out of the third carrier matrix
and placed into a well of a microtiter plate with the top of the
fleece facing up. In a luminescence microtiter plate reader
(Luminoscan), 100 .mu.l of substrate solution (Lumiphos 530,
preparation of disodium 3-{methoxyspiro
1,2-dioxetane-3,2'-tricyclo[3.3.1-
.1..sup.3,7]decane}phenylphosphate, Boehringer Mannheim) are added.
The luminescence is measured over a period of 10 min.
[0145] In order to determine the blank value, the carriers are
immersed into buffer solution, allowed to chromatograph and
analyzed without cocaine gas being aspirated
[0146] Measurement Results
2 Amount of cocaine applied Luminescence [pg] [rel. units/60 sec]
Blank 60.46 1 76.86 10 106 100 134.4 1000 244.7
[0147] The resulting calibration curve is shown in FIG. 5.
EXAMPLE 4
[0148] Instead of the cocaine gas used in the test stand described
in connection with example 2b, a gas containing cocaine particles
is aspirated onto the first matrix (immune adsorber) from example
2a and/or the carrier of example 3. Samples were taken at 1 cm
above a polyethylene surface which had previously been contaminated
with a powder-like mixture of 1 part of cocaine and 1000 parts of
lactose. 5 mg of this mixture were spread over surface area of 220
cm.sup.2. While the samples were taken, the surface was also
exposed to a pressurized air current of 5 l/min generated by a
nozzle with approximately 1 mm in diameter from a distance of
approximately 1 cm in order to support the removal of particle-like
contamination. All tests produced measurements corresponding to a
cocaine amount of smaller than 1 ng.
* * * * *